Experimental Study On Bone Regeneration With BMP-2 And VEGF Transfected Mesenehymal Stem Cells Seeded On Calcium Phosphate Cement

BackgroundBone defect results from many clinical problem,such as trauma,tumor resection and infection.Autografts and allografts have been used to heal bone defects,but these treatments have limitations.With the rapid development of bone tissue engineering,it provides a new idea for bone defect reconstruction.Studies have shown that calcium phosphate cement has the incomparable advantage of other inorganic materials as a biological substitute materials,it is a material of physical and chemical quality and biological characteristics of the animal and human bones veryclose,so it is always a hot research area about bone defect reconstruction.As a tissue engineering scaffold materia,calcium phosphate cement can provide support structure for osteoblast and appropriate external environment of cells because of that it is favorable in the exchange of nutrients for cell growth,attachment,proliferation and differentiation.Acting as template with a three-dimensional bracket structure,calcium phosphate cement can guided tissue regeneration and organization structure repair,meanwhile,as a ideal tissue engineering scaffold materia,it has chemical surface for tissue regeneration and higher mechanical strength except of degradation speed matching tissue regeneration.But some studies showed that the porosity and pore size limited the degradation of calcium phosphate artificial bone.So,to improve the gap between the connectivity in three-dimensional bracket structure of calcium phosphate cement could be get a ideal balance between mechanical strength and tissue regeneration.Baesd on that,we producted the porous calcium phosphate cement with directional crystal template method in previous study,the three-dimensional bracket of calcium phosphate has an advantage of improved gap between the connectivity and satisfied mechanical strength.With the requirements to further improve the performance of scaffolds,single-phase biological scaffolds have been difficult to meet clinical requirements,and thus the different nature of the material compound in order to obtain excellent clinical performance bio materials has become the hotspot of current materials research in the field.This kind of biomaterial had satisfactory biocompatibility and osteoconduction but lack of osteoinduction,the composite seed cells are the effective ways to solve this problem.Stem cells have the multidirectional differentiation and can be induced to differentiate into osteoblast cells.It has been a hot area of research because of the quantities which it comes from a variety of sources and conveniently.Bone tissue engineering with seed cells of mesenchymal stem cells(MSCs)got rather satisfactory effects in bone defect reconstruction in vivo.Organism activity gene plays an important role in regulating the mechanism of bone repair.BMP induces differentiation of mesenchymal-type cells into osteoblast cells,BMP-2 was an important growth factor associated with transforming growth factor-B(TGF-?)and paly a role in the treatment of bone defection which can improve cell growth and alkaline phosphatase activity.Many studies showed that BMP-2 with or without carrier can increase osteoblast.The proliferation,differentiation and survival of seed cells base on blood supply,but the key technology of vascularization hasn't been solved ideally yet.VEGF is the the strongest effect and high specificity angiogenesis factor.Growth factors added to bracket structure were lack of local effective therapeutic concentrations as short half the period.Slow release method with operational complexity and lower encapsulation efficiency limits application.With the development of genetic engineering technology,seed cells with gene transfection of targetting growth factors in vitro is to be a effective methord which keep local effective therapeutic concentrations of growth factors.Compared to the limited function of sole growth factor,compined growth factors work synergistically.Thus we try to improve pore size and porosity of calcium phosphate scaffold and loaded MSCs with gene transfection of BMP-2 and VEGF,thereby enhancing osteoinductivity and promote bone formation.We fabricated a calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold based on that.The physical and chemical properties,mechanical properties,biocompatibility and osteogenesis of the scaffold were investigated,it provides a experimental basis for clinical applications.Objectives1.To investigate the preparation and characterization of the calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold.2.To investigate the MSCs with gene transfection of BMP-2 and VEGF biocompatibility of the composite scaffold.Methods1.Preparation and characterization of calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold.