Development of donor/graft quality assessment methods and cryopreservation protocols to improve the efficiency of osteochondral allograft transplantation

Abstract

Articular cartilage (AC) is a resilient layer of smooth, white connective tissue that envelops the articulating surfaces of bones within a joint. It plays a crucial role in facilitating the proper functioning of joints and serves as an essential constituent of the musculoskeletal system. The regenerative capacity of cartilage is constrained by its inadequate vascularization and the restricted responsiveness of specialized cell populations to damage. Therefore, AC injuries present a unique and challenging medical problem. The existing clinical surgical interventions for the repair of articular cartilage encompass Microfracture, Autologous chondrocyte implantation, Autologous osteochondral transplantation, and Osteochondral allograft (OCA) transplantation. The OCA transplantation procedure has several advantages compared to alternative surgical therapies. Some of these benefits include the ability to fix bigger problems (>3 cm2), avoid problems at the donor site, shorten surgery times, and fix cartilage problems and subchondral bone lesions at the same time. According to reports, it is typically recommended that OCAs be implanted within 28 days following harvest because the chondrocyte viability of the graft decreases significantly after 14–28 days. Therefore, OCA transplantation also has limitations. First, the lack of quality assessment methods for screening grafts leads to lower graft availability and, thus higher costs. Second, there is a lack of preservation solutions for OCA. Thus, the purpose of this study was to develop OCA quality assessment methods and cryopreservation protocols to improve the therapeutic efficacy of OCA transplantation. In chapter I, we introduce a non-destructive analysis method utilizing micro-computed tomography (μCT) to quantify the GAG content inside the extracellular matrix (ECM) of cartilage. This method is employed to assess the functional efficacy of OCA transplantation by comparing GAG levels before and after the transplantation process. The rabbit model OCA was treated with different degrees of chondroitinase to obtain grafts containing different GAG concentrations. The effect of the transplantation surgery was evaluated by μCT and histological analysis 4 weeks and 12 weeks after transplantation. The results show that as the GAG content of OCA before transplantation decreases, the transplantation success efficiency of OAT decreases. Hence, it is hypothesized that the diminished GAG content of the graft in the rabbit model may impact the functional outcome of OCA following transplantation. Moreover, the utilization of non-destructive μCT analysis is a potential means to assess the efficacy of OCA transplantation. In chapter II, we analyzed the changes in ECM content of donor cartilage tissue at different ages, including GAG and collagen type 2 (COL II), and proposed a non-destructive testing method for donor cartilage tissue. A total of 18 human donors with an age range of 22–79 years were analyzed for the characteristics of lateral femoral condyle cartilage tissue. The contents of GAG and COL II in each cartilage tissue were evaluated through biochemical, histological, and micro-computed tomography (μCT) analyses. The cell viability of each cartilage tissue was assessed by live and dead staining analyses. The biochemical analysis results of each cartilage tissue showed that, compared with the 20s–50s, the GAG content in the 60s and 70s groups decreased significantly, with statistical significance. In the comparison of COL II content compared with other groups, it decreased significantly in the 70s group, which was statistically significant. Also in the histological analysis, the positive staining intensity of Safrain-O and COL II histological staining decreased in the 70s group. For the correlation regression analysis between μCT and Safranin-O histological evaluation, the R2 value was 0.845, which means there was a high correlation. The present study demonstrated that in normal human knee joint cartilage tissue, cell viability, GAG, and COL II content decrease with age, especially after the age of 60, when all indicators decline significantly. Therefore, GAG content may become a standard for evaluating the preoperative quality of OCA. Moreover, the biological characteristics of cartilage tissue at different ages (22–79 years old) can be inferred through non-destructive μCT scanning, indicating the possibility of clinical application. In chapter III, to improve the transplantation efficiency of OCAs, we propose a new cryo preservation protocol for human OCAs. OCAs were obtained from the lateral femoral condyle of donors of different ages and were divided into the 20s, 30s, 40s, 50s, 60s, and 70s groups according to age. Assess the permeability of cryoprotectant agents (CPA) to tissues by µCT analysis. Changes in the biological properties of each graft before and after cryopreservation were assessed by live and dead staining, biochemical, and histological analyses. The results of cell live and death staining analysis showed that in the 20s to 50s group, the cell survival rate after cryopreservation for one month was over 70%, while in the 60s and 70s groups, the cell survival rate was about 60%. Similarly, the GAG content of grafts in the 20–50s group maintained the level before cryopreservation but decreased significantly in the 60s and 70s groups. Analysis by culture using chondrocyte pellets showed that the production of GAG and COL II of the grafts in the 20-50s group was similar to that before cryopreservation but decreased in the 60s and 70s groups. This study reports the cryopreservation process of human articular osteochondral grafts, demonstrating that osteochondral grafts from 20 to 50 years of age can be successfully frozen by using this cryopreservation protocol. Keywords: Osteochondral allografts, Glycosaminoglycan, Extracellular matrix, Cryopreserva tion, µCT.