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No / The prevalence of suboptimal outcome for surgical interventions in the treatment of full-thickness articular cartilage damage suggests that there is scope for a materials-based strategy to deliver a more durable repair. Given that the superficial layer of articular cartilage creates and sustains the tribological function of synovial joints, it is logical that candidate materials should have surface viscoelastic properties that mimic native articular cartilage. The present paper describes force spectroscopy analysis by nano-indentation to measure the elastic modulus of the surface of a novel poly(vinyl alcohol) hydrogel with therapeutic potential as a joint implant. More than 1 order of magnitude decrease in the elastic modulus was detected after adsorption of a hyaluronic acid layer onto the hydrogel, bringing it very close to previously reported values for articular cartilage. Covalent derivatization of the hydrogel surface with fibronectin facilitated the adhesion and growth of cultured rat tibial condyle chondrocytes as evidenced morphologically and by the observance of metachromatic staining with toluidine blue dye. The present results indicate that hydrogel materials with potential therapeutic benefit for injured and diseased joints can be engineered with surfaces with biomechanical properties similar to those of native tissue and are accepted as such by their constituent cell type.

Cartilage tissue specimens were obtained from the flexor surface of the navicular bone and distal radiocarpal bone articular surface (controls) from 8 horses 2 to 5 years old. Water, DNA, total collagen, total glycosaminoglycans, chondroitin sulphate, and keratan sulphate contents were determined. The results from each site were compared and the differences were analyzed by paired t-test (P < 0.05). Significant differences were determined between the water content of the navicular bone flexor surface cartilage (68.32&#177; 3.46 % ) and the distal radiocarpal bone articular surface cartilage (60.60&#177; 4.09%). The total DNA content, total glycosaminoglycan content, total chondroitin sulphate content, and total keratan sulphate for the flexor surface of the navicular bone was: 524.51&#177; 92.89 ng, 0.1533&#177; 0.0338 mg, 0.1018&#177; 0.0197 mg 0.0800&#177; 0.0176 mg, and 0.0092&#177; 0.0037 mg per mg of dry weight cartilage, respectively. The total DNA content, total glycosaminoglycan content, total chondroitin sulphate content, and total keratan sulphate for the distal radiocarpal articular surface cartilage was: 508.80&#177; 70.16 ng, 0.1686&#177; 0.00838 mg, 0.0919&#177; 0.0191, 0.0615&#177; 0.0109 mg, and 0.0074&#177; 0.0029 mg per mg dry weight cartilage, respectively. Not significant differences were determined between these values. We concluded that the cartilage of the flexor surface of the navicular bone is biochemically similar to hyaline articular cartilage, but differs from previous descriptions of fibrocartilage. Further studies are needed to determine types and proportions of collagen types of the flexor surface of the normal navicular bone. These findings establish a basis of comparison to assess navicular cartilage in aging, disease, and repair. / Master of Science

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.

