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The influence of tissue geometry on compaction and contractile properties of ovine myofibroblasts

BMTE 09.21

Chiara Tamiello

January 2009 – May 2009

Coaches:

Anita Driessen-Mol

Marijke Van Vlimmeren

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ABSTRACT

Background: Currently, tissue engineered heart valves are cultured with the leaflets attached to each other as

this has shown to provide good tissue formation because of the generated stress within the scaffold. However,

when detaching the leaflets and exposing them to physiologic systemic flow conditions the stress generated

within the tissue is released resulting in shrinkage of the tissue, referred as tissue compaction, which, on its turn,

results in loss of functionality of the heart valve. It is hypothesized that the geometry of the tissue itself

influences the degree of stress generated in the tissue and, thereby, the resulting compaction after release of the

constraints.

In this study effect of the geometry of the scaffold on tissue compaction, cell orientation and contractile

properties of the cells are investigated.

Methods: Strips, rectangular, triangular and circular scaffolds of rapid-degrading polyglycolic acid (PGA)

meshes coated with poly-4-hydroxybutyrate were seeded with saphenous vein derived myofibroblasts. These

tissues were cultured under static constraint for four weeks (n = 4 constrained constructs and n = 1 unconstrained

construct per group). Subsequently, constraints were released partially for strips, rectangular and triangular

constructs and completely for the circular constructs. Compaction was assessed from photographs of the released

constructs obtained over 48 hours. Finally, cell orientation and contractile properties of the cells were

qualitatively investigated by immunostaining of tissues sections along different directions while tissue formation

was analyzed by means of histology.

Results: Quantification of the compaction during the culture demonstrated that the rectangular constructs

compacted more than the other geometries. Culturing the constructs unconstrained resulted in clumped tissues.

After release of the constraints, all geometries continued to compact over 48 hours. All geometries, except the

circular construct, compacted in an unpredictable way. In contrast, the circular construct compacted in the

direction of its radius for the first 24 hours, starting to curl after that time. Immunostaining demonstrated that in

the circular scaffold cells are randomly oriented before and after the release of the constraints. Results did not

allow for a qualitative assessment of the contractile properties of the cells. Histology showed a sufficient tissue

formation in the constructs, except in the rectangular construct.

Conclusion: The results indicate that compaction is a continuous process. The circular tissues compact in a more

predictable way than the other geometries. This is most likely due to being either constrained in all directions, or

only partially. This study may provide insights towards the design of a heart valve in which geometry is

adjusted to the expected compaction in order to compensate for the loss in the functionality that usually occurs.

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TABLE OF CONTENTS

INTRODUCTION ................................................................................................................................................................... 4

2 MATERIALS AND METHODS ......................................................................................................................................... 8

2.1 CULTURING SHEEP VENA SAPHENA CELLS ......................................................................................................... 8 2.2 PREPARATION OF THE CONSTRUCTS ..................................................................................................................... 8 2.3 SEEDING ...................................................................................................................................................................... 10 2.4 EXPERIMENTAL DESIGN ......................................................................................................................................... 11 2.5 RELEASE OF CONSTRAINTS OF THE CONSTRUCTS ........................................................................................... 11 2.6. ASSESSMENT OF DEGREE OF COMPACTION ..................................................................................................... 12

2.6.1 IMAGE PROCESSING .......................................................................................................................................... 12 2.7 TISSUE PROCESSOR AND EMBEDDING ................................................................................................................ 13 2.8 IMMUNOSTAINING AND HYSTOLOGY ................................................................................................................. 14

3. RESULTS ........................................................................................................................................................................... 15

3.1 COMPACTION OF THE CONSTRUCTS .................................................................................................................... 15 3.1.1 COMPACTION OF THE CONSTRAINED CONSTRUCTS DURING THE CULTURING PERIOD.................... 15 3.1.2 COMPACTION OF THE UNCONSTRAINED CONSTRUCTS DURING THE CULTURING PERIOD .............. 16 3.1.3 QUALITATIVE EVALUATION OF COMPACTION DURING 48 HOURS AFTER RELEASE OF CONSTRAINTS

........................................................................................................................................................................................ 17 3.2 CELL ORIENTATION AND CONTRACTILE PROPERTIES ................................................................................... 19

3.2.1 Strip ........................................................................................................................................................................ 19 3.2.2 Rectangle................................................................................................................................................................ 19 3.2.3 Triangle .................................................................................................................................................................. 20 3.2.4 Circle ..................................................................................................................................................................... 20

3.3 TISSUE FORMATION ................................................................................................................................................. 22

4. DISCUSSION ..................................................................................................................................................................... 24

5. CONCLUSION .................................................................................................................................................................. 26

REFERENCES ...................................................................................................................................................................... 28

APPENDIX 1: CULTURING SHEEP VENA SAPHENA CELLS ................................................................................... 29

APPENDIX 2: PREPARATION OF SCAFFOLD FOR TISSUE ENGINEERING CONSTRUCTS ........................... 32

APPENDIX 3: AREA MEASURAMENT BY MEANS OF IMAGEJ SOFTWARE ...................................................... 37

APPENDIX 4: SECTIONING PARAFFIN BLOCKS CONTAINING SCAFFOLD MATERIAL ............................... 39

APPENDIX 5: IMMUNO-FLUORESCENT STAINING VIMENTIN, DESMIN AND Α-SMA ................................... 41

APPENDIX 6: HEMATOXYLIN & EOSIN (H&E) STAINING ..................................................................................... 43

APPENDIX 7: RESULTS – COMPACTION DURING CULTURE ................................................................................ 46

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INTRODUCTION

Surgical replacement of diseased heart valves with biomechanical and bioprosthetic valve substitutes is

the most common treatment for end-stage valvular heart diseases, with approximately 85,000 valve

replacements performed each year in the US and 275,000 worldwide [1]. Replacement of diseased

valves reduces the morbidity and mortality associated with valvular disease, but come at the expense of

risking complications unique to the implanted prosthetic device. These complications include primary

valve failure (valve durability), prosthetic valve endocarditis (PV), prosthetic valve thrombosis (PVT),

thromboembolism, anticoagulant-related hemorrhage, and mechanical hemolytic anemia [2]. These

problems have been attributed to the design as well as the durability and biological incompatibility of

materials used in mechanical valves.

Recent advances in tissue engineering have demonstrated the potential of in the development of

customized living and autologous replacement body parts. The advantages of a tissue-engineered

implant include the ability to regenerate over prosthetic and mechanical implants and grow. Further, the

risks related to immunological complication and pathogen transfer are diminished. [3]. In the case of

tissue engineered heart valves, the feasibility of the autologous tissue engineering concept for heart

valve application has been investigated, demonstrated and improved for the last decade[4]. The

approach involves the seeding of autologous cells onto a porous synthetic polymer or biological

material configured in the shape of the heart valve. The cells are subsequently stimulated to develop a

complete autologous, living heart valve replacement [2].

In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are

recognized as being of utmost importance. The optimal functionality of a tissue is strictly correlated to

its appropriate histological organization. It is known indeed that abnormal tissue structures resulting

from various pathologies are responsible for functional defects [5].

In previous studies, good tissue formation in tissue engineered heart valve leaflets was provided when

culturing them constrained and attached to each other. Due to contractile properties of the seeded

myofibroblasts, contractile forces develop within the tissue, which has been demonstrated to be

beneficial for the tissue architecture and functionality. However, before implantation the leaflets have

to be separated and due to the stress release within the tissue coupled to the removal of the constraints,

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the leaflets compacted resulting in leakage of the valve, thus in its loss of functionality [6].This is

clarified in Fig. 1.

Fig. 1. The effects of stress generation. Schematic image representing the positive (left) and negative (right) effects of stress

generation within the tissue in a tissue engineered heart valve cultured with the three leaflets attached to each other: tissue

architecture is optimized while the release of the stress after separation of the leaflets results in compaction and, therefore,

in leakage of the heart valve.

