Indiana University-Purdue University Indianapolis (IUPUI) / Articular cartilage (AC) is a strong but flexible connective tissue that covers and protects the end of the long bones. Although cartilage has excellent friction and wear properties that allow smooth joint function during daily activities, these properties are not fully understood. Many material properties of cartilage are anisotropic and vary with anatomic location and the composition of the tissue, but whether this is also true for cartilage friction and wear has not been previously determined. Furthermore, cartilage disease and injury are major health concerns that affect millions of people, but there are few available treatments to prevent the progression of cartilage degeneration. Collagen crosslinking may be a potential treatment to reduce cartilage wear and slow or prevent the progression of cartilage disease. The objectives of this thesis were to investigate the relationships between the friction/wear characteristics of cartilage and the orientation of the preferred fiber direction, the anatomic location of the tissue, the composition of the tissue, and exogenous photochemical crosslinking. In the superficial zone, AC has preferential fiber direction which leads to anisotropic material behavior. Therefore, we hypothesized that AC will show anisotropic behavior between longitudinal and transverse direction in an accelerated, in vitro wear test on bovine cartilage in terms of friction and wear. This hypothesis was proven by the quantification of glycosaminoglycans released from the tissue during the wear test, which showed that more glycosaminoglycans were released when the wear direction was transverse to the direction of the fibers. However, the hydroxyproline released from the tissue during the wear test was not significantly different between the two directions, nor was the coefficient of friction. The material properties of AC can also vary with anatomic location, perhaps due to differences in how the tissue is loaded in vivo. We hypothesized that cartilage from a higher load bearing site will give better wear resistance than cartilage from lower load bearing regions. However, no differences in friction or wear were observed between the different anatomic locations on the bovine femoral condyles. The concentration of collagen, glycosaminoglycans, cells and water in the tissue was also quantified, but no significant differences in tissue composition were found among the locations that were tested. Although wear did not vary with anatomic location, variation in the wear measurements were relatively high. One potential source of variation is the composition of the cartilage. To determine whether cartilage composition influences friction and wear, a correlation analysis was conducted. An accelerated, in vitro wear test was conducted on cartilage from bovine femoral condyles, and the tissue adjacent to the wear test specimens was analyzed for collagen, glycosaminoglycan, cell, and water content. Because wear occurs on the cartilage surface, the superficial zone of the cartilage might play an important role in wear test. Therefore, composition of the adjacent cartilage was determined in both the superficial zone and the full thickness of the tissue. A significant negative correlation was found between wear and collagen content in the full thickness of the tissue, and between the initial coefficient of friction and the collagen content in the superficial zone. This correlation suggests that variation in the collagen content in the full thickness of the cartilage partially explains differences in amount of wear between specimens. The wear resistance of cartilage can be improved with exogenous crosslinking agents, but the use of photochemical crosslinking to improve wear resistance is not well understood. Two photochemical crosslinking protocols were analyzed to improve the wear resistance of the cartilage by using chloro-aluminum phthalocyanine tetrasulfonic acid (CASPc) and 670nm laser light. The cartilage treated with the two crosslinking protocols had lower wear than the non-treated group without changing the friction properties of the cartilage.

