An Experimental Study Of Two Methods For Repairing Annulus Fibrosus Defect With The Aid Of Intervertebral Disc Mirror

Section 1 Preparation of platelet-rich plasma and experimental study of the role of mesenchymal stem cells in bone marrowObjective:Discussion Isolation and identification of research methods goat bone marrow mesenchymal stem cells(bone marrow-derived mesenchymal stem cell,BMSCs),while the study was between PRP goat bone marrow mesenchymal stem cell proliferation.Methods : Inter goat bone marrow mesenchymal stem cells gradient centrifugation,isolated and cultured between purified goat bone marrow mesenchymal stem cells by using adherent culture,cultured passaged to cell proliferation were observed between the different generations of goat bone marrow mesenchymal stem cells.Using immunocytochemistry methods among which the 3rd and 4th generation goat bone marrow mesenchymal stem cell surface markers CD29 antigen expression,CD34 and CD44 were identified.Inter above method of goat bone marrow mesenchymal stem cells grew well selected primary cells were randomly divided into two groups,A group obtained by adding different proportions goat blood centrifugation second time with a good rich in culture medium plasma,and group B is not joined goat PRP group formed the control group,were observed cell proliferation.Results : Vitro cultured goat bone marrow mesenchymal stem cells,and to achieve success passaged cell proliferation,cells were observed generations,we found that the first generation of 3-4 cells multiply rapidly,vigorous growth.The method of cellular immunity showed between goat bone marrow mesenchymal stem cells CD29,CD44 expression was positive for CD34 expression was negative,confirmed the identification between goat bone marrow mesenchymal stem cells.A group added 0.1volume fraction of the first four days of platelet-rich plasma goat,goat BMSCs proliferation activity increased,showing rapid growth,increasing growth curve rise,doubling time of about 50 h.By comparison,the addition of 0.2 volume fraction of goat PRP the first four days,the proliferation of goat BMSCs increased vigor than the 0.1 group,and the relatively high amplitude growth curve,doubling time shortened to 35 h.And on the fourth day was added 0.3 volume fraction of PRP goat,goat BMSCs growth activity is the highest,the biggest rise in the growth curve,doubling time is the shortest 25 h.Goat BMSCs cultured on day 4,respectively,at the concentrations of 0.3,0.2 and 0.1 body PRP experimental group and the control group,the difference since there are significant(P <0.05,P <0.01).Conclusion Between applications gradient centrifugation extraction cultured goat bone marrow mesenchymal stem cells persented the spindle,fibrous growth,strong ability to clone,and 3rd and 4th generation when the most active,the fastest growth,while goat platelet-rich plasma for goatbone marrow proliferation of mesenchymal stem cells have a role in promoting the greater the volume fraction and the platelet-rich plasma is more obvious for goat bone marrow mesenchymal stem cell proliferation.Section 2 Experimental study of intervertebral disc mirror assisted two ways to repair the disc annulus defect goatObjective By comparison between gelatin sponge carrier goat bone marrow mesenchymal stem cells combined with platelet-rich plasma graft method and direct suture repair of the annulus defect goat intervertebral disc defect,explore the effectiveness and feasibility of two repair methods.Methods Extraction and separation of the cells were identified when mesenchymal stem cells,and subcultured cultured to the third generation from the iliac goat.Then blood from the jugular vein of goats using two degrees centrifugation into platelet-rich plasma.Select 12 6-month-old goat beat lumbar lumbar spine X-ray and MRI T2 WI sagittal scan.The 12 goats L1 / 2-L6 / 7 discs,a total of 72,were randomly divided into mesenchymal stem cells to repair group(A),the suture group(group B)and control group(group C),each 24 disc.A group by the method of the first portion of the extract between goat bone marrow mesenchymal stem cells by adding PBS,cell suspension.In a sterile environment the prepared cell suspension into the gelatin sponge.Then goats under artificial damaged annulus posterior discectomy surgery with disc knife to open a 0.5cm gap,gelatin sponge complex containing goat BMSCs breakage placed in the upper layer of sponge covered with a layer of plastic jelly-like platelet-rich plasma.In thesame way for groups B and C next fibrosis defect posterior discectomy surgery,group B annulus defect using sutures.Group C of the annulus defect without any treatment.Then fascia,subcutaneous and skin sutured.After conventional antibiotic therapy.After 3 weeks,6 weeks,12 weeks each time point were randomly selected four goats shooting lumbar lumbar spine X-ray and MRI T2 WI sagittal scanning using disc height index(DHI)Evaluation and Improvement Pfirrmann grade disc degeneration Happening.Goats were sacrificed after three periods according to three different groups after removing damaged tissue growth,respectively,followed by histological examination.Finally,using SPSS statistical software for different groups of various methods were analyzed.Results:In Masson trichrome staining gelatin,over time,A group can be seen a large number of vacuolated chondrocytes,cells are large,blue-stained nuclei.Cell growth neat,dense.There are plenty of surrounding cells and cartilage matrix collagen fibers,into a large blue dye.And it contains a large number of mature bone lacuna around Aizen trabecular,immature red dye and a small amount of trabecular bone.Trabecular bone around the red dye contains a large number of mature muscle fibers to repair connective tissue,generally close to the normal structure of the structure of the control group,form a complete,state of repair over.Group B: at the endplate fiber thick,regular shape.Fiber ends incision tapering gradually small arc outward tilt,focus,outer 1/3 see fiberaggregation.Cut the outer visible scar tissue,blood vessels visible near to the incision.The inside of the fiber and the nucleus boundary distinguished.Group C: see shrinkage fiber morphology,messy,irregular fracture,incision fiber gradually move closer but no significant central tendency,the inner nucleus pulposus and annulus no boundaries,we can see a large crack.And part of the endplate separation,the inner and outer annulus boundaries unclear.There was no significant difference(P> 0.05)in each group preoperative disc height index,after 3 weeks,6 weeks and 12 weeks in each group disc height index DHI A group> Group B> C group,the difference was statistically significant(P < 0.05);preoperative groups lumbar MRI T2 WI modified Pfirrmann score was no significant difference(P> 0.05),after 3 weeks,6 weeks and 12 weeks in each group lumbar MRI T2 WI modified Pfirrmann Rating a group <B <C group,the difference was statistically significant(P <0.05);after 3 weeks,6 weeks and 12 weeks histology Masson staining,degeneration scores A group <B <C group,the difference was statistically significant(P <0.05).Conclusion:From the X-ray,magnetic resonance imaging examination and Masson trichrome staining gelatin,by grouping comparison,gelatin sponge inter-carrier successfully transplanted bone marrow mesenchymal stem cells combined with platelet-rich plasma and annulus repair defects,and with time,chondrocytes and an increase in the number of myofibroblasts,an increase in the content of type ? collagen,increase the degree of repair.Defects can also direct suture repair of the annulus defect,but lower than the stem cells repair method is poor.