| BackgroundTitanium metal scaffold has been widely applied in clinic because of its light quality, good biocompatibility and resistant corrosion. Porous titanium showed advantages among massive bone defect, bone joint prosthesis fixation, femoral head necrosis bracket, osteoporosis implants, which can enhance ingrowth into the pores to achieve better fixed effects. However bio-inert titanium itself is not conducive into cells and tissue growth. Researches before focused on modification of porous titanium such as surface coating and composite sustained-release growth factor microspheres. But cells in the stent are still stuck in the two-dimensional growth pattern. In this study, we are committed to creating a three-dimensional growth model in porous titanium, which is suitable for cell and tissue.Objective1. Preparation of porous titanium metal with similar extracellular structure and the study of its characterization and biological activity.2. Detecting of osteogenic ability and biocompatibility of the composite scaffold.3. Tissue ingrowth of complex scaffold in vivo study.Methods1. According to micro-CT data and tissue anatomical measurement, internal porous titanium stent suitable for cell culture and animal implantation were prepared using 3D printing technology. Using gelatin thermal crosslinking method, porous gelatin scaffold was constructed in the pores with platelet adhered on gelatin surface to mimic the natural extracellular matrix.2. MC3T3 cells were seeded onto the composite scaffolds. CCK-8 cytotoxicity test was used in the cytotoxicity of the composite scaffold. The expression of alkaline phosphatase (ALP) and type I collagen (Collagen) cells were detected and the osteogenic ability of the composite scaffold was verified by the expression of alkaline phosphatase and collagen I.3. The model of skull defect and necrosis of femoral head were established. By micro-CT scanning and pathological tissue section method, the tissue repair and the internal structure of porous titanium stent were verified.Results1. Through thermal crosslinking method, gelatin cross-linked scaffold can be effective created in the 3D printing porous titanium. Scanning electron microscopy found that gelatin pore size ranged between 100um to 300um and platelets can also be adhered to the gelatin surface. Cytokines release assay presented that composite scaffold could stably and effectively release growth factors at least 21 days.2. MC3T3 was seeded into the composite scaffold. CCK-8 experiment showed that the inner cells of the composite scaffolds grew rapidly with good biocompatibility. The expression of osteoblasts was detected at multiple time points, and the secretion of alkaline phosphatase and type I collagen cell supernatant was significantly increased compared with that in control group (p<0.05).3. The New Zealand rabbit model of skull defect and femoral head necrosis were established successfully. After implantation of the composite scaffold, micro-CT showed that the experimental group had a significant difference in the skull defect and the defect of the femoral head. Meanwhile there was a significant difference in bone volume between the experimental group and other group (p<0.05). The hard tissue sections of the skull defect were found to be effective in the formation of mature bone and new blood vessel.ConclusionCross-linked gelatins with surface adhered platelets were constructed in3D printing porous titanium stent mimicking extracellular matrix, which could be effective in low dose and long-term sustained release growth factors. Composite scaffold has the ability of osteogenesis, which can effectively repair large bone defect and necrosis of femoral head. |
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