Preservation Effect On Rabbit Osteochondral Allografts Cultured In Different Tissue Solutions

ObjectiveRabbit osteochondral allografts(OCA)were cultured to 35 d by two different tissue solutions(Tsmu-A and Tsmu-B)in vitro,and to compare of preservation effects according to chondrocyte viability,histological outcomes and other indicators.Then OCA cultured by solution were transplanted into defect of rabbit knee,the results of transplantion evaluated according to results of the general condition,range of motion,imaging,chondrocyte viability,histology,biomechanics of OCA and immunology in vitro during six months,and the better tissue solution was selected at the end.Methods1.Preservation effect of rabbit osteochondral tissue stored in three solutions in vitro.120 osteochondral allografts which were 5mm x 6mm in weight-bearing area of femoral condyles from New Zealand rabbit were harvested by ARTHREX under sterile condition,then divided randomly into four groups.Fresh: fresh OCA untreated;Tsmu-A: cultured for 35 d at 4°C by Tsmu-A solution and changed every 2 days;Tsmu-B: cultured for 35 d at 4°C by Tsmu-B solution and changed every 2 days;Basic: cultured for 35 d at 4°C by basic solution and changed every 2 days.Chondrocyte viability of cartilage graft was examined via FDA-EB fluorescence staining;gross assessment and histological score was performed to evaluate the morphologic change of allografts;IHC of type II collagen were performed to check contents in ECM.Moisture content was tested.At last,to compare of preservation effects of both solutions according to these indicators.2.Outcome of rabbit osteochondral allografts transplantation in vivo.24 osteochondral allografts were divided randomly into three groups and each group contains 8 allografts.Tsmu-A: cultured for 35 d at 4°C by Tsmu-A solution and changed every 2 days;Tsmu-B: cultured for 35 d at 4°C by Tsmu-B solution and changed every 2 days;Fresh: fresh and untreated;and the allografts was implanted by mosaic technique to rabbit knee joint to repair defect which was created about 5mm x 5mm in left knee.Negative group and Sham group was added.Five groups of 40 knees was set in the part.Preoperative range of motion(ROM)was detected;radiographs and MRI were observed after two weeks;at 6th months postoperative,the testing index of rabbit model was evaluated by gross evaluation,ROM,radiographs and MRI evaluation,chondrocyte viability,histology change,biomechanical of OCA and immunology testing;and to ultimately select a better tissue solution according to results.Result1.In vitro:(1)Chondrocyte viability: cell viability of each preservation group were lower than fresh group and there were significant differences between the groups in Day 35(P <0.05);Tsmu-B was more than 70% but Tsmu-A was not as good as 60%.(2)Histological: Makin scores of each preservation group were higher fresh group and there were significant differences between the groups in Day 35(P <0.05);proteoglycan,acid mucopolysaccharide and collagen II content and of preservation groups decreased in Day 35,but Tsmu-B slightly reduced.Acid mucopolysaccharide and collagen II content between Tsmu-A and Tsmu-B were statistically significant(P <0.05),and Tsmu-A was more than Tsmu-B.(3)Moisture content: moisture content of each preservation group were higher than fresh group and there were significant differences between the groups in Day 35(P <0.05);Tsmu-B increase slightly.Therefore,preservation effect of Tsmu-B was best in vitro.2.In vivo:(1)General condition and ROM: morphology and function of joint all animals transplanted were changed to some degree at 6th months postoperative;ROM of Tsmu-A and Tsmu-B were less than fresh and sham group,and Tsmu-A,between Tsmu-A and Tsmu-B were statistically differences(P <0.05).(2)Imaging: osteochondral allograft was located in the defect at 2 weeks,good alignment,indicating that the transplantation was no problem;there are healing tendencies between host bone and allografts bone and different degrees of fracture and signal between the host cartilage and allografts cartilage at 6th months postoperative.Tsmu-B was better.(3)General observation: compared with fresh control group,the score of Tsmu-A and Tsmu-B were low and there were significant differences between the groups at 6th months postoperative;the score was high in Tsmu-B indicating its observation was the best.(4)Chondrocyte viability: cell viability of each preservation group were lower than before and fresh,there were significant differences among the groups at 6th months postoperative(P <0.05);Tsmu-A was 17.22 ± 2.56% and Tsmu-B was 49.34 ± 3.29%;both groups were lower than 70%.(5)Histological: there were some fracture between allograft groups at 6th months postoperative;Tsmu-B and fresh was not clear,but Tsmu-A was apparent.Histological scores were significant differences between two groups(P <0.05).The content of proteoglycans,acid mucopolysaccharide,collagen II was lower than before and fresh,there were significant differences between the groups(P <0.05)and Tsmu-B was higher.(6)Biomechanical: Young's modulus and moisture content of Tsmu-A and Tsmu-B were less than fresh at 6th months postoperative,and there were significant differences between two groups(P <0.05).The values of Tsmu-B were closer to normal.(7)Immunology: IL-6 level of Tsmu-A and Tsmu-B were less than fresh,but Tsmu-A and Tsmu-B had no significant(P >0.05).Conclusions1.A better preservation of osteochondral allografts and transplantion outcomes were Tsmu-B solution.2.Tissue culture method for preserving osteochondral allografts may reduce immunology rejection.RelevenceThe research explored preservation effects on rabbit osteochondral allografts by using different tissue solutions in vivo and vitro,ultimately to find an optimal solution prolonging preservation time according to a variety of scientific evaluation.It will help to make the foundation for the establishment and development of osteochondral tissue bank.