Fabrication Of Structurally And Functionally Optimized Bone Repair Materials And Investigation Of Their Biological Properties

At present,bone tissue repair materials used in clinic have prominent problems such as low bioactivity,slow bone regeneration,and poor repair quality.By optimizing their chemical composition,physical structure and biological function,the effect of clinical bone repair can be improved.This paper focused on the goal of"accelerating bone tissue rapid repair".By simulating the three-dimensional multipolar structure of natural bone and the self-healing process of bone trauma,multiple drugs controlled release system was introduced into the scaffold of tissue engineering to construct artificial bone substitute materials with special biological functions,realizing multistage loading and multistage controlled release of bioactive factors or molecules so as to play a synergistic role between biological factors,thereby improving the bone regeneration ability of artificial bone substitute materials.The main contents include the following aspects:Since many complex physiological processes are controlled by multiple biomolecules,comprehensive regulation of bone tissue regeneration process may be more effectively achieved by administration of more than one type of biofactor.Thus,in the second chapter,we proposed a novel bone tissue engineering scaffold incorporating a multiple peptide-based drugs delivery vehicle for the accelerated bone regeneration.Pore-closed poly?lactic-co-glycolic acid??PLGA?microspheres with a surface structure of multilayer polyelectrolytes??Ha-Cs?₂-Hep-BMP-2-Hep-?Cs-Ha?₂?were prepared as the multi-barrier microcarriers for osteogenic growth peptide?OGP?and BMP-2 loading by a pore-closing process and a layer-by-layer?LBL?assembly technique,followed by immobilization on the surface of a highly interconnected porous hydroxyapatite?HA?scaffold.On the basis of such a construction,a sequential delivery of OGP and BMP-2 was unfolded in time in a coordinated manner through an orchestrated sequence of spatial changes,targeting different bone healing stages.The in vitro studies showed that the OGP release was very minimal?<11.7%?in the first 15 d but accelerated remarkably thereafter,while at least 56.3%of the BMP-2 payload had been released at this time and subsequent only marginal releasing.Additionally,the scaffolds carrying dual-biofactor exhibited a stronger ability to induce bone marrow mesenchymal stem cells?MSCs?differentiation toward osteoblasts than those incorporating OGP or BMP-2 alone and factor-free scaffolds,in terms of alkaline phosphatase?ALP?activity and osteogenic genes and proteins?Runx2,COL I,and OCN?expression.The results of in vitro cell culture demonstrated the roles of BMP-2 in osteogenic differentiation early as well as the effect of OGP on the accelerated proliferation and maturation of osteoblast precursors at a later stage.Further in vivo osteogenesis studies also revealed that the dual biofactor-loaded scaffold manifested the best repair efficacy due to a potential synergistic effect of BMP-2 and OGP.Taken together,our findings suggested that such a dual delivery system may provide a therapeutic strategy sequentially targeting multiple events or mechanisms during bone healing and was proved to be a promising therapeutic scaffold for future use in bone tissue regeneration.In the process of bone regeneration,relatively early biological events including inflammatory response,angiogenesis,or stem cell homing,help the accompanying target actions of cell differentiation and calcification.In the third chapter,we proposed a novel cell-guided tissue engineering system based on a surface-functionalized porous hydroxyapatite?HA?scaffolds with the ability to recruit cells and accelerate the differentiation of them along the osteoblastic lineage for optimizing large-sized bone defect repair.Inspired by microstructural properties of natural bone,HA scaffolds similar to the trabecular bone structure were prepared via a sugar sphere leaching technique,in which the inter-pore opening size was controllable.Dexamethasone?Dex?-loaded hydroxypropyl-?-cyclodextrin microspheres?Dex@CDMs?and stromal cell derived factor-1?SDF-1?were uniformly immobilized onto HA surface by a cross-linked alginate coating.The resulting scaffold?SDF-1/Dex@CDMs-HA?enabled the controlled dual-delivery of SDF-1 and Dex.In vitro cell culture assays showed that initially released SDF-1 markedly stimulated the migration of mesenchymal stem cells?MSCs?to the deep interior of the scaffold,providing abundant target cells for the function of Dex which was subsequently released.Osteogenic differentiation potential of these cells was also further facilitated via a synergistic action of SDF-1 and Dex.Additionally,in vivo studies demonstrated that the cell-guided system effectively improved the early cell recruitment and vascularization within the deep interior of scaffold and significantly accelerated the extensive formation of osteoid and mineralized tissue compared with controls.Accordingly,such a microsphere coating-decorated multifunctional scaffold shows a promising potential for cell-free bone tissue engineering applications.In clinical use,the titanium?Ti?based implants were still faced with the problem of loosening and failure of the implants caused by the low osseointegration rate and the implant infection.In the fourth chapter,a porous template packed with sugar spheres was used as a pore forming agent to prepare porous Ti scaffolds similar to natural trabecular bone structure.The method can accurately control macro-pore structure,including pore shape,size and interconnectivity.Vancomycin?Van?loaded albumin nanoparticles?Van-BNPs?were prepared by solvent extraction.Based on a modified biomimetic mineralization process and the adhesion mechanism of mussels,a silicon doped calcium phosphate?pSiCaP@Van-pBNPs/Pep?coating?pSiCaP@Van-pBNPs/Pep?was designed and constructed on the surface of three-dimensional metal titanium scaffold,and then cell adhesion peptide?GFOGER?and Van-BNPs were immobilized on this coating,simulating the microenvironment of the extracellular matrix?ECM?of natural bone matrix.In vitro and in vivo experiments showed that such pSiCaP@Van-pBNPs/Pep composite coating promoted cell adhesion,spreading,proliferation and osteogenic differentiation,and improved the ectopic osteogenesis ability of Ti scaffold.Antibacterial experiments showed that this biofunctional Ti scaffold exhibited excellent antibacterial activity,thereby reducing the risk of bacterial infection at the bone healing site.This study provides an effective strategy to enhance the bioactivity of metal implants.The physical crosslinked polyvinyl alcohol?PVA?gel,due to the small size of the internal pore,poor cell affinity and low biological activity,presents the problem that cells,tissues and blood vessels are difficult to migrate and grow into the material and PVA implants cannot be tightly bind to the surrounding tissues in vivo.In the fifth chapter,PVA and Cu doped HA powders were used as raw materials and wax balls were serve as pore forming agents to prepare a three-dimensional interconnecting PVA/CuHA composite scaffold with similar to natural trabecular bone structure and with a surface like ECM network structure.Based on the adhesion mechanism of mussels,the peptide-1?BFP-1?was immobilized on the surface of PVA/CuHA by polydopamine?pDA?coating to construct a biofunctional PVA/CuHA-pDA-pep scaffold.In vitro and in vivo experiments showed that the composite scaffold has good biocompatibility,osteoinductive activity,antibacterial activity and the ability to promote vascularization,and has a potential application in the repair of infected bone defects.