Abstract Type IX collagen is a quantitatively minor component of cartilage collagen fibrils. Although a few mutations have been associated with multiple epiphyseal dysplasia, recent evidence suggests involvement of type IX collagen in a wider spectrum of phenotypes. The functional role of this molecule remains undetermined, in part due to difficulties in obtaining high amounts of intact protein. To facilitate more efficient mutation screening and comparison of the genomic organization of the human genes encoding the α1(IX), α2(IX) and α3(IX) polypeptides, their genomic structures were characterized. Complete nucleotide sequences were determined for the COL9A2 and COL9A3 genes along with sequences for all the exon boundaries in the COL9A1 gene. Putative transcription control elements were identified and the alternative promoter region was characterized in the human and mouse COL9A1 genes. Mutation screening was performed for the COL9A3 gene and two apparently neutral 9-bp deletions within the COL1 domain were identified. These are the first deletions within a triple-helical domain of any collagen that are not associated with a disease phenotype. An insect cell expression system with an exogenous source of prolyl 4-hydroxylase was used to produce heterotrimeric human type IX collagen. The recombinant protein consisted of the three a chains in a 1:1:1 ratio and showed correct folding and high thermal stability. Up to 10 mg of secreted protein could be purified from a litre of culture medium. The expression system was used to analyze the chain association of type IX collagen in cellulo. Although the chains are capable of homotrimerization, a preference for heterotrimer formation was noted.The neutral deletion was characterized further using the insect cell system. Mutant α3(IX) chains carrying a deletion of one Gly-X-Y triplet were shown to form correctly folded heterotrimers with the wild-type α1(IX) and α2(IX) chains. The results suggest a function for the NC2 domain in neutralizing the effect of the deletion. This work provides a novel means for the analysis of type IX collagen mutations and their protein-level effects, and should enable future studies to be made of the structure-function relationship in type IX collagen.

Acetabular labral tears are associated with hip osteoarthritis. A current surgical treatment strategy for a torn labrum, labral resection, has recently shown poor patient outcomes with radiographic signs of osteoarthritis two-years post-operation. Since mechanical factors play a role in the etiology of osteoarthritis, identifying the mechanical role of the labrum may enhance current surgical treatment strategies. In this pilot study, we assessed the relationship between mean cartilage strain, maximum cartilage strain and the three-dimensional cartilage strain distribution in six human cadaver hips with various pathologic conditions of the labrum. We developed a novel technique of mapping cartilage strain using quantitative magnetic resonance imaging (qMRl). qMRl provides a non-invasive means of quantifying the cartilage strain distribution in the hip in three dimensions. Each specimen was assessed first with an intact labrum, then after surgically simulating a longitudinal peripheral labral tear, then after arthroscopically repairing the tear, and after labral resection. We validated the precision of the technique through use of an additional specimen which served as a control. To minimize motion artifact in the high-resolution MR images, we determined that 225 minutes was required for cartilage to reach a steady-state thickness under load. We also determined 16.5 hours was required for cartilage to recover to a steady-state unloaded thickness. The difference in mean and maximum cartilage strain when the labrum was repaired and resected was assessed using a paired t-test. We found that the resected group had an increased mean and maximum cartilage strain of 4% and 6%, respectively and the 3D cartilage strain distribution was elevated throughout the region of interest. When the condition of the intact labrum was compared to the torn labrum, we found no change in mean and maximum cartilage strain, and little obvious change in the 3D pattern of cartilage strain distribution. Based on our findings of increased cartilage strain after labral resection when compared to labral repair, we hypothesize that the labrum’s contribution of additional surface area assists in load distribution, which spares cartilage from excessive loads. We therefore recommend that the longitudinal peripheral torn labrum should not be resected if it is possible to be repaired, because in vivo, labral resection may create an environment with increased articular cartilage strain, which is thought to be associated with cartilage degeneration. / Applied Science, Faculty of / Mechanical Engineering, Department of / Graduate

No description available.

