The Preparation And Biological Activity Evaluation Of Electrospun PBS/PPC Nanofiber Membrane Carrying Rh-BMP2

Objective and background：Along with the developing of implant technology,implant restoration hasbecome a very mature technique. And the implant denture is popular amongthe patients for it resembles the natural teeth in beauty, comfortableness andthe mastication efficiency. But because the long-time agomphosis, periodontaldisease, tumor and trauma, some patients suffer the insufficient bone mass inimplantation area which has a direct impact on the stability of implant in theinitial period, even leads to the failure of the implant surgery. Guide BoneRegeneration (GBR) technique is a common clinical method to solve theproblem of insufficient bone mass, while the key element lie in the applicationbiological membrane.At present, the common biological membranes applied in clinic are dividedinto absorbable membranes and non-absorbable membranes. The former oneis not only expensive but has poor mechanical strength which would lead tocollapse and could not provide sufficient osteogenesis space, and highdegradation velocity; the later one has enough strength to maintain a certainbone-forming space,But there are still some problems during the application, such as dehiscence, exposure and infection of soft tissue caused bynon-absorbable membrane,and the patient would suffer more pain due to asecond operation. Therefore, it is urgent to developed one compoundmembrane with good plasticity, spatial maintainment ability and biologicalactivity, is also the hot issue among the researches concerning GBR.Electrospining technology plays an important role in the synthesis ofnanofiber scaffold material. The electrospun scaffold material has a largerspecific surface area and three-dimensional vesicular structure similar to theextracellular matrix being favorable to the adherence and extension of cellsand its vesicular structure is good for the local circulation which brings nutritionto the cells in bone defect areas. Given this, the application of electropiningtechnique is one of the best chose in the preparation of tissue engineeringscaffolds material.Poly (butylene succinate)(PBS) is a new degradable macromolecularmaterial with stable property and a typical polymer material of completebiodegradation which are easy to be resolved and metabolized into CO2andH2O as end-products by microorganism and enzymes. By improving andmodifying the structure which has a better biocompatibility and hydrophilie,PBS has an extensive application prospect in biomedical field.Poly (propylene carbonate)(PPC) is a full biodegradable materialconsidered as an ideal medical material. It can be degraded without pollutionand has good biocompatibility, temperature memorability and high barrier property.Bone morphogenetic protein-2(BMP-2) has the highest osteogeniccapability and it improves osteogenesis by inducing undifferentiatedmesenchymal stem cells to proliferate and differentiate in the direction ofosteoblast cross membrane, thus it can be used alone. But it has a shorthalf-life period and would be attenuated and metabolized soon in localapplication. A small dose can hardly achieve the expected osteogenesis effect,while a large dose is expensive and wasteful in local application. Therefore,there is a need to mix it with carrier of slow attenuation, which could slow downthe hydrolysis velocity and contribute to the continual inducement ofosteogenesis.Therefore, this study compounds the PBS/PPC nanofiber membrane andcompound biological membrane (PBS/PPC/rh-BMP2) by means ofelectrospining. Scan electron microscope, contact angle meter and universaltester are applied to inspect the morphology, hydrophile and mechanicalproperty of scaffold material and study the degradation property and theprotein attenuation ability. In the in-vitro cell experiment, implant theosteogenic sarcoma cell MG63on the surface of scaffold material, theninspect what influence that the material exerts on the proliferation of MG63byCCK-8method, use Laser confocal inverted microscope to observe the growthsituation of MG63after DAPI colouration and inspect the cell differentiation byALP method. Method:Ⅰ. The preparation of biological membraneTo prepare the PBS/PPC、PBS/PPC/rh-BMP2and PBS/PPC/BSAbiomembrane by electrostatic spinning method.