Construction Of Tissue Engineering Tendon By Biocomplex Synovialization Combined With Decellularized Tendon Scaffold Regeneration

Background and Objective:Hand tendon injury and hand trauma are especially common in daily life,which not only affects the patient's physical and mental health,but also greatly affects the patient's ability to work,resulting in huge economic losses for individuals and society.The clinical standard procedure after flexor tendon injury is immediate repair surgery and early recovery of finger activity to prevent postoperative adhesions.However,due to frequent multiple injuries,it is impossible to carry out early rehabilitation training,resulting in serious adhesion to surrounding tissues,which seriously affects hand function;or tendon injury is serious,the defect is large,and one-stage repair surgery cannot be performed.All above are the indications for tendon graft reconstruction.However,autografts have limited resources and can cause defects in the graft donor site,causing pain and other complications after surgery,so clinical application is greatly limited.Xenografts have outstanding ethical problems,and patients are less accepted,making allografts possible as an alternative strategy.However,the immune rejection that may be caused by postoperative surgery cannot be ignored.Therefore,it is urgent to construct a graft suitable for the flexor tendon injury.With the continuous development of tissue engineering,although many artificial tendon materials are gradually derived,such as polylactic acid,silk and collagen,due to the complex composition of extracellular matrix(ECM)and its special biomechanical properties,there is still no developed synthetic materials that can replace natural tendons.Flexor digitorum profundus tendon belongs to the intrasynovial tendon.Compared with the extrasynovial tendon,its surface is smooth and covered with a thin mucopolysaccharide.The main components are hyaluronic acid(HA)and lubricin,which can be effectively lubricated.Therefore,how to simulate the physiological micro-environment of flexor digitorum profundus tendon and construct a tissue engineering scaffold that adapts to long-term repair after reconstruction and has better structural function,further reducing wear,improving repair function and reducing rejection,has yet to be resolved.In addition,as a major component of flexor digitorum profundus tendon,tenocytes have an irreplaceable role in the function and structural integrity,but it is also the key to solve the problem of graft rejection.Our previous studies have confirmed that although the incidence of rejection after grafting is significantly reduced,the internal structure and post-repair function are affected,especially the biomechanic characteristics change.Therefore,how to re-cellular the decellularized tissue engineering scaffold,how to choose the appropriate seed cells to replace the function of tenocytes,how to further achieve its structural remodeling is an important difficulty that still needs to be overcome.The purpose of this project is to construct a tissue-engineering scaffold from intrasynovial allograft suitable for the repair of flexor digitorum profundus tendon injury in the body,using decellularization technology to reduce its rejection,and revitalize through seed cell selection,planting,induction and so on.Activation was performed to verify the effect of the microenvironment of the tendon scaffold in the allogeneic decellularized intrasynovial scaffold on seed cells.At the same time,a synovial surface modifying substance derived from autologous joint fluid was constructed to observe the effect of improving the surface sliding resistance of the tendon scaffold in the decellularized synovial scaffold.And by mimicing the in vivo mechanical environment,the role and mechanism of post-transplant scaffold remodeling were further explored.Method:1.Construction of tissue engineering scaffold from decellularized allograft intrasynovial tendon(1)Animal study:The 12-month-old experimental dogs were subjected to anesthesia,disinfection,and surgery to obtain the bilateral flexor digitorum profundus tendon of the fore paw,and the sliding friction force was directly measured by the sliding resistance measuring instrument at the interface between the Zone ? region of the flexor digitorum profundus tendon and the A2 pulley ligament.The tissue structure was observed by HE staining,and the structural function of the allograft intrasynovial tendon was evaluated as a tissue engineering scaffold.The decellularized intrasynovial tendon scaffold was further constructed by physical deep cryolysis.(2)Seed cell acquisition:Autologous bone marrow mesenchymal stem cells were isolated by density gradient centrifugation,surface markers were identified,and their multi-directional differentiation ability was identified under induction conditions.The extracellular matrix microenvironment of decellularized tendon scaffolds was obtained by urea extraction method.The effect on the proliferation and differentiation of seed cells were examined.2.Study on the role and mechanism of structural remodeling and function optimization of decellularized allogeneic intrasynovial tendon scaffold(1)Surface modification of the decellularized allogeneic intrasynovial tendon scaffold:the joint fluid was extracted from the bilateral knee joints of the experimental dog,and the cross-linking reaction of the carbodiimide derivative was carried out to prepare the autologous joint fluid containing the biological surface modification activity for decellularization.The allogeneic intrasynovial tendon scaffold was surface modified.