Osteoarthritis (OA) is the most common type of joint disease that affects nearly 5 million people in Canada alone. OA involves degenerative processes affecting the integrity of articular cartilage, a thin soft tissue at the surface of subchondral bones in joints that facilitates smooth and friction less movement. This disease also affects the other tissues of the joint, including the synovium and its resident cells the fibroblast-like and macrophage-like synoviocytes. Consequences of this pathology include painful movement and stiff joints resulting in loss of range of motion. A broad number of factors may contribute to the development of OA, including obesity, injuries, infections, genetic predispositions and aging. Although there are a number of medications used for the treatment of OA, these only serve to manage its symptoms. An actual cure is yet to be developed. Inorganic polyphosphate (polyP) has previously been identified as a potentially interesting biomolecule for the treatment of OA because of its ability to stimulate tissue formation by chondrocytes - the cells found in the articular cartilage. In this thesis, we first aimed to evaluate the potential of polyP as a therapeutic for joint diseases such as OA further, by characterizing its effects on a number of cell processes (e.g., proliferation, metabolic activity, migration, matrix accumulation) in fibroblast-like synoviocytes (FLS) - specialized cells found in the synovium encapsulating the synovial joints. The synovium is an important tissue for joint physiology and OA pathogenesis; it is thus essential that any therapeutic introduced in the joint not impact this tissue negatively. These studies showed that polyP significantly inhibits FLS proliferation. This effect is interesting in the context of OA as FLS proliferation is associated with progression of the disease. PolyP also increased FLS migration rate and caused changes in metabolic activity, although the trends were inconsistent over time. We also optimized a new protocol for the synthesis of sub-micron calcium-polyP particles. Nanoparticle drug delivery for OA treatment has been gaining importance in recent years as a way to access the cells in cartilage through the small pores in the extracellular matrix (ECM) and increase drug retention time in the joint. The calcium-polyP particles were synthesized by drop-wise addition of polyP into a calcium solution at controlled pH, drop rate and mixing rate. Particles size and stability before and after sterilization were measured by dynamic light scattering. We showed that the addition of sodium citrate dihydrate as a capping agent largely prevented particle agglomeration and increased particle stability. Control over particle size, particularly in the nanometer scale, remains to be achieved; however, this work is a first step towards the development of polyP delivery systems for the treatment of OA.

Indiana University-Purdue University Indianapolis (IUPUI) / Osteoarthritis affects millions of people of different age groups around the world. With very few treatment options and the highly restricted capacity of cartilage to repair, new treatment options are needed. The objective of this thesis was to develop a repeatable cartilage testing protocol, which could be used to test cartilage properties and determine if crosslinking can be used as a potential treatment for osteoarthritis. Previous studies have shown CASPc can be used as a photo-sensitizer to obtain collagen crosslinking through a secondary process. The ability to perform cartilage crosslinking by light-activation, which could be done arthroscopically is especially attractive as this would allow the surgery to be minimally invasive. The indentation protocol developed for a stress-relaxation test was able to achieve 95% repeatability, meaning the error in determining cartilage properties stayed within 5% of the average for tests performed at different times. Results of photo-chemical crosslinking demonstrated no change in cartilage stiffness when compared with control specimens. The spherical indenter chosen to indent the cartilage was suspected to apply less strain on cartilage as a result of its profile, which only compressed the cartilage instead of stretching its surface. The stiffness of CASPc control specimen was observed to be increasing when compared with no-CASPc control, as a result of added viscosity of CASPc solution. This elevated stiffness was observed to diminish over time due to the diffusion of CASPc from cartilage.

A Thesis Submitted to the Faculty of Science University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy Johannesburg 1976 / This thesis describes the results of experimental investigation of aspects of the endocrine regulation of growth in the rabbit, rat and man. In particular the role of serum somatomedin and cartilage responsiveness to somatomedin have been examined. Throughout a relationship between the physiological control mechanisms, namely the stimulus and the degree of end- organ responsiveness, and the velocity of growth has been sought. / IT2017