Ⅱ. The test of physical and chemical property1.Observe the surface topography of the material by scanning electronmicroscopy.2.Detect the hydrophile by contact angle meter.3.Test the tensile mechanical property by universal tester.Ⅲ. The test on the degradation and slow release property.1.Evaluate the degradation situation by testing its weight loss ratio, PH valueschange and water absorption.2.Test the release amount of protein which reflects the in vitro release situationby BCA.Ⅳ. The osteoblast compatiblity of the membrane1.CCK-8experimentInspect the influence exerted by material on the proliferation of MG63byCCK-8method. 2.The growth situation of MG63on the surface of biological membrane.Observe the growth situation of MG63on the surface of material after DAPIcoloration by Laser confocal inverted microscope.3.ALP activity experiment.Detect the influence exerted by material on the proliferation of MG63by ALPactivity experimentResult:Ⅰ. The inspection of representation and physical and chemical property.1.Scanning electron microscopeThe Biological membranes prepared have vesicular structure and eventexture, the average fibre diameter of PBS/PPC is880nm, close to the value910nm of PBS/PPC/rh-BMP22.PorosityThe porosity of PBS/PPC is78.3%and79.6%for PBS/PPC/rh-BMP2.3.Contact angleContact angle test result showed that PBS/PPC has good wettability,and thecontact angle is87°，and the contact angle of PBS/PPC/rh-BMP2is79°.4.Mechanical propertyThe tensile strength of PBS/PPC is2.31±0.35MPa and2.05±0.47MPa for PBS/PPC/rh-BMP2. The breakage elongation of PBS/PPC is31.63%and22.24%for PBS/PPC/rh-BMP2.Ⅱ. The test on the degradation and slow release property.1.Weight loss ratioPBS/PPC is slow in degradation and the weight loss ratio is6.04%after12weeks. The degradation ratio of PBS/PPC/rh-BMP2is higher than PBS/PPC,and the weight loss ratio is14.38%after12weeks.2.PHThe PH values of PBS/PPC and PBS/PPC/rh-BMP2have slight dropsduring degradation.3.Water absorptionThe water absorption of PBS/PPC/rh-BMP2is44.4%for the first week and67.1%for the12thweek,and the water absorption of PBS/PPC is only36.5%for the first week and53.1%for the12thweek.4.Protein release ratio.The release rate is29.3%for the first week, and43.6%for the third weekwhich is a quick release period and the accumulative release rate is51.1%.Ⅲ.The influence of biological membrane on the proliferation, adherence anddifferentiation of MG63.1.CCK-8The results show that the Haiao bio-membrane group proliferates much faster than PBS/PPC and PBS/PPC/rh-BMP2group. In the first day, there isno obvious difference between the PBS/PPC and PBS/PPC/rh-BMP2groupand the pure cells group; and in the fourth and seventh day, the proliferationrates in PBS/PPC and PBS/PPC/rh-BMP2group is obviously higher than thatof pure cells group but there is no obvious difference between the PBS/PPCgroup and PBS/PPC/rh-BMP2group.2.DAPI colorationAfter one-hour cultivation, the amount of cells adhered of PBS/PPC/rh-BMP2group and Haiao bio-membrane group are larger than that of PBS/PPC groupand the cells is tight in arrangement. And the cells not only adhere on thesurface of the material but grow into the material.3.ALPIn the first day,there is no obvious difference between all groups, in thefourth day, the ALP activities of MG63in PBS/PPC、PBS/PPC/rh-BMP2、andHaiao bio-membrane group is increasing compared with that in the pure cellsgroup, Haiao bio-membrane improves the activity of ALP is most obvious.Andin the seventh day,the ALP activity of MG63in PBS/PPC/rh-BMP2is betterthan Haiao bio-membrane group.Conclusion:1.The electrospun scaffold material has a larger specific surface area andthree-dimensional vesicular structure，which has a better porosity beingfavorable to the circulation and nutrition supply and has a good mechanical strength as well. It is the ideal biological material.2.PBS/PPC can be degraded slowly, which could slow down the release ofprotein.It can be a kind of controlled release carrier for protein.3.PBS/PPC and PBS/PPC/rh-BMP2are good for cell proliferation andadhesion,and the PBS/PPC/rh-BMP2could promote the differentiation ofosteoblasts as well.