FGF-2 Lentiviral Vector-mediated Human Amnion Mesenchymal Stem Cells Combined With Rabbit Autologous Platelet-rich Plasma Promote Rabbit Tendon-bone Healing

Objective:(1)To explore the effects of different concentrations of FGF-2 solutions on the ligament formation and osteogenesis of human amnion mesenchymal stem cells.(2)To explore the feasibility of FGF-2 lentivirus vector transfected hAMSCs combined with rabbit autologous platelet rich plasma(PRP)to promote tendon-to-bone healing.Methods:(1)The hAMSCs were extracted by enzymatic digestion(twice trypsin digestion and once type II collagenase digestion)and subcultured.The cell morphology of hAMSCs was observed under inverted microscope and hAMSCs were passed to third generation(P3 generation hAMSCs)for subsequent experiments.Identification of P3 generation hAMSCs by immunofluorescence and flow cytometry.(2)P3 generation hAMSCs were induced with different concentrations of FGF-2solutions(0,10,20,40 ng / ml)and CCK8 was used to detect the effects of different concentrations of FGF-2 solution on cell proliferation;After 14 days,qRT-PCR was used to detect the expression of ligament-related and osteogenic genes of hAMSCs induced by different concentrations of FGF-2 solutions;Western blot/immunofluorescence were used to detect the expression of ligament-related and osteogenic proteins of hAMSCs induced by different concentrations of FGF-2solutions;Picrosirius red staining was used to detect collagen expression of hAMSCs after induction with different concentrations of FGF-2 solutions.The P3 generation hAMSCs were transfected with FGF-2 lentivirus vector,the expression of green fluorescent protein was observed under a fluorescence microscope,and the transfection efficiency of the lentivirus vector was detected by quantitative real-timefluorescent PCR(qRT-PCR)and Western blot.(3)Rabbit autologous PRP was prepared by two centrifugation method,and PRP was activated with bovine thrombin and Cacl2.(4)Preparation of external joint tendon-bone healing model: New Zealand white rabbits were anesthetized with 3% sodium pentobarbital at a dose of 0.5 mL/kg.After anesthesia,skin preparation and disinfection were performed.An incision was made along the Achilles tendon of left hindlimb and an Achilles tendon with a length of about 2 cm was harvested as a graft.Cut the skin layer by layer on the inside of the upper side of the right tibia and fully expose the upper end of the tibia.A bone tunnel was made in the proximal tibia of the right hindlimb at the longitudinal axis of the tibia by a 2.5-mm-diameter drill.The removed Achilles tendon was transplanted into the bone tunnel,and the two ends of the Achilles tendon were left about 0.5 cm and fixed on the surrounding soft tissue for postoperative biomechanical detection.After Achilles tendon transplantation to bone tunnel and fixation,the following treatments were carried out: the first group: no special treatment(control group);the second group: injection of auto-activated PRP into the bone tunnel(PRP group);the third group: injection of auto-activated PRP mixed with hAMSCs into the bone tunnel(PRP+hAMSCs group);the fourth group: injection of auto-activated PRP mixed with FGF-2 lentivirus vector-transfected hAMSCs into bone tunnel(PRP+FGF-2-hAMSCs group).The incision was sutured layer by layer,and disinfected again,and 200,000 U penicillin was injected intramuscularly for 7 consecutive days.The animals were sacrificed 3 months after operation,and the tibia-graft complexes were taken for gross observation,histological examination(HE staining,safranin O/fast green staining,toluidine blue staining,Masson's trichrome staining,picrosirius red staining),biomechanical examination,radiographic examination to observe tendon-to-bone healing.Results:(1)The primary hAMSCs were circular or elliptical,and gradually grew into vortex and adherent growth after subculture.Immunofluorescence resultssuggested that P3 generation hAMSCs highly expressed vimentin and did not express Cytokeratin 19(CK19);Flow cytometry results showed that the P3 generation hAMSCs highly expressed the phenotypic molecules CD44,CD73,CD90,and CD105 of mesenchymal stem cells,and hardly expressed CD34,CD11 B,CD19,CD45,HLA-DR.