L’arthrose est une maladie dégénérative des articulations et représente la deuxième cause d’invalidité en France. En raison des liens entre l’obésité et l’arthrose concernant à la fois les articulations portantes et non portantes, nous faisons l’hypothèse que des protéines produites par le tissu adipeux, les adipokines, constituent des facteurs clés impliqués dans cette arthropathie. En premier lieu, nous avons montré que l’expression de la leptine, de l’adiponectine et de leurs récepteurs évolue de façon inverse et dépend fortement de l’état de différenciation des chondrocytes. Dans une seconde étude, nous avons comparé la production des adipokines par le ligament adipeux de Hoffa à celle mesurée dans la graisse sous-cutanée et avons ainsi mis en évidence des différences entre les 2 tissus adipeux. Les travaux réalisés ensuite ont permis de préciser le rôle des adipokines dans l’arthrose. Ainsi, la production d’adiponectine par les chondrocytes augmente lorsque le cartilage se dégrade et apparaît directement reliée à celle de la MMP-13 et du TGF-[bêta]. En revanche, l’expression de son récepteur AdipoR1 est associée à l’expression d’éléments matriciels et d’un facteur de transcription spécifique du cartilage impliqué dans la synthèse de ces éléments. Le traitement des chondrocytes à l’adiponectine a permis de confirmer in vitro les données observées in vivo chez les patients atteints d’arthrose, à savoir que l’adiponectine induit l’expression du TGF-[bêta]et de la MMP-13. Les résultats obtenus avec la leptine indiquent par ailleurs que l’obésité influence fortement la réponse des chondrocytes à cette adipokine. Elle semble ainsi protéger le cartilage chez les patients non obèses en stimulant l’expression de l’IGF-1, du collagène de type 2 et du TIMP-2, mais contribue au processus dégénératif chez les patients obèses en augmentant l’expression de la MMP-13. Enfin l’induction d’une arthrose expérimentale chez le rat Zucker n’ayant pas de récepteur fonctionnel à la leptine a montré que cette adipokine est susceptible de préserver l’articulation des modifications du cartilage et surtout de l’os sous-chondral / Osteoarthritis (OA) is a degenerative joint disease and represents one of the most frequent and disabling disease. There is a positive association between obesity and OA, and not only for knee joints but also for non-weight-bearing joints suggesting that adipose-derived proteins, namely adipokines, may be some keys factors in OA pathophysiology. First, we found that leptin and adiponectin expression and their receptor evolves in an opposite way and depend on differenciation stage of chondrocyte. The production of adipokines were then compared according to adipose tissue and some differences were found between, the infrapatellat fat pad and subcutaneous adipose tissue. After this work, we aimed to further characterize the role of leptin and adiponectin in OA. Adiponectin production by chondrocytes increases when cartilage is damaged and seems to be directly related with MMP-13 and TGF-[bêta] expression. AdipoR1 expression is associated with the expression of matrix components and with Sox9, a transcription factor involved in their synthesis. Adiponectin treatment confirms data in OA patient, that is adiponectin can induce TGF-[bêta] and MMP-13. Then, we showed obesity influences the chondrocyte responsivness to leptin. This adipokine seems to protect cartilage collected from normal or overweight patient by stimulating IGF-1, type 2 collagen and TIMP-2 expression while leptin increases MMP-13 expression for obese patients. Finally, experimental OA in Zucker rat deficient in leptin receptor, showed the protective effect of leptin on cartilage and on subchondral bone