To overcome the leaking of tissue engineered valves cultured with the leaflets attached to each other, it

is relevant to understand the different aspects that influence the compaction of the leaflets. It is

hypothesized that the geometry of the tissue itself influences the degree of stress generation and

thereby, the resulting compaction after release of the constraints [5]. The degree of stress generated in

the tissue cannot be measure directly, while analysis of the compaction and of the contractile properties

of the cells can provide an indirect measure of it. When the influence of geometry on the compaction is

known, the design of a heart valve could perhaps be adjusted to take this compaction into account.

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The aim of this study is to investigate the effects of the geometry of the scaffold on the degree of

compaction of the tissue after release of the constraints applied during culture. The correlation of the

contractile cell properties and cell orientation within the tissue to the compaction of the tissue are also

studied together with an evaluation of the tissue formation in the different constructs.

For this purpose, four different scaffold geometries were studied: strip, rectangle (shorter and wider

scaffolds compared to strips), triangle and circle.

The scaffolds cut off of rapid-degrading polyglycolic acid (PGA) meshes were coated with poly-4-

hydroxybutyrate and seeded with saphenous vein derived myofibroblasts. These tissues were cultured

under static constraint for four weeks (n = 4 constrained constructs and n = 1 unconstrained construct

per group). Subsequently, constraints were released partially for strips, rectangular and triangular

tissues and completely the circular tissues and the compaction was assessed from photographs obtained

over 48 hours. Further, orientation and contractile properties of the cells were qualitatively investigated

by means of immunostaining of tissue sections along different directions: staining of the cytoskeleton

with phalloidin was used to visualize cell orientation while α-Smooth Muscle Actin (α-SMA)

expression was used to assess the contractility of the cells [7]. Finally, the density of the tissue in the

different scaffolds was assessed by means of the Hematoxylin and Eosin (H&E) staining.

Differences in the compaction and in the cell orientation were expected especially between

strips/rectangles and circles/triangles because of the differences in the stress generation [5]. In the

circular scaffold, compactions after release of the constraints was expected in all directions, while in

the strips and rectangular constructs shrinkage was expected mostly along the longitudinal direction.

Further, in the case of the triangular constructs compaction was expected along the base and height of

the scaffold, if the base was released from the constraining ring.

Cell orientation was expected random in the constrained and released circular scaffold. In the

rectangles and in the strips constrained to the ring, cells were expected to align mostly along the

longitudinal direction, namely the direction along which stress developed (axis of tension).On the other

hand, in the released sample a random orientation was expected because of stress release along several

directions. The triangular scaffold was expected to have a random orientation when being constrained

as well as after release of the constraints.

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α-SMA expression was supposed to be higher in the constrained samples than in the released ones.

During constrained culture, myofibroblasts tend to develop contractile forces and pull on the

constraining frame, while contractility is supposed to decrease when the stress is released because of

releasing of the constraints.

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2 MATERIALS AND METHODS

2.1 CULTURING SHEEP VENA SAPHENA CELLS

Ovine vena saphena cells (isolation I, passage 3) were cultured using regular cell culture methods

[Appendix 1]. The medium used for cell culture consisted of DMEM Adanced (Gibco/Invitrogen Corp)

supplemented with 10% Lamb serum, 1% GlutaMAX (Gibco, UK), 1% PenStrep. Cell cultures were

maintained in a humidified incubator at 37° C and 5% CO2 and medium was replaced every 3 days.

Cells were cultured until passage 6.

2.2 PREPARATION OF THE CONSTRUCTS

For each geometry, 5 scaffolds (Table 1), were cut out of rapid-degrading nonwoven polyglycolic acid

meshes (PGA; thickness 1.0 mm, specific gravity 70 mg/cm3, lot# MD00645 and MD00283) and

coated with a thin layer of poly-4-hydroxybutyrate (P4HB (1.75 % w/v)) dissolved in tetrahydrofuran

(THF; Fluka; Germany).[Appendix 2]

Geometry Constrained Not constrained

strip 25* 5 mm 18*5 mm

rectangle 22*11 mm 18*11 mm

triangle b = 20 mm, h = 20 mm b = 20 mm, h = 20 mm

circle r = 12.5 mm r = 9 mm

Table 1. Scaffold measures. b, base of the triangle; h, height of the triangle; r, radius of the circle

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Note that the unconstrained constructs are smaller than the constrained ones because the seeding area

only is taken into consideration. For the triangle the same measures were kept in order to maintain the

geometry of the construct; however, the actual area seeded was the same.

The constructs were glued with polyurethane (PU) glue (20% w/v ; DSM, Netherlands) to a stainless

steel ring (inner diameter 16 mm, outer 25mm), (Fig. 2a). The strips and the rectangular constructs

were glued at both ends, the triangles at the three vertices and the circles along their perimeter. A

custom-built set up was required for the circular scaffold in order position the tissue near the surface of

the medium to provide good oxygen perfusion (Fig. 2b). This set up consisted of two white tips

(Greiner Bio, Austria) where glued onto each other and attached to the ring at opposite positions with

PU. The white tips are free from DNase, RNase and human DNA, free from endotoxin (pyrogen) and

are non-cytoxic.

(a)

(b)

Fig. 2. Set up used in the experiment. PGA-Scaffold glued to a stainless steel ring (a): this set up was used for strips,

rectangular and triangular constructs. Set-up for the circular scaffold (b): the white tips were glued onto each other and

attached to the ring with PU. The circular scaffold was already glued to the stainless steel ring.

The samples were dried over night in the vacuum oven to allow for vaporization of the leftover THF.

The white tips in the circular set-up enlarged in the contact points with the ring. The cause is not

known, but this was probably caused by a reaction between PU, plastic and stainless steel.

Thereafter, the rings with strips, rectangular and triangular scaffolds were placed in 6-wells plates

while the circular constructs were put in medium tubes as a different amount of medium was required

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because a density of 1.5x106 cells per cm

3 was used to seed the scaffolds and 0.25x10

6 cells are usually

cultured in 1 ml of medium. After sterilization with 70% ethanol and washing with PBS, the wells were

filled with TE medium and placed in the incubator overnight.

2.3 SEEDING

The seeding with ovine vena saphena myofibroblasts was performed the day after sterilization of the

scaffold constructs, as described previously [Appendix 2]. Briefly, the cells (passage 6) were

enzymatically detached with trypsin and after a centrifuging step they were counted and resuspended in

a thrombin solution. Subsequently, fibrinogen was added and the cells were evenly distributed over the

scaffolds at a density of 1.5x106 cells per cm

3.

The seeding area (Table 2) was equal in the constrained and not constrained constructs. Therefore the

number of cell used was also the same in each group. The constructs were placed in the incubator for

30 minutes to let the fibrin gel further polymerize. After that, Tissue Engineering medium (TE

medium) made with DMEM Advanced and supplemented with additional 1% lamb serum, 1%

Pen/Strep, 1% Glutamax and 130 mg L-ascorbic acid 2-phosphate (0.26 mg/mL, Sigma, Germany) was

added in order to promote extracellular matrix production. The amounts of TE medium used are

summarized in Table 2.The constructs were cultured for four weeks and the medium was changed

every 3 to 4 days.

Table 2. Seeding area, amount of cells seeded and amount of medium for each geometry.

geometry Seeding area

(cm2)

Cells (x106) Medium (mL)

strip 0.9 1.35 5

rectangle 2.0 3.0 12

triangle 1.8 2.7 10

circle 2.5 3.8 15

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2.4 EXPERIMENTAL DESIGN

Four experimental groups were studied. Each group (n = 5 samples) was representative for a different

geometry: strips, rectangular, triangular and circular geometry. For each group, n = 4 samples were

cultured constrained to a stainless steel ring (constrained constructs, Table 3), while one of them was

cultured not constrained (unconstrained constructs, Table 3). In order to study the compaction of

different geometries, three out of four constrained constructs were released partially or completely from

the constraining ring (released constructs, Table 3). -SMA expression and cell orientation were

analyzed in slices from a released construct and a constrained construct used as a control.

