Characterization and Development of Optimization Strategy for the Processing of

Allogenic and Xenogenic Bone and Pericardium

Submitted to

The Faculty of Engineering at the Friedrich-Alexander University of

Erlangen-Nuremberg

to obtain the degree

DOKTOR-INGENIEUR

presented by

Mohannad Qasim Mustafa Marashdeh

Erlangen 2007

As dissertation approved by The Faculty of Engineering Science of the Friedrich-Alexander University of Erlangen-Nuremberg

Day of submission: 17.04.2007

Day of examination: 06.06.2007

Dean: Prof. Dr.-Ing. A. Leipertz

Examiners: Prof. Dr. R. Buchholz

Prof. Dr. P. Greil

Charakterisierung und Entwicklung einer Optimierungsstrategie für die Prozessierung von allogenen und xenogenen Knochen und Perikard

Der Technischen Fakultät

der Friedrich-Alexander Universität Erlangen-Nürnberg

zur Erlangung des Grades

DOKTOR-INGENIEUR

vorgelegt von

Mohannad Qasim Mustafa Marashdeh

Erlangen 2007

Als Dissertation genehmigt von der Technischen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der Einreichung: 17.04.2007

Tag der Promotion: 06.06.2007

Dekan: Prof. Dr.-Ing. A. Leipertz

Berichterstatter: Prof. Dr. R. Buchholz

Prof. Dr. P. Greil

Acknowledgment

Acknowledgment I would like to express my gratitude to all those who gave me the possibility to complete this thesis. First of all, I want to thank Prof. Rainer Buchholz and Prof. Thomas Neeße for their constant guidance, support and encouragement. I am deeply indebted to my supervisor Dr. Roman Breiter whose motivations and stimulating suggestions helped me during the research and writing phases of this thesis. I would like to thank the Examination board (Prof. Greil and Prof. Pischetsrieder) for their valuable criticism and evaluating the present work. Special thanks to the company Tutogen Medical GmbH for the financial support during this thesis. My sincere thanks go to Dr. Dueck and Dr. Georgiadis for their valuable advices and support during the work. I’m also grateful for Silke Schwarz, Ludwig Körber and Ana Herakovic for the helpful collaboration during the research phase. I would like to thank the staff of LUR and BVT Erlangen as well as of Tutogen Medical GmbH for their help and support. I am deeply indebted to those who have participated in reviewing the present work, especially Khaled Abderrzaq and his wife Rana Al-Rabei. Finally, I would like to express my deepest, warmest and endless gratitude to my parents, brothers and sisters for their patience, enthusiastically supporting and unlimited encouragement

Abstract

I

Abstract Allografts and xenografts are used as alternatives to autografts, however the concern

about the immunological reaction and the transmission of host diseases are the main

limitations coupled with the use of these grafts. Therefore these grafts have to be

preprocessed before being used. Unfortunately, the preprocessing treatments could

destruct the biological and structural integrity of the tissues. Tutoplast® process is a

comprehensive process for the conservation of the allo- and xenografts.

During this work, the influence of Tutoplast process on the stability of collagenous

tissues was examined. For this purpose, measurements of the fraction of denatured

collagen (DC), measurements of isotonic shrinkage temperature, SDS-PAGE

investigations and the mechanical properties were used to evaluate the quality of the

tissues.

It was proved that the processing induces certain structural destruction or worsening of

the quality of tissues. Therefore, it was reasonable to follow the contribution of each step

in the process in this destruction.

It was found that the 1 N NaOH treatment in the process induces amino acids

modification yielding tissues with lower thermal stability, however this could be

reversible. It was observed that treating the tissues with 1 N CH3COOH is not suitable to

restore the tissues to their physiological state. The best variant was to treat the tissues

with 0.1 N CH3COOH followed by 1-2 10-min water baths.

The 3 % H2O2 had little effect on the quality of the tissues. Furthermore, 10 % H2O2

could be used to guarantee the oxidation of soluble proteins and the inactivation of

viruses without almost further worsening of the quality of tissues.

The pure acetone treatment used in the process was found to be more effective than the

graded acetone treatment as dehydrating agent; however the 2-week treatment is too long.

It was observed that the tissues were fully dehydrated after 2 days. Further treatment with

acetone leads to avoidable volume shrinkage of the tissues.

The structural heterogeneity and the fiber orientation were dominant during the

characterization of the mechanical properties, which made the analysis of the results

complicated.

Summary

II

Summary The autograft is considered as the gold-standard graft in the medical field because it

contains viable cells and growth factors, which stimulates the healing of the graft.

However, the limited availability and the additional morbidity are the main disadvantages

associated with the transplantation of autografts. Therefore, alternative grafting materials

have been always used to fulfill the increasing demand for grafts in the medical field.

Allografts and xenografts are used as alternatives to autografts, however the concern

about the immunological reaction and the transmission of host diseases are the main

limitations coupled with the use of these grafts. Therefore these grafts have to be

preprocessed before being used. Unfortunately, the preprocessing treatments could

destruct the biological and structural integrity of the tissues.

The present work aims to examine the possibilities to optimize a process for the

conservation and processing of bone and soft tissue allo- and xenografts (the Tutoplast®

process). In order to perform an optimization of the process, first the influence or the

modifications induced by the process were defined carefully. Second, the effect of each

step of the process on the stability of the tissues was studied separately. Finally, time-

concentration modifications or alternative steps were evaluated.

During this work, measurements of the fraction of denatured collagen (DC) after

selective enzymatic digestion technique, measurements of isotonic shrinkage

temperature, SDS-PAGE investigations and the mechanical properties were used to

evaluate the quality of the tissues.

The bovine bones, used in this work, were first pulverized under liquid nitrogen to

accelerate the demineralization of the bones, which is necessary before the enzymatic

digestion of bone samples. The bone powder from three different types of mills was

examined based on the measurements of DC. The results showed that the ball mill

induced significantly the least destruction to the collagen structure in comparison to the

micro-dismembrator and milling machine. Interestingly, the damage caused by micro-

dismembrator and milling machine is reversible after 1-week storage at 8 °C. It is

expected that the triple helix is unfolded but the polypeptide chains are still fixed in their

positions, which enable the recovery of the native triple helix by building hydrogen

bonds.

Summary

III

The thermal stability of the collagenous tissues was considered as a crucial assessment

parameter because it is sensitive to any structural destruction or modification. The

thermal denaturation of collagen induces unfolding of triple helix into random coils by

breaking the hydrogen bonds; the ability of collagen to resist this unfolding is an

indication of its “healthiness”. The tissues were treated thermally in the range of (55-200

°C) for 1 h in furnace and then incubated with α-chymotrypsin to determine the fraction

of denatured collagen (DC).

The DC for Tutoplast-processed bovine cancellous bone remained unchanged till the

temperature 90 °C, and then it started to increase linearly with increasing temperature.

Regarding the thermal stability of bovine pericardium, the measurements of DC showed

higher thermal stability of the native lyophilized (initial water content 7%) and Tutoplast-

processed pericardium (initial water content 1.7%) in comparison with the native

pericardium (initial water content 85%). The DC for native lyophilized and Tutoplast-

processed pericardium remained unchanged until 135 and 150 °C respectively, whereas

for the native pericardium, it started to increase from 55 °C. This could be attributed to

the water content, according to the polymer in a box mechanism; dehydration reduces the

lateral dimensions of the lattice, constrains the number of possible configurations,

reduces the free-volume available for denaturating α-chains, reduces the configuration

entropy and thereby increases the thermal stability of collagen.

A reduction of DC at high temperatures (185-200 °C) has been observed with the

Tutoplast-processed and the lyophilized pericardium but not with the native pericardium.

This could be attributed to the formation of heat generated advanced glycation end

products (AGEs), which hinders the enzymatic digestion of collagen. The

spectrophotometric measurements of the extent of browning proved the formation of

AGEs. The absence of AGEs with the native pericardium could be ascribed to the higher

moisture content that delays the formation of AGEs.

The dominance of water content during the measurements of DC made it necessary to

exclude the effect of the water content by performing measurements of isotonic shrinkage

temperature at fully hydrated conditions in water bath, in which only the structure

integrity and healthiness plays a role in shaping the thermal stability.

Summary

IV

It was seen obviously the Tutoplast-processed pericardium has lower thermal stability or

thermoelasticity to resist the thermal shrinkage as indication of structural modification or

destruction caused by the processing. The Tutoplast-processed started to shrink from 42

°C, whereas the native lyophilized and the native pericardium from 64 and 65 °C

respectively. Furthermore, the shrinkage process of Tutoplast-processed strips was slow

and took place over relatively wide temperature range (42-70 °C), in comparison to that

of the lyophilized and the native pericardium that took place with 5 degrees. The

presence of residual ions in the Tutoplast-processed pericardium resulted from the

processing, which was confirmed by the measurements of the conductivity, resulted in

swelling of the tissues during the shrinkage process and consequently to thick samples,

which shrunk too slowly.

The analysis of the DC and shrinkage temperature measurements draws the conclusion

that the Tutoplast-process induces two contradictory factors, stabilizing factor,

represented by the dehydration that dominant under dry conditions, and destabilizing

factor, which may represented by structural modification that becomes visible under fully

hydrated conditions. The next challenge was to investigate the role or the effect of each

step of Tutoplat process and its contribution in the structural modification caused by the

process.

The sodium hydroxide treatment in the process is used as a protection against creutzfeldt-

Jakob disease and is scientifically recognized as an acceptable and effective methodology

for reducing prion infectivity by six log. The effect of the sodium hydroxide treatment on

the stability of bovine pericardium process was examined. It was proved the amount of

dissolved or hydrolyzed collagen after 150 min 1 N NaOH treatment at room temperature

was not significant and lower than 1 % of the original dry weight this could be attributed

to the fact that NaOH treatment doesn’t destroy the helical structure of collagen. It was

shown that 1 N NaOH treatment induces swelling of the pericardium tissues,

modification of some amino acids and destruction of intra-and intermolecular collagen

cross-links leading to significantly lower shrinkage temperature, approximately 25

degrees lower than the native untreated samples. Furthermore the shrinkage of the

NaOH-treated strips was too slow and occurred over wide temperature range,

Summary

V

approximately 30 degrees, because the samples are thick and swelled from the action of

the alkali, which prevented smooth shrinkage of the samples.

In order to treat or to remove the effect caused by the NaOH treatment, several treatment

possibilities have been tested. The aim of this treatment was to restore the pericardium

strips to their physiological state and pH. The volume of NaOH as well as of CH3COOH

was chosen to have dully submerged strips, for example, for the treatment of 10 and 5

strips 100 and 50 ml were used respectively. It is seen that the 1 N CH3COOH treatment

shifts the pH value of the strips too rapidly from the basic to the acidic region causing

damage also. 5 and 15 min treatment was enough to shift the pH to 5 and 3 respectively

yielding pericardium strips with thermal shrinkage starting at 50 and 45 °C respectively.

Therefore it was reasonable to test the effect of lower concentrations of CH3COOH. The

15-min treatment with 0.1 N CH3COOH shifted the pH value of the pericardium strips to

6, resulting in thermal shrinkage starts at 62 °C, which was close to that of the native

untreated pericardium.

Also as alternative to the acid treatment, the efficiency of different washing or rinsing

fluids, such as distilled water and phosphate buffer (pH 7.4) has been tested. It is

observed that submerging or washing the NaOH-treated samples once with water or

phosphate buffer is not sufficient to reduce the pH value of the samples due to the limited

capacity of the water or buffer to wash the ions. A complete neutralization of the samples

could be achieved by intensive washing with distilled water baths for 90 min, in which

the water bath has to be changed every 10 or 15 min.

The best variant has been achieved by treating the NaOH treated strips with 1 N

CH3COOH for 15 min followed by one or two 10-min distilled water washing bath. With

this variant, the pericardium strips has a final pH value 8 and a thermal shrinkage starts at

64 °C.

The measurements of DC show no significant influence of the NaOH treatment. This

could be attributed to the fact that α-chymotrypsin is not active to digest collagen at

highly alkaline conditions and only active at neutral to slightly alkaline ranges

The hydrogen peroxide step in the process has been found to be effective against the

human immunodeficiency virus (HIV). Through this treatment, soluble proteins are

Summary

VI

eliminated, remaining viruses are inactivated and the potential for graft rejection is

minimized. The effect of H2O2 on the stability of the pericardium was studied.

It was seen that the treatment with 3% H2O2 (pH = 5.52) is almost not destructive to the

collagen structure. This was confirmed by the high thermal stability assessed by the

measurements of isotonic shrinkage temperature. The samples treated with 10% H2O2

(pH = 4.13) had almost similar thermal stability to those treated with 3% H2O2. In

contrast to the 3 and 10% treatment, the treatment with 30% (pH = 2.40) was extreme

destructive and resulted in completely destructed pericardium strips, which can’t be

further tested with the isotonic shrinkage technique. Hydrogen peroxide is a relatively

nonspecific oxidizing agent, under acidic conditions the primary reaction is the

conversion of methionine residues to sulfoxide. Oxidation of Met residues is associated

with the loss of the biological activity for many proteins. It is expected the treatment with

H2O2 under mild concentrations and pH doesn’t lead to complete oxidation of the Met

residues and consequently to the complete destruction of the collagen, which was

observed at extreme concentration and pH.

Regarding the DC measurements, as discussed with the NaOH treatment, α-chymotrypsin

couldn’t attack the tissues under acidic conditions because it is inactive in this pH range.

Acetone treatment is used in Tutoplast process to inactive the remaining prion and

viruses and to dehydrate the tissues. The effect of acetone treatment on the stability of

pericardium was examined. In the current study the pure acetone was compared with the

graded acetone. After 18 days, both of them have almost the same weight loss and

shrinkage, however with different curve course. The pure acetone is much more effective

to dehydrate the tissues; the maximum weight loss has almost been achieved within the

first two days, whereas the maximum weight loss has been reached at the tenth day

during the graded acetone treatment.

Regarding the shrinkage, during the first 2 days of the pure acetone treatment, the tissue

shrunk 22.68% of their volume. Further shrinkage was also observed until the maximum

shrinkage reached at the sixteenth day, 52.91%.

The functions of the acetone treatment in the Tutoplast-process are tissue dehydration and

inactivation of any prions and viruses. Therefore it is recommended to check if the 2-day

pure acetone treatment is sufficient to inactivate prions and viruses.

Summary

VII

The acetone treatment has no significant influence on the collagen denaturation. The DC

values of pure acetone as well as graded acetone treated samples are almost similar to

those of the native untreated samples.

The ideal grafting material should not only be adequately osteogenic, -conductive, and -

inductive but also mechanically stable and disease free. Therefore the effect of the

processing on the mechanical properties of the tissues was examined.

The analysis of ultimate strength and elastic modulus values after thermal treatments of

bone cubes gives no clear relationship or statement about the temperature-dependency of

the mechanical properties of the bone samples. The influence of the structure

heterogeneity (non-uniform mineral distribution) and the fiber orientation could be

behind the scattering of the results and the absence of convenient statement about the

mechanical properties of bones.

The analysis of the mechanical properties of pericardium did not lead to any conclusion

about the influence of the processing of the mechanical stability. It was expected, despite

the separation between the left and the right side of the sac, that the effect of anisotropy

was dominant over the influence of the processing. It can be concluded that only SALS-

selected samples can be used to assess the effect of the processing on the mechanical

properties of the pericardium.

Nomenclature

VIII

Nomenclature Symbol Description Units

G∆ Gibbs free energy kJ/mol

H∆ van’t Hoff enthalpy kJ/mol

S∆ Entropy of transition kJ/mol.K

A The frequency factor sec-1

B Heating rate parameter -

DC Fraction of denatured collagen %

E The activation energy kJ/mol

k The kinetic first order constant sec-1

K The equilibrium constant -

/K The apparent first order constant sec-1

L The length of the fiber or tissue mm

0L The initial length of the fiber mm

∞L The length of the completely shrunken fiber mm

l The length of the collagen molecule mm

Dl The length of the denatured collagen molecule mm

Nl The length of the native collagen molecule mm

AAm Mass of aromatic amino acids µg

Cm Collagen mass µg

n The number of collagen molecules -

Nn The number of native collagen molecules -

Dn The number of denatured collagen molecules -

q The heating rate °C/min

R The ideal gas constant J/K. mol

2/1t The time of half shrinkage sec

∞t The time of maximum shrinkage sec

x Temperature parameter -

UX The fraction of unfolded collagen %

Nomenclature

IX

Symbol Description Units

FX The fraction of final denatured collagen %

NX The fraction of native collagen %

SX The fraction of collagen in the supernatant %

z Adaptation parameter -

Abbreviations Symbol Description

AGEs Advanced Glycation End Products

BSE Bovine Spongiform Encephalopathy

CEL Carboxyethyl)lysine

CJD Creutzfeldt - Jakob disease

CML Carboxymethyllysine

DBM Demineralized Bone Matrix

DHLN Dihydroxylysinonorleucine

DSC Differential Scanning Calorimetry

Gly Glycine

HA Hydroxyapatite

HIV Human Immunodeficiency Virus

HLKNL Hydroxylysino-5-ketonorleucine

HLN Hydroxylysinonorleucine

HP Hydroxylysyl Pyridinoline

Hyp Hydroxyproline

LKNL Lysino-5-ketonorleucine

LP Lysyl Pyridinoline

MOLD Methylglyoxal-Lysine Dimer

PE Polyethylene

PTFE Polytetrafluoroethylene

rER Rough Endoplasmic Reticulum

Table of contents

X

1 Introduction................................................................................................................. 1

2 State of the art ............................................................................................................. 2

2.1 Bone grafting ...................................................................................................... 2

2.1.1 Graft types................................................................................................... 2

2.1.1.1 Autograft ................................................................................................. 2

2.1.1.2 Allograft.................................................................................................. 2

2.1.1.3 Xenograft ................................................................................................ 3

2.1.1.4 Alloplastic ............................................................................................... 3

2.1.2 Bone healing ............................................................................................... 4

2.1.3 Graft processing .......................................................................................... 6

2.1.3.1 Graft Processing Techniques used in the Medical Field......................... 6

2.1.3.2 Tutoplast® process ................................................................................. 7

2.2 Collagen ............................................................................................................ 10

2.2.1 Collagen types........................................................................................... 10

2.2.2 Collagen Synthesis.................................................................................... 12

2.2.3 Collagen Cross-links................................................................................. 14

2.2.4 The role of hydroxyproline in collagen stabilization................................ 16

2.2.5 The Thermal stability of collagen ............................................................. 18

2.2.6 Advanced Glycation End Products (AGEs).............................................. 19

2.3 Bone .................................................................................................................. 20

2.3.1 Bone Composition .................................................................................... 20

2.3.2 Bone Hierarchy ......................................................................................... 21

2.4 Pericardium....................................................................................................... 23

3 The Objectives .......................................................................................................... 26

4 Materials and Methods.............................................................................................. 27

4.1 Materials ........................................................................................................... 27

4.1.1 Bovine Bones ............................................................................................ 27

4.1.2 Bovine Pericardium .................................................................................. 28

4.2 Methods............................................................................................................. 29

4.2.1 Preparation Steps ...................................................................................... 29

Table of contents

XI

4.2.1.1 The pulverization of the bones.............................................................. 29

4.2.1.2 The Demineralization of Bones ............................................................ 30

4.2.2 The Determination of Denatured Collagen (DC)...................................... 30

4.2.2.1 A Selective Digestion Method .............................................................. 31

4.2.2.2 Spectrophotometeric Determination of the DC .................................... 32

4.2.3 The Measurements of the Extent of Browning ......................................... 34

4.2.4 The Measurements of the Isotonic Shrinkage temperature....................... 34

4.2.5 SDS-PAGE ............................................................................................... 35

4.2.6 Characterization of the Mechanical Properties ......................................... 39

4.2.7 The measurements of the Thermal Conductivity...................................... 39

4.3 Different physical and chemical treatment ....................................................... 39

4.3.1 The thermal treatment of collagenous tissues........................................... 40

4.3.1.1 The thermal treatment of bovine bone .................................................. 40

4.3.1.2 The thermal stability of bovine pericardium......................................... 40

4.3.2 The Sodium hydroxide treatment and the corresponding neutralization .. 41

4.3.2.1 Sodium hydroxide treatment................................................................. 41

4.3.2.2 The neutralization of the tissues after the NaOH treatment.................. 42

4.3.3 The hydrogen peroxide treatment ............................................................. 43

4.3.4 The acetone treatment ............................................................................... 43

4.3.5 The determination of water content .......................................................... 44

5 Results Interpretations .............................................................................................. 45

5.1 The Effect of Bone Pulverization on the Collagen ........................................... 45

5.1.1 The Measurements of DC ......................................................................... 45

5.1.2 Discussion of the Results .......................................................................... 46

5.2 The Thermal stability of Collagenous Tissues.................................................. 47

5.2.1 Analysis of the Thermal Stability with the Measurements of DC............ 47

5.2.1.1 The Thermal Stability of Tutoplast-Processed Bovine Bone ............... 47

5.2.1.2 The Thermal Stability of Native Bovine Pericardium .......................... 48

5.2.1.3 The Thermal Stability of Tutoplast-Processed Bovine Pericardium .... 49

5.2.1.4 The Thermal Stability of the Lyophilized Bovine Pericardium............ 50

5.2.1.5 The Measurements of the Extent of Browning ..................................... 51

Table of contents

XII

5.2.1.6 Discussion of the Results ...................................................................... 52

5.2.1.7 Modeling of the Thermal Denaturation of Pericardium ....................... 59

5.2.2 Measurements of Isotonic Shrinkage Temperature .................................. 65

5.2.2.1 The Results............................................................................................ 66

5.2.2.2 Discussion of the Results ...................................................................... 66

5.2.2.3 Modelling of the Thermal Shrinkage of Pericardium........................... 68

5.2.3 SDS-PAGE investigations ........................................................................ 78

5.2.3.1 Results................................................................................................... 78

5.2.3.2 Discussion ............................................................................................. 81

5.3 The Effect of Different Steps in the Tutoplast® Process.................................. 83

5.3.1 The Effect of Sodium Hydroxide Treatment ............................................ 83

5.3.1.1 The Hydrolysis of Collagen Amino Acids............................................ 84

5.3.1.2 The Measurements of the Shrinkage Temperature ............................... 86

5.3.1.2.1 The effect of NaOH Solution.......................................................... 86

5.3.1.2.2 The Effect of the Neutralization Step ............................................. 87

5.3.1.3 The Measurements of DC ..................................................................... 91

5.3.1.4 SDS-PAGE Investigations .................................................................... 92

5.3.1.5 Discussions ........................................................................................... 94

5.3.2 The Effect of Hydrogen Peroxide Treatment............................................ 98

5.3.2.1 The Shrinkage Temperature Measurements ......................................... 98

5.3.2.2 Measurements of DC ............................................................................ 98

5.3.2.3 SDS-PAGE Investigations .................................................................... 99

5.3.2.4 Discussion of the Results .................................................................... 100

5.3.3 The Influence of Acetone Treatment ...................................................... 101

5.3.3.1 The Extent of Drying and Shrinkage .................................................. 102

5.3.3.2 The Measurements of DC ................................................................... 103

5.3.3.3 Discussion of the Results .................................................................... 104

5.4 The Mechanical Properties of the Collagenous Tissues ................................. 105

5.4.1 The Mechanical Properties of Bovine Bones.......................................... 105

5.4.2 The Mechanical Properties of Bovine Pericardium................................ 106

5.4.3 Discussion of the Results ........................................................................ 107

Table of contents

XIII

6 Optimization of Tutoplast Process.......................................................................... 110

6.1 The Sodium Hydroxide treatment................................................................... 110

6.2 The Hydrogen Peroxide treatment .................................................................. 111

6.3 The Acetone treatment.................................................................................... 111

7 Literature................................................................................................................. 112

1. Introduction

1

1 Introduction Transplantation has become world wide one of the most important innovations in the

medical field. Organs save lives and tissues improve the quality of the life for millions of

people. A single donor can help more than 50 persons awaiting an organ or transplant.

Bone and tissue donations give a new meaning to life for many patients each year. While

most people are familiar with organ donor programs, tissue donation is a relatively new

concept.

Various grafting material including autografts, allografts, xenografts and synthetic

materials have been used clinically. The autograft is considered as the gold-standard graft

because it contains viable cells and growth factors, which stimulate the healing of the

graft. However, the limited availability and the additional morbidity are the main

disadvantages associated with the transplantation of autografts. Therefore, alternative

grafting materials have been always used to fulfill the increasing demand for grafts in the

medical field.

Allografts are the surgeon’s second choice, however the concern about the

immunological reaction and the transmission of host diseases are the main limitation

coupled with the use of Allografts.

Several preprocessing treatments have been applied to deal with allografts limitations.

However, achieving the two goals, suppressing the immunological reactions and avoiding

the transmission of host diseases, requires a combination of different preprocessing

treatments. It was proved that the preprocessing treatments could destruct the biological

and structural integrity of the tissues.

