Ice-free Cryopreservation with Nanowarming for Banking of Viable Meniscal Transplants

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SUMMARY Nearly a million Americans suffer from meniscus tears every year, and 80% of patients who undergo partial or total meniscus removal subsequently develop pain, transient effusion, and knee osteoarthritis. Currently, meniscal allograft transplantation (MAT) with human cadaver tissues is considered medically necessary for treatments with symptomatic post meniscectomy knees with major meniscus loss. Although MAT enables favorable outcomes in the long term, the challenge of donor-recipient size-matching strongly limits broad implementation of MAT since ineffective preservation techniques restrict the number of available fresh grafts. Our ultimate, long-term objective is to maintain cell viability, tissue structure and biomechanical integrity of meniscal allografts for the purpose of long-term storage, thereby overcoming the size-matching challenge. Our previous studies have demonstrated that ice-free cryopreservation by vitrification combined with nanowarming is a promising preservation method for preserving living cells and matrix structure in large avascular tissues. However, this method has encountered critical challenges when applied to meniscal tissues, the major hurdle being the transport limitation of cryoprotectant (CPA) molecules in the meniscus. The large and complex meniscal structure and ECM composition prevents uniform distribution of CPA throughout the tissue, thereby causing ice crystal formation during cooling, which damages living cells and tissue structure. To overcome the CPA transport limitation, two critical knowledge gaps need to be filled: 1) location-specific mass transport mechanism of CPA molecules in the whole meniscus, 2) location-specific effects of CPA concentration and exposure time (CPA toxicity) on meniscal viability. Herein, we hypothesize that meniscal allografts loaded with adequate and toxically tolerable CPAs can be preserved without the loss of viability and functionality by vitrification integrated with nanowarming compared to conventionally cryopreserved allografts. Specifically, we aim to 1) determine location-specific porcine meniscal structure- and composition-dependent CPA transport properties in the whole porcine meniscus, 2) investigate location-specific effects of CPA concentration and exposure time on meniscal viability and optimize both parameters to achieve optimal viability after vitrification, and 3) evaluate in vivo functionality of vitrified meniscal allografts in a 4-month preclinical transplant study using a porcine model. Collectively, these aims will demonstrate our concept that the vitrification method can be used to preserve living cells and tissue structures in meniscal allografts in a pig model. Future work will combine the developed methodologies and our computational model to optimize viability of human meniscal allografts after CPA addition and vitrification to identify the optimal CPA loading and vitrification protocol for each donor graft. Further, by incorporating patient-specific meniscus anatomies and morphology, this work will break therapeutic limitations in meniscal replacement.