Bone Decalcification, Conventional Decalcification, Rapid ... · Bone Decalcification, Conventional...

Chapter 1: Introduction 1

Keywords

Bone Decalcification, Conventional Decalcification, Rapid Decalcification,

Histology, Decalcification Endpoint

2 Chapter 1: Introduction

Abstract

Bone decalcification is a time consuming process for conducting routine histological

analysis of specimens in research and pathology. Numerous approaches have been

trialled to increase the rate of decalcification time without compromising

histological quality. In this study a laboratory microwave was used to increase the

temperature and perfusion of decalcification solutions in mice, rat and sheep bone.

Bone was decalcified with 10% Ethylenediaminetetraacetic acid, 10% formic acid,

10% Hydrochloric acid and 5% nitric acid, both conventionally (4°C & 25°C) and in a

KOS-milestone microwave (37°C). Decalcification was monitored by micro-

computed tomography analysis to determine the endpoint of decalcification of

bone. Specimens in a microwave decalcified on average 50 – 80% faster than

conventional methods in all species when EDTA was used. Furthermore, in formic,

hydrochloric and nitric acids decalcification in the microwave was 40 – 50% quicker

than the respective conventional conditions. Histology results from the EDTA and

formic acid groups showed preservation of antigens and tissue morphology in all

species, in contrast to the hydrochloric and nitric acid groups were the evidence of

tissue damage and a loss of antigenicity were observed. In conclusion, specimens

decalcified in the microwave using EDTA and formic acid, maintained tissue

morphology and antigenicity which was similar to the conventional conditions. This

study shows that a laboratory microwave is a valuable appliance that could be used

to decrease the time of decalcification of bone tissue while maintaining histological

accuracy.

https://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid


Weight Loss .................................................................................................. 53

4.3 Discussion ..................................................................................................... 57

Chapter 5: Histological evaluation of various decalcification conditions ..... 61

5.1 Histological staining of bone ......................................................................... 61 5.1.1 Mice staining ........................................................................................ 61 5.1.2 Rat staining .......................................................................................... 67 5.1.3 Sheep staining ...................................................................................... 74

5.2 Histological Discussion ................................................................................. 80 5.2.1 Tissue morphology ............................................................................... 80 5.2.2 Immunohistochemistry ......................................................................... 81

Chapter 6: Conclusion .................................................................................... 84

6.1 General Conclusion ....................................................................................... 84

6.2 Study Limitations & Future Work ................................................................. 85

Bibliography ......................................................................................................... 87

Chapter 7: Appendix A .................................................................................. 93


List of Figures

Figure 2-1: Mechanism of EDTA chelation of calcium, calcium is chelated by the

oxygen molecules……………………………………………………………………19

Figure 3-1: Sectioning and preparing specimens for decalcification………………27

Figure 4-1: Pilot study results demonstrating the total amount of time required to

decalcify bone ………………………………………………………………………38

Figure 4-2: Representative 3D µCT images of the pilot study……………………..39

Figure 4-3A: Representative 3D µCT images of mice tibia decalcified in EDTA and

formic acid at indicated time points…………………………………………………45

Figure 4-3B: Representative 3D µCT images of mice tibia decalcified in HCl and

nitric acid at indicated time points…………………………………………………..46

Figure 4-4A: Representative 3D µCT images of rat tibia decalcified in EDTA at

indicated time points………………………………………………………………...47

Figure 4-4B: Representative 3D µCT images of rat tibia decalcified in formic acid at


Figure 4-4C: Representative 3D µCT images of rat tibia decalcified in HCl at


Figure 4-4D: Representative 3D µCT images of rat tibia decalcified in nitric acid at


Figure 4-5A: Representative 3D µCT images of sheep tibias decalcified in EDTA

and formic acid at indicated time points…………………………………………….51

Figure 4-5B: Representative 3D µCT images of sheep tibias decalcified in HCl and

nitric acid at indicated time points…………………………………………………..52

Figure 4-6: Graphical representation of the change in mice tibia weight, pH of

decalcification solution and rate of bone volume loss over time……………………54

Figure 4-7: Graphical representation of the change in rat tibia weight, pH of


Figure 4-8: Graphical representation of the change in sheep tibia weight, pH of


Figure 5-1: Hematoxylin & Eosin staining in mice tibia…………………………...62

Figure 5-2: Safranin O & Fast green staining in mice tibia for cartilage and bone...63

Figure 5-3: Collagen-1 immunohistochemistry in mice tibia for bone matrix……..65


Figure 5-4: vWF immunohistochemistry in mice tibia for vasculature and serum

proteins………………………………………………………………………………66

Figure 5-5: Hematoxylin & Eosin staining in rat tibia……………………………..68

Figure 5-6: Safranin O & Fast green staining in rat tibia for cartilage and bone…..70

Figure 5-7: Collagen-1 immunohistochemistry in rat tibia for bone matrix………..71

Figure 5-8: vWF immunohistochemistry in rat tibia for vasculature and serum

proteins………………………………………………………………………………73

Figure 5-9: Hematoxylin & Eosin staining in sheep tibia………………………….76

Figure 5-10: Collagen-1 immunohistochemistry in sheep tibia for bone matrix…...77

Figure 5-11: vWF immunohistochemistry in rat tibia for vasculature and serum

proteins………………………………………………………………………………79


List of Tables

Table 2-4: Summary of rapid decalcification attempts and tissue morphology

preservation…………………………………………………………………...…20-21

Table 2-5: Summary table of microwave decalcification experiments……...….23-24

Table 3-1: Pilot study µCT scanning schedule for decalcifying specimens……28-29

Table 3-2: Decalcification specimen groups, species and conditions………............29

Table 3-3: Micro CT time points for assessing decalcification progress……….30-31

Table 3-4: Protocols for tissue processing specimens for paraffin embedding……32

Table 3-5: Protocols for H&E, Safranin O and IHC staining…………………..34-36


List of Abbreviations

°C Degrees Celsius

µCT Micro Computed Topography

BV Bone Volume

Col-1 Collagen Type 1

DAB 3, 3 – diaminobenzidine

EDTA Ethylenediaminetetraacetic Acid

HCl Hydrochloric Acid

H&E Hematoxylin and Eosin

HRP HorseRadish-Peroxidase

IHC Immunohistochemistry

Mins Minutes

OCT Optimal Cutting Temperature

Rpm Revolutions per Minute

Saf-O Safranin O

Secs Seconds

vWF von Willebrand Factor


QUT Verified Signature


Acknowledgements

“Alone we can do so little, together we can do so much” – Helen Keller

This research project could not have been possible without the support of many

who I am grateful to have encountered along my journey.

I would firstly like to thank my supervisory team

Dr Vaida Glatt – Primary supervisor

Associate Professor Mia Woodruff – Secondary supervisor

Professor Mark Pearcy – Tertiary supervisor

I appreciate all the support that my supervisory team provided me with along the

way. I would like to especially acknowledge Dr Vaida Glatt for her tutelage in

improving my scientific literacy and helping me push myself to levels of

achievement I did not believe was possible. This project would not have been

possible without her belief in my abilities and her tenacity to encourage me to

achieve more.

I would like to thank my family, my mother and sister in particular for the emotional

and financial support they provided me throughout the project when things got

tough. My dog Coco for always being cheerful when skies were grey, if I could learn

to embrace her optimism and enthusiasm for every second of life I would be a

better person for it.

I would like to thank my IHBI friends for their lunchtime conversations about

science and non-related issues. Dr Keith Blackwood, Dr Caroline Grant, Emily Bailey,

Pelin Tufecki, Nicole Loechel, Stephanie Fountain, Michal Bartnikowski, Sha Pather,

Nick Quirk, Jess Thompson, Daniel Singh, Cosmo Hutmacher, Chandan Mager, Emily

Bryan, Stephanie Fay and Flavia Savi all offered me friendship and support I am

grateful for.

Finally I would like to achknowledge the Faculty of Science and Engineering at QUT

for providing me with the faculty scholarship and financial assistance required to

complete this project. Furthermore, I would like to thank QUT for providing me with

this opportunity to further my education, career and provide me with this chance to

further discover and explore myself as a person and as a scientist.


Chapter 1: Introduction

“I do not think there is any thrill that can go through the human heart like that felt by the inventor as he sees some creation of the brain unfolding to success... such emotions make a man forget food, sleep, friends, love, everything..” Nikola Tesla

1.1 OVERVIEW

Histological analysis is the microscopic analysis of two-dimensional tissue sections

obtained by a process of fixation, embedding, sectioning and staining. It is one of

the most commonly used outcome measures to evaluate tissue morphology of in

vivo animal models in orthopaedic research as well as for clinical diagnostic

evaluations. There are two methods that are routinely used to process bone

specimens for histological analysis, it is either embedding in paraffin wax or

polymeric resins. Paraffin sections are usually 5 µm in thickness, which is ideal not

only for examining of individual cells, but also for immunohistochemistry (IHC) in

the detection of target proteins. However, paraffin also has its limitations allowing

for only smaller sized specimens (25mm x 30mm x 5mm) to be used in the

embedding process, and does not provide a sufficiently hard matrix for cutting the

thinner slices needed for electron microscopy (typically 80-100 nm thick), which

require embedding in, epoxy or acrylic resins.

Resin embedding can generate an entire overview of the specimen and can be used

to analyse calcified tissue. However, the polymerisation process produces a

significant amount of heat, which is potentially negative to the antigenicity of bone

proteins, making IHC analysis difficult. For this reason, paraffin embedding is a more

commonly used method, being cheaper and less time consuming than resin

embedding. Paraffin also has an added advantage over resin as it has a larger range

of staining options. Moreover, because of the dense nature of bone tissue,

sectioning paraffin embedded bone specimens is not possible due to the lack of

infiltration of paraffin wax into the specimen, which requires the removal of calcium

ions from hydroxyapatite in bone. This time consuming process is known as

decalcification, and depending upon the method used to reach complete

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decalcification, the amount of time and quality of outcome varies considerably.

This project aims to address this issue by investigating multiple methods to increase

the rate of decalcification of bone explants from in vivo studies while at the same

time preserving the tissue and antigen integrity post decalcification for IHC analysis.

1.2 AIMS & SIGNIFICANCE

The novelty of this project was to determine a faster method to decalcify bone

using a microwave that is specifically intended for this purpose. This is extremely

important, as it greatly reduces the time required to complete histological analysis.

The methods evaluated during this project could potentially be adopted by research

and pathology labs that routinely decalcify bone.

Hypothesis

Bone that is rapidly decalcified in a microwave in EDTA will retain

antigenicity and tissue morphology.

Aims

1. To monitor the rate of decalcification using a micro computed

tomography (µCT) system

2. To investigate if rapid decalcification has a detrimental effect on the

tissue morphology and antigenicity

1.3 THESIS OUTLINE

Chapter 2: Literature Review

This chapter includes a comprehensive literature review on bone decalcification

methods and their relativity to current histology techniques. Briefly, this chapter

explores the mechanisms behind bone decalcification and the development of

methods to decrease the time required to achieve endpoint. Microwave methods

become a focal point in the review as this method is the most promising method to

achieve aim 2.

Chapter 3: Methodology

This chapter outlines the research design and experimental methods used in this

study to test the hypothesis and aims. The collection of bone tissue, processing,


decalcification, µCT analysis and histology is all outlined in detail and chronological

order.

Chapter 4: Results of decalcification times by Micro CT analysis

This chapter focuses on measuring mineral bone loss throughout decalcification

using a µCT system. An exact endpoint time for each decalcification treatment for

each species of bone was determined with a reproducible degree of accuracy.

Chapter 5: Evaluation of various decalcification conditions on histological staining

This chapter outlines the histological findings from the study. Staining results were

assessed by the degree of tissue preservation observed when compared to the

conventional control method.

Chapter 6: Conclusion

This chapter summarises the results and forms conclusions about the results from

the study in relation to the hypothesis and aims. This chapter also comments on the

study limitations and proposed future work for this area of research.

Chapter 2: Literature Review 15

Chapter 2: Literature Review

2.1 PREFACE

This chapter reviews the current techniques used in the decalcification process of

bone for histological analysis. A brief overview of histological techniques is

presented, as well as an outline of the problems encountered with current

decalcification methods. Rapid techniques for decalcification are introduced and

reviewed with a focus on microwave assisted decalcification, which is investigated

in this thesis.