Articular cartilage (AC) provides an interface between bones within joints that serves to minimize friction, absorb and distribute load along the joint, and facilitate movement. Cartilage has poor self-repair abilities following injury, in part due to the limited migration of chondrocytes and the avascular nature of the tissue which impedes the ability of progenitor cells to reach the site of injury. As a result, cartilage defects are at risk of progressing into osteoarthritis (OA), a degenerative disease of the whole joint. OA affects almost 5 million Canadians and can cause pain, severely reduce joint mobility, and negatively impact quality of life. Cartilage tissue engineering is a field that aims to develop strategies to repair cartilage defects with a combination of cells, biomaterials, and/or cues, either biochemical or biophysical in nature, to guide tissue formation. Tissue engineering strategies can include an in vitro maturation phase to create cartilage constructs with sufficient mechanical properties to withstand the cyclic loads present in the joint upon implantation. Identifying optimal culture conditions during this in vitro maturation phase is key for the generation of functional cartilage constructs. Typical tissue engineering strategies use supraphysiological glucose concentrations to ensure there is sufficient glucose in the media for energy production and proteoglycan synthesis between media changes; however, studies have found that these elevated glucose concentrations may elicit catabolic processes in the chondrocytes. We hypothesized that culturing constructs at physiological glucose concentrations in larger media volumes, to prevent glucose depletion, would generate cartilage constructs with superior biochemical properties. To test this hypothesis, primary chondrocytes were cultured in physiological (5 mM) and supraphysiologic (25 mM) glucose concentrations at low (2 mL) and high (11 mL) media volumes. The composition of the tissue and the different metabolic pathways conducted by chondrocytes were then evaluated. Our results indicated that high media volumes generate constructs with significantly higher proteoglycan and collagen content, the two major components of the extracellular matrix. Physiological glucose concentrations had no apparent effect on matrix accumulation; however, histological sections suggest that this culture condition may provide improved cell morphology. The glucose consumption rate was comparable for all four media conditions which suggests that the constructs may have similar matrix synthesis rates. Lactate concentration was significantly higher in low media volumes which may lead to a more acidic environment. The levels of both bioenergetic molecules quantified in the constructs, adenosine triphosphate (ATP) and inorganic polyphosphate (polyP), follow similar trends as the levels of matrix components; however, the relationship between ATP and polyP remains poorly understood. This thesis provides insight into the optimal culture conditions for engineered cartilage by demonstrating that media volume is an important culture parameter for matrix accumulation. Future work is required to understand the mechanisms behind this effect of media volume, to characterize effects of glucose concentration at the cellular level, and to identify key nutrients that will form functional cartilage constructs.

Le chitosan est un polysaccharide linéaire dérivé de la chitine formé de monomères de glucosamine et de N-acétyl-D-glucosamine. Utilisé comme implant dans un nouveau modèle de régénération du cartilage qui est basé sur la technique de la microfracture, ce biopolymère induit un recrutement massif des neutrophiles humains (PMN) en plus d'améliorer significativement la réparation du cartilage hyalin par rapport aux autres techniques actuellement utilisées en médecine régénérative. Ce projet de maîtrise avait pour objectif premier de définir le phénotype des PMN en présence de chitosan 80M. une préparation déacétylée à 80% qui est utilisée dans le modèle de régénération présenté. Puisque le degré de déacétylation est une caractéristique structurelle importante qui confère en partie au chitosan ses effets biologiques, deux préparations de chitosan ont été utilisées afin de comparer les phénotypes obtenus (80M et 95M). L'étude a démontré que le chitosan 80M est chimiotactique pour les PMN contrairement au chitosan 95M. De plus, les PMN ne produisent pas de réactifs toxiques de l'oxygène et ne libèrent pas le contenu de leurs granules en présence de chitosan, contrairement aux agonistes classiques tels que le fMLP ou le LPS. Ces résultats suggèrent que le chitosan aurait un effet positif sur les PMN en empêchant ces cellules d'être néfastes suite à l'utilisation de leurs armes antimicrobiennes, ce qui permettrait ainsi d'avoir une meilleure réparation du cartilage.

Indiana University-Purdue University Indianapolis (IUPUI) / This study investigated the response of the condylar cartilage subjected to a small bone defect as a local stimulus in the rat mandible, in order to clarify one of the mechanisms of condylar cartilage growth. Fourteen male Wistar albino rats weighing 200 to 260 gm were divided into two groups, with two animals in a pilot study and 12 animals in the experimental group. Five male Wistar albino rats weighing 400 to 540 gm were used in a third group, which provided a basis for comparisons with the experimental group. To evaluate bone growth changes of the rat mandible, a superimpositioning method of tracings of the left and the right mandibles was done using the molars, incisors, anterior and inferior borders of the mandible. A sliding micrometer was used to measure each distance between the posterior edge of mental foramen (MF) and the posterior border of condylar process (CP) or angular process (AP). In the pilot study, one of two animals showed a decreased angular process and an increased condylar process, and the results suggested that the decreased activity of the masseter, resulting from dissecting a part of the masseter, may play an important role in changing the size or the shape of the rat mandible. In the experimental study, the masseter was not dissected and the results indicated that a hole drilled at the condylar notch did not stimulate condylar growth enough to be measurable. The third group showed that there were minor asymmetries between the left and the right mandible in normal rats, ranging from 0.49 to .0297 mm. Therefore, a larger sample size would be needed to arrive at definite conclusions.

No description available.