Osteoarthritis is a joint disease associated with the irreversible breakdown of articular cartilage in joints, causing pain, impaired mobility, and reduced quality of life in over 27 million Americans and many more worldwide. The tolls by osteoarthritis (OA) on the workforce and healthcare system represent significant economic burdens. An attractive strategy for treating OA is cartilage tissue engineering (CTE). CTE strategies have been promising at producing cell-scaffold constructs at small sizes (3-5 mm in largest dimension), but OA often does not present symptoms until lesions reach 25 mm in diameter. Using bovine chondrocytes seeded in agarose, our lab has produced small CTE constructs with native cartilage levels of compressive stiffness and proteoglycan content. As construct dimensions are increased, however, the resulting tissue suffers from extreme heterogeneity of deposited matrix due to nutrient transport limitations. The ability to successfully scale up constructs to clinically relevant sizes is a major goal in CTE research. Another major and largely unresolved obstacle is the translation of successes from animal cell models to CTE systems with human cells, which is ultimately necessary for clinical treatment of OA. In this dissertation, experiments are placed forth which seek to address the nutrient limitations in large cartilage constructs and to help bridge the gap from animal cells to human cells for CTE. The growth of CTE constructs is limited by the poor availability of nutrients at construct centers due to consumption by cells at the construct periphery. The first series of studies in this dissertation sought to identify nutrients in culture media that are consumed by cells and are critical for matrix production, and to characterize their transport behavior. Among several candidate nutrients, glucose proved to be the most indispensable; little to no growth transpired in constructs when glucose fell below a critical threshold concentration. A subsequent study provided a system-specific glucose consumption rate. These parameters were informative for computational models of construct growth, which helped predict transport and growth phenomena in constructs and suggest improved culture techniques for later experiments. The cultivation of tissue constructs of increasing size presents logistical challenges, as the constructs’ requirements for nutrients, growth factors, and even sizes of culture vessels increase. The addition of nutrient channels to constructs to improve nutrient transport and tissue growth is a promising strategy, but more sophisticated casting and culture techniques are required for constructs with channels, particularly as construct size is increased. We first designed casting and culture devices for cylindrical 10 mm × 2.3 mm (diameter × height) constructs with 1 mm diameter nutrient channels. With information gleaned from computational models predicting glucose availability in constructs, we refined our culture system and demonstrated beneficial effects of nutrient channels on construct mechanical properties and extracellular matrix contents. This was the most successful instance to date of the use of nutrient channels in CTE, and is highly promising for channels’ ability to mitigate transport limitations in constructs. We next sought to optimize key parameters for culturing channeled constructs. The addition of channels is an optimization problem: greater numbers of closer-packed channels increase nutrient availability within the construct but simultaneously detract from the construct’s initial volume and cell population. Furthermore, we suspected that uneven swelling of 10 mm diameter constructs was a side effect of transient treatment with 10 ng/mL TGF-β, a highly effective and commonly-employed technique for elevating construct functional properties. By increasing channel densities in 10 mm diameter constructs, we identified a channel spacing that yielded optimal construct functional properties. In constructs with this channel spacing, reducing the TGF-β dosage by tenfold resulted in similar or elevated properties by constructs. These experiments supplied us with optimal parameters for further scaling up our constructs to clinically-relevant sizes, a practice that can be adapted for any CTE culture system for large constructs. The ability to treat severe OA by entirely resurfacing diseased joints with CTE would be highly desirable, yet this ability remains elusive, as efforts to grow constructs of such size have thus far been stymied by nutrient transport limitations. We scaled up our culture system for 10 mm diameter constructs, employing previously optimized culture conditions and channel spacing, and cultured articular surface-sized (40 mm diameter, 2.3 mm thick) constructs. These constructs were 100× the size of our small constructs, yet they still attained similar functional properties, reaching native cartilage levels of compressive stiffness and proteoglycan content. These are the largest CTE constructs to ever achieve such favorable properties. These results demonstrate that with nutrient channels, CTE constructs have the potential to replace entire joint surfaces that have been compromised by OA. Finally, we began to explore the feasibility of translating techniques from our bovine and canine model systems into human cells. We harvested adult human chondrocytes from expired osteochondral allografts and cast them in small (3 mm diameter) constructs, culturing the constructs under various conditions that have been previously successful for animal constructs. We observed similarities between human versus bovine and canine constructs, most notably that high initial cell seeding density led to marked increases in functional properties, in some cases approaching mechanical and biochemical properties of native human cartilage. Human constructs also exhibited poor GAG retention and long-term growth relative to animal constructs. By establishing successful techniques for human constructs in addition to identifying new challenges, we provided an in-depth characterization of human chondrocytes in agarose that is promising overall for eventual clinical translation. The body of work presented in this dissertation followed a methodical approach to scaling up CTE constructs to the sizes of entire joint surfaces, through experimentation with nutrient channels in constructs and with the support of predictive computational models. The principle behind nutrient channels is fundamental and therefore can be applied to CTE systems using other scaffold and cell types. By incrementally increasing the scale of bovine chondrocyte-laden constructs and by performing initial studies with small human CTE constructs, we have laid down groundwork for future studies seeking to grow articular surface-sized human engineered cartilage.

Thesis (M.S.)--Mississippi State University. Department of Agricultural and Biological Engineering. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Boyan, Barbara; Committee Co-Chair: Schwartz, Zvi; Committee Member: Bonewald, Lynda; Committee Member: Jo, Hanjoong; Committee Member: Sambanis, Athanassios. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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