2.2 BONE HISTOLOGY OVERVIEW

Histology is defined as the study of the microscopic structure of biological tissues.

The histology process involves the collection, fixation, sectioning and staining of

tissues for microscopic analysis. Scientists and pathologists routinely perform

histological study of tissue to understand microbiology and microstructures [1]–[3].

It is performed on two different tissue types, soft tissue and hard tissue. Soft tissue

is defined as a non-mineralised tissue that can be embedded with paraffin wax and

sectioned into thin slices, e.g., liver, kidney, brain, thyroid, tendon, muscle, etc.

Hard tissue is defined as tissue that is mineralised and is too dense for infiltration

with paraffin wax, therefore, this tissue requires demineralisation prior to paraffin

wax embedding so the specimen can be sectioned - examples of hard tissues are

bone, tooth enamel and atherosclerotic plaques [4].

Fixation

Fixation is defined as the process of using a preservative solution to prevent tissue

decomposition for further analysis; improper fixation of a specimen will result in

tissue artefacts or an irreversible loss of cellular quality [5]. The two most

commonly used solutions to fix tissues are either 10% neutral buffered formalin

(NBF) or 4% para-formaldehyde (PFA) [6]. The time of fixation depends on the

16 Chapter 2: Literature Review

density of tissues and the sample size. For example, denser tissues like bone

require longer fixation times due to the lack o f solution penetration into the

tissue. Whereas softer tissue, like kidney, fix quicker due to the easier

penetration of fixative into the deeper layers of tissue.

Decalcification

Tissue decalcification is the process of removing calcium ions from the

extracellular matrix to allow for the tissue to be histologically investigated.

Tissue is submerged into a decalcification solution that causes calcium ions

to dissolve into solution. The process of decalcification is covered in more

detail in section 2.3 of this thesis.

Tissue Processing

Processing is the final step in tissue preparation prior to paraffin embedding,

and is carried out by immersing tissues in graded ethanol solutions of

increasing concentrations ranging from 50 -100% to dehydrate the

specimen. Xylene is then used to clear the tissue of residual ethanol as

paraffin wax and ethanol are immiscible. Specimens are then immersed in

molten paraffin wax to ensure complete penetration throughout the

specimen so that embedding is uniform.

Embedding

A hard tissue like bone can be embedded using one of three methods: a

Methyl- methacrylate (MMA) resin, flash freezing of a fresh tissue in OCT

(optimal cutting temperature) compound for cryosectioning or embedding a

decalcified tissue into paraffin wax [7]. Resin embedding is less frequently

used by research and pathology labs as it is expensive, requires special

equipment and training, and may potentially degenerate target proteins or

enzyme activity due to the exothermic nature of the embedding process

[8]. If cryosectioning is used to process bone then decalcification of tissue is

not required, however, the quality of the sections produced often have poor

results of tissue morphology [9], [10]. An advantage of cryosectioning is it

can be used to achieve rapid results; however, a drawback of this method


is the expense, difficulty to achieve good outcomes and requires

specialized training and extensive experience. Embedding specimens in

paraffin wax is the most common method used to prepare specimens for

microtome sectioning. Paraffin embedding is a technique that does not

require extensive training, is cost effective and specimens can be orientated

in various planes. This method allows for multiple sections to be cut with

ease and the tissue morphology is well preserved.

2.3 BONE DECALCIFICATION METHODS FOR HISTOLOGY

There are two phases of bone, the organic and inorganic phase [11]. The organic

phase of bone consists of blood vessels and bone specific proteins, which lets bone

interact with its surrounding environment. The inorganic phase of bone is the

mineralised part of bone which is made up of hydroxyapatite [12]. Calcium and

phosphate ions are part of the hydroxyapatite molecule that forms the crystalline

structure of bone. Calcium is one of the key components in giving bone its

mechanical properties and crystalline matrix [12]. To perform paraffin histology on

bone it is vital to remove the calcium from the hydroxyapatite matrix so that the

bone can be sectioned by microtomy [13]. Demineralisation of bone occurs by using

either a chelating agent, mineral acid or an organic acid to remove the calcium

component from the tissue [14], [15].

In order to decalcify bone, an acid free solution was originally proposed in 1923,

however, this method was deemed impractical as it required special apparatus,

training and a laborious process [16]. Subsequently, a method using trichloroacetic

acid was trialled in 1923 [16], however, this method was quickly abandoned as it

also failed to decalcify bone. Since then, various acids such as nitric, formic,

hydrochloric (HCl) and ethylenediaminetetraacetic acid (EDTA) have been

successfully used to decalcify bone. [13], [16], [17]. Acidic solutions were favoured

because they were able to decalcify bone without any specific apparatus, although

in the early stages of research there was a lack of agreement over an optimal

method. For this reason, many research laboratories developed ‘in house’ protocols


from the studies on acids to meet their requirements, and as a result limited

publications were available on this research subject. As the need for diagnosing

bone disorders increased along with the use of animal models in pre-clinical testing,

the standardization for decalcification of bone became a more focused research

topic. Consequently, more research was conducted on bone decalcification and a

consensus within the research community was reached over reliable decalcification

solution and conditions [13], [17]–[19].

The ideal decalcification solution had to rapidly decalcify the bone while not

interfering with the gross tissue morphology [20], [21]. These criteria were intended

to ensure that any histological staining performed on processed specimens was not

compromised by the decalcification solution affecting the tissue integrity. This was

particularly important for immunohistochemistry analysis where chromogen tagged

antibodies are used to stain proteins of interest, and where using more acidic

decalcification solutions have been shown to cause damage to tissue [18]–[22]. For

this reason, the importance of preserving tissue quality for histological analysis has

resulted in the vast majority of solutions based on 1 of the 4 most commonly used

acids in decreasing order of strength: nitric, hydrochloric, formic and EDTA [15]. The

strength of each decalcification solution is defined by the mechanism in which

calcium is removed from bone.

Mineral acids (nitric and hydrochloric acid), and organic acid (formic acid) use

hydrogen ions to break the bond between calcium ions and hydroxyapatite. This

then causes the dissolution of hydroxyapatite into calcium ions that form salt and

allows inorganic phosphate to become soluble, which is shown in the following

equation.

Ca (10) (PO4)6(OH) 2(s) + 8H+(aq) 10Ca2+

(aq) + 6HPO42-

(aq) +2H2O(l)

On the contrary, a compound that works via the chelation approach decalcifies

bone on a different principle. The chelation of calcium from bone is achieved using

EDTA, and is demonstrated in Figure 1 below. Chelation is the compound’s ability to


bind calcium from bone without having to use hydrogen ions and it works by

binding calcium from hydroxyapatite due to the sharing of electrons [18], [27].

Calcium has a higher affinity for EDTA molecules than it does for hydroxyapatite,

therefore, it is able to chelate calcium out of bone. Contrary to acidic

decalcification, it is also dependent on pH, where the more basic the pH, the more

rapid the chelation of calcium from bone [26].

Figure 2-1: Mechanism of EDTA chelation of calcium, calcium is chelated by the

oxygen molecules (Adapted from Stanley Smith papers series 15/5/50 Organic

chemistry chapter 12 1999).

To balance the need for reduced rate of decalcification and to obtain qualitative

histology data, decalcification solutions started to incorporate acids like nitric,

hydrochloric, EDTA and formic [15], [26]. The paradigm of decalcification is now

focused on the removal of calcium with the highest possible preservation of tissue

morphology and antigenicity of proteins after tissue processing for histological

analysis. Therefore, researchers have been using the chelation approach to remove

calcium from bone, which requires EDTA solution [13], [18], [22], [24], [28]–[34]. To

determine whether a bone is fully decalcified includes methods such as x-rays,

precipitate formation (using calcium oxalate), weighing of bone, using a pin prick as

well as bending by hand to determine the tissue resistance [35]. While all these

methods have their own limitations, nevertheless they are helpful tools to

determine the end of the decalcification process.

+


While advances in histological techniques have led to a form of standardisation, the

chelation approach (EDTA) used in bone research is limited by the slow process of

bone decalcification. For example, bone harvested from small animals such as mice

and rats can take weeks to decalcify, whereas bone harvested from larger animals

like sheep and bovine can take months [13], [15], [19], [26], [36]. Therefore, finding

a new approach to increase the rate of the decalcification process and to maintain a

high degree of tissue morphology has become a focus in this research area [25].

2.4 DEVELOPMENTS IN RAPID DECALCIFICATION

The most commonly reported method found in literature to decalcify bone is the

use of 10% EDTA at 4°C (refrigeration) or 25°C (room temperature) changing the

solution on a weekly basis [23], [25], [37], [38]. Nevertheless, due to its nature, the

process of decalcification is time consuming, and depending on the specimen size,

can take from several weeks up to several months to complete [39]. For this reason,

there have been attempts to develop a rapid decalcification procedure in order to

reduce the time required for decalcification, and at the same time not cause

significant damage to the tissue morphology. Even though the process for

developing a rapid decalcification technique dates back as far as the 1966 (first use

of a conventional microwave) [17], it is evident from the reports in the literature

that there is a lack of standardisation on the specimen size and tissue used for

experimentation. As a result, it is difficult to determine the most appropriate

method to increase the rate of decalcification while preserving morphology of

tissue (Table 2.4) [15], [20], [22], [26], [28], [39]–[41].

Table 2-4: Summary of rapid decalcification attempts and morphology

preservation

Author Methodology Findings

[15] R. A. Skinner et al. Formic acid at room

or fridge temperature

Formic acid did not negatively

affect staining results.

[20] V.J. Madden et al. EDTA in a microwave

and room

temperature

Microwave decalcification at

37°C did not effect

microstructure of cells compared

to routine 4°C method

[22] S. Jimson et al. EDTA, HCl, nitric acid,

formic acid

Concludes formic acid is the

most practical acid for speed


and quality

[28] Y. Shabita et al. EDTA, Formic acid,

Nitric acid & HCl

Formic acid and EDTA achieve

the best preservation of tissue.

Nitric acid and HCl cause

extensive damage to DNA

[39] C. Sanderson et

al.

EDTA with additives

at room temperature

Decalcification time with

additive decreased, morphology

remained intact.

[40] S. D. Cunningham

et al.

EDTA in a microwave

vs routine EDTA

Microstructure was better

preserved in microwave

decalcification compared to

routine method

[41] S. L. Gruntz Routine Formic acid

vs microwave formic

acid

Staining outcomes from the

microwave group were

favourable to the routine group

Chemical methods have been used to increase the rate of decalcification with

varying success. For instance, ammonium hydroxide and DMSO (Dimethyl sulfoxide)

in EDTA has been reported to reduce the time to decalcify bone by enhancing EDTA

infiltration into the specimen while maintaining an equivalent level of cellular detail

[39]. However, a study by Kito et al. reported that the tissue morphology showed

evidence of damage when DMSO was used [42]. In addition, this method hasn’t

been widely adopted, most likely due to safety concerns (toxicity) regarding DMSO

use. Another investigated method was the comparison of EDTA to ETDA

(Association of hydrochloric acid (12%), EDTA (0.07%), sodium tartrate (0.014%),

sodium and potassium tartrate (0.8%) and water [36]. The authors of this study

conclude that ETDA is faster than EDTA and the tissue retains its morphology at an

equivalent level. The same study also appears contradictory as the authors’ state

that mineral acids have damaging effects on specimen tissue and proceed to use

HCl as the key component of their ETDA decalcification solution. Chemical additives

to decalcification solutions aren’t routinely used and other methods of improving

the rate of decalcification have been trialled.

The use of ovens, microwaves or ultrasonic cleaners to improve the rates of

decalcification has become an area of research focus. Conventional ovens and


microwave ovens are used to increase the temperature of decalcification and

therefore increase the diffusion rate of solution through bone [43]. This will be

covered in more detail in section 2.5. Similarly, rapid decalcification using an

ultrasonic cleaner with formic acid has also been trialled [44]. Ultrasonic cleaners

and microwaves use the same principle to increase the rate of decalcification,

however, in the ultrasonic cleaner the creation of waves increases the molecular

activity of the solution resulting in more interactions between the bone and

decalcification solution [44]. A study by Hideki et al. reported that an ultrasonic

cleaner with formic acid and 30% mannitol decalcified bone retaining its antigens,

but this study did not compare their findings with a control group (standard

histology protocol). This study also acknowledged the limitations of ultrasonic

cleaners, stating that temperature distribution is not uniform and requires frequent

recalibration [44]. Based on these studies, the goal of this current study is to

develop an optimal rapid decalcification method using a microwave to reduce the

time required for bone decalcification without compromising tissue morphology.

