BMP-2 Loaded Microspheres Incorporated Nanofibrous Scaffold For Bone Tissue Engineering

As one of the most challenging problem in medicine area,the repair and functional reconstruction of bone defect has always been the hotspot of scientific research.The traditional method to restore bone defect includes autograft and allograft transplantation,but the source of autologous transplantation is limited,and autologous transplantation also faces the extra cost of anesthesia surgery and pain in clinic.Allograft is also restricted by the immunological rejection.Alternatively,by integrating bioactive factors with scaffold to promote cell growth and differentiation,the bone tissue engineering provides new sights for bone repair.Bone morphogenetic proteins are widely used as the osteogenic growth factors for bone tissue engineering.However,the directly encapsulation of BMP into scaffold would easily impair their bioactivity.Fortunately,the problem would be effectively circumvented by introducing microcarriers into the polymer scaffolds,which could not only maintain the bioactivity of growth factors,but also facilitate the long-term release of factors for enhanced osteogenesis.Based on the forementioned principles,in this study,we established a biomimetic bone composite scaffold,in which the BMP-2 loaded PLGA microspheres were incorporated into PLLA/PLGA/PCL nanofiber scaffold for bone tissue engineering.We investigated the physicochemical properties of the composite scaffold and its ability to induce osteogenesis in vitro and in vivo.The main contents are as follows:(1)The preparation and characterization of BMP-2 loaded PLGA microspheres: First of all,we prepared PLGA microspheres using the double emulsion method(W/O/W)and studied the morphology and particle size of microspheres by scanning electron microscope(SEM),then we studied the release profile of PLGA microspheres.The SEM results showed that the size of most microspheres was between 20 to 39 ?m,and the surface of PLGA microspheres was porous.Bovine serum albumin(BSA)was selected as model drug and loaded into PLGA microspheres to stimulate the release profile of BMP-2 loaded PLGA microspheres.The results confirmed that protein molecules could slowly release from PLGA microspheres.Therefore,the prepared BMP-2 loaded PLGA microspheres probably possess the biological activity and great potential for bone tissue engineering.(2)We prepared the PLLA/PLGA/PCL nanofibrous scaffold by thermal induced phase separation(TIPS),the PLGA microspheres was then combined with nanofibrous scaffold to obtain PLGA-PLLA/PLGA/PCL composite scaffold: First,the PLGA microspheres were evenly dispersed in the mixture solution of n-hexane and tetrahydrofuran(THF).THF would slightly dissolve the surface of nanofiber scaffold and PLGA microspheres to bind them together.Through the morphology observation of PLLA/PLGA/PCL nanofibrous scaffold,the phase separated scaffold exhibited a porous network structure with interconnected micropores and continuous nanofibrous architecture.The SEM images also confirmed that the PLGA microspheres can disperse on the surface and channel of the nanofibrous scaffold.The obtained composite scaffolds can still maintain the three dimensional porous and nanofibrous structure.From the in vitro degradation study of PLGA-PLLA/PLGA/PCL composite scaffolds,it can be concluded that the composite scaffolds could degrade in simulated body fluid environment.With the structural decomposition of the composite scaffold,the composite scaffold is probably conducive to promote cell growth and tissue regeneration.(3)Next,we evaluated the biological activity of composite scaffolds in terms of biocompatibility,induced osteogenesis in vitro and osteogenesis ability in vivo.Results showed that the composite scaffolds have good biocompatibility and the porous structure,which make them suitable for osteoblast adhesion and proliferation.The loading of BMP-2 growth factor can significantly promote the cell growth,differentiation and mineralization.The results of animal tests showed that the released BMP-2 could continuously exert the osteogenesis function and promote new bone matrix secretion,thus accelerating the bone matrix mineralization and formation of new bone.Overall,the BMP-2 loaded PLGA microspheres were successfully incorporated into PLLA/PLGA/PCL nanofibrous scaffold to construct a bioactive composite scaffold for bone repair application.The composite scaffold can promote osteoblast adhesion and proliferation as well as osteoblast differentiation in vitro.The in vivo ectopic osteogenesis and bone defect repair in situ were also demonstrated.Therefore,this prepared composite scaffold may be a suitable alternative for bone tissue engineering applications.