The Study On The Osteoinductivity Of BMP-2 Incorporated Biomimetic Coatings On Three Polymers: Polyactive,Ethicon,PLGA

Bone defect is one of the most common and troublesome diseases of human.Although extensive researches have been done,reconstruction of bone defect remains unsolved. Autografting and allografting suffer from a variety of drawbacks.To address the problems, numerous studies have tried to synthesize ideal bone substitute as an alternative to autologous and allogenic bone graft.As a strategy to replace bone grafts,synthetic polymers are being widely used and continuously developed for a variety of biomedical applications.But the low conductivity and lack of osteoinductivity limited their application in reconstruction of large-area bone defect. Mineralization of synthetic polymers has been repeatedly shown to enhance their osteoconductivity and was thought to be a promising strategy.Whereas some methods to prepare the coatings,always concern organic solvent,which may be harmful to transplanted cells or host tissues,Furthermore,other methods preincorporating ceramic particles into polymers instead of on the surface,will inevitably decreases the contact chance between the osteogenic cells and the bioactive ceramics.Biomimetic coatings methods also have some deadly drawbacks.Currently,the osteogenic activity to polymers is conferred mainly by the adsorption of bioactive agents,such as BMP-2.Whereas such superficially adsorbed molecules were released so fast that BMP-2 could exhibit osteogenic activities at a large amount,which thus make the products expensive and limit their clinic application.Increasing the BMP-2 dose or the surface area was proved to be not successful.Part One:The influence of Polyaetive,Ethicon,PLGA's geometries, surface chemistries on physicochemieal characteristics of biomimetic calcium phosphate coatings.The present study was to determine the influence of polymers'geometries,surface chemistries on the morphology,chemical groups,and chemical components of biomimetic calcium phosphate coatings. Materials and methods:Three polymers such as polyactive,ethicon and PLGA were selected and collagen was adopted as control material.Coating was prepared biphasicly:immersion of four carriers into 5 times simulated body fluid to prepare seeding layer;immersion into supersaturated calcium phosphate solution to prepare the protein-carrying layer on the basis of seeding layer.The geometry and surface topography of four carriers,morphology of coatings were examined by scanning electron microscopy(SEM).The chemical groups of the four carriers and coating were examined by fourier-transform infrared spectroscopy,(FTIR)..Calcium phosphate ratio of coating was gauged through energy dispersive X-ray analysis(EDAX).The component of coating was examined by X-ray dispersive analysis(XRD).Results:SEM showed that the geometries of the four carriers were different from one another. Polyactive and collagen were porous,whereas ethicon and PLGA were fibrous.The morphologies of coating prepared on the four carriers were the same:seeding layer showed amorphous hollow spherical structure and the protein-carrying layer showed a crystalline.The chemical groups of the four carriers were different,whereas the chemical groups' peaks of coating prepared on them were the same.EDAX indicated that the calcium phosphate ratio of coating changed to the volume of superatured calcium phosphate solution.XRD suggested the chemical component of seeding layer was amorphous calcium phosphate(ACP)and the protein-carrying layer was calcium-deficient hydroxyapatite(CDHA).Conclusions:1.The biphasic coating preparation method could form unanimous coatings on the polymers.2.The coatings bore the same morphologies and chemical groups.Part Two:The incorporation of protein into biomimetic calcium phosphate coating and its controlled releaseMaterials and methods:When the protein-carrying layer was prepared,bovine serum albumin was added into supersaturated calcium phosphate solution and incorporated into coating.The influence of incorporated protein on the coating chemical groups was examined by fourier-transform infrared spectroscopy.Using bovine serum albumin linked fluoresceine isothiocyanate(FITC) as indicater to gauge the in vitro release kinetics of incorporated protein.The coating was labeled by dual-fluorescence(rhodamine to indicate the location of seeding layer and FITCBSA to indicate the location of protein-carrying layer),the micro-location of biphasic coatings and the distribution of incorporated proteins were examined by laser confocal scanning microscopy(LCSM).Results:The incorporation of protein doesn't change the position of characteristic peaks of coating.On position of amino I,the peaks of coating and the amino I of protein overlapped and shifted to a higher position.The release of the incorporated protein could last over 35 days.The release patterns of