Table 3. Overview of the experimental design.

2.5 RELEASE OF CONSTRAINTS OF THE CONSTRUCTS

After four weeks, three out of four constrained constructs were released from the ring (released

constructs). Following the inner diameter of the rings, the strips and the rectangles were cut from one

side, by means of a scalpel. For the triangular constructs 2 vertices were cut loose while the circles

geometries Tot.

Unconstrained

constructs

Constrained

constructs

Released

constructs

Cell orientation (slices

from)

α-SMA expression(

slices from)

circle

n = 5 n =1 n = 4 completely cut

loose

n=3

1 released construct

+

1 constrained construct

(control)

1 released construct +


(control)

rectangle n = 5 n=1 n = 4 one side cut

loose

n=3


+


(control)



(control)

triangle n = 5 n=1 n = 4 2 vertices cut

loose

n=3


+


(control)



(control)

strip n = 5 n=1 n = 4 one side cut

loose

n=3


+


(control)



(control)

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were completely cut loose from the ring. One construct for each group was left constrained to the ring

in order to be used as a control for further analysis. After assessing the degree of compaction over 48

hours (2.6) in the released constructs, the samples were stored overnight at 4°C in 3.7% formalin. After

24 hours they were put in PBS and stored in the 4°C fridge until use for embedding and sectioning as

described in 2.7.

2.6. ASSESSMENT OF DEGREE OF COMPACTION

During culture, due to the contractile properties of the seeded myofibroblasts, contractile forces

develop within the tissue resulting in reorganization of the tissue and compaction of the construct. In

order to quantify the degree of compaction during the culturing period, at the end of every culture

week, digital photographs of the TE constructs were taken with a digital camera (Canon, Japan). The

amount of compaction was measured with the help of ImageJ software and results were represented as

the relative compaction of the constrained constructs by comparing the surface area at the end culturing

week (t = 4 week) with the seeding area (t = 0).

After releasing the constraints, photographs of the TE constructs were taken at each time point (i.e. t=0,

25, 45 min. and t =1, 1.5, 2, 2.5, 3, 6, 8, 24, 48 hours from release of the constraints) for macroscopic

appearance. The camera was placed on a stative to keep magnification and position the same in all

photographs. The background used was a black paper to avoid the effects of shadows in the

photographs. A scale (1 mm) for calibration was also present in the image.

2.6.1 IMAGE PROCESSING

ImageJ software was used in order to measure the surface area of the constrained constructs at the end

of culturing (t = 4 weeks).The inner perimeter of the ring was selected and the image was then

transformed in 8-bit level. Then, a threshold operation was performed and the Region of Interest (ROI)

was assigned to a red mask. After entry of the Known Distance, which was “1”, and the Unit of Length,

which was “mm”, the area was calculated by the software and the results displayed the area in mm2.

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Finally, the results were represented as the relative compaction of the TE construct by comparing the

calculated surface area relative at week 4 to the seeding area at t = 0 (Table3, seeding area).The

procedure is described in Appendix 3.

2.7 TISSUE PROCESSOR AND EMBEDDING

After fixation, one released construct representative for each geometry and the relative constrained

construct used as a control were completely detached from the rings and put in the tissue processor

(Adamas) overnight. Tissue processing dehydratated the constructs and then removed the dehydrant

with a substance that was miscible with the embedding medium (paraffin). Thus, the constructs were

embedded in paraffin. The analysis of cell orientation needed to be studied along at least two different

directions. For this reason the samples were cut before embedding following the plan that is described

in Fig.3.

(a)The rectangular scaffolds and the strips were cut along

directions y and x. Thus along NO and MO segments. The

embedding was carried out in order to obtain sections

parallel y and x directions.

(b)The triangular scaffold was analyzed along two

directions: y direction and z direction. Thus, the scaffold was

cut along AO and the BO segments and the embedding was

carried out in order to obtain sections parallel to y and z

directions.

(c)The circular scaffold was cut along AO and BC segments.

Thus, the two quarters of the circle were embedded in order

to obtain a section parallel to y and x directions while the

remnant half of the circle was embedded so that the scaffolds

could be sectioned through the plane parallel to its surface.

Thus, three different sections were available for this

construct.

Fig.3. Directions considered for the embedding and the sectioning. Strips and rectangular constructs (a) were cut and

embedded along y and x directions, triangular constructs (b) were cut and embedded along y and z directions and circular

constructs (c) were cut and embedded along y and x directions and on the plane parallel to the surface.

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2.8 IMMUNOSTAINING AND HYSTOLOGY

10µm-thick sections were cut from the embedded samples and air-dried (Appendix 4). In order to

visualize the cytoskeleton for cell orientation and the expression of α-SMA, sections were

deparaffinized, blocked with PBS containing 1% bovine serum albumin (BSA) and incubated with

rhodamine-conjugated phalloidin (1:200, Sigma), with a mouse monoclonal antibody (DAKO, Canada)

directed against α-SMA (1:500), and with a secondary antibody coupled to Alexa 488(goat anti-mouse,

1:300) as described in Appendix 5.As a control, sections were additionally stained, the primary

antibody was omitted. Nuclei were counterstained with DAPI. The sections were viewed with a

fluorescence microscope (Zeiss, Germany); particularly, the intensity of the green signal (α-SMA

expression) was used as measure of α-SMA expression. Further, sections of the control constructs

were histologically examined by Hematoxylin and Eosin stain (H&E stain) in order to have a visible

look at the nucleus of the cells and their state of activity (tissue formation) (Appendix 6). The sections

were viewed with an optical microscope (Zeiss, Germany).

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3. RESULTS

3.1 COMPACTION OF THE CONSTRUCTS

3.1.1 COMPACTION OF THE CONSTRAINED CONSTRUCTS DURING THE

CULTURING PERIOD

By means of pictures taken during the 4 culturing weeks it was possible to assess the compaction of the

different geometry constructs. Obviously, circular construct could not compact because they were

constrained, thus they were not taken into account for this analysis.

Compaction during the culturing period was investigated in order to get some insight into changes in

compaction over time and to elucidate differences between the groups. It was observed that compaction

for two out of three rectangles, started at the end of week 2. One of the rectangles was not properly

fixated, so one end detached from the constraining ring during the culture. For strips and triangles,

compaction started during the third week of culturing. Compaction was not observed in one of the

triangles, which therefore was excluded for further analysis. Overall, the tendency towards compaction

was visible for all the geometries over the four weeks of culturing, starting after week 2.

Quantification of compaction performed with the help of ImageJ software is provided in Table 4 and in

Appendix 5. It was pointed out that the strips and rectangular constructs compacted to a greater extent

(37 % and 41%), compared to the triangular constructs (7%). Overall, rectangles compacted more than

the other constructs, with the lowest SD as can be seen in Table 4.

geometry Average relative compaction SD

Strip 37 % 5.0

Rectangle 41% 0.5

Triangle 7 % 7.5

Table4. Compaction during culturing

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3.1.2 COMPACTION OF THE UNCONSTRAINED CONSTRUCTS DURING THE

CULTURING PERIOD

For the unconstrained constructs, it was macroscopically visible that at the end of week 2 strips and

rectangular unconstrained constructs (n = 1) started to bend while the triangle and the circles had not

changed their shape. One week later, the circular tissue bended and tended to form a cylindrical

construct while for the triangle, only one vertex started to curl. For the strip and the rectangle

compaction continued until the end of the fourth week of culturing when they reduced to a clump; the

triangle was also completely curled into a clump and the circle formed a structure similar to a tube, as

can be seen in Fig.3. Results are summarized in Table 5.

Strip rectangle triangle Circle

Week1 NO NO NO NO

Week2 + + NO NO

Week3 ++ ++ + +

Week4 +++ +++ +++ ++

Table 5. Compaction of the unconstrained constructs during the culturing period. +, starting of the compaction; ++, the

degree of compaction increases, +++, the construct reduces to a clump.

