Validation of the Application of Backscattered Ultrasound and

Photoacoustic Signals for Assessment of Bone Collagen Contents Using

Hydroxyprline Assay

by

Shendu Ma

998736293

A thesis submitted in conformity with the requirements for the degree of

BACHELOR OF APPLIED SCIENCE AND ENGINEERING

Department of Mechanical and Industrial Engineering

University of Toronto

Supervisor: Professor A. Mandelis

April 23, 2012

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i

ABSTRACT

In the previous research on the application of backscattered ultrasound (US) and

photoacoustic (PA) signals for the assessment of bone collagen and mineral contents,

Center for Advanced Diffusion-Wave Technologies (CADIFT) has identified that both PA

and US are sensitive to mineral content changes. In addition, PA is also sensitive to changes

in the collagen content of the bone, but US is not significantly sensitive to these changes.

This thesis project focuses on using hydroxyproline (HP) collagen assay to validate the

sensitivity of US and PA on collagen content assessment in bone samples. Results from

the HP collagen assay qualitatively support the conclusion from previous research.

Effectiveness and efficiency of HP collagen assay on measuring total collagen content level

for the samples used in previous research settings is confirmed with positive match between

the predicted and measured collagen content levels. Ongoing process and further direction

involve building quantitative correlation between specific collagen content level and US

and PA signal.

3

ii

ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to Professor Andreas Mandelis for

his continuous support and encouragement since my summer research at CADIFT in 2014.

In September 2015, Professor Andreas Mandelis kindly gave me the opportunity to

participate in this thesis project as part of his ongoing project that could potentially lead to

publication. Throughout the entire process of this thesis project, Professor Andreas

Mandelis has generously provided me with abundant research resources, precious personal

time and inspiring academic support.

I would like to thank Dr. Bahman Lashkari for his supervision and instruction on

this thesis project. As my closest colleague and most helpful teacher in the lab, Dr. Bahman

Lashkari has always been patient and helpful throughout this project.

Lastly, I would like to thank Professor Craig Simmons, Professor Edmund Young

and Professor Michael Sefton from the Department of Mechanical and Industrial

Engineering at University of Toronto for providing me useful information and authorizing

me to use their lab facilities to finish this project.

4

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................................ 5

LIST OF TABLES .............................................................................................................. 6

1.0 INTRODUCTION ........................................................................................................ 7

2.0 BACKGROUND ........................................................................................................ 11

2.1 COLLAGEN CONTENT IN BONES ............................................................................... 11

2.2 DECOLLAGENIZATION OF BONE TISSUE ................................................................... 12

2.3 UA AND PS ASSESSMENT ........................................................................................ 13

3.0 LITERATURE REVIEW ........................................................................................... 16

3.1 ELISA COLLAGEN ASSAY ....................................................................................... 17

3.2 SIRIUS RED COLLAGEN ASSAY ................................................................................ 17

3.3 HYDROXYPROLINE COLLAGEN ASSAY ..................................................................... 18

4.0 EXPREMENTAL PROCEDURE .............................................................................. 20

4.1 LIST OF MATERIAL ................................................................................................... 20

4.2 SAMPLE PREPARATION – HYDROLYSIS .................................................................... 21

4.3 ASSAY PROCEDURE .................................................................................................. 22

5.0 RESULTS ................................................................................................................... 23

5.1 ASSUMPTIONS AND CONDITIONS: ............................................................................. 23

5.2 RAW RESULTS OF COLORED PLATE ......................................................................... 24

5.3 CALCULATIONS AND DATA HANDLING .................................................................... 27

6.0 DISCUSSION ............................................................................................................. 31

6.1 REPRODUCIBILITY AND REPEATABILITY .................................................................. 31

6.2 AGREEMENT TO DECOLLAGENIZATION GROUP SAMPLES ........................................ 31

6.3 DEFICIT FOR DEMINERALIZATION GROUP SAMPLES ................................................ 34

6.4 OTHER SOURCES OF ERROR ..................................................................................... 34

7.0 ONGOING PROCESS AND FUTURE DIRECTION ............................................... 38

8.0 CONCLUSION ........................................................................................................... 39

REFERENCES ................................................................................................................. 41

FIGURES AND TABLES ................................................................................................ 52

11.0 APPENDICES .......................................................................................................... 53

11.1 APPENDIX A: COMPARISONS BETWEEN COLLAGEN ASSAY METHOD AND KIT OFF-

THE-SHELF ..................................................................................................................... 53

11.2 APPENDIX B: HYDROXYPROLINE COLLAGEN ASSAY PROTOCOL FROM CHONDREX

INC. ................................................................................................................................ 57

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LIST OF FIGURES

Figure 1: Color Comparison between First Batch of Samples (on the right) and Second

Batch of Samples (on the left). ......................................................................................... 24

Figure 2: Raw Results before Adding Dyeing Agents...................................................... 25

Figure 3: Hydroxyproline Assay Results for Tested Samples. ......................................... 26

Figure 4: Sample Taken from Different Sites on the Same Bone Tissue. ........................ 33

Figure 5: Cap Deformation due to Over-heating during Incubation................................. 36

Figure 6: Cap Screw Thread Corrosion due To Volatilized HCl during Incubation. ....... 36

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LIST OF TABLES

Table 1: Materials used in Hydroxyproline Collagen Assay ............................................ 20

Table 2: Content in the Wells on the Plate. ...................................................................... 27

Table 3: Semi-quantitative Results for Tested Samples (units all in μg/ ml). .................. 28

Table 4: Translated Results in Weight Percentage. .......................................................... 29

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1.0 INTRODUCTION

In the previous research on the application of backscattered ultrasound (US) and

photoacoustic (PA) signals for the assessment of bone collagen and mineral contents

conducted by the Center for Advanced Diffusion-Wave Technologies (CADIFT), the

backscattered US and back-propagating PA signals from trabecular bones, and their

variations with reduction in bone minerals and collagen content were examined (1). The

results showed that both PA and US are sensitive to reduction of the mineral content of

bone. Moreover, PA is also sensitive to changes in the collagen content of bone, but US is

not significantly sensitive to these changes (1).

In the previous research, the samples were washed and kept in saline solution for

up to 2 days to dissolve the blood inside the pores. The samples were treated either with

ethylenediaminetetraacetic acid (EDTA) or with hypochlorite solution (NaOCl). The first

group was demineralized with 50% solution of EDTA in distilled water (pH=7.7) for

decalcification simulating the osteoporosis disease. This solution produces a very slow and

gentle demineralization (2, 3). The extent of the demineralization depends on solution

concentration and exposure duration as well as on the exposed area and bone compactness.