2.5 MICROWAVES IN RAPID DECALCIFICATION

In recent years microwaves have been introduced as a new method to increase

decalcification rates in bone. Several studies have shown that a microwave used

under certain conditions can result in rapidly decreased decalcification times while

still preserving the integrity of the studied tissues [19], [20], [26], [29], [40], [44].

The temperatures used in microwave studies vary from 25°C, 37°C, 45°C, 60°C

[43][45][46] [21] or haven’t been specifically recorded [22], [37]. For instance, even

though previous reports suggested that using strong mineral acids such as

hydrochloric or nitric for decalcification were detrimental to tissue morphology

[20], [22], [24], [26], [44], other studies have reported that the use of the same

acids can yield acceptable results when used in combination with a conventional

microwave [26], [12]. EDTA has also been explored using a microwave to increase

the rate of decalcification. These studies showed no detrimental effect on tissue

morphology, however, they only focused on one antigen for IHC or only one

temperature for decalcification [20], [21], [24], [26], [34], [37], [38], [40], [41], [44]–


[47]. It should be also mentioned that the antigens investigated by those studies

were robust and highly specific to the type of bone being tested. A study performed

on mice skulls used EDTA to assess temperatures and their effects on nucleic

stability and β-Galactosidase activity at 37°C, 42°C and 50°C [48], the authors

concluded that at 42°C nucleic acid and amino acid stability was achieved. Although

microwaves have become the most reliable method to decrease the decalcification

time of bone [22], [49], despite that, these studies have a narrow focus on the

conditions, acids and bone specimens used. Studies need to be performed that

include a range of antigens from matrix proteins to highly specific cell markers.

Investigating these antigens would provide more insight into the potential benefits

and implementation of microwave induced rapid decalcification. The

aforementioned suggests the rapid decalcification method using a microwave

shows promise, but requires further investigation, which forms the merit for the

thesis.

Table 2 – 5: Summary table of microwave decalcification experiments

Author Methodology Findings

[21] E. Keithley et al. Routine EDTA vs

Microwave EDTA

Immunohistochemistry was not

effected by the microwave

compared to the routine method

[24] V.J. Madden et al. EDTA in a microwave

and room

temperature

Microwave decalcification at

37°C did not effect

microstructure of cells compared

to routine 4°C method

[26] G. Callis et al. EDTA, HCl, nitric acid,

formic acid

Concludes microwave

decalcification is faster and

staining is adequate

[37] G. Travlos EDTA and mineral

acids

Mineral acids cause damage to

tissue morphology, EDTA is

favourable however, heat

smearing of nuclei may occur

[38] J. Neves et al. EDTA and nitric acid

in a microwave

EDTA decalcification showed

well preserved tissue


morphology, nitric acid showed

evidence of tissue damage.

[40] S. D. Cunningham

et al.

EDTA in a microwave

vs routine EDTA

Microstructure was better

preserved in microwave

decalcification compared to

routine method

[41] S. L. Gruntz Routine Formic acid

vs microwave formic

acid

Staining outcomes from the

microwave group were

favourable to the routine group

[44] H. Hatta et al. Formic acid in

sonicator

Staining results for both H & E

and IHC showed evidence of

well-preserved tissue and

antigen morphology

[45] E. C. Weisberger

et al.

Microwave

decalcification for in

house use

Staining results were adequate

for clinical use and microwaves

reduced decalcification time

[46] P. Tinling EDTA microwave vs

routine method

Microwaves had no observable

negative consequences on tissue

morphology

[47] W. Howat et al. Formalin fixation of

fresh tissue in a

microwave

Microwave use did not have any

negative effects on tissue

morphology or DNA integrity.

Chapter 3: Research Design 25

Chapter 3: Research Design

3.1 PREFACE

This chapter describes the research design and methodology that was used in

testing the hypothesis of this study.

3.2 RESEARCH DESIGN

3.2.1 Aim 1: To Monitor the Rate of Bone Decalcification Using a Micro CT

system

Sections of female Merino sheep, the intact rats and mice tibiae (n = 3 per group)

were decalcified using conventional and rapid decalcification methods. Tibae were

chosen as a model because it is commonly used in bone research [50], [51]. Intact

tibiae were harvested from sheep, and then sectioned into smaller fragments to

ensure each experimental group consisted of tibia sections from 3 different animals.

To minimize variability, specimens from Wistar rats and C57Bl6 mice, were matched

by age and gender. Groups of specimens were decalcified in 10% EDTA, 5% nitric,

10% hydrochloric, and 10% formic acid solutions as they are the most commonly

used acids for decalcification. For a rapid decalcification study groups of specimens

were decalcified at 37°C in a microwave designed for this purpose (KOS Milestone,

Abacus) and compared to the specimens decalcified using the standard protocol at

4°C and 25°C methods (control groups). The rate of the decalcification was

monitored using µCT. First, a pilot study was performed to determine the frequency

of time points for µCT scanning as shown in section 3.3 of this chapter (Table 1).

Results from this pilot study were used to conduct the main study.

3.2.2 Aim 2: To Investigate if Rapid Decalcification Has a Detrimental Effect on

the Tissue Morphology & Antigenicity

After Aim 1 was complete the tissue integrity of the specimens was analysed using

histological analyses. Specimens were embedded in paraffin wax and sectioned. To

determine the overall tissue morphology all specimens were stained with

26 Chapter 3: Research Design

hematoxylin and eosin (H & E), and safranin-O staining was used for cartilage

viability. The IHC analysis focused on protein viability using collagen-1 and vWF

antibodies. Collagen type 1 (col-1) is the main structural protein in bone matrix, this

protein gives bone its tensile strength and serves as an organisational framework

for the mineral structure. VonWillebrand factor (vWF) is part of the clotting cascade

and is used in orthopaedic studies to assess vasculature throughout bone as part of

the mineralisation and remodelling process, it is highly specific to activated

epithelial cells. These markers served as quality control for the tissues that undergo

rapid decalcification. Results from the rapidly decalcified groups were compared to

the staining of the current gold standard (EDTA 4°C).

3.3 MATERIALS & METHODS

3.3.1 Specimen Harvesting & Fixation

All animal samples were acquired through the tissue use notification program of

the local animal ethics committee, Queensland University of Technology,

Brisbane, QLD (# 1400000377).

Mice, rat and sheep tibias were harvested from sacrificed animals, mice and rat

tibiae were left fully intact and placed straight into 4% paraformaldehyde solution

(Sigma-Aldrich, Castle Hill, NSW) immediately after the sacrifice. A total of 3 tibias

were collected from different animals per treatment group. Sheep tibias were

sectioned into thin slices using an EXAKT 310 diamond cutter band saw and weighed

with a microbalance to make sure that the weight of each specimen was

approximately 1.65 grams. Excess soft tissue was stripped away from the bone to

allow for greater surface area and perfusion of fixative solution into the bone to

maximise antigen preservation. Fixation was done at 4°C placed on agitation

equipment; fixation times were: for mice 24 hours, for rats 48 hours and for sheep

96 hours. Specimens were stored in 70% ethanol post fixation at 4°C to preserve

antigenicity.


3.3.2 Pilot Study

All samples undergoing decalcification were weighed and photographed, and then

placed into the individual specimen containers with 50mL of respective

decalcification solution. A specimen from each group was scanned at selected

intervals to determine the total time required to reach complete decalcification. A

pilot study was performed using one specimen from each group to get an

approximate estimate of the time points that need to be monitored before

performing the study on a larger scale. Briefly, specimens were placed either into

EDTA, formic acid, hydrochloric acid or nitric acid at 4°C, 25°C using agitation, and at

25°C and 37°C in a KOS microwave (KOS rapid microwave tissue processor,

Milestone) at 20% power (150 watts) while being stirred at 400rpm. An additional

study comparing agitation and non-agitated decalcification was tested at 4°C for the

acid groups for the sheep pilot study to assess if it decreased the rate of

decalcification. Sheep, rat and mice tibia were scanned by µCT (µCT40; Scanco

Medical, Bassersdorf, Switzerland) with an isotropic voxel size at 30µm, 16µm, and

12µm respectively, using 70kV energy and 200-ms integration time. The frequency

of specimen scanning is indicated in the Table 3-1. All bones were

deemed decalcified when bone volume (BV) had reached 0 (%) value as measured

by µCT.

Figure 3-1: An example of how a sheep tibia was sectioned. Bone from the

diaphysis was stripped of soft tissue and sectioned into slices that were

approximately the same weight and size.


Table 3-1: Pilot study µCT scanning schedule for decalcifying specimens

Species Decalcification condition Scanning frequency

Mice 37°C EDTA 1 day time points

Mice 4°C Formic Acid

25°C Formic Acid

25°C Formic Acid

37°C Formic Acid

4 hour time points

4 hour time points

4 hour time points

2 hour time points

Mice 4°C Hydrochloric Acid

25°C Hydrochloric Acid



3 hour time points

3 hour time points

3 hour time points

2 hour time points

Mice 4°C Nitric Acid

25°C Nitric Acid

25°C Nitric Acid

37°C Nitric Acid

60 minute time points




Rat 37°C EDTA 1 day time points

Rat 4°C Formic Acid

25°C Formic Acid

25°C Formic Acid

37°C Formic Acid

12 hour time points

12 hour time points

12 hour time points

6 hour time points

Rat 4°C Hydrochloric Acid




4 hour time points

4 hour time points

4 hour time points

3 hour time points

Rat 4°C Nitric Acid

25°C Nitric Acid

25°C Nitric Acid

37°C Nitric Acid





Sheep 37°C EDTA 1 week time points

Sheep 4°C Formic Acid

25°C Formic Acid

25°C Formic Acid

37°C Formic Acid

8 hour time points

8 hour time points

8 hour time points

4 hour time points

Sheep 4°C Hydrochloric Acid


2 hour time points

2 hour time points




2 hour time points

1 hour time points

Sheep 4°C Nitric Acid

25°C Nitric Acid

25°C Nitric Acid

37°C Nitric Acid





3.3.3 Decalcification & Micro CT Analysis

After fixation specimens were divided up into their respective groups for

decalcification as shown in Table 3-2.

Table 3-2: Decalcification specimen groups, species and conditions

Temperature Species Acids Specimen Number

4° Conventional

Decalcification

Benchtop method

Mouse

Rat

Sheep

10% EDTA

10% Formic Acid

10% HCL

5% Nitric Acid

n = 3 per acid group

and temperature.

e.g. 4°C EDTA n = 3

25° Conventional

Decalcification

Benchtop method

Mouse

Rat

Sheep

10% EDTA

10% Formic Acid

10% HCl

5% Nitric Acid


and temperature.


25° Rapid

Decalcification

Microwave

Method

Mouse

Rat

Sheep

10% EDTA

10% Formic Acid

10% HCl

5% Nitric Acid


and temperature.


37° Rapid

Decalcification

Microwave

Method

Mouse

Rat

Sheep

10% EDTA

10% Formic Acid

10% HCL

5% Nitric Acid


and temperature.


Prior to each time point specimens were weighed, photographed and the pH of the

decalcification solution was measured before the specimen was scanned by µCT


with the set parameters as indicated in the section 3.3.2. After each micro CT scan

the specimens were placed back into their decalcification container until the next

time point, and the entire procedure was repeated. Specimens in EDTA had their

decalcification solutions changed on weekly basis, acid groups were scheduled to be

changed if the pH was measured to be greater than 5.0, however, this did not occur

in this study and as a result acids were not replenished. All bones were

deemed decalcified when bone volume (BV) had reached 0 (%) value as measured

by µCT. After reaching endpoints the nitric, HCL and formic acid specimens were

neutralised in their specimen containers using sodium bicarbonate powder until the

pH was neutral. Specimens were then washed in running tap water for 4 hours and

placed into 70% ethanol in preparation for paraffin wax embedding.