Strip (a) Rectangle (b) Triangle (c) Circle (d)

Fig.3. Unconstrained constructs at the end of the 4th

week of culturing. The strip (a), the rectangle (b) and the triangle (c)

compacted and reduced to a clump; the circle formed a structure similar to a tube (d).

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3.1.3 QUALITATIVE EVALUATION OF COMPACTION DURING 48 HOURS AFTER RELEASE OF CONSTRAINTS

Compaction was investigated macroscopically from photographs of the released constructs during 48

hours after release of the constraints.

Strips

At t = 25 min. after sacrifice the strips started to curl and to compact, as can be seen in Fig. 4a. The

compaction along the axis of tension, as expected, was common to all the constructs and continued

over 48 hours, as depicted in Fig. 7b and Fig. 7c.

Rectangles

Compaction started soon after the release of the constraints, as can be seen in Fig. 5d (t = 25 min.) and

continued until the time point t = 48 hours (Fig. 4e -4f). The TE constructs compacted along the axis of

tension as expected and the longer edges curled towards the centre of the construct.

Triangles

Compaction was observed already at t = 25 min. (Fig. 4g).At time points t = 24 and t = 48 hours, two

out of 3 triangles completely bend on themselves while continuing to compact. An example is shown if

Fig. 4h-4i. The triangle that did not compacted during culturing did compact during the 48 hours along

the height and base directions, while maintaining its own shape.

Circles

All the circular constructs showed a similar behaviour during 48 hours: they started to compact in the

radial direction soon after release from the constraining ring (Fig. 4l, t = 25 min.) maintaining their

circular shape until t = 24h when the edges visibly started to curl (Fig. 4m). Shrinkage and curling

continued until t = 48h (Fig. 4n)

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t=25min.(a) t =24 (b) t=48h (c)

t = 25min. (d) t=24 h (e) t = 48h (f)

t = 25min. (g) t = 24h (h) t = 48 h (i)

t =25min. (l) t = 24h (m) t = 48h (n)

Fig. 4. Photographs of the released construct an overview of the compaction during 48 hours after release of the

constraints. Strips (a, b, c): at t = 25 min. after sacrifice the strip starts to curl and to compact (a); comparison of (b)and

(c) points out that compaction continues until t = 48h. Rectangles (d, e, f): compaction starts soon after the release of the

constraints (d, t = 25min.).The shrinkages continues until the time point t = 48 hours (e, f).Triangles (g, h, i ) : compaction

is observed already at t = 25 min. (g).At t = 24h the triangle depicted in (h) bends on itself and continues to fold until t =

48h (i).Circles (l, m, n): the circle after release of the constraints, maintains its shape while compaction in the radial

direction, as depicted at t = 25min (l). At t= 24h (m) its edges start to curl. Shrinkage and curling are more pronounced at

time point t = 48h (n).

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3.2 CELL ORIENTATION AND CONTRACTILE PROPERTIES

Sections were double immunostained for cell orientation analysis and with α-SMA as a measure for the

contractile properties of the cells. Controls confirmed the suitability of the stainings.

3.2.1 Strip

When studying in the x direction, both surface layers were aligned in the released construct (Fig. 5a),

while only one of them was aligned in the control (constrained construct). This could be due to the fact

that the cut along that direction was not orthogonal to the width of the sample. However, the alignment

was more pronounced (Fig. 5b). Along the y direction, the released construct showed a slight alignment

of cells along the surface layers (Fig. 5c), while in the control the cells were more randomly oriented

(Fig. 5d). When comparing the constrained construct along y (Fig. 5d) and x (Fig. 5b) directions, it was

visible that the cell nuclei were more rounded along the y direction while they were more stretched

along the x direction. The cells were indeed expected to align mostly along the axis of tension, namely

the x direction in the constrained construct, while in the released one any cellular alignment was not

predictable.

-SMA expression was observed but differences were not noticed between control and the released

construct, even if the same settings for exposure time were used.

3.2.2 Rectangle

Results about rectangle were not obtained because the control section along y direction detached from

the poly-L-lysine coated slides during deparaffinization. This could be due to the size of the section or

the fair tissue formation. The analysis along x direction did not provide any insight in the orientation

and in the α-SMA expression

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3.2.3 Triangle

Cell alignment was visible in both samples along the y direction: in the released construct a thick layer

of aligned cells was present (Fig. 5f), while in the control the cells clearly had a different orientation

within some micrometers as can be seen in Fig. 5g.

Along the z direction, the cells had no organization in the released construct (Fig. 5h).In contrast, the

control showed a strong orientation of the surface layer (Fig. 5i).

A lot of scaffold (red autofluorescence) was still visible in the middle of the control (Fig. 5i); while

fewer scaffold was observed in the released construct (Fig. 5h).

Results did not allow for view of differences in the expression of α-SMA between the two constructs.

3.2.4 Circle

The comparison between the directions x and y pointed out that, in both directions, the cells are much

more aligned in the control while, in the released construct, they had a more random orientation, as can

be seen in Fig. 5m and Fig. 5n.The scaffold was really compacted in the middle of the released

construct (Fig. 5m), while in the constrained construct there was more empty space in the middle of the

construct (Fig. 5n). The slices cut in the plane parallel to the surface showed very little scaffold

remaining in the control and cells randomly oriented, as expected (Fig. 5q). The released construct had

much more scaffold and randomly oriented cells as well (Fig. 5r). The cells showed some alignment

along the edge but this result is not reliable because the circle curled after 24 hours making it

impossible to have a useful section containing all the surface of the scaffold.

α-SMA expression, based on the intensity of the green signal seen through the microscope, appeared to

be higher in the control

21

Released construct Constrained construct (control)

Dir.x

(a)

(b)

(e)

Dir.y

(c)

(d)

Dir. y

(f)

(g)

(l)

Dir. z

(h)

(i)

22

Dir.y

(m)

(n)

(q)

Dir. p

ara

llel to su

rface

(o)

(p)

Fig. 5. Cross sections of the released and constrained (control) constructs stained with phalloidin and immunolabeled with

antibodies directed against α-SMA (magnification x200).Strip: along the x direction, (a) cells are organized predominantly

in the surface layer in the released construct,(b) the same happens in the constrained construct (control )but the alignment

is more pronounced. Along the y direction cells are randomly oriented in both constructs (c, released construct) and (d,

constrained construct). (e), directions considered for sectioning the strips. Cross sections of triangular construct

demonstrates that, along the y direction, (f) cells organize themselves in a thick layer of aligned cells in the released

construct while, in the control, one of the surface layers shows that cells have a different orientation within some

micrometers from the surface (g). Along the z direction, (h) cells are not organized in the released construct, while a strong

alignment is visible on the surface layers of the constrained construct (i). (l), directions considered for sectioning the

triangular constructs. Circular construct: along the y direction, (m) cells of the released construct demonstrate that they

have a slight organization; (n) in the control, there is a much stronger alignment. (o) and (p) show sections cut in the plane

parallel to the surface: a lot of scaffold (red autofluorescence) and random cell orientation can be seen in the released

construct (o), very little scaffold and random cell orientation in the constrained construct (p). (q), directions considered for

sectioning the circular constructs.

3.3 TISSUE FORMATION

Histology (hematoxylin and eosin staining) confirmed that cells were successfully seeded in the

constructs but tissue formation resulted in some slight differences among the constructs. Strip tissue

appeared to be dense and almost uniform over the cross section (Fig. 6a).There was organization of the

23

tissue predominantly in outer layer. The rectangular construct showed holes that were probably just a

cutting artifact due to the fair density of the tissue. The black line is probably caused by an air bubble

(Fig. 5 b). In the triangular construct the cells were clearly visible and the tissue was almost uniform

across the section (Fig. 5c). Finally, cross section of the circular construct showed denser and more

organized tissue formation near the surface layers than in the middle (Fig. 5d).Overall, sufficient tissue

formation was shown, except for the rectangular construct.