The second group was treated with sodium hypochlorite solution to decollagenize the

sample (4, 5). The exposure duration for samples treated with EDTA was 5 hours except

for sample 1 which was demineralized for 10 hours; and the samples treated with

hypochlorite solution which was demineralized for 3 hours except for sample 2 which was

decollagenized for 6 hours.

The major issue with the demineralization and decollagenization method is that it

does not provide the exact weight percentage of the mineral or collagen content remained

8

in the samples after treatments. In order to obtain these numbers and quantitatively

correlate the percentage of mineral or collagen loss with US and PA results, additional

measurement is needed. The other minor issue with this demineralization and

decollagenization method is that the desired degree of demineralization or

decollagenization is obtained by changing solution concentration, exposure duration as

well as exposed areas and bone compactness. The operation is cumbersome and the

accuracy is hard to maintain. Since the objective of the previous research is to propose the

integrated application of US and PA assessment and to examine the relative sensitivity of

it, the desired resolution on the demineralization and decollagenization control level is

relatively low. However, as the research moves forward, a motivation to quantitatively

correlate the results of US and PA assessment to variations in collagen and mineral content

in the samples is encouraged. A more accurate quantitatively controlled demineralization

and decollagenization is also needed for improvement on the resolution of US and PA

assessment.

Due to the complexity of the composition of bone content (6, 7), solving the latter

issue would require more time and resources beyond the scope of this thesis. Moreover,

the current controlling methods of demineralization and decollagenization have advantages

of easy to use, efficient and reliable. It is of a lower priority in terms of validating the results

from previous research compared to the second issue. Although bone mineral density

(BMD) is definitely a major factor in the strength of bones, new studies suggest the chance

of bone fracture even without BMD deficit (6-8). Bone minerals are responsible for

compression strength of the bone and a vital factor for bone integrity. On the other hand,

the organic phase of bones which is mainly collagen type I provides the bone with tensile

9

strength and ductility due to its viscoelastic properties. The reduction of collagen content

with aging (9-11) could be an important factor increasing fracture risk without decrease in

bone minerals. The changes of collagen cross-links during osteoporosis have been the

subject of several studies (12-14). There are still many unclear issues and more research is

needed on the variation of the organic phase of bones with aging and as a result of diseases

such as osteoporosis and diabetes. Therefore, any method or modality that can assess either

the collagen content, or collagen cross-linking, or both, may assist in better understanding

of bone diseases, their diagnosis and even the selection of therapeutic strategies. Some

studies have proposed the assessment of collagen cross-linking by analyzing the urine or

serum (11, 12).

To conclude, because of the reasons stated above, it is not included in this thesis to

optimize the control of demineralization and decollagenization and to validate the bone

mineral content result of the US and PA assessment. The focus of this thesis is to find an

effective and efficient method to measure the bone collagen content level in both treated

and untreated samples. The requirement of the solution is open to destructive method. The

solution could be mechanical, biochemical, chemical etc. The solution should also be able

to support the results from the previous research on US and PA assessment on bone density

detection. The results of measured collagen content level using the final solution proposed

in this thesis should match the results from previous research. Furthermore, after

comparison and analysis over the candidate solutions, a detailed list of material,

experimental procedure, results calculation and analysis of the final solution should be

documented. Then, a verification run of the final solution on the samples used in the

previous research is performed. The results from the verification run is analyzed. In the

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discussion section, deficit between results from the final solution and the previous research

is compared and source of error is analyzed. The thesis is concluded with future direction

and improvement.

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2.0 BACKGROUND

In order to explore the possible solutions that can measure the bone collagen content level,

the role of collagen content in bones, characteristic of collagen content, and reactions with

collagen content need to be reviewed. Secondly, since samples from both before and after

demineralization and decollagenization treatment are tested, the treatment itself and its

effect on the collagen content needs to be evaluated. Lastly, as the validation is required to

match the result from the US and PA methods, they need to be briefly investigated.

2.1 Collagen Content in Bones

Bone is a highly complicated tissue that is capable of adapt itself to mechanical

environment. According to its density, bone can be divided into cortical bones and

trabecular bones. Bone matrix is the smallest unit of bone tissue which consists of the

mineral phase and the collagen fibers. The mineral phase contributes to the stiffness of the

bones while the collagen fibers contributes to the toughness of the bones (7). The

anisotropic structure of bones and the material properties of bone tissues endows the

function of resisting mechanical loads. Bone strength is related to five factors: geometry of

bones, microarchitecture of trabecular bones, the turnover, the mineral and the collagen.

Bone appears to be the only tissue that contains a significant pool of immature crosslinks

(15). Several studies conclude that bone strength is strongly related to tissue mass and

stiffness, which is determined by the mineral phase (16-18), whereas the collagen matrix

contributes mainly to bone toughness (19-24).

Most of the non-cartilaginous tissues including the bone tissues contain both Type

I and Type III collagen (25-27). The individually banded collagen fibers in bone tissues

are also likely to contain Type V collagen (28). Tendon and bone have been considered

12

exceptions to the foregoing generalizations, and the banded fibers of bone are believed to

be composed almost exclusively of Type I collagen. According to evidences from different

biochemical studies such as the studies of bone collagen chemical composition (29),

characterization of the biosynthetic products of bone-derived cell cultures (30), and indirect

immunofluorescent localization (31); it is concluded that bone is an exception to the

foregoing generalization of Type I collagen. Type V collagen has been extracted directly

from bone (32). It was validated and concluded by Douglas R. K.et al. that Type III

containing collagen fibers are detected at all ages examined, from 30 fetal weeks to 80

years. Type VI collagen is present in fetal bone in discrete fibrils separate from Type III

collagen, and becomes restricted to the margins of bone cells and the bone surface by 7

years (33). Therefore, in order to obtain the total collagen content level within cattle bone

samples, the collagen assay must be able to detect collagen Type I to Type VI so that it

will cover all possible types of collagen existed in bone tissues.