(2)After hAMSCs were induced by different concentrations of FGF-2 solutions,the CCK8 results suggested that the cell proliferation in each group showed an S-shaped curve.The proliferation rate of cells in the 10 ng/ml FGF-2 induction group was not significantly changed compared with the control group,while the proliferation ability of the 20 ng/ml and 40 ng/ml FGF-2 induction group was significantly improved compared with the control group,of which 10 ng/ml is the most obvious.14 days after induction,qRT-PCR results showed that the expression of ligaments-related and osteogenesis genes was significantly up-regulated in the different concentrations of FGF-2 induction groups,and the ligament-related and osteogenesis genes were most significantly up-regulated in the 10 ng/ml FGF-2 induction group.Western Blot/immunofluorescence results suggested that the expression of ligaments-related and osteogenesis proteins in the group induced by different concentrations of FGF-2was significantly up-regulated,and the ligament-related and osteogenesis proteins were most significantly up-regulated in the group of 10 ng/ml FGF-2.Picrosirius red staining results suggested that collagen secretion could be seen in the cells of each group,and collagen was mainly secreted in the cytoplasm around the nucleus.The control group showed a small amount of collagen secretion.The collagen secretion of the FGF-2 induction groups with different concentrations was significantly higher than that of the control group.Among them,the collagen secretion of the 10 ng/ml FGF-2 induction group had the largest collagen secretion.FGF-2 lentivirus vector was transfected into P3 generation hAMSCs with the optimal MOI(MOI=50).The expression of green fluorescent protein was seen in the lentiviral transfection group and the unloaded plasmid group after 24 hours,and the expression of greenfluorescent protein was the strongest and stable after 96 hours.No expression of green fluorescent protein was seen in the non-transfected groups at all time points.The results of qRT-PCR and Western Blot indicated that the expression levels of FGF-2mRNA and protein in the lentiviral transfection group were significantly higher than those in the unloaded plasmid group and the untransfected group(P<0.05),while there was no statistical difference in the expression levels of FGF-2 mRNA and protein in the unloaded plasmid group and the untransfected group(P>0.05).(3)After the rabbit venous blood was centrifuged for the first time,the whole blood is divided into three layers,the upper layer is the plasma layer,the middle layer is the PRP layer,and the lower layer is the cell layer(red blood cells).The upper layer of plasma,the middle PRP,and a section of the lower layer were transferred into a new centrifuge tube and centrifuged again.PRP was deposited on the bottom of the centrifuge tube.The upper 3/4 of the plasma was removed after the second centrifugation,and the lower 1/4 of PRP was retained.The PRP at this stage was liquid and had no biological activity.After adding an activator(bovine thrombin/Cacl2),the inactive PRP could be activated,and PRP changed from a liquid state to a gel state.At this stage,the PRP had biological activity and a large number of growth factors were activated.(4)At 3 months after operation,a clear gap was seen between the graft and the bone tunnel in the control group,and abnormally hyperplastic tissues were seen around the bone tunnel;The gap between the graft and the bone tunnel in the PRP group and the PRP+hAMSCs group tended to narrow gradually compared with the control group,but the color and gloss of the tissue around the bone tunnel were significantly different from normal tissues;The gap between the graft and the bone tunnel in the PRP+FGF-2-hAMSCs group almost disappeared,and the color around the bone tunnel was close to normal tissue.Histological test results showed that there was almost no gap between the graft and the bone tunnel in the PRP+FGF-2-hAMSCs group,no inflammatory cells were present,new fibrocartilage and a large number ofneatly arranged collagen fibers were seen.The tendon-to-bone healing was better than other groups.Radiographic examination results suggested that the formation of new bone could be seen in the bone tunnels in each group,and the diameter of the bone tunnels in the PRP+FGF-2-hAMSCs group was significantly reduced compared with other groups.Biomechanical test results suggested that the mechanical strength of the PRP+FGF-2-hAMSCs group was significantly higher than the other groups.Conclusions :(1)hAMSCs were successfully isolated and cultured;(2)The autologous PRP was successfully prepared by double centrifugation method;(3)FGF-2 lentiviral vector-mediated hAMSCs combined with rabbit autologous platelet-rich plasma can promote rabbit tendon-bone healing.