Fig. 6. Histology of the constrained constructs by H&E stain (magnification x100). (a), strip tissue is organized near the

outer layer and the density is almost uniform throughout the section. (b), cross section of the rectangular construct

demonstrates fair tissue formation that resulted in cutting artifacts (holes in the tissue). (c), cross section of the triangular

construct shows dense tissue throughout the section. (d), tissue of the circular construct is not composed of dense tissue in

the middle of the construct, while more organized and dense tissue is shown on the surface layers.

a b

c d

24

4. DISCUSSION

The described analysis conducted on the degree of compaction of the constructs gave some insight into

the trend of compaction during culturing time for different geometries of constructs cultured either

constrained to a ring or not. Differences between the compaction of different geometries were also

studied after release of the constraints.

The overall results indicate that the shrinkage of the tissue during culturing started during the third

week of culturing. Taken together, the results suggest that the PGA scaffold starts to degrade after 2

weeks allowing the cells to pull on the extracellular matrix (ECM). During culturing, compaction

occurred mainly in the constrained rectangular constructs. This can be caused by the fair density of the

tissue formation that has been found by means of histology. The even distribution of the generated

stress in the triangular constructs could explain a less pronounced compaction in the constrained

triangular construct.

In this study it was also proved that it is not possible to obtain functional tissue when culturing

unconstrained constructs. Differences in compaction over time suggest also that the orientation of the

fibers in the PGA scaffold might influence the tendency to compaction of geometrically different

constructs. The constructs that were cut parallel to the PGA fibres started to curl one week earlier than

the ones that were not cut along the fiber direction. This could be further investigated in future studies.

One of the most interesting finding of this study is that compaction does not stop but continues until 48

hours from the release of the constraints for all the geometries taken into consideration. Further studies

are needed in order to establish whether compaction continues beyond that point.

It was also relevant to note that circular constructs started to bend after 24 hours from sacrifice had

passed. All the released circular constructs maintained their flattened shape while compacting in the

radial direction and compacted to the same extent during the first day after release of the constraints, as

expected. Therefore, there is a good possibility that the shrinkage of this kind of scaffold is predictable

and can be taken into account before culturing a heart valve with the three leaflets attached to each

other.

25

The results from immunofluorescence staining were restricted to strips, triangular and circular

constructs. It is clear that orientation of the cells can be predicted in the case of the circular scaffold:

the cells of the control appeared to be locally stretched along the directions taken into account in this

study, but the overall orientation is random because it is likely that the same results could be found in

all the every radial directions. In contrast, in the experimental sample, the cells are not stretched

because they released the stress generated within the tissue during culturing. Thus, they have a local

and overall random orientation. In the strip and in the triangular construct the unpredictable bending

and compacting, thus the remodelling of the tissue is influencing the orientation of the cells within the

tissue. However, based on this experiment it was important to note that the results for strips were in

agreement with the expectations only in the case of the constrained construct: alignment was more

pronounced in the longitudinal direction and cells were not randomly oriented like in the circle not only

in the constrained construct but also in the released sample. This is likely to be due to stress release

along the axis of tension, which is the direction along which contractile forces developed during

constrained culture. Further, no other major findings can be derived.

One of the limitations of this study is clear: the assessment of -SMA expression is not reliable.

Nevertheless, the exposure time used was the same for the experimental sample and the control, it was

impossible to determine from the fluorescent green signal clear differences in the expression of -SMA

expression. This limitation could be overcome using ELISA analysis, which allows a more reliable

quantification of -SMA expression. In this way, the trend of -SMA expression after release of the

constraints could be elucidated. Further, it could be relevant to achieve a quantitative measurement of

the compaction in the different scaffold in order to provide a more accurate indirect measure of the

stress generated in the tissue engineered heart valve leaflet.

Finally, if further studies will be conducted on the contractile properties of cells within tissue from

different scaffolds it should be taken into consideration that in this study it was not possible to achieve

a proper sectioning in the plane of the surface because the released samples tended to curl and bend.

Therefore, it was not possible to have the scaffold completely parallel to the blade in the microtome.

Further, when performing the cuts along different directions, the cuts must be performed perpendicular

to each other in order to have useful sections to stain for cell orientation.

26

5. CONCLUSION

The aim of this study was to investigate the effects of the geometry of the scaffold on the compaction

of the tissue and the correlation of the contractile cell properties and cell orientation within the tissue to

the compaction. This was done by culturing four different PGA-scaffold geometries seeded with ovine

myofibroblasts constrained to a stainless steel ring. Compaction of the tissue during the culturing

period and during 48 hours after partial or total release of the constraints was assessed by means of

photographs. The contractile properties of the cells and their orientation were investigated by means of

immunostaining, while histology (H&E stain) gave some insight about tissue formation in the

constructs.

In this study it was shown that geometry indeed influences the degree and the way of compaction of the

constructs. The circular construct appeared to have the most predictable way of compacting during 48

hours after detachment from the constraining ring. This might be connected to the random organization

of the cells in the tissue that appeared to be denser on the surface layers. Immunostaining pointed out

that the randomness in the orientation was maintained during compaction after release of the

constraints but the cells were much less stretched in the released construct due to stress release. The

behaviour of the circular construct suggests that culturing the scaffold completely attached to a frame

lead to a predictable compaction after detachment. It is likely that this kind of scaffolds are more

attractive for the optimization of heart valve cultured with the three leaflets attached to each other in

order to avoid leakage after their detachment [6].

A remarkable finding of this research is that compaction is continuing at least until 48 hours after

sacrifice of the constructs. More research is needed in order to quantify the amount of compaction over

a longer period in order to elucidate when it eventually stops.

-SMA expression is also an important parameter that should be objectively quantified by ELISA in

order to elucidate the correlation with the differences in the geometry and the stress generated within

the tissue.

In summary, this study describes the influences of the tissue geometry on the compaction of PGA

scaffolds seeded with ovine myofibroblasts. Results demonstrated that circular constructs do not curl

27

and compact in a predictable way during the first 24 after release of the constraints. This finding can

give some useful insights to help the design of a scaffold for a tissue engineered heart valve cultured

with the leaflets attached to each other in order to avoid any loss of functionality due to compaction of

the tissue.

28

REFERENCES [1] E. Rabkin and F.J. Schoen. Cardiovascular tissue engineering. Cardiovasc Pathol, 11:305-317, 2002

[2] Eric Kardon, MD, FACEP, Associate Staff, Division of Emergency Medicine, Athens Prosthetic

Heart Valves, Feb 26, 2007; http://emedicine.medscape.com/article/780702-overview

[3] Tissue engineered tri-leaflet heart valve-preliminary fabrication of biodegradable polymer

scaffolds. Teoh, S.H.; Lan, C.Y.; Ranawake, M.; Hutmacher, D.W.; Chew, Y.T.; Sim,

K.W.[Engineering in Medicine and Biology, 1999. 21st Annual Conf. and the 1999 Annual Fall

Meeting of the Biomedical Engineering Soc.] BMES/EMBS Conference, 1999. Proceedings of the

First Joint Volume 2, Issue, Oct 1999 Page(s): 748 vol.2

[4] Driessen - Mol, Functional Tissue Engineering of Human Heart Valve Leaflets, PhD. Thesis, 2005,

Eindhoven University of Technology

[5] Grenier Guillaume; Rémy-Zolghadri Murielle; Larouche Danielle; Gauvin Robert; Baker Kathleen;

Bergeron François; Dupuis Daniel; Langelier Eve; Rancourt Denis; Auger François A; Germain Lucie

Tissue reorganization in response to mechanical load increases functionality. Tissue engineering 2005;

11(1-2):90-100

[6] J. Kortsmit , N. J. B. Driessen, M. C. M. Rutten and F. P. T. Baaijens. Real Time, Non-Invasive

Assessment of Leaflet Deformation in Heart Valve Tissue Engineering. Annals of biomedical

engineering (Ann Biomed Eng); 2009-Mar; vol. 37 (issue 3): pp 532-41

[7] Guillaume Grenier, Murielle Rémy-Zolghadri, François Bergeron, Rina Guignard, Kathleen Baker,

Raymond Labbé, François A. Auger, Lucie Germain. Mechanical Loading Modulates the

Differentiation State of Vascular Smooth Muscle Cells. Tissue Engineering. November 2006, 12(11):

3159-3170.

