Treatment of articular cartilage defects in the goat with frozen versus fresh osteochondral allografts: effects on cartilage stiffness, zonal composition, and structure at six months.

BACKGROUND: Understanding the effectiveness of frozen as compared with fresh osteochondral allografts at six months after surgery and the resultant consequences of traditional freezing may facilitate in vivo maintenance of cartilage integrity. Our hypothesis was that the state of the allograft at implantation affects its performance after six months in vivo. METHODS: The effect of frozen as compared with fresh storage on in vivo allograft performance was determined for osteochondral allografts that were transplanted into seven recipient goats and analyzed at six months. Allograft performance was assessed by examining osteochondral structure (cartilage thickness, fill, surface location, surface degeneration, and bone-cartilage interface location), zonal cartilage composition (cellularity, matrix content), and cartilage biomechanical function (stiffness). Relationships between cartilage stiffness or cartilage composition and surface degeneration were assessed with use of linear regression. RESULTS: Fresh allografts maintained cartilage load-bearing function, while also maintaining zonal organization of cartilage cellularity and matrix content, compared with frozen allografts. Overall, allograft performance was similar between fresh allografts and nonoperative controls. However, cartilage stiffness was approximately 80% lower (95% confidence interval [CI], 73% to 87%) in the frozen allografts than in the nonoperative controls or fresh allografts. Concomitantly, in frozen allografts, matrix content and cellularity were approximately 55% (95% CI, 22% to 92%) and approximately 96% (95% CI, 94% to 99%) lower, respectively, than those in the nonoperative controls and fresh allografts. Cartilage stiffness correlated positively with cartilage cellularity and matrix content, and negatively with surface degeneration. CONCLUSIONS: Maintenance of cartilage load-bearing function in allografts is associated with zonal maintenance of cartilage cellularity and matrix content. In this animal model, frozen allografts displayed signs of failure at six months, with cartilage softening, loss of cells and matrix, and/or graft subsidence, supporting the importance of maintaining cell viability during allograft storage and suggesting that outcomes at six months may be indicative of long-term (dys)function. CLINICAL RELEVANCE: Fresh versus frozen allografts represent the "best versus worst" conditions with respect to chondrocyte viability, but "difficult versus simple" with respect to acquisition and distribution. The outcomes described from these two conditions expand the current understanding of in vivo cartilage remodeling and describe structural properties (initial graft subsidence), which may have implications for impending graft failure.

The in vivo performance of osteochondral allografts in the goat is diminished with extended storage and decreased cartilage cellularity.

BACKGROUND: Currently, osteochondral allografts (OCA) are typically used after 4°C storage for prolonged durations (15-43 days), 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. PURPOSE: To determine the effect of 4°C storage duration (14, 28 days) 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 15 recipient goats and analyzed at 12 months. 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 similar after 14 and 28 days of storage, and both 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 nonoperated controls. However, cellularity at the articular surface and matrix fixed charge in 4°C-stored OCA were lower than fresh, by ~55% (95% confidence interval [CI], 32%-76%) and ~20% (CI, 9%-30%), respectively. In frozen OCA, cellularity and matrix fixed charge were lower than 4°C-stored OCA, by ~93% (CI, 88%-99%) and ~22% (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 to 5 times lower density than goat, repair outcomes may be related to absolute minimum number of cells rather than density.

The proteoglycan metabolism of articular cartilage in joint-scale culture.

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 (37 degrees C vs. 4 degrees C) 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 37 degrees C incubation for 28 days, but decreased by approximately 30% with 4 degrees C incubation. Concomitantly, with 37 degrees C incubation, cartilage sGAG was depleted by approximately 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 4 degrees C decreasing release by approximately 79% compared with 37 degrees C incubation, and cycloheximide inhibition of cell metabolism at 37 degrees C decreasing release by approximately 47%. These results indicate that the articular cartilage of joint-scale grafts have enhanced chondrocyte viability with incubation at 37 degrees C, but may need anabolic stimuli or catabolic inhibitors to maintain sGAG content.

Chondrocyte viability is higher after prolonged storage at 37 degrees C than at 4 degrees C for osteochondral grafts.

BACKGROUND: Osteochondral allografts are currently stored at 4 degrees C for 2 to 6 weeks before implantation. At 4 degrees 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: The objective of the study was to determine the effects of prolonged 37 degrees C storage compared with traditional 4 degrees 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) 14 days at 4 degrees C including 10% fetal bovine serum, (iii) 28 days at 4 degrees C including 10% fetal bovine serum, (iv) 28 days at 37 degrees C without fetal bovine serum, (v) 28 days at 37 degrees C including 2% fetal bovine serum, or (vi) 28 days at 37 degrees C including 10% fetal bovine serum. 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: The 37 degrees C storage maintained high chondrocyte viability compared with 4 degrees C storage. Viability of samples after 28 days at 37 degrees C was approximately 80% at the cartilage surface en face, approximately 65% in the superficial zone, and approximately 70% in the middle zone, which was much higher than approximately 45%, approximately 20%, and approximately 35%, respectively, in 4 degrees C samples after 28 days, and slightly decreased from approximately 100%, approximately 85%, and approximately 95%, respectively, in fresh controls. Cartilage thickness, glycosaminoglycan content, and collagen content were maintained for 37 degrees C and 4 degrees C samples compared with fresh controls. CONCLUSION: The 37 degrees 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 a physiologic temperature of 37 degrees C may prolong storage duration, improve graft availability, and improve treatment outcomes.