Objectives: Osteochondral allografts (OCA) are currently stored at 4˚C for an average of 24 days after procurement. However, chondrocyte viability, the major determinant of graft performance in-vivo, decreases and matrix integrity deteriorates with time under such conditions; for instance, chondrocyte viability falls below the acceptable level of 70% by 28 days. Hydrostatic pressure (HP) is a significant component in the mechanical loading environment and its application in vitro (0.5 - 15 MPa) elicited favorable effects on cartilage and bone. The purpose of this study was to investigate the effects of HP during osteochondral storage. Methods: Osteochondral (OC) explants (6x8 mm) were harvested from bovine humeral heads purchased from a local slaughterhouse (Fig. 1). OCs were randomly assigned to one of 4 groups: freshly harvested; stored at 4°C and atmospheric pressure (4˚C AP); stored at 37°C and AP (37˚C AP); and stored at 37°C with cyclic HP at 0-0.5 MPa, 0.5 Hz (37˚C HP). The explants were stored for 7, 14, 21, or 28 days in Dulbecco’s modified eagle medium/Ham’s nutrient mixture F12 (1:1), with antibiotics and with or without fetal bovine serum (FBS). Chondrocyte viability and density were assessed using LIVE/DEAD® staining in the entire tissue section and in the superficial, middle, and deep zones using the Image J program. In addition, we stained OC explants with safranin-O fast green; hematoxylin and eosin, sudan IV and antibodies against proliferating cell nuclear antigen (PCNA), keratan sulfate (KS). Furthermore, the microstructure and material composition of subchondral bone in the explants was quantified using micro-computed tomography (micro-CT, Scanco uCT 35) at a 12 micron nominal resolution. Results: Cartilage thickness was maintained at 37°C HP, significantly increased at 37°C AP and significantly decreased at 4°C AP (P < 0.05) with/without FBS at 28 days. Full-thickness chondrocyte viability was significantly higher at 21 and 28 days in the 37˚C HP group compared to 4°C or 37°C AP regardless of FBS (P<0.05). Specifically, chondrocyte viability in the superficial zone was maintained with 37˚C HP (Fig. 3) versus not in the other culture conditions. Cartilage matrices were similar between the conditions by 14 days (Fig. 4). However, surface fibrillation and degeneration were demonstrated in the 37 °C AP. KS seemed to be maintained in the superficial zone with HP by 28 days but decreased under other conditions (Fig. 5). More proliferating cells with 37°C HP were observed in the superficial zone compared to other conditions at 28 days. Utilization of FBS further increased the number of proliferating cells in each condition. The fatty granules were observed and distributed evenly throughout the subchondral bone and trabecular bone under all conditions at 28 days (Fig. 6). However, in the 4°C and 37°C AP groups, fewer fatty granules were observed in the subchondral bone and more fat in the trabecular bone (Fig. 6). Micro-CT evaluation revealed that the conditions produced similar tissue mineral density (Fig. 7). However, the trabecular number was significantly lower with 37°C AP compared to the other conditions (P<0.05). Conclusion: Osteochondral explants stored with 37°C HP maintained chondrocyte viability, histologically determined cartilage integrity, and subchondral bone quality. [Figure: see text]
Trabecular Bone Image Segmentation Using Wavelet and Marker-Controlled Watershed Transformation