Tutoplast® process is a comprehensive and validated conservation and sterilization

process, been used for 30 years, aims to eliminate the antigenicity of allografts as well as

the possibility of disease transmission without affecting the mechanical and biological

properties of the tissues. The process deactivates, destroys and removes all unwanted

materials, such as fats, cells, viruses and microbes.

In this dissertation, the influence of the Tutoplast® process on the thermal and

mechanical properties of the tissues have been assessed in order to establish a

background profile for the possible future plans of the process optimization.

2. State of the Art

2

2 State of the art 2.1 Bone grafting Bone grafting is a surgical procedure where the bone is taken from donor site and

implanted into the patient to improve the function or strengthen a damaged part. Bone

graft is the second most common transplantation tissue after blood (Boyce T 1999). More

than 500,000 bone grafting procedures are happening annually in the United States and

2.2 million worldwide in order to repair bone defects in orthopaedics, neurosurgery and

dentistry (Lewandrowski, D. Gresser et al. 2000). Bone-grafting is usually required to

stimulate bone-healing. In addition, spinal fusions, filling defects following removal of

bone tumors and several congenital diseases may require bone grafting (Giannoudis,

Dinopoulos et al. 2005).

2.1.1 Graft types

There are four classes of bone grafts (Hoexter 2002): autograft, allograft, xenograft and

alloplastic.

2.1.1.1 Autograft

The autograft is defined as the tissue transplanted from one site to another within the

same individual. The gold standard of bone-grafting is harvesting autologous cortical and

cancellous bone from the iliac crest (Giannoudis, Dinopoulos et al. 2005). Although

autologous bone can be harvested from the tibia, fibula, olecranon, distal radius, and ribs,

the iliac crest remains the most common donor site (Laurie, Kaban et al. 1984). Despite

the best success rates in bone grafting achieved with autografts, harvesting autologous

bone from the iliac crest has, however, several downsides as it lengthens the overall

surgical procedure and is usually complicated by residual pain and cosmetic

disadvantages (Summers and Eisenstein 1989; Giannoudis, Dinopoulos et al. 2005).

Furthermore, it may fail in clinical practice as most of the cellular (osteogenic) elements

do not survive transplantation (Sandhu, Grewal et al. 1999).

2.1.1.2 Allograft

Allografts are tissues taken from individuals of the same species as the hosts. Allograft is

the most frequently chosen bone substitute and is regarded as the surgeon's second option

(Carter 1999). Its use has increased 15-fold during the past decade and accounts for about

one-third of bone grafts performed in the United States (Boyce T 1999). The concern

2. State of the Art

3

about the immunological response and the transmission of host diseases is the main

obstruction to use allografts. The processing of allograft tissue lowers the risk of

transferring viral diseases but, that can significantly deteriorate the biological and

mechanical properties of the graft (Bos, Goldberg et al. 1983; Giannoudis, Dinopoulos et

al. 2005).

2.1.1.3 Xenograft

The xenograft is defined as a tissue taken from other species (i.e. bone of bovine origin).

Like allografts, xenografts are available in large amounts but have to be preprocessed to

avoid the immunological reactions.

2.1.1.4 Alloplastic

Alloplastic materials, synthetic materials, offer the potential for "off-the-shelf" solutions

to reconstructive tissue needs (replacement and/or augmentation), which avoid donor

scars and morbidity and typically simplify the operative procedure in terms of time and

complexity of technique (Eppley 1999). The wide range of implantable synthetic

materials can be divided into a few categories: carbon-based polymers, non–carbon-based

polymers, metals, and ceramics (MacRae 2005). The chemical composition, physical

form, and differences in surface configuration result in varying levels of bioresorbability

(Hoexter 2002). MacRae (MacRae 2005) has evaluated the application of alloplastic

grafts:

Non-carbon-based polymers

The silicone, as a non–carbon-based polymer, is highly resistant to degradation and has a

high degree of chemical inertness due to its silicon-oxygen bonds. Silicone implants

retain their strength and flexibility though a wide range of temperatures and easily can be

sterilized. However, silicone has a tendency to fragment and deteriorate, when subjected

to repeated movement with mechanical loading. The solid silicone is considered as inert,

however, in the liquid or gel form, the silicone is not as inert and can incite a chronic

inflammatory reaction. As with other alloplastic implants, one complication of silicone

implants is extrusion.

Carbon-based polymers

Carbon-based polymers have been used in various forms and include

polytetrafluoroethylene (PTFE), polyethylene (PE), aliphatic polyesters, and

2. State of the Art

4

methylmethacrylate. PTFE is nonresorbable and highly biocompatible with no tendency

for chronic inflammatory reaction, whereas the aliphatic polyesters are used extensively

for the advantage of being resorbable. In general, the surgical requirement or need

determine if the material should be resorbable or not.

Metals

Metals have been used in maxillofacial plating for their strength and durability. Stainless

steel no longer is used due to its tendency to corrode. Titanium, an elemental metal, has

had no reports of allergy, toxicity, or tumorigenesis.

Ceramics

Hydroxyapatite (HA) is calcium phosphate salt, the principal inorganic compound in

bone matrix. It can be produced synthetically in a dense form, but this dense form of HA

was found to be difficult to shape and prone to migration and extrusion. Porous forms of

HA have been much more successful and are based on the calcium carbonate structure of

marine corals. Their porosity permits fibrovascular and osseous ingrowth. However, HA

implants cannot tolerate significant load bearing and tend to crack and fracture.

One major limitation of alloplastic implants is their susceptibility to infection. A variety

of factors contribute to this risk, including the location of the implant, vascularity of the

pocket, operative technique in handling and placing the implant, and the ability of

bacteria to adhere to and penetrate the material.

In general, it is not easy to decide which graft type is better; the decision must be taken

for each case separately depending on the surgical requirement and the patient medical

history.

2.1.2 Bone healing

Bone is unique tissue because it is continuously metabolically active and thus subject to a

variety of systemic and local factors throughout life (Pilitsis, Lucas et al. 2002). Bone

consists of the following bone cells (Felsenberg 2001; Pilitsis, Lucas et al. 2002):

osteoblasts, osteoclast and osteocytes, which are essential for the bone formation process

Osteoblasts

Osteoblasts or the bone-forming cells synthesize and secrete unmineralized ground

substance, the osteoid, and found in areas of high metabolism within the bone (Van De

Graaff 1998). Osteoblasts are involved in matrix development as well as calcification [3],

2. State of the Art

5

As the process of bone deposition ends, some osteoblasts remain on the periosteum and

endosteum, whereas others become osteocytes (Kalfas 2001).

Osteoclasts

Osteoclasts or the bone resorbing cells are very important for bone growth, healing, and

remodeling. Osteoclasts secrete proteolytic enzymes that able to dissolve both the

inorganic and organic osseous matrices, resulting in the formation of erosive pits called

Howship lacunae and the release of calcium and phosphate (Kalfas 2001).

Osteocytes

Mature bone cells, made from osteoblasts, maintain healthy bone tissue by secreting

enzymes and controlling the bone mineral content. They also control the calcium release

from the bone tissue to the blood.

The ability of bone healing and fusions formation by transplantation of bone grafts is

based on three key concepts (Pilitsis, Lucas et al. 2002): osteogenesis, osteoconduction

and osteoconduction.

Osteogenesis

Osteogenesis, defined as the ability to produce new bone, is determined by the presence

of osteoprogenitor cells and osteogenic precursor cells in the area. Both fresh autografts

and bone marrow cells contain osteogenic cells.

Osteoconduction

Osteoconductive properties are determined by the presence of a scaffold that allows for

vascular and cellular migration, attachment, and distribution (Helm, Dayoub et al. 2001).

Osteoconduction may be achieved through the use of autografts, allografts, demineralized

bone matrix (DBM), hydroxyapatite, and collagen.

Osteoinduction

Osteoinduction is defined as the ability to stimulate stem cells to differentiate into mature

cells through stimulation by local growth factors (Subach, Haid et al. 2001). Bone

morphogenetic proteins and DBM are the most potent osteoinductive materials, although

allo- and autografts have some osteoinductive properties (Kalfas 2001).

2. State of the Art

6

The terms osteogenic, osteoconductive, and osteoinductive are not absolute, and are best

understood when used in the context of a comparative study (Bauer and Muschler 2000).

The ideal grafting material should be not only osteogenic, -conductive, and -inductive but

also mechanically stable and disease free (Kalfas 2001; Pilitsis, Lucas et al. 2002)

Autografts possess these properties. However, their use is limited by the morbidity

associated with the process of obtaining them and by the often-insufficient amount of

graft (Helm, Dayoub et al. 2001). Allografts are osteoconductive, weakly osteoinductive,

not osteogenic (Pilitsis, Lucas et al. 2002).

2.1.3 Graft processing

As mentioned in the previous section, the use of allograft and xenograft is restricted by

the concern about the immunological reactions and the transmission of host diseases.

Bone allografts are different from most solid organ transplants in that cells are removed

intentionally as thoroughly as possible to minimize immunologic rejection (Bauer and

Muschler 2000). Many experimental studies have shown that in general, bone graft

materials show optimum incorporation with the host when histocompatibility differences

are minimized by either matching tissue types, or processing the allografts with

techniques that reduce immunogenicity (Bonfiglio and Jeter 1972; Blives 1975;

Goldberg, Powell et al. 1985). The osteoinductive, osteoconductive, and biomechanical

properties of different allografts vary depending on the methods of graft processing

(Bauer and Muschler 2000; Hofmann, Konrad et al. 2005).

2.1.3.1 Graft processing techniques used in the medical field

Several studies showed that deep-frozen or freeze-dried allogeneic tendons could be used

without invoking an immunological reaction (Shino, Inoue et al. 1988; Horibe, Shino et

al. 1991; Toritsuka, Shino et al. 1997). However, the transmission of viral diseases with

allografts processed by these methods is not excluded (Fideler, Vangsness et al. 1994).

Regarding the risk of disease transmission, such as hepatitis and human

immunodeficiency virus (HIV), a careful donor-screening has to be performed but some

diseases can be undetectable during the incubation period (Steelman 1994). Therefore

secondary sterilization, such as γ-irradiation is desirable for safer clinical use of allografts

(Toritsuka, Shino et al. 1997). Unfortunately high levels of gamma irradiation are

required to inactivate viral contamination. Fideler et al. (Fideler, Vangsness et al. 1994)

2. State of the Art

7

found that some HIV-infected human patellar tendon-bone allografts remained infected

when irradiated to levels less than 3 Mrad (30,000 Gy). Several studies showed that deep

freezing as well as freeze drying cause no significant change in the mechanical integrity

of the graft (Pelker, Friedlaender et al. 1984; Woo, Orlando et al. 1986; Paulos, France et

al. 1987), whereas γ-irradiation generates free radicals creating deterioration in the

mechanical properties, especially in a dried condition (Cheung, Perelman et al. 1990;

Maeda, Inoue et al. 1993; Salehpour, Butler et al. 1995). Dose-dependent destruction

caused by gamma sterilization was illustrated (Gibbons, Butler et al. 1991). The

allografts were not significantly affected by 2 Mrad of gamma irradiation but weakened

significantly after 3 Mrad of irradiation. It was also confirmed that freeze dying followed

by gamma irradiation reduces the tensile strength of patellar tendons significantly,

whereas reversing the order, gamma irradiation followed by freeze drying, changes the

tensile strength minimally (Haut and Powlison 1989). This suggests that irradiation of dry

tissue causes severe changes in the mechanical properties due to reduced protection

against the action of oxygen and the lack of water which absorbs energy (Maeda, Inoue et

al. 1993).

2.1.3.2 Tutoplast® process

The Tutoplast® process has been available for over 30 years to sterilize and preserve

tissues utilized in all surgical disciplines, including dentistry, neurosurgery, orthopedics,

ophthalmology, otolaryngology, gynecology, urology and pediatric surgery (Schoepf

2006). In contrast to the deep freezing and freeze drying, which decrease the viral titers

but do not guarantee complete eradication (Jackson, Grood et al. 1988), Tutoplast®

process is a comprehensive sterilization and preservation method that aims to eliminate

the antigenicity of allograft as well as the possibility of disease transmission without

affecting the mechanical and biological properties of the tissues. It was reported that the

human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other viruses are

inactivated during Tutoplast® process (Diringer and Braig 1989; Deinhardt 1991;

Koschatzky and Wolfel 1992). Furthermore, neither viral transmission during clinical use

nor immune response during experimental study has occurred using Tutoplast®

processed dura matter (Stoess and Pesch 1977). Regarding the biomechanical analysis, it

was reported that Tutoplast® processed fascia lata has significantly higher stiffness than

2. State of the Art

8

freeze dried one (Hinton, Jinnah et al. 1992). Maeda et al (Maeda, Inoue et al. 1993) have

examined the effect of gamma irradiation on canine tendons before Tutoplast® process

and after the process. They showed that irradiation before Tutoplast® process affected

the mechanical properties minimally, whereas reversing the order had deep effects on the

mechanical properties.

The process consists of different main steps; the whole process is shown in fig. 2.1:

Figure 2.1: Tutoplast® Process for soft and hard tissues

Processing steps

Donor screening

Delipidization

Osmotic treatment

NaOH treatment

H2O2 oxidation

Acetone dehydration

Primary Packaging

γ sterilization

Hard tissues Soft tissues

Final packaging

Tutoplast® Process

2. State of the Art

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Delipidization

Lipids are removed in an ultrasonic acetone bath. Lipids removal is important because

they may interfere with healing process and form cytotoxic product when irradiated

(Moreau, Gallois et al. 2000). This step also prepares the tissue so that the subsequent

steps could penetrate the graft more effectively.

Osmotic treatment

In this step a series of alternating hypertonic saline and distilled water baths are used.

This step ruptures the cell membrane, kills bacteria, washes out cellular debris and

removes antigens.

Hydrogen peroxide treatment

This step has been found to be effective against the human immunodeficiency virus

(Hinton, Jinnah et al. 1992). Hydrogen peroxide is a relatively nonspecific oxidizing

agent, which reacts with a wide variety of organic compounds. It can modify thioether,

indole, sulfhydryl, disulfide, imidazole and phenolic at the neutral or slightly alkaline

conditions (Manning, Patel et al. 1989). Under acidic conditions the primary reaction is

the conversion of methionine residues to sulfoxide (Neumann and Timasheff 1972).

Oxidation of Met residues is associated with the loss of the biological activity for many

proteins (Manning, Patel et al. 1989). Through this treatment, soluble proteins are

eliminated, remaining viruses are inactivated and the potential for graft rejection is

minimized

Acetone dehydration

The acetone wash, followed by vacuum extraction, dehydrates the tissues allowing them

to be room temperature storable. During this step any residual prions are removed and

enveloped viruses are inactivated.

Sodium hydroxide treatment

The soft tissues, not bones, are received 1 N NaOH for 1 h at room temperature to reduce

the prion infectivity as protection step against Bovine Spongiform Encephalopathy (BSE)

and Creutzfeldt - Jakob disease (CJD). It was confirmed that this step reduces prion

infectivity by six log (Brown, Rohwer et al. 1986).

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After Tutoplast® process, tissues are cut, packed and sterilized using limited-dose

gamma irradiation (17.8-25 kGy) to eliminate any microbial contaminations that may

result from handling and packaging.

2.2 Collagen Collagen is derived from the Greek words kolla and gennan, meaning to produce glue.

The collagens constitute a family of related proteins that are assembled in a variety of

supramolecular structures in extracellular matrices. These structures illustrate how the

basic motif of collagens was utilized to generate a diversity of supramolecular matrix

network to accomplish an equally diverse number of functions in the tissues of

multicellular organisms (Vuorio and De Crombrugghe 1990). Collagen, a fibrous protein,

is the most abundant protein in animals, accounting for 30 % of all proteins in mammals

(Patino, Neiders et al. 2002). Collagen is the major protein of the bone, teeth, tendon,

ligaments, cornea and cartilage. It is composed of three chains and has a unique Gly-X-Y

repeating sequence, where X and Y are frequently proline and hydroxyproline

respectively. The chemical characterization of collagen was an impasse for many years

because of the insolubility of collagen fibers. The breakthrough came when it was found

that collagen from the tissues of young animals can be extracted in soluble form because

it is not yet cross-linked (Stryer 1988).

2.2.1 Collagen types

There are 20 collagen types, type I is the most abundant structural protein found in

vertebrate. Based on their supramolecular structures, the collagens are divided into two

main classes (Vuorio and De Crombrugghe 1990; Patino, Neiders et al. 2002): fibril-

forming (or fibrillar) collagens and non-fibril-forming collagens. The fibrillar collagens,

contain type I, II, III, V and XI collagens, form highly organized fibers and fibrils

providing the structural support for the body in skeleton, skin, blood vessels, nerves and

the fibrous capsules of organs (Vuorio and De Crombrugghe 1990). The non-fibril

forming collagens, all the collagen falling outside the fibril-forming collagens, are very

heterogeneous structurally and functionally and have been further classified according to

their molecular characteristics, supramolecular structures, and the types of extracellular

networks that they form into: basement membrane collagens, short chain collagens and

fibril-associated collagens (Hulmes 1992; Patino, Neiders et al. 2002). A list of the

2. State of the Art

11

collagen types, their constituent α-chains, and the tissue distribution is presented in Table

2-1.

Table 2-1: Collagen types (modified from (Patino, Neiders et al. 2002))

Type α chains Tissue Distribution

I α1(I), α2(I) Connective tissues (bone,

tendon, skin, etc)

II α1(II) Cartilage

III α1(III) Extensible connective

tissues (skin, lung)

IV α1(IV), α2(IV), α3(IV),

α4(IV), α5(IV)

Basement membranes

V α1(V), α2(V), α3(V) Tissue containing collagen

I, minor component

VI α1(VI), α2(VI), α3(VI) Most connective tissues,

including cartilage

VII α1(VII) Basement membrane

associated anchoring fibrils

VIII α1(VIII), α2(VIII) Product of endothelial and

various tumor cell lines

IX α1(IX), α2(IX), α3(IX) Tissue containing collagen

II, minor component

X α1(X) Hypertrophic zone of

cartilage

XI α1(XI), α2(XI), α3(XI) Tissue containing collagen

II, minor component

XII α1(XII) Tissue containing collagen

I, minor component

XIII α1(XIII)

XIV α1(XIV) Tissue containing collagen

I, minor component

2. State of the Art

12

Type I collagen is the most abundant animal protein, covers 80% to 99% of total collagen

(Burgeson and Nimni 1992), and forms the matrix of bone, skin, pericardium and other

collagenous tissues. Type I collagen molecules are composed of three polypeptide chains,

two identical α I chains and one α II, each consists of 1300-1700 amino acid residues, the

majority (~ 1000) are organized into a central triple helix configuration (Shirley and

Boot-Handford 1998; Wright and Humphrey 2002). Mutations in type I collagen,

deletions, insertions, and single amino acid substitution, result in many diseases, such as

osteogenesis imperfecta, Ehlers Danlos syndromes and many degenerative diseases

(Patino, Neiders et al. 2002)

2.2.2 Collagen synthesis

Intracellular events

Procollagen molecules have been identified as the precursors of collagen molecules

(Burgeson and Nimni 1992). The synthesis of procollagen molecules takes place within

the fibroblast. The first step in the process of collagen synthesis is the formation of

collagen specific messenger-RNA (Vuorio and De Crombrugghe 1990). After the gene

transcription, functional mRNA are formed and transported to the cytoplasm and

translated on membrane-bound polysomes to the rough endoplasmic reticulum (rER)

(Burgeson and Nimni 1992). As the collagen polypeptides are synthesized in the rER,

important post-translational events accompany this process. Prolyl and lysyl hydroxylases

mediate the hydroxylation of proline and lysine. Thereafter glycosylation takes place, the

hydroxylysine residues are covalently bound to carbohydrate units (Stryer 1988).

Glycosylations are catalyzed by two specific enzymes, a galactosyl transferase and

glucosyl transferase. The importance of post-translational modification of the collagen is

illustrated in heritable disorders of collagen. In Ehlers-Danlos syndrome type IV, the

hydroxylation is reduced causing dysfunctions of the connective tissue, conversely,

overhydroxylation results in osteogenesis imperfecta. Once the hydroxylation and

glycosylation are completed, the individual α-chains align to form the triple helix. After

the formation of triple helix, the procollagen molecules move from rER towards Golgi

apparatus. In the Golgi, procollagen molecules are packed in Golgi-derived vesicles and

carried toward the cellular membrane by cytoskeletal movements.

2. State of the Art

13

Extracellualr events

Once the newly synthesized procollagen molecules are secreted by fibroblasts, the

propeptides procollagen are cleaved by specific proteases called procollagen peptidases,

forming tropocollagen molecules, as shown in fig 2.2. For each type of collagen, there is

one protease for the amino-terminal propeptide and another protease for the carboxyl-

terminal propeptide. This proteolytic cleavage of propeptides is required for the fiber

formation because they prevent the premature formation of the fiber. Defective removal

of propeptides can lead to generalized disorders of connective tissue (Stryer 1988).

Figure 2.2: Schematic diagram of the conversion of procollagen into tropocollagen

by the excision of the amino-and carboxyl terminals modified from

(Stryer 1988)

After the cleavage of propeptides, the tropocollagens are arranged in staggered fashion

according to the Hodge-Petruska scheme to form collagen fibers (Hodge and Petruska

1963). Tropocollagens are 300 nm long, separated in one row by 40 nm gaps and the

adjacent rows are displaced by 67 nm, as shown in fig 2.3. The 40-nm gap between

adjacent tropocollagen molecules in a row is important in enabling collagen to become

cross-linked after the fiber forms. This gap may also play a role in bone formation (Stryer

N-propeptide Procollagen

Procollagen Peptidases

Tropocollagen C-telopeptide

C-propeptide

N-telopeptide

2. State of the Art

14

1988). The last step in collagen synthesis is the formation of intramolecular covalent

cross-links, which will be discussed in details in the next section.

Figure 2.3: Schematic representation of the basic structural design of a collagen

fiber, modified from (Stryer 1988).

2.2.3 Collagen cross-links

Although cross-linking occurs extracellularly, the nature of cross-links also depends on

previous intracellular post-translational modifications to the collagen molecule, in

particular hydroxylation of lysine residues (Knott and Bailey 1998). The collagen is

cross-linked by a unique mechanism based on aldehyde formation from lysine or

hydroxyl lysine side chains by the enzyme lysyl oxidase. Lysyl oxidase (EC 1.4.4.13), the

only enzyme required for cross-link formation, converts the amine side chains of specific

lysine and hydroxylysine residues into aldehyde. Inhibition of this enzyme has deep

effects on the strength of bone and many other tissues because of the subsequent

reduction in cross-linking. Two pathways of cross-linking can be defined in the fibrillar

collagens (Eyre 1984), one based on allysine, the lysine-derived aldehyde, the other on

hydroxyallysine, the hydroxylysine-derived aldehyde. The latter pathway is dominant in

most connective tissues except skin. 3-Hydroxypyridinium residues are the adult cross-

links on the hydroxyallysine route. Two forms of 3-hydroxypyridinium have been

Tropocollagen molecule

40-nm gap 300 nm

67 nm

2. State of the Art

15

identified, hydroxylysyl pyridinoline, HP, and lysyl pyridinoline, LP. The location of

cross-linking residues are evident in molecules of type I, II and III collagens (Miller

1971; Knott and Bailey 1998). There are four cross-linking sites, one in each of

telopeptide (residues 9N and 16C) and two sites in the helix (residues 930 and 87).

Figure 2.4: The hydroxyallysine route of collagen cross-linking, modified from (Eyre

1984).

The immature hydroxylysine aldehyde derived cross-links

After the formation of hydroxyallysine, hydroxylysine derived aldehyde, the immature

cross-links are derived from hydroxyallysine. These cross links can be identified in vitro

with borohydride reduction technique. Two Schiff bases (aldimines), borohydride

reducible cross-links, are formed from the condensation reaction, followed by bohydride

reduction, of hydroxyallysine with lysine (hydroxylysinonorleucine, HLN) or with

hydroxylysine (dihydroxylysinonorleucine, DHLN), as shown in fig 2.4. These Schiff

bases are unstable and undergo immediate, spontaneous Amadori rearrangement to form

ketoamines, lysino-5-ketonorleucine (LKNL) and hydroxylysino-5-ketonorleucine

(HLKNL). Borohydride reduction of these ketoamines gives also HLN and DHLN,

making their individual quantification difficult (Mechanic, Kuboki et al. 1974). All forms

Hydroxylysine

Lysyl oxidase

Hydroxylysine

∆HLN ∆DHLN

HLKNL LKNL

HP and LP

DHLN HLN

NaBH4

NaBH4NaBH4

NaBH4

+ Hyl+ Lyl

2. State of the Art

16

of reducible cross-link fall in concentration as connective tissues mature (Fujii and

Tanzer 1974).