2.2 Decollagenization of Bone Tissue

In the previous research, three cattle femurs (Angus, Canadian) were purchased

from a local butcher. Ten trabecular bone samples were cut from the femurs. Samples were

cut with a saw to produce flat measurement areas without any cortical over-layer. The

samples were washed and kept in saline solution for up to 2 days to dissolve the blood

inside the pores. The samples were treated either with ethylenediaminetetraacetic acid

(EDTA) or with hypochlorite solution (NaOCl). The first group was demineralized with

50 % solution of EDTA in distilled water (pH=7.7) for decalcification simulating the

osteoporosis disease. This solution produces a very slow and gentle demineralization (34,

35). The extent of the demineralization depends on solution concentration and exposure

13

duration as well as on the exposed area and bone compactness. The second group was

treated with sodium hypochlorite solution to decollagenize the sample (35-37). For ease of

reference to the samples, those demineralized with EDTA are identified with odd numbers

and the ones which were decollagenized with hypochlorite solution are classified with even

numbers. The exposure duration for samples treated with EDTA was 5 hours. Exceptions

were: Sample 1 which was demineralized for 10 hours; and the samples treated with

hypochlorite solution which were demineralized for 3 hours except for sample 2 which was

decollegenized for 6 hours (sample numbering is consistent from previous research to this

thesis project).

The decalcification treatment should not have any interaction with the

decollagenization treatment since only non-organic composition is removed in the

decalcification treatment. This decollagenization method removes collagen content in

samples regardless of the types of collagen. However, when EDTA binds with metallic

ions, it can also act like antioxidant. Therefore, for samples that are only treated with

EDTA, they could possibly reflect antioxidant properties. This effect could potentially lead

to an elevation of collagen content in EDTA treated samples compared to untreated

samples as the organic composition of such samples may have higher resistance to organic

oxidation over time (34-37).

2.3 UA and PS Assessment

The use of ultrasound for diagnosis of osteoporosis started in the 1980s and

essentially depends on the measurement of the speed of sound (SOS) and on normalized

broadband ultrasonic attenuation (nBUA) (38-42). New approaches such as fast and slow

wave detection and backscattered ultrasound were also introduced recently (42-45) and

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clinical instruments based on these parameters were proposed (43, 46). Although they did

not reduce the dominance of SOS and nBUA measurements in quantitative ultrasound

(QUS), these alternative approaches introduce parameters that may reveal more

information about the state of health of trabecular bones. The large number of mechanical

parameters affecting the ultrasonic response, as well as the substantial variation of human

bone tissue and complexity of its structure, are the major challenges of QUS in offering a

reliable diagnostic method for osteoporosis. Nevertheless, the backscatter method has the

advantage of facilitating measurements at crucial sites like hip or spine where the risk of

fracture is high. Several parameters have been introduced and applied to quantify bone

backscattered ultrasound. Some typical parameters are the frequency dependent

backscatter coefficient (BSC or η(f)) (47-54), the apparent integrated backscatter (AIB)

(55-58), and the broadband ultrasound backscatter (BUB) (59-62).

In the previous study, Lashkari et al. measured both the US backscattering and also

the photoacoustic (PA) back-propagating signal. The dependence of the PA signal on

optical properties of the tissue provides more specific information about bone composition

and structure. In their previous studies (63-66), it was shown that laser light can penetrate

at least as deep as 1.5 mm in cortical bone and 3 mm in cancellous bone and can generate

a detectable PA signal from those depths. It was shown that the PA back-propagating signal

is sensitive to controlled changes of bone minerals. The PA signals also indicate the

sensitivity to variation in bone composition. PA signal could also be detected as “coherent

structure backscattering” in a way very similar to ultrasound in frequencies above 1 MHz.

PA was also used to generate guided ultrasound waves in long bones for bone assessment

(66, 67).

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To conclude, the signal measured as AIB by US and PA in dB reflects the level of

mineral or collagen content in bone samples, the negative sign of the results refers to a

demineralization or decollagenization treatment. The larger the magnitude, the more

sensitive the assessment is on the specific sample.

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3.0 LITERATURE REVIEW

Appropriate methods that fits the objective of quantify collagen content in bone

samples are reviewed in this section. The most effective biochemical application is to use

the chemicals that selectively bind to specific bonds in unique amino acids existing in

specific type of collagen. Antibodies that bind to specific pathogens on the amino acid or

the collagen fiber can also satisfy such application through similar mechanism. Based on

this theoretical background, the following collagen assay could be our candidate solution:

ELISA for specific types of collagen (68, 69)

ELISA for specific pro-domains of collagen (68, 69)

Western blotting using specific collagen antibodies (69, 71)

Sirius Red based assays for soluble collagen (71-73)

Tissue hydrolysis followed by analysis of Hydroxyproline residues (either by a

colorimetric kit or by HPLC) (74-76)

Based on the feature of these methods and the requirements for our application,

since the photoacoustic and ultrasonic signal detects collagen content regardless of its type,

an assay with no discrimination on collagen type should be chosen. Thus, both Sirius red

and hydroxyproline methods are suitable candidate for our experiment. However, since our

sample would be solid and non-cultured after been tested by photoacoustic and ultrasonic

detection, hydroxyproline method would only require tissue hydrolysis while Sirius red

would require another sample solubilisation beforehand. Theoretical background will be

shortly introduced in the next two sections. Additionally, the best fitted candidate assay

17

methods are evaluated from the time consumption for each array. The final selection should

have the smaller time consumption to achieve best efficiency. The detailed comparison

including manufacturer comparison and economic comparison can be found in Appendix

A.

3.1 ELISA Collagen Assay

Various species and types of collagen can be used as an antigen in ELISA for

studying antibody specificity and cross-reactivity (68, 69). Since collagen is a rigid

fibrillary protein with unique physical and chemical properties and differs from other

globular proteins, special attention for handling this protein is required. For example,

immunoglobulins in human and animal sera bind to polymeric and fibrillary collagen non-

specifically, and create significantly high false-positive reaction in ELISA (68).

Similarly, a secondary antibody in ELISA also binds to fibrillary collagen non-

specifically and creates high background values. Furthermore, these forms of collagen

affect the assay sensitivity significantly due to the formation of excess collagen layer on

the surfaces of ELISA plate (69). In general, ELISA collagen assay is more appropriate for

collagen assay on specific type of collagen and it would require a summation of several

assays on different collagen types in order to obtain the total collagen content in a sample.

Therefore, it is not the best candidate for our objective although it is most commonly used

in biochemical researches.