Die Degeneration des Gelenkknorpels ist Hauptursache für chronische Schmerzen und eine dadurch bedingte Einschränkung der Lebensqualität. Für die Sozialversicherungssysteme ist dies mit steigenden Kosten verbunden. Gegenwärtige Behandlungsoptionen wie die Mikrofrakturierung oder die (matrix-assoziierte) Autologe Chondrozytentransplantation (M-) ACT führen zu einem minderwertigen Reparaturgewebe aus Faserknorpel mit unzureichenden mechanischen Eigenschaften an der Defektstelle. Es besteht ein Bedarf an der Entwicklung und Testung neuer Knorpeltherapien, die ein funktionelles Reparaturgewebe für nachhaltige Beschwerdefreiheit erzeugen. Das hier verwendete kürzlich etablierte osteochondrale Ex vivo-Testsystem (EVTS) eignet sich zur Evaluation unterschiedlicher zellbasierter Behandlungsansätze für die Knorpelregeneration. Aus der medialen Femurkondyle von Schweinen wurden zylindrische 8 mm große osteochondrale Explantate (OCE) isoliert. Es wurden Knorpel-Knochendefekte und reine Knorpeldefekte kreiert und mit autologen Schweine-Chondrozyten (CZ) bzw. einer Mischung aus CZ und mesenchymalen Stammzellen (MSC) gefüllt, die in Kollagen Typ I Hydrogel eingebettet waren. Nach vierwöchiger Kultivierung wurden die Proben histologisch und immunhistochemisch gefärbt (Safranin-O-Färbung, Kollagen Typ II, Aggrekan), die Zellvitalität (Lebend-Tot-Färbung) überprüft und die extrazelluläre Matrixproduktion analysiert. Nach vierwöchiger Kultur im EVTS in Normoxie und Hypoxie zeigten sich die in Kollagen-I-Hydrogel eingebetteten Zellen lebensfähig. Die Auswertung der verschiedenen Ansätze erfolgte über den standardisierten ICRS-II-Score der International Cartilage Repair Society (ICRS) mit drei unabhängigen Bewertern. Insgesamt resultierten bessere Ergebnisse im Hinblick auf die Matrixsynthese in den Monokulturen aus CZ im Vergleich zu den Co-Kulturen aus CZ und MSCs. Da dieser Unterschied nicht groß war, könnten MSCs zur Einsparung autologer CZ eine Alternative in der Behandlung von Knorpeldefekten darstellen. Hypoxie spielte eine Rolle bei reinen Knorpeldefekten, nicht bei Knorpel-Knochendefekten. Dies bestätigt die Bedeutung des physiologischen hypoxischen Milieus des Gelenkknorpels, das einen niedrigen Sauerstoffgehalt von 2-5 VII % aufweist. Die Ergebnisse zeigen, dass die unterschiedlichen Faktoren aus Zellkombination, Knorpeldefektgröße und Kultivierung in Hypoxie oder Normoxie Einfluss auf die Ausbildung der extrazellulären Matrix haben. Weiterhin fehlt jedoch das Verständnis für die genauen Mechanismen des Knorpelregenerationsverhaltens. Ex vivo-Testsysteme können dabei helfen ein weiteres Verständnis zu erlangen und entsprechende Behandlungsstrategien zu evaluieren. / Degeneration of articular cartilage is a major cause of chronic pain - impairing the quality of life and rising health care costs. Current treatment options like microfracture, ACT or MACT result in fibrocartilaginous repair tissue with insufficient mechanical properties at the defect site. Hence, new cartilage therapies generating functional repair tissue need to be developed and tested. Here we used a recently established ex vivo osteochondral model to evaluate the therapeutic potential of several cell-based cartilage regeneration approaches. Reproducible cylindrical 8 mm osteochondral explants (OCE) were isolated from porcine medial condyles. Full-thickness and cartilage-only defects were created and filled with autologous porcine chondrocytes respectively a mixture of chondrocytes and mesenchymal stem cells, embedded in collagen type I hydrogel. After static culture for four weeks, samples were analyzed for cell viability (live/dead staining) and extracellular matrix production, using immunohistochemical staining (Safranin-O-staining, collagen type II, aggrecan). Embedded cells remain viable after four weeks culture in ex vivo osteochondral model. Outcome of different cartilage regeneration approaches were compared using the recommended guidelines proposed by the International Cartilage Repair Society (ICRS) and ICRS-II-score with three independent evaluators. Overall, the monocultures from CZ performed better than the co-cultures from CZ and MSCs. Since this difference was not large, MSCs could be an alternative in the treatment of cartilage defects to save autologous CZ. Hypoxia played a role in pure cartilage defects, but not in cartilage-bone defects. This confirms the importance of the physiological hypoxic milieu of the articular cartilage, which has a low oxygen content of 2-5 %. The results show that the different factors from cell combination, cartilage defect size and cultivation in hypoxia or normoxia influence the formation of the extracellular matrix. However, there is still no understanding of the exact mechanisms of cartilage regeneration behavior. Ex vivo test systems can help to gain further understanding and to evaluate appropriate treatment strategies.