After the time points were established in the pilot study the main study was

conducted. Mice, rat and sheep tibial specimens were organised into groups of 3

specimens as shown in Table 3-1 & 3-2. Microwave and µCT protocols were the

same as for the pilot study described above. Results in chapter 4.2.1 demonstrate

that decalcification at 25°C in the KOS milestone microwave was the same as 25°C

on the benchtop. Therefore, the use of a microwave at 25°C was discontinued from

the remainder of the study as it did not offer any differences to the benchtop

decalcification. Table 3.3 shows an amended experimental plan taking into account

the removal of the 25°C microwave group.

Table 3-3: Micro CT time points for assessing decalcification progress

TIMEPOINTS TO BE SCANNED TO MONITOR DECLALCIFCATION PROGRESS

Species Acid Temperature Time points

Mice 10%

EDTA

4°C

25°C

37°C

t= 0 & every 24 hours



Mice 10%

Formic

4°C

25°C

37°C




Mice 10% HCl 4°C

25°C




37°C t= 0 & every 2 hours

Mice 5% Nitric 4°C

25°C

37°C




Rats EDTA 4°C

25°C

37°C

t= 0 & every 1 week

t= 0 & every 1 week

t= 0 & every 1 day

Rats Formic 4°C

25°C

37°C




Rats HCl 4°C

25°C

37°C




Rats Nitric 4°C

25°C

37°C

t= 0 & every 1 hour

t= 0 & every 1 hour

t= 0 & every 30 mins

Sheep EDTA 4°C

25°C

37°C

t= 0 & every 2 weeks

t= 0 & every 2 weeks

t= 0 & every 1 week

Sheep Formic 4°C

25°C

37°C




Sheep HCl 4°C

25°C

37°C



t= 0 & every 1 hour

Sheep Nitric 4°C

25°C

37°C

t= 0 & every 1 hour

t= 0 & every 1 hour

t= 0 & every 30 mins

3.3.4 Histology

Specimens were processed in an Excelsior S3 Tissue processor (Thermoscientific,

Scoresby Vic) by dehydrating through graded ethanol solutions, cleared in xylene

and infiltrated with paraffin wax (Table 3-4). After processing specimens were


embedded in paraffin wax blocks using a Shandon Histocentre 3 (Thermoscienfitic,

Scoresby, VIC). Embedded paraffin blocks were sectioned at a thickness of 5 µm

using a Leica RM2265 microtome (Leica, Mount Waverly Vic) with a Feather S35

microtome blade (HD scientific, Sydney) and collected onto poly-l-lysine microscope

slides and oven dried at 55°C for 16 hours. Prior to sectioning specimens were

cooled for 16 hours and soaked in ice water to ensure good quality sections.

Table 3-4: Protocols for tissue processing specimens for paraffin embedding

Reagent Time

50% Ethanol 30 minutes






100% Ethanol II 60 minutes

Xylene I 45 minutes

Xylene II 45 minutes

Xylene III 60 minutes

Paraffin Wax I 45 minutes

Paraffin Wax II 60 minutes

Paraffin Wax III 60 minutes

Three slides from each group was stained either with H&E, Saf-O (Table 3-5) or IHC

analysis was performed for the col-1 and vWF antigens. Briefly, all specimens were

dewaxed and rehydrated through xylene and graded ethanol. Bone tissue and

ceullular morphology was analysed by Hematoxylin and Eosin staining (H&E),

hematoxylin (H) stains for cell nuclei (Mayers for H&E) and Eosin (E) was used to


stain cellular cytoplasm. For cartilage staining fast green and Safranin-O (Saf-O,

Weigerts) was used to assess the bone and cartilage. Antigenicity of the tissue was

assessed by immunohistochemistry for matrix (collagen 1) and cellular markers

(vWF).

Briefly, IHC was performed for Collagen type 1 and vWF on the slides as

described in table 3.4. All washing steps in the procedure were performed

with pH7.4 Tris HCL, and Bovine Serum Albumin (BSA) was dissolved in PBS

(Phosphate Buffered Saline). Antigen retrieval incubation was performed using

ready-to-use ProteinaseK (DAKO,S3020) at room temperature for 5 minutes

(mice) and 10 minutes (rats and sheep). Peroxidase blocking was performed

using 3% H2O2 for 15 minutes. Slides were then serum blocked using 2%

bovine serum albumin (Sigma Aldrich, Brisbane) at room temperature for 60

minutes. Primary antibodies were applied to the slides at room temperature

for 1 hour using a dilution of 1:500 rabbit polyclonal Collagen 1 antibodies

(Abcam ab34710) and 1:200 polyclonal rabbit anti-human vWF antibodies

(DAKO IR527). Following primary antibody incubation, slides were incubated

with a DAKO secondary antibody for 1 hour at room temperature and DAB

(DAKO K3468) developed for 5 minutes. Counterstaining with hematoxylin,

dehydration and coverslipping was then performed to complete the staining

routine.

Negative controls were performed for each staining run. Isotype controls were

performed on each mice and rat staining run.

Negative control: A negative control is performed on every tissue section

undergoing immunohistochemistry. This process does not include a primary

antibody incubation of the tissue section. Secondary antibody is added and colour

developed with DAB solution. Expected results for this control should be complete

negative staining, any indication of positive staining indicates cross reactivity with

the secondary antibody which indicates that staining results are inaccurate.

Isotype control: An isotype control is utilised when working with monoclonal

antibodies. The tissue section is incubated with a non-immune immunoglobulin of

the same isotype (for example, IgG1, IgG2A, IgG2B, IgM) and concentration as the


primary monoclonal antibody. This is performed to identify if positive staining was

caused by non-specific interactions of immunoglobulin molecules with the tissue

section. Positive staining using this control indicates antibody cross reactivity with

the tissue type which indicates a potential to interpret the results as a false positive.

This is only performed if the origin of the antibodies used has cross reactivity

potential with the target species.

Results were not interpreted if the negative controls or isotype control failed. After

staining slides were then dehydrated through graded ethanol and cleared with

xylene and finally were mounted using Eukitt mounting medium (Sigma-Aldrich,

Castle Hill, NSW) and coverslipped using a 52 x 20mm coverslip (Thermoscientific,

Scoresby, VIC).

Table 3-5: Protocols for H&E, Safranin O and IHC staining

Hematoxlyin and Eosin staining protocol

Reagent Time

Xylene I & II 6 mins I then 6 mins in II

Ethanol 100% -> 100% -> 90% -> 70% -> 50% 2 mins for each ethanol wash

Tris HCl 2 mins

Mayer’s Hematoxylin 2 mins

Running tap water (warm) 5 mins

Eosin 30 secs

Running tap water 15 seconds

Ethanol 70% -> 90% -> 100% -> 100% 2 mins for each ethanol wash

Xylene III & IV 6 mins in III and 6 mins in IV

Safranin O staining protocol

Reagent Time



Ethanol 100% -> 100% -> 90% -> 70% ->50% 2 mins for each ethanol wash

Running tap water 2 mins

Weigerts Iron Hematoxylin 2 mins

2% Acid alcohol 30 secs


0.05% Fast Green FCF 5 mins

1% Acetic Acid 30 secs

0.1% Safranin O 5 mins

Ethanol 100% -> 100% -> 100% 2 mins for each ethanol wash


Immunohistochemistry staining protocol

Reagent Time


Ethanol 100% -> 100% -> 90% -> 70% -> 50% 2 mins for each ethanol wash

Tris HCl I 2 mins

Proteinase K antigen retrieval 5 mins

Tris HCl I -> Tris HCl II -> Tris HCl III 2 mins for each wash

3% H2O2 blocking 20 mins


2% Bovine Serum Albumin antigen blocking 1 hour

Antibody (Collagen 1 or vWF) 1 hour


Secondary antibody 1 hour



DAB substrate 5 mins


Mayers hematoxylin 2 mins


Ethanol 70% -> 90% -> 100% -> 100% 2 mins for each ethanol wash


Chapter 4: Results of decalcification times determined by Micro CT analysis 37

Chapter 4: Results of decalcification times

determined by Micro CT

analysis

4.1 PREFACE

This chapter investigates an approximate time frame for complete decalcification of

specimens. Briefly mice, rat and sheep specimens were decalcified and scanned by

micro-CT at determined intervals.

4.2 MICRO CT DATA & DECALCIFICATION TIMES

4.2.1 Pilot Study

A pilot study was performed to estimate an approximate rapid decalcification time

for formic, hydrochloric and nitric acid using one specimen per group from all

species. All specimens were first decalcified and scanned using µCT as described in

chapter 3 section 3.3.2. Results from the pilot study demonstrated that there was

no difference between microwave and benchtop decalcification at 25°C and as a

result this particular group was discontinued from further analysis of the main

study. The results from the pilot study confirmed the selection of time points and

these were used for the remainder of the study (Figure 4-1).

Sheep specimens were subjected to an additional test during the pilot study, they

were decalcified at 4°C with and without agitation. Results from this study

demonstrated that agitation reduced the time required to reach complete

decalcification (Figure 4-2). Therefore, for the remainder of this study agitation was

applied to all the specimens to reduce decalcification time.

38 Chapter 4: Results of decalcification times determined by Micro CT analysis

Figure 4-1: Pilot study results demonstrating the total amount of time

required to decalcify bone (n =1). Results show that mice tibia require 4 – 8

hours to decalcify while sheep and rat specimens require 5 to 40 hours

respectively, relative to the acid used. Microwave and conventional

decalcification methods required the same amount of time at 25°C for all acids

tested.


0 hours 2 hours 4 hours 0 hours 2 hours 4 hours 5 hours

0 hours 4 hours 8 hours 10 hours 0 hours 6 hours 12 hours 15 hours

0 hours 24 hours 40 hours 0 hours 24 hours 40 hours 54 hours

Figure 4-2: Representative 3D µCT images of the pilot study. Decalcification of

sheep tibia at 4°C in nitric acid, hydrochloric acid and formic acid at selected time points

– agitated specimens (left), static decalcification (right).

FORMIC

ACID

HCL

NITRIC

ACID

AGITATED GROUPS STATIC GROUPS


4.2.2 Monitoring of Specimen Decalcification Time using the Micro CT system

Mice, rat and sheep specimens were evaluated using micro CT. Specimens

decalcified using the microwave were found to be the fastest method for all species

types. A graphical representation of the specimen weight, change in decalcification

solution pH and µCT results is shown in figures 4-3 (mice), 4-4 (rats), 4-5 (sheep).

Results from the µCT examination show that the rate of decalcification was

proportional to the strength of the acid. This trend was observed in both the pilot

study and main study.

Mice

Bone volume (BV) was calculated using µCT and is presented as a percentage,

where the initial volume of the intact bone was considered as 100%. Decalcification

was determined to be complete when BV had reached 0%.

In EDTA complete decalcification of tibia was achieved at 3 days, 2 days and 30

hours for 4°C, 25°C and 37°C, respectively. In the microwave group (37°C), at 12

hours only 25% of BV remained, thereafter it slowly declined until full

decalcification (30 hours) was reached (Figure 4-6C). In contrast, when the

microwave group (37°C) had reached 0% of BV the 25°C and 4°C groups were at

45% and 67% of BV, respectively (Figure 4-6C).

Decalcification in formic acid was completed in 4 hours and 8 hours for microwave

(37°C) and conventional groups (4°C and 25°C), respectively. At 2 hours in the

microwave (37°C), only 25% of BV remained, complete decalcification occurred

after 4 hours (Figure 4-6F). Comparatively, decalcification in conventional (4°C &

25°C) groups at 4 hours still had 45% and 55% of BV, respectively, at 8 hours the

conventional groups had completely lost all calcium (Figure 4-6F).

Mice tibia in HCl decalcified in 3 hours using the microwave (37°C), whereas it took

5 hours in the conventional groups (4°C and 25°C). After 2 hours in the microwave

(37°C) only 18% of BV was remaining, reaching complete decalcification at 3 hours

(Figure 4-6I). Decalcification in the conventional groups (4°C and 25°C) at 4

hours tibia had 15 - 20% of BV, slowly declining thereafter with complete loss of

calcium at 5 hours (Figure 4-6I).


