The Effects Of Autologous Mesenchymal Stem Cells Transfected By BMP-2or HGF Gene On Tendon-bone Healing

Purpose: To evaluate the effects of local application of HGF or BMP-2genetransduced autologous mesenchymal stem cells (MSCs) on tendon-bone healing afteranterior cruciate ligament reconstruction(ACLR) in rabbits.Methods: The MSCs of rabbit were harvested by bone marrow aspiration, thenisolated by density centrifuge, identified and serial subcultivated. MSCs weretransfected with an adenoviral vector encoding BMP-2or HGF. The efficiency ofinfection were detected by flow cytometry (FCM), and expression of HGF or BMP-2were detected by enzyme-linked immunoadsorption assay (ELISA). The surfacemarkers, differentiation potential and proliferative capacity were tested to evaluate theeffects of gene modification on rabbit bone marrow MSCs. Eighty New Zealand whiterabbits were randomly divided into four groups-Control group, MSCs group,MSCs-HGF group and MSC-BMP2group. Normal ACL were transected andreconstructed using autologous digital longextensor tendon in bilateral knee of eachrabbit. We injected0.1ml fibrin glue (FG) into the tendon-bone interface of tibial sideon Control group,5×106MSCs+100ul fibrin glue on MSCs group,5×106MSCs-HGF+100ul fibrin glue on MSCs-HGF group and5×106MSCs-BMP2+100ulfibrin glue on MSCs-BMP2group. Histological assessment was done at4,8, and12weeks using HE and toluidine bluestains. Biomechanical testing of force and stiffnessduring loading to ultimate failure was performed at12weeks.Results:①Isolated rabbit bone marrow MSCs were homogeneous withfibroblast-like morphology and strong proliferative capacity. MSCs were positive forCD29, CD44, CD73, and were negative for CD31, CD45. Induced differentiation test showed that rabbit bone marrow MSCs have the ability to differentiat into osteoblastand adipocyte.②Adenovirus vector can successfully transfect rabbit bone marrowMSCs with high efficiency. Almost all of the cells expressed GFP when transfected atthe MOI of150. Target proteins were expressed persistently and the highest expressionlevel was at48hours. Target gene transfection does not affect cell proliferation anddifferentiation ability, does not change the surface markers of MSCs.③In theMSCs-BMP2group, there were large areas of cartilage cells at the tendon-bone junctionat4week and8weeks. At12weeks, junctional tissue, characterized by a continuous4-layer structure of tendon, fibrocartilage, calcified cartilage, and bone, was regeneratedby a direct insertion. In the MSCs-HGF group, there were some new-born vessels at4weeks. The vessels gradually decreased at8week. At12weeks, the tendon-to-boneconnected tightly. A large number of orderly arranged collagen fibers were parallel tothe load axis. At12weeks, the MSCs-BMP2group had significantly higher failure loadand stiffness than other groups(P<0.05). The MSCs-HGF group and MSCs group hadsignificantly higher failure load than control group(P<0.05). There was no statisticaldifference of stiffness between MSCs-HGF group, MSCs group and control group(P>0.05).Conclusions: Autologous transplantation of MSCs modified by BMP-2gene canaccelerate tendon bone healing process and resulted in healing by an intervening zone ofa direct insertion rather than collagen fibers and scar tissue. MSCs modified by HGFgene can promote new-born vessels formation and the remodeling of the collagen fibersin the tendon bone interface.