3.2 Sirius Red Collagen Assay

Sirius Red specifically binds to the [Gly-X-Y]n helical structure of fibrillary collagens such

as Type I to V collagen, and is used for detecting all types and species of collagen, whereas

Fast Green binds to non-collagenous proteins (75, 76). Because this assay does not require

18

collagen solubilisation, it is widely used for the measurement of total collagen content in

various tissues (77-81). Since Sirius Red and Fast Green have absorptions at 540 nm and

605 nm respectively, the OD values of the extracted dyes can be used for the calculation

of collagen and non-collagenous protein content in each section. For general histological

studies in which tissue sections are 10-20 μm thick, the assay sensitivity for collagen and

non-collagenous proteins is greater than 3 μg/section and 50 μg/section, respectively.

Although Red Sirius is selectively targeted for Type I to V collagen, it was discussed in

the previous section that this range should cover the collagen types contained in bone

tissues. On the other hand, the sensitivity and reliability of Sirius Red collagen assay is

improved if the sample is soluble. In order to adapt this assay to our sample condition,

additional pre-process on the samples are required.

3.3 Hydroxyproline Collagen Assay

Due to its highly restricted distribution in collagen, the hydroxyproline content accurately

reflects the amount of collagen in the sample. Therefore, quantitating hydroxyproline has

been utilized for evaluating tissue fibrosis or collagen deposition (82-84). However, classic

hydroxyproline assays are not useful since it requires cumbersome procedures and special

tools (84). Hydroxyproline collagen assay works for quantitation of total collagen of any

type and species in tissue specimens and tissue homogenates (83). More importantly, this

collagen assay is suitable for both soluble and both colored and colorless samples. The

sample preparation is also simpler than Sirius Red collagen assay as it involves only a

hydrolysis process. Thus, hydroxyproline collagen assay is selected as the most appropriate

collagen assay for our objective. Its efficiency and effectiveness is validated through

19

verification experiment on the same samples used in the previous research by Lashkari B.

et al.

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4.0 EXPREMENTAL PROCEDURE

As hydroxyproline collagen assay is selected, a detailed list of material and procedure is

documented in this section. The hydroxyproline assay kit (Catalog #6017) from Chondrex

Inc. is used. Procedures in this section are altered or adapted from the hydroxyproline assay

protocol provided by Chondrex Inc. to fit the conditions of our lab such as availability of

equipment, replacement of material with similar function etc. The original protocol can be

found in Appendix B.

4.1 List of Material

The following table summarizes information of all the material used in the hydroxyproline

assay, the provider of the material and storage instruction are also included for future

reference and budget concern.

Table 1: Materials used in Hydroxyproline Collagen Assay

Item Quantity Amount Storage Provider

Hydroxyproline Standard 1 vial 4 mg/ml x 0.5

ml -20°C Chondrex

10X Chloramine T

Concentrate 1 vial 1 ml -20°C Chondrex

2X DMAB

(dimethylaminobenzaldehyde)

Concentrate

1 vial 5 ml -20°C Chondrex

Solution A - Chloramine T

Dilution Buffer 1 bottle 10 ml -20°C Chondrex

Solution B - DMAB Dilution

Buffer 1 vial 5 ml -20°C Chondrex

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96-well Plate 1 plate

96-well (8-

well strips ×

12)

-20°C Chondrex

Concentrated HCl (12.1N) 1 bottle 500 ml 25°C BioShop

Glass screw-thread vial (2 ml)

with Teflon cap 1 package

100

vials/package 25°C VWR

4.2 Sample Preparation – Hydrolysis

Since all of our samples are in solid state, sample preparation steps for soluble samples

are omitted. Form the trial experiment, no solid residue is observed, samples can be

completely dissolved. Thus, the steps of centrifuge is also omitted. The remaining steps

are listed as below:

1. Weigh 10 mg of a tissue sample in a glass screw-thread vial.

2. Add 100 μl of distilled water.

3. Mash the tissue sample with a small spatula.

4. Add 100 μl of concentrated HCl (10N), and tightly screw on the Teflon cap.

5. Incubate at 120°C for 8 hours. Mix the sample periodically during

incubation.

6. Cool down.

7. If hydrolyzed black residue is still present in the sample, transfer to a micro

centrifuge tube and spin at 10,000 rpm for 3 minutes.

It is a violation of lab safety protocol at the University of Toronto to have the oven or hot

bath turned on without supervision. The 24 hour incubation is unachievable. However, it

has no significant effect on the results of sample hydrolysis if samples are not incubated

for 24 hours. Most samples can be completely dissolved within 8 hours of incubation.

Changes to the specific step is made as presented.

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4.3 Assay Procedure

From the trial experiment, it is observed that most of the samples after sample hydrolysis

are colored but without any solid residue. Therefore, steps for handling colorless samples

are omitted in this section.

1. Prepare Standard Dilutions: Take 100 μl of Hydroxyproline (HP) Standard

and add to 900 μl of distilled water to make 400 μg/ ml of the diluted HP

standard; then serially dilute it with distilled water. For example, mix 500 μl of

the standard (400 μg/ml) with an equal volume of distilled water to make a 200

μg/ml solution, and then repeat it five more times to make 100, 50, 25, 12.5,

and 6.3 μg/ ml standards.

2. Prepare Sample Dilutions: The hydrolyzed samples can be used undiluted. If

necessary, the samples can be diluted with 5N HCl. If your sample has color (is

not clear), Sample Blank wells should be prepared due to the higher background

color. See steps 4 and 5 for this process.

3. Prepare Chloramine T solution: Mix 10 μl of 10X Chloramine T solution and

90 μl of Solution A for each well. For example, 10 samples, 7 point standard,

one blank (all in duplicate) will require 3.6 ml of the 1X Chloramine T solution.

Mix 360 μl of 10X Chloramine T solution with 3.24 ml of Solution A. Add

Standards and Samples: Choose 4-1 or 4-2 depending on your samples.

4. Add Reference Wells: Add 10 μl of standards, distilled water (blank, B) into

the first two columns on the plate, and samples into the remaining empty wells

in duplicate.

5. Add 1X Chloramine T Solution: Add 100 μl of the 1X Chloramine T solution

into the purple and orange wells, and add 100 μl of Solution A into the green

wells. Incubate at room temperature for 20 minutes.

6. Prepare DMAB solution: Mix 50 μl of 2X DMAB solution and 50 μl of

Solution B for each well. For example, 10 samples, 7 point standard, one blank

(all in duplicate) will require 3.6 ml of the 1X DMAB solution. Mix 1.8 ml of

2X DMAB solution with 1.8 ml of Solution B.

7. Add 1X DMAB solution: Add 100 μl of 1X DMAB solution into all wells and

incubate at 60°C for 30 minutes.

