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

  • JIN YONGJUN (JIN YONGJUN, 아주대학교 일반대학원)

초록/요약

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.

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목차

BACKGROUND １
1.1. Articular cartilage ２
1.1.1. Chondrocyte ２
1.1.2. Glycosaminoglycan ３
1.2. Osteochondral allograft (OCA) transplantation ３
1.2.1. The standards of OCA quality assessment ４
1.2.2. The analysis method of OCA quality assessment ４
1.2.3. The cryopreservation of OCA ５
1.3. Aim of study ５
CHAPTER I: Effects of Glycosaminoglycan content in donor cartilage extracellular matrix on the functional properties of osteochondral allograft as evaluated by μCT non-destructive analysis ６
2.1. Introduction ７
2.2. Materials and methods １０
2.2.1. Experimental design １０
2.2.2. Preparation and characterization of OCA １２
2.2.3. Radiographic iinvestigation of OCA １２
2.2.4. The quantification of GAG levels in OCA １３
2.2.5. Compare the correlation between OCA's µCT value and GAG content １３
2.2.6. Cellular characterization of OCA １３
2.2.7. The histological examination of OCA １４
2.2.8. Analysis of OCA biomechanics １４
2.2.9. Preoperative analysis of OCA １４
2.2.10. OCA transplantation animal model. １５
2.2.11. Gross morphological characteristics of postoperative OCA １５
2.2.12. The postoperative OCA was subjected to µCT and GAG quantitative analysis １５
2.2.13. Statistical analysis １６
2.3. Results １７
2.3.1. Analysis of GAG content and cell viability of OCA １７
2.3.2. Assess OCA characteristics through imaging, biomechanical and histological analysis ２０
2.3.3. The preoperative assessment of OCAs ２３
2.3.4. Gross morphology assessment after OCA transplantation ２５
2.3.5. Biomechanical assessment after OCA transplantation ２５
2.3.6. Histological assessment after OCA transplantation ２５
2.3.7. Quantitative analysis of OCA GAG content and HU value after surgery ２９
2.3.8. Quantitative comparison of functional changes at postoperative OCA transplant sites ３１
2.3.9. Cell viability at the OCA transplantation site after surgery ３３
2.4. Discussion ３５
CHAPTER II: To analyze the biological characterization of human femoral condyle cartilage in aging variation, with a non-destructive measurement method ３９
3.1. Introduction ４０
3.2. Materials and methods ４２
3.2.1. Experimental design ４２
3.2.2. Radiographic analysis ４４
3.2.3. Biochemical analysis ４６
3.2.4. Compare correlation between OCA's µCT and GAG content ４６
3.2.5. Cell viability analysis ４６
3.2.6. Histologic and Immunohistochemical analysis ４６
3.2.7. Statistical analysis ４７
3.3. Results ４８
3.3.1. Evaluation of chondrocyte viability in OCA in different age groups ４８
3.3.2. The histological assessment of OCAs across several age cohorts ５１
3.3.3. Evaluation of OCAs Safranin-O and μCT images in different age groups ５３
3.3.4. COL II immunohistochemical evaluation of OCAs in different age groups ５８
3.4. Discussion ６０
CHAPTER III: Cryopreservation of human articular cartilage using Controlled-Rate Freezers: Assessment of cryoprotectant penetration by µCT ６３
4.1. Introduction ６４
4.2. Materials and methods ６６
4.2.1. Experimental design ６６
4.2.2. Preparation and cryopreservation of graft ６８
4.2.3. CPA treatment of grafts ６８
4.2.4. Cryopreservation of grafts ６８
4.2.5. The process of graft thawing ６８
4.2.6. Quality assessment and analysis of grafts ６９
4.2.7. Radiographic Analysis of Grafts ６９
4.2.8. Cellular characterization of OCA ６９
4.2.9. Biochemical analysis of grafts ６９
4.2.10. Histologic analysis of grafts ７０
4.2.11. Immunohistochemical analysis of grafts ７０
4.2.12. Statistical analysis ７１
4.3. Results ７２
4.3.1. Evaluation of CPA distribution by μCT analysis ７２
4.3.2. CRF cryopreservation protocol ７４
4.3.3. Evaluation of chondrocyte survival after cryopreservation ７６
4.3.4. Evaluation of ECM content in cartilage after cryopreservation ７９
4.3.5. Evaluation of ECM production by isolated chondrocytes from cryopreserved grafts ８２
4.4. Discussion ８５
CONCLUSIONS ８８
REFERENCES ９０

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