[8] Visual Guide IV: Measuring cells with ImageJ, http://naranja.umh.es/~atg/tutorials/VGIV-

MeasuringCellsImageJ.pdf

http://emedicine.medscape.com/article/780702-overview

29

APPENDIX 1: CULTURING SHEEP VENA SAPHENA CELLS

MEDIUM:

DMEM Advanced (500 ml)

10% Lamb Serum (50 ml)

1% Glutamax (5 ml)

1% Penicillin/Streptomycin (5 ml)

THAWING THE CELLS

Normally the cells are frozen in an amount of 3x106

cells per vial. This is a good amount to start with in

a T150 flask. The passage that is on the vial is the passage at which they were frozen, so add a passage

when you set up the cells.

Add 25 ml of medium to a T150 flask

Take a vial out of the liquid nitrogen

Warm up between your hands

Carefully open the vial to let the N2 escape

Transfer the content of the vial to the flask and rinse the vial once with medium from the flask.

Mix the cells with the medium gently and place the flask in the incubator

Change the medium one day after thawing to remove dead cells.

MEDIUM CHANGE

The medium should be changed every three or four days. Most convenient is to do this every

Thursday and Friday or every Monday and Tuesday.

Discard the medium

30

Rinse the cells once with PBS

Add new medium up to 25 ml per flask.

SUBCULTURING THE CELLS

Sheep cells grow rather fast in comparison to human cells and they are much smaller so more cells fit

in one flask. After about 5 days the flask is confluent and the cells are ready to be subcultures. When

confluent there are about 20x106 cells in one flask .You can transfer these cells 1:6 (approximately

3x106 per flask). Do never let the cells grow too confluent as this will influence their growth.

Discard the medium

Rinse the cells twice with PBS

Add trypsin (2.5 ml per flask) and distribute evenly over the bottom of the flask.

Place the flask in the incubator for 7 minutes

Tap the flask several times and check whether all the cells are rounded up and de-attached. If

not placed the flask into the incubator for a few more minutes.

Add 5ml of medium to the flask and mix the cells and trypsin

Transfer the medium to a centrifuge tube

Add 10 ml of PBS to the flask and pipette up and down to the walls of the flask to get all the

leftover cells transferred into the centrifuge tube

Check the flask microscopically for leftover cells. If there are still many cells in there rinse the

flask again with PBS and add this to the centrifuge tube.

Centrifuge 7 minutes at 1000rpm(or 5 min at 1500 rpm)

Discard the supernatant and resuspend the cells in 4 ml medium in order to count them.

After counting, divide them the cells over 6 new flasks.

Fill each flask up to 25 ml with medium and place the flasks in the incubator.

31

NB. Ovine sheep vena cava cells can be subculture up to passage 8-9, after that passage they will not

be the same cells anymore as it can be noticed in a changed morphology and a different growth speed.

COUNTING THE CELLS

Transfer 1 to a eppendorf tube

Add 50 of buffer A

Spin the vial for 5 seconds

Add 50 of buffer B

Spin the vial for 5 seconds

Use the nucleon counter to count the cells

32

APPENDIX 2: PREPARATION OF SCAFFOLD FOR TISSUE

ENGINEERING CONSTRUCTS

Introduction:

This is a standardized protocol for the preparation of the scaffold for Tissue Engineering control strips,

prior to the seeding of the cells onto the scaffold. Polyglycolic acid (PGA) is used for the scaffold

material and poly-4-hydroxybutyrate (P4HB) is used for the coating of the scaffold. The rectangular

scaffold strips are attached to RVS rings using polyurethane (PU). Two sizes of rings may be used but

the use of small rings is advised. This protocol is based on the original protocol (Tissue Engineering of

tissue strips in 6-wells plate) written by M. Stekelenburg, which is based on the tissue engineering

protocol, written by A.Mol.

Precautions

Perform operations and materials handling according to safe microbiological procedures.

Several hazardous chemicals are being used in this protocol. Use the appropriate personal protection

(gloves, fume-hood, etc) and read the concomitant MSDS’s.

Requirements

1. PGA

2. P4HB solution dissolved in THF

3. Polyurethane (PU) dissolved in THF

4. RVS rings (OD 25 mm, ID 16 mm)

5. 6 wells-plate

6. 70% ethanol

7. PBS(sterile)

8. TE medium

33

Preparation of reagents

The medium used for tissue engineering differs from the medium used for cell culture

TE medium sheep

DMEM Advanced(500 ml)

1% Lamb serum (5 ml)

1% Glutamax (5 ml)

1% Penicillin/Streptomycin(5 ml)

L-ascorbic acid 2-phosphate, Sigma(130 mg)

N.B: L-ascorbic acid 2-phosphate has to be solved in DMEM Advanced and sterile filtered: therefore,

add 130 mg of L-ascorbic acid 2-phosphate to a 50 ml tube and add 10 ml of DMEM Advanced, put

this for several minutes in the warm water bath (in warm medium L-ascorbic acid 2-phosphate

dissolves quicker). After about 10 minutes it will be dissolved, sterile filter it into the rest of DMEM.

Then add the rest of the components to the medium.

PH4B solution

Dissolve 1.75 gram of P4HB in 100 ml THF to obtain a 1.75 w/v solution

Procedures

Day1: COATING AND GLUEING OF THE PGA

Cut the scaffolds out of the PGA with the desired measured (Remember to cut the strips parallel

to the fibres in the PGA sheet).

Coat the PGA scaffolds with P4HB by dipping the scaffold into the solution and discard the

P4HB solution afterwards (in the fume -hood).

Leave the scaffold to dry on a glass plate in the safety cabinet until P4HB is white.

34

The scaffolds can then be mounted onto the RVS rings. Put some PU solution in a glass beaker.

Put a droplet of the viscous PU solution on either sides of the PGA scaffold edges or vertices

that you intend to glue. Make sure that the PU runs from the scaffolds ends in one streak over

the side and bottom of the RVS ring. This will ensure attachment of the scaffold to the ring.

This procedure is necessary as the PU does not adhere to RVS.

Let the scaffolds dry in the safety cabinet for approximately 24 hours.

Day2: DRYING IN THE VACUUM OVEN

Let the scaffolds further dry in the vacuum stove overnight.

Day 3: STERILIZING AND PREPARING THE SCAFFOL FOR SEEDING

Fill the wells with 70% ethanol and leave it for 30 minutes(work in the LAF cabinet)

Remove the alcohol and rinse thoroughly with PBS.

Fill the wells with TE medium and place in an incubator overnight.

Day 4: SEEDING OF THE SCAFFOLD

Prepare the thrombin solution: weight about 1-1.5 mg of thrombin and transfer to a centrifuge

tube.

Add an amount of TE medium to obtain a concentration of 10 IU/ml

Shake the solution and put on ice for about 10 minutes

Sterile filter the solution with the syringe and the sterile filter

Store the sterile solution on ice until use

Prepare the fibrinogen solution : weight about 50 mg of fibrinogen and transfer to a centrifuge

tube

Add an amount of TE medium to obtain a concentration of 10 mg actual protein per lm medium

35

Mix (gently shake) the solution for a couple of minutes until (almost all) the fibrinogen is

dissolved and sterile filter this.

Store the sterile solution on ice until use.

Before you start , calculate the amount of cells you need .The seeding density should be

Rinse the desired amount of flasks with the cells twice with PBS

Add 2.5 ml of trypsin and distribute evenly over the bottom.

Place the flask for 7 minutes in the incubator

Check whether all the cells are rounded up and de-attached. If not place the flask back in the

incubator for a few more minutes

Add 5ml of medium to the flasks and mix the medium with the cells and trypsin

Transfer the medium with cells to a centrifuge tube

Rinse the flask with PBS and add to the tube

Centrifuge 7 minutes at 1000 rpm

Discard the supernatant and resuspend the cells in 5 ml of medium for counting.