In nitric acid mice tibia decalcified in 90 minutes using the microwave (37°C),

whereas it took 3 hours using conventional conditions (4°C and 25°C). The

microwave group (37°C), had only 10% of BV remaining after 1 hour, and reached

complete decalcification after 90 minutes. Furthermore, at 2 hours the conventional

groups (4° and 25°C) had 50% and 37% of BV remaining, respectively. Conventional

groups reached their endpoints at 3 hours.

The data was also confirmed by uCT images. In EDTA, formic and nitric acids

specimens had segments of bone still present in the distal, proximal and diaphysis

in late stages of decalcification (Figure 4-3A & B). In contrast, specimens in HCl at 4

hours, proximal and distal ends of the bone were still present, whereas at 5 hours

were completely decalcified (Figure 4-3B). Not surprisingly, the fastest

decalcification times were achieved in specimens subjected to microwave

conditions (37°C) regardless of solution used. In summary nitric acid decalcification

was achieved in 1.5 – 3 hours which was faster compared to HCL (3 – 5 hours),

formic acid (4 – 6 - 8 hours) and EDTA (30 - 50 - 72 hours).

Rats

Rat tibias in EDTA were completely decalcified in 5 days using the microwave (37°C),

contrastingly it took 5 and 4 weeks at 4°C and 25°C, respectively. In the microwave

group (37°C) the rate of BV% loss was rapid when compared to the conventional

groups (4°C & 25°C). At 1 week tibias in the microwave group had completely

decalcified whereas in the conventional groups 50% of BV was remaining (Figure 4-

7C). At 2 weeks the conventional groups (4°C & 25°C) had 25% and 15% of BV

remaining, respectively (Figure 4-7C). Gradually the remaining bone was lost in the

conventional groups until decalcification was complete.

Formic acid decalcified rat tibias in 24 hours using a microwave (37°C), 36 hours at

25°C and 48 hours at 4°C. At 12 hours 15%, 40% and 65% of BV remained for the

37°C, 25°C and 4°C groups (Figure 4-7F). Complete decalcification occurred after 24

hours in the microwave group (37°C), however, in conventional (4°C & 25°C) groups

still had 20% and 15% of BV, respectively (Figure 4-7F). At approximately 25% of BV

the rate of bone loss slowed for all groups.


In HCl complete decalcification was achieved at 8 hours, 12 hours and 15 hours for

the 4°C, 25°C and 37°C correspondingly. After 8 hours using the microwave (37°C)

rat tibia was completely decalcified, however, the conventional groups had 10% and

20% of BV remaining, respectively (Figure 4-7I). Decalcification of the remaining

bone was gradual with the 25°C group requiring 12 hours and the 4°C group

needing 15 hours (figure 4-7I).

Nitric acid decalcification of rat tibias was achieved in 2 hours using the microwave

(37°C), whereas it took 3 hours at 25°C and 4 hours at 4°C. At 1 hour the microwave

group (37°C) reached 10% of BV remaining, conversely 50% at 4°C and 45% at 25°C

of BV was present (Figure 4-7L). Complete decalcification occurred at 3 hours and 4

hours for the 25°C group and 4°C group, respectively (Figure 4-7L).

Data from Figure 4-7 was confirmed by µCT images in Figure 4-4. Broadly a trend in

all specimens was observed. In each tibia, the proximal end was the final part of the

bone to decalcify (Figure 4-4A, 4B, 4C, 4D). Interestingly in formic acid (Figure 4-4B)

a coral like appearance of bone was observed in the 25°C and 37°C groups. This was

not observed in any other rat group. Clearly the fastest decalcification times were

achieved in the microwave groups (37°C) irrespective of the solution used. Overall

nitric acid decalcified tibia in 2 – 4 hours, HCl required 8 – 15 hours, formic acid was

completed in 24 – 48 hours and EDTA achieved decalcification in 5 days – 5 weeks.

Sheep

Sheep tibia in EDTA were decalcified in 2 weeks using a microwave (37°C),

comparatively the conventional groups (25°C & 4°C) required 6 and 8 weeks,

respectively. At 2 weeks the conventional groups (25°C & 4°C) 25% and 40% of BV

was remaining respectively, whereas the microwave group (37°C) was at 0% of BV

(figure 4-8C). Decalcification progressed in the conventional groups (4°C & 25°C) at

a steady rate. After 6 weeks the 25°C group was completely decalcified, however,

the 4°C group had 8% of BV remaining (figure 4-8C). Decalcification was complete in

the 4°C group at 8 weeks (Figure 4-8C).

Decalcification in formic acid was achieved at 16, 24 and 40 hours by 37°C, 25°C and

4°C groups, respectively. At 16 hours the microwave group (37°C) had completely


lost all calcium, however, in the conventional groups (25°C & 4°C) only 22% and 30%

of BV remained, respectively (Figure 4-8 F). At approximately 50% of BV the

conventional groups experienced a steady decline of calcium loss. After 24 hours

the 25°C group had achieved decalcification, contrastingly the 4°C group still had

20% of BV remnant (Figure 4-8F). At 40 hours the 4°C group was completely

decalcified (Figure 4-8F).

In HCl decalcification was achieved in 3 hours by microwave treatment (37°C),

whereas it took 8 hours at 25°C and 10 hours at 4°C. Bone decalcification at 37°C

occurred at 3 hours, in comparison at 4 hours the conventional groups (25°C and

4°C) had 30% and 45% of BV remaining, respectively (Figure 4-8I). After 8 hours the

25°C group had achieved complete calcium loss, at this stage the 4°C had 12% of BV

remaining (Figure 4-8I). At 10 hours the 4°C group had completely decalcified

(Figure 4-8I).

Using nitric acid sheep tibia decalcified in 1, 3 and 4 hours at 37°C, 25°C and 4°C,

respectively. After 1 hour in the microwave (37°C) sheep tibia was completely

decalcified, contrastingly the conventional groups had approximately 53% of BV

remaining (Figure 4-8L). Decalcification was achieved at 3 hours for the 25°C group,

whereas the 4°C had 15% of BV remaining. At 4 hours the 4°C had completely

decalcified.

Images obtained from µCT confirmed the BV loss data (Figure 4-5A, 5B). Broadly

sheep tibia decalcification rates were found to follow a similar trend in all

decalcification conditions. Microwave groups were relatively rapid compared to

conventional groups; furthermore the BV loss curves showed that decalcification

slowed down as less BV remained. Furthermore, µCT images showed intact tibias

gradually diminishing where bone went from a thick annular shape to a thin

crescent until decalcification was complete. Interestingly Formic acid at 37°C had a

coral like surface morphology at 4 and 8 hours, this occurrence was not observed in

any other group in sheep. As expected the microwave groups managed to decalcify

bone considerably faster than the conventional conditions for each solution. To

summarise decalcification of sheep tibia was achieved in nitric acid decalcification in


1 – 4 hours followed by HCL (3 – 10 hours), formic acid (16 - 40 hours) and EDTA (2-

8 weeks).


FORMIC ACID EDTA

0 hrs 12 hrs 24 hrs 30hrs

0 days 1 day 2 days 3 days

0 hrs 2 hrs 4 hrs

0 hrs 4 hrs 8 hrs

4°C

25°C

37°C

Figure 4-3A: Representative 3D µCT images of mice tibia decalcified in EDTA (left) and

formic acid (right) at indicated time points. Mineralised bone matrix appears white and

decalcified bone is represented by black.


4°C

25°C

37°C

NITRIC ACID HYDROCHLORIC ACID

0hrs 2hrs 3 hrs

0 h 1 hr 2 hrs

0 hrs 4 hrs 5 hrs

0 hrs 2 hrs 3 hrs

Figure 4-3B: Representative 3D µCT images of mice tibia decalcified in HCl (left) and

nitric acid (right) at indicated time points. Mineralised bone matrix appears white and

decalcified bone is represented by black.


EDTA

0 weeks 1 week 2 weeks 3weeks 4weeks 5weeks

0 days 1 day 2 days 3 days 4 days 5 days

37°C

25°C

4°C

Figure 4-4A: Representative 3D µCT images of rat tibia decalcified in EDTA at indicated

time points. Mineralised bone matrix appears white and decalcified bone is represented by

black. Proximal ends of the tibia took the longest time to decalcify in each specimen.


0 hrs 12 hrs 24 hrs 36 hrs 48 hrs

0 hrs 6 hrs 12 hrs 18 hrs 24 hrs

FORMIC ACID

37°C

25°C

4°C

Figure 4-4B: Representative 3D µCT of rat tibia decalcified in formic acid at indicated

time points. Mineralised bone matrix appears white and decalcified bone is represented

in black. Proximal ends of the tibia took the longest time to decalcify in each specimen.


HYDROCHLORIC ACID

0 hrs 4 hrs 8 hrs 12 hrs 15hrs

0 hrs 3 hrs 6 hrs 8 hrs

37°C

25°C

4°C

Figure 4-4C: Representative 3D µCT images of rat tibia decalcified in hydrochloric acid

at indicated time points. Mineralised bone matrix appears white and decalcified bone is

represented in black. Proximal ends of the tibia took the longest time to decalcify in each

specimen.


4°C

25°C

NITRIC ACID

37°C

25°C

4°C

0 hrs 1 hr 2 hrs 3hrs 4hrs

0 mins 1 hr 2 hrs

Figure 4-4D: Representative 3D µCT images of rat tibia decalcified in nitric acid at

indicated time points. Mineralised bone matrix appears white and decalcified bone is

represented in black. Proximal ends of the tibia took the longest time to decalcify in

each specimen.


EDTA

4°C

25°C

37°C

0 weeks 2 weeks 4 weeks 6 weeks 8weeks

0 weeks 1 weeks 2 weeks 2.5 weeks

FORMIC ACID

0 hrs 8 hrs 16 hrs 24 hrs 32 hrs 40 hrs

0 hrs 4 hrs 8 hrs 16 hrs

37°C

25°C

4°C

Figure 4-5A: Representative 3D µCT images of sheep tibias decalcified in EDTA and formic

acid at indicated time points. Mineralised bone matrix appears white and decalcified bone is


specimen.


Figure 4-5B: Representative 3D µCT images of sheep tibias decalcified in HCl and nitric acid

at indicated time points. Mineralised bone matrix appears white and decalcified bone is


specimen.

4°C

25°C

37°C

0 hrs 1 hr 2 hrs 3hrs 4hrs

0 mins 30 mins 60 mins

NITRIC ACID

HYDROCHLORIC ACID

4°C

25°C

37°C

0 hrs 1 hr 2 hrs 3hrs

0 hrs 2 hrs 4 hrs 6 hrs 8hrs 10hrs


4.2.3 Measuring Specimen Weight Loss & pH during decalcification

Weight Loss

Weight loss was measured during decalcification as a robust method to determine

when specimens had reached the endpoint. Intact mice and rat tibias were found to

have lost approximately 40% of their initial starting weight, regardless of the

decalcification method used (Figures 4 - 6 & 7). Likewise, sheep tibia sections lost

approximately 60% of their starting weight when specimens had completely

decalcified (Figure 4 – 8). However, an accurate endpoint could not be determined

based solely on specimen weight loss as decalcification was terminated when BV%

reached 0.

pH

Acids that decalcified bone by a pH drop showed an increase in the pH over time. In

all cases, pH changes (pH 7.4 – 7.4) were not observed across all EDTA groups for all

species (figures 4-6B, 4-7B, 4-8B). A loss of acidity was observed in formic, HCl and

nitric acid solutions. The final pH when specimens were decalcified varied on the

decalcification condition and acid. For example, in rats the formic acid in the

microwave group had a final pH of ~2.0 in comparison to conventional methods,

which had ~1.90 and ~1.85, respectively. Correlation between a final pH for all

species using any of the acids was not observed. Therefore, decalcification endpoint

could not be accurately determined based on the measurable changes in pH of the

solutions.


Figure 4-6: Graphical representation of the change in mice tibia weight, pH of decalcification

solution and rate of bone volume loss over time. Error bars indicate the standard error of the

mean (n=3). Blue represents 4°C, green 25°C and red 37°C

A B C

D E F

G H I

J K L


Figure 4-7: Graphical representation of the change in rat tibia weight, pH of decalcification

solution and rate of bone volume loss over time. Error bars indicate standard error of the

mean (n=3). Blue represents 4°C, green 25°C and red 37°C.

