Biological Properties Of EDC Cross-linked Acellular Tendon Of Banna Mini-pig Inbred-lines Abstract

Object To observe the histological and structural changes of EDC cross-linked tendon of Banna mini-pig inbred-lines and detect its degradation, cytotoxicity, immunogenicity and biomechanics in vitro, then evaluate the feasibility and validity of porcine-derived xenogeneic tendon graft.Method (1) Extensor digitorum tendons harvested from Banna mini-pig inbred-lines were divided into fresh tendon group,deep-freezed tendon group,simply decellularized tenodon group and EDC cross-linked acellular tendon group.The histological and structural changes treated by different methods were observed via HE staining and scaning electron microscopy and compared between groups to evaluate the histological and structural influence of EDC cross-linking on tendons; (2) Samples from different treated groups were immerged into PBS containing collagenase typeⅠ(60ug/ml)and the degradation rate in 1,2,3,4,5,6,7d were computed.The degradation rates were compared between groups to evaluate the influence of EDC cross-linking on tendon degradation; (3) Fibroblasts were co-cultured with infiltrated fluid from different treated tendons and cell corresponding proliferation rates were detected by MTT assay in 1,2,3,4,5,6,7d to evaluate the cytotoxicity of each group; (4) Infiltrated fluid of different group tendons was co-cultrued with lymphocytes as stimulating antigen source and lymphocyte proliferation test was used to evaluate the immunogenicity of each group tendon.Samples of different treated tendons were embedded subcutaneously in the backs of SD rats and inflammations were observed and compared between groups through HE staining in 1,2,3,4w after operation; (5) Ultimate tensile strength, breaking elongation and Young's modulus of each group was detected respectively and properties of biomechanics were compared between groups.Results (1) Fresh tendon and deep-freezed tendon were similar in histology and structure, the fiber arrangement was in order and fibers connected tightly with adjacent ones,tendoncytes were detected in intervals of fibers; fiber bundles of simply decellularized tenodon were inhomogeneous in diameters and arranged in disorder,the inter-connection between bundles were loosed and irregular pores in various shapes and sizes were detected, no cell components were observed. Bundles of EDC cross-linked acellular tendon became smooth and tightly inter-connected with each other, and the pores were seen in similar shape and size; (2) In 7d the degradation rates of fresh tendon, deep-freezed tendon, simply decellularized tenodon and EDC cross-linked tendon were respectively 25.560±0.948%, 25.950±0.890%, 72.154±0.926% and 23.550±0.890%. As far as the degradation rate was concerned, there were no significant differences between fresh tendon, deep-freezed tendon and EDC cross-linked tendon (P>0.05),while each of them was different significantly to simply decellularized tenodon (P<0.05); (3) ANOVA showed no significant difference between deep-freezed tendon group, simply decellularized tenodon and EDC cross-linked acellular tendon group; (4) The immunogenicity between deep-freezed tendon and simply decellularized tenodon was no significant difference (P>0.05).After cross-linked by EDC, immunogenicity of simply decellularized tenodon was decreased and lower significantly than deep-freezed tendon.There was no significant difference between EDC cross-linked acellular tendon group and the control group; (5) Biomechanics test illuminate that ultimate tensile strength and Young's modulus of simply decellularized tendon were significantly lower than deep-freezed tendon while the breaking elongation were higher dramatically (P<0.05). After cross-linking by EDC, all the above mechanical parameters of acellular tendon were not different significantly to those of deep-freezed tendon (P>0.05).Conclusions (1) EDC cross-linking modification made the connection of acellular tendon bundles more close and tight, the arrangement more orderly and porous structure more regular in shape and size; (2) EDC cross-linking modification enhanced the resistance of acellular tendon to degradation; (3) The tendon scaffold cross-linked using EDC showed no apparent cytotoxicity; (4) EDC cross-linking modification decreased the immunogenicity of acellular tendon; (5) EDC cross-linking modification enhanced the ultimate tensile strength and Young's modulus of acellular tendon while decreased the breaking elongation.