The mature enzymatic cross-links (Hydroxypyridinium)

Hydroxylysyl pyridinoline, HP, the most abundant form of the mature cross-links found

in a wide variety of tissues, was first discovered by Fujimoto et al. in bovine Achilles

tendon (Fujimoto, Akiba et al. 1977; Fujimoto, Moriguchi et al. 1978). It is composed of

two hydroxyallysine residues and a helical hydroxylysine and predominant in highly

hydroxylated collagens such as type II in cartilage. A less abundant form, Lysyl

pyridinoline (LP) is found primarily in calcified tissues and is thought to form from two

hydroxyallysine residues and a lysine residue (Eyre 1984). Robins and Duncan proposed

that HP is formed from the condensation of an HLKNL cross-link with hydroxyallysine

(Robins and Duncan 1983). LP is thought to occur through the reaction of LKNL with a

hydroxyallysine (Ogawa, Ono et al. 1982).

N+

OH

NH2

O OH

NH2

O OH

O

OHNH2

N+

OH

NH2O

OH

NH2

OOH

NH2

O OH

(a) (b)

Figure 2.5: The mature collagen cross-links Hydroxylysyl pyridinoline, HP (a) and

Lysyl pyridinoline, LP (b)

2.2.4 The role of hydroxyproline in collagen stabilization

Collagen has a unique Gly-X-Y repeating sequence, where X and Y are frequently

proline and hydroxyproline (Hyp) respectively. It is known in the literature that Hyp

brings stabilization for the collagen structure, but the way how Hyp stabilizes collagen is

2. State of the Art

17

still debatable. The post-translational modification of proline in the Y position to Hyp has

been shown to grant significant additional stability (Kielty, Hopkinson et al. 1993). A

number of studies have been performed to investigate the stabilizing role played by Hyp.

Gustavson (Gustavson 1955) suggested that Hyp residues stabilize collagen by

participating in direct interchain hydrogen bonding. Later, the collagen model was

proposed from fiber diffraction data (Rich and Crick 1961) and it became clear that steric

considerations would not permit direct hydrogen bonding between the Hyp hydroxyl

groups and peptide carbonyls in the same chain, between different chains in the same

molecule, or between different molecules (Bella, Brodsky et al. 1995). In the Rich and

Crick II (RCII) triple helix structure (Rich and Crick 1961), only one hydrogen bond is

formed directly between amide groups (Gly:NH ··· O=C:X) for each tripeptide unit. This

leaves two carbonyl groups and any amide groups present in the X or Y positions (when

these residues are not imino acids) available for hydrogen bonding. The inability of the

Hyp hydroxyl to form direct hydrogen bonds with backbone carbonyl groups together

with the examinations supporting specific binding of water molecules to collagen chains

led to the idea that stabilization of the collagen triple helix by Hyp residues took place

through intramolecular water bridges, involving those carbonyl and amide groups which

were not participating in the interchain RCII hydrogen-bonding pattern (Ramachandran,

Bansal et al. 1973; Suzuki, Fraser et al. 1980; Bella, Brodsky et al. 1995).

Holmgren et al. (Holmgren, Taylor et al. 1998) disagreed with the suggestion that Hyp

stabilizes collagen by providing sites for hydrogen bonding of water and challenged this

by demonstrating that substitution of 4(R)-fluoroproline for 4(R)-hydroxyproline in

collagen-like peptides further increases the thermal stability of the triple helices, despite

the fact that 4-(R)-fluoroproline does not provide a site for hydrogen bonding of water.

They concluded that the stability granted by Hyp cannot be explained by the additional

water bridges but resulted from an electron withdrawing inductive effect of the hydroxyl

or fluoro group that favors the trans configuration of the peptide bond required for

formation of the triple helix. These results were consistent with previous observations

that Hyp residues enhanced the thermal stability of collagen-like peptides even in a

completely anhydrous environment (Engel, Chen et al. 1977) and also with X-Ray

diffraction analysis, which ascertained that the structure of proline is affected by the

2. State of the Art

18

inductive effect elicited by the hydroxyl group of Hyp (Panasik, Eberhardt et al. 1994).

Furthermore Engel and Prockop (Engel and Prockop 1998) concluded that the water

bridges in collagen do not contribute significantly to the stability of the structures.

Instead, the water molecules may simply be innocent bystanders.

2.2.5 The thermal stability of collagen

One of the best criteria to study the way how the collagen molecule held in its triple helix

is to examine conditions under which the stabilization breaks down (Miles and Bailey

2004). The thermal denaturation of collagen induces unfolding of triple helix into random

coils by breaking the hydrogen bonds (Privalov 1982); the ability of collagen to resist this

unfolding is an indication of its “healthiness”.

The shrinkage or contraction of collagen fiber is used as a gross metric of collagen

denaturation (Weir 1949; Gustavison 1956; Hoermann and Schlebusch 1971). There are

two classes of shrinkage temperature measurements (Lee, Pereira et al. 1995): (1)

hydrothermal isometric tension (HIT) and (2) isotonic shrinkage temperature. Verzar

(Verzar 1964) borrowed ideas from muscle physiology and introduced in 1960 the

hydrothermal isometric tension (HIT). In HIT test, the sample is held at constant length in

an aqueous bath while increasing its temperature linearly with time. HIT tests measure

changes in the force that are needed to maintain the tissue at its fixed length during

heating; the force increases when the tissue tries to shrink against the fixed constraint

(Humphrey 2003). In the isotonic shrinkage temperature, the sample is held either

unconstrained or under isotonic constraint; simultaneous measurements of temperature,

time and length are recorded.

It was proved that the level of dehydration as well as the mineral content affects the

thermal stability of collagen. Kronick and Cooke (Kronick and Cooke 1996) showed that

increasing the mineral content enhances the thermal stability of bone. They found that the

denaturation temperature for fully mineralized, partially mineralized and demineralized

bone was 155, 113 and 63 °C respectively.

Much work has been done to elucidate the state of water in biological systems. Rochdi et

al (Rochdi, Foucat et al. 1999) have studied the dependence of the enthalpy and

temperature of denaturation of collagen on the water content using differential scanning

calorimetry (DSC). They found that increasing the water content increases the

2. State of the Art

19

denaturation enthalpy and decreases the denaturation temperature. Miles and Ghelashvili

(Miles and Ghelashvili 1999) utilized the equations of the entropy of a polymer in a box

(Doi and Edwards 1986) to examine the effect of solvent concentration on the collagen

denaturation. They proposed that dehydration brings stabilization mainly by reducing the

lateral dimensions of the lattice and consequently the entropy of activation. They

suggested that dehydration resulting in stripping away of water bridges that connected via

hydrogen bonds to the triple helix. This stripping away reduces the enthalpy of

denaturation because fewer hydrogen bond need to be broken. This explains the increase

of denaturation enthalpy with increasing the water content.

2.2.6 Advanced glycation end products (AGEs)

Non-enzymatic glycation is a common posttranslational modification of proteins

(DeGroot 2004). Maillard reaction or non-enzymatic glycation of proteins is initiated by

the reaction of sugars with lysine and arginine residues in proteins, and eventually leads

to the formation of advanced glycation end products (AGEs) such as

(carboxymethyl)lysine (CML), (carboxyethyl)lysine (CEL), and cross-links, such as

pentosidine, methylglyoxal-lysine dimer (MOLD), and threosidine (Verzijl, DeGroot et

al. 2002). It is now apparent that the AGE formation can also be initiated by lipid

peroxidation (Januszewski, Alderson et al. 2003).

AGEs accumulate with increasing age (Verzijl, DeGroot et al. 2000), results in increased

stiffness of the cartilage collagen network and subsequently leads to increased

susceptibility of the collagen network to mechanical failure (brittleness). Thus,

accumulation of AGEs could be a molecular mechanism that causes age to be a major

predisposing factor for the development of Osteoarthritis (OA) (Verzijl, DeGroot et al.

2002).

In addition the in vivo AGEs, the heat-generated AGEs can be formed in common foods

during the spontaneous reactions between reducing sugars and proteins or lipids (Baynes

and Thorpe 2000). These AGEs are formed in the presence of heat much more rapidly

and in greater concentrations than the in vivo AGEs (Vlassara, Cai et al. 2002).

Therefore, eating high temperature cooked foods will increase the AGEs content in the

body and consequently accelerates aging.

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2.3 Bone Bone is a living, dynamic connective tissue, which has been evolved to fulfill two

functions (Einhorn 1996): the provision of mechanical integrity for both locomotion and

protection, and involvement in the metabolic pathways associated with mineral

homeostasis. Bone refers to a family of materials each with a somewhat different

structural motif, but all having in common the basic building block, the mineralized

collagen fibril (Weiner and Wagner 1998). This family of materials also contains other

members, dentin, the material that constitutes the inner layers of teeth; cementum, the

thin layer that binds the roots of teeth to the jaw; and mineralized tendons. The diversity

of structures within this family reflects the fine-tuning or adaptation of the structure to its

function (Weiner and Wagner 1998).

2.3.1 Bone composition

Water, Hydroxyapatite and collagen Type I are the major components of the bone family

of materials (Weiner and Wagner 1998). The solid phase of the bone consists of organic

(30%) and inorganic (70%) part (Felsenberg 2001). Hydroxyapatite constitutes 95 % of

the inorganic part, whereas the rest 5 % consists of Mg, K and Na-chlorides and

fluorides. Collagen Type I constitutes 95 % of the organic part and 5 % is represented by

the non-collagenous proteins. The composition of bone is shown in fig 2.6.

66.5%28.5%

Hydroxyapatite

Non-collagenous proteins

Collagen Type I

Chloride, Fluoride

3.5%

1.5%

Figure 2.6: The composition of bone, modified from (Felsenberg 2001)

2. State of the Art

21

2.3.2 Bone hierarchy

Bone has a very complex hierarchical structure and is optimized to achieve a remarkable

mechanical performance (Fratzl, Gupta et al. 2004). In order to understand the

mechanical properties of bone material, it is important to understand the mechanical

properties of its component phases, and the structural relationship between them at the

various levels of hierarchical structural organization (Weiner and Traub 1992; Landis

1995). The levels of bone hierarchy, shown in fig 2.7, are (Rho, Kuhn-Spearing et al.

1998)

Macrostructure

At the macrostructure level, bone is distinguished into the cortical (or compact) and

cancellous (or trabecular) types. Cortical bone is a dense solid mass, represents nearly

80% of the skeletal mass (Berne and Levy 1993). It provides a strength where bending

would be undesirable and it is predominant in the appendicular skeleton. Trabecular

bone, represents 20% of the skeletal mass, is less dense, more elastic, has a higher

turnover rate, and much more porous than cortical bone.

Microstructure

The building block of cortical bone, osteon, consists of different sheets (lamellae, 3-7

µm) of mineralized collagen fibers, which wrap in concentric layers (3–8 lamellae)

around a central canal to form what is known as an osteon or a Haversian system.

Cancellous bone is made of an interconnecting framework of trabeculae in a number of

combinations, all comprising the following basic cellular structures: rod–rod, rod–plate,

or plate–plate. A trabecular rod is about 50–300 µm in diameter.

2. State of the Art

22

Figure 2.7: Hierarchical structural organization of the bone, modified from (Rho,

Kuhn-Spearing et al. 1998)

Sub-microstructure

Bone lamellae are 3–7 µm thick (Marotti 1993), but the arrangement and orientation of

the substance of a lamella is not well known (Rho, Kuhn-Spearing et al. 1998). There

may be differences in the lamellae encountered in cortical and cancellous bone. The

osteonal lamellae are wrapped around a central canal, and sequential concentric lamellae

have fiber orientations alternating with each other, spiraling around the central canal.

Parallel lamellae are arranged to form the trabeculae, the building block of the trabecular

bone.

Nanostructure and sub-nanostructures

The most prominent structures seen at this Nano-scale are the collagen fibers, surrounded

and infiltrated by mineral. The three main materials at the sub-nanostructures are crystals,

Cancellous bone

Cortical bone

Osteon

Lamella Collagen fiber

Collagen molecule

Collagen fibril

Bone crystals

10-500 µm 3-7 µm

0.5 µm 1 nm

Microstructure

Sub-microstructure

Nanostructure

Sub-nanostructure

2. State of the Art

23

collagens, and non-collagenous organic proteins. The mature crystals are plate-shaped

(Weiner and Traub 1992) found within the discrete spaces within the collagen fibrils,

thereby limiting the possible primary growth of the mineral crystals, and forcing the

crystals to be discrete and discontinuous. The average lengths and widths of the plates are

50×25 nm. Crystal thickness is 2–3 nm (Landis 1995). The primary organic component

of the matrix is Type I collagen. Collagen molecules secreted by osteoblasts self-

assemble into fibrils with a specific tertiary structure having a 67 nm periodicity and

40 nm gaps or holes between the ends of the molecules, as shown in fig. 2.3. Non-

collagenous organic proteins, including phosphoproteins, such as osteopontin,

sialoprotein, osteonectin, and osteocalcin, may function to regulate the size, orientation,

and crystal habit of the mineral deposits. Through chelation of calcium or enzymatic

release of phosphorus from these proteins, they may serve as a reservoir for calcium or

phosphate ions for mineral formation. However, additional studies are needed to

conclusively define their actions and mechanisms (Rho, Kuhn-Spearing et al. 1998).

2.4 Pericardium The pericardium is a fibro-serous dense connective tissue membrane which encloses the

heart, providing both an internally lubricated protective scaffold for the contracting

myocardium and a metabolically active membrane which can greatly influence cardiac

performance by participating in local prostaglandin metabolism (Simionescu and

Kefalides 1991). Structurally the pericardium is composed of an inner, serous (visceral)

membrane that lines the pericardial cavity and an outer, fibrous (parietal) layer composed

of densely packed wavy collagen fibers among which flat, elongated fibroblasts are

scattered. The major protein constituents known to date are type I collagen, a low

molecular weight dermatan sulphate proteoglycan (decorin) and elastin (Simionescu,

Deac et al. 1994).

Bovine pericardium is widely used as a biomaterial in the fields of reconstructive and

replacement surgery (Sacks, Chuong et al. 1994). Despite the advantages of bovine

pericardium, represented by the availability and immunology, a major difficulty lies in its

intrinsic structural and mechanical variability (Crofts and Trowbridge 1989; Lee, Haberer

et al. 1989). The structure variability of the pericardium made the characterization and

2. State of the Art

24

modeling of the mechanical properties difficult. Therefore the mechanical properties of

the bovine pericardium is poorly characterized (Sacks, Chuong et al. 1994).

Bovine pericardium is generally considered to be mechanically anisotropic (Lee,

Courtman et al. 1984; Zioupos and Barbenel 1994). Small angle light scattering (SALS)

has been used to quantify the collagen fiber architecture of bovine pericardium (Sacks,

Chuong et al. 1994; Mirnajafi, Raymer et al. 2005). SALS maps indicated large animal-

to-animal variability in fiber architecture (Hiester and Sacks 1998; Hiester and Sacks

1998), precluding the use of an anatomic location as a simple guideline for selecting

structurally consistent specimens. Previous studies on the relation between bovine

pericardium biaxial mechanical properties and tissue structure suggested that the local

collagen fiber architecture plays a major role in determining the degree of mechanical

anisotropy (Sacks, Chuong et al. 1994). This suggests the necessity to select structurally

uniform specimens to avoid the mechanical variability (Sacks and Chuong 1998).

Sacks and Chuong (Sacks and Chuong 1998) suggested a procedure for the selection of

specimens, in which first large sections were scanned to select almost uniform regions, as

shown in fig 2.8. The selection process was aided by a simultaneous display of the

regional averaged preferred fiber directions and orientation index (OI), which facilitated a

quantitative assessment of the uniformity of the collagen fiber architecture. Regions of

good structural consistency were removed, and then rescanned to select smaller and more

uniform regions for the mechanical test.

2. State of the Art

25

Figure 2.8: Overview of the pericardium sorting procedure. (a) A course SALS scan

of an anterior section of the pericardium sac showing where the 50 mm

× 75 mm rectangular cutouts regions were extracted, (b) a rescan of the

cutout showing where the 25 mm × 25 mm biaxial test specimen was

selected, and (c) high spatial resolution scan of the biaxial test specimen

overlaid on gray scale OI values demonstrating high uniformity of both

fiber preferred directions and OI, along with definition of the PD and

XD axes (Sacks and Chuong 1998).

3. The Objectives

26

3 The Objectives The demand for the biological implants is rising continuously due the necessity to use

these implants in many surgical and clinical applications. In order to fulfil the increasing

market demand, the production capacity must be increased in the near future. These facts

show how urgent should the Tutoplat process be intensified or optimized without

affecting the quality of the processed tissues.

In order to intensify or optimize the process, the influence of the whole process as well as

of each step of the process on the structural stability of the processed tissues has to be

examined and analysed carefully. This analysis must be the basis of future optimization

trials.

First the influence of Tutoplast process on the thermal stability of processed tissues has to

be studied because the thermal stability is a good indication of the ‘healthiness’ of the

tissues. Furthermore examining the thermal stability is important to understand the effect

behind the thermal treatment of tissues during some surgical operations. Native and

processed samples are exposed to thermal treatment in furnace, followed by a selective

enzymatic digestion method for the measurements of denatured collagen (DC). Analysis

of the isotonic shrinkage temperature (thermoelasticity) as well as SDS-PAGE is used

also to assess the influence of the Tutoplast-process on the thermal stability of the

processed tissues.

After investigating the influence of the whole process, the influence of each step has to be

examined to check its contribution in the probable modification caused by the process.

For this purpose, native samples were exposed to NaOH, H2O2 and acetone treatment

separately with different variations in the treatment concentration and duration. The

investigation of the influence of each step is followed by measurements of the isotonic

shrinkage temperature, measurements of DC and SDS-PAGE.

The effect of the Tutoplast-process on the mechanical properties of the processed tissues

has to be also investigated using tensile and compression tests.

4. Materials and Methods

27

4 Materials and Methods 4.1 Materials For the execution of the experiments, different bovine materials have been used:

4.1.1 Bovine Bones

Femoral head cancellous bone samples, provided from Tutogen Medical GmbH, were

taken from Norwegian sources in the age of 18-25 months. To avoid the risk of the

bovine spongiform encephalopathy (BSE), the scientific steering committee (SCC)

assessed the geographical BSE risk (GBR) of different countries and came to the

conclusion that it is highly unlikely that BSE could be present in Norway (report on the

assessment of the geographical BSE risk of Norway, July 2000). Therefore the samples

used in the experiments were taken exclusively from Norway.

The bone samples were either kept native or processed according to the Tutoplast®

process, described in section 2.1.3.1. For the mechanical analysis, cubes 1×1×1 cm were

sawed from the native bones and stored in 26% NaCl solution at 8 °C.

The Tutoplast-processed bones were pulverized under liquid nitrogen with a ball mill, as

will be discussed in section 4.2.1.1, sieved and the fractions (< 250 µm), shown in fig 4.1,

were stored at -20 °C for the further experiments and examinations.

Figure 4.1: Tutoplast-processed bovine cancellous bone powder (< 250 µm)

4. Materials and Methods

28

4.1.2 Bovine Pericardium

Native bovine pericardium specimens were taken from Norwegian sources in the age of

18-25 months, mechanically defatted and then either kept as native or processed

according to the Tutoplast® process or lyophilized, as shown in fig 4.2.

The native pericardium was stored in 26% NaCl solution at 8 °C, whereas the Tutoplast-

processed pericardium was stored at room temperature. Furthermore a native pericardium

was washed with distilled water to remove the traces of NaCl, lyophilized for 48 h in a

Christ Alpha 1–4 freeze-drying system (Christ, Osterode am Harz, Germany) and then

stored at room temperature.

(a) (b)

(c)

Figure 4.2: Bovine pericardium (a) native, (b) Tutoplast-processed, (c) native

lyophilized

4. Materials and Methods

29

4.2 Methods For the assessment of the quality of the Xenografts, different quality assurance tests have

been used.

4.2.1 Preparation Steps

For the analysis of bone samples different preparation steps were necessary before the

analysis, whereas no preparation step was done for the analysis of the pericardium

samples.

4.2.1.1 The pulverization of the bones

Demineralization is necessary for the analysis of the bone collagen, especially during the

enzymatic digestion to allow better diffusion of the enzyme. The demineralization of

bone is a slow process which takes several weeks. For the acceleration of the

demineralization, the bones can be pulverized to enhance the diffusion of the

demineralizing solutions and consequently to guarantee faster demineralization of the

bones.

Femoral head cancellous bones were pulverized using three types of mills; milling

machine, micro-dismembrator and ball mill.

The milling machine

The milling machine FP1 (Deckel, Bielefeld, Germany) was used to pulverize the bone

samples. It was possible to pulverize relatively large specimens (in the range of 5×2× 1

cm3) in relatively short time (less than 1 minute). During the grinding process, liquid

nitrogen was always refilled to avoid thermal stresses resulted from the milling process.

The milling process was executed under the following parameters, 1200 rpm and feed

rate of 75 mm/min.

Micro-dismembrator

Micro-dismembrator (Sartorius, Goettingen, Germany) was used also to pulverize the

bone samples. In contrast to the milling machine, it was only possible to pulverize small

samples (in the range of 5×5× 5 mm3); however it takes relatively long time (10-20 min).

The bone samples, the Teflon-shaking flask as well as the 4 mm-stainless steel balls were

cooled for 10 minutes in liquid nitrogen, afterwards the shaking flask, filled with the bone

specimens and the liquid nitrogen, was closed and attached to the micro-dismembrator

4. Materials and Methods

30

before the grinding starts. The grinding process was performed under the following

conditions: 2000 rpm swing frequency and 16 mm swing amplitude.

The ball mill

The ball mill analysette 3 pro (Fritsch, Idar-Oberstein, Germany) was used to pulverize

the bone samples. Relatively thin samples (5 mm), regardless of the length and width,

could be pulverized within relatively short time (2-3 min). The pulverization takes place

with 50 mm stainless steel ball under continuous filling of liquid nitrogen.

For the evaluation of the effect of milling process on the bone structure, especially

collagen, samples taken fresh after the milling as well as after 1-week storage at 8 °C,

were used for the measurements of denatured collagen fraction (DC), as shown in table 4-

1.

Table 4-1: The number of bone powder samples taken from the different mills for

the analysis of DC

Number of samples

Mill type Fresh After 1-week storage

Ball mill 11 12

Milling machine 6 6

Micro-dismembrator 6 6

4.2.1.2 The Demineralization of Bones

For efficient diffusion of the enzymatic digestion, the bones have to be demineralized

(Wang, Bank et al. 2001). For this purpose, approximately 1 g of bone powder (<250 µm)

were demineralized using 50 ml 0.5 M EDTA solution (pH 7.4) in a beaker for 72 h with

stirring at room temperature. The beaker was covered with a watch glass.

4.2.2 The Determination of Denatured Collagen (DC)

A selective enzymatic digestion method has been used for the determination of the

fraction of denatured collagen (DC) for the bone and pericardium samples. It consists of

the following steps:

4. Materials and Methods

31

4.2.2.1 A Selective Digestion Method

Intact triple helical collagen molecules are highly resistant to proteolytic enzymes,

whereas degraded (unwound) collagen is easily digested. This fact was exploited to

develop a simplified method for the quantification of the amount of degraded collagen in

the collagen network of connective tissues. This method involves selective digestion of

degraded collagen by α-chymotrypsin (Bank, Krikken et al. 1997).

After certain treatment step, either thermally or chemically, the demineralized bone

powder samples (~ 10 mg for each) as well as the pericardium (~ 8 × 8 × 0.6 mm3 for

each) samples were digested in microcentrifuge tubes overnight at 37 °C with 0.5 ml

incubation buffer containing 1 mg α-chymotrypsin/ml. The incubation buffer is made of

0.1 M Tris HCl, pH 7.3, containing the proteinase inhibitors, 1 mM iodoacetamide, 1 mM

EDTA, 10 µg/ml Pepstatin-A. α-chymotrypsin digests the denatured collagen exclusively

without affecting the intact collagen, as shown in fig 4.3.

After the α-chymotrypsin digestion step, the supernatant, including the denatured

collagen, is separated from the residue, including the intact collagen by centrifugation

step. The centrifugation is done with micro-centrifuge Biofuge pico (Heraeus, Hanau,

Germany) with 10000 rpm for 5 min.

The supernatant was removed quantitatively and diluted 1:1 with 12 N HCl, whereas the

residue was immersed in 6 N HCl. The supernatant and residue were hydrolyzed at 121

°C for 20-24 h in a heating block MBT 250 (Kleinfeld, Gehrden, Germany). The

hydrolyzates were dried using vacuum exsiccator (Häberle, Gaggenau, Germany)

supported with vacuum pump and cold trap (Häberle, Gaggenau, Germany).

4. Materials and Methods

32

Figure 4.3: The principle of the α-chymotrypsin selective digestive method, modified

from (Bank, Krikken et al. 1997)

4.2.2.2 Spectrophotometeric Determination of the DC

The spectrophotometric determination was done according to SOP prepared by Dr. C.C.

Clark (Department of Orthopaedic Research, University of Pennsylvania) as a

modification of previous method (Switzer and Summer 1971). The method is based on

the determination of the hydroxyproline content (HYP) taking into consideration that

hydroxyproline constitutes 14% of the collagen (Elgawish, Glomb et al. 1996).