A B C

D E F

G H I

J K L


Figure 4-8: Graphical representation of the change in sheep tibia weight, pH of

decalcification solution and rate of bone volume loss over time. Error bars indicate

standard error of the mean (N = 3). Blue represents 4°C, green 25°C and red 37°C.

A B C

D E F

G H I

J K L


4.3 DISCUSSION

Monitoring bone decalcification by µCT is a novel and effective approach. Based on

current literature only one other study has used µCT to analyse specimen

decalcification [52]. Gold standard methodology for examining mineral content in

bone has been reported as measuring radio opacity by x-rays or µCT scanning. In

this study it was crucial to precisely determine the exact endpoint of bone

decalcification to prevent excess damage to the specimen. The data clearly shows

the point at which the bone becomes radio-opaque and calcium is removed from

the tissue without any ambiguity at the resolution scanned. Previously reported

methods of decalcification testing have been noted for being inconclusive and

subjective [26]. In comparison to currently reported empirical methods it is evident

that µCT systems have an advantage in accurately assessing bone decalcification.

During the decalcification process bend testing was done by hand in the lab to

determine specimen softening. Prick testing with a needle was not performed as

excess tissue damage would interfere with tissue morphology analysis. Midway

through decalcification the rat and mice tibia appeared decalcified due to the ease

of bending through the diaphysis however Figures 4-4 and 4-5 demonstrate that

there is still bone present in the proximal and distal ends. Prick testing may have

detected calcified bone in the proximal and distal ends. However, the resulting

damage to the tissue would have interfered with histological analysis. Embedding of

the bone based on the empirical bending tests would have resulted in difficulty

embedding and sectioning specimens. Therefore, due to the dense bone in the

proximal and distal ends bend testing is insufficient to correctly determine

decalcification

An alternate method for determining bone decalcification was to measure the

weight of the bone during the decalcification process [19], [39]. Testing for

complete decalcification by measuring the specimen weight didn’t accurately

predict when a specimen was reaching end point. Figures 4-3, 4 – 4 & 4 -5

demonstrate this as the rate of bone loss was fairly constant throughout

decalcification. In rat and mice groups the rate of weight loss decreased as

decalcification reached completion for all acids, however as the specimen weight


did not plateau an endpoint could not be determined. Sheep tibia specimens did

not follow this trend and a steady rate of weight loss was observed in all groups. A

potential explanation of the consistent weight loss might be due to the continual

loss of bone marrow by acidic digestion [53]. Decalcification solution pH was also

monitored simultaneously with the specimen weight. As expected the pH of the

acidic decalcification solutions became less acidic as calcium disassociated from the

specimens. This is due to the requirement of H+ ions to encourage phosphate

groups to disassociate from bone and form a salt, as a result of this reaction calcium

dissolves into solution. Chelation based decalcification (EDTA) did not show any

change in pH as H+ ions aren’t required for chelation. Broadly it was observed that

as the specimen weight decreased the pH of the decalcification solution became

less acidic within the acid groups. In summary it was not possible to accurately

determine the decalcification endpoint based on the weight of the specimen and

observable pH changes in the solution.

Endpoint times determined by µCT were consistent among each specimen in each

group of the study (n=3). Minimal variance in completion times was observed which

suggests µCT evaluation of decalcification is an accurate method. Mice, rat and

sheep decalcification times were dependant on the temperature. With the acid

groups it was noted that rapid decalcification groups would be completed in half

the time of a 4°C conventional group for rats and mice and one third of the time for

sheep. This observation was unexpected as decalcification rates were thought to be

non-linear. Data suggests that the rate of decalcification may follow an exponential

pattern. An explanation into this occurrence may be the pH dependant mechanism

of acidic decalcification. The more acidic the decalcification solution the more

uniform the decalcification is as calcium is likely to disassociate from hydroxyapatite

in low pH conditions [54]. Decalcification times with EDTA was more random as

rapid decalcification was found to be twice as fast in mice and 3 to 4 times faster in

rats and sheep, respectively. As a chelation based method EDTA is limited by the

saturation of molecules with calcium ions. Further research into calcium

disassociation from hydroxyapatite would provide further insight into

decalcification mechanisms.


Mice tibia decalcification times in this study were comparable to times reported in

previous studies [41][55]. Gruntz et.al found mice tibia to decalcify in EDTA and

formic acid at approximately the same time found in this study. For EDTA

decalcification times reported by Masahiko et.al are 3 days longer. Times for nitric

and hydrochloric acid aren’t reported for mice tibia [41], [55], [56]. The methods of

determining endpoint in these studies are not mentioned, however, it is likely that

bone bending and pricking was used. The decalcification times in this study are

more likely to be accurate because of µCT use to determine endpoint.

Decalcification times for rat tibias in this study were found to be faster for all acids

and EDTA reported in recent literature [15], [18], [22], [26], [30], [36], [41], [56]–

[58]. Over estimation of rat tibia decalcification was found throughout the

literature. This would suggest that one of two things is occurring with rat tibia

decalcification, endpoint determination in these studies was insufficient at

estimating an endpoint or that rat tibia decalcification is quite variable. Data from

the µCT results demonstrate consistent repeatability in decalcification times; this

suggests that endpoint determination techniques in previous studies were

insufficient. Over decalcification with acid results in deleterious effects for cell

nuclei, cytoplasm and tissue swelling if done by EDTA [20], [26]. The results from rat

tibia analysis by µCT would indicate that over decalcification of specimens is

common throughout academic literature.

Sheep tibia decalcification patterns in this study were consistent across all four acid

groups. Within each group the lower temperature (4°C) followed the same pattern

as the 25°C and 37°C but at a slower rate. Formic acid decalcification resulted in a

coral like structure of the remaining bone in each specimen. This could be due to

the less acidic pH of formic acid and the organic acid mechanism of H+ ion release.

Sheep decalcification times are under reported throughout literature as mice and

rat tibias are commonly studied due to ease of access to the animals. Bovine, canine

and porcine decalcification times are reported [26], [41], [44] however, the

comparability of these species is unknown [50]. In one study an intact sheep tibia

was used to determine the decalcification time in EDTA, this type of specimen is not

representative of specimens in research or pathology. Intact sheep tibia times were


found to be 30 days by constant EDTA replacement decalcification [59]. Other

studies by Gruntz et.al and Callis et.al both used dog specimens for their

decalcification times. Callis et.al found dog tibias of a similar size to the sheep tibia

used in this study varied from 8-24 hours for microwaved specimens (all acids) to 10

– 20 days for conventionally decalcified specimens [26] in EDTA as determined by X-

ray. A major limitation of the Callis et.al study is the lack of specimen replicates for

each treatment group (n=1), in contrast, in this study all tested specimens (n=3)

found a similar decalcification pattern within each group. Results from Gruntz et.al

did not compare to the endpoint times determined by µCT analysis in this study.

Sheep tibia were found to decalcify faster in formic acid than dog tibia, however,

Gruntz et.al reported faster decalcification times in EDTA for canine bone then the

sheep tibia in this study. Further research on large animal bone decalcification

would prove useful to determining appropriate endpoint times for large animal

studies.

The advantage of the µCT based approach is its accurate detection of calcium in

specimens. In contrast, pin pricking, bending and weight loss measuring tests lack

this specificity [35]. Nevertheless, there are limitations to this approach, for

instance while it would be possible to scan at a higher resolution to identify cellular

detail, this study demonstrated that the resolutions used were sufficient enough to

show the decalcification process of the mineral matrix. Conversely, the main

limitations of using µCT to monitor decalcification are the time required, the

associated costs of scanning specimens, and the availability of µCT scanners in

laboratories.

In summary, the results from this study demonstrate the accuracy of µCT

determining complete decalcification and show that decalcification in literature is

potentially overestimated. The results are likely to be relevant for research and

pathology labs looking for guidelines in time required to decalcify bone for

histological studies.

Chapter 5: Histological evaluation of various decalcification conditions 61

Chapter 5: Histological evaluation of

various decalcification

conditions

5.1 HISTOLOGICAL STAINING OF BONE

5.1.1 Mice staining

Tissue morphology by H&E

Mice tibia sections were stained with H&E and examined by light microscopy. Tissue

morphology analysis revealed good tissue preservation in EDTA and formic acid

decalcified specimens for all conditions (Figure 5 – 1 A – F). Cement lines as well as

lamellar bone could be identified in the EDTA and formic acid groups. Epithelial cell

and erythrocyte staining was observed in vascular tissue. In addition, staining of cell

nuclei did not show evidence of deterioration for all conditions in EDTA or formic

acid. Damage to the tissue morphology was observed in specimens decalcified in

HCl or nitric acids for all conditions (Figure 5 -1 G – L). Cement lines and vasculature

tissue architecture staining was not observed or showed evidence of damage.

Osteocyte nuclei staining had a smudged appearance in both groups which resulted

in a loss of cellular detail. Evaluation with H&E stain revealed that tissue

morphology was well preserved when EDTA or formic acid was used.

Cartilage by Safranin O

Cartilage and bone morphology was well preserved in specimens decalcified in

EDTA and formic acid for all conditions (Figure 5 – 2 A - F). Tissue preservation of

cartilage and bone was observed for all decalcification conditions in HCL and nitric

acids, however, non-specific nucleic staining was observed in the cartilage matrix. In

the HCl and nitric acid groups nuclei appeared to stain green rather than black in

cartilage and bone (Figure 5-2 G – L). Furthermore, in the acid groups shrinkage of

chondrocytes and lacunae was observed (Figure 5-2).

62 Chapter 5: Histological evaluation of various decalcification conditions

A B C

D E F

G H I

J K L

Figure 5-1: Hematoxylin & Eosin staining in mice tibia. Bone matrix stains various shades

of pink and purple with cellular nuclei staining blue. Cement lines in the cortical bone

matrix are clearly visible in the formic acid and EDTA groups as indicated by arrows. Note

that in the HCl and nitric acid groups cement lines are either unclear or absent.

Magnification x20 scale bar = 50µm.


Figure 5-2: Safranin O & Fast green staining in mice tibia for cartilage and bone. Cartilage tissue

stains red and bone stains green. The chondrocytes and nuclei in EDTA are well preserved in

comparison to the acids. Magnification x20 scale bar 50µm.

A B C

D E F

G H I

J K L


Collagen type I in bone matrix

Tissue antigenicity was well preserved in the EDTA and formic acid decalcified

specimens for all conditions. Mice tibia in EDTA had positive staining of collagen

type I in the bone matrix (Figure 5 – 3 A – F). Tissue architecture was well preserved

and bone remodelling sites could be identified from the staining in all EDTA and

formic acid groups. In contrast, specimens decalcified by HCl showed evidence of

tissue degradation (Figure 5 – 3 G, H & I). Cement lines were stained in the

conventional groups (4°C & 25°C) and absent in the microwave group (37°C). Mice

tibia decalcified in nitric acid showed evidence of reduced antigenicity regardless of

the condition used (Figure 5 – 3 J, K & L). Cortical bone did not stain positively

throughout the matrix and positive staining was observed on the edges of the

tissue. Mice tibia specimens decalcified in EDTA with a microwave or formic acid

had similar antigenicity preservation and staining patterns compared to the

conventional 4°C EDTA condition.

vWF staining for serum proteins

Mice tibias in EDTA had well preserved vascular tissue for all decalcification

conditions (Figure 5 – 4 A, B &C). Blood vessel endothelium (epithelial lining) and

serum proteins stained positive for vWF. Vascular tissue was also well preserved in

formic acid for all conditions with positive vWF staining observed inside blood

vessels and the endothelium (Figure 5 – 4 D, E & F). Vascular tissue antigenicity was

retained in all HCl and conventional (4°C & 25°C) nitric acid groups as evidenced by

positive vWF staining. Epithelial cells in the endothelium had lost cellular detail as

nuclei and cytoplasm staining was unable to be identified (Figure 5 – 4 G – K).

