| Objectives:The range of tooth movement in orthodontic treatment is limited by the thickness of alveolar bone.Thin alveolar bone has a great risk of bone fenestration and dehiscence in orthodontic treatment.Subperiosteal injection is a minimally invasive method for bone augmentation.Laponite(Lap),an innovative inorganic nanomaterial,is able to promotes bone differentiation in the absence of exogenous growth factors.This study aims to explore a minimally invasive bone augmentation method through Lap composite hydrogel subperiosteal injection.Methods:1.The Lap composite hydrogels were prepared by solution-gel method,and the Lap contents were 0%,0.05%,0.1%,and 0.2%,respectively.Material characterization and detection for each group of composite hydrogels were proceeded.2.Rat cranial periosteum-derived mesenchymal stromal cells(P-MSCs)were isolated and cultured by enzyme digestion method.P-MSCs were co-cultured with Lap composite hydrogel extracts from each group respectively.The cytocompatibility of Lap composite hydrogel was evaluated by fluorescence staining of live/dead cells and CCK8 assay.The effects of Lap composite hydrogel on osteogenic differentiation were evaluated by several methods,including alkaline phosphatase staining&quantitative analysis,alizarin red staining and osteogenic related genes(ALP,COL1A1)expression level.Therefore,the optimal Lap concentration could be selected.3.The subperiosteal hydrogel injection model over scull was established in rats.A negative control group(NC group),Lap group and a positive control group(BMP-2 group)were set.The skull samples were harvested at 4,8,and 12 weeks after surgery,respectively.The effect of bone augmentation through Lap composite hydrogel subperiosteal injection was evaluated by micro-CT,Hematoxylin-Eosin staining,Masson’s trichrome staining,osteocalcin(OCN)immunohistochemical staining and tartrate-resistant acid phosphatase(TRAP)staining.Results:1.The Lap composite hydrogel was successfully prepared by sol-gel method.Fourier transform infrared spectroscopy(FTIR)shows that different components in the composite gel were connected by nonchemical bonding.Along with the increase of Lap content in the composite gel,the pore diameter of the freeze-dried scaffold enlarged gradually.The swelling property decreased and the degradation property increased(P<0.05),however,no significant difference was observed in compression property(P>0.05).2.P-MSCs were isolated and cultured successfully.Lap composite hydrogel showed good cytocompatibility.0.1%Lap hydrogel group had better performance than other groups in alkaline phosphatase activity and osteogenic gene expression tests(P<0.05).Obvious mineralized nodules were found in no group by alizarin red staining.0.1%Lap composite hydrogel has relatively better performance in promoting bone differentiation than others.3.After subperiosteal injection of composite hydrogel over the skull,new bone formed under the periosteum which achieved minimally invasive bone augmentation.From week 4 to week 12,the area of new bone formation increased in each group(P<0.05).At week 12,the bone mass of Lap group and BMP-2 group was significantly higher than that of NC group(P<0.05).OCN expression was found in the new bone formation area but not in the old bone tissue.TRAP positive cells were observed near the gel injection area and the inner side of the bone marrow cavity.Conclusions:The addition of Lap changed the material properties of the composite hydrogel.0.1%Lap composite hydrogel performed a favorable ability to promote bone differentiation in vitro.The subperiosteal injection of Lap composite hydrogel could effectively achieve bone augmentation. |
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