8. Read Plate: Read the OD values at 530-560 nm. If the OD values of samples

are greater than the OD values of the highest standard, re-assay the samples at

a higher dilution.

23

5.0 RESULTS

5.1 Assumptions and Conditions:

Due to the unavailability of the colorimeter or a plate reader, the semi-quantitative results

are obtained at this stage while quantitative results are under its way by using facilities

from other labs. This means that there is no fully quantitative calculation based on the

optical density (OD) values as proposed by the assay protocol. By conducting the assay

procedure to sample 1, sample 2, sample 4, sample 6 and sample 3 on both treated and

untreated samples, raw results are shown is Figure 1. For this batch of samples, since the

24 hours of incubation under 120 °C was not maintained, instead, in order to completely

dissolve solid samples, it took 5 days of incubation under room temperature. The prepared

samples are colorless. However, when the incubation condition is strictly maintained for 8

hrs, sample solutions tuned in to brown color. It may be due to the reaction between the

organic composition and HCl solution. The comparison between two batches of samples

after hydrolysis is illustrated in Figure 1, although second batch of samples are prepared

for further fully-quantitative analysis involving colorimeter measurement of OD level in

the supplementary document of this thesis. Therefore, for the results being analyzed in this

report, procedure for colorless samples were followed.

24

Figure 1: Color Comparison between First Batch of Samples (on the right) and Second Batch of Samples

(on the left).

5.2 Raw Results of Colored Plate

Although the fully-quantitative results is under an ongoing process and will be

submitted in the form of supplementary document, the semi-quantitative results are still of

significant value in terms of validate the effectiveness and efficient of hydroxyproline

collagen assay. Figure 2 shows the raw results without dyeing agents added. A slight

difference in color density/optical density is noticeable in the raw results. For example,

solution in A3 and A4 is significantly higher in optical density compared to solution in B3

and B4. C3, C4 and D3, D4 differentiate with each other in the similar fashion. All samples

are duplicated to evaluate the repeatability and reproducibility of the result. This means

25

that color for column 1 should be the same as column 2, and so are column 3 and column

4, column 5 and column 6.

Figure 2: Raw Results before Adding Dyeing Agents.

The Chloramine T Solution and the DMAB solution function as dyeing agents in

the collagen assay. They do not add any other effect to the binding of hydroxyproline and

collagen amino acid. After dyeing operations and incubation, the results of the colored

plate is shown in Figure 3 on the next page.

1 2 3 4 5 6

A

B

C

D

E

F

G

H

26

Figure 3: Hydroxyproline Assay Results for Tested Samples.

In Figure 3, column 1 and column 2 are references indicating total hydroxyproline

concentration in the solutions in the wells from low to high. From row A to H, as the color

of the solution becomes deeper, reference hydroxyproline concentration becomes higher.

Notice that for this specific result, well E1 is significantly different from this trend. This

could be a result from false addition of solutions or pollution of solution in this specific

well. However, since all the references are added in duplicate, column 2 along is sufficient

to serve as a complete reference array. Column 3 to column 6 contain all the samples being

tested. The allocation of samples is summarized in Table 2.

1 2 3 4 5 6

A

B

C

D

E

F

G

H

27

Table 2: Content in the Wells on the Plate.

1 2 3 4 5 6

A Ref. 6.25

μg/ ml

Ref. 6.25

μg/ ml

Sample 2

Treated

Sample 2

Treated

Sample11

Treated

Sample11

Treated

B Ref. 12.5

μg/ ml

Ref. 12.5

μg/ ml

Sample 2

Untreated

Sample 2

Untreated

Sample11

Untreated

Sample11

Untreated

C Ref. 25

μg/ ml

Ref. 25

μg/ ml

Sample 4

Treated

Sample 4

Treated

D Ref. 50

μg/ ml

Ref. 50

μg/ ml

Sample 4

Untreated

Sample 4

Untreated

E Ref. 100

μg/ ml

Ref. 100

μg/ ml

Sample 6

Treated

Sample 6

Treated

F Ref. 200

μg/ ml

Ref. 200

μg/ ml

Sample 6

Untreated

Sample 6

Untreated

G Ref. 400

μg/ ml

Ref. 400

μg/ ml

Sample 1

Treated

Sample 1

Treated

H Ref. 800

μg/ ml

Ref. 800

μg/ ml

Sample 1

Untreated

Sample 1

Untreated

5.3 Calculations and Data Handling

By comparing the color density of the samples with the references, semi-

quantitative results of hydroxyproline concentration can be easily obtained by pair-wise

comparison. For example, B3 and B4 have deeper color than A1 and A2, therefore, the

concentration of B3 and B4 is between 12.5 μg/ml to 25 μg/ml. Notice that the lowest

hydroxyproline concentration is 6.25 μg/ml instead of 0 μg/ml in the protocol. The

consequence resulted from the alteration is that if the color in the samples is lighter than

the level of 6.25 μg/ml, it does not need the 0 μg/ml to set the lower limit to conclude that

it is within the 0 to 6.25 μg/ml range. The examples are A3, A4, C3, C4, E3 and E4. Their

colors are all lighter than A1 and A2, therefore, their hydroxyproline concentration should

be within the range of 0 to 6.25 μg/ml. The semi-quantitative results of all wells are

tabulated in Table 3.

28

Table 3: Semi-quantitative Results for Tested Samples (units all in μg/ ml).

1 2 3 4 5 6

A Ref. 6.25

μg/ ml

Ref. 6.25

μg/ ml

0-6.25

μg/ ml

0-6.25

μg/ ml

6.25-12.5

μg/ ml

6.25-12.5

μg/ ml

B Ref. 12.5

μg/ ml

Ref. 12.5

μg/ ml

6.25-12.5

μg/ ml

6.25-12.5

μg/ ml

0-6.25

μg/ ml

0-6.25

μg/ ml

C Ref. 25

μg/ ml

Ref. 25

μg/ ml

0-6.25

μg/ ml

0-6.25

μg/ ml

D Ref. 50

μg/ ml

Ref. 50

μg/ ml

6.25-12.5

μg/ ml

6.25-12.5

μg/ ml

E Ref. 100

μg/ ml

Ref. 100

μg/ ml

0-6.25

μg/ ml

0-6.25

μg/ ml

F Ref. 200

μg/ ml

Ref. 200

μg/ ml

6.25-12.5

μg/ ml

6.25-12.5

μg/ ml

G Ref. 400

μg/ ml

Ref. 400

μg/ ml

6.25-12.5

μg/ ml

6.25-12.5

μg/ ml

H Ref. 800

μg/ ml

Ref. 800

μg/ ml

0-6.25

μg/ ml

0-6.25

μg/ ml

These results are in the unit of μg/ ml which requires a translating calculation to the

weight percentage of the sample. The following formulas are the necessary translating

calculations:

