| Research background:At present,the schemes relied on autologous bone graft are the "gold standard" for the repair and treatment of critical bone defects,but these treatment schemes easily lead to complications in the donor site and are limited by the limited donor site.Injectable hydrogel provides a new therapeutic option for bone repair.It can not only integrate the advantageous functions of each component through the selection of different components,but also load growth factors,cells,and drugs into it to enrich it in the bone defect area and play a role in promoting bone repair.Research purposes:The injectable hydrogel carrier was constructed with low molecular weight sodium alginate,hydroxyapatite and type Ⅰ collagen,and used to carry bone marrow mesenchymal stem cells(BM-MSCs)expanded in vitro.The injectable hydrogel is filled to the bone defect by injection.Through the slow degradation of the composite hydrogel and the osteogenic effect of the BM-MSCs loaded therein,the local conditions of the critical bone defect are improved,and the bone regeneration in the bone defect is promoted,thereby completing the bone defect repair.To verify the promotion effect of injectable BM-MSCs/ hydrogel composite system on critical-sized bone defect repair and explore the effect of collagen content in composite hydrogel on the osteogenesis of the composite system.This study will provide a reference for clinical stem cell treatment of large segmental bone defects and defective nonunion.Research methods:1.Nano-hydroxyapatite was prepared by aqueous deposited polymerization,and then mixed with collagen solution and sodium alginate powder to prepare alginate/hydroxyapatite/collagen composite hydrogel.The surface morphology and microporous structure characteristics of the material were observed by scanning electron microscope.2.The degradation performance of the composite hydrogel in animals was studied by subcutaneous injection of the composite hydrogel into rats.3.In vitro study,the biocompatibility of the composite system was verified by cell activity detection(CCK-8 method)and live/dead cell staining after coculture with BMMSCs,and the morphology of cells loaded in the composite hydrogel was observed by phalloidin /DAPI staining.4.The effects of the composite hydrogel on the osteogenic differentiation of BMMSCs were verified by real-time quantitative polymerase chain reaction through alkaline phosphatase color reaction and alkaline phosphatase activity analysis.5.In vivo study,this work established the New Zealand White rabbit model of critical-sized bone defect by radial osteotomy and divided it into five groups: a simple critical-sized bone defect group and treatment groups loaded with BM-MSCs composite hydrogel,wherein the collagen content in the composite hydrogel was different.The group with simple critical-sized bone defect was the negative control group.The X-ray observation,Micro-CT scanning and reconstruction were conducted to analyze the effect of BM-MSCs/ hydrogel composite system on bone repair in the environment of critical-sized bone defects.Findings:1.In this study,alginate/hydroxyapatite/collagen composite hydrogel was successfully prepared.The results of scanning electron microscope showed that the composite hydrogel had a three-dimensional network structure similar to the natural extracellular matrix,which could be used as a temporary external environment for cells to support and produce extracellular matrix.2.During the 61 days of experimental observation,the composite hydrogel can degrade slowly in rats,and it will not cause inflammatory reaction of the surrounding tissues.3.In vitro cell experiment,the living/dead cell staining experiment,phalloidin/DAPI(4’,6-diamidino-2-phenylindole,4’,6-diamidino-2-phenylindole)staining experiment and cell viability test showed that all the composite hydrogels had good cell compatibility and could maintain the normal morphology and viability of cells.4.Compared with the group without collagen,the composite hydrogel containing collagen promoted the osteogenic differentiation of BM-MSCs,and up-regulated the expressions of alkaline phosphatase and osteocalcin.5.In vivo experiment,the critical-sized radius defect of New Zealand white rabbits was successfully established by osteotomy.Micro-CT results after 8 weeks of implantation of the composite hydrogel-cell system in each group showed that compared with the negative control group,the composite hydrogel group loaded with BM-MSCs promoted bone regeneration in the radius defect,and this promotion was most significant at a collagen content of 25mg/m L.Research conclusions:Collagen can promote the osteogenic differentiation of BM-MSCs loaded in composite hydrogel,and the composite hydrogel loaded with BM-MSCs can promote the repair of critical bone defect in radius of New Zealand white rabbits. |
