Preparation And Evaluation Of Anti-adhesion Membrane Derived From Decellularized Tendon

Objective: Tendon is the dense connective tissue connecting skeletal muscle and bone.Tendon injury is a common orthopedic disease caused by trauma,age-related degeneration or overuse.Although the incidence of tendon adhesion decreases with the improvement of surgical technique and postoperative activities,it is still the main complication after tendon repair,which often leads to pain,limited joint movement,and even the need for reoperation.The decellularized matrix derived from tendon has the similar composition to natural tendon,good biocompatibility and low immunogenicity.Therefore,this study attempts to use tendon decellularized matrix material to prepare decellularized tendon membrane(DTM)to prevent the tendon adhesion after injury.1.To evaluate the effect of the optimized tendon decellularization method,as well as the physical and chemical properties of fresh tendon,decellularized tendon and DTM.2.The biocompatibility of DTM in vitro was tested by cytotoxicity test,hemolysis test and cell affinity test.3.The membrane was implanted subcutaneously into the back of rats to detect the degradation of membrane and the induced immune response in vivo.4.The effect of DTM on tendon derived cells was detected at cell level.5.Animal experiments were conducted to analyze the effect of DTM in preventing adhesion and promoting repair.Methods: 1.After optimizing the decellularization method,the effect of tendon decellularization was verified by HE staining,DNA quantification and agarose electrophoresis.Masson staining and Sirius red staining were used to qualitatively analyze the tissue components in the transverse sections of the fresh and decellularized tendons.The collagen content and glycosaminoglycan content of the fresh and decellularized tendons under dry weight were detected.Above these were used to detect the effect of decellularization method on tendon tissue.DTM was prepared with decellularized matrix material of tendon,and the porosity of membrane was detected by scanning electron microscope.The protein components of fresh tendon,decellularized tendon and DTM were extracted and analyzed by proteomics.2.The biocompatibility of the membrane in vitro was tested by cytotoxicity test,hemolysis test and cell affinity test.The membrane was implanted subcutaneously into the back of rats,and then HE staining and immunofluorescence were used to detect the degradation and immune response of the membrane in vivo.The effect of membrane on tendon derived cells was detected by PCR and immunofluorescence.3.The membrane was used to repair Achilles tendon injury in rabbits,and the tendon was obtained after 3,6 and 12 weeks.The transverse sections of the tendons were stained with HE staining and Masson staining,and the adhesion score,ankle flexion and tendon mechanics were measured to evaluate the effect of DTM in preventing adhesion and promoting repair.Results: 1.He staining,DNA quantitative test and agarose electrophoresis showed that the method optimized by microsection technology was effective.Specifically,no visible nuclei were found in DECM tissue sections stained by HE staining;in the qualitative DNA experiment of agarose gel electrophoresis,there was no residual DNA fragment in DECM;in the double chain DNA(dsDNA)quantitative experiment,the dsDNA content in dry DECM was 40.46 + 4.58 ng/mg,less than 50 ng/mg.After decellularization,the whole structure of DECM is similar to that of natural tissue,but the structure of DECM is looser and has many holes.Masson trichrome staining showed that DECM and natural tissue were composed of collagen fibers.The contents of collagen in DECM and natural tissue were915.26 ± 42.75 μg/mg and 926.91 ± 19.28 μg/mg,respectively.There was no significant difference between the two groups(P > 0.05),indicating that there was no obvious damage of collagen during decellularization.Sirius red staining showed that there was only type 1collagen(COL1)but no type 3 collagen(COL3).The content of glycosaminoglycan in DECM and normal tissues was 0.96 ± 0.04 μg/mg and 0.97 ± 0.03 μg/mg,respectively,with no significant difference(P > 0.05).DTM is white and opaque in wet state,colorless and transparent in dry state.The SEM results show that the structure of DTM structure is uniform and dense,without visible pores.Proteomics results showed that COL1A1,COL2A1 and many other structural proteins,such as collagen,laminin,fibrinogen,fibrillary protein,fibronectin and so on,existed in fresh tendon,decellularized tendon and DTM.After decellularization treatment,the relative content of these structural proteins in the total protein in the decellularized tissue increased.This phenomenon can be attributed to the removal of cell-related proteins in the tissue after decellularization treatment,resulting in the further purification of structural proteins in the extracellular matrix.In contrast,after decellularization of the fresh tendon tissue,the relative content of growth factors such as pigment epithelium derived factor(PEDF),von Willebrand factor,and angiopoietin like growth factors in the total protein decreased,which indicates that the growth factors were lost in the process of decellularization.In the process of preparing DTM from decellularized matrix,the relative content of 41 proteins increased,while the relative content of 40 proteins decreased.Compared with the total number of 1381 proteins detected,these proteins accounted for a small proportion,which proved that the preparation conditions of DTM were relatively mild.2.In vitro biocompatibility tests included cytotoxicity test,hemolysis test and cell affinity test.The cytotoxicity test showed that the relative proliferation rate of L929 cells treated with DTM within 1 day was higher than100% of the control group.From the first day to the third day,the relative proliferation rate of L929 cells gradually decreased to nearly 100%,which indicated that DTM was not only non-toxic to L929 cells,but also promoted cell proliferation.In hemolysis test,normal saline was used as negative control and distilled water as positive control.The upper liquid of DTM extract group was transparent,similar to that of normal saline group,indicating that DTM did not cause hemolysis.Tendon derived cells can fully extend on DTM,and the cell morphology is good,which indicates that DTM has good cytocompatibility.After subcutaneous implantation in rats,the degradation of DTM in vivo was studied with HE staining,and the immune response of tissue was studied by HE staining and immunofluorescence.All rats survived to the scheduled time point without complications.HE staining showed the morphological changes and degradability of DTM.DTM showed the red band after HE staining.In general,there was no obvious change of membrane structure in the first three weeks.A large number of cells could be seen in the membrane from the sixth week,and the membrane disappeared completely at the twelfth week.HE staining and immunofluorescence method showed the changes of the type,number and distribution of inflammatory cells after membrane implantation.The results showed that no macrophages were found from the first week to the twelfth week after membrane implantation.At the first week after implantation,there were a few lymphocytes around the membrane.In the second week,some lymphocytes and a large number of plasma cells appeared around the membrane.In the third week,in addition to some plasma cells and lymphocytes,some eosinophils were also found around the membrane.In the fourth week,some neutrophils were found around the membrane,and the number of plasma cells and lymphocytes decreased significantly;a large number of plasma cells and lymphocytes were found in the outer layer of the membrane,but no cells were found in the middle layer of the membrane.At the sixth week,plasma cells and lymphocytes gradually spread into the membrane,and eosinophils were still seen around the membrane,but the number of eosinophils was much less than that at the third week.No residual DTM was found in the twelfth week,and all inflammatory cells disappeared.There was no significant difference between the DTM group and the control group,indicating that DTM is a safe biomaterial,and will not cause serious inflammatory reaction and immune rejection.3.The experimental results of rabbit Achilles tendon repair showed that the ankle flexion,adhesion score,mechanical strength and histology were better than those of the control group,indicating that DTM can not only effectively prevent the occurrence of adhesion after tendon repair,but also improve the quality of tendon repair.Conclusions: An anti-adhesion membrane,i.e.,decellularized tendon matrix membrane(DTM)was successfully prepared by an optimized tendon decellularization method from bovine extracellular matrix.The physical and chemical properties and biological functions of DTM were evaluated.The physical and chemical properties,biological evaluation and experimental results of rabbit Achilles tendon repair show that DTM can not only prevent tendon adhesion,but also improve the quality of tendon repair,hence demonstrating its great application potential.