Count the cells

Mix gently the cells and the medium

Divide the cells in vials according to the number of cells needed by each scaffold.

Remove the medium from the well and aspirate the remaining medium from the strip. The

scaffold must be nearly dry before seeding.

Decide the amount of thrombin and fibrinogen you want to use to seed the scaffold. Add the

desired amount of thrombin to the cells Take the fibrinogen in the pipette tip and put the pipe to

the final volume estimated for each scaffold. When fibrinogen is added to the thrombin, the

solution starts to gel very quickly. Therefore you need to adjust the volume of the pipette before

you start mixing. The total volume of fibrinogen and thrombin is not enough because the cells

have a volume as well. Not to loose the cells, take a larger volume.

36

Mix the cell/thrombin solution gently with the fibrinogen for several times and the immediately

pipette the mixture at several spots of the scaffold.

After seeding all the scaffolds, place them with the cells in the incubator for about 30 minutes

to let the fibrin gel further polymerize

Carefully add medium to the constructs and place them back into the incubator on a shaking

table to allow mixing of the medium.

37

APPENDIX 3: AREA MEASURAMENT BY MEANS OF IMAGEJ

SOFTWARE

The method that will be described in this section was used to measure the surface area of the

constrained constructs at the end of culturing (t = 4 weeks) by means of ImageJ software. In order to

improve the representation in the display and enhance the contrast the colours can be redistributed over

the full colour range. Further, the equalization of an image allows a much better visual discrimination

of image details and features. ImageJ provides such functionalities via the Enhance Contrast-function

from the Process-menu (equalization option selected, 0.5% saturation of pixel) [8]. This was performed

on the photographs. Therefore, the selection of the region of interest for all further image analysis/

measurement was facilitated (Fig.7a).

Subsequently, the inner perimeter of the ring was selected via the elliptical or brush selection and the

outside was cleared (Edit-menu / Clear outside-function) (Fig.7b). The image was cropped to allow a

better visualization (Fig.7c) and then inverted (Edit-menu/Invert-function). (Fig7d).

Contrast enhanced picture (a) Inner diameter selected and

outside cleared (b)

Cropped image (c) Inverted image(d)

Fig.7. First steps of the image processing

The image was then transformed in 8-bit level via the Type-function from the Image-menu(Fig. 8a)

This resulted in an image showing white colour (corresponding to 255) out of the region of interest

(ROI), while the ROI was represented by high levels of grey (close to 0). If some regions out of the

ROI were still incorporated in the image because of the non-uniform luminance texture of the image,

38

they were discarded carefully selecting the areas with the freehand selection tool after zooming on the

image (Fig.7b).

In order to measure the area a threshold operation was needed. Selecting the Red entry in the pop up

menu inside the Threshold dialogue from Image/Adjust menu it was possible to assign the ROI to a red

mask. As a result of that, the ROI was the only masked area (Fig.7c).

Once the ROI was selected with the Wand-tool from the ImageJ toolbar, the contour of the selection

was displayed in the image with a yellow line.

Before calculating the area the scale must be set. For this purpose the Set Scale-function from the

Analyze menu of ImageJ had to be selected and the reference scale in the picture (1cm) was used to

draw a correspondent straight line with the tool in the toolbar.

After entry of the Known Distance, which was “1”, and the Unit of Length, which was “mm”, the

Analyze Particles–function was chosen from the Analyze menu. Previous window settings were not

changed and the Outline entry in the pop up menu was selected in order to double check which areas

were measured (Fig.7d). The results displayed the area in mm2. Finally, the results were represented as

the relative compaction of the TE construct by comparing the calculated surface area relative at week 4

to the seeding area at t = 0 .

8 bit image (a) Regions out of ROI have been

cleared (b)

Red masked ROI (c) Outline of the surface area (d)

Fig.8. Final steps of the image processing

39

APPENDIX 4: SECTIONING PARAFFIN BLOCKS CONTAINING

SCAFFOLD MATERIAL

Sectioning paraffin blocks containing scaffold material, such as in tissue engineered tissues is quite

difficult due to differences in stiffness between the paraffin, tissue, and the scaffold material.

Successful sectioning depends on many factors:

- Sectioning technique: this protocol describes the optimal settings for the microtome to be used

when scaffold material is present. With this protocol and a lot of patience you should be able to

obtain some nice sections.

- Type and amount of scaffold: blocks with tissues based on PGA and P4HB can be sectioned.

When the coating percentage increases, sectioning will be more difficult, but still possible. PCL

is too stiff to be sectioned using paraffin and this still can be better embedded in plastic. Do not

section pure scaffolds as these will not adhere to the slides. You need tissue to prevent the

sections from being lost during staining.

- Amount of tissue: blocks with scaffolds with good tissue formation are always easier to cut

compared to scaffolds with less tissue. Keep that in mind when sectioning static controls!

- Temperature of the block and sectioning speed: the block needs to be very cold to diminish the

differences in stiffness between the various components. Therefore, always store the blocks at -

20C and cool them frequently during sectioning. Furthermore, the slower you perform the

sectioning, the better the sections.

Preparation:

- Turn on the cold plate, the microtome and the hot plate.

- Take the blocks out of the -20C one for one for sectioning and not all at the same time. They have

to be really cold when you start. Place the block on the cold plate.

- Set the thickness for trimming at 30 µm and for sectioning at 10 µm.

- Place a new blade in the microtome. Always start sectioning at the most left side of the blade. Make

sure the angle of the blade is set at 10.

Trimming the block:

- Make sure the handle is locked and place the block in the microtome holder. Try to use the

orientation in which the construct will be divided equally over the blade.

- Make sure the block is positioned properly with respect to the blade and adjust the holder if

necessary. Make sure the blade holder is fixed before starting sectioning!

To give you an idea about the time required for sectioning:

If you would like to have 6 slides with 2 good quality sections per slide from one paraffin

block it will take between 30 and 60 minutes, depending on the amount of times you cool

the block in between.

40

- Set the sectioning window

- Set the microtome for trimming and use continuous mode. Start trimming at speed 2-5. Let the

microtome trim until you have reached the block. Then slow down the speed to about 1 and check

the sections if you are already in your sample (use the pins to prevent the sample from curling). If

this is the case stop trimming and place the block back on the cold plate.

Sectioning the block:

- Prepare some poly-L-lysine coated slides by writing down the sample name (use a crayon as other

pens will be washed off in stainings) and putting on water.

- Place the block back into the microtome holder in the same position as used for trimming.

- Set the microtome for sectioning and use continuous mode. Start sectioning at speed 1-2 until you

are in the sample again (this normally takes 3 to 4 sections). When you are in the sample again stop

sectioning.

- Now use single mode and set the speed at near to zero (really near to zero). Start sectioning and use

the pins to prevent the section from curling. When finished, transfer the section to the glass slide on

top of the water. You can in this way cut some sections (varying from 2-6) which are useful. You

will notice it immediately when the block is not cold enough anymore and you will need to place it

on the cold plate again and repeat the sectioning part for more sections (see comments in textbox).

- Transfer the slides containing the water and the sections to the hot plate and see the sections being

stretched. As soon as stretching is complete remove the water using a tissue. Do not touch the

coupes.

- Leave the slides drying in the special slide holder overnight.

41

APPENDIX 5: IMMUNO-FLUORESCENT STAINING VIMENTIN, DESMIN

AND α-SMA

Precautions

- Perform operations and materials handling according to safe microbiological procedures.

- Several hazardous chemicals are being used in this protocol. Use the appropriate personal

protection (gloves, fume-hood, etc) and read the concomitant MSDS’s.

