| Objective:The injury of anterior cruciate ligament(ACL)is a commo n kind of knee joint disease,which offen leads to the instability of knee,osteoarthritis and meniscus injury.Due to the limited capacity of regeneration,the graft is the main means of reconstruction.Autograft,allograft and artificial materials are offen clinically used for transplantation and revision,associated with the problems of high demands and limited sources.While the artificial ligament reconstruction is not biological,it shows fatigue,creep and mechanical failure in the long term.The native ligament-bone junction is typical four-layered structure(collagen fiber,fiber cartilage,calcified fibrous cartilage and bone).The traditional single-phase-structure scaffold shows direct connection in the bone tunnel after implantation.There is no four-layer structure formed,which may be the main reason for the biological fixation failure.In this study,we intend to develop a kind of Tri-phase(ligament,cartilage and bone area)silk scaffold.It is manufactured with silk fibrion,transforming growth factor-β3(TGF-β3)gene,and bone morphogenetic protein-2(BMP-2)gene,which can induce the differenation of bone marrow mesenchymal stem cells(BMSCs)into fibroblast,chondrocyte and osteoblast.This might result in the formation of tissue-engineered ligament-bone junction and stable bio-fixation.Methods:1.The silk mesh was woven,and silk fibroin was extracted.The three-phase scaffold was modified with silk fibroin,chondroitin sulfate,hyaluronic acid sodium,hydroxyapatite(HA)and phosphatidylserine(PS).The parameters including pore size,porosity,water content,surface element,surface roughness and biomechanical property were determined.2.Lentivirus which loading TGF-β3 and BMP-2 gene was immorbilized in cartilage and bone area of the scaffold as experimental group.After BMSCs seeding,the cell’s proliferation,adhesion and expression of related genes were observed by fluorescence microscope,scanning electron microscope(SEM)and real-time PCR(RT-PCR).3.After ACL excision in rabbit,the scaffolds were implanted.The samples were harvested at 4 weeks,8 weeks and 12 weeks after operation.The regereration of ligament-bone junction were evaluated by radiological,histological and biomechanical examination.Results:1.After modification,the porosity of the scffold was 78.44 + 1.75% and the water content was 79.72 + 1.36%.The surface element analysis showed the Ca element in area C,S and Na elements in area B were all higher than those in area A.The P elements increased significantly after PS modification.Analysis of surface roughness showed the profile of the arithmetic average deviation has been increased significantly after HA modification.The tensile and fatigue tests showed that the scaffold could meet the basic mechanical requirements of ACL in rabit.2.BMSCs had good adhesion and proliferation on scaffold by SEM and laser scanning confocal microscope examination.Chondrocyte specific gene expression was found to be increased in area B,and osteoblast specific gene expression increased in area C over time.3.Micro-CT detected that the amount of bone ingrowth in the experimental group was higher than that of the control group,expecially in areas C.Histological observation showed that the fibrous connective tissue has filled in between the bone and scaffold in the control group.On the contrary,bone like tissue and cartilage like tissue ingrowth w ere found in the experimental group,appearing a gradient distribution and good healing.Conclusions:Three phease gene modified silk scaffold has good biological compatibility.Compared with none modifided scaffold,the gene modifeded scaffold has the better regeneration of the ligement-bone junction. |
PDF Full Text Request