Likewise, nitric acid decalcification of mice tibia in the microwave also revealed a

similar loss in vascular tissue antigenicity and cellular detail. Background (non-

specific) staining was observed in the bone matrix in all acid groups (formic, HCl &

Nitric). Differences in antigenicity and tissue morphology were noted to occur

between EDTA and the acid groups. Compared to EDTA, the acid groups showed

evidence of morphological damage and reduced antigenicity.


Figure 5-3: Collagen-1 immunohistochemistry in mice tibia for bone matrix. Positive staining for

collagen appears brown and cell nuclei appear blue. Specific positive staining for collagen type 1 is

observed in specimens decalcified in EDTA, formic acid and HCl. Specimens decalcified in nitric acid

showed a lack of positive staining throughout the bone matrix. Magnification x20 scale bar 50µm.


Figure 5-4: vWF immunohistochemistry in mice tibia for vasculature and serum proteins.

Positive staining for vWF appears brown in blood vessels and cell nuclei appear blue. Endothelium

lining of vasculature is well preserved and stains positive in all groups. Serum proteins within the

blood vessels are also positive. Magnification x20 scale bar 50µm.

A B C

D E F

G H I

J K L


5.1.2 Rat staining


Examination of the H&E stained sections revealed differences in staining for

specimens decalcified in EDTA and acid groups. Cellular morphology showed

evidence of deterioration for all conditions in HCl and nitric acid (Figure 5 – 5 G – L).

Osteocyte staining in the cortical bone was observed to be faded in all the HCL

groups. In contrast, osteocyte staining in the nitric acid groups was observed for all

conditions. Specimens decalcified in HCl and nitric acid showed evidence of

deterioration as nuclei staining was not observed in the bone marrow, regardless of

the condition. Tissue architecture was lost as cement lines were not observed in the

microwave groups, however, in conventional groups despite a smudged

appearance, they could be identified as indicated by arrowheads in Figure 5 - 5.

Cells in the bone marrow were characterised by pink staining which is indicative of

eosin uptake for all groups. Furthermore, vascular tissue architecture showed signs

of damage as epithelial cell and erythrocyte staining appeared smudged for all HCl

and nitric conditions. In contrast, specimens decalcified in formic acid and EDTA had

well preserved tissue morphology for all conditions used. Staining of cell nuclei was

observed in bone and vascular tissue (Figure 5 – 5 A – F). Furthermore, cement lines

and remodelling sites were clearly defined in bone matrix. Interestingly specimens

decalcified in EDTA using a microwave showed preservation of tissue structure and

staining patterns similar to the routine condition (EDTA 4°C).


Figure 5-5: Hematoxylin & Eosin staining in rat tibia. Bone matrix stains various shades of pink

and purple with cellular nuclei staining blue. Cement lines in the cortical bone matrix are clearly

visible in the formic acid and EDTA groups as indicated by arrows, however, in HCl and nitric

they were not well preserved.. Note that in the HCl and nitric acid groups cell nuclei staining is

absent and tissue matrix appears dark. Magnification x20 scale bar = 50µm.

A B C

D E F

G H I

J K L


Cartilage by Safranin O

Cartilage and bone morphology was well preserved in the EDTA and formic acid

decalcified specimens as evidenced by Safranin O staining. Epiphyseal cartilage and

bone tissue stained bright red and green, respectively, in specimens for all

decalcification conditions. Specimens decalcified in HCl and nitric acid showed signs

of tissue degradation and cytological damage (Figure 5 – 6 G – L). Cell nuclei were

observed to stain green instead of black in chondrocytes for the 4°C conventional

HCl and all nitric acid groups. Cell nuclei stained black in all other decalcification

conditions in rats. Chondrocyte and cartilage lacunae shrinkage appeared to occur

in the HCL and nitric acid groups, however, the shrinkage was less severe in the

microwave HCL group. Furthermore signs of shrinkage were observed in the formic

acid group, despite this the cellular morphology still appeared intact. Bone

morphology staining with fast green did not show any discernible changes between

the acid groups. Cartilage tissue morphology was well preserved when decalcified

by EDTA regardless of the condition used, formic acid revealed minor damage to

cytological structures and tissue damage was observed in HCl and nitric acid groups.


Collagen type I antigenicity was preserved in rat specimens decalcified in EDTA and

formic acid (Figure 5 – 7 A – F). Positive immunoreactivity was observed throughout

the bone matrix as indicated by brown staining of the tissue matrix. Tissue

morphology of the formic acid and EDTA groups had specific positive staining

throughout the bone matrix. Specimens in HCl and nitric acid showed signs of tissue

matrix damaged as evidenced by the loss of tissue morphology and non-specific

collagen type I staining. Deterioration of the lamellar bone matrix was observed in

the nitric acid and HCL microwave groups (Figure 5 – 7 I – L). Furthermore, damage

was also observed in the conventional HCl groups, although it was possible to

distinguish lamellar bone morphometry (Figure 5 – 7 G, H). Staining of cell nuclei

was observed in EDTA and formic acid groups, however, in the HCL and nitric acid

groups cell nuclei staining was absent. Antigenicity of collagen type 1 in specimens

decalcified by EDTA in the microwave and all formic acid groups did not show any

differences to the routine EDTA condition (4°C).


Figure 5-6: Safranin O & Fast green staining in rat tibia for cartilage and bone. Cartilage

tissue stains red and bone stains green. The chondrocytes and nuclei in EDTA are well

preserved in comparison to the acids. Magnification x20 scale bar 50µm.


Figure 5-7: Collagen-1 immunohistochemistry in rat tibia for bone matrix. Positive staining for

collagen appears brown and cell nuclei appear blue. Specific positive staining for collagen type

1 is observed in specimens in all decalcification solutions. Specimens decalcified in nitric acid

showed a lack of detail in the tissue matrix as indicated by arrows. Tissue matrix was well

preserved in EDTA and formic acid. Conventional HCl conditions (J, H) showed evidence of

tissue preservation, however, cell nuclei staining was absent. Magnification x20 scale bar 50µm.

A B C

D E F

G H I

J K L



Vasculature morphology and serum protein antigenicity of vWF was well preserved

in specimens decalcified by EDTA and formic acid regardless of the decalcification

conditions (Figure 5 – 8 A – F). Positive staining was localised to the blood vessels

and the surrounding endothelium. Circulating vWF within the blood vessels stained

positive indicating that serum protein antigenicity was intact for all conditions in

EDTA and formic acid. Staining for vWF in HCL decalcified specimens revealed

similar staining patterns to EDTA and formic acid, however, non-specific background

staining was seen throughout the bone matrix (Figure 5 – 8 G – I). In addition

positive staining within the vasculature was more intense, although this staining

appeared smudged which hindered identification of endothelium. While positive

staining was observed in the nitric acid group, the staining intensity appeared

diminished or was absent in blood vessels (Figure 5 – 8 J - L). Furthermore, detail in

cellular morphology was lost as nuclei staining was not observed in the

endothelium. Non-specific background staining was observed in the tissue matrix.

Decalcification of rat tibias in EDTA or formic acid demonstrated vasculature tissue

preservation, however, specimens in HCl or nitric acid showed signs of damage as

evidenced by vWF staining.


Figure 5-8: vWF immunohistochemistry in rat tibia for vasculature and serum proteins.

Positive staining for vWF appears brown in blood vessels and cell nuclei appear blue.

Endothelium lining of vasculature is well preserved and stains positive in all groups. Serum

proteins within the blood vessels is also positive. Magnification x20 scale bar 50µm.

A B C

D E F

G H I

J K L


5.1.3 Sheep staining


Examination of H&E stained sections revealed differences in tissue preservation

related to the decalcification solutions. Good preservation of the tissue morphology

was observed in specimens decalcified in EDTA regardless of the conditions used

(Figure 5 – 9 A – C). Osteon morphology was well preserved in the EDTA and formic

groups and is indicated by double headed arrows in Figure 5 – 9. In addition cement

lines between osteon borders were visible and are identified using arrow heads.

Haversian canals were observed in the centre of osteons and the epithelial lining

was well preserved (Figure 5 – 9). Specimens decalcified in formic acid showed

similar staining patterns to EDTA, however, cement lines were only observed in

specimens decalcified in the microwave group and not in the conventional groups.

Staining patterns for specimens decalcified in HCl was observed to be similar to

formic acid. In HCl cement lines were absent in the conventional groups, however,

for the microwave group they were distinguishable. Tissue morphology showed

signs of damage in the nitric acid groups with a loss of tissue matrix architecture

observed in the osteons. Cell nuclei and cement line staining was absent or faint in

the tissue matrix. Light microscopy analysis of H&E stained sheep tibia slices

revealed tissue was well preserved when decalcified in EDTA or formic acid for all

conditions, in contrast, specimens in HCl or nitric acid showed evidence of tissue

degradation.


Bone tissue matrix in sheep specimens was stained for collagen type I. Sheep tibia

specimens decalcified in EDTA and formic acid demonstrated good tissue and

antigenicity preservation. Positive staining was observed throughout the bone

matrix and tissue architecture was intact in all EDTA groups. Furthermore, cement

lines, osteons, Haversian canals and lamellar bone were visible throughout the bone

matrix. Specimens decalcified in formic acid had similar staining results to the EDTA

groups, however, tissue morphology detail was lost. Remodelling sites, osteons and

cement lines were not observed in the conventional 4°C group compared to the

25°C conventional group and microwave group. Tissue morphology of specimens


decalcified in HCl or nitric acids showed signs of deterioration. Positive staining was

observed in the bone matrix for HCl, however, tissue architecture was unable to be

identified in all groups. Likewise, in nitric acid positive staining was observed,

however it was inconsistent throughout the bone matrix with some regions

remaining unstained for all groups. Cell nuclei staining in the formic, HCl and nitric

acid groups were either diminished or absent.


Figure 5-9: Hematoxylin & Eosin staining in sheep tibia. Bone matrix stains various shades of

pink and purple with cellular nuclei staining blue. Cement lines in the cortical bone matrix are

clearly visible in the EDTA and formic groups as indicated by arrows, however, in HCl and

nitric they were not well preserved. Osteons are indicated by double headed arrows and were

well preserved in the EDTA groups and rapid formic and HCl groups. Note that in the HCl and

nitric acid groups cell nuclei staining is absent and tissue matrix appears dark. Magnification

x20 scale bar = 50µm.

A B C

D E F

G H I

J K L


Figure 5-10: Collagen-1 immunohistochemistry in sheep tibia for bone matrix. Positive staining

for collagen appears brown and cell nuclei appear blue. Specific positive staining for collagen

type 1 is observed in specimens in all decalcification solutions. Specimens decalcified in HCl and

showed a lack of detail in the tissue matrix. Nitric acid staining was patchy and inconsistent in the

bone matrix as indicated by arrows. Tissue matrix was well preserved in EDTA and formic acid

with positive staining observed throughout the bone matrix. Magnification x20 scale bar 50µm.

A B C

D E F

G H I

J K L



Sheep tibia specimens decalcified in EDTA had well preserved vascular tissue

irrespective of the conditions tested. Immunohistochemistry revealed intact

endothelium and circulatory proteins. Specimens decalcified in formic acid had

similar staining patterns to EDTA, however, however, some cytological detail was

lost in all decalcification conditions. Endothelium and serum proteins in blood

vessels did not stain as intensely as what was observed in the EDTA groups.

Furthermore, some endothelial tissue appears to have been lost during the

decalcification or staining process, most notably in the 4°C conventional group.

Positive staining is diminished for endothelial tissue and serum proteins in

specimens decalcified by HCl. A loss of endothelium is evident in blood vessels,

furthermore, a loss of antigenicity is observed in serum proteins as partial positive

staining is observed. This same trend is noted when HCl was used in the microwave,

although the intensity of positive staining is greater when compared to the

conventional (4°C & 25°C) groups. Tissue vasculature is partially preserved in tissue

decalcified in nitric acid. Endothelium tissue is clearly visible lining blood vessels in

all groups. However, serum protein antigenicity has been lost as indicated by partial

positive staining in the blood vessels for all groups.


Figure 5-11: vWF immunohistochemistry in sheep tibia for vasculature and serum proteins.

Positive staining for vWF appears brown in blood vessels and cell nuclei appear blue. Endothelium

lining of vasculature is well preserved and stains positive in all groups. Serum proteins within the

blood vessels is also positive in EDTA and formic acid as indicated by arrow heads. Magnification

x20 scale bar 50µm.