First a calibration line, using stock solution (10 mg/l), was constructed to determine the

amount of hydroxyproline, as shown in table 4-2.

Incubation with α-chymotrypsin at 37 °C for 24 h

fragments in the supernatant

denatured collagen intact collagen

no fragments in the supernatant

4. Materials and Methods

33

Table 4-2: The quantities used to construct the calibration line of hydroxyproline

content

Aliquot from stock µg hydroxyproline Volume H2O

0 µl 0 2.30 ml

100 µl* 1.0 2.20 ml

250 µl* 2.5 2.05 ml

350 µl* 3.5 1.95 ml

500 µl* 5.0 1.80 ml

Regarding the determination of HYP content for the samples, after evaporating the HCl,

1ml water is added to each sample, placed in ultrasonic bath for 1 min to disrupt the

samples and then a stock solution is prepared for each sample.

The samples are diluted with water with the factor (1:500) and placed in capped tubes to

begin with the assay:

Assay

• The assay is started with adding 0.5 ml potassium borate buffer to all samples

• 2 ml freshly prepared 0.2 M Chloramine-T is added. In this step, the

hydroxyproline will be oxidized to pyrrole-2-carboxylic acid (Berg 1982)

• After 25 min, 1.2 ml of 3.6 M sodium thiosulfate is added to stop the oxidation

• 1.5 g KCl is added

• In hood, 2.5 ml toluene is added. Shaking by hand to initially disperse KCl is

required, and then shaking is continued for 4 min in automatic shaker

• Centrifugation for 5 min at 1500 rpm in

• Using Pasteur pipette, the toluene (upper) layer is removed and discarded with

being careful not to disturb lower layer.

• The capped tubes are placed in boiling water bath for 30 min. This step will

convert the pyrrole-2-carboxylic to Pyyrole

• After cooling the samples to room temperature, 2.5 ml toluene is added and

shaken again with hand and automatic shaker

4. Materials and Methods

34

• Centrifugation for 5 min at 1500 rpm in

• Carefully 1.5 ml of the toluene (upper) layer is removed and placed in a tube

• To each 2-ml toluene aliquot, 0.6 ml Ehrlich’s reagent is added. The Pyyrole

forms with Ehrlich reagent a chromophore

• After 30 min, the absorbance at 560 nm is read

The content of hydroxyproline is measured in the residue and supernatant and converted

to collagen content. Relating the amount of collagen in the supernatant to the amount in

the residue leads to the determination of DC.

4.2.3 The Measurements of the Extent of Browning

5 samples (8×6 mm2) were taken from native, Tutoplast processed, rehydrated Tutoplast

processed and lyophilized pericardium, heated for 1 h at 185, 200 °C in addition to

unheated control samples and then hydrolyzed in 6 N HCl for 24 h. The extent of

browning was detected spectrophotometrically at 420 nm (Friedman and IMolnar-Per

1990) with Specord 210 (Analytik Jena, Jena, Germany).

4.2.4 The Measurements of the Isotonic Shrinkage temperature

For the evaluation of the quality of the bovine pericardium, the technique of the isotonic

shrinkage has been used, as shown in fig 4.4. Pericardium strips (4×1.5 cm) were strained

between two holders in a vessel of water, one holder is fixed and the other is movable

attached with isotonic load. Heating of the aqueous phase is accomplished with a Bunsen

burner with an approximately average heating rate, 2.5 °C/min. The change in the

temperature and specimen length are simultaneously recorded using PT100 (labfacility,

Teddington, UK) and incremental optical encoder (Agilent Technologies, Santa Clara,

CA, USA) respectively. The encoder contains a single lighting emitting diode (LED) as a

light source. Opposite the emitter is the integrated detector circuit. By shrinkage or

change in length, the code wheel rotates between the emitter and the detector, causing the

light beam to be interrupted by the pattern of spaces and bars on the code wheel. The

analysis and representation of the data is done by program developed on Testpoint

software based on the simultaneous acquisition of the experimental data and the graphical

representation.

4. Materials and Methods

35

4cmφ =

Figure 4.4: Schematic setup of the isotonic shrinkage technique

4.2.5 SDS-PAGE

SDS-PAGE is a technique used in biochemistry, genetics and molecular biology to

separate proteins according to their molecular size. The analysis consists of the following

steps:

Preparation of polyacrylamide Gels (PAA)

Previous examinations (Schwarz 2006) have shown that 15% acrylamide gel with silver

stain staining was the best variant for the current objectives.

Two glass plates with two spacers are used to make a single cassette (Peqlab,

Biotechnologie GmbH, Erlangen Germany). Regardless of the system, preparation

requires casting two different layers of acrylamide between the glass plates. The lower

layer is separating gel and the upper layer is stacking gel.

First the separating gel was prepared, all the constituents of the fine-pored gel (pH 8.2)

were mixed, except N, N, N’, N’-tetramethylethylenediamine (TEMED) and ammonium

persulfate (APS). Thereafter, TEMED and APS are added to the mixture, as shown in

table 4-3.

4. Materials and Methods

36

Table 4-3: The composition of 15% PAA separation gel

Substances/gel volume 10 ml 15 ml

H2Odeionized 2.3 3.4

30% bisacrylamide 5.0 7.5

1.5 M Tris (pH 8.8) 2.5 3.8

10 % SDS 0.1 0.15

10 % APS 0.1 0.15

TEMED 0.004 0.006

The gel must be mixed quickly, poured, overlaid with 1 ml distilled water and allowed to

polymerize for 25-30 min. The water has to be decanted after the polymerization.

The next step is to prepare the stacking gel, during the preparation of stacking gel; all the

constituents are added in the order used in the preparation of the separation gel, as shown

in table 4-4.

Table 4-4: The composition of 5% PAA stacking gel

Substances/gel volume 2 ml 3 ml

H2Odeionized 1.4 2.1

30% bisacrylamide 0.33 0.5

1.5 M Tris (pH 6.8) 0.25 0.38

10 % SDS 0.02 0.03

10 % APS 0.02 0.03

TEMED 0.002 0.003

After the addition of TEMED and APS, the stacking gel is poured on the separating gel,

followed by insertion of combs. The protein lysates are passed through the large-pored

stacking gel (pH 6.8), and gathered at the boarders with the separating gel and

consequently penetrate into the separating gel.

4. Materials and Methods

37

Preparation of the Samples

In order to separate the fragments of the collagen, the samples have to be mixed with a

sample buffer with the volume ratio (1:4). The composition of sample buffer is shown in

table 4-5

Table 4-5: The composition of sample buffer

Substances Quantities required in 10 ml water

Tris (pH 6.8) 0.38 g

SDS 1 g

glycerol 2.5 ml

D,L-dithiothreitol (DTT) 0.38 g

Bromphenol blue 5.0 mg

The objectives of sample preparation are to put the proteins into a denaturing buffer,

rendering them suitable for electrophoresis, and to adjust the concentrations of sample so

that an appropriate amount of protein can be loaded onto a gel.

Assembling, Loading, and Running Gels

The assembly of a gel running stand varies with the type of apparatus. The top of the

cassette must be continuous with an upper buffer chamber and the bottom must be

continuous with a lower chamber so that current will run through the gel itself.

The samples, including standard Mark 12® (Invitrogen, Karlsruhe, Germany) and α-

chymotrypsin as reference, are loaded. The anode must be connected to the bottom

chamber and the cathode to the top chamber. The negatively-charged proteins will move

toward the anode. Gels are usually run at a voltage that will run the tracking dye to the

bottom as quickly as possible without overheating the gels, in this case 300 V.

Disassembly and staining

When the dye front is nearly at the bottom of the gel, it is the time to stop the run. The

plates are separated and the gels are washed shortly with distilled water. Subsequently the

4. Materials and Methods

38

gels are incubated in fixation solution overnight at 4 °C. The composition of the fixation

solution is shown in table 4-6.

Table 4-6: the composition of the fixation solution

Substances Quantities (µl)

Ethanol 100 % 100

Acetic acid 20

H2Odeionized 80

Formalin 37 % 0.1

After the incubation, the gels are washed twice with a washing solution (50 % ethanol)

for 25 min. the gels are then stained using silver stain for 20 min; the composition is

shown in table 4-7.

Table 4-7: The composition of silver stain

Substances Quantities

AgNO3 0.2 g

H2Odeionized 100 ml

Formalin 37% 75 µl

After that they are washed with distilled water and incubated in developer solution for 3-

5 min until the bands become visible. Finally the gels are laid in stop solution for 10 min

until the end of the development reaction.

Documentation

The gels are laid on converter screen plate, illuminated with white light; black-white

photo was taken and finished with the help of the program Vision Capt (Peqlab,

Biotechnologie GmbH, Erlangen Germany) (Koerber 2006).

4. Materials and Methods

39

4.2.6 Characterization of the Mechanical Properties

The mechanical testing machine Instron model 5569 (Instron Ltd, High Wycombe, UK)

in association with Instron Merlin software has been used for the characterization of the

mechanical properties of the bone and pericardium at room temperature.

The bones are tested using the compression test, first the sample was placed on the

fastening plate beneath the load cell. The load cell starts to descend and compress the

sample. The compression proceeds and the force acting on the sample increases until it

reaches the maximum value then it decreases. The test can be stopped when the force

reaches a pre-adjusted value below the maximum, e.g. 50%. The data is analyzed using

the Merlin software to construct the required stress-strain diagram.

The temperature dependent mechanical properties of the bone were examined. For this

purpose, 9 (1×1×1 cm3) samples were taken and heated for 1 h at each temperature of the

following temperatures 37, 60, 80 and 100 °C, before being mechanically tested.

The pericardium sample (8×1.5 cm2 strip) is tested using the tensile test, in which the

load cell used in the compression test is removed. Alternatively, the sample is inserted

between two clamps, upper and lower. The upper clamp starts to pull the sample causing

extension of the sample. The test is stopped when the sample is extended to pre-adjusted

value, e.g. 200%. The data is analyzed using the Merlin software to construct the required

stress-strain diagram.

The influence of Tutoplast processing on the mechanical properties of the pericardium

was examined. For this purpose, 10 (8×1.5 cm2) strips were cut from the left side of the

pericardium sac and 10 strips from the right side. From each side 5 strips were processed

and the other 5 kept unprocessed as control.

4.2.7 The measurements of the Thermal Conductivity

5 (8×6 mm2) native as well as 5 processed samples were submerged in 30 ml distilled

water for 1 h, thereafter the conductivity of the water was measured using conductivity

meter WTW LF95 (WTW, Weilheim, Germany).

4.3 Different physical and chemical treatment During the current work the following chemical and thermal treatments have been

executed:

4. Materials and Methods

40

4.3.1 The thermal treatment of collagenous tissues

4.3.1.1 The thermal treatment of bovine bone

Bovine cancellous bone samples, taken from Norwegian sources in the age of 18-25

months, were processed according to Tutoplast process in January 2005, pulverized with

milling machine under liquid nitrogen and the fraction (< 250 µm) was stored at -20 °C in

March 2005. The thermal stability of bone powder was examined during April and May

2005 and also September and October 2005.

The bone powder samples were heated for 1 h at each temperature in the range between

(55-200 °C), as shown in table 4-8.

Table 4-8: The samples used for the thermal treatment of bovine bone

Temperature

(°C)

25 55 90 120 135 150 160 170 200

No. of

samples

6 5 6 9 9 9 9 9 6

For temperatures lower than 100 °C; the samples were heated in a water bath, whereas at

temperatures higher than 100 °C, the samples were incubated for 15 min in boiling water

bath and then heated in a furnace to the corresponding temperature. Afterwards, the

samples were demineralized with 0.5 M EDTA (pH 7.4) for three days, incubated with α-

chymotrypsin overnight at 37 °C for the determination of DC.

4.3.1.2 The thermal stability of bovine pericardium

For the evaluation of the thermal stability of bovine pericardium, bovine pericardium

specimens from Norwegian sources in the age of 18-25 months were stored under 26 %

NaCl solution at 8 °C in January 2006. The thermal stability of the native and Tutoplast-

processed pericardium sample was examined during February and March 2006, whereas

the thermal stability of lyophilized pericardium during August and September 2006.

4. Materials and Methods

41

For the measurements of DC

For the evaluation of the thermal stability of native bovine pericardium, 5 samples (8×6

mm2) were heated at each temperature of the following temperatures (25, 55, 70, 80, 90,

100, 110, 120, 135, 150, 160, 170, 185 and 200 ° C) for 1 h in furnace.

For the evaluation of the thermal stability of Tutoplast-processed bovine pericardium, a

part of a native pericardium was processed with Tutoplast process. 5 samples (8×6 mm2)

were heated at each temperature of the following temperatures (25, 55, 70, 80, 90, 100,

110, 120, 135, 150, 160, 170, 185 and 200 ° C) for 1 h in furnace.

For the evaluation of the thermal stability of the native lyophilized bovine pericardium, a

part of a native pericardium was lyophilized for 48 h in a Christ Alpha 1–4 freeze-drying

system (Christ, Osterode am Harz, Germany). 5 samples (8×6 mm2) were heated at each

temperature of the following temperatures (25, 55, 70, 80, 90, 100, 110, 120, 135, 150,

160, 170, 185 and 200 ° C) for 1 h in furnace.

For the investigation of the kinetic and thermodynamic parameters, isothermal

experiments have been performed. For this purpose, samples of Tutoplast processed and

lyophilized pericardium were taken and heated for 5, 10, 15 and 20 min in furnace at

temperatures 145, 160 and 175 °C (3 samples for each time at each temperature).

After the thermal treatment the samples were incubated with α-chymotrypsin overnight at

37 °C for the determination of DC.

SDS-PAGE

At each temperature from the following temperatures, 25, 55, 90, 120, 135, 150, 160, 170

and 185 °C, 2 (8×6 mm2) native and 2 Tutoplast-processed pericardium samples were

heated for 1 h. After the thermal treatment, the samples were incubated with α-

chymotrypsin overnight at 37 °C. The supernatant, containing the denatured collagen

part, was taken for SDS-PAGE investigations.

4.3.2 The Sodium hydroxide treatment and the corresponding neutralization

4.3.2.1 Sodium hydroxide treatment

For the evaluation of the sodium hydroxide treatment, bovine pericardium specimens

from Norwegian sources in the age of 18-25 months were stored under 26 % NaCl

solution at 8 °C in May 2006.

4. Materials and Methods

42

Measurement of the extent of hydrolysis

For the measurement of the extent of hydrolysis at room temperature, 10 native and 10

Tutoplast-processed (8×6 mm2) pericardium samples, with average dry weight of 16.8

and 12 mg respectively, were treated with 1 N NaOH for different durations (0, 30, 60,

90, 120 and 150 min) in August 2006. The extent of hydrolysis was measured

spectrophotometrically at 280 nm based on the content of the aromatic amino acids. For

this purpose, a calibration line for the content of aromatic amino acids was constructed

using a stock solution of 2 mg aromatic amino acids in 100 ml water. 5 samples contain

the following weights (0, 2.5, 5, 7.5, 10, 12.5, 15, 20 µg aromatic amino acids) were used

to construct the calibration line (Koerber 2006).

Shrinkage temperature measurements

5 Strips (4×1.5 cm) were cut from a native pericardium in June 2006 and treated with 50

ml 1 N NaOH for 1 h, shortly washed with water and immersed in 0.9% NaCl solution

for 24 h before being tested. 5 control untreated strip were also tested.

Measurements of DC

5 (8×6 mm2) native pericardium samples were treated with 40 ml 1 N NaOH for 1 h and

incubated with α-chymotrypsin for the determination of DC. 5 control untreated samples

were also tested

SDS-PAGE

At each concentration and each time from the following 0.5 N (30 min, 1 h, 2 h), 1.0 N

(30 min, 1 h, 2 h) and 2.0 N (30 min, 1 h, 2 (8×6 mm2) native pericardium samples were

treated with NaOH. After the treatment, the samples were incubated with α-chymotrypsin

overnight at 37 °C. The supernatant, containing the denatured collagen part, was taken for

SDS-PAGE investigations.

4.3.2.2 The neutralization of the tissues after the NaOH treatment

In order to neutralize the pericardium samples after the NaOH treatment, 10 native

pericardium strips (4×1.5 cm) were treated with 100 ml 1 N NaOH for 1 h, 5 samples of

them were treated with 50 ml 1 N CH3COOH for 5 min and the other 5 samples with 50

ml 1 N CH3COOH for 15 min. This treatment was performed in June 2006.

For the examination of the influence of the concentration CH3COOH, 10 native strips

(4×1.5 cm) were treated with 100 ml 1 N NaOH for 1 h, 5 of them neutralized with 50 ml

4. Materials and Methods

43

1 N CH3COOH for 15 min and the other 5 with 50 ml 0.1 N CH3COOH for 15 min. This

treatment was performed in November 2006.

For the evaluation of the efficiency of washing solutions after the NaOH treatment, 15

native strips (4×1.5 cm) were treated for 1 h with 150 ml NaOH, 5 of them were

immersed for 30 min in 50 ml distilled water, another 5 were washed under stirring for 30

min with 50 ml 100 mmol phosphate buffer (pH 7.4) and the other 5 washed under

stirring for 90 min in 50 ml distilled water, in which water was changed every 15 min.

This treatment was performed in September 2006.

For the evaluation of efficiency of acid treatment step followed by washing step after the

NaOH treatment, 10 strips were treated for 1 h with 100 ml 1 N NaOH and treated with

100 ml 0.1 N CH3COOH for 15 min, subsequently 5 of them were washed under stirring

twice for 10 min with 50 ml 100 mmol phosphate buffer and the other 5 with 50 ml

distilled water. This treatment was performed in November 2006.

4.3.3 The hydrogen peroxide treatment

Shrinkage temperature measurements

12 native bovine pericardium strips (4×1.5 cm) were treated with H2O2 for 48 h, 4 of

them with 3 %, another 4 with 10 %, and the other 4 with 30 % H2O2, immersed in 0.9 %

NaCl solution for 24 h before being tested with the isotonic shrinkage technique in June

2006.

Measurements of DC

15 (8×6 mm2) native pericardium samples were treated H2O2 for 48 h, 5 of them with 3

%, another 5 with 10 %, and the other 5 with 30 % H2O2 and incubated with α-

chymotrypsin for the determination of DC. 5 control untreated samples were also tested

4.3.4 The acetone treatment

For the evaluation of the acetone treatment, bovine pericardium specimens from

Norwegian sources in the age of 18-25 months were stored under 26 % NaCl solution at 8

°C in April 2006.

The extent of drying and volume shrinkage

Two native bovine pericardium groups were exposed to acetone treatment in May 2006

for 18 days, the first group with 100% acetone and the second with acetone series starting

from 30% until 100%, every two days increased by 10% (Koerber 2006). Each 2 days, 5

4. Materials and Methods

44

samples 24×26×1.0 mm3 were taken for the measurements of the extent of drying and 1

sample for the volume shrinkage measurements from both groups (pure acetone and the

acetone series)

The measurements of DC

9 native bovine pericardium samples were subjected to 100% acetone and 6 to acetone

series treatment for 18 days, in addition to 10 samples untreated, incubated overnight

with α-chymotrypsin for the determination of DC (Koerber 2006).

4.3.5 The determination of water content

For the determination of water content, samples taken from bovine bone, native

pericardium, Tutoplast-processed pericardium, and lyophilized pericardium and heated

for 1 h at 105 °C, as shown in table 4-9. The weight loss was considered as the water

content

Table 4-9: The samples used in determining the water content

Substance Native

pericardium

(8×8 mm2)

Tutoplast-

processed

pericardium

(8×8 mm2)

Lyophilized

pericardium

(8×8 mm2)

Bone powder

No. of samples 10 10 10 5

5. Results Interpretations

45

5 Results Interpretations 5.1 The effect of bone pulverization on the collagen As described in section 4.2.1.1, the digestion of bone samples with α-chymotrypin

requires bone demineralization to guarantee the diffusion of α-chymotrypin (Wang, Bank

et al. 2001). In order to accelerate the bone demineralization, the bone samples were

pulverized; three types of mills have been used to pulverize femoral head cancellous

bones, milling machine, micro-dismembrator and ball mill. Subsequently, the powder

was sieved and the fraction (< 250 µm) was taken for the further examinations.

5.1.1 The measurements of DC

The analysis of bone powder using the measurements of denatured collagen (DC) shows

that pulverizing the bones either with the milling machine or with the micro-

dismembrator is destructive represented by the high values of DC, they have DC, mean ±

95% confidence interval, 56.6 % ± 6.2 and 68.4 % ± 20.7 respectively. In contrast to the

milling machine and the micro-dismembrator, ball mill is significantly the least

destructive mill and has relatively low DC values, 16.6 % ± 4.6. Interestingly the

destruction or denaturation caused by the milling machine and the micro-dismembrator is

temporary. It is confirmed that storing the bone powder for 1 week at 8 °C yields

significantly lower values of DC, 34.1 % ± 5.0 and 27. 0 % ± 6.7 respectively, as seen in

fig 5.1. The DC for the samples pulverized with the ball mill is almost unaffected by the

1 week-storage, 17.9 % ± 2.9.

5. Results Interpretations

46

DC

(%)

0

20

40

60

80

100

ball mill

micro-dismembrator

milling machine

fresh after 1 week Figure 5.1: Investigation of the effect of bone pulverization on the DC (error bars

indicate 95 % confidence interval for 6-12 samples)

5.1.2 Discussion of the results

It is reasonable to test the influence of bone pulverization on the stability of bone and

collagen for two reasons: first, to avoid any interfering influences on further experimental

results done with the bone powder, second because grinding is one the processing steps

for powder and granulate biological implants. It is known that the mechanical grinding or

pulverization can lead to changes in the structure of collagen and to mechanical

denaturation (Segalova, Dubinskaya et al. 1981). Day (Day 2005) has observed that

micro-dismembrator doubled the DC during the pulverization of osteaoarthritic bones. In

the present work pulverization has been done under liquid nitrogen to avoid thermal or

mechanical stress resulted during the process, which could destruct or denature the

collagen molecules in bone. However it was not enough to prevent the denaturation of

bones pulverized with the milling machine as well as with the dismembrator.

Surprisingly, a kind of ‘renaturation’ has been observed after storing the bone powder for

1 week at 8 °C. It is expected that the relatively short time of pulverization can create

5. Results Interpretations

47

moderate heating that results in a local unfolding within the protein, which appears to

regain its native structure upon the restoration of normal temperatures. This unfolding

may be due to the breaking of a small number of consecutive hydrogen bonds (Wright

and Humphrey 2002), in this case, the triple helix is unfolded but the polypeptide chains

are still fixed in their positions (Hoermann and Schlebusch 1971), which enable the

recovery of the native triple helix by building hydrogen bonds.

5.2 The thermal stability of collagenous tissues An excellent mean of studying the way in which the collagen molecule is held in its triple

helix is to examine the conditions under which the stabilization breaks down, helix coil

transition (Miles and Bailey 2004). The thermal denaturation of collagen induces

unfolding of the triple helix into random coils by breaking the hydrogen bonds (Privalov

1982); the ability of collagen to resist this unfolding is an indication of its “healthiness”.

Many imaging techniques such as, micro-computed tomography (Laib, Barou et al. 2000;

Bagi, Hanson et al. 2006) and transmission electron microscope (Porter, Nalla et al. 2005;

Sahar, Hong et al. 2005) can detect structural modification or destruction in µm or nm

scale. In contrast to these techniques the thermal stability covers higher level of

hierarchy.

5.2.1 Analysis of the thermal stability with the measurements of DC

5.2.1.1 The thermal stability of Tutoplast-processed bovine bone

The collagen denaturation induced by the thermal treatment is obviously shown in the

measurements of DC. It is observed, as seen in fig 5.2, that DC for bone powder remains

unchanged till the temperature 90 °C, and then it starts to increase linearly with

increasing temperature until almost 90 % denaturation is reached at temperature 160 °C.

Thereafter the DC is almost unchanged between 160 and 170 °C and then it decreases

slightly between 170 and 200 °C.

5. Results Interpretations

48

Temperature (°C)

0 20 40 60 80 100 120 140 160 180 200 220

DC

(%)

0

20

40

60

80

100

6 56

9

9

9

99

6

Figure 5.2: The temperature dependency of DC for Tutoplast-processed cancellous

bovine bone powder (error bars indicate 95% confidence interval for 5-

9 samples)

5.2.1.2 The thermal stability of native bovine pericardium

The DC of native pericardium (initial water content ~ 85.7%) is sensitive to the

temperature increase, as shown in fig. 5.3. It remains unchanged until 55 °C and then it

starts to increase progressively with increasing temperature until 150 °C. After that it

reaches a plateau between 150 and 200 °C. A significant reduction of the DC has not

been observed up to 200°C.