Research Report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirement for the Degree of Masters of Medicine in Otorhinolaryngology Johannesburg October 2017 / AIM: This study aimed to assess the anatomical and functional success rate of Butterfly Cartilage Inlay Graft myringoplasties done at Chris Hani Baragwanath Academic Hospital using an oto-endoscope. Size of perforation was assessed as a possible predictor of success. METHODS: The study comprised of a retrospective review of all records from the ENT Department at Chris Hani Baragwanath Academic Hospital of all patients who underwent BCIG myringoplasty from January 2009 to December 2013. 85 of the 160 patients who had BCIG’s at CHBAH met the inclusion criteria for this study. Data was collected on a data collection sheet and analysed using standard statistical methods. RESULTS: 85 patients were included in the study of ages 5 years – 67 years with a mean age (SD) of 19,2 years (16,3). 61% were children (<13 years), 39% adults (14 – 49 years) and only 6% were >50 years. There were 30 (35%) Female patients and 55 (65%) Male patients. The data presented in this study show an anatomical success rate of 90,6% for Butterfly Cartilage Inlay Grafts at Chris Hani Baragwanath Academic Hospital. The anatomical success rate of this study is equal to the success rates reported in the literature for the same procedure. 87% of patients experienced hearing improvement post operatively. The average hearing improvement in this study post Butterfly Cartilage Inlay Graft is 15dB. Finally, perforation size does not influence both anatomical and functional success rates in this study. CONCLUSION: Endoscopic BCIGs performed at Chris Hani Baragwanath Academic Hospital, for small, medium and large perforations, show anatomical and functional success rates similar to those reported in the literature, performed with both microscope and endoscope. Size of perforation is not a predictor of anatomical and functional success for this procedure. / MT2018

Articular cartilage provides a near frictionless surface for the articulating ends of bones. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If damaged, whether by traumatic injury or disease, cartilage lacks the ability for self-repair. This study explores the production of scaffoldree cartilage and investigates the effect of Tissue Growth Technologies’ CartiGen Bioreactor on the cartilage. Chondrocyte and bone marrow-derived stem cell (BMSC) attachment to chitosan is also investigated in hopes of producing a bilayered construct for osteochondral repair. Results demonstrate that culturing of scaffoldree cartilage in the CartiGen bioreactor resulted in an enhancement of the scaffoldree cartilage’s biomechanical and biochemical properties and that the chitosan microspheres were able to successfully support porcine chondrocyte and BMSC attachment. Results from both studies are encouraging for future work involving tissue engineered cartilage.

Although advances have been made toward understanding the complex mechanisms that regulate the process of DNA transcription, the specific mechanisms of activation for many individual genes remain unknown. In this study, we focus on the role the transcription factor SOX9 plays in activating cartilage-specific genes, specifically Col9a1 and Cartilage Link Protein (CRTL1). Previously, enhancers of these genes containing single SOX9 binding sites were shown to be activated through SOX9 binding. However, the hypothesis was made that in cartilage-specific genes dimeric SOX9, as opposed to monomeric SOX9, is necessary for activation. We identified a putative binding site adjacent to each of the known single SOX9 binding sites in the Col9a1 D and E enhancers and in the CRTL1 enhancer. Electrophoretic Mobility Shift Assays (EMSAs) were performed to determine whether SOX9 bound to these putative sites. Transient transfections were then performed using wild-type and mutant enhancer- reporter plasmids to determine whether these putative SOX9 binding sites were important for activation in vivo. Although dimeric SOX9 bound to each of the enhancers in vitro, several different effects were seen in vivo. In the presence of the wild-type Col9a1 D enhancer, no activation was seen. However, when the enhancer was extended to include an additional pair of newly found SOX9 binding sites, expression was increased 10-fold. When any of the four SOX9 binding sites within this enhancer were mutated, expression was completely eliminated, suggesting that interdependent dimers or a tetramer of SOX9 is necessary for the activation of transcription. The weaker Col9a1 enhancer E was found to increase gene expression minimally through binding of either dimeric or monomeric SOX9. However, dimeric SOX9 was required for the activation of gene expression by the CTRL1 enhancer. Through this study we validate the importance of not just monomeric but of dimeric and possibly tetremeric SOX9 as an activator of cartilage-specific gene expression.