A B C

D E F

G H I

J K L


5.2 HISTOLOGICAL DISCUSSION

5.2.1 Tissue morphology

Hematoxylin & Eosin

Tissues are routinely stained with H&E in diagnostic and research laboratories to

provide a histological overview of specimens. Staining with H&E in this study

demonstrated the effects decalcification had on bone in mice, rats and sheep.

Specimens that were decalcified in EDTA, both conventionally and using a

microwave had well preserved tissue morphology. In formic acid decalcified

specimens showed intact tissue and specific nuclei staining. This is in the agreement

with previous reports in the literature that formic acid is an effective decalcification

solution that does not compromise tissue morphology [15], [22], [37], [44], [58],

[60]–[62]. Results from this study demonstrate that formic acid can be used as a

decalcification solution to achieve similar results to decalcification by EDTA. In

mineral acids (HCl & Nitric) decalcified bones had deleterious effects on tissue

morphology with cell nuclei and tissue matrix staining showing signs of damage.

DNA in the cell nuclei could have been degraded because of the acidic conditions in

both the HCl and nitric acid solutions. This phenomenon has also been reported in

other studies [15], [25], [40], [48], [49], [58]. Furthermore, the bone matrix in rats

and sheep decalcified in mineral acids appeared darker than the formic and EDTA

groups. When mineral acids are used to decalcify bone, it may cause the pH of the

tissue matrix to become more acidic degrading proteins within the cytoplasm and

tissue matrix. Therefore, the increased exposure time that rat and sheep tibia had

to acid may have resulted in degradation of the tissue. As a consequence, affinity of

the tissue for eosin staining is reduced [63]. Consequently, acid decalcification may

cause a decrease in pH which may result in an increased affinity for hematoxylin

uptake in the bone matrix, although further testing would be needed to confirm this

occurrence.

Safranin O

Fracture healing and osteoarthritis studies routinely investigate cartilage tissue, this

often requires the decalcification of bone to allow for specimen processing. Staining

for bone and cartilage in the specimens decalcified in EDTA showed preservation of


cartilage morphology. It has been previously reported that EDTA chelation can

affect the proteoglycan structure of cartilage and, therefore compromise staining

[22], [26], [64], however this was not observed in this current study. Formic acid is

often chosen in cartilage studies as a decalcification solution, because it has fast

decalcification time as well as it is known to have minimal effect on cartilage tissue

morphology [65]. Staining results in both mice and rat tibia sections confirmed the

selection of formic acid for bone decalcification in cartilage studies. In this study,

HCl and nitric acid showed evidence of damage to the tissue morphology in

cartilage and bone. However, the observed tissue shrinkage in Figure 5.2 may have

been artefact due to dehydration caused by prolonged exposure to ethanol post-

fixation for mice specimens [5] An additional cause for chondrocyte shrinkage may

have occurred during fixation due to an osmolarity imbalance of the fixative [66].

Chondrocyte nuclei stained green instead of black this would be characteristic of

acidic degradation during decalcification, whereas, osteocyte staining was either

absent or diminished in the bone matrix. However, the overall morphology

remained intact and the staining results could still be interpreted. In contrast to this

study, Hoemann et.al advocated the use of HCl to decalcify bone as tissue

morphology and showed that osteochondral specimens were adequately preserved

[67]. Microwave decalcification did not appear to result in negative outcomes for

rat and mouse tibias when compared to their relative conventional condition using

EDTA and formic acid. This would suggest that microwaves can be used for

decalcification using EDTA or formic acid solutions for tissue undergoing cartilage

analysis by safranin-O.

5.2.2 Immunohistochemistry

Collagen type I

The goal of achieving good quality immunohistochemistry staining becomes difficult

given the special considerations bone processing requires. [58]. Staining with col-1

staining allowed for analysis of the bone matrix and the effects different

decalcification conditions had on the tissue in mice, rats and sheep. As expected,

tissue preservation was not compromised in EDTA at 4° and 25°C for all species.

Furthermore, decalcification using a microwave (37°C) showed comparable levels of


tissue preservation and staining specificity to the conventional decalcification

groups (4°C & 25°C). In previous work using microwave EDTA decalcification of

temporal bones by Cunningham and Madden showed that microwave

decalcification (45°) preserved and enhanced the tissue microstructure [20], [40].

Further evidence suggests that decalcification of specimens in EDTA at higher

temperatures (37 - 45°C) is possible having tissue and morphology antigenicity

comparable to conventional conditions (4°C and 25°C) [46]. Targeted chelation of

calcium by EDTA may be the reason why the effect on tissue antigenicity is minimal,

regardless of the temperature [56]. Adequate fixation of the tissue would guard the

antigens from any potential deleterious effects from heat when microwave is used

to decalcify bones [47].

Decalcification by organic (formic) and mineral acids (HCl and nitric) produced

mixed results regarding the preservation of tissue morphology and retention of

antigens in routine staining and immunohistochemistry, respectively. Formic acid

has been previously reported to be acceptable for use by pathology and research

labs as it is able to decalcify bone without causing damage to the tissue morphology

and antigenicity[14], [23], [44]. The results from this study agree with the previous

reports in the literature suggesting that formic acid is generally acceptable as a

decalcification solution when speed is prioritised over optimal staining and

morphology. Previous studies have also reported the acceptability of results

obtained from HCl and nitric acid decalcification [23], [26], [38]. In contrast, Neves

et.al found that the morphology of the tissue was poor despite observing specific

positive staining [38]. The results and conclusions drawn from this study are in

agreement with Neves et.al where they concluded that HCl acid should be avoided

for bone decalcification if preservation of bone matrix and antigenicity is desired.

Von Willebrand Factor

Staining for vWF was performed to assess if the decalcification procedures in this

study had an effect on the cells and serum proteins in the tissue. Staining in EDTA

decalcified groups clearly showed epithelial cells and circulating protein within

blood vessels. Previous studies reported the suitability of EDTA successfully

preserving vasculature antigenicity regardless of the conditions used [68], [69].


Microwave decalcification in EDTA in this study showed similar tissue morphology

and staining specificity when compared to the conventional groups.

Formic acid decalcification results showed specific staining for vasculature and the

tissue morphology appeared intact, however, the staining resolution was slightly

smeared. Below optimal staining resolution may be caused by formic acid

denaturing vasculature and serum proteins during decalcification, as it diffuses

throughout the sample. Hatta et.al reports that tissues decalcified by formic acid in

an ultrasonic cleaner was able to retain antigens, although a comparison against

EDTA was not conducted in this study [44]. Studies have reported that fixation was

a key factor for antigen preservation when decalcifying with formic acid [70], [71].

The hydrochloric and nitric acid groups had background staining in the bone matrix

and unclear resolution of positive staining within the vasculature for all species.

Tissue morphology and antigenicity appears to be negatively affected by the

mineral acids. In this study the time of exposure to mineral acids was shorter

compared to the reports found in the literature, which may be the reason why

positive staining and tissue preservation was observed in some HCl and nitric acid

groups. [13], [22], [26]. Athanasou et al reported that when sufficient fixation and

controlled decalcification in HCl and nitric acid was used no negative effects were

observed on diagnostic markers e.g vWF, Prostate Specific Antigen[23]. This was in

contrast to our study, which demonstrated that HCl or nitric acid decalcification,

despite being carefully controlled had caused the damage to the tissue morphology

and antigenicity. Based on the results from this study even with minimal

decalcification times and acid exposure, the deleterious effects of mineral acids on

the tissue were very evident.

84 Chapter 6: Conclusion

Chapter 6: Conclusion

6.1 GENERAL CONCLUSION

This study showed that it was possible to accurately determine an endpoint time of

bone decalcification using µCT, which was based on the BV parameter reaching 0

(%). This method is advantageous compared to weighing, chemical and mechanical

testing methods as it has a greater degree of accuracy. Furthermore, this method

does not cause any damage to the specimens.

Decalcification times for mice, rat and sheep were greatly decreased when using a

microwave (37°C) compared to conventional methods (4°C & 25°C). For EDTA, the

time required to achieve decalcification using a microwave (37°C) was 50% - 80%

shorter than conventional methods (4°C & 25°C) for all species. Comparatively,

when formic, HCl and nitric acids were used in the microwave, decalcification time

was decreased by approximately 40 – 50% compared to the respective conventional

methods.

Antigenicity and tissue morphology decalcified in EDTA were well preserved for all

conditions and in all species. Likewise, formic acid also showed preservation of

tissue morphology and antigens for all conditions in all species. Reliable results can

be obtained rapidly if specimens are decalcified in formic acid. Damage to the tissue

morphology and a loss of antigenicity was observed in specimens for all

decalcification conditions in HCl and nitric acid. Therefore, it is advisable to avoid

using HCl or nitric acid for decalcification. To conclude, decalcification in EDTA and

formic acid can be performed using laboratory microwaves with no observable

negative consequences on bone.

This study demonstrates the value a laboratory microwave has in increasing the

efficiency of histological processing. Decalcification times were greatly reduced

allowing for a more rapid processing of specimens from dissection and fixation

through to embedding, sectioning and staining. Furthermore, it was demonstrated

that when using a microwave to decalcify bone specimens in EDTA or formic acid,

Chapter 6: Conclusion 85

the standard of histology results were comparable to the respective conventional

methods.

6.2 STUDY LIMITATIONS & FUTURE WORK

While this study had taken into account routinely used stains found in literature for

bone and cartilage testing, it would be of value to determine what effect the

decalcification methods has on the tissue morphology and antigenicity of other

stains and antibodies commonly used in bone research.

For example for immunohistochemistry antigens such as Osteocalcin, Collagen – II,

Alkaline Phosphatase and CD34 and histological stains like alcain blue and toluidine

blue should be performed to assess antigenicity, cartilage morphology and bone

matrix. Furthermore, investigating the accuracy of the scout view feature of µCT to

determine the decalcification endpoint would be of benefit as well, as this would

decrease the scanning time and cost per sample. Moreover, evaluation of human

tissue would also be of interest as the decalcification times and protocols would be

directly applicable to clinical settings.

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93

Chapter 7: Appendix A

Mice Collagen type 1 negative control

Mice Collagen type 1 isotype control

Mice vWF negative control

Mice vWF isotype control

Rat Collagen type 1 negative control

Rat Collagen type 1 isotype control

Rat vWF negative control

Rat vWF isotype control

Sheep Collagen type 1 negative control

Sheep vWF negative control

Representative images of positive vWF stained blood vessels

Representative images of positive vWF stained cells in the bone marrow

94

Appendix 1a: Representative images of negative control results for Collagen 1

staining in mice. Positive staining was not observed in the tissue

95

Appendix 1b: Representative images of isotype control results for Collagen 1 staining

in mice. Positive staining was not observed in the tissue

96

Appendix 1c: Representative images of negative control results for vWF staining in

mice. Positive staining was not observed in the tissue

97

Appendix 1d: Representative images of isotype control results for vWF staining in

mice. Positive staining was not observed in the tissue

98

Appendix 1e: Representative images of negative control results for Collagen 1

staining in rats. Positive staining was not observed in the tissue

99

Appendix 1f: Representative images of negative control results for vWF staining in

rats. Positive staining was not observed in the tissue

100

Appendix 1g: Representative images of isotype control results for Collagen 1 staining

in rats. Positive staining was not observed in the tissue

101

Appendix 1h: Representative images of isotype control results for vWF staining in

rats. Positive staining was not observed in the tissue

102

Appendix 1i: Representative images of negative control results for Collagen 1 staining

in sheep. Positive staining was not observed in the tissue

103

Appendix 1j: Representative images of negative control results for vWF staining in

sheep. Positive staining was not observed in the tissue

104

Appendix 1k: Representative images of positive vWF stained blood vessels. Note the positive

lining and nuclei around the circumference of the vessels can be observed as epithelial cell lining

105

Appendix 1l: Representative images of positive vWF stained cells in the bone marrow.

Megakaryocytes in the bone marrow express vWF. The staining is specific for these large

multinucleated cells.

Bone Decalcification, Conventional Decalcification, Rapid ... · Bone Decalcification, Conventional...

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