(1)Preparation of porous calcium phosphate scaffold and calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold.Porous calcium phosphate scaffold(S1)was prepared by using calcium phosphate powder with directional crystal template method.The calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold(S2)was prepared by using porous calcium phosphate scaffold(S1).The crystalline phase of the obtained nacre nanoparticles was analyzed with an XRD.The morphology of the nacre nanoparticles was examined by a TEM.(2)Crystalline phase of calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffoldThe crystalline phase of the obtained porous calcium phosphate scaffold(S1)and calcium phosphate composite scaffold loaded MSCs with gene transfection of BMP-2 and VEGF(S2)were analyzed with an XRD.The morphology of the calcium phosphate powder was examined by a TEM.The morphology and structure properties of the scaffolds were investigated by scanning electron microscope(SME)and mechanical property testing.The porosity of the scaffold was measured by the Archimedes method.2.Evaluation of the MSCs with gene transfection of BMP-2 and VEGF biocompatibility of the composite scaffold.MSCs with gene transfection of BMP-2 and VEGF cultured on porous calcium phosphate scaffold(S1)for two weeks was classified as experimental group,MSCs with gene transfection of BMP-2 and VEGF cultured without scaffold was classified as control group,respectively.At the indicated time,MSCs morphology was observed by inverted phase contrast microscope and scanning electron microscope.In addition,MTT assay and ALP activity test were used to evaluate the MSCs abilities of proliferation and osteogenetic-differentiation respectively.The amount and area of calcium nodules in each group were observed by alizarin red staining.Results1.The diffractogram from XRD analysis of the calcium phosphate powder was indicated that the samples were phase-pure and well crystallized.TEM micrographs of the calcium phosphate powder were shown that crystals were spherical shape,with a diameter range from 45 to 95 nm.Porous calcium phosphate scaffold(S1)and calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold(S2)were prepared,they are all cylinder shape(5mmx5mmx 15mm).2.Two scaffolds possessed porous structure,the pore sizes of the two scaffolds were(330.0±47.2)?m and(325.1±50.6)?m.There was no significant di:fference between them(P=0.141).The porosities of the these scaffolds were 82.1±0.6%and 80.0±0.5%.There was no significant difference between them(P=0.211).The diffractogram from XRD analysis of the two scaffolds indicated:diffraction peak of the porous calcium phosphate scaffold(S1)was similar to that of the calcium phosphate powder;compared to scaffold S1,diffraction peak of the calcium phosphate composite scaffold(S2)loaded MSCs with gene transfection of BMP-2 and VEGF cultured combined indicated diffraction peak characteristics of amorphous.The compressive strength of the two scaffolds were(4.4±0.3)Mpa and(4.5±0.2)MPa.There was no significant difference between S1 and S2(P>0.05).3.The MSCs with gene transfection of BMP-2 and VEGF adhered and grew well on the surface of the porous calcium phosphate scaffold(S1).The MSCs adhesion rate get higher as the hours pass away.There was no significant difference of the MSCs adhesion rate after 2h and 4h culture(P=0.072);but there were all significant differences of the MSCs adhesion rate after 8h?12h culture compared to previous time points,repectively(P<0.001).MTT detection showed that MSCs were well-grown,the OD value in each group was higher than that in previous time points,there were all significant difference(P<0.001).Compared to control group,the OD value in expremental group were similar after ld(P=0.179)and 3d 3d(P=0.114)culture(P>0.05),but there was significant difference after 7d culture(P<0.001).The expression of the ALP in each group was higher than that in previous time points.There was no significant difference of the ALP expression after 1d(P=0.083)compared to control group,there was significant difference of the ALP expression in expremental group after 3d(P=0.002)and7d(P<0.001)culture(P<0.05).After 3d of the MSCs with gene transfection of BMP-2 and VEGF cultured with porous calcium phosphate scaffold,the number of mineralized nodules and area of expremental group was more obvious than control group.4.Conclusions1.Calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold has an appropriate structure,suitable compressive strength as a tissue engineering scaffold materials.2.Calcium phosphate loaded MSCs with gene transfection of BMP-2 and VEGF composite scaffold is non-cytotoxic biological materials,which has good biocompatibility,and the differentiation and minerlization of MSCs with gene transfection of BMP-2 and VEGF is regular.The composite scaffold met the needs of bone tissue engineering.