Cryopreserved, Thin, Laser-Etched Osteochondral Allograft Maintains the Functional Components of Articular Cartilage After Two Years of Storage

Abstract

Background : Despite improvements in treatment options and techniques, articular cartilage repair continues to be a challenge for orthopedic surgeons. This study provides data to support that the 2-year Cryopreserved, Thin, Laser-Etched Osteochondral Allograft (T-LE Allograft) embodies the necessary viable cells, protein signaling, and extracellular matrix (ECM) scaffold found in fresh cartilage in order to facilitate a positive clinical outcome for cartilage defect replacement and repair. Methods: Viability testing was performed by digestion of the graft, cells were counted using a trypan blue assay. Growth factor and ECM protein content was quantified using biochemical assays. A fixation model was introduced to assess tissue outgrowth capability and cellular metabolic activity in vitro . Histological and immunofluorescence staining were employed to confirm tissue architecture, cellular outgrowth, and presence of ECM. The effects of the T-LE Allograft to signal bone marrow-derived mesenchymal stem cell (BM-MSC) migration and chondrogenic differentiation were evaluated using in vitro co-culture assays. Immunogenicity testing was completed using flow cytometry analysis of cells obtained from digested T-LE Allografts and fresh articular cartilage. Results: Average viability of the T-LE Allograft post-thaw was found to be 94.97 ±3.38%, compared to 98.83 ±0.43% for fresh articular cartilage. Explant studies from the in vitro fixation model confirmed the long-term viability and proliferative capacity of these chondrocytes. Growth factor and ECM proteins were quantified for the T-LE Allograft revealing similar profiles to fresh articular cartilage. Cellular signaling of the T-LE Allograft and fresh articular cartilage both exhibited similar outcomes in co-culture for migration and differentiation of BM-MSCs. Flow cytometry testing confirmed the T-LE Allograft is immune-privileged as it is negative for immunogenic markers and positive for chondrogenic markers. Conclusions: Using our novel, proprietary cryopreservation method, the T-LE Allograft retains excellent cellular viability, with native-like growth factor and ECM composition of healthy cartilage after two years of storage at -80 o C. The successful cryopreservation of the T-LE Allograft alleviates the limited availably of conventionally used fresh osteochondral allograft (OCA), by providing a readily available and simple to use allograft solution. The results presented in this paper supports clinical data that the T-LE Allograft can be a successful option for repairing chondral defects.