The In Vivo And In Vitro Study Of Premoting The Healing Of Anterior Cruciate Ligament Autogenous Graft With GAM Materials

A variety of autologous tendon grafts for reconstruction of anterior cruciate ligament (ACL) can not attain complete healing even after relatively a long period of time, there still exist serious complications like relaxation and breakage of the autologous tendon graft.Either autologous or allogeneic grafts had similar healing and remodeling process after reconstruction and experienced four phases including graft necrosis, cellular re-migration, vascular reconstruction and remodeling. semitendinosus tendon was observed after removal of semitendinosus tendon. Histological and immunohistochemical results confirmed that the regenerated tissues were new tendons because the tissues had normal morphology and organizational structure of the tendon and were mainly composed by typeⅠcollagen. Nevertheless, the new tendons contained significantly lower levels of glycosaminoglycans and collagen than normal tendons, with disarranged cells at muscle-tendon junction and smaller diameter than contralateral tendon. In the meantime, the new tendons were attached to different sites of the tibia, with certain bias in comparison with the normal attachment sites. The regenerated tendon could transmit the external force through the muscle-tendon junction, while the transmission rate was significantly lower than normal tendons, with maximum anti-traction force for only 32% of the normal, which resulted in significantly weaker biological function than normal tendon.Some research showed using extraneous growth factor locally could increase ligament stiffness and anti-breake energy. But growth factor is macromolecule protein and its metabolism is faster, so its biological availability is lower which make it not feasible to use growth factor locally in tissues internal environment. Gene plasmid which code growth factor is stable in vivo for it is not easily hydrolyzed by a variety of proteolytic enzyme, so its curative effect is more lasting as to express strongly for several weeks。Traditional gene transfection is mainly to culture host seed cells in vivo for a time, transfect cultured cells with target gene and then transplant these cells back into body。The method has a high economic costs, complicated process and manipulate period, so is not fit for acute ligament trauma reparation.Recently, some scholar put forward the concept of "in situ tissue engeneering" for feasible solution and the new technique had been an important research aspect in gene medicine and tissue engineering. The basical concept of in situ tissue engeneering is make a complex of biological materials and DNA plasmid which is so called "gene activated matrix, GAM".when the biological materials is placed in tissue, the cells which capture those gene plasmid would be transflected and express the target growth factor to premote tissue repairing. On another way, the in vivo local reactor can be named local gene delivery carrier.Author think gene activated matrix technique could be a hopeful solution for ligament trauma reparing. Comparative evaluation of ACL autologous tendon graft from morphological and bio-mechanical levels as well as through long-term animal experiments studies will help comprehensively ascertain in vivo outcome of autologous grafts, provide information to search new method for optimizing tendon autograft to ligaments, thereby reduce incidence of relaxation and breakage of autografts after ACL reconstruction.Objective1. To explore the ability of transfection of cultured rabbit Gingival Fibroblasts with rabbit Transforming Growth Factor-β1 Gene and stable expression in Vitro.2. To built up and test the rabbit ACL reconstruction model, find out the actual performance of tendon graft which was dealed with GAM material during deplore the difference of the tissue and biomechanical strength during healing process, and gain biomechanical information for clinical use of ACL reconstruction surgery.3. To learn the histological changes of rabbit anterior cruciate ligament reconstruction model, determine the difference between two groups on collagen express and miscrostrcture observation.Methods1. A eukatyotic expression vector for EGFP-N1-hTGF-β1 was contructed by use of recombitnant DNA technique.Rabbit gingival fibroblasts were cultured in vitro and the cultured gingival fibroblasts were transfected with the complexes of Lipofectamine reagent and EGFP-N1-TGF-β1 in the ratio of 3:1 (μL:μg) in vitro. Positive clones were selected by G418. The expression was verified by using RT-PCR and fluorescence microscope. Mink lung cell bioassay was carried out to examine the biological activity of hTGF-β1.2.48 New Zealand white rabbits were divided into two groups:the experiment group and control group. Every groups conclude 24 animals. The rabbit's ACL were resected and autogenous semitendinous was taken for transplantment. Prepare bone tunnel, import the tendon graft, fix the graft and finally complete ACL reconstruction model building. The control group weren't implemented any intervention, while the experiment group were dealed with GAM material being injected in bone tunnel. specimens were gained at four observation stage:1 month,3 month and 6 month. Observe the tendon graft in general configuration, test their biomechanical index which conclude the maximum broken load (peak load), the displacement of maximum load, and calculated stress, strain and stiffness. Acording to the data, changes relationship chart was made over time.3. Establish the rabbit rabbit anterior cruciate ligament reconstruction model according to the procedure it mentioned previously. In experimental group, TGF-β1 was locally injected in the bone tunnel but in the control group the wound was only sutured. The samples were taken and studied by light miscroscopy and electron miscroscopy at 1,3,6 months after opreration, through that we can observe the changes of collagen express and miscrostrcture.Results1. The eukatyotic vector which contain EGFP-N1-TGF-β1 was constructed successfully. The hTGF-β1 gene transfected Gingival Fibroblasts cells showed prominently elevated mRNA expression of hTGF-β1 by RT-PCR. fluorescence microscope detect target gene were transfected into Gingival Fibroblasts cells. Transfected GF showed strong posotive activity and lasted for more than 4 weeks. The culture solution of transfeced GF could inhibit the proliferation of Mink lung cells.2. The specimens of survived animals were observed in. configuration. Just one tendon graft was broken in control group and all other was fine actually, so the general success rate of graft transplant was 97.9%. at the interval of one month, there was no difference between two group on anybiomechanical index. at the interval of 3 month and 6 month, the maximum broken load and stiffness of tendon graft in experiment group was significantly higer than that of control group(p<0.05)while the other biomechanical index between two group shows no difference.3. By light miscroscopy we see the number of fibroblasts in group B was less than that in group A, and the fibers were arranged irregularly at every observation internal. in group A, the number and appearance of fibroblasts were similar to normal strcture.there were more fibroblasts and collagen which became larger and arranged regularly. By electron miscroscopy we found the karyokinesis of cell miscrostructure was more active and some fibroblasts had an vibrant metabolism in group A. there were plenty of endoplasm and mitochondria in cytoplast and more extracellularmatrix were filled in fibers. Conclusions:1. The recombinant vector was constructed correctly, and the mRNA of rabbit TGF-β1 could be prominently expressed in Gingival Fibroblasts cells. Transfected Gingival Fibroblasts could effectively express the active TGF-β1 and produce biological efficiency.2. Because our procedure of rabbit ACL reconstruction model-made was similar to clinical surgery, the tendon graft has a good result postoperatively. Some biomechanical characteristic of experiment group was better than that of control group, which demonstrate GAM material can increase the tendon graft healing quality when it was dealed with target gene.3. In TGF-β1-treated animals, the tendon graft healing process was more quickly and its tissue strcture was better, so TGF-β1 can promote the healing of reconstrctured ligament.