The Preparation And Properties Of Loading Recombinant Human Bone Morphogenetic Protein-2 Nanometer Stent Materials

The number of traditional bone grafts is reducing while the quantity demanded is rising gradually.Tissue engineering technology is urgent to develop appropriate substitutes to clinical application,as drug delivery system electrospinning polymer nanofiber mats with potential applications can protect the drug from inactivation.The electrospun nanofiber mats which have core-shell structure are able to overcome the problem of drug initial burst release,also protect unstable biological agents,so that different release rates of drugs from shell and core layer structure can be obtained and they efficiently act on the tissue of patient.BMP have an effect on inducing ectomesenchymal cells including animal and human body to differentiate into nerve tissue and cartilage.The ideal tissue engineering scaffold materials must meet certain biological requirements.In this experiment,zein?PLLA and PLGA which were biodegradable?good biocompatibility and non-toxic were selected to fabricate scaffolds.Among the materials,shell-core materials of nanofibers were Zein and PLLA respectively,PLLA provided mechanical support,PLGA was used as the carrier material of core-shell structure composite microspheres.DEX loaded in nanofibers had the effect on promoting the growth and proliferation of rat bone marrow mesenchymal stem cells(RMSCs),rhBMP-2 had the effect on inducing RMSCs to differentiate into osteoblast.The purpose of this study was to establish two drug loaded scaffolds by electrospun technology.One scaffold was zein/PLLA nanofiber mat that loaded with rhBMP-2 and DEX,the other was PLGA microsphere loaded rhBMP-2,in addition,process parameters of fabrication and relevant characteristics were studied on the two carriers loaded with rhBMP-2.Zein/PLLA ? zein-DEX/PLLA ? zein/PLLA-rhBMP-2(the concentrations ofrhBMP-2 were 12 ? 30 ? 48?g/ml respectively)? zein-DEX/PLLA-rhBMP-2(the concentrations of rhBMP-2 were 12?30?48?g/ml respectively)nanofiber mats that fabricated by coaxial electrospun technology without irregular phenomenon such as beaded defects all had rough surface,among them zein/PLLA nanofiber mat had a relatively smooth surface.The diameter size and distribution of nanofibers had no significant change after adding different types and quality of drugs,the average diameter size were about 300 nm.The addition of DEX with small molecular weight led to the decrease of solution viscosity,further influenced the increase of diameter size and distribution of zein/PLLA nanofiber.Rough surface of the scaffolds influenced by the addition of drugs provided a strong support for cell adhesion and growth.Among the fabricated nanofiber mats,zein/PLLA nanofiber mat had the highest tensile strength and modulus of elasticity,also the longest breaking elongation,zein-DEX/PLLA nanofiber mat took second place.The tensile strength of zein/PLLA-rhBMP-2 nanofiber mats was slow,nanofiber mats loaded with dual drugs had the minimum tensile strength,there was no significant effect on tensile strength on account of different concentrations of rhBMP-2.For zein/PLLA scaffold there was little difference on young's modulus after adding drugs.The accumulative release profile of different nanofiber mats in vitro indicated that the release rate of the drug from single drug loaded nanofiber was faster than dual drugs loaded nanofiber.DEX loaded in zein(shell layer)had an initial burst release while rhBMP-2 loaded in PLLA(core layer)presented a slow and steady release.In combination with the release results,the whole period of drug release was up to 21 days and ultimately the cumulative release results of DEX and rhBMP-2 were 55%-65% and 5%-35%respectively,it indicated that the two drugs may release for a longer time.The release system of rhBMP-2 and DEX loaded in nanofibers fabricated by coaxial electrospun technology was controllable and contributed to promote RMSCs to differentiate into osteoblast.In the cell cultivation the effect on promoting proliferation of zein-DEX/PLLA nanofiber mat was more prominent than zein-DEX/PLLA-rhBMP-2nanofiber mats the 6th day,but the results contrast the 9th day.Results shown that DEX and rhBMP-2 significantly effected on cell proliferation,and the promotingeffect of nanofiber mats loaded with dual drug was more significant.The optimal process parameters of shell-core structure PLGA microspheres that fabricated by electrospun technology(electrospray)were studied: the high voltage was 25 KV,the receive distance was 16 cm,the fitting speeds of core layer and shell layer were 0.1 and 1 ml/h respectively.There was no filamentation on the obtained PLGA microspheres surface with groove-like structure.The pure PLGA microspheres had the minimum average diameter 4.43?m compared with other rhBMP-2 loaded microspheres.PLGA microspheres loaded with higher concentration rhBMP-2 had a narrower size distribution and smaller standard deviation,The standard deviation decreased gradually from 1.02 ?m,0.86 ?m,0.78 ?m to 0.69 ?m.Drug release profiles in vitro indicated that rhBMP-2 had an initial burst release at 6h,after that the release rate was smaller.Ultimately the cumulative release of rhBMP-2 was 41%?34% and 27%(the concentrations of rhBMP-2 were 12?24?48?g/ml respectively).In this study,PLGA microspheres loaded with higher concentration of drug might cause a smaller accumulative release rate could be inferred by the release curve of rhBMP-2.In vitro degradation,initial quality of composite PLGA microspheres slowly reduced by almost 11% in the first three weeks,the quality decreased obviously in the following weeks.The final remained quality of the microspheres was about 15% after exposure to PBS buffer for 7 weeks.Surface topography had changed after contacting with PBS for different time during degradation process,surface gradually collapsed over time.Although the cell proliferation was not significant as before the 7th day,the rhBMP-2-PLGA microspheres were all able to promote proliferation,and cytotoxicity of microspheres in vitro cell cultivation was very slight.The results declared rhBMP-2-PLGA microspheres had great potential in the application of bone tissue engineering,simultaneously microspheres loaded with drug with groove-like surface that fabricated by electrospray had wide development prospects in the field of drug release.These two kinds of drug carrier systems,which fabricated in this study,will have a potential application prospect in the field of bone tissue engineering.