The Experimental Study Of Growth Factor Gene Active Material Improving The Healing Of ACL Autogenous Graft: In Vitro And In Vivo

part 1:Synthesis of gene active material of human platelet-derived growth factor-B and it's bioactivity testObjective:To construct the eukaryotic expression plasmid of human platelet-derived growth factor-B (hPDGF-B), and explore the ability of transfection of cultured rabbit Gingival Fibroblasts with platelet-derived growth factor-B in vitro.Methods:To construct and verify the eukaryotic expression vector for EGFP-N1-hPDGF-B by recombinant DNA technique and restriction enzyme.Rabbit gingival fibroblasts were cultured in vitro and the cultured gingival fibroblasts were transfected with the complexes of LipofectAMINE reagent and EGFP-N1-hPDGF-B in the ratio of 3:1 (μL:μg) in vitro. Positive clones were selected by G418 screening. The expression of hPDGF-B was detected by RT-PCR and fluorescence microscope. MTT test was done to examine the bioactivity of hPDGF-B and draw a growth curve of fibroblasts.Results:The eukaryotic vector containing EGFP-N1-PDGF-B was constructed successfully. The expression of PDGF-B on the level of mRNA was tested by PCR. The process that the target cell was transfected by target gene can be observed under fluorescent microscope. Fibroblasts cells showed prominently elevated mRNA expression of hPDGF-B and lasted over 4 weeks.Conclusions:The recombinant vector was constructed correctly, and the mRNA of rabbit hPDGF-B could be efficiently and stably expressed in Gingival Fibroblasts cells. Transfected Gingival Fibroblasts could effectively express the active hPDGF-B, take biological effects and apparently improve growth. fibroblastPart 2:The histological evaluations of experiment of reconstructive anterior cruciate ligament with PDGF gene material in vivoobjective To determine the difference between two groups on collagen expression and microstructural observation according to histomorphological method, including light microscope and transmission electron microscope. To evaluate the function of PDGF-B with lipofectMINE gene transfected system to improve the healing of transplant in the rabbit anterior cruciate ligament reconstructive model.Methods Twenty-four New Zealand adult rabbits were randomly divided into 2 groups, the experimental group and the control group. The rabbit anterior cruciate ligament reconstructive model was established according to the procedure as mentioned previously. PDGF-B was locally injected into the reconstructive location in the experimental group while only normal saline (NS) was administered in the control group. The samples were taken and studied under light microscope and electron microscope 1,3,6 months after operation respectively. Through which we can observe the changes of collagen expression and the microstrcture of cells.Results 1.The amounts of fibroblasts and collagen, the diameter and arrangement of collagen fiber in the experimental group are all superior to those in the control group. And the histological morphology was approaching the normal structure 6 month after operation under light microscope.2. The karyokinesis of cell miscrostructure was more active in the experimental group than that in the control group, indicating a prosperous recovery metabolism. There were plenty of endoplasm and mitochondria in cytoplast and copious extracellular matrix between fibers Conclusion The reconstructive tendon transfected by PDGF was better than that in the control group, indicating that PDGF gene plasmids using lipofectMINE as vector have favorable transfected biological effects in the healing location of resconstructive anterior cruciate ligament and have a specific practical prospect.Part 3:Biomechanical test of reconstructive anterior cruciate ligament graft with PDGF gene material in vivoObjective:To observe the liposome-PDGF gene transfected system's function in improving the biomechanical strength of reconstructive ligament in animal models.Methods:Twenty-four New Zealand adult rabbits were randomly divided into two groups, the experimental group and the control group. Each group concludes 12 animals. The rabbit anterior cruciate ligament reconstructive model was established, PDGF-B was locally injected into the reconstructive location in the experimental group while only NS was administered in the control group. Specimens were obtained 1 month,3 months and 6 months after operation, to observe the tendon graft in general configuration, test their biomechanical indexes including the maximum broken load (peak load), the displacement of maximum load, and calculated stress, strain and stiffness, and form a time-dependent relationship chart according to data above.Results:1. The animals showed no obvious abnormality by observation. There is one case experiencing rupture of ligament in control group while no such case was found in the experimental group. The total successful rate of reconstructive surgery in animal models is 97.9%.2. There was no apparent difference between two groups on each biomechanical index one month after operation. The maximum broken load and stiffness of tendon graft in the experimental group was significantly higher than that in the control group, p≤0.05, while other biomechanical indexes between two groups show no difference 3 months and 6 months after operation respectively.Conclusion:Liposome-PDGF gene transfected system has a positive promoting effect in the wound healing of anterior cruciate ligament reconstruction in rabbits, and can enhance biomechanical efficiency in the process of reconstruction of ligament graft.