5. Results Interpretations

49

Temperature (°C)

0 20 40 60 80 100 120 140 160 180 200 220

DC

(%)

0

20

40

60

80

100

Figure 5.3: The temperature dependency of DC for native bovine pericardium

(error bars indicate 95% confidence interval for 5 samples)

5.2.1.3 The thermal stability of tutoplast-processed bovine pericardium

The Tutoplast-processed pericardium (initial water content ~ 1.9%) is extremely

thermally stable, as shown in fig 5.4. The DC is almost constant until 150 °C and then it

jumps too sharply to reach almost complete denaturation at 160 °C. The DC is almost

unchanged in the temperature range between 160 and 170 °C. One of the most interesting

remarks in these results is the sudden decrease of DC in the range (185-200 °C).

5. Results Interpretations

50

Temperature (°C)

0 20 40 60 80 100 120 140 160 180 200 220

DC

(%)

0

20

40

60

80

100

Figure 5.4: The temperature dependency of DC for Tutoplast-processed bovine

pericardium (error bars indicate 95% confidence interval for 5

samples)

5.2.1.4 The thermal stability of the lyophilized bovine pericardium

As observed with the Tutoplast-processed pericardium, the lyophilized pericardium

(initial water content ~ 7%) is also extremely thermally stable, as shown in fig. 5.5. The

DC is almost unchanged until 135 °C and then it increases but smoothly with increasing

temperature until 170 °C. An extreme reduction of DC in the range (185-200°C) has also

been shown with the lyophilized pericardium.

5. Results Interpretations

51

Temperature (°C)

0 20 40 60 80 100 120 140 160 180 200 220

DC

(%)

0

20

40

60

80

100

Figure 5.5: The temperature dependency of DC for the native lyophilized bovine

pericardium (error bars indicate 95% confidence interval for 5

samples)

5.2.1.5 The measurements of the extent of browning

The analysis of extent of browning, measured at 420 nm, shows that the absorbance of

the lyophilized and Tutoplast processed pericardium is slightly higher than that of the

native and rehydrated Tutoplast processed at 185 °C, as shown in fig. 5.6. The extent of

browning of the lyophilized and Tutoplsat processed pericardium becomes significantly

higher at 200 °C

5. Results Interpretations

52

Abs

orba

nce

(-)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

RT 185 °C 200 °C

rehydrated processed

processed

native lyophilized

native

Figure 5.6: The measurements of the extent of browning at 420 nm for native, native

lyophilized, rehydrated Tutoplast and Tutoplast-processed bovine

pericardium at room temperature, 185 and 200 °C (error bars indicate

± standard deviation for 5 samples)

5.2.1.6 Discussion of the results

As seen in figures 5.2-5.5 and 5.7, there was always an initial value of DC (around 16-22

%) even before the thermal treatment. The detection of denatured collagen in the native

pericardium at the beginning can be probably justified by the presence of soluble

collagen molecules, such as the newly synthesized and the other non-cross-linked

collagen (Bank, Krikken et al. 1997). Furthermore the effect of mechanical defatting and

the cutting of samples can’t be excluded. The initial value of DC for Tutoplast-processed

pericardium is possibly resulted from the processing, whereas contribution of soluble

proteins in this initial value is not expected because such proteins must be eliminated

during the hydrogen peroxide oxidation step in the process (Schoepf 2006). The initial

DC value of the lyophilized pericardium is quite higher than those of the native and the

5. Results Interpretations

53

solvent-processed. It is expected that the lyophilization cleaves some terminal collagen

domains, makes them accessible for α-chymotrypsin.

The denaturation or melting temperature (at DC = 0.5) is an important parameter to

assess the thermal stability of collagen. According to the results of the thermal stability of

Tutoplast-processed bovine cancellous bone, the denaturation temperature is

approximately 135 °C, as shown in fig 5.2, which differs from values got by other

research groups. The degree of mineralization (Kronick and Cooke 1996) as well as the

water content (Miles and Ghelashvili 1999) could cause this difference in denaturation

temperature. Kronick et al (Kronick and Cooke 1996) proved the influence of the degree

of mineralization on the denaturation temperature of collagen using differential scanning

calorimetry (DSC) measurements, they observed a denaturation temperature of 155 °C

for fully mineralized bone, while 113 °C and 63 °C for partially mineralized and

demineralized bone respectively. However, they didn’t mention the absolute mineral

content of the samples. Fratzl et al (Fratzl, Gupta et al. 2004) suggested that bone is not

uniformly mineralized but composed of bone packets, each having its own mineral

content. Therefore the sampling site could also affect the denaturation temperature. In the

current work, the bones are pulverized to avoid the effect of the heterogeneity in mineral

distribution; however the mineral content of the samples has not been determined.

Regarding the water content, Trebacz and Wojtoowicz (Trebacz and Wojtowicz 2005)

have tested the influence of water content on the denaturation temperature of cortical and

trabecular bone samples using DSC and observed for samples of water content between

0-8% a hydration-dependent peak within a range from 154 to 161 C. Wang et al (Wang,

Bank et al. 2001) observed a denaturation temperature of 160 °C for cortical bone

samples after selective digestion with α-Chymotrypsin without statement about the water

content. In this work, the powder samples are heated in boiling water bath for the

temperatures lower than 100 °C. In order to have relatively similar conditions for all the

samples regarding the effect of moisture content on the thermal stability, the samples for

the temperatures above 100 °C are heated for 15 min in boiling water bath before being

heated in furnace. This preheating step in water bath increases the water content of the

samples to approximately 59%.

5. Results Interpretations

54

Concerning the thermal stability of bovine pericardium, The DC curve of native,

Tutoplast-processed and lyophilized pericardium shows a similar sigmoidal behavior.

The curve consists of 3 regions; the length of each region is different from one tissue to

another depending on the water content and the preprocessing. The initial region of the

curve, where almost no change is observed, is followed by a region, where DC

approximately increases linearly with temperature. The final region is represented by

plateau or decrease of DC depending on the preprocessing.

The DC of the native pericardium (initial water content ~ 85.7%) shows higher sensitivity

to temperature change than that of the Tutoplast-processed (initial water content ~ 1.9%)

and the lyophilized (initial water content ~ 7%). It starts to increase linearly from 55 °C

until it reaches a plateau at 150 °C. The denaturation temperature (at DC = 0.5) is

approximately 115 °C, which differs from the reported values of shrinkage temperature

of native bovine pericardium in the literature (60-65 °C) (Pasquino, Pascale et al. 1994;

Moore, Chen et al. 1996). The measurements of shrinkage temperature as indication of

collagen denaturation are performed under fully hydrated conditions in water bath,

whereas the measurements of DC are taken after thermal treatment in furnace, in which

the thermal stability behaves differently, as will discussed later in the polymer in a box

mechanism.

Over a temperature range from 150 to 200 °C the DC is temperature independent. The

logical explanation for this behaviour is that the thermal denaturation of collagen is

kinetically controlled. A same level of heating damage can be obtained by combinations

of heating time and temperature level (Wright and Humphrey 2002). It is expected that

the 1-h heating was too long to examine the effect of increasing temperature on the DC,

in other words, with this relatively long time, the maximum limit of damage can be

reached in this range regardless of the temperature.

5. Results Interpretations

55

Temperature (°C)

0 20 40 60 80 100 120 140 160 180 200 220

DC

(%)

0

20

40

60

80

100

native

native lyophilized

processed

Figure 5.7: The temperature dependency of DC for native, Tutoplast-processed and

native lyophilized bovine pericardium (error bars indicate 95%

confidence interval for 5 samples)

A possible reason for not reaching complete denaturation or complete enzymatic

digestion at high temperatures could be the presence of proteoglycans, which prevent α-

chymotrypsin to diffuse easily (Bank, Krikken et al. 1997).

In contrast to the native pericardium, the Tutoplast-processed and lyophilized

pericardiums are extremely thermally stable. A relatively large initial region, where DC is

almost unchanged, is ended by a sharp denaturation. A final region, characterized by the

reduction of DC, has been observed in both cases.

The Tutoplast-processed pericardium is thermally slightly more stable than the

lyophilized one; the DC for Tutoplast-processed samples remained constant until

approximately 150 °C, whereas for the lyophilized samples, it was almost unchanged

until 135 °C. Dehydration in the lyophilized and Tutoplast-processed pericardium

5. Results Interpretations

56

obviously gives the samples a kind of protection against thermal denaturation. According

to the polymer in a box mechanism (Doi and Edwards 1986; Miles and Ghelashvili 1999)

dehydration reduces the lateral dimensions of the lattice, constrains the number of

possible configurations, reduces the free-volume available for denaturing α-chains

(Trebacz and Wojtowicz 2005), reduces the configuration entropy and thereby increases

the thermal stability of collagen. The slight preference for the Tutoplast-processed against

the lyophilized pericardium could be attributed to the lower water content of the

Tutoplast-processed pericardium. The dehydration constrains the movement of molecules

to be denatured and appeared to be confined within a “box”, as shown in fig 5.8. It is

supposed that the less the water content is, the more the collagen within this box is

confined, i.e. the Tutoplast-processed is more confined and consequently thermally more

stable.

Figure 5.8: schematic illustration shows the therma activation of collagen molecules

under higher and lower water content according to polymer in a box

mechanism, modified from (Miles and Ghelashvili 1999)

According to polymer in a box mechanism, the water content determine the size of the

box, the higher the water content, the larger the box. Therefore, the collagen molecules in

Low water content

Thermal activation

High water content

Thermal activation

5. Results Interpretations

57

the native pericardium are confined within large box, which couldn’t restrict the

movement of molecules to be denatured. During the thermal treatment of the native

pericardium, the molecules begin to denature with simultaneous reduction of water

content due to the evaporation. This reduction of water content leads to reduction of the

dimensions of box confining the molecules. After certain time, depending on the

temperature, the water is totally evaporated and the collagen molecules are confined

within small box. At this time, the denaturation of collagen proceeds with a rate lower

than that at the beginning of the heating but still higher than that of the originally dry

tissues (in this case Tutoplast-processed and lyophilized) because this box constrains

partly denatured or disordered molecules, whereas the box in the case of the dry tissues

contains intact and well-ordered molecules.

Understanding the polymer in a box mechanism is helpful to differentiate between

heating wet samples in furnace and heating wet samples in water bath. During the heating

in furnace, the dimensions of the box become smaller due to the evaporation of water,

whereas no evaporation of water takes place during the heating in water bath and

consequently no reduction of the box dimensions. Therefore the tissues are thermally less

stable when they heated in water bath.

A very remarkable observation was the extremely sharp increase of DC for the Tutoplast-

processed pericardium within 10 degrees (160-170 °C), in comparison with the relatively

smooth increase for that of lyophilized pericardium (135-170 °C). It is thought that

dehydration restricts the movement of collagen molecules and become confined within a

“box”, which protects collagen molecules from thermal denaturation. The molecules are

located within this hypothesized box. In order to denature collagen molecules, the

“protective box” has to be overcome and then the molecules remain unprotected against

thermal denaturation. In the case of Tutoplat-processed pericardium, after the supposed

removal of the protective box, the pericardium, which contains partly modified collagen

molecules, has lower ability to resist the thermal denaturation, which explains the sharp

increase of DC in the range 160-170 °C, as shown in fig 5.7. In contrast to Tutoplast-

processed pericardium, the lyophilized pericardium contains relatively “healthy” and

intact collagen molecules that could resist the thermal denaturation causing smooth not

sharp denaturation in the range 135-170 °C.

5. Results Interpretations

58

It was seen in general that lyophilized and Tutoplast-processed pericardium has sharper

denaturation in comparison with the native pericardium. As the collagen further

dehydrated, water molecules are stripped away causing an increase of the thermally-labile

domain and consequently a sharper denaturation (Miles and Ghelashvili 1999).

One of the most interesting remarks in DC results is the sudden decrease of DC for the

Tutoplast-processed and lyophilized pericardium in the range 185-200 °C. There are two

possible reasons behind the reduction of DC or the inability of α-chymotrypsin to digest

the denatured collagen at high temperatures, the breaking of covalent cross-links or the

formation of heat-generated advanced glycation end products (AGEs). Trebacz and

Wojtowicz (Trebacz and Wojtowicz 2005) have recognized two endothermal peaks

during DSC study of bones and tendons, the first endotherm was in the range from 155 to

165 °C for bones and from 118 to 137 °C for tendons, whereas the second endotherm was

in the range from 245 to 290 °C for bones and from 200 to 285 °C for tendons. They

supposed that that second peak, which accompanied with brown bones, is related to the

breaking of covalent cross-links. In the other hand, Maillard (browning) reaction or non-

enzymatic glycation is initiated in vivo from the reaction of reduced sugar with lysine and

arginine residues in protein (Verzijl, DeGroot et al. 2002). Furthermore AGEs formation

can be initiated by lipid peroxidation (Januszewski, Alderson et al. 2003). In contrast to

the in vivo AGEs, the heat generated AGEs, generated during the spontaneous reaction

between reducing sugars and proteins or lipids, form much rapidly and in greater

concentrations (Vlassara, Cai et al. 2002). In this study, it is expected the formation of

heat-generated AGEs, not the breaking of cross-links, was behind the reduction of DC.

The presence of proteoglycans in the pericardium tissues (Simionescu and Kefalides

1991; Simionescu, Deac et al. 1994) leads to the formation of AGEs at high temperatures,

which hinders the enzymatic degradation of collagen (DeGroot, Verzijl et al. 2001). The

reduction of DC was observed at relatively low temperatures in this study, from 185 °C,

in comparison with the supposed reported temperatures for the breaking cross-links

(Trebacz and Wojtowicz 2005). Furthermore the samples were characterized by a light

brown colour not dark brown as one expected in case of organic decomposition.

Spectrophotometrical measurements of the extent of browning prove that the lyophilized

and Tutoplast-processed pericardium formed heat-generated AGEs in the range (185-200

5. Results Interpretations

59

°C). The absorbance of browning for the lyophilized and Tutoplast-processed was

slightly higher than that of the native and rehydrated Tutoplast-processed at 185 °C and

become significantly higher at 200 °C as indication of the formation of heat generated

AGEs. The reason behind the absence of AGEs with the native pericardium (water

content ~ 85.7 %) as well as rehydrated Tutoplast-processed (water content ~ 94.0 %)

could be ascribed to the higher moisture content, which probably delays the reaction of

AGEs formation (Vlassara, Cai et al. 2002). It is supposed that the thermal denaturation

under high moisture-content proceeds too quickly preventing the formation of AGEs,

whereas under dry condition, the denaturation doesn’t proceeds quickly due the higher

resistance against the thermal denaturation, which allows the formation of heat-generated

AGEs. These results could explain the sudden reduction of DC at higher temperatures

with the Tutoplast-processed and the lyophilized but not with the native pericardium. The

current results confirm that the Proteoglycans are not removed during Tutoplast-process.

The slight not sharp reduction of DC, observed with the bone powder at high

temperature, could be attributed to the moderate water content, 59%.

5.2.1.7 Modeling of the thermal denaturation of pericardium

The thermal stability of collagen is one of the controversial subjects whether the collagen

denaturation is an equilibrium process or kinetic process. The Lumry and Eyring model

was established in 1954 to study the conformation changes in globular proteins, with

intermediate state that could either refold to the original native state or convert

irreversibly to the final denatured state (Lumry and Eyring 1954). The simplest form of

the Lumry and Eyring model can be presented by the following scheme (Sanchez-Ruiz

1992):

FUNkK→⇔ (5.1)

where N , U , and F are the native, unfolded and final (irreversibly denatured) states of

the protein, respectively.

Sanchez-Ruiz assumed that there is a reversible chemical equilibrium between N and U

and the unfolding enthalpy is constant. The van’t Hoff enthalpy H∆ can be obtained

from the temperature dependency of the equilibrium constant between the native and

unfolded states:

5. Results Interpretations

60

2

lnRT

HdT

Kd ∆= (5.2)

where R is the ideal gas constant. Assuming that H∆ is temperature independent, the

equilibrium constant K can be expressed as (Sanchez-Ruiz 1992):

[ ][ ] [ ]

⎭⎬⎫

⎩⎨⎧

−∆−=== mN

U TTRH

XX

NUK /1/1exp (5.3)

where xuand xN

are the molar fractions of unfolded and native states and Tm is the

temperature at which K = 1.0

Sanchez-Ruiz assumed also that the irreversible step ( FU → ) is described by the

kinetics of a first order reaction. The rate equation for the irreversible formation of F is

given in the following equation:

][][ UkdtFd

= , or uF Xk

dtdX = (5.4)

where X F is the molar fraction of the final state

The temperature dependency of the rate constant k, can be expressed with the Arrhenius

equation as:

[ ]⎭⎬⎫

⎩⎨⎧ −−= */1/1exp TT

REk , (5.5)

where E is the activation energy and T* is the temperature at which =k 1.0 min-1

(Sanchez-Ruiz 1992). Taking equation (5.2) into consideration and 1=++ XXX FUN ,

then:

)1(1 XK

kKdt

dXF

F −+

= (5.6)

Equation (5.6) shows that, at constant temperature, X F changes with time after first-order

kinetics with an apparent rate constant equal to 1+K

kK

Privalov (Privalov 1979) developed a theory of the temperature-induced changes of small

globular proteins based on equilibrium thermodynamics. He checked the validity of two-

state model and suggested that if the van’t Hoff enthalpy is identical to the calorimetric

enthalpy, then the denaturation is considered as a two-state process in equilibrium, i.e. the

5. Results Interpretations

61

protein presents a single cooperative unit. Later Privalov (Privalov 1982) showed that

collagen doesn’t present a single cooperative unit. This indicates that collagen melting is

an extremely cooperative process and the number of residues forming a “cooperative

block” that melts as a single structural unit can be found from the ratio between the van’t

Hoff enthalpy and the calorimetric enthalpy. Privalov proposed that the denaturation of

collagen is a slow process. Therefore calorimetric studies of collagen must be carried out

only at low heating rate, and the transition temperature can be obtained by extrapolating

to zero heating rate. Davis and Bächinger (Davis and Bachinger 1993) examined the

triple helix-coil transition of type III collagen solution using optical rotary dispersion and

ascertained that the transition is a reversible equilibrium process. They observed a

hysteresis in the helix-coil transition, where the midpoint of the refolding transition is 6-7

°C lower than that of the unfolding transition. They agreed that collagen, in their case the

cooperative length or block was 95 tripeptide units. Engel and Bächinger (Engel and

Bächinger 2000) examined Type III collagen solution and suggested that a strong

argument against the kinetic model of the collagen denaturation is the hysteresis

accompanied “reversibility” of the process, which they observed in their investigation.

They fitted their experimental data with the equilibrium model by least square fit with

equation 3, taking into consideration that 1=+ XX UN , neglecting the state F. They used

the equation of Gibbs free energy, equation (5.7) to evaluate the thermodynamic

quantities: 000 ln STHKRTG ∆−∆=−=∆ , (5.7)

where ∆S0 is the entropy of transition.

Leikina et al (Leikina, Mertts et al. 2002) suggested using combination of ultra-slow

differential scanning calorimetry (DSC) with isothermal circular dichroism for Type I

collagen solution that the collagen denaturation is reversible. They ascertained that

collagen type I is thermodynamically stable accompanied by large hysteresis with

equilibrium time from several hours (rats) to several days perhaps months or years

(humans) and the apparent irreversibility of collagen denaturation at the time scale of

several hours is simply manifestation of hysteresis. They proposed that denaturation is an

extreme slow equilibrium process and collagen is overheated by at least several degrees

above the equilibrium in the reported denaturation experiments. Denaturation of

5. Results Interpretations

62

overheated collagen occurs much faster than renaturation creating an appearance of an

irreversible rate limited process. Also Persikov et al (Persikov, Xu et al. 2004) have

observed the reversibility in the thermal transition of collagen-like peptides.

In contrast to the authors mentioned above, Miles (Miles 1993) and Miles et al (Miles,

Burjanadze et al. 1995) have shown that the thermal denaturation of collagen is governed

by irreversible rate process in which collagen is transformed to denatured state via a

highly temperature dependent kinetic rate constant, not by equilibrium thermodynamics,

as in equation (5.8).

NTkdtdN )(−= (5.8)

They examined the thermal stability of lens capsules (non-fibrillar Collagen IV) and rat

tail tendon (fibrillar collagen I) with differential scanning calorimetry (DSC). They

observed no evidence of denaturation endotherm after holding heat-denatured tendons for

up to five days as indication of the irreversibility in the short and medium term. Miles

observed that the native collagen content declined according to a first order kinetics after

storing lens capsules isothermally. He observed also that the rate constant was highly

temperature dependent increasing about one order of magnitude every 2.3 °C rise.

Recently Miles and Bailey (Miles and Bailey 2004) have examined solutions of collagen-

like peptides using DSC and shown also that the dentauration is kinetic controlled. In the

last study they showed that endotherms could be classified in one of three regions: the

equilibrium region, the mixed region or the rate (kinetic) region, dependent on the

polymer concentration and the scanning rate. They suggested that holding collagen at

constant temperature will not stop the denaturation as it would if the system were in

equilibrium. Also as an argument against the equilibrium model, they proposed that

reducing the temperature will continue the denaturation but with a slower rate, in contrast

to the equilibrium where some helix would be recovered. They concluded, in agreement

with other studies (Miles 1993), (Miles, Burjanadze et al. 1995), (Leikina, Mertts et al.

2002) that the denaturation temperature is increasing logarithmically with the scanning

rate. Therefore it is impossible to extrapolate to zero scanning rate to obtain the

hypothetical equilibrium temperature.

5. Results Interpretations

63

Miles and Bailey (Miles and Bailey 2004) ascertained that the validity of kinetic model

does not depend whether the triple helix is irrecoverable or recoverable, and the

renaturation of molecular type I collagen, if it is possible at all, is very slow in relation to

the time scale of experiment.

The aim of this work is to examine the effect of the conservation process on the thermal

stability of bovine pericardium and to analyze the results with the thermodynamic and

kinetic models found in the literature. To assess the thermal stability, measurements of

the fraction of denatured collagen (DC) after selective digestion with α-chymotrypsin

were performed.

For the investigation of kinetic and thermodynamic parameters, it was assumed that the

thermal denaturation consists of two steps one reversible step followed by an irreversible

one, as described in equation (5.1). It was assumed also that α-chymotrypsin digests both

U and F fractions leaving them together in the supernatant. Therefore a mathematical

equation was derived from equation 5.1, 5.3, 5.4 and 5.6 for the summation of U and F

and it was called S, where S the fraction in the supernatant, as following

dtdX

dtdX

dtdX FUS += (5.9)

dtXXd

dtdX NUF )1( −−

= . Taking into consideration that K

XX UN = yields:

UU X

KkK

dtdX

+−=

1 (5.10)

Substituting equations (5.6) and (5.10) in equation (5.9):

)1(1

)1(1 SUF

S XK

kKXXK

kKdt

dX−

+=−−

+= (5.11)

It is assumed that the chemical equilibrium of the reversible reaction UNK⇔ is a very

slow reaction in comparison to the irreversible reaction; consequently the rate of F

formation will be determined by apparent first order reaction.

FNK

→/

(5.12)

For this purpose, isothermal experiments of Tutoplast-processed and lyophilized

pericardium with time variation have been performed to determine /K .

5. Results Interpretations

64

Temperature (°C)

140 145 150 155 160 165 170 175 180

K´

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Tutoplast-processed

Native lyophilized

Figure 5.9: The temperature dependency of /K for lyophilized and Tutoplast-

processed bovine pericardium

It can be seen that the denaturation of the Tutoplst-processed pericardium is faster than

that for the lyophilized pericardium in the range of the examined temperatures (145-175

°C), as shown in fig 5.9. The /K values can give an indication about the progress of the

thermal denaturation and consequently the resistance to this denaturation. The /K value is

too low at 145 °C for both the lyophilized and the solvent because the reaction is too slow

at this temperature. Increasing the temperature to 165 °C leads to 4.5-fold increase of the /K value of the Tutoplast-processed, whereas the /K value of the lyophilized is

increased 1.3 fold. This indicates that the lyophilized pericardium is healthier and has

higher resistance to thermal denaturation than the Tutoplast-processed. The largest jump

in the /K value of the lyophilized pericardium is observed at 175 °C, the temperature

increase from 160 to 175 °C leads to 2.6-fold increase of the /K value; however it is still

far away from that of the Tutoplast-processed pericardium. The thermal denaturation of

the solvent-processed-pericardium is sharp due to the lower thermal stability and

5. Results Interpretations

65

associated with an activation energy 118.95 kJ/mol, whereas the thermal denaturation of

the lyophilized pericardium is associated with an activation energy 61.02 kJ/mol.

It was not possible to get useful information from the isothermal experiments performed

with the native pericardium because it contains high moisture content (≈ 85.7%) that

dominates the behavior of the thermal stability. It is found that the high water content

accelerates the thermal denaturation of the pericardium. The water content is normally

evaporated within the first 5-10 min, as shown in fig 5.10, within this time fast

denaturation takes place. As long as the water is evaporated, the denaturation proceeds

but with different kinetics.

