The AMECM/DCB Scaffold Prompt Successful Whole Meniscus Regeneration In A Rabbit Total Meniscectomy Model

Objective:The meniscus lacks self-repair ability because of its unique structural features, so the treatment of meniscus lesion is a big challenge all the time. The aim of this study is to:a) construct a composite meniscus scaffold with accellular meniscus extracellular matrix (AMECM) and decalcified cancellous bone (DCB) (AMECM/ DCB scaffold), with AMECM scaffold and DCB scaffold as control, and then test their physicochemical characteristics in vitro and immunological properties in vivo; b) utilize the meniscus fibrochondrocytes to evaluate the cytotoxicity, cytocompatibility, and the cell-scaffold interaction of the three different scaffolds; c) we respectively implant the three different scaffolds in the New Zealand rabbits that underwent total meniscectomy, and evaluate the meniscus regeneration and articular cartilage protective effect of the three different groups.Method:a. We prepared the acellular meniscus extracellular matrix through physic-chemical method, and then treated the decalcified cancellous bone through the improved Urist method, and finally, we constructed the three different meniscal scaffolds by infusing and lyophilization, respectively; b. we detected the physicochemical characteristics of the three different scaffolds, including micro-structure analysis through SEM(scanning electron microscope), the scaffold composition detection through histological and biochemistry analysis, and mechanical property testing with BOSE mechanical testing machine; c. we implanted the three different scaffolds subcutaneously in the rats to assess the immunological rejection of the scaffolds; d. we seeded the meniscus fibrochondrocytes into the three scaffolds and then observed the microstructure with SEM to, tested the cytotoxicity of scaffolds through live/dead cell staining, detected the glycosaminoglycan(GAG) and collagen content secreted by the fibrochondrocytes in the three scaffolds after 3,7,14 days; e. we respectively implanted the three different scaffolds into the New Zealand rabbits which underwent total meniscectomy, and then evaluated the meniscus regeneration and cartilage protective effect of the three different groups through macroscopic observation, histological analysis, X-RAY, MRI, biomechanics test and RT-PCR at 3 and 6 months time points.Results:a. The SEM result showed that all the three different scaffolds possessed three-dimensional porous structure and good porosity. The GAG content of AMECM/DCB scaffold and AMECM scaffold was higher than that of the DCB scaffold (P<0.05). The biomechanics property of AMECM/DCB scaffold was superior to those of the other two scaffolds (compressive modulus, tensile modulus) (P<0.05). And no significant immunoreaction was observed in the rats for all the three different scaffolds. b. The rabbit meniscus fibrochondrocytes can grow well in all the three scaffolds, and the live/dead cell staining result showed that there were no significant difference in the three scaffolds, however, the fibrochondrocytes seeded in AMECM/DCB scaffold and AMECM scaffold could secrete more GAG and collagen than DCB scaffold. c. The in vivo repairexperiment showed that, there was no significant meniscus regeneration in control group and AMECM group, and just a little synovium growth-in, while the neomeniscus appeared in AMECM/DCB group and DCB group, and the semiquantitative histologic score, Ishida score, results showed that the neomeniscus in AMECM/DCB group were better than those of DCB group(P<0.05), and the Mankin score of relevant femur condyles cartilage and tibial plateau cartialge in AMECM/DCB group was more higher than those in other two groups(P<0.05). Both the X-ray result (K-L grade score) and MRI result (WORMS score) showed that the repair effect of AMECM/DCB group was better than those of the other two groups (P<0.05). The tensile modulus of neomeniscus at 3 month was higher than that of 6 month groups, while at the same time point, the tensile modulus of neomeniscus in AMECM/DCB group was higher than that of DCB group(P<0.05). The RT-PCR result showed that aggrecan, Sox9, and collagen ? expression at 3 month is higher than those of 6 month groups, while at the same time point, aggrecan, Sox9, and collagen ? expression of neomeniscus of AMECM/DCB group was higher than that of DCB group(P<0.05).Conclusion:The AMECM/DCB scaffold has good three-dimensional porous structure. better biomechanical property compare to the AMECM scaffold and DCB scaffold, and can promote GAG and collagen secretion of fibrochondrocytes. The in vivo repair experiment results indicated that the AMECM/DCB scaffold could promote meniscus regeneration and restrain the osteoarthritis (OA) progress. The AMECM/DCB scaffold is a good choice for meniscus tissue engineering.