Yes / Poly(vinyl alcohol) (PVA)/graphene oxide (GO) nanocomposite hydrogel as artificial cartilage replacement was prepared via freezing/thawing method by introducing polyethylene glycol (PEG). Efficient grafting of PVA molecules onto GO surface was realized by formation of hydrogen bonding, resulting in exfoliation and uniform distribution of GO in PVA matrix. By introduction of appropriate content of GO, the increased crystalline regions of PVA and the formation of GO centered second network structure led to the increase of the storage modulus and effective cross-linking density. And therefore the mechanical strength and toughness of the composite hydrogel were improved simultaneously: the tensile strength, elongation at break, and compressive modulus showed approximately 200%, 40%, and 100% increase of the neat PVA hydrogel. Besides, for the sample with 1.5 wt % GO content, the maximum force retention and dynamic stiffness were improved remarkably in the process of sinusoidal cyclic compression, and the compressive relaxation stress also increased significantly, indicating the enhancement of the compressive recoverable and antifatigue ability, and resistance to compressive relaxation by formation of high load-bearing, dense, and reinforcing double network structure. Moreover, more than 50% decrease in coefficient of friction was obtained for the composite hydrogel, and the worn surface presented relative smooth and flat features with sharp decreasing furrow depth, confirming the lubrication effect of GO-PEG. This study shows promising potentials in developing new materials for cartilage replacement with simultaneous combination of high mechanical property and excellent lubrication.

Studies have investigated and dismissed the effect of freeze-thaw cycles on both skeletal muscle and on trabecular bone, but have failed to properly address the effects of these storage methods on the integrity of articular cartilage. Preventing cartilage injury is important in minimizing the long term debilitating effects of osteoarthritis. Accurate subfracture injury prediction must take into account the possible effects that freeze thaw cycles may have on the mechanical properties of cartilage tissue. This paper addresses this concern with matched pair testing of various low temperature storage techniques against fresh control groups. Controlled mechanical indention tests were performed on bovine articular cartilage-on-bone specimens to compare stiffness, peak stress, and loading energy of the cartilage. Findings showed that a slow freeze thaw or flash freeze cycle caused cartilage stiffness to decrease by 37% and 31% respectively. Compressive stress at this strain was also lowered by 31% with a single freezing process. These results may be indicative of a weakened extracellular matrix structure caused by the freeze-thaw process. It is still unclear whether these changes in mechanical properties will result in a change in injury susceptibility for articular cartilage. / Master of Science

Cheuk, Yau Chuk. / Thesis submitted in: Dec 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-144). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.iii / PUBLICATIONS --- p.v / ACKNOWLEDGEMENT --- p.vi / LIST OF ABBREBIVIATIONS --- p.vii / INDEX FOR FIGURES --- p.x / INDEX FOR TABLES --- p.xiv / TABLE OF CONTENTS --- p.xv / Chapter CHAPTER ONE - --- INTRODUCTION / Chapter 1.1 --- "Joint function, structure and biochemistry" / Chapter 1.1.1 --- Function of joint --- p.1 / Chapter 1.1.2 --- Types of cartilage --- p.1 / Chapter 1.1.3 --- Composition and structure of articular cartilage --- p.2 / Chapter 1.1.4 --- The subchondral bone --- p.3 / Chapter 1.1.5 --- Maturation of articular cartilage and subchondral bone --- p.3 / Chapter 1.2 --- Osteochondral defect / Chapter 1.2.1 --- Clinical problem --- p.6 / Chapter 1.2.2 --- Spontaneous repair --- p.7 / Chapter 1.2.3 --- Current treatment strategies --- p.7 / Chapter 1.2.4 --- Limitations of current treatment strategies --- p.8 / Chapter 1.2.5 --- Treatments under development --- p.11 / Chapter 1.2.6 --- Potential and limitations in cell therapies --- p.14 / Chapter 1.3 --- The 3-D scaffold-free cartilage / Chapter 1.3.1 --- Fabrication of scaffold-free cartilage --- p.16 / Chapter 1.3.2 --- Scaffold-free cartilage for chondral / osteochondral defect repair --- p.18 / Chapter 1.3.3 --- Scaffold-free bioengineered chondrocyte pellet from our group --- p.20 / Chapter 1.3.4 --- BCP as a possible treatment for OCD --- p.21 / Chapter 1.4 --- The objectives of the study --- p.22 / Chapter 1.5 --- The study plan / Chapter 1.5.1 --- Design of the study --- p.23 / Chapter 1.5.2 --- Choice of animal model --- p.23 / Chapter 1.5.3 --- Selection of evaluation time points --- p.24 / Chapter 1.5.4 --- Choice and modification of histological scoring system --- p.24 / Chapter CHAPTER TWO - --- METHODOLOGY / Chapter 2.1 --- Preparation of reagents and materials for tissue culture and histology --- p.26 / Chapter 2.2 --- Creation of osteochondral defect model --- p.28 / Chapter 2.3 --- Synthesis of scaffold-free cartilage using 3-D chondrocyte pellet culture / Chapter 2.3.1 --- Isolation of rabbit costal chondrocytes --- p.31 / Chapter 2.3.2 --- Three-dimensional chondrocyte pellet culture --- p.31 / Chapter 2.3.3 --- BrdU labeling for cell fate tracing --- p.32 / Chapter 2.4 --- Further characterization of the 3-D scaffold-free chondrocyte pellet / Chapter 2.4.1 --- Gross appearance --- p.35 / Chapter 2.4.2 --- Cell viability / Chapter 2.4.2.1 --- Alamar blue reduction assay --- p.35 / Chapter 2.4.3 --- Preparation of samples for histology --- p.36 / Chapter 2.4.4 --- General morphology and histomorphology / Chapter 2.4.4.1 --- H&E staining --- p.36 / Chapter 2.4.5 --- Cartilage properties / Chapter 2.4.5.1 --- Safranin O /Fast Green staining --- p.37 / Chapter 2.4.5.2 --- Immunohistochemistry of type II collagen --- p.37 / Chapter 2.4.5.3 --- Immunohistochemistry of type I collagen --- p.38 / Chapter 2.4.6 --- Angiogenic properties / Chapter 2.4.6.1 --- Immunohistochemistry of VEGF --- p.40 / Chapter 2.4.7 --- Osteogenic properties / Chapter 2.4.7.1 --- ALP staining --- p.40 / Chapter 2.5 --- Implantation of scaffold-free cartilage into osteochondral defect model / Chapter 2.5.1 --- Surgical procedures --- p.41 / Chapter 2.5.2 --- Experimental groups --- p.42 / Chapter 2.6 --- Assessment of osteochondral defect healing / Chapter 2.6.1 --- Macroscopic evaluation --- p.43 / Chapter 2.6.2 --- Preparation of samples for histology --- p.43 / Chapter 2.6.3 --- Histology for general morphology / Chapter 2.6.3.1 --- H&E staining --- p.45 / Chapter 2.6.4 --- Histological scoring / Chapter 2.6.4.1 --- Modification of the scoring system --- p.45 / Chapter 2.6.4.2 --- Procedures of scoring and validation --- p.45 / Chapter 2.6.5 --- Cell proliferation / Chapter 2.6.5.1 --- Immunohistochemistry of PCNA --- p.49 / Chapter 2.6.6 --- Cartilage regeneration / Chapter 2.6.6.1 --- Safranin O /Fast Green staining --- p.49 / Chapter 2.6.6.2 --- Immunohistochemistry of type II collagen --- p.49 / Chapter 2.6.6.3 --- Immunohistochemistry of type I collagen --- p.50 / Chapter 2.6.6.4 --- Polarized light microscopy --- p.50 / Chapter 2.6.7 --- Expression of angiogenic factor / Chapter 2.6.7.1 --- Immunohistochemistry of VEGF --- p.50 / Chapter 2.6.8 --- Bone regeneration / Chapter 2.6.8.1 --- μCT analysis --- p.50 / Chapter 2.6.9 --- Histomorphometric analysis of cartilage and bone regeneration --- p.53 / Chapter 2.6.10 --- BrdU detection for cell fate tracing --- p.55 / Chapter 2.6.11 --- Statistical analysis --- p.55 / Chapter CHAPTER THREE - --- RESULTS / Chapter 3.1 --- Further characterization of the 3-D chondrocyte pellet culture / Chapter 3.1.1 --- Gross examination --- p.57 / Chapter 