𝐻𝑦𝑑𝑟𝑜𝑥𝑦𝑝𝑟𝑜𝑙𝑖𝑛𝑒 (𝜇𝑔 𝑚𝑙⁄ ) × (𝐷𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝑊𝑎𝑡𝑒𝑟 𝑉𝑜𝑙𝑢𝑚𝑒 𝑚𝑙 + 𝐻𝐶𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 𝑚𝑙)

𝑆𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑚𝑔)= 𝐻𝑦𝑑𝑟𝑜𝑥𝑦𝑝𝑟𝑜𝑙𝑖𝑛𝑒 𝑙𝑒𝑣𝑒𝑙 (𝜇𝑔 𝑚𝑙⁄ ) 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒

𝐻𝑦𝑑𝑟𝑜𝑥𝑦𝑝𝑟𝑜𝑙𝑖𝑛𝑒 𝑙𝑒𝑣𝑒𝑙 (𝜇𝑔 𝑚𝑙⁄ ) 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 ×100

13.5= 𝑐𝑜𝑙𝑙𝑎𝑔𝑒𝑛 𝑙𝑒𝑣𝑒𝑙 (𝜇𝑔 𝑚𝑔⁄ )

The total solution volume added to each well are 200 μl in each well. And according to the

procedure, 10 μg of solid bone samples are added. Therefore, for a 6.25 μg/ml

hydroxyproline concentration, it is translated into:

29

𝐶𝑜𝑙𝑙𝑎𝑔𝑒𝑛 𝑙𝑒𝑣𝑒𝑙 =6.25(μg ml⁄ ) × 0.2(ml)

10 (mg)×

100

13.5= 9.2593 𝜇𝑔 𝑚𝑔⁄ = 0.926%

Therefore, raw data in Table 3 can be translated into final weight percentage range for each

sample as tabulated in Table 4.

Table 4: Translated Results in Weight Percentage.

1 2 3 4 5 6

A 0.926% 0.926% 0.926%-

1.852%

0.926%-

1.852%

1.852%-

3.704%

1.852%-

3.704%

B 1.852% 1.852% 1.852%-

3.704%

1.852%-

3.704%

0.926%-

1.852%

0.926%-

1.852%

C 3.704% 3.704% 0.926%-

1.852%

0.926%-

1.852%

D 7.407% 7.407% 1.852%-

3.704%

1.852%-

3.704%

E 14.815% 14.815% 0.926%-

1.852%

0.926%-

1.852%

F 29.630% 29.630% 1.852%-

3.704%

1.852%-

3.704%

G 59.260% 59.260% 1.852%-

3.704%

1.852%-

3.704%

H 118.519% 118.519% 0.926%-

1.852%

0.926%-

1.852%

For sample 2, sample 4, and sample 6, since these samples are decollagenized, the

treated samples should have nearly zero collagen content. The results of A3, A4, C3, C4,

E3, and E4 match the theoretical reasoning. In contrast, untreated samples should contain

a certain level of collagen content, which is supported by results for B3, B4, D3, D4, F3

and F4.

However sample 1 and sample 3 which are EDTA treated indicate completely

different results compared to sample 2, 4 and 6. EDTA treated samples have higher

30

collagen content than untreated samples. The level of collagen content is similar for all the

samples regardless of treatment type and treatment existence.

31

6.0 DISCUSSION

6.1 Reproducibility and Repeatability

According to the comparison between duplicated samples, the results are consistent for all

the samples. However, for the experiment run analyzed specifically in this report, the

references are not perfectly reproducible (solution in well E1 was not properly dyed).

Although this deficit between references did not affect any of the results, the similar error

could occur to the samples. The following factors may lead to this deficit and should be

paid with extra attention for the samples in later experiments:

1. During preparation of the standard dilutions, carefully label solutions for each

concentration as they are all colorless and of the same volume after the preparation.

2. When adding the dyeing agents, make sure the pipette tip does not make any contact

with the solution in the wells as they will affect the color of the solution and

therefore affect the final result.

3. Strictly follow the 60°C incubation temperature and 30 minute incubation time after

adding DMAB solution as DMAB is very sensitive to temperature over 80°C (83).

6.2 Agreement to Decollagenization Group Samples

For the results of sample 2, 4 and 6, which are the group of samples underwent

decollagenization with NaOCl, untreated samples have higher collagen content level

(1.852%-3.704%) compared to treated samples (0%-1.852%). The comparison between

treated and untreated samples matches the prediction. Theoretically, NaOCl should take

away all organic composition of the bone tissue, results from treat samples has almost zero

collagen content which shows a positive match. Although the difference between treated

and untreated samples is identifiable, the range of collagen content level in the untreated

32

samples is nearly at the lower boundary of the prediction. The collagen content level could

be higher compared to the collagen level in cattle bone tissues in vivo, which is 5%-15%

(79). Several reasons could explain the low level of collagen content in our samples:

1. Location where the sample is taken from the bone pieces. As illustrated in Figure

4, the 10 mg sample dissolved into HCl solution is taken from different location on

the bone pieces in order to avoid bias, however, it is impossible to have bias free

samples as collagen content level varies with location on the bones.

As indicated in Figure 4, red square represents the part that is treated

(surface looks more trabecular compared to untreated part on the left), orange

circles are the sites where solid samples are taken. Notice that the color of bone

tissues differentiate with the depth from the bone outer surface to inner marrow

region on the untreated site (circled in blue square), collagen content level is

different for these regions. Although treatment has made the appearance difference

unable to identify, we try to take samples from as various sites on the same bone

piece as possible in order to avoid such bias.

33

Figure 4: Sample Taken from Different Sites on the Same Bone Tissue.

2. Insufficient incubation temperature leads to incomplete hydrolysis of collagen

content, since this batch of samples is prepared with modified incubation conditions

(5 days under room temperature vs. 24 hrs under 120 °C).

3. Collagen loss during frequent frosting and defrosting when taking the samples off.

In general, for samples treated with NaOCl decollagenization process only, the

hydroxyproline process is validated for the assay of collagen content despite the instability

due to improper operations. Quantitative reliability and statistic relationship can be further

34

established if the optical density values becomes handy with availability of equipment like

colorimeter.