Time (min)0 5 10 15 20 25

wei

ght l

oss

(%)

0

20

40

60

80

100

Figure 5.10: The time dependency of weight loss of native bovine pericardium at 105

°C

5.2.2 Measurements of isotonic shrinkage temperature

As discussed in the previous section, the moisture content is the deciding factor in

shaping the thermal stability during the measurements of DC. Therefore it was reasonable

to exclude the effect of the moisture content by performing measurements of isotonic

shrinkage temperature at fully hydrated conditions in water bath, in which only the

structure integrity and healthiness plays a role in shaping the thermal stability.

5. Results Interpretations

66

5.2.2.1 The results

4 (4×1.5 cm2) strips from the native, native lyophilized and Tutoplast-processed

pericardium were used for the measurements of shrinkage temperature. The Tutoplast-

processed pericardium starts to shrink approximately from 42 °ّC until the maximum

shrinkage is reached at 70 °C, as shown in fig 5.11. The lyophilized and the native

pericardium have almost similar progress in the shrinkage curve. The shrinkage intervals

are (65-73 °C) and (64-72 °C) respectively.

Temperature (°C)

20 30 40 50 60 70 80

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

100

processed native lyophilized

native

Figure 5.11: The shrinkage curve for the native, native lyophilized and Tutoplast-

processed pericardium (average of 4 samples, each curve contains

approximately 1000 points, 95% confidence interval lies between ± 2-

17)

5.2.2.2 Discussion of the results

There is no absolute shrinkage temperature (Weir 1949), the shrinkage temperature must

be defined at certain constant heating rate and constant isotonic load. Furthermore some

authors defined the shrinkage temperature as the temperature at which the shrinkage

5. Results Interpretations

67

begins (Lennox 1949), while others as the temperature at which the tissue shrinks to 50%

of its maximum shrinkage (Rasmussen, Wakim et al. 1964; Danielsen 1990). The

shrinkage process of Tutoplast-processed strips was slow and takes place over relatively

wide temperature range (42-70 °C). Therefore, considering the shrinkage temperature as

the temperature at which the tissue shrinks to 50% will overestimate the shrinkage

temperature of Tutoplast process pericardium. The presence of residual ions in the

Tutoplast-processed pericardium resulted from the processing was confirmed by the

measurements of the thermal conductivity; 5 (8×6 mm2) native samples in 30 ml water

had thermal conductivity 12.6 µs/sec, whereas the processed 138 µs/sec. The presence of

the residual ions leads to swelling of the tissues during the shrinkage process and

consequently to thick samples, which shrink too slowly.

Lennox (Lennox 1949) has examined the effect of moisture content on the shrinkage

temperature. He concluded that increasing the soaking period increases the moisture

content and reduce the shrinkage temperature. Furthermore Weir (Weir 1949) observed

that dry specimens elongate before shrinkage occurs, but after soaking 1 h or longer in

water no preliminary elongation was observed. Therefore, in this work in order to

exclude any effect of moisture content or any preliminary elongation, the strips were

rehydrated in 0.9% NaCl solution for 24 h to have fully hydrated strips.

The bovine pericardium is known to be mechanically anisotropic, which affects the

mechanical properties of the pericardium (Crofts and Trowbridge 1989; Lee, Haberer et

al. 1989). The mechanical anisotropy was proved to be irrelevant for the initiation of

thermal denaturation (Lennox 1949). In this study, despite the well-known non-uniform

orientation of the pericardium, the randomly chosen strips have almost the same

shrinkage temperature.

Regarding the extent of the shrinkage, Lennox (Lennox 1949) observed two different

groups with different extent of shrinkage along the fatty layers. In this study, the extent of

shrinkage is different from one sample to another and no conclusion can be drawn about

the effect of temperature or processing on the extent of shrinkage. Therefore the

shrinkage for each sample is related to the maximum shrinkage, which gives almost

homogenous results.

5. Results Interpretations

68

The results of shrinkage temperature confirmed the conclusion drawn from the

measurements of DC that the dehydration or the moisture content has governed the

thermal stability. Under fully hydrated conditions, the thermal stability or the

thermoelasticity of the Tutoplast-processed pericardium to resist shrinkage was relatively

low in comparison with that for the native and the native lyophilized pericardium. It is

believed that structural modification or destruction induced by the processing causes this

reduction of thermal stability.

A conclusion has been drawn during the measurements of DC that the native lyophilized

pericardium samples are relatively intact undamaged. This is drawn from the smooth

denaturation in the temperature range (135-170 °C) during the DC measurements. This

conclusion has been also verified by the shrinkage temperature measurements. The

shrinkage temperature or the shrinkage curve was almost similar to that of the native

pericardium. Both of them were thermally unaffected until 64 and 65 °C respectively and

for both of them, the shrinkage proceeds quickly within 5 degrees.

5.2.2.3 Modelling of the thermal shrinkage of pericardium

The shrinkage curve consists of three different regimes (Chen, Wright et al. 1997; Chen,

Wright et al. 1998): an initial slow shrinkage regime, a rapid, large-shrinkage regime, and

finally a slow continuing-shrinkage regime.

Weir (Weir 1949) assumed that the shrinkage of collagen is described by a first order

kinetics as the following:

kLdtdL

−= (5.13)

Rearranging and integrating yields:

∞∞ +−−= LkteLLL )( 0 (5.14)

where

L is the length of the tissue at time (t), ∞L the completely shrunken length, 0L the initial

length and k the shrinkage rate constant.

Plotting ln( ∞− LL ) against t will yield a straight line of slope –k. Weir (Weir 1949)

disagreed with the use of such relation to obtain the rate constant (k) due to the negligible

5. Results Interpretations

69

linearity at the initial region of the shrinkage curve. Instead of that, he suggested to use

the time of half-shrinkage t1/2.

2/1/2ln tk = (5.15)

The model described in equation (5.13) is absolutely empirical and doesn’t explicitly

describe the progress of the denaturation of collagen molecules. In the current work, an

alternative model has been developed to describe the isothermal and the non-isothermal

isotonic shrinkage of collagen, in which the progress of the shrinkage is described

mathematically more in details and related to the number of denatured collagen

molecules.

The progressive denaturation of individual molecules

It is assumed that the collagen fiber consists of n collagen molecules; and that all the

molecules have the same length l with the total fiber length L .

At the beginning, where no shrinkage or denaturation occurs, there are only native

molecules and therefore:

NlnL ⋅= 00 (5.16)

where

0n is the initial number of collagen molecules and Nl the length of native molecules

If denaturation starts at t > 0, the hydrogen bonds will be broken, some molecules will

become denatured, as shown in Fig. 5.12, and consequently the fiber will begin to shrink

having the length )(tL at time t:

DDNN lnlntL +=)( (5.17)

where Nn is the number of native molecules at time t, Dn the number of denatured

molecules at time t and Dl the length of the denatured molecules. It can be assumed that

the total number of the molecules within a fiber is always constant, and therefore:

DN nnn +=0 (5.18)

5. Results Interpretations

70

Figure 5. 12: Schematic representation of the native and denatured collagen. Nl and

Dl represents the length of the native and denatured collagen molecule

respectively

Combining equations (5.16-5.18) yields:

)()( 0 DND llnLtL −−= (5.19)

The last equation is valid for both the isothermal and non-isothermal shrinkage of

collagen.

Modeling of isothermal shrinkage

As previously assumed (Weir 1949; Miles 1993; Miles, Burjanadze et al. 1995), the

shrinkage or denaturation of collagen follows first order kinetics, rearranging equation

(5.13) in terms of n:

nTkdtdn )(−= (5.20)

Integrating equation (5.20) yields:

tTkennN)(

0−= (5.21)

Taking equation (5.18) into consideration:

))(1(0tTkennD

−−= (5.22)

Substituting equation (5.22) in equation (5.19) gives:

Nl

Dl

5. Results Interpretations

71

))()(1()( 00 DN lltTkentLL −−−=− (5.23)

The last equation describes the time-dependence progress of shrinkage mathematically.

The t1/2 is defined at 50% of the maximum shrinkage. Therefore it is reasonable to relate

the shrinkage at time t to the maximum shrinkage at ( ∞t or endt ):

∞∞−−

−−=

−−

tTke

tTketLLtLL

)(1

)(1)(

)(

0

0 (5.24)

Due to the asymptotic behavior of the shrinkage curve, ∞t can be defined as the time at

which the shrinkage rate is reduced to 1% of its rate in the linear region, therefore

equation (5.23) is differentiated to obtain the rate of the shrinkage and consequently:

01.0)(

0

=−=⎟⎠⎞

⎜⎝⎛

⎟⎠⎞

⎜⎝⎛

∞∞ tTke

dtdLdtdL

t

t (5.25)

In this case it is assumed that 0t is the time at which the linear region of the shrinkage

curve begins. Rearranging equation (5.25) yields an equation for the determination of ∞t :

)(100lnTk

t =∞ (5.26)

The time of maximum shrinkage ∞t can related to the time of the half shrinkage 2/1t :

2/164.6 tt =∞ (5.27)

Temperatures 65, 66.5, 68, 69.5 °C are selected to perform the isothermal experiments

with the bovine pericardium. These temperatures are selected based on previous

examinations in the last section, which showed that these temperature lie in the shrinkage

range of the pericardium. The current results prove that the shrinkage of pericardium is

kinetically controlled, the higher the temperature, the faster the shrinkage. Assuming that

the shrinkage follows first order kinetics, the time of the half shrinkage (t1/2) is used for

the determination of the kinetic constant (k), as shown in table 5-1.

5. Results Interpretations

72

Table 5-1: The kinetic parameters of the mean shrinkage curve of the native

pericardium (4-5 samples)

Temperature (°C) t1/2 (sec) k (sec-1)

65 385 0.0018

66.5 64 0.0108

68 32 0.0221

69.5 25 0.0277

For the determination of the kinetic parameters, Arrhenius equation was used:

)/exp( RTEAk −= (5.28)

Plotting ln (k) against (1/T), as shown in Fig 5.13, yields the slope (E/R) and the intercept

(ln A). The activation energy, E, and the frequency factor, A, for the bovine pericardium

examined in this study are 565.95 kJ/mol and 8.64×1084 sec-1 respectively.

1/T*103 (1/K)

2.91 2.92 2.93 2.94 2.95 2.96 2.97

ln (k

)

-8

-7

-6

-5

-4

-3

-2

Figure 5.13: Plot of the natural logarithm of the kinetic constant k against the

inverse of the temperature (error bars indicate 95% confidence interval

for 4-5 samples)

5. Results Interpretations

73

The current values lie close to the kinetic parameters of collagen denaturation reported in

the literature, which supposed to be different from tissue to another. The E values in the

literature vary between 102-103 kJ/mol, whereas A between 1030-10105 sec-1 (Vijverberg,

Pearce et al. 1993; Agah, Pearce et al. 1994; Pearce and Thomsen 1995; Moran,

Anderson et al. 2000).

The experimental data obtained from the different isothermal curves are modeled using

equation (5.24). The proposed model has fitted the experimental data well, as shown in

Fig 5.14. The largest deviation is observed at lower temperatures. This could be attributed

to the relatively long lag phase or initial shrinkage region. As a limitation of the model,

the delay or the initial slow region at low temperatures can’t be modeled or fitted well

because the model describes the progress, as one single step, exponentially. It is

concluded that the model fits the higher temperatures better and the deviation becomes

almost negligible because the initial region is too short at higher temperatures to be

detected.

Time (sec)0 200 400 600 800 1000 1200 1400

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

100

65 °C

66.5 °C

68 °C

ModelExperimental

Figure 5.14: The measured and modeled isothermal shrinkage curve of the bovine

pericardium (average for 5 samples)

5. Results Interpretations

74

Modeling of non-isothermal shrinkage

In the normal shrinkage temperature experiment, the temperature changes with time

according to a constant heating rate (q). Therefore rearranging equation (5.13):

nTkqdT

dn )(1−= (5.29)

)/exp()( RTEATk −= (5.30)

∫ −−=∫T

T

n

ndTRTE

qA

ndnN

00

)/(exp (5.31)

where, A is the frequency factor and E the activation energy

The integration of the term dTRTE∫ − )/(exp in equation (5.31) can be expressed in the

form (Senum and Yang 1977):

[ ])()()/(exp 0xfxfREdTRTE −=∫ − (5.32)

where RTEx /=

There are different degrees of rational approximations for )(xf (Senum and Yang 1977).

The first degree of approximation is considered as the following:

21.)exp()(+

−=

xxxxf (5.33)

Assuming that )( 0xf is negligible, therefore:

⎥⎦⎤

⎢⎣⎡

+−−

=2/

1./

)/(expln0 RTERTE

RTERE

qA

nnN (5.34)

)2/

)/(exp(exp0 ⎥⎦⎤

⎢⎣⎡

+−−

=RTE

RTEqTAnnN (5.35)

))2/

)/(exp(exp1(0 ⎥⎦⎤

⎢⎣⎡

+−−

−=RTE

RTEqTAnnD (5.36)

Substitution of equation (5.35) in equation (5.19) yields:

)())2/

)/(exp(exp1()( 00 DN llRTE

RTEqTAnTLL −⎥⎦

⎤⎢⎣⎡

+−−

−=− (5.37)

The last equation describes the temperature-dependence progress of the shrinkage of

collagen. Analogously to the isothermal shrinkage, the shrinkage at temperature T is

related to the maximum shrinkage at temperature ∞T :

5. Results Interpretations

75

)2/

)/(exp(exp1

)2/

)/(exp(exp1

)()(

0

0

⎥⎦

⎤⎢⎣

⎡+

−−−

⎥⎦⎤

⎢⎣⎡

+−−

−=

−−

∞

∞∞∞

RTERTE

qAT

RTERTE

qTA

TLLTLL (5.38)

Some authors defined the shrinkage temperature as the temperature at which the

shrinkage begins (Lennox 1949), while others as the temperature at which the tissue

shrinks to 50% of its maximum shrinkage (Rasmussen, Wakim et al. 1964; Danielsen

1990).

The experimental data obtained from the non-isothermal shrinkage curve are fitted with

the proposed model using equation (5.38), after the determination of E and A from the

isothermal experiments. The suggested model has also fitted the experimental data

satisfactorily, as shown in Fig 5.15. Small deviation from the experimental data is also

observed at the initial and final regions. The shrinkage temperature, taken at 50% of the

maximum shrinkage, is 68.0 °C.

Temperature (°C)40 50 60 70 80

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

100

Model

Experimental

Figure 5.15: The measured and modeled non-isothermal shrinkage curve of the

bovine pericardium under heating rate 2.5 °C/min (average for 5

samples)

5. Results Interpretations

76

Little deviation is observed at the initial region as well as at the final slow continuing

shrinkage region. The model, based on the kinetic parameters obtained from the

isothermal experiments, fits a one-step shrinkage curve with an exponential form, which

deviates slightly from the measured shrinkage curve, due to the relatively long initial and

final regions observed with the measured curve.

The denaturation or shrinkage temperature of collagen is heating rate dependent (Weir

1949; Miles 1993; Miles, Burjanadze et al. 1995), therefore it is reasonable to establish a

relationship between the heating rate and the shrinkage temperature. For this purpose, a

dimensionless heating rate parameter is introduced, EAqRB = in addition to the

dimensionless temperature parameterRTEx = . Rearranging equation (5.38) yields

)2

)(exp1(exp1

)2

)(exp1(exp1

)()(

0

0

⎥⎦

⎤⎢⎣

⎡+−−

−

⎥⎦⎤

⎢⎣⎡

+−−

−=

−−

∞

∞

∞

∞

xx

Bx

xx

xBTLLTLL (5.39)

Introducing ⎥⎦⎤

⎢⎣⎡

+−

=2

)exp(1x

xx

z results in:

)/exp(1)/exp(1

)()(

0

0

BzBz

TLLTLL

∞∞ −−−−

=−− (5.40)

The shrinkage temperature T50 is defined as the temperature at which the tissue shrinks to

50% of its maximum shrinkage. Therefore, substituting 0.5 in the left side of equation

(5.40) under constant B leads to the determination of 2/1z and consequently 2/1x .

Variation of the heating rate, q , and consequently B leads to variation of 2/1z and 2/1x .

A relationship between 2/1x and B , which shows the effect of varying the heating rate in

the range (0.1-50 °C/min).on the shrinkage temperature, is shown in Fig 5.16.

5. Results Interpretations

77

B=qR/EA *1090 (-)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

x 1/2

=E/R

T 50

(-)

196

197

198

199

200

201

202

203

204

0.1

2

5

502520

10

Figure 5.16: The influence of varying the heating rate parameter B on the

temperature parameter x in the range of heating rate (0.1-50 °C/min)

An exponential-like relationship has been observed. It is seen that the shrinkage

temperature is sensitive to the change of heating rate especially at lower heating rates.

The largest drop in temperature parameter 2/1x is observed in the range (0.1-2 °C/min),

i.e. the shrinkage temperature is increased approximately by five degrees, from 62.5 to

67.4 °C. Afterwards, with increasing the heating rate the shrinkage temperature will

increase but not as significant as the case at lower heating rates. Interestingly, the

modelled shrinkage temperature at the heating rate used in the current work, 2.5 °C/min,

taken from fig 5.16 is very close to the experimental shrinkage temperature. The

modelled shrinkage temperature is 67.8 °C, whereas the experimental is 68 °C.

5. Results Interpretations

78

5.2.3 SDS-PAGE investigations

As discussed previously, thermal denaturation induces unfolding of triple helix into

random coils, which makes collagen susceptible for the enzymatic degradation. SDS-

PAGE was used to evaluate the thermal stability of collagen by analyzing the α-

chymotrypsin digested fraction of collagen after the thermal treatment. Determining the

fragments size during the SDS-PAGE investigation can provide information about the

extent of denaturation.

5.2.3.1 Results

Figure 5.17 shows SDS-PAGE of native and Tutoplast-processed pericardium at room

temperature and 55 °C, taken from (Koerber 2006). At room temperature, clear large

bands or fragments are observed in the range of 100, 66, 55, 13, 15 and 10 kD with the

native pericardium, whereas only band in the range of 150 kD is detected with the

Tutoplast processed pericardium.

At 55 °C the native pericardium has weak bands in the range of 120, 97, 22 and 14 kD,

whereas the Tutoplast processed pericardium has the same fragment, 150 kD, found at

room temperature.

Figure 5.17: SDS-PAGE for standard (lane 1), native pericardium at room

temperature (2, 3), processed at room temperature (4, 5), native at 55

°C (6, 7), processed at 55 °C (8, 9) and α-chymotrypsin (10)

5. Results Interpretations

79

The number of large fragments becomes less for the native pericardium at 90 °C, as

shown in fig 5.18. Bands in the range of 31, 21.5, 15 and 13 kD were detected. For the

Tutoplast-processed pericardium, as observed at room temperature and 55 °C, only a

large fragment at 110 kD is observed.

The bands, observed with the native pericardium at 90 °C, are also detected at 120 °C but

with lower intensity. No further large fragments have been detected at 120 °C for the

Tutoplast-processed pericardium.

Figure 5.18: SDS-PAGE for standard (lane 1), native pericardium at 90 °C (2, 3),

processed at 90 °C (4, 5), native at 120 °C (6, 7), processed at 120°C (8,

9) and α-chymotrypsin (10)

No large fragments were detectable neither with the native nor with the Tutoplast-

processed pericardium at temperatures 135 and 150 °C, as shown in fig 5.19.

5. Results Interpretations

80

Figure 5.19: SDS-PAGE for standard (lane 1), native pericardium at 135 °C (2, 3),

processed at 135 °C (4, 5), native at 150 °C (6, 7), processed at 150°C (8,

9) and α-chymotrypsin (10)

Also no large fragments were detectable neither with the native nor with the Tutoplast-

processed pericardium at temperatures 160 and 170 °C, as shown in fig 5.20.

5. Results Interpretations

81

Figure 5.20: SDS-PAGE for standard (lane 1), native pericardium at 160 °C (2),

processed at 160 °C (3), native at 170 °C (4, 5), processed at 170°C (6,

7), native at 185 °C (8, 9) and α-chymotrypsin (10)

5.2.3.2 Discussion

The denaturation of collagen triple helix occurs through a melting of the hydrated

crystallites, involving rupture of hydrogen bonds and rearrangement of the triple helix

into random chain configuration (Miles and Bailey 2001). The denaturation of collagen

increases the susceptibility to proteolytic degradation. During the denaturation the triple

helix unwinds leaving the cleavage sites accessible for enzymatic degradation. SDS-

PAGE separates the proteins or the fragments according to their size. The determination

of the fragment size for enzymatic digested proteins gives an impression how accessible

the cleavage sites were.

Regarding the SDS-PAGE at room temperature, large fragments have been detected with

the α-chymotrypsin digested fraction of native pericardium. It is expected that these

fragments represent the soluble and the new synthesized non cross-linked collagen, which

α-chymotrypsin can digest (Bank, Krikken et al. 1997). In the case of the Tutoplast-

5. Results Interpretations

82

processed pericardium, it is assumed that the soluble proteins are already eliminated

during Tutoplast-process (Schoepf 2006). This explains the absence of fragments on the

gel except only one band in the range of 150 kD. This band could represent the processed

and modified terminal domains outside the triple helix.

At 55 °C the number of large fragments in the native pericardium becomes less.

According to the measurements of DC discussed in section 5.2.1.2, the denaturation of

native pericardium starts at 55 °C. It is supposed that at this temperature, domains of the

triple helix start to unfold through heating, which makes them susceptible to α-

chymotrypsin. It is supposed that no large fragments of the soluble proteins still

detectable. In the Tutoplast-processed pericardium, the band is the same as that obtained

at room temperature. A simple explanation for this behavior is that the terminal domains

still resistant at this temperature against heating but can be digested in large fragments by

α-chymotrypsin. The triple helix, according to the results of DC measurements and the

effect of dehydration discussed previously, can’t be attacked yet during the heating and

consequently it is not susceptible to α-chymotrypsin.

At 90 °C the denaturation goes on with the native pericardium allowing more cleavage

sites to be susceptible to α-chymotrypsin leading to the reduction of large fragments in

the SDS-PAGE. For the Tutoplast processed pericardium, the terminal collagen starts to

be affected from heating causing fragmentation and consequently leading to smaller

fragments, however still large. Also the triple helix is still not attacked.

At 120 °C, in consistency with the measurements of DC, the denaturation reaches almost

close to the maximum denaturation with the native pericardium. More cleavage sites

become accessible for α-chymotrypsin, which lowers the intensity of the large fragments.

For the Tutoplast-processed no large fragments are detectable. It is expected that the

terminal domains are extremely denatured and fully digested by α-chymotrypsin. It is

thought also that the triple helix in not affected.

No fragments are detected at temperatures 135 and 150 °C, neither for the native nor for

the Tutoplast-processed. It is assumed at these temperatures that the triple helix of the

native pericardium is fully accessible for α-chymotrypsin, and the triple helix of the

Tutoplast-processed pericardium is still not accessible, which explains the absence of

large fragments on the SDS PAGE.

5. Results Interpretations

83

No large bands are detected in the range 160-185 °C, neither with the Tutoplast-

processed nor with the native pericardium.

The absence of bands is observed during the SDS-PAGE investigations of the Tutoplast

processed pericardium either at low temperatures or at high temperatures (above 160 °C).

It is not easy, using SDS-PAGE only, to judge how the extent of denaturation is, because

during the investigations of SDS-PAGE, only the denatured fractions are tested without

obtaining any information about the intact fractions. Consequently, the analysis and the

assessment of the SDS-PAGE investigation could be better understandable with the help

of the previous analysis of the measurements of DC.

During the measurements of DC for the Tutoplast-processed pericardium, The DC is

almost unchanged until 150 °C, thereafter, sharp transition from the triple helix to

random coils is observed in the range 160-170 °C. Therefore, the absence of large

molecules in the Tutoplast-pericardium could be attributed to the fully inaccessible

cleavage sites at temperatures up to 160 °C, and to the fully accessible cleavage sites at

temperatures above 160 °C.

5.3 The effect of different steps in the Tutoplast® process The analysis of the results of the thermal stability draws the attention to many crucial

conclusions, which can help to understand the effect induced by Tutoplast-process. It was

concluded the processing induces two contradictory factors, stabilizing factor,

represented by the dehydration, and destabilizing factor, which may represented by

structural modification. The next challenge was to investigate the role or the effect of

each step of Tutoplat process and its contribution in the structural modification caused by

the process.

5.3.1 The effect of sodium hydroxide treatment

The NaOH treatment in the process is scientifically recognized as an acceptable and

effective methodology for reducing prion infectivity by six log (Brown, Rohwer et al.

1986; Schoepf 2006). However treating the collagenous tissues with NaOH leaves

detrimental side effects (Kearney and Johnson 1991).

5. Results Interpretations

84

5.3.1.1 The hydrolysis of collagen amino acids

The extent of hydrolysis at room temperature was checked by measuring the amount of

the aromatic amino acids dissolved in the NaOH solution spectrophotometrically at 280

nm.

