Background: To date, conventional freezing and cryopreservation of articular cartilage has had limited success due to the mechanical injury of cells resulting from uncontrolled ice crystal propagation. Frozen then thawed grafts show a total lack of viable articular cartilage cells and weakened matrix. Directional freezing using a precise velocity offers a new approach to the process of freezing, enabling cryopreservation of articular cartilage for long term storage and implantation.
Hypothesis: Cryopreservation of articular cartilage using directional freezing maintains significant chondrocyte viability and extra cellular matrix quality.
Study Design: Controlled Laboratory Study.
Methods: Articular Cartilage, collected from 20 porcine hind legs harvested immediately after slaughter, was transferred to the processing laboratory for cryopreservation and analysis. Cryopreservation was performed using a directional freezing system (MTG 1315). During preparation for freezing cryoprotectants were injected into the matrix using an array of 20 micron needles. Thirty 15mm cylindrical grafts were examined for cell viability and cell density using fluorescent and confocal microscopy and proteoglycan synthesis via 35SO4 uptake. Biomechanical assessment was performed on a second set of 9 grafts to determine the matrix instantaneous dynamic modulus of elasticity.
Results: Chondrocyte viability (53%±9%), viable cell density (18900 ± 4100 cell/mm3, 68%±5.7% viability) and 35SO4 uptake (59% compared to fresh control) were achieved. Biomechanical measures were mildly impaired (62%±5.2%) compared to fresh control due to the injection of cryoprotectants. In addition, chondrocyte viability in the cryopreserved allograft was preferentially maintained in the superficial zone. Similar results were obtained in human in-vitro studies.
Conclusion: Cryopreservation using directional freezing enables the preservation of viable cells within the collagen matrix. These cells are embedded in the supporting hyaline cartilage matrix with good mechanical stability. The behavior of cryopreserved cartilage after transplantation as indicated in sheep transplantations favors the generation of new, healthy hyaline cartilage during one year follow-up. The high percentage of viable cartilage cells, the quality of the matrix following freezing and thawing, and the ability to store these grafts in a hospital facility, are encouraging to meet the growing demand of such allografts in human cartilage repair procedures.