6.3 Deficit for Demineralization Group Samples

For samples treated with EDTA demineralization process (sample 1 and sample 3),

untreated samples and treated samples are supposed to have similar collagen content level

as only mineral content is removed from the treatment. The results have indicated a deficit

that treated samples have higher collagen content level than untreated samples.

Recall that it was mentioned in the background section, using EDTA as

demineralization agent could bring antioxidant effect to samples which slows down organic

composition loss (including collagen loss) due to oxidation over time. This effect can

explain why EDTA treated samples have higher collagen content than untreated samples.

However, the values of the differences are questionable.

Overall, qualitatively, it is validated that hydroxyproline collagen assay is able to

distinguish treated and untreated collagen level within the demineralization group of

samples. The explanation is reasonable and the result is consistent with our prediction.

6.4 Other Sources of Error

Except for the factors discussed above, there are several other sources of error for the

hydroxyproline collagen assay. They may not result in significant deficit but they are still

worth analyzing in order to achieve higher accuracy of the hydroxyproline collagen assay.

Causes of the error and actions to avoid such error are discussed.

35

1. DMAB solution and Chloramine T solution are not stable and cannot be stored and

reused for multiple assays. Moreover, to ensure the effectiveness of them, it is better

to use them within 2 hrs after their preparation (74).

2. HCl at 10 N concentration volatilize rapidly under room temperature and pressure.

As in our research, we dilute reagent level HCl (highest concentration, 12.1N, 37%)

into 10N HCl, the dilution process is also highly volatilizing, the actual HCl used

might be at lower concentration than the designed 10N. The best action is to buy

the 10N HCl off-the-shelf or to use the diluted HCl right after preparation.

3. During the incubation of the samples, Teflon cap could potentially deform and lead

to a leakage of HCl vapor under such high temperature. It will ultimately lead to

incomplete hydrolysis process. To avoid this, make sure all the vial cap is securely

tightened before incubation starts and do not use plastic cap instead of Teflon cap

since it is easier to deform and potentially melt with the reaction between the cap

and HCl. Figure 5 and Figure 6 shows the failure of a vial cap during one trial

experiment.

36

Figure 5: Cap Deformation due to Over-heating during Incubation.

Figure 6: Cap Screw Thread Corrosion due To Volatilized HCl during Incubation.

37

4. Make sure to use a new pipette tip after each use with different solution. Since the

collagen content level in our samples is supposed to be lower than 20%, and only

10 μl of each prepared sample solution is used, slight pollution from pipette tip

contact with other samples could cause a significant deficit in the result.

38

7.0 ONGOING PROCESS AND FUTURE DIRECTION

At the current stage, we have contacted Prof. Craig Simmons from the MIE

department who referred us to Prof. Michael Sefton’s Sefton Lab in the CBBR facility that

is capable of providing optical density measurement for our research. Communication with

Prof. Michael Sefton is initiated and preparation for the optical density measurement is

finished. By the time this thesis report is being marked, a fully-quantitative results on the

second batch of samples (with more samples and more comprehensive sample preparation

including properly monitored 8 hr, 120°C incubation) should be available. It will be handed

in to Prof. Andreas Mandelis in the form of a thesis supplementary document in addition

to this report.

Furthermore, over the summer of 2015, I will be continuing related data analysis

and quantitative correlation between collagen content level and US and PA signals to

complete validation of the previous research in order to get the output of this thesis to the

publication stage.

A transition report is also expected in the summer in order to document the standard

procedure for such collagen assay for any later uses.

39

8.0 CONCLUSION

This thesis project aimed to find an effective method to measure the collagen

content level in cattle bone tissues. Samples are subjected to decollagenization and

demineralization process using NaOCl and EDTA as treatment agents respectively. The

assay plan chosen must be able to provide accurate measurement of the weight percentage

of the collagen content in specific bone samples. The assay plan should be easy to conduct,

efficient and reliable.

Several candidate collagen assay methods are evaluated and justified. The

dominating factor in this project is that only a total collagen content level is needed instead

of collagen content level for specific collagen types. Based on this assumption, Sirius Red

collagen assay and hydroxyproline collagen assay were the best candidates. Among these

two assay plans, Sirius Red collagen assay requires a more cumbersome process including

cell culture, collagen isolation and collagen concentration. In contrast, hydroxyproline

collagen assay is suitable for both solid and liquid samples. Hydroxyproline collagen assay

is chosen as the experimental plan with modified procedures.

Two batches of samples are prepared. The first batch of samples are assessed to

provide semi-quantitative verification run to evaluate the effectiveness and efficiency of

hydroxyproline collagen assay for our requirement. The second batch of samples are

prepared for a fully-quantitative results using facility that supports optical density

measurement.

The results from the first batch of samples confirms the ability of hydroxyproline

assay to quantify collagen content level within a range of weight percentage using the

reference arrays. Results showed that for the decollagenized group, untreated samples have

40

collagen content at 1.852%-3.704% while treated samples have collagen content within

0%-1.852%. For the demineralized group, untreated samples have collagen level at 0%-

1.852% compared to the range of 1.852%-3.704% for the treated samples. The result

matches the prediction that decollagenization process should remove most of the collagen

content and the EDTA demineralization agent can delay the oxidation of collagen content

and result in a slightly higher collagen level in treaded samples within its group.

The values of the weight percentage obtained from the first batch of samples are

lower than expectation. This is mainly because the first batch of samples used a alternative

sample preparation approach of incubating for 5 days under room temperature instead of

24 hrs under 120 °C, which leads to incomplete hydrolysis. Despite of the deficit from this

error, the hydroxyproline collagen assay is validated as a suitable method to satisfy the

objective.

41

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Ultrasonic Backscatter and Transmission Parameters at the Os Calcis in

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52

FIGURES AND TABLES

53

11.0 APPENDICES

11.1 Appendix A: Comparisons between Collagen Assay Method and Kit Off-the-

Shelf

Collagen Assay Methods and Kits Comparison 1. Collagen Assay Methods:

Currently for the analysis of collagen, various types of assays exist [1]:

- ELISA for specific types of collagen

- ELISA for specific pro-domains of collagen

- Western blotting using specific collagen antibodies

- Sirius Red based assays for soluble collagen

- Tissue hydrolysis followed by analysis of Hydroxyproline residues (either by a

colorimetric kit or by HPLC)

Based on the feature of these methods and the requirements for our application, since the

photoacoustic and ultrasonic signal detects collagen content regardless of its type, an assay

with no discrimination on collagen type should be chosen. Thus, both Sirius red and

hydroxyproline methods are suitable candidate for our experiment. However, since our

sample would be solid and non-cultured after been tested by photoacoustic and ultrasonic

detection, hydroxyproline method would only require tissue hydrolysis while Sirius red

would require another sample solubilisation beforehand. Theoretical background will be

shortly introduced in the next two sections.

