Experimental Study Repairing Bone Defect With Seed Cell Modulated By Herb Medicine Compound With Biomaterial

Aim:A desirable clinical orthopedic requirement is the healing of segmental bone defect.with the development of technology and theory,a promising approach is the development of tissue engineering artificial bone that compound seed cell and absorbed biomaterial play the role of inducement,conduction even directly formatted bone.According to TCM,bone union after fracture get delayed if the kidney is deficient at nourishing bone marrow thus the treatment of promoting the union of fracture always start with invigorating the kidney and strengthen the bone.If it could be verified that Chinese medicine of invigorating the kidney and strengthen the bone have the function of enhancing the marrow stoma cells(MSC) to proliferate or differentiate or both,so the specific mechanism and target could be explained from the point of cytology about bone growing.This research is expected to answer the following questions:1.if the cytological mechanism of drynaria(Chinese herb Gusuibu) on promoting the union of fracture is the procedure from MSC to osteoblast to bone growing then to the union of fracture.Thus some theoretical support could be found to confirm the theory of "if the bone marrow get well nourished then the bone get strengthened."2.If MSC interfered with Chinese medicine have the ability of stabilized proliferation and passage.3.If MSC interfered with Chinese medicine can differentiate to osteoblast.4.If there is dose-effect relationship during the cell culture interfered with extraction of drynaria.5.Is there a way to combine this specific cultured seed cells with biological matrix materials to make some organic tissue engineering artificial bone? 6.How is the efficacy of such artificial bone when it is implanted into bone defect area?Part 1:the influence to proliferation of bone stromal cells with Gu suibu extractMethods:1.grouping:①blank controlled groups(standarding culture)②induce controlled groups(condition culture fluid)③herbⅠmedia(gu suibu 20mg/ml)④herbⅡ(contain gu suibu 2mg/ml)⑤herbⅢ(contain gu suibu 0.2mg/ml).Bone marrow stromal cell were established from adult new Zealand white rabbits and culture in above culture solution respectively.Results:1.Situation of cell growthAdherent cell of Chinese medicine groupⅠhad a small number,Slow growths, morphology slender,less cytoplasm and cell shrinkage loss.Most cells of blank group were long spindle,other cells in each group gradually increased,extended pseudopodia,were fusiform,stellate,polygonal and etc.There were more small intracytoplasmic particles,nucleolus obviously,and cell basicly covered and there was colony a week or so.2.Cell viability and growth curve of the measured resultsThere is no significant difference between induction groups with blank group. The low and middle concentration of extract can promote the proliferation of bone marrow stromal cell and the low concentration had a greater role than the middle concentration,while high concentration of extract can inhibit the proliferation of bone marrow stromal cells and lead to apoptosis.3.Cell viability and growth curve of the measured resultsBy measurement mitotic index records,mapping it into a curve,and observing the HE staining of the cell line,we drew the conclusion that Chinese medicine group significantly inhibited cell growth,and had the low curve.Conclusion:The low and middle concentration of extract can promote the proliferation of bone marrow stromal cell,while high concentration of extract can inhibited the proliferation of bone marrow stromal cells and lead to apoptosis.the influence of conditional media to cell proliferation was unobvious.Part 2,the influence to differentiation of bone stromal cells with Gusuibu extractMethods:bone marrow stromal cells culture same above.Results:1.Inverted phase contrast microscope and scanning electron microscopyCells of ChineseⅠgroup floated off the wall,the number of cells significantly reduced,cells edge became rough and shrinked,and nuclear condensation,cracking. Cells oftraditi0nal Chinese MedicineⅡ,Ⅲgroups and the induction control group were uniform,polygonal,and kept growing into complex layer after integration into a single layer and formatted several dense cells groups like islands,in which the closely middle cell were gradually embedded by empty vesicles and dark particles and turned into the matrix of white calcified nodule formation.Blank control group,contacted inhibition after the integration of the single layer,and ultimately to stop proliferation.2.Scanning electron microscopeMost cells of Chinese MedicineⅡ,Ⅲgroups were single-cell structure, spindle or polygonal cells with multiple processes,and different in the number of ranges,the length and the thickness.We can see the formation of long filaments between