incorporated protein on the four polymers were the same,which contains the initial burst release and the following slow release.The burst release kinetics was associated with the geometries of carriers:the release rate from fibrous polymers was higher than the porous polymers.The release kinetics of slow release was associated with the dissolution of coating, its rates were the same from the four polymers.LSCM showed the two-layer structure of the biphasic coating along the surface of polymers.The incorporated protein distributed homogenously within the protein-carrying layer instead of adsorption into polymer.Conclusions:1.Protein could be incorporated into coatings.2.The controlled release of incorporated proteins could last over 5 weeks.3.The release patterns of incorporated protein on the four polymers were the same.Part Three:The subcutaneous ectopie osteoinduction of BMP-2 incorporated biomimetic coatings on Polyactive,Ethicon,PLGAMaterials and methods:The osteoinductivity was conferred to polymer through the BMP-2 incorporation incorporation into coating under sterile condition.The BMP-2 loadings were examined by Enzyme-linked immunosorbant assay,ELISA.The prepared samples were implanted in rat's subcutaneous bone induction model for 5 weeks.Three control groups were set up: carrier+coating;carrier only;carrier+adsorbed BMP-2.The samples were retrieved after 5 weeks,fixed,embedded in methyl methacrylate(MMA),hard tissue slided,through random systematic sampling and stereologically analyzed.The volume and distribution of newly formed bone were examined.The coated carriers were also directly coated,slided and stereologically analyzed with the parameter of surface area density.Results:New bone could be found in carrier+coating+incorporated BMP-2 group and carrier+adsorbed BMP-2 group,whreas none could be found in carrier only group and the carrier+coating group.For each polymer,the bone volume of carrier+coating+incorporated BMP-2 group was higher than the corresponding carrier+adsorbed BMP-2.The BMP-2 osteoinductive efficacy of carrier+coating+incorporated BMP-2 group was also higher than that of polymer+adsorbed BMP-2 group.The bone volume densities of carrier+coating+incorporated BMP-2 at 5 weeks were proportional to the surface area density of polymer at time 0.Conclusions:1.The incorporated BMP-2 could maintain its bioactivities and induced bone formation in rat's subcutaneous ectopic bone induction model.2.The osteoinductive efficacy of incorporated BMP-2 was higher than the adsorbed BMP-2.3.The surface area density of polymer at time 0 was the main modulating factor on osteoinductive activities.Part Four:The influence of BMP-2 incorporated coatings on Polyaetive,Ethieon,PLGA on their biodegradability and the bioeompatibilityMaterials and methods:The samples retrieved from the rat's ectopic osteoindution model were analyzed through the stereological methology.The volume of Foreign-body giant cell(FBGC)was used to indicate the influence of BMP-2 incorporated biomimetic coating on the biocompatibility of polymers. The remaining polymer volumes were analyzed to indicate the influence of BMP-2 incorporated biomimetic coating on the biodegradability of polymer.The distribution and volume of fat tissue were used to indicate the growth character of fat tissue in the process of osteoinduction.Sub-capsular soft tissue in groups of polymer was also analyzed to thoroughly evaluate the bio-function of the functionalized polymers. Results:No difference could be found between the remaining polyactive volumes of different groups at 5 weeks and that of time 0.In different groups of collagen,Ethicon and PLGA,the remaining polymer volume of carrier+coating group was the highest;the remaining polymer volumes of polymer only group and the carrier+adsorbed BMP-2 group were similar; compared to the group of carrier+coating,the remaining polymer volume of carrier+coating+BMP-2 group decreased with or without stastic significance.The subcapsular total tissue volume of carrier+coating+BMP-2 group was higher than the other three groups.Except the two totally degraded groups of collagen,the volume density of foreignbody giant cell of carrier+coating+BMP-2 group of each polymer was the lowest and that of carrier+coating group was the highest.The newly formed fat tissue grew accompanying the bone tissue.In carrier+coating+BMP-2 group,the volume of newly formed fat tissue was significantly higher than polymer only group and polymer+coating group.Conclusions:1.The biodegradation of polymer was the summary results of multi-factors.The biodegradability of polymer was the determing factor,on the basis of which,incorporated BMP-2 could accelerate the degradation of polymer and the coating could protect the polymer from degradation.2.The BMP-2 incorporated biomimetic coating could favor the osteoinductive activities through attenuating the foreign-body giant cell response.