3.1.2 --- Cell viability --- p.57 / Chapter 3.1.3 --- Cartilage properties --- p.61 / Chapter 3.1.4 --- Angiogenic properties --- p.63 / Chapter 3.1.5 --- Osteogenic properties --- p.64 / Chapter 3.2 --- Implantation of scaffold-free cartilage and assessment / Chapter 3.2.1 --- Gross examination --- p.65 / Chapter 3.2.2 --- General morphology --- p.67 / Chapter 3.2.3 --- Histological scores --- p.71 / Chapter 3.2.4 --- Cell proliferation --- p.75 / Chapter 3.2.5 --- Cartilage regeneration --- p.78 / Chapter 3.2.6 --- Expression of angiogenic factor --- p.90 / Chapter 3.2.7 --- Bone regeneration --- p.93 / Chapter 3.2.8 --- Histomorphometric analysis on cartilage and bone regeneration --- p.96 / Chapter 3.2.9 --- Cell fate tracing --- p.100 / Chapter CHAPTER FOUR - --- DISCUSSION / Chapter 4.1 --- Summary of key findings / Chapter 4.1.1 --- Further characterization of BCP and determination of implantation time --- p.102 / Chapter 4.1.2 --- Implantation of BCP in OCD --- p.102 / Chapter 4.2 --- Spontaneous healing in osteochondral defect / Chapter 4.2.1 --- Findings from the current study --- p.104 / Chapter 4.2.2 --- Comparison with other studies --- p.104 / Chapter 4.2.3 --- Factors affecting spontaneous healing --- p.105 / Chapter 4.3 --- Fabrication and further characterization of the 3-D chondrocyte pellet / Chapter 4.3.1 --- Comparison of different methods of producing scaffold-free cartilage construct --- p.106 / Chapter 4.3.2 --- Cartilage phenotype of the BCP --- p.107 / Chapter 4.3.3 --- Angiogenic and osteogenic potential of the BCP --- p.108 / Chapter 4.3.4 --- Role of mechanical stimulation on tissue-engineered cartilage --- p.109 / Chapter 4.4 --- Repair of osteochondral defect with allogeneic scaffold-free cartilage / Chapter 4.4.1 --- Advantages of the current scaffold-free chondrocyte pellet --- p.111 / Chapter 4.4.2 --- Remodeling of BCP after implantation --- p.111 / Chapter 4.4.3 --- Effect of BCP on cartilage repair --- p.112 / Chapter 4.4.4 --- Effect of BCP on bone regeneration / Chapter 4.4.4.1 --- Findings in the present study --- p.113 / Chapter 4.4.4.2 --- Possible reasons of slow bone repair --- p.114 / Chapter 4.4.4.3 --- Effect of BCP on bone region peripheral to defect --- p.115 / Chapter 4.4.5 --- Immunorejection-free properties of the BCP --- p.116 / Chapter 4.4.6 --- Comparison with other animal studies using scaffold-free cartilage --- p.117 / Chapter 4.4.7 --- Possibility of implanting a BCP cultured for shorter or longer period --- p.118 / Chapter 4.4.8 --- Scaffold-free cartilage construct and construct with scaffold for OCD repair --- p.119 / Chapter 4.4.9 --- Chondrocytes and stem cells for OCD repair --- p.120 / Chapter 4.5 --- Limitations of the study / Chapter 4.5.1 --- Animal model --- p.122 / Chapter 4.5.2 --- Histomorphometric analysis --- p.122 / Chapter 4.5.3 --- Lack of quantitative data analysis --- p.122 / Chapter 4.5.4 --- BrdU labeling of cells --- p.123 / Chapter 4.5.5 --- Lack of biomechanical test --- p.123 / Chapter 4.5.6 --- Small sample size --- p.123 / Chapter CHAPTER FIVE - --- CONCLUSION --- p.124 / Chapter CHAPTER SIX - --- FUTURE STUDIES / Chapter 6.1 --- Identification of factors affecting bone repair after OCD treatment --- p.125 / Chapter 6.2 --- Modifications of BCP treatment --- p.125 / Chapter 6.3 --- Alternative cell source --- p.126 / Chapter 6.4 --- Alternative cell tracking methods --- p.126 / Chapter 6.5 --- Inclusion of biomechanical test --- p.126 / APPENDICES / Appendix 1. Conference paper 1 --- p.129 / Appendix 2: Conference paper 2 --- p.130 / Appendix 3: Animal experimentation ethics approval --- p.131 / BIBLIOGRAPHY --- p.132

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (M.S.)--University of Missouri--Columbia, 2004. / "July 2004." Typescript. Includes bibliographical references (leaves 63-70). Also issued on the Internet. Also available on the Internet.