(Hazardous) Chemicals

- Triton-X-100 is harmful; R22,41/S26,36,39

- Ethanol is highly flammable and harmful; R11,20,21,22,36,37,38,40/ S7,16,24,25,36,37,39,45

- Acetone is highly flammable; R11,36,41,66,67/S9,16,26

- Hydrochloric acid (HCl) is toxic, corrosive and dangerous to the environment;

R23,24,25,34,36,37,38/S26,36,37,39,45

- DAPI is mutagenic, reprotoxic, eye irritant; R36/S26

- Paraclear (Xylene) is harmful and highly flammable; R10,20,22,36,37,38

Requirements

- PBS

- 1% BSA in PBS

- TwPBS (0.1%Tween 20 in PBS)

- 1% Triton-X-100 in PBS

- Antigen retrieval solution

- Your primary and secondary antibodies

- Monoclonal Mouse anti-αSMA (IgG2a,DAKO, M0851, 1:500)

- Rhodamin-conjugated phalloidin (1:200, P1951, Sigma)

- Anti-mouse goat IgG2a alexa 488 (1:300).

- DAPI, to stain the nuclei as well

- Mowiol embedding agent (ready made)

- Cover glasses

Procedures

1. Deparaffinize slides (25min):

42

1. 2 x 5 min Paraclear (Xylene)

2. 2 x 2 min Ethanol 100%

3. 2 min Ethanol 96%




2. 5 min PBS

3. Incubate 20 min. with epitope retrieval (Tris-EDTA Buffer cook it till 100˚C than incubate 20min.

at RT)

4. Incubate 30 min. with 1% BSA in PBS

5. Wash 5 minutes in 0.1% Tween 20 PBS agitate gently

6. Wash 5 minutes in 1% Triton X-100 agitate gently

7. Wash 5 minutes in PBS agitate gently

8. Dry the glasses carefully and draw limiting circles with the wax-pen.

9. Incubate 2 hours at room temperature with primary antibody solution in 1% BSA in PBS

- 1:500 aSMA …… l aSMA + …… l 1% BSA in PBS

10. Wash 5 minutes in 0.1% Tween-20 PBS agitate gently

11. Wash 3 x 5 minutes in PBS agitate gently

12. Incubate 30 minutes with secondary antibody with Rhodamin-conjugated phalloidin

- 1:200 Rhodamin-conjugated phalloidin

- 1:300 alexa 488 IgG2a

- …… l phalloidin + …… l Alexa 488 + ……. l PBS

13. Wash 5 minutes in 0.1% Tween 20 PBS agitate gently

14. Wash 3 x 5 minutes in PBS agitate gently

15. Incubate 5 minutes with DAPI in PBS (1:1000)

16. Wash 5 minutes in 0.1% Tween-20 PBS

17. Wash 3 x 5 minutes in PBS

18. Embed in Mowiol

19. Store slices in the dark at 4°C.

43

APPENDIX 6: HEMATOXYLIN & EOSIN (H&E) STAINING

Introduction

In routine histology the hematoxylin and eosin stain (better known as the ‘H&E’ stain) gives a visible

look at the nucleus of the cells and their present state of activity.

The H&E stain uses two separate dyes: hematoxylin is a dark purplish dye that will stain the

chromatin (nuclear material) within the nucleus, leaving it a deep purplish-blue color and eosin is an

orange-pink to red dye that stains the cytoplasmic material including connective tissue and collagen,

and leaves an orange-pink counterstain. This counterstain acts as a sharp contrast to the purplish-blue

nuclear stain of the nucleus, and helps identify other entities in the tissues such as cell membrane

(border), red blood cells, and fluid.

Performing the H&E stain: After the tissue has been paraffin embedded, sectioned, placed on a slide

and the slide dried in an oven, the slide is taken through brief changes of Xylene, alcohol and water to

‘hydrate’ the tissue. This process is called ‘running the slides down to water’ and must be done to

give the cells an affinity for the dyes. The slides are then stained with the nuclear dye (hematoxylin)

and rinsed, then stained in the counterstain (eosin). They are then rinsed, run in the reverse manner

from the run down (taken back through water, alcohol, and Xylene), then coverslipped.

Precautions

- Perform operations and materials handling according to safe microbiological procedures.

- Several hazardous chemicals are being used in this protocol. Use the appropriate personal

protection (gloves, fume-hood, etc) and read the concomitant MSDS’s.

(Hazardous) Chemicals

- Xylol/Xylene is harmful and highly flammable; R10,20,22,36,37,38

- Hematoxylin is harmful; R22,36,37,38

- Eosin is harmful and irritant; R36,37,38/S26,36

- Ethanol is highly flammable and harmful; R11,20,21,22,36,37,38,40/S7,16,24,25,36,37,39,45

- Acetic Acid is harmful and corrosive; R10,35/S23,26,45

- Entallan contains Toluene which is highly flammable, toxic and teratogenic;

R11,23,24,25/S16,25,29,33

- You are working with (very) toxic, harmful and flammable solutions; so wear gloves and

labcoat and work as much as possible in the fumehood!

44

Requirements

* Reagents and solutions:

- Xylol (Xylene, see chemical list)

- 100% Ethanol (EtOH, see chemical list)

- Diluted EtOH, 96% and 70%

- Hematoxylin solution (Mayers’, Sigma, cat# MHS16), can be re-used several times; don’t throw

back in original solution!

- Eosine Y solution (aqueous, Sigma, cat# HT110-2-16), before use slowly add 1 ml glacial acetic

acid per 200 ml Eosine Y solution to acidify the solution. Can be re-used several times; don’t

throw back in original solution!

- Tap water, MilliQ water

- Entallan (contains Toluene, see chemical list)

* Equipment and disposables:

- Dewaxing/ hydration/ dehydration series

- cover slips

Procedures

Before starting procedure fill in the administration list: date, name, which sequence, staining, number

of slides! After about 1000 slides or when staining become faint the hematoxylin and eosin should be

changed.

Check the “Dewaxing/ hydration/ dehydration series” if there are still enough solutions in the buckets

(EtOH and Xylol evaporate during time) and whether the solutions are clean (e.g. redness in the

dehydrating range caused by rests of eosin). If not: refill and/or change solutions.

1) Dewax and rehydrate the sections:

a) 2x 5 minutes Xylol

b) 3x 2 minutes 100% EtOH

c) 1x 2 minutes 96% EtOH

d) 1x 2 minutes 70% EtOH

e) 1x 2 minutes MilliQ water

2) Stain 10 minutes in Mayer’s hematoxylin solution

3) Wash in slow running tap water for 5 minutes

4) Stain in acidified aqueous eosin Y solution for 30 sec-3 minutes (depends on tissue and thickness of

the section, 30 sec 20 dips)

5) Wash in slow running tap water for 1 minute

6) Dehydrate, mount and cover the sections:

a) 10 dips in 70% EtOH

b) 10 dips in 96% EtOH

c) 3x 10 dips in 100% EtOH

45

d) 2x 3 minutes Xylol

e) Entallan and coverslip

f) Let dry O/N in fumehood

Expected results

Nuclei: Purple, Purple/ Black

Cytoplasm, extracellular matrix: different shades of pink till orange

References

- Sigma-Aldrich, H&H Informational Primer, “Hematoxylin & Eosin” (The Routine

Stain) by H. Skip Brown, BA, HT (ASCP)

- Administratieruimte: Book “Theory and practice of histological techniques”

- Internet: www.Histosearch.com/histonet

46

APPENDIX 7: RESULTS – COMPACTION DURING CULTURE

Construct Area seeded

(cm2)

Surface area at week 4

(cm2)

Compaction % Average

compaction

SD

Strip1 0.9 0.534 40

37

(+)

5.0

Strip2 0.9 0.510 43

Strip3 0.9 0.600 33

Strip4 0.9 0.605 33

Rectangle1 1.98 1.17 41

41

(++)(excluding R2)

0.5

Rectangle2 1.98 0.756 62

Rectangle3 1.98 1.15 42

Rectangle4 1.98 1.144 42

Triangle1 1.8 1.8 0

6.75

7.5

Triangle2 1.8 1.764 2

Triangle3 1.8 1.607 11

Triangle4 1.8 1.501 16

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