connected cells,the cell surface and cell gap can be seen the deposition of calcium salt crystallization.3.Alkaline phosphatase stainingThe positive rate of staining of cells of traditional Chinese MedicineⅡ,Ⅲgroups and the induction control group was 35.6%,31.4%and 72.4%,however blank group was only 3.6%.Induced group,blank group and each Chinese medicine group had a very significant difference(P＜0.001).Chinese MedicineⅡ,Ⅲgroups and blank group had also a very significant difference(P＜0.001).There is no significant difference between the two Chinese medicine groups(P＞0.05).4.TypeⅠcollagen immunohistochemistry Each group of typeⅠcollagen both in the cells and matrix were positive staining,induced group and the blank group was significant difference(P＜0.001). Chinese MedicineⅡ,Ⅲgroup,although obviously not as good as induced group (P＜0.05),but significantly higher than the blank group(P＜0.05,P＜0.01),while there is no significant difference between the two Chinese medicine groups(P＞0.05).5.Calcium nodules stainingSpecimens of Chinese MedicineⅡ,Ⅲgroups can be seen on the golden calcium nodules,low-fold vision of an average of 1～2,tess than the average 3 to 4 of inducted group,and the blank group can be seen nodules of calcium only OccasionalConclusion:high concentration of extract can inhibited the proliferation of bone marrow stromal cells and leads to apoptosis or death,went against to differentiate to osteoblast.The low and middle concentration of extract can promote the differentiation of bone marrow stromal cell,Part 3 the biocompatibility study of osteoblast with multiple-hole scaffoldMethod:The passaged rabbit osteoblasts cell suspension were adjusted to density of 5×10~4/ml,and put in Scaffold materials in the culture plate,so that the cell suspension can fully infiltrate into porous scaffolds with co-culture.Added conditioned medium after 8 hours,replaced the culture medium the next day,and continuously cultured.Results:1.Inverted phase contrast microscope and scanning electron microscopyAfter 7 days culture,osteoblasts closely attached to the stent surface differentiated and proliferated,and connected into a film,most cells were long spindle and triangle, extending pseudopodia,matrix enveloping the cells.Cells filled the pores around the stent material in which calcium nodules can be seen.2.ALP(alkaline phosphatase) staining:Osteoblasts cells can be seen in the cytoplasm of light brown to brownish-black particles after Ca-Co-culture 2 weeks.3.Osteoblast survival and proliferation ability on scaffoldsTest value 0.221±0.017,contrast is 0.140±0.015,(P＜0.01),Showed:scaffold was propitious to the adhesion and proliferation of osteobtast.Conclusion:Osteoblast cell distribution in biomaterial scaffold was uniform,strong adhesion,differentiation and secrete new matrix,bioglass that provide survive space and carrier as bone tissue engineering scaffolds have good biocompatibility with osteoblast.Part 4 Experimental study of repairing bone defect with tissue engineering artificial bone,Methods:24 new Zealand white rabbits were randomly divided into 4 groups, making 1.5 cm bone defect models in middle segment of radius and implant different material,ⅠArtificial bone,Ⅱbioglass,Ⅲauto iliac graft,Ⅳblank contrast。Fixed with muscle and skin,Penicillin im everyday after operation.Routine feed.At 8,12 weeks after implantation,the effectiveness of bone formation was evaluated by means of gross,histological observation and scanning electronic microscope(SEM) examination and biomechanical analysis.Results:1.Gross observe:12 weeks:bone defect region completely repaired was same to normal bone inⅠgroups,primary repairing little consecutive callus through inⅡgroups,a lot of consecutive callus through inⅢgroups,pseudoarthrosis formed inⅣgroups.2.X-ray observes:12weeks,completely renovated inⅠandⅢsubstantial compacted and marrow cavity formatted,thickness of substantial Compact closely to normal bone.little consecutive callus across breaking gapingⅡgroups,sclerosis and separated form pseudoarthrosis in defect region.3.Histological observe:12 weeks:bone bridge and girder structure has been established between grafts grains,marrow cavity dredged,restore characteristic of natural bone in groupⅠ. Little new bones formation,bone bridge link sparsely.Trabeculae inconspicuous. Between graft grains. 4.Scanning electron microscope:12 weeks,dense link have shaped in groupsⅢ,obvious repairing has been seen, link wasn't thick in groupsⅡ,all of region is fiber tissue,and crack has been seen on middle in groupsⅣ.5.Biomechanical test:Torsion intensity of GroupⅢwas same as that groupⅠ,higher than any other two groups.Results:repairing ability of tissue-engineering artificial bone of osteoblast and bioglass almost same that auto iliac-implant,excel that any other tow groups.And this artificial bone can achieve the repairing bone defect.