The Effection Of PLGA Scaffold Contain The Simvastatin In Construction Of Vascularize Artificial Bone

With the development of the tissue-engineering's technique and theory, more and more people use the tissue-engineering bone to repair the defect of jaw bones. Tissue-engineering bones have many advantages especially can increase histocompatibility and plasticity, convenient to obtain the materials and can repair the defect quickly. Artificial bone is tissue-engineering bone,it means under the condition vitro , seed cells who have the potential bone formation be separated cultured amplification and induced to osteoblast-like cells,after several passages, the cells seeded on the 3-dimensional scaffold, in vitro culture 2-3 weeks formation artificial bones tissue which function and structure is similar to autogenous bone with the cells adhered and amplified and the inducement of extracellular matrix.But in tradition tissue-engineering bone , nutrition is mainly depend on the diffusion so a tissue with more than 0.7cm in volume can not survive by diffusion and requires formation of new blood capillaries to supply essential nutrients and oxygen.Especially when ingraft to the body ,the internal cells maybe degeneration and necrosis because lack of nutrition and lead to the transplant is failed.So it use in repairing large area defect and reconstruction organ is confined.With the development of tissue-engineering vascularization,we can formation of new blood capillaries in tissue-engineering bone,so the nutritional support of the tissue-engineering bone is improvedso the survival rate and volume confine can be solved . Tissue-engineering bone vascularization have extensive application perspective.The key of construction tissue-engineering bone is the origin of the seed cells and the selection of the scaffold.Mesenchymal stem cells have enormous plasticity of differentiation.Under different induce condition ,it possess a broad differentiation capacity, possibly beyond tissues of mesodermal origin.Otherwise, the quantity of mesenchymal stem cells is parum (l/104-105single cells), but they have high reproductive activity ,after passage can maintain stable cell phenotype so we can through culture and passage to obtain a great quantity cells in short period. Mesenchymal stem cells action as the seed cells in tissue-engineering bone have absolutely preponderance.The scaffold material that have the proper strength , factor of porosity and degradation velocity can provide seed cells enough adhere and amplification space and through pore offer various kinds of correlated signaling molecule, nutrient substance , other cell which also participate the ostosis, cell factor and growth factor et al.lt also can take away the metabolism trash of seed cells and cellular necrosis product to maintain region dynamic microenvironment.During people research kinds of materials,found that the copolymer of the poly lactic acid (PLA) and polygly colic acid (PGA) have the same degradation velocity with biological metabolism,proper factor of porosity and enough support intensity,so more and more peopleuse it as medicine or vetor of artifical bone.The medicine of Statins contains Simvastatin and pravastatin et al,they are the HMG-CoA reductase inhibitors and be discovered in 1980s,be used to depress blood plasma cholesteryl,regulate blood fat,treat heart-blood diseases.Be considerd to rival the penicillin and called the epoch of Statins.In recently years,found except hyperlipoidemia cure it also can reduce the incidence of osteoporosis, stimulate the bone formation,increase osteocalcin level and reduce the incidence of bone fracture.In all kinds of Statins ,the function of Simvastatin is most obvious. Simvastatin have the synergistic action with BMP familiy to induce MSCs differentiation to osteoblast,^ provide the basis for its application in bone tissue- engineering and therapeutics of osteoporosis.But the exact signal pathway is still unknown.In addition people also found after use Simvastatin to treat the ischemic heart disease, ischemic cardiac muscle's capillary density is obviously increase and indicated Simvastatin have the capability to promote heart muscle capillary neogenesis and then protect the cardiac muscle. Maybe this is one reason of why clinically earlier period use Statins can decrease death rate of myocardial infarction patient.Others consider Simvastatin through vascular endothelial growth factor or NO sythase to activate endotheliocytes or mobilize the endotheliocytes origined from bone marrow in the circulation system take part in the blood avessel neogenesis in the ischemic region.But there is no report is about Simvastatin will synergistic effect with which signalingmolecule or if it have the direct relation with endotheliocyte's amplification and activation.In our experiment,we used the mesenchymal stem cells which is separated from rat as the seed cells,after passage we get enough cells.Then the cells were separated to two group ,one culture in the osteoblast conditioned medium contains Simvastatin and the other in vascular endothelial cell conditioned medium, so the mesenchymal stem cells can induced to osteoblast and vascular endothelial cell.We observe their function and morphous.Use the immunohistochemistry and transmission electron microscope to determine the founction of Simvastatin in vascular endothelial cell induced differentiation.In the domestic,we first synthesis the PLGA scaffold contain the Simvastatin and used it as the 3D direct culture vetor to culture the osteoblast and the vascular endothelial cell and then be planted to the rat leg's muscular sac.The control group use the PLGA scaffold that did not contain the Simvastatin.We used the soft X-ray and specimen slice method to approach under the function of Simvastatin ,the formation efficiency of vascularize artifical bone .To provide new way to construction the vascularize artifical bone . and the clinical application of Simvastatin.Result: In vitro culture condition, under the effect of Simvastatin, mesenchymal stem cells can differentiate into osteoblast and vascular endothelial cell.Compare to the the control group Simvastatin can inhibit cell proliferation andpromote its differentiation. ALP dyeing of osteoblast display the extracellular calcification nodus is obviously increase in experimental group. The expression of vWF protein which is the specificity mark of vascular endothelial cell is also increase ,in the endochylema there is a lot of buffy particles and the difference had statistical significance(P<0.05) .In vivo experiment,we use soft X-ray to observe the aberrance osteogenesis function of PLGA scaffold which contain the Simvastatin is manifest in the experimental group.Specimen HE dyeing ,found the area of the angiopoiesis in experimental group is obviously larger than the control group, and the difference had statistical significance(P<0.05).Conclusion : Used density gradient centrifugation and the difference of adherence capability can separate and extract pure mesenchymal stem cells.After passage we obtain bulk of vigor stable seeds cell.With the function of differentiate culture fluid, mesenchymal stem cells can differentiate into osteoblast and vascular endothelial cell.During this process Simvastatin have the co-induction function,ALP dyeing and expression vWF protein in the experimental group were higher than the control group.But we found the Simvastatin have the inhibitory action to cell proliferation.