During open orthopaedic surgical procedures, the articular cartilage covering the exposed joint surfaces can be exposed to air for prolonged periods. This exposure facilitates cartilage drying, characterised by changes to the extracellular matrix and chondrocyte death. Due to cartilage’s limited capacity for regeneration, it has been proposed that this may lead to post-operative joint degeneration. It has been proposed that chondrocyte death occurs as a result of both drying and nutritional deficiency. It is known that death during drying correlates with the drying interval and is initiated in the superficial chondrocytes, progressing to deeper layers at higher intervals. Additionally, it is well established that periodic rewetting (e.g. using 0.9% saline solution) can reduce chondrocyte death. The work presented in this thesis aimed to characterise chondrocyte death during drying, with particular reference to the chondrotoxic/protective effect of environmental variables (airflow) and surgical interventions (irrigation solutions), mechanism of injury and cell death, and the effect of in vivo drying on joint health. An ex vivo model of cartilage drying was developed and carried out on bovine and human intact cartilage and osteochondral explants, while varying environmental factors (drying interval, airflow velocity, oxygen concentration) and interventions (irrigation solutions and protective coverings. Throughout the study, cartilage drying was assessed in terms of 1) cartilage macroscopic appearance, 2) percent chondrocyte death (PCD), 3) cartilage water content, and 4) chondrocyte morphology. Histologically and fluorescently labelled samples were imaged using light and confocal laser scanning microscopy respectively, which formed the basis of the qualitative and quantitative assessments. Experimental drying at high airflow velocities had a more severe effect on cartilage appearance, PCD, and water content than in static air. This relationship was apparent in dried intact joints and osteochondral explants and in bovine and human samples. This suggests that the effects of surgical drying (where ventilation systems and airflow are routine) may be more pronounced than previously suggested and demonstrates a correlation between PCD and water-loss. Irrigation solutions supplemented with glucose (25-100 mM) had no significant effect on the PCD or water content in dried samples. Additionally, PCD was minimal in osteochondral explants cultured in the absence of glucose, even after 24 hr. This suggests that nutritional deficiency is unlikely to contribute to PCD during drying. However, chondrocyte death (in intact bovine cartilage) was reduced when drying was carried out at an oxygen concentration more reflective of the in vivo environment (5 %), which suggests that cell death during drying may be facilitated by a hyperoxic shock. Finally, in vivo cartilage drying was carried out on murine cartilage. Compared to sham operated controls, dried cartilage demonstrated a loss of surface integrity (4 weeks post-surgery) and fibrillations (8 weeks) and an increased modified Mankin score (at 4 and 8 weeks). Microscopically, an altered cartilage thickness, and chondrocyte density and arrangement were visible. These changes are comparable with changes in osteoarthritis. The results of this study demonstrate the importance of maintained cartilage hydration in order to avoid unnecessary chondrocyte death articular cartilage degeneration.

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Thèse de doctorat : Sciences physiques et de l'ingénieur. Mécanique : Bordeaux 1 : 2008. / Titre provenant de l'écran-titre.

Thesis (M.S.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-186).

This study aimed to characterize the in-situ mechanical property and morphology of individual collagen fibril in osteoarthritic (OA) cartilage using indentation-type atomic force microscopy (IT-AFM). The specimens with intact articular cartilage (AC), mild to severe degenerated OA cartilage were collected with informed consent from the postmenopausal women who underwent hip or knee arthroplasty. The fresh specimens were cryo-sectioned by layers with 50m thick for each from the articular surface to calcified cartilage, and then processed for AFM imaging and nanoindentation test. For each layer, a total of twenty collagen fibrils were randomly selected for testing. AFM tips with the nominal radius less than 10 nm were employed for probing the individual collagen fibril, and the obtained cantilever deflection signal and displacement were recorded for calculating its elastic modulus. Besides AFM nanoindentation, AFM and scanning electron microscopy (SEM) images, haematoxylin & eosin (H&E) staining and micro-indentation were performed on AC to study the changes of ultrastructure and composition between intact AC and OA cartilage. Results showed that an intact AC exhibited a gradation in elastic modulus of collagen fibrils from surface region (2.65±0.31GPa) to bottom region (3.70±0.44GPa). It was noted in the initial stage of OA cartilage that the coefficient of variation for mechanical properties of collagen fibers, ranging from 25~48%, significantly increased as compared with intact one (12%). The thickened and stiffened collagen fibrils initially occurred at either surface region (3.11±0.91GPa) or bottom region (5.64±1.10GPa) with OA progression. Besides thickens, alteration of D-periodic banding patterns of collagen fibrils was observed. It was echoed by fibrotic changes of surface region and tidemark irregularities. On the contrast, the micromechanical properties of cartilage decreased while AC suffered from OA. This result revealed the different approachs of nano and micro-mechanical properties changes in AC. In summary, the alteration of mechanical properties of collagen fibrils started from calcified cartilage as well as articular surface during OA onset, and the low compliance of thickened collagen fibrils deteriorated along disease progression. This study also reveals that the outstanding ability by AFM, in investigating the structure and mechanical properties of collagen fibrils and AC in nanometer scale, is impressive and this nanotechnological instrument is worth to be expected in further development for clinical use. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

