Highly Open Porous Gelatin Microspheres With MC3T3-E1 Cells For Repair Of Skull Defects Study

BackgroundSkull defect caused by trauma surgery,has always been a clinical problem of the surgeon.At present,the commonly used materials for skull defect repair include autologous bone,allogeneic bone,xenogeneic bone,plexiglass,bone cement,titanium mesh and titanium alloy,etc.Autologous bone grafting is still considered the gold standard for skull defect repair.However,these materials are not ideal due to the limited availability of materials,secondary donor damage,immune rejection,susceptibility to infection,complex preparation processes,high manufacturing costs,and poor plasticity.Tissue engineering provides a new way to repair skull defects,and cell carriers have always been a research hotspot in the field of tissue engineering.MC3T3-E1 is a commonly used model cell for bone tissue engineering research because of its simple culture,fast proliferation and stable osteogenic ability.ObjectiveBy combining gelatin microspheres,porous gelatin microspheres,HA-gelatin composite porous microspheres with MC3T3-E1 cells,and transplanting them into nude mice subcutaneously and SD rat skull defect models,it was verified that the cellular carrier porous microsphere scaffolds can be used in rats.Osteogenesis in vivo and in vitro,combined with paraffin sections,gene expression,ALP detection,quantitative detection of type I collagen,quantitative detection of calcium,and qualitative and quantitative analysis of it,provide a reference for the study of skull defect repair.Method1、Using collagenase to degrade microspheres in vitro;2、Perform cell proliferation experiments on the cells in the microspheres;3、Cell culture;4、Prepare scaffolds of CCG-MSs,HOPG-MSs and HA-OPG-MSs loaded with cells,check whether they are scaffolds,stain actin filaments,and evaluate CCG-MSs,HA-OPG-MSs and HOPG-MSs scaffolds through the F-actin staining kit;5、Analysis of cell viability;6、Quantitative analysis of the expression of type Ⅰ collagen,alkaline phosphatase activity,and calcium content;7、Hematoxylin is used to stain the nucleus inside the microsphere.H&E and immunohistochemistry were used to stain the scaffolds collected in the first and sixth weeks in vivo,and the in vitro scaffolds on day 14 and the in vivo scaffolds on week 1were stained with Alizarin Red S.In order to perform immunohistochemical analysis,the samples in the body for 6 weeks are decalcified,and then immunohistochemical analysis is performed with other samples;8、The in-vivo CT imaging was performed on the 6th and 12 th week using a high-resolution CT scanner.Then the image is three-dimensional imaging.ResultsGelatin has been employed to prepare highly open porous microspheres with particle sizes of 100–300 μm.Highly open porous gelatin microspheres(HOPG-MSs)have 2–70 μm of open pores and interconnected paths.HOPG-MSs has the advantage of MSs and scaffolds,and could be used as a carrier for osteoblast transplantation.In addition to convenient transplantation into defective tissue and in contrast to the poor performances of covalently crosslinked gelatin microspheres(CCG-MSs)and traditional open porous gelatin/hydroxyapatite composite microspheres(HA-OPG-MSs),HOPG-MSs present suitable surface pores and interconnected passages,thereby promoting cell adhesion and cell proliferation in vitro,and improving the differentiation of mouse embryonic osteoblast precursor cells(MC3T3-E1 cells).HOPG-MSs also greatly promote osteoblast regeneration compared with traditional CCG-MSs and HA-OPG-MSs in ectopic bone formation mouse models.HOPG-MSs can not only absorb nutrients from outside the scaffold but also protect cells during transplantation so that more live cells are allowed to proliferate and migrate to the damaged tissue.ConclusionIn conclusion,HOPG-MSs can greatly improve osteogenic differentiation of MC3T3-E1 and up-regulate biological markers during osteogenesis.These results may have significant implications for osteogenesis and bone generation in the repair of skull defects.However,further studies are needed to evaluate the therapeutic potential of cell-laden 3D highly porous microsphere scaffolds.These results provide new insights into the use of controlled degradable cell-laden HOPG-MSs as a scaffold to promote osteogenesis in further trials of skull defect repair.