(2)The role and mechanism of scaffold structure remodeling:seed cells were planted by mechanical perforation technique,and three-dimensional culture was carried out by mechanical recirculation of bioreactor,and structural remodeling of recellularized scaffolds was observed by HE staining;The gap junction GJA1,tendon component collagen COLI and collagen remodeling related gene MMP-9 expression changes were examined by PCR;and GJA1 specific blocker was given to further verify its mechanism of scaffold structure remodeling.3.Application of tissue engineering intrasynovial tendon scaffold in the reconstruction of flexor digitorum profundus tendon(1)The establishment of the rerupture model of flexor digitorum profundus tendon:the distal flexor tendon of the second finger distal interphalangeal joint of the experimental dog was cut,and the 3 mm tendon was removed at the distal end to increase the tension of the tendon repair,and the modified Kessler technique was performed.The tendon suture is performed and the distance between the repair ends is regularly monitored.(2)The effect of allogeneic intrasynovial tendon scaffold on the repair of tendon injury:the autologous extrasynovial tendon and allogeneic scaffolds were transplanted respectively,and the effects of autologous extrasynovial tendon on the repair of flexor digitorum profundus tendon injury were compared with those of allogeneic scaffold after recellularization and surface modification.the autologous synovial tendon.The mechanical repair test was used to judge the effect of transplant repair,and the biocompatibility was compared by tissue section staining.Results:1.Construction of tissue engineering scaffold from decellularized allograft intrasynovial tendon(1)The flexor digitorum profundus tendon was selected as the main structural source of the intrasynovial tendon scaffold.The histological characteristics,surface gliding resistance characteristics and structural components of the obtained tendon samples were consistent with the requirements of the intrasynovial tendon scaffold.(2)The decellularization effect of the obtained intrasynovial tendon scaffold was good,but the surface gliding performance was impaired.The obtained bone marrow mesenchymal stem cells can stably proliferate and have good diferentiation pluripotency.The matrix microenvironment of the decellularized tendon scaffold facilitates the proliferation and tenogenesis of seed cells.2.Study on the role and mechanism of structural remodeling and function optimization of decellularized allogeneic intrasynovial tendon scaffold(1)After the autologous joint fluid in the cross-linking reaction with the carbodiimide derivative,the surface resistance of the decellularized allogeneic intrasynovial tendon scaffoldwas significantly reduced.(2)Through the bioreactor biomimetic three-dimensional cultured tendon scaffold complex,under the stimulation of cyclic axial mechanical stress,the inner fiber structure of the scaffold is more compact,and the alignment direction is consistent with the direction of the tensile stress.At the same time,seed cells can migrate better inside the scaffold,and the genes related to tenogenesis are more efficiently expressed,and it is confirmed that the mechanical stress stimulation stimulates the gap-associated gene GJA1 between the seed cells in the scaffold,and further regulates the genes involved in collagen remodeling.The expression of MMP-9 promotes the synthesis and degradation of collagen components in the extracellular matrix and promotes the remodeling of the scaffold tissue structure.3.Application of tissue engineering intrasynovial tendon scaffold in the reconstruction of flexor digitorum profundus tendon:The decellularized intrasynovial tendon scaffold with biofilm complex synovial modification and seed cell replantation is applied in flexor digitorum profundus tendon reconstruction to compare with autologous extrasynovial tendon graft.It has less adhesion to surrounding tissues,lower surface sliding resistance,better joint activity after reconstruction,and good biocompatibility with the host's natural tendon,but it still needs to be improved in connection with the bone end of the host.In this study,the allogeneic intrasynovial tendon scaffold was successfully constructed by allogeneic flexor digitorum profundus tendon.Decellularization treatment was carried out by physical deep cryogenic technique,seed cells were obtained by autologous bone marrow mesenchymal stem cells,recellularized by mechanical punching,and structural remodeling with functional optimization was successfully completed by surface modification and cyclic mechanical stress.Through the three-dimensional culture in bioreactor and GJA1 specific blocker,the feedback regulation mechanism of gap junction between seed cells on MMP-9 was elucidated.Furthermore,in vivo,the effect of decellularized intrasynovial tendon scaffold with biofilm complex synovial modification and seed cell replantation was applied to tendon reconstruction.It provides a new treatment strategy for clinical tendon injury.