One major barrier that prevents cartilage integration following mosaic arthroplasty is the presence of a zone of chondrocyte death (ZCD) that is generated upon osteochondral graft harvest, which can extend up to 400 μm into the cartilaginous portion of the graft. In order for cartilage integration to occur, chondrocytes must be present at the graft periphery; however chondrocyte migration through the ZCD to the graft periphery is inhibited by the dense extracellular matrix (ECM) of cartilage. The purpose of this study was to develop a method for increasing the number of chondrocytes within the ZCD and at the periphery of a cartilage graft. This method used a combination of collagenase treatment (as a means of degrading the ECM within the ZCD) and chondrocyte chemotaxis (as a means of improving chondrocyte migration into the ZCD and to the cartilage periphery). Results indicate that treating bovine articular cartilage with 0.6 % collagenase for 10 min decreased with extent of the ZCD by approximately 35% (collagenase: 109 ± 13 μm; control: 175 ± 13 μm). Each of the chemotactic agents tested (PDGF-bb, bFGF, and IGF-I) were found to induce bovine chondrocyte chemotaxis at concentrations of 25 ng/mL in modified Boyden chamber experiments. However, in bovine articular cartilage samples that were pre-treated with collagenase (0.6% for 10 min), supplementation with 25 ng/mL of either PDGF-bb or bFGF had no apparent effect on the ZCD relative to samples treated only with collagenase (PDGF-bb: 85 ± 10 μm; bFGF: 88 ± 10 μm). Alternatively, bovine articular cartilage samples pre-treated with collagenase (0.6% for 10 min) and supplementation with 25 ng/mL IGF-I resulted in an approximately 65% reduction in the ZCD relative to samples treated only with collagenase (IGF-1: 38 ± 5 μm). Thus, treating osteochondral grafts with collagenase and IGF-1 induces chondrocyte repopulation of the zone of chondrocyte death generated by osteochondral graft harvesting, and could enhance cartilage integration after implantation. / Thesis (Master, Chemical Engineering) -- Queen's University, 2009-12-21 20:16:05.815

Tissue engineering holds great promise for developing functional tissue to repair damaged articular cartilage. However, cartilaginous tissues formed in vitro typically possess poor mechanical properties in comparison to native tissue due to the inability of chondrocytes to accumulate adequate amounts of extracellular matrix (ECM). While mechanical stimuli can enhance the properties of engineered cartilage, it may be more efficient to harness the underlying mechanotransduction pathways responsible. Substantial evidence suggests that the mechanotransduction signaling cascade is initiated by rapid adenosine 5’-triphosphate (ATP) release from chondrocytes (purinergic receptor pathway). Thus, the purpose of this study was to investigate the effects of exogenous ATP supplementation on the biochemical and mechanical properties of tissue engineered cartilage. Primary bovine articular chondrocytes, seeded on MilliporeTM filters, were grown in the presence of 0, 62.5 or 250 µM ATP for a period of four weeks. Both anabolic and catabolic effects were examined and a therapeutic dose range of ATP was determined. ATP stimulation (62.5 - 250 µM) enhanced ECM synthesis by 23 - 43% and long-term collagen accumulation by 16 - 26%, in a dose-dependent manner; however, long-term proteoglycan accumulation decreased as a result of 250 µM ATP. In addition, ATP supplementation significantly improved the mechanical properties of the developed tissues (5- to 6.5-fold increase in tissue stiffness). Interestingly, high doses of ATP (250 µM) also elicited a 2-fold increase in MMP-13 gene expression, 39% increase in MMP-13 activity, and 54% more extracellular inorganic pyrophosphate (ePPi) – an ATP degradation product. Dose-dependent increases in MMP-13 activity suggested that catabolic effects were occurring alongside anabolic effects, which initiated the investigation of a therapeutic dose of ATP. Doses of 31.25 µM and 125 µM ATP were added to cartilaginous tissues and investigated in terms of MMP-13 activity and ECM synthesis. Tissues supplemented with 62.5 – 125 µM ATP exhibited a balance between anabolic and catabolic effects. By harnessing the purinergic receptor pathway, anabolic effects of mechanical stimuli were achieved in the absence of externally applied forces. Understanding how the catabolic effects of ATP are manifested would be valuable in order to further maximize the therapeutic potential of ATP stimulation. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2010-09-22 14:08:28.114

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