Background Osteochondral allografts are currently stored at 4°C for 2–6 weeks before implantation. At 4°C, chondrocyte viability, especially in the superficial zone, deteriorates starting at 2 weeks. Alternative storage conditions could maintain chondrocyte viability beyond 2 weeks, and thereby facilitate increased graft availability and enhanced graft quality. Purpose Determine effects of prolonged 37°C storage compared to traditional 4°C storage on chondrocyte viability and cartilage matrix content. Study Design Controlled Laboratory Study Methods Osteochondral samples from humeral heads of adult goats were analyzed (i) fresh, or after storage in medium for (ii) 14d at 4°C including 10% FBS, (iii) 28d at 4°C including 10% FBS, (iv) 28d at 37°C without FBS, (v) 28d at 37°C including 2% FBS, or (vi) 28d at 37°C including 10% FBS. Portions of samples were analyzed by microscopy after LIVE/DEAD® staining to determine chondrocyte viability and density, both en face (to visualize the articular surface) and vertically (overall and in superficial, middle, and deep zones). The remaining cartilage was analyzed for sulfated-glycosaminoglycan and collagen. Results 37°C storage maintained high chondrocyte viability compared to 4°C storage. Viability of samples after 28d at 37°C was ~80% at the cartilage surface en face, ~65% in the superficial zone, and ~70% in the middle zone, which was much higher than ~45%, ~20%, and ~35%, respectively, in 4°C samples after 28d, and slightly decreased from ~100%, ~85%, and ~95%, respectively, in fresh controls. Cartilage thickness, glycosaminoglycan content, and collagen content were maintained for 37°C and 4°C samples compared to fresh controls. Conclusion 37°C storage of osteochondral grafts supports long-term chondrocyte viability, especially at the vulnerable surface and superficial zone of cartilage. Clinical Relevance Storage of allografts at physiological temperature of 37°C may prolong storage duration, improve graft availability, and improve treatment outcomes.

Background Currently, osteochondral allografts (OCA) are typically used after 4°C storage for prolonged durations (15-43days), which compromises chondrocyte viability, especially at the articular surface. The long-term in vivo performance of these fresh-stored allografts, in association with variable cellularity, is unknown. Hypothesis/Purpose Determine the effect of 4°C storage duration (14, 28days) versus the best (fresh) and worst (frozen) conditions of chondrocyte viability on structure, composition, and function of cartilage in the goat, and the association of retrieved chondrocyte cellularity with those tissue properties. Study Design Controlled Laboratory Study Methods The effect of allograft storage on in vivo repair outcomes was determined for OCA transplanted into fifteen recipient goats and analyzed at 12months. Repair outcomes were assessed by examining cartilage structure (gross, histopathology), composition (cellularity by depth, matrix fixed charge), and biomechanical function (stiffness). Relationships between cellularity and structural scores, matrix fixed charge, and stiffness were assessed by linear regression. Results Repair outcomes in 4°C-stored OCA were inferior to fresh OCA, and were accompanied by diminished cellularity at the surface, matrix fixed charge, and histopathological structure. Overall, cellularity by depth and matrix fixed charge in cartilage of fresh OCA were similar to non-operated controls. However, cellularity at the articular surface and matrix fixed charge in 4°C-stored OCA were lower than fresh, by ~55% (95%CI, 32-76%) and ~20% (95%CI, 9-30%), respectively. In frozen OCA, cellularity and matrix fixed charge were lower than 4°C-stored OCA, by ~93% (95%CI, 88-99%) and ~22% (95%CI: 10-35%), respectively. Cellularity correlated negatively with cartilage health indices, including structural scores, and positively with matrix fixed charge and stiffness. Conclusion Reduced cellularity at the articular surface, resulting from 4°C storage, was associated with variable long-term outcomes, versus consistently good repair by fresh allografts. Cellularity at the articular surface was an important index of biological performance. Clinical Relevance Normal chondrocyte density in vivo, especially in the superficial region of cartilage, is important for maintaining long-term cartilage function and matrix content. In human cartilage, containing cells at ~3-5× lower density than goat, repair outcomes may be related to absolute minimum number of cells rather than density.

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Understanding and controlling chondrocyte and cartilage metabolism in osteochondral tissues may facilitate ex vivo maintenance and application, both for allografts and tissue-engineered grafts. The hypothesis of this study was that maintenance of chondrocyte viability and matrix content and release of sulfated glycosaminoglycan (sGAG) in the articular cartilage of joint-scale osteochondral fragments are temperature and metabolism dependent. The aims were to assess, for adult goat joints, the effects of incubation temperature (378C vs. 48C) on cartilage chondrocyte viability and tissue matrix content and mechanical function, and the effects of temperature and cellular biosynthesis on sGAG release. Chondrocyte viability was maintained with 378C incubation for 28 days, but decreased by *30% with 48C incubation. Concomitantly, with 378C incubation, cartilage sGAG was depleted by *52% with the lost sGAG predominantly unable to aggregate with hyaluronan, whereas collagen content, tissue thickness, and tissue stiffness were maintained. The depletion of sGAG was diminished by slowing metabolism, with 48C decreasing release by *79% compared with 378C incubation, and cycloheximide inhibition of cell metabolism at 378C decreasing release by *47%. These results indicate that the articular cartilage of joint-scale grafts have enhanced chondrocyte viability with incubation at 378C, but may need anabolic stimuli or catabolic inhibitors to maintain sGAG content.