First a calibration curve for the content of aromatic amino acids is constructed using a

stock solution of 2 mg aromatic amino acids in 100 ml water, as discussed in section

4.3.2.1. Different dilution series have been performed to obtain the calibration line shown

in fig 5.21 (Koerber 2006).

m AA (µg)

0 5 10 15 20 25

Abs

orba

nce

280

nm (-

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Figure 5.21: The calibration line of the aromatic amino acids. The absorbance

measured at 280 nm, error bars indicate 95% confidence interval for 5

samples (y = 0.0259 x + 5.46.10-3, r2 = 0.9964)

The amount of collagen dissolved or hydrolyzed in the NaOH solution can be

determined, taking into consideration that the aromatic amino acids constitute 2.84 % of

the collagen (Fietzek and Kuehn 1976).

Fig 5.22 shows the time-dependent hydrolysis of the collagen in the native pericardium at

room temperature. The amount of dissolved or hydrolyzed collagen is increased linearly

5. Results Interpretations

85

with the time of treatment. However, the amount is too low. Treating the native

pericardium with 1 N NaOH for 150 min yields an average amount of dissolved collagen

of 137 µg, which is lower than 1% of the original dry weight of the sample.

Time (min)

0 20 40 60 80 100 120 140 160

mc

diss

olve

d (%

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Figure 5.22: Time-dependent hydrolysis of collagen during the treatment of native

pericardium with 1 N NaOH at room temperature, error bars indicate

95 % confidence interval for 10 samples

The same linear time-dependence hydrolysis of collagen is almost observed with the

Tutoplast-processed pericardium samples, as shown in fig 5.23. Also the amount of

dissolved collagen in the NaOH solution is neglected in comparison with the original dry

weight.

5. Results Interpretations

86

Time (min)

0 20 40 60 80 100 120 140 160

mc

diss

olve

d (%

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Figure 5.23: Time-dependent hydrolysis of collagen during the treatment of

processed pericardium with 1 N NaOH at room temperature, error bars

indicate 95 % confidence interval for 10 samples

5.3.1.2 The measurements of the shrinkage temperature

For the assessment of the effect of the NaOH solution treatment and the subsequent

neutralization step on the quality of native bovine pericardium, measurements of isotonic

shrinkage temperature were used and analyzed.

5.3.1.2.1 The effect of NaOH solution

The destruction or the structural modification induced by the NaOH treatment is

obviously illustrated by the extreme reduction of the shrinkage temperatures or the

thermoelasticity to resist the shrinkage, as shown in fig 5.24. The NaOH treated samples

starts to shrink approximately at 42 °C, whereas the control samples at 67.5 °C. The

shrinkage of the swelled NaOH-treated samples is slow and takes place over a relatively

wide temperature range (~ 30 degrees). The shrinkage process of the native untreated

samples is too fast and occurred mostly within 5 degrees.

5. Results Interpretations

87

20 30 40 50 60 70 80 90

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

100

NaOH

native

Figure 5.24: The influence of 1 N NaOH treatment on the shrinkage temperature of

the pericardium (average of 5 samples)

5.3.1.2.2 The effect of the neutralization step

The efficiency of acetic acid treatment to restore the tissues to their physiological state

was tested. Fig 5.25 shows the influence of treating the pericardium strips with 1 N

CH3COOH after the 1 N NaOH treatment. It is observed that the 5-min treatment results

in tissues with pH of 5 and thermal shrinkage starting from 50 °C, whereas the 15 min-

treatment yields tissues with pH 3 and thermal shrinkage starting from 45 °C.

5. Results Interpretations

88

Temperature (°C)

20 30 40 50 60 70 80 90

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

100

5 min

15 min

Figure 5. 25: The effect of varying the duration of the acetic acid neutralization step

on the shrinkage temperature of NaOH-treated pericardium (average

for 5 samples)

It was reasonable to check the effect of lowering the CH3COOH concentration. Under

constant treatment duration, 15 min, the 0.1 N CH3COOH leaves tissues with a pH 6 and

thermal shrinkage starting from 62 °C, where as the 1 N CH3COOH results in tissues

with pH 3 and thermal shrinkage starting from 45 °C, as shown in fig 5.26.

5. Results Interpretations

89

Temperature (°C)

40 50 60 70 80 90

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

1001 N CH3COOH

0.1 N CH3COOH

Figure 5.26: The effect of varying the concentration of the acetic acid neutralization

step on the shrinkage temperature of NaOH-treated pericardium

(average of 5 samples)

As alternatives to acetic acid, the influence of rinsing solutions to restore the tissues to

the physiological state was tested. Immersing the NaOH-treated strips in distilled water

(pH = 7.8) without stirring for 30 min does nothing to reduce the pH value of the strips.

The strips preserve their pH value 14 with thermal shrinkage starts at 48 °C, as shown in

fig 5.30. The capacity of phosphate buffer (pH = 7.4) is insufficient to reduce the pH

value of the strips. After 30 min stirring in 100 mmol phosphate buffer (pH 7.4), the final

pH of strips is 10.5. The thermal shrinkage starts at 54 °C. Fig. 5.27 shows that stirring

the tissues in distilled water (pH = 7.8) for 90 min, in which water is changed every 15

min, is the best variant to treat the tissues. The strips have a final pH value of 8.0 and

thermal shrinkage starting at 62 °C.

5. Results Interpretations

90

Temperature (°C)

20 30 40 50 60 70 80 90

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

100stirred/changed water 75 min

stirred phosphate buffer 30 min

immersed in water 30 min

Figure 5.27: The effect of different buffering systems on the shrinkage temperature

of NaOH-treated pericardium (average of 5 samples)

Figure 5.28 shows that treating the NaOH-treated strips with 1 N CH3COOH followed by

1-2 10-min water or phosphate buffer baths results in tissues with shrinkage curve close

to that one of the native untreated samples. The tissues begin to shrink from 63 and 64 °C

after the CH3COOH/phosphate buffer and CH3COOH/distilled water treatment

respectively, whereas 65 °C for the native untreated samples, as discussed in section

5.2.2.

5. Results Interpretations

91

Temperature (°C)

20 30 40 50 60 70 80 90

Nor

mai

lized

Shr

inka

ge (%

)

0

20

40

60

80

100

water

phosphate buffer

Figure 5.28: The Influence of the 0.1 N acetic acid treatment step followed by

distilled water or phosphate buffer bath (pH 7.4) on the thermal

shrinkage of NaOH-treated pericardium (average of 5 samples)

5.3.1.3 The measurements of DC

The measurements of DC, seen in fig 5.29, show no significant influence of the 1 N

NaOH treatment for 1 h. The native samples have DC, average ± 95 % confidence

intervals, 11.6 % ± 1.3, whereas the NaOH treated samples 12.0 % ± 3.3.

5. Results Interpretations

92

Native NaOH

DC

(%)

8

10

12

14

16

18

Figure 5.29: The influence of 1 N NaOH treatment for 1 h on the DC of bovine

pericardium, error bars indicate 95 % confidence interval for 5 samples

5.3.1.4 SDS-PAGE investigations

The α-chymotrypsin digested fraction of NaOH-treated pericardium was taken for the

SDS-PAGE investigation. Regardless of the concentration or the duration of the NaOH

treatment, no large fragments can be detected, as shown in fig. 5.30 and 5.31, as

indication of the extreme denaturation

5. Results Interpretations

93

Figure 5.30: SDS-PAGE for standard (lane 1), 30 min 0.5 N NaOH-treated

pericardium (2, 3), 1 h 0.5 N NaOH-treated pericardium (4, 5), 2 h 0.5 N

NaOH-treated pericardium (6, 7), 30 min 1 N NaOH-treated

pericardium (8, 9) and α-chymotrypsin (10)

Figure 5. 31: SDS-PAGE for standard (lane 1), 1 h 1 N NaOH-treated pericardium

(2, 3), 2 h 1 N NaOH-treated pericardium (4, 5), 30 min 2 N NaOH-

treated pericardium (6, 7), 1 h 2 N NaOH-treated pericardium (8, 9)

and α-chymotrypsin (10)

5. Results Interpretations

94

5.3.1.5 Discussions

The treatment of pericardium strips with NaOH results in swelling of the strips, as shown

in fig 5.32.

Figure 5.32: Swelled NaOH-treated bovine pericardium

The swelling of collagen and other fibrous proteins in acid and alkaline solutions is

governed by the osmotic pressure differences arising between the protein phase and the

external solution as a result of the formation of protein salts (Donnan membrane effect)

and by the cohesion of the protein i.e. the forces opposing swelling, such as interweaving

of the fibres and intermolecular forces, first observed by (Procter 1914). The degree of

swelling will depend on the balance of these two factors. In contrast to the acid swelling,

which decreases below pH 2.0, the alkaline swelling increases progressively with

increasing pH and shows no decrease at high pH (Bowes and Kenten 1950). The increase

in alkaline swelling at high pH could be attributed to the reduction of cohesion forces as a

result of breaking of structural features (Bowes and Kenten 1950).

The alkaline hydrolysis of collagen at high temperatures is a well-known method for a

complete degradation or hydrolysis (Kang, Dixit et al. 1975; Mann, Gaill et al. 1992).

However very little attention has been paid to the partial degradation of proteins by low

concentrations of alkali at low temperatures (Berry, Hong et al. 1989). In the current

work the extent of collagen hydrolysis in 1 N NaOH treated native as well as Tutoplast-

Swelled Native

5. Results Interpretations

95

processed pericardium samples at room temperature was checked based on the content of

the aromatic amino acids. The analysis of the results shows a time-dependent hydrolysis

of the collagen in the NaOH solution; however the content of dissolved or hydrolyzed

collagen is low. After 150 min treatment the amount of dissolved collagen is lower than 1

% of the original dry weight. These results confirm with previous investigations that

showed that NaOH could hydrolyze the intra- and intermolecular cross-links without

destroying the helical structure of the collagen molecule (Fujii 1969; Hattori, Adachi et

al. 1999). Kearney and Johnson (Kearney and Johnson 1991) extracted 55.6 µg/mg dry

weight collagen during 18 h treatment with 1 N NaOH, which is considered as low

content of extracted collagen.

The shrinkage temperature analysis shows an extreme reduction in the thermal stability or

in the thermoelasticity to resist the shrinkage. The NaOH-treated strips start to shrink at

42 °C, whereas the native untreated strips at 67.5 °C. Furthermore the shrinkage of the

NaOH-treated strips is too slow and occurrs over wide temperature range, approximately

30 degrees, because the strips are thick and swelled from the action of the alkali, which

prevents smooth shrinkage of the strips. This reduction of the thermal shrinkage is

expected due the well-known action of alkali on the collagen, represented by amino acids

modification and destruction of intra-and intermolecular collagen cross-links. This alkali

action is explained in details in the literature; Hattori et al (Hattori, Adachi et al. 1999)

have shown that the extractability of collagen by the alkaline treatment for 14 day at

room temperature was much higher than that by pepsin or acetic acid, especially from an

aged samples. They justified the higher extractability by the ability of alkaline treatment

to remove the telopeptide involved in cross-linking of collagen molecule and to break

some additional cross-links in the triple helical region, which resistant to the pepsin

treatment. Previous studies (Rauterberg and Kuhn 1968; Fujii 1969; Hattori, Adachi et al.

1999) proved amino acid modifications induced by the alkaline treatment represented by

the deamination of acid amides of Asn and Gln. Hattori et al (Hattori, Adachi et al. 1999)

detected that the isoelectric point of collagen was lowered from 9.3 to 4.8 because of the

conversions of Asn and Gln to Asp and Glu, which reduced the thermal stability of

collagen.

5. Results Interpretations

96

The damage caused by NaOH treatment could be reversed or eliminated by a following

and immediate treating step.

After treating 10 pericardium strips in 100 ml 1 N NaOH, the NaOH solution is decanted

and the strips were treated with 1 N CH3COOH, 5 strips of them with 50 ml for 5 min

and the other 5 strips with 50 ml for 15 min. The aim of this treatment was to restore the

pericardium strips to their physiological state and pH. It is seen that this treatment shifts

the pH value of the strips too rapidly from the basic to the acidic region causing damage

also. 5 and 15 min treatment with 50 ml 1 N CH3COOH is enough to shift the pH to 5

and 3 respectively yielding pericardium strips with thermal shrinkage starting at 50 and

45 °C respectively. Not only alkaline reduces the thermal stability of collagen but also

acids because it cleaves hydrogen bonds (Gustavison 1956). The volumes of NaOH as

well as of CH3COOH used to treat the strips are chosen to have fully submerged strips.

For the treatment of 10 and 5 strips, 100 and 50 ml are used respectively. Therefore lower

concentrations of CH3COOH not lower volumes are further tested to treat the strips

without inducing secondary damage. 0.1 N CH3COOH has been used to treat the NaOH-

treated tissues. The 15-min treatment with 0.1 N CH3COOH shifts the pH value of the

pericardium strips to 6, resulting in thermal shrinkage starts at 62 °C, which is close to

that of the native untreated pericardium.

Also as alternative to the acid treatment, the efficiency of different washing or rinsing

fluids, such as distilled water and phosphate buffer (pH 7.4) has been tested. It is

observed that submerging or washing the NaOH-treated samples once with water or

phosphate buffer is not sufficient to reduce the pH value of the samples due to the limited

capacity of the water or buffer to wash the ions. A complete neutralization of the samples

could be achieved by intensive washing with distilled water baths for 90 min, in which

the water bath has to be changed every 10 or 15 min.

The best variant has been achieved by treating the NaOH treated strips with 1 N

CH3COOH for 15 min followed by one or two 10-min distilled water washing bath. With

this variant, the pericardium strips has a final pH value 8, the same pH as distilled water,

and a thermal shrinkage starts at 64 °C.

The idea of treating the damage induced by the alkaline treatment is well known in the

literature and has been borrowed from the treatment of skin or eye alkaline burns. The

5. Results Interpretations

97

damage caused by alkaline agent continues until the pH values returns to a physiological

level (Gruber, Laub et al. 1975). The use of acid neutralization in the treatment of skin

alkaline treatment is debatable. Some authors (Bromberg, Song et al. 1965; Wolfort,

DeMeester et al. 1970; Mozingo, Smith et al. 1988) suggested that alkaline burns should

be treated by water irrigation alone and the using of acid neutralization step to treat skin

alkaline burns produces exothermic reaction with the alkali that may extent the depth of

skin burn by increasing the temperature. Recently Andrews et al (Andrews, Mowlavi et

al. 2003) challenged the belief that the acid neutralization step causes secondary tissue

damage. They demonstrated using Wistar rats that the neutralization with acetic acid

offers a more rapid return to physiologic pH, less severe tissue damage, and improved

wound healing in comparison to those treated with water. Furthermore, they recorded the

skin temperatures that were below the rat body temperature, 33°C, from induction to

completion of the experiment. They suggested that although an exothermic reaction may

have been present, any heat produced was dissipated by the hypothermic room

temperature, 25°C, of the solutions used.

In the current work, it is observed that only washing with water to restore the tissues to

their physiological state takes long time and therefore an acetic acid treatment would be

helpful for quick restoration of the tissues but with moderate concentration and duration

to avoid a secondary damage.

The measurements of DC show no significant influence of the NaOH treatment. This

could be attributed to the fact that α-chymotrypsin is not active to digest collagen at

highly alkaline conditions and only active at neutral to slightly alkaline ranges (Moe and

Birkedal-Hansen 1979). Washing the samples with water until neutralization before being

digested with α-chymotrypsin is not a practical solution because as observed with the

shrinkage temperature measurements, the effect of NaOH is reversible by restoring the

tissues to their physiological neutral state.

Regarding the SDS-PAGE investigation, no large fragments are detectable with the α-

chymotrypsin digested fraction of the NaOH-treated samples. This gives indication how

strong the denaturation of collagen is and how accessible almost the cleavage sites for α-

chymotrypsin are. These results are consistent with previous study (Berry, Hong et al.

5. Results Interpretations

98

1989), which shows that treating collagen starting with a concentration of 0.25 N NaOH

yields heterogeneous small peptides (<20 kD).

5.3.2 The effect of hydrogen peroxide treatment

This step in the Tutoplsat® process has been confirmed to activate viruses, including

enveloped and non-enveloped DNA and RNA viruses (Schoepf 2006). However, the

oxidation with H2O2 leads to the modification of protein amino acids (Neumann and

Timasheff 1972).

5.3.2.1 The shrinkage temperature measurements

It is seen that the 3% treatment caused a little reduction in the thermal shrinkage, as

shown in fig 5.33. The 3%-treated pericardium starts to shrink from 60 °C, whereas the

native untreated samples at 66 °C. Furthermore, the shrinkage curve of the 10% treated

pericardium is almost very close; it starts to shrink at 58 °C.

Temperature (°C)

40 50 60 70 80 90

Nor

mal

ized

Shr

inka

ge (%

)

0

20

40

60

80

10010%

3%

w/o

Figure 5.33: The Influence of the varying the H2O2 concentration on the shrinkage

curve of the pericardium (average of 4 samples)

5.3.2.2 Measurements of DC

DC measurements show no significant influence of the H2O2 treatment regardless of the

concentration, 3, 10 and 30%, as shown in fig 5.34. The native untreated samples have

5. Results Interpretations

99

DC, average ± 95 % confidence interval, 11.6 ± 1.3, whereas the 3, 10 and 30% treated

samples 11.4 ± 3.3, 10.5 ± 3.8 and 9.9 ± 3.7 respectively.

Native 3% 10% 30%

DC

(%)

0

2

4

6

8

10

12

14

16

Figure 5.34: The influence of H2O2 treatment of the DC of native bovine

pericardium, error bars indicate 95 % confidence intervals of 5 samples

5.3.2.3 SDS-PAGE investigations

Native as well as Tutoplast-processed samples were treated with 3, 10 or 30% H2O2 and

digested overnight with α-chymotrypsin at 37 °C. The digested fraction was analyzed

using SDS-PAGE (Koerber 2006), as shown in fig 5.35.

Regarding the native 3%-treated sample, clear bands are detected in the range 97.4- 116.3

kD. Also weak bands are detected in the range of 40 and 56 kD, whereas with the

processed treated with 3%, only fragments larger than 100 kD are detected.

The native pericardium treated with 10% H2O2 is almost similar to the 3% treated

sample. It has large clear bands in the range of 100 kD and also weak bands in the range

between 45 and 56 kD. Large bands above 100 kD are observed with the processed 10%-

treated pericardium.

5. Results Interpretations

100

Regarding the 30% treatment, only clear bands in the range of 100 kD and weak bands

between 55 and 66 kD are found with the native pericardium.

Figure 5. 35: SDS-PAGE for standard (lane 1), 3% H2O2-treated native pericardium

(2), 3% H2O2-treated processed pericardium (3), 10% H2O2-treated

native pericardium (4), 10% H2O2-treated processed pericardium (5),

30% H2O2-treated native pericardium (6), 30% H2O2-treated processed

pericardium (7) and α-chymotrypsin (9)

5.3.2.4 Discussion of the results

The hydrogen peroxide treatment in the Tutoplast process has been found to be effective

against HIV (Hinton, Jinnah et al. 1992). However, the oxidation with H2O2 leads to the

modification of protein amino acids (Neumann and Timasheff 1972).

One of the most common pathways of protein degradation is the oxidation of amino acids

(Met, Tyr, Trp, Cys, and His). This oxidation can occur through photolytic or chemical

reactions, and is dependent on such factors as the temperature, pH, the presence of certain

excipients, heavy metals and the presence of molecular oxygen (Manning, Patel et al.

1989; Duenas, Keck et al. 2001). Hydrogen peroxide is a relatively nonspecific oxidizing

agent, which reacts with a wide variety of organic compounds. It can modify thioether,

5. Results Interpretations

101

indole, sulfhydryl, disulfide, imidazole and phenolic at the neutral or slightly alkaline

conditions (Manning, Patel et al. 1989). Under acidic conditions the primary reaction is

the conversion of methionine residues to sulfoxide (Neumann and Timasheff 1972).

Oxidation of Met residues is associated with the loss of the biological activity for many

proteins (Manning, Patel et al. 1989). Restoration of the biological activity was found to

be achieved with the reduction of Met sulfoxide to Met (Caldwell, Luk et al. 1978). In the

current work it is seen that the treatment with 3% H2O2 (pH = 5.52) is almost not

destructive to the collagen structure. This was confirmed by the high thermal stability

assessed by the measurements of isotonic shrinkage temperature. The samples treated

with 10% H2O2 (pH = 4.13) have almost similar thermal stability to those treated with

3% H2O2. In contrast to the 3 and 10% treatment, the treatment with 30% (pH = 2.40) is

extreme destructive and leads completely destructed pericardium strips, which can’t be

further tested with the isotonic shrinkage technique. It is expected the treatment with

H2O2 under mild concentrations and pH doesn’t lead to complete oxidation of the Met

residues and consequently to the complete destruction of the collagen, which is observed

at extreme concentration and pH.

Regarding the DC measurements, as discussed with the NaOH treatment, α-chymotrypsin

couldn’t attack the tissues under acidic conditions because it is inactive in this pH range.

SDS-PAGE Investigations are consistent with the measurements of the shrinkage

temperature. At mild concentrations, 3 and 10%, large fragments are detected with the α-

chymotrypsin digested fraction native pericardium as indication of the inaccessibility of

the cleavage sites to α-chymotrypsin. It is expected that the absence of large fragments

with the Tutoplast-processed pericardium could ascribed to the processing not to the

H2O2 treatment. An extreme reduction of the intensity and the size of the fragments have

been observed with the 30%-treated samples as a signal of an extreme degradation.

5.3.3 The influence of acetone treatment

The acetone treatment in the Tutoplast-process aims to remove any residual prions and to

inactivate any enveloped viruses (Schoepf 2006). The acetone wash, followed by vacuum

extraction, dehydrates the tissues to be storable at room temperature.

5. Results Interpretations

102

5.3.3.1 The extent of drying and shrinkage

Figure 5.36 shows that the weight loss, through dehydration, increases almost linearly

with increasing the duration of the acetone series treatment until the tenth day, thereafter

no significant change has been observed. The shrinkage in the volume of the sample is

approximately parallel to the weight loss; it is linear until the tenth day and then it

remained constant.

Time (day)

0 2 4 6 8 10 12 14 16 18 20

Wei

ght l

oss

(%)

0

20

40

60

80

100

Shrin

kage

(%)

0

10

20

30

40

50

60

Figure 5.36: The time-dependence weight loss and volume shrinkage during the

acetone series treatment of native pericardium

In contrast to the acetone series, the loss of the weight is much more quickly by the

treatment with 100% acetone. The samples lose approximately 76% of their weight

within the first 2 days. From the second until the eighteenth day only further 2% weight

is lost, as shown in fig 5.37. Regarding the shrinkage, it behaves differently; it has the

largest shrinkage within the first 2 days, 22.68%, then it increases slowly but significantly

until it reaches a constant value at the sixteenth day, 52.91%.

5. Results Interpretations

103

Time (day)

0 2 4 6 8 10 12 14 16 18 20

Wei

ght l

oss

(%)

0

20

40

60

80

100

Shrin

kage

(%)

0

10

20

30

40

50

60

Figure 5.37: The time-dependence weight loss and volume shrinkage during the

100% acetone treatment of native pericardium

5.3.3.2 The measurements of DC

It is seen obviously that the acetone treatment either 100% or series has no significant

influence on the DC, as shown in fig 5.38. The average DC ± 95 % confidence interval of

the 100%-acetone treated samples is 17.9 % ± 3.2, whereas 15.4 % ± 2.3 for the acetone

series treated samples. The average DC of the control untreated samples is 12.7 % ± 2.1.

5. Results Interpretations

104

DC

(%)

8

10

12

14

16

18

20

22

24

26

w/o series 100 % Figure 5.38: The influence of acetone treatment on the fraction of denatured

collagen, DC, (error bars indicates 95% confidence interval for 6-10

samples)

5.3.3.3 Discussion of the results

Acetone is used in Tutoplast process to inactive the remaining prion and viruses and to

dehydrate the tissues. In the current study the pure acetone was compared with the graded

acetone. After 18 days, both of them have almost the same weight loss and shrinkage,

however with different curve course. The pure acetone is much more effective to

dehydrate the tissues; the maximum weight loss has been achieved within the first two

days almost, whereas the maximum weight loss has been reached at the tenth day during

the graded acetone treatment.

Regarding the shrinkage, during the first 2 days of the pure acetone treatment, the tissue

shrinks 22.68% of their volume. Further shrinkage is also observed until the maximum

shrinkage is reached at the sixteenth day, 52.91%.

5. Results Interpretations

105

The shrinkage of the tissue during the graded acetone treatment is almost parallel to the

weight loss. The shrinkage increases with increasing the treatment duration until it

reached the maximum shrinkage at the tenth day.

During the dehydration process, water is replaced with polar solvents with low H-

bonding abilities, such as acetone, which removes hydrogen bonded water bridges, allows

more direct hydrogen bonding between the molecules and consequently brings the

collagen fibrils closer causing shrinkage (Pashley, Agee et al. 2001; Pashley, Agee et al.

2003; Nalla, Balooch et al. 2005).