1.1 Sirius Red Soluble Collagen Assay [1]

This assay recognizes soluble or (acid/pepsin) solubilized collagen. The assay is

colorimetric, has a 96-well plate format and is based on precipitation of collagen with

Sirius-Red, an anionic dye with sulphuric acid groups. This dye can bind the side-chain

groups of basic amino acid residues. The dye is released from the precipitated complex at

high pH followed by colorimetric detection. The assay is optimized such that other proteins

(such as albumin) do not interfere. Gelatin (unfolded collagen) is not recognized by this

assay.

Application: The assay is used for the measurement of (soluble) collagen in e.g. cell culture

media, and (acid or acid/pepsin) solubilized collagens e.g. from cell culture extracts. The

assay is less suitable for the determination of collagen in tissues, since in tissues most of

the collagen is cross-linked and therefore often only a low percentage of the collagen is

solubilized upon extraction.

1.1 Hydroxyproline Total Collagen Assay [1]

This assay recognizes all types of collagen (mature, immature, procollagen, degraded

collagen, cross-linked collagen, collagen from various sources). The assay is colorimetric,

has a 96-well plate format, and is based on the quantification of hydroxyproline, an amino

acid exclusively occurring in collagen. Hydroxyproline is released from collagen upon acid

hydrolysis of the collagen containing sample. Hydrolysis is carried out at 95 C, and the

product can directly be used for hydroxyproline analysis, without washing or drying steps.

This analysis is based on Chloramine T/DMBA.

Application: The assay is used for the measurement of total collagen. This includes all

procollagen, unfolded collagen, mature collagen as well as collagen degradation products

54

of all collagen types present in the sample. Since the first step is complete hydrolysis of

the sample, difficulty in extraction of collagen plays no role. The assay is applicable for all

types of samples, including tissue.

[1] http://www.quickzyme.com/wp-content/uploads/2012/07/Application-note-How-to-

choose-your-collagen-assay.pdf

2. Collagen Assay Kits:

There are three major companies specialized in collagen detection products. They are

QuickZyme form Netherland, Chondrex Inc. and Sigma-Aldrich from the States. They all

provide products and service on hydroxyproline total collagen assay that is applicable to

solid non-cultured samples. A detailed comparison between these products are shown in

the following table:

Company, Product

QuickZyme,

Total Collagen

Assay Kit

Chondrex Inc.,

Hydroxyproline

Assay Kit

Sigma-Aldrich,

Hydroxyproline

Assay Kit

No. of Tests Per Kit 100 40 100

Price Per Kit € 292

(CAD $ 542.60) USD $ 357.50 CAD $ 429.50

Approximate Price

Per Test

€ 292

(CAD $ 5.43) USD $ 8.94 CAD $ 4.30

Components Not in

Kit

1. 12M and 6M HCl

for sample

hydrolysis

2. 4 M HCl for

sample dilution

3. Single and/or

multichannel

pipettes

4. Eppendorf

centrifuge

5. Incubator (or

thermos-block or

oven) for heating

at 95oC

6. Incubator (or

oven) for heating

at 60oC

7. Microplate reader

capable of

measuring at a

wavelength

between 540 and

580 nm, 570 nm

preferred.

8. Microplate shaker

1. Concentrated HCl

(10N)

2. A glass screw-

thread vial (1-2

ml) with a Teflon

cap (example:

National

Scientific B7999-

1)

1. 96 well flat-

bottom plate – It

is recommended

to use clear plates

for colorimetric

assays.

Spectrophotometr

ic multiwell plate

reader

2. Concentrated

(37% or ~12 M)

HCl, Catalog

Number 320331

or equivalent)

3. Activated

charcoal (Catalog

Number 242276

or 97876, or

equivalent).

4. 120 °C heating

block.

5. Pipette

compatible with

concentrated HCl.

6. Centrifugal

Evaporator or 60

°C oven.

55

7. Pressure-tight vial

with PTFE-lined

cap, or 2 mL

polypropylene

vial.

Purchasing Website goo.gl/2WJuLC goo.gl/MDnWV4 goo.gl/0QB1kw

Tech. Spec. Website goo.gl/HVYrd6 goo.gl/h6sMU0 goo.gl/0QB1kw

Manual Doc. goo.gl/sCAqan goo.gl/h6sMU0 goo.gl/bZ9jma

QuickZyme and Sigma-Aldrich’s kits are relatively cheaper in terms of cost per test.

Sigma-Aldrich’s kit does not include the significant 96 flat plate component which are

included for the other kits.

Chondrex’s kit requires the least components outside the kit. QuickZyme and Sigma-

Aldrich have Canadian distributors while Chondrex does not have a Canadian distributor.

More shipping time is expected for Chondrex product.

Product Component Price Per Unit Quantity Per

Unit

Purchasing

Website

QuickZyme

12M HCl USD $7.50 100 mL goo.gl/z4EIHx

6M HCl USD $8.20 500 mL goo.gl/v2HgEv

4M HCl CAD $47.90 1 L goo.gl/3XoZVl

Single Pipette USD $199.00 1 Qty goo.gl/mg8GJf

Total CAD $270.00 Grand Total CAD $812.60

Chondrex

10N HCl USD $67.91 100 mL goo.gl/XIfr4y

Glass Screw

Thread Vial

(National

Scientific B7999-

1)

Approx. USD

$40.00 100 Qty goo.gl/BVCBjZ

Total CAD $120.00 Grand Total CAD $477.50

Sigma-Aldrich 96 flat bottom

plate CAD $176.00 1 Qty goo.gl/fYT5HI

56

12M HCl CAD $75.20 500 mL goo.gl/ocqkkl

Activated charcoal CAD $32.00 250 g goo.gl/EE2tNI

2 mL

polypropylene vial CAD $102.00 100 Qty goo.gl/yoWygb

Total CAD $ 385.20 Grand Total CAD $ 814.70

57

11.2 Appendix B: Hydroxyproline Collagen Assay Protocol from Chondrex Inc.

58

59

60