Experimental Study Of Repairing Rats Supraspinal And Interspinal Ligaments By The Method Of Tissue Engineering

objectivePatients with spinal fractures often injury supraspinal and interspinal ligaments. But after the ligaments' injury,they may not heal or heal with scar.That often leads to obvious a loss of joints' function and pain.And even though the ligament injury can heal,ligament ultrastructure and biochemical composition change a lot after the healing.Even in a number of years after ligament healing,its biomechanical properties are still inferior to normal ligament.Domestic and foreign scholars use the biological methods to try to promote the healing of ligaments in recent years.The main methods include cytokine therapy,gene therapy and tissue engineering method.Mesenchymal stem cells(MSCs),as a kind of stem cells,have multi-directional differentiation potential.This experiment uses tissue engineering method with MSCs as seed cells to promote the healing of ligaments.And to observe effects on promoting healing of rats supraspinal and interspinal ligaments.MethodMSCs culture in vitroSeparate and harvest rat femur MSCs by density gradient centrifugation.Inoculate to DMEM culture medium by the density of 2.0×10~5/mL.When primary cells covered about 80%of the bottom,subculture by the ratio of 1:3.Preparation of the cell-collagen sponge complexTag the MSCs by adding 3 mg/L BrdU to the culture medium of the third generation of cells for 24 hours.Digest MSCs by trypsin,and made the cells suspended.Then diluted the suspension with DMEM culture medium into 10×10~6/mL concentration.Drip it on the collagen sponge(disinfected with UV).Put it into 5%CO2 incubation box with 37℃constant temperature for 24 hours,so that cells will adsorp on collagen sponge and become cell-collagen sponge complex.Construction of animal modelCut off the rats supraspinal and interspinal ligaments between L4 to L5.Transplant cells-collagen sponge complex to the experimental group,and only collagen sponge to the control group.HE stainThe animals were killed on the 4th week after transplantation.Get the specimens for histological examination.Select a section of each specimen for HE staining. immunohistochemical stainSelect the sections of each specimen again to do immunohistochemical staining. The cells with brown nuclei were positive staining cells.ResultsMSCs culture in vitroObserved under inverted microscope could see that MSCs partly had coverage 3 days after the cells inoculated.Cells growed into a monolayer after cultivated 12-14 days.After passage,MSCs could significantly speed up the proliferation and only took 9-10 days to integrate into a single layer.the results of animal model.20 animals all survived.histological observations4 weeks after transplantation,6 of experimental group could be seen formed ligament-like tissue and we could see patterns in line with the direction,which was close to normal tissue fibroblasts and similar to the normal ligament organization. While in control group,new organizations were formed by more adipose tissue.The cells disordered,and the collagen fibers looked like loosely filamentous Net.The difference of better healing percentage between the two groups was significant(P＜0.05).the results of immunohistochemical stainCells of brown positive nuclei could be seen from the slices of experimental group. Control group had no positive staining cells. DiscussionMSCs suit as seed cells.It is also reasonable to use of type-I collagen as a carrier. Some of these tissues that repaired the damage of the ligaments came from the transplanted MSCs.Using MSCs without adding exogenous growth factor,can repair ligaments effectively in vivo.In the experimental group,ligaments had better restoration.In the control group,the recovery was relatively poor.ConclusionIt's feasible to repair the injury of supraspinal and interspinal ligaments by tissue engineering technology with the seed cells of MSCs.This method can promote the healing of ligaments,and the healing was better than the effect of natural healing.