The results shows that the pure acetone treatment was more effective to dehydrate the

tissues, however taking into consideration that almost all the water content is removed

within the first two days, it is recommended to stop the pure acetone treatment after the

second day to avoid further shrinkage of the tissue. During the acetone change, the tissues

are taken out from the acetone solution and placed in the air before being weighed and

submerged again in the next acetone solution. In this case, Acetone could be evaporated

from the tissue quickly before being submerged again in the acetone solution. This causes

a gradient in acetone concentration between inside and outside the tissue and

consequently a gradient in the polarity. This could be probable explanation for the further

shrinkage despite the removal of water content.

The functions of the acetone treatment in the Tutoplast-process are tissue dehydration and

inactivation of any prions and viruses. Therefore it is recommended to check if the 2-day

pure acetone treatment is sufficient to inactivate prions and viruses.

The acetone treatment has no significant influence on the collagen denaturation. The DC

values of pure acetone as well as graded acetone treated samples are almost similar to

those of the native untreated samples.

5.4 The mechanical properties of the collagenous tissues

5.4.1 The mechanical properties of bovine bones

The ideal grafting material should not only be adequately osteogenic, -conductive, and -

inductive but also mechanically stable and disease free (Kalfas 2001). Biomechanical

concerns must be carefully considered in the bone healing process during fusion. It has

been appreciated for more than a century that bone forms in places where stress requires

its presence (Pilitsis, Lucas et al. 2002).

5. Results Interpretations

106

The analysis of ultimate strength and elastic modulus values after thermal treatments of

bone cubes, shown in fig 5.39, gives no clear relationship or statement about the

temperature-dependency of the mechanical properties of the bone samples. The 95%

confidence interval for each temperature is very wide as indication of the extreme

scattering of the data. The compressive ultimate strength for the samples treated at 37, 60,

80 and 100 °C are 7.6 ± 1.3, 8.7 ± 3.1, 8.2 ± 2.0 and 10.6 ± 4.2 (mean ± 95% confidence

interval) respectively, whereas the elastic modulus are 25.6 ± 8.8, 42.6 ± 21.2, 38.5 ±

13.2 and 51.9 ± 26.4 respectively.

37 °C 60 °C 80 °C 100 °C 37 °C 60 °C 80 °C 100 °C

Elas

tic M

odul

us (M

Pa)

0

20

40

60

80

100

120

Ulti

mat

e St

reng

th (M

Pa)

0

5

10

15

20

Figure 5.39: The temperature-dependence mechanical properties of bovine

cancellous bone (error bars indicate 95 % confidence interval of 9

samples)

5.4.2 The mechanical properties of bovine pericardium

The mechanical properties of the pericardium reflect the ‘the healthiness’ and the

structural integrity. Any modification or destruction induced by processing should be

detected in a biomechanical analysis.

5. Results Interpretations

107

As observed with the bone samples, Figure 5.40 does not show any reasonable

relationship or conclusion about the influence of the processing on the mechanical

stability of the pericardium samples. Wide 95% confidence intervals have been observed,

which prevent any assessment of the effect of the processing. The tensile ultimate

strength for the left side native and processed, and the right side native and processed

were (mean ± 95% confidence interval) 9.2 ± 4.4, 12.5 ± 3.5, 8.9 ± 8.9 and 6.8 ± 1.7

respectively, whereas the elastic modulus were 113.5 ± 53.4, 115.6 ± 52.8, 93.0 ± 87.9

and 80.8 ± 24.5 respectively.

Ulti

mat

e ST

reng

th (M

pa)

-5

0

5

10

15

20

Elas

tic M

odul

us (M

Pa)

0

50

100

150

200

N P

N P

N P

N P

Left

Left

Right

Right Figure 5.40: The influence of Tutoplast process on the mechanical properties of

bovine pericardium (error bars indicate 95 % confidence interval of 5

samples)

5.4.3 Discussion of the results

The bone has several functions in the body but at least two key biomechanical roles (Burr

and Turner 2003); bones shield vital organs from trauma and serve as levers against

5. Results Interpretations

108

which muscles contact. Bone is a natural composite, in which Minerals are responsible

for the bone stiffness and collagen for the bone toughness (Wang, Bank et al. 2001; Burr

and Turner 2003).

The bone samples were heated in the range 37-100 °C before being tested to induce

collagen denaturation, which supposed to be important in determining the mechanical

properties of the bones. The results do not show any relationship between the temperature

and the mechanical properties. Wang et al (Wang, Bank et al. 2001) heated cortical

samples in the range 37-200 °C before being tested in three-point bending configuration.

They showed that heating induces denaturation, which lead to a significant decrease in

the toughness of bone but has little effect on the stiffness of the bone. The deviation of

the current results from those of Wang et al could be attributed to different reasons;

Wang et al didn’t observe any influence of the thermal denaturation on the mechanical

properties up to 160 °C. Furthermore they used the three-point bending configuration,

which combination between tension and compression (Einhorn 1992), whereas in this

study compression test was used.

Assuming that heating in the range used in this study has no influence on the mechanical

properties, the mechanical properties of all the samples should be almost identical

however, it is not the case and the results are scattered.

The influence of the structure heterogeneity and the fiber orientation could be behind the

scattering of the results and the absence of convenient statement about the mechanical

properties. Fratzl et al (Fratzl, Gupta et al. 2004) suggested that the bone matrix is not

uniformly mineralized, but shows a pronounced local variation. Therefore the bone

material is composed of bone packets, each having its own mineral content corresponding

to its tissue age. In the current work, taking the samples without previous knowledge of

the mineral content could be a reason for the scattering of the results.

Furthermore, Peterlik et al (Peterlik, Roschger et al. 2006) found that the fracture energy

changes by two orders of magnitude depending on the collagen orientation, and the angle

between collagen and crack propagation direction is decisive in switching between

different toughening mechanisms. In the current study 1×1×1 cm3 cubes were cut from

the same direction, however the confusion of the orientation during the thermal treatment

5. Results Interpretations

109

and then during the mechanical test as experimental error is not excluded, which may

contribute in the scattering of the results.

The pericardium is a viscoelastic material (Paez and Jorge-Herrero 1999), i.e. the stress

strain relationship is non linear. Therefore the Hooke’s law is normally not considered for

the analysis of the mechanical properties of the pericardium. Despite this fact, Hooke’s

law has been used to describe the linear region of the stress strain diagram (Zioupos and

Barbenel 1994). In this study, it was assumed that Hooke’s law is valid in the linear

region of the stress stain diagram.

It is known in the literature that Bovine pericardium is generally considered to be

mechanically anisotropic (Lee, Courtman et al. 1984; Zioupos and Barbenel 1994). To

overcome the mechanical anisotropy of the pericardium, some researchers use the small

angle light scattering (SALS) to quantify the collagen fiber architecture and to select

samples with minimum variability to be used in the mechanical tests (Hiester and Sacks

1998; Hiester and Sacks 1998; Sacks and Chuong 1998; Mirnajafi, Raymer et al. 2005).

Others separated between the left and the right side of the pericardial sac (Garcia Paez,

Jorge-Herrero et al. 2001). In the current work, native as well as Tutoplast-processed

samples from the left and the right sides of the pericardium were taken for the analysis of

the mechanical properties.

The analysis of the results does not lead to any conclusion about the influence of the

processing of the mechanical stability. It is expected, despite the separation between the

left and the right side of the sac, that the effect of anisotropy was dominant over the

influence of the processing. It can be concluded that only SALS-selected samples can be

used to assess the effect of the processing on the mechanical properties of the

pericardium.

6. Optimization of Tutoplast Process

110

6 Optimization of Tutoplast process The optimization of the process aims to achieve one or more of the following goals

• Improving the quality of the processed tissues

• Shortening the process duration

• Reduction of the processing costs.

Any kind of optimization that saves time and costs with worsening the quality of the

tissues will not be taken into consideration.

The effect of Tutoplast processing of the stability of bovine pericardium was studied. It

was concluded that the processing induces structural destruction. The contribution of

each step in the process was followed; analyzed and practical solutions were suggested as

the following:

6.1 The sodium hydroxide treatment Tutoplast process

During Tutoplast process, the tissues are treated with 1 N NaOH for 1 h. This treatment

induces osmotic swelling of the tissues and reduction of the thermal shrinkage. The

action of alkali on the collagen is represented by amino acids modification and

destruction of intra-and intermolecular collagen cross-links.

In order to restore the tissues to their physiological state, the tissues are treated with 1 N

CH3COOH for 15 min. This treatment shifts the pH value of the strips too rapidly from

the basic to the acidic region (from 14 to 3) causing swelling and lower thermal stability

also.

After the 15-min 1 N CH3COOH, the tissues are submerged in RO-water for 30 min,

which was found inefficient to restore the tissues to their physiological state.

Alternatives

The 1 N NaOH treatment for 1 h is a validated step against CJD, therefore it was kept

unchanged. As alternatives to the 15-min 1 N CH3COOH as neutralization step, the 15

min 0.1 N CH3COOH was more efficient and yielded tissues with pH 6 and thermal

shrinkage close to that of the native untreated tissues.

6. Optimization of Tutoplast Process

111

Restoring the tissues to their physiological state after the NaOH treatment by washing

with water bath is possible but it takes long time, at least 90 min, with changing the water

bath every 10-15 min.

The best variant was to treat the NaOH-treated strip with 0.1 N CH3COOH for 15 min

followed by one or two 10-min water baths.

6.2 The hydrogen peroxide treatment Tutoplast

The tissues are treated during the process with 3 % H2O2 for 72 h. This step induces little

reduction of the thermal shrinkage of the strips.

Alternatives

Using 10 % H2O2 could be more effective to oxidize the proteins and to inactivate the

viruses. It was seen that the 10 % H2O2 treatment results in tissues have almost the

similar shrinkage temperature as that of the 3 % H2O2 treated tissues. Therefore using the

10 % will almost not induce further destruction

6.3 The acetone treatment Tutoplast

During the process the tissues are treated with pure acetone for two weeks. This steps

aims to dehydrate the tissues and inactivate the remaining prions

Alternatives

The pure acetone used in the process was found to be more efficient than graded acetone

as dehydrating agent. However, the 2-week treatment is too long and caused avoidable

shrinkage. It was found that after 2 days the tissues were fully dehydrated.

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1 Introduction................................................................................................................. 1 2 State of the art ............................................................................................................. 2

2.1 Bone grafting ...................................................................................................... 2 2.1.1 Graft types................................................................................................... 2

2.1.1.1 Autograft ................................................................................................. 2 2.1.1.2 Allograft.................................................................................................. 2 2.1.1.3 Xenograft ................................................................................................ 3 2.1.1.4 Alloplastic ............................................................................................... 3

2.1.2 Bone healing ............................................................................................... 4 2.1.3 Graft processing .......................................................................................... 6

2.1.3.1 Graft processing techniques used in the medical field............................ 6 2.1.3.2 Tutoplast® process ................................................................................. 7

2.2 Collagen ............................................................................................................ 10 2.2.1 Collagen types........................................................................................... 10 2.2.2 Collagen synthesis .................................................................................... 12 2.2.3 Collagen cross-links.................................................................................. 14 2.2.4 The role of hydroxyproline in collagen stabilization................................ 16 2.2.5 The thermal stability of collagen .............................................................. 18 2.2.6 Advanced glycation end products (AGEs) ............................................... 19

2.3 Bone .................................................................................................................. 20 2.3.1 Bone composition ..................................................................................... 20 2.3.2 Bone hierarchy .......................................................................................... 21

2.4 Pericardium....................................................................................................... 23 3 The Objectives .......................................................................................................... 26 4 Materials and Methods.............................................................................................. 27

4.1 Materials ........................................................................................................... 27 4.1.1 Bovine Bones ............................................................................................ 27 4.1.2 Bovine Pericardium .................................................................................. 28

4.2 Methods............................................................................................................. 29 4.2.1 Preparation Steps ...................................................................................... 29

4.2.1.1 The pulverization of the bones.............................................................. 29 4.2.1.2 The Demineralization of Bones ............................................................ 30

4.2.2 The Determination of Denatured Collagen (DC)...................................... 30 4.2.2.1 A Selective Digestion Method .............................................................. 31 4.2.2.2 Spectrophotometeric Determination of the DC .................................... 32

4.2.3 The Measurements of the Extent of Browning ......................................... 34 4.2.4 The Measurements of the Isotonic Shrinkage temperature....................... 34 4.2.5 SDS-PAGE ............................................................................................... 35 4.2.6 Characterization of the Mechanical Properties ......................................... 39 4.2.7 The measurements of the Thermal Conductivity...................................... 39

4.3 Different physical and chemical treatment ....................................................... 39 4.3.1 The thermal treatment of collagenous tissues........................................... 40

4.3.1.1 The thermal treatment of bovine bone .................................................. 40 4.3.1.2 The thermal stability of bovine pericardium......................................... 40

4.3.2 The Sodium hydroxide treatment and the corresponding neutralization .. 41

4.3.2.1 Sodium hydroxide treatment................................................................. 41 4.3.2.2 The neutralization of the tissues after the NaOH treatment.................. 42

4.3.3 The hydrogen peroxide treatment ............................................................. 43 4.3.4 The acetone treatment ............................................................................... 43 4.3.5 The determination of water content .......................................................... 44

5 Results Interpretations .............................................................................................. 45 5.1 The effect of bone pulverization on the collagen.............................................. 45

5.1.1 The measurements of DC.......................................................................... 45 5.1.2 Discussion of the results ........................................................................... 46

5.2 The thermal stability of collagenous tissues ..................................................... 47 5.2.1 Analysis of the thermal stability with the measurements of DC .............. 47

5.2.1.1 The thermal stability of Tutoplast-processed bovine bone ................... 47 5.2.1.2 The thermal stability of native bovine pericardium.............................. 48 5.2.1.3 The thermal stability of tutoplast-processed bovine pericardium......... 49 5.2.1.4 The thermal stability of the lyophilized bovine pericardium................ 50 5.2.1.5 The measurements of the extent of browning....................................... 51 5.2.1.6 Discussion of the results ....................................................................... 52 5.2.1.7 Modeling of the thermal denaturation of pericardium.......................... 59

5.2.2 Measurements of isotonic shrinkage temperature..................................... 65 5.2.2.1 The results............................................................................................. 66 5.2.2.2 Discussion of the results ....................................................................... 66 5.2.2.3 Modelling of the thermal shrinkage of pericardium ............................. 68

5.2.3 SDS-PAGE investigations ........................................................................ 78 5.2.3.1 Results................................................................................................... 78 5.2.3.2 Discussion ............................................................................................. 81

5.3 The effect of different steps in the Tutoplast® process .................................... 83 5.3.1 The effect of sodium hydroxide treatment................................................ 83

5.3.1.1 The hydrolysis of collagen amino acids................................................ 84 5.3.1.2 The measurements of the shrinkage temperature.................................. 86

5.3.1.2.1 The effect of NaOH solution........................................................... 86 5.3.1.2.2 The effect of the neutralization step................................................ 87

5.3.1.3 The measurements of DC...................................................................... 91 5.3.1.4 SDS-PAGE investigations .................................................................... 92 5.3.1.5 Discussions ........................................................................................... 94

5.3.2 The effect of hydrogen peroxide treatment............................................... 98 5.3.2.1 The shrinkage temperature measurements............................................ 98 5.3.2.2 Measurements of DC ............................................................................ 98 5.3.2.3 SDS-PAGE investigations .................................................................... 99 5.3.2.4 Discussion of the results ..................................................................... 100

5.3.3 The influence of acetone treatment......................................................... 101 5.3.3.1 The extent of drying and shrinkage .................................................... 102 5.3.3.2 The measurements of DC.................................................................... 103 5.3.3.3 Discussion of the results ..................................................................... 104

5.4 The mechanical properties of the collagenous tissues .................................... 105 5.4.1 The mechanical properties of bovine bones............................................ 105 5.4.2 The mechanical properties of bovine pericardium.................................. 106

5.4.3 Discussion of the results ......................................................................... 107 6 Optimization of Tutoplast process .......................................................................... 110

6.1 The sodium hydroxide treatment .................................................................... 110 6.2 The hydrogen peroxide treatment ................................................................... 111 6.3 The acetone treatment ..................................................................................... 111

7 Literature................................................................................................................. 112

Einleitung Verschiedene Transplantationsmaterialien, einschließlich Autografts, Allografts,

Xenografts und synthetische Materialien, im klinischen Bereich werden eingesetzt. Das

Autograft wird als das Goldstandardtransplantat betrachtet, weil es intakte Zellen und

Wachstumsfaktoren enthält, welche die Heilung des Transplantates anregen. Jedoch sind

die begrenzte Verfügbarkeit und die zusätzliche Morbidität die Hauptnachteile, die mit

der Verwendung von Autografts verbunden sind. Deshalb müssen alternative

Transplantationsmaterialien verwendet werden, um die zunehmende Nachfrage nach

Transplantaten im medizinischen Bereich zu erfüllen.

Allografts werden zwar häufig eingesetzt aber das Risiko immunologischer Reaktionen

und die Übertragung von Krankheiten schränken deren Verwendung ein.

Die Risiken bei der Verwendung von Allografts und Xenografts (Antigenität und

Infektionsgefahr) können durch verschiedene Behandlungen verringert werden. Leider

führen solche Behandlungen oft zu Veränderungen der mechanischen und biologischen

Gewebeeigenschaften.

Der Tutoplast® Prozess stellt einen kompletten und validierten Konservierungs- und

Sterilisationsprozess, der seit 30 Jahren verwendet wird. Dieser Prozess zielt auf saubere

und sichere Transplantate durch die Beseitigung von Antigenität sowie viralen

Krankheiten, ohne die mechanischen und biologischen Eigenschaften der Gewebe zu

verändern. Der Tutoplast® Prozess deaktiviert, zerstört und entfernt alle unerwünschten

Materialien aus den prozessierten Geweben, wie Fette, Zellen, Viren und Mikroben.

In der hier vorliegenden Dissertation wird der Einfluss des Tutoplast® Prozesses und die

Auswirkung jeder seiner Einzelschritte auf die thermischen und mechanischen

Eigenschaften von Knochen und Perikard beurteilt. Dies stellt die Grundlage für die

zukünftigen Pläne einer Prozessoptimierung dar.

Zielsetzung Die Nachfrage nach den biologischen Implantaten steigt stetig an. Um die zunehmende

Marktnachfrage zu erfüllen, muss die Produktion an Implantaten in naher Zukunft erhöht

werden. Diese Tatsachen weisen zwingend auf eine notwendige Optimierung des

Tutoplast Prozesses hin, ohne dabei die Produktqualität (prozessierte Gewebe) zu

verschlechtern.

Für eine Prozessoptimierung bedarf es einer vollständigen Überprüfung und Analyse

eines jeden Prozesseinzelschrittes. Dies stellt die Basis für künftige

Optimierungsversuche dar.

Zuerst wird der Einfluss des Tutoplast Prozesses auf die thermische Stabilität der

prozessierten Knochen und Perikard untersucht, da die thermische Stabilität ein guter

Hinweis auf die Unversehrtheit der Gewebe ist. Weiterhin ist die Kenntnis der

thermischen Stabilität wichtig, um den Einfluss einer thermischen Behandlung der

Gewebe während medizinischer Therapien zu verstehen.

Native und prozessierte Perikardproben werden thermisch behandelt und anschließend

enzymatisch verdaut, um den Anteil an denaturiertem Kollagen (DC) zu bestimmen. Zur

Überprüfung der jeweiligen Prozesseinzelschritte werden native Proben separat mit

NaOH, H2O2 und Aceton bei unterschiedlichen Bedingungen (Konzentration,

Einwirkdauer) behandelt und anhand von Messungen der isotonischen

Schrumpfungstemperatur sowie DC Messungen und SDS-PAGE bewertet.

Der Einfluss des Tutoplast-Prozesses auf die mechanischen Eigenschaften der

prozessierten Knochen und Perikard wird durch Druck- und Zugfestigkeitsversuche an

entsprechenden Proben ermittelt.

Zusammenfassung Die hier vorliegende Arbeit konzentriert sich auf das Aufzeigen von

Optimierungsmöglichkeiten für den Tutoplast® Prozess. Die Optimierungsversuche

wurden durch unterschiedliche Qualitätssicherungstests evaluiert. Um eine

Prozessoptimierung des Prozesses durchzuführen, musste zunächst der Einfluss des

Prozesses auf die Materialien Knochen und Perikard sorgfältig definiert werden.

Weiterhin war der Einfluss eines jeden Einzelschrittes separat zu untersuchen und durch

die gültigen Qualitätssicherungstests zu beurteilen.

Die thermische Stabilität der Kollagengewebe wurde als entscheidender Parameter

betrachtet. Die Perikardproben wurden im Bereich von 55 bis 200 °C für 1 h im

Trockenschrank thermisch behandelt und anschließend mit α-Chymotrypsin verdaut, um

den Anteil an denaturiertem Kollagen (DC) zu bestimmen. Die DC Messungen zeigten

bei nativen lyophilisierten (Wassergehalt 7%) und Tutoplast-prozessierten Perikard

(Wassergehalt 1.7%) eine höhere thermische Stabilität, im Vergleich zu nativem Perikard

(Wassergehalt 85%). Der DC Anteil für das native lyophilisierte und Tutoplast-

prozessierte Perikard blieb bis 135 °C unverändert, während sich der DC Anteil bei

nativem Perikard bereits ab 55 °C zu erhöhen begann. Dies konnte auf den Wassergehalt

im Perikard zurückgeführt werden. Nach dem Polymer in a Box Mechanismus, begrenzt

eine Dehydratisierung die Anzahl möglicher Konfigurationen, verringert damit die

Konfigurationsentropie und erhöht dadurch die thermische Stabilität des Kollagens.

Der Einfluss des Wassergehalts während der DC Messungen wurde dadurch eliminiert,

dass die Schrumpfungstemperaturmessungen in einem Wasserbad durchgeführt werden.

durch Schrumpfungstemperaturmessungen lassen sich Destruktionen oder Änderungen in

der Gewebestruktur zu erkennen.

Es wurde festgestellt, dass das Tutoplast-prozessierte Perikard eine niedrigere

Schrumpfungstemperatur aufweist. Ein Hinweis auf strukturelle Gewebeveränderungen

bzw. Zerstörungen, welche durch die Prozessierung verursacht werden. Tutoplast-

prozessiertes Perikard beginnt ab 42 °C zu schrumpfen, während der Schrumpfprozess

bei nativem lyophilisiertem und nativem Perikard ab 64 bzw. 65 °C einsetzt.

Die Ergebnisse aus den DC- und Schrumpfungstemperaturmessungen führen zu der

Schlussfolgerung, dass der Tutoplast-Prozess strukturelle Gewebeänderungen induziert,

die unter trockenen Bedingungen nicht nachweisbar sind. Nachfolgend wurde der Beitrag

eines jeden Prozesseinzelschrittes auf die strukturelle Gewebeänderung untersucht.

Hinsichtlich der Natriumhydroxidbehandlung wurde festgestellt, dass 1 N NaOH eine

Modifizierung der Aminosäuren, und eine erhebliche Senkung der

Schrumpfungstemperatur verursacht. Des Weiteren wurde nachgewiesen, dass eine

NaOH Behandlung bei Raumtemperatur zu einer Hydrolyse des Kollagens führt, die

jedoch als nicht signifikant eingestuft werden kann.

Um den Einfluss der NaOH Behandlung zu eliminieren, wurden einige

Neutralisationsmöglichkeiten geprüft. Als beste Variante stellte sich eine 15-minütige

Behandlung der Proben mit 0,1 N CH3COOH und zwei anschließenden 10-minütigen

Wasserspülungen dar.

Im Fall der Wasserstoffperoxidbehandlung (H2O2) wurde nachgewiesen, dass eine 48-

stündige der Gewebe Behandlung mit 3 und 10% H2O2 Lösungen kaum zu nachteiligen

Gewebeveränderungen führt. Die Schrumpfungstemperaturen der so behandelten Proben

lagen um 5 bzw. 7 Grad niedriger als die der unbehandelten Proben. Die Behandlung mit

30% H2O2 Lösung war extrem destruktiv, so dass die Proben für weitere Messungen nicht

mehr herangezogen werden konnten.

Hinsichtlich des Acetondehydratisierungschrittes wurde gezeigt, dass eine Behandlung in

reinem Aceton einen effektiveren Prozessschritt darstellt als eine aufsteigende

Acetonreihe. Jedoch führt die lange Behandlungsdauer zu unerwünschter Schrumpfung.

Die DC- Messungen zeigten in beiden Fällen (reines Acetons/Acetonreihe) keinen

Einfluss auf die Stabilität des Kollagens.

Die mechanischen Eigenschaften der Knochen und Perikard wurden ebenfalls

charakterisiert. Im Temperaturbereich von 37 bis 100 °C konnte keine signifikante

Abhängigkeit der Druckfestigkeit boviner Knochen nachgewiesen werden. Der Grund

hierfür könnte in der strukturellen Heterogenität und der Faserorientierung dieses

Materials liegen.

Ein Einfluss des Tutoplast-Prozesses auf die Zugfestigkeit des Perikards konnte nicht

nachgewiesen werden. Hier könnte der Grund in der mechanischen Anisotropie des

Materials liegen.