| Background and Objective:Facing the challenge of implant surgery with insufficient remaining bone in the maxillary posterior region,maxillary sinus floor elevation is considered an effective way to address this challenge by filling the space formed by the original sinus floor and the elevated sinus floor mucosa with bone graft material to increase the available bone height.Recent clinical studies have found that elevation of the maxillary sinus mucosa alone without the implantation of any bone replacement material can achieve an increase in intra-sinus bone height,suggesting that intra-sinus osteogenesis is achieved by the osteogenic microenvironment formed by the enclosed space between the elevated maxillary sinus mucosa and the primitive bone wall.Current studies have shown that the maxillary sinus mucosa has MSCs that express various MSC surface markers including Stromal cell antigen-1(Stro-1)and have the ability to differentiate in multiple directions,called Maxillary Sinus Membrane-derived Mesenchymal Stem Cells(MSMCs).derived Mesenchymal Stem Cells(MSMSCs),which can express a variety of osteogenic related proteins including Alkaline Phosphatase(ALP)under osteogenesis induction.Neural Epidermal Growth Factor-like 1(NELL-1)protein is a novel osteogenic secretory protein that plays an important role in the osteogenesis process.NELL-1protein is preferentially expressed in tissues of neural crest origin and therefore has specific effects on the development and regeneration of craniofacial tissues.NELL-1is also a regulator of the Wnt/β-catenin signaling pathway,which increases nuclear accumulation of β-catenin by activating the Wnt/β-catenin pathway,thereby stimulating Runt-related transcription factor 2(Runt-2).NELL-1 has been shown to promote the differentiation of osteoblasts and the bone regeneration process.The effect of NELL-1 on osteogenic differentiation of various stem cells such as adipose mesenchymal stem cells and periodontal stem cells has been studied,therefore it is hypothesized that NELL-1 may also have an important effect on osteogenic differentiation of human MSMSCs and its mechanism.The purpose of this study was to investigate the effect of NELL-1 on the biological behavior of human MSMSCs through in vivo and ex vivo experiments,and to further investigate whether NELL-1 can regulate the osteogenic differentiation of human MSMSCs through the Wnt/β-catenin pathway,thus adding a theoretical basis for the elucidation of the osteogenic mechanism of human MSMSCs,and providing a new idea for the application of NELL-1 protein in the clinical treatment of bone volume deficiency in the maxillary posterior region.The results showed that the osteogenesis of human MSMSCs is regulated by the catenin pathway.Methods:1.In vitro isolation and identification of human MSMSCs The cells were isolated and cultured using enzyme digestion combined with tissue block apposition method,microscope observe the cell morphology and growth pattern,flow cytometry to identify their stem cell surface markers,and flow cytometry to detect their multidirectional differentiation potential using alizarin red staining to evaluate their osteogenic differentiation potential,oil red 0 staining to evaluate their lipogenic differentiation potential,and alizarin blue staining to evaluate their chondrogenic differentiation potential,respectively.The chondrogenic differentiation potential was evaluated by alizarin blue staining.2.In vitro study of the effect of NELL-1 protein on the biological behavior of human MSCs using NELL-1 protein(10,100,1000ng/m L)to induce human MSCs,crystalline violet staining to observe cell morphology and proliferation pattern,CCK-8 assay to detect cell proliferation,live-dead cell staining to detect cell viability;ALP staining and activity to detect changes in osteogenic differentiation ability of cells The m RNA expression of osteogenic factors was detected by quantitative Real-Time Polymerase Chain Reaction(q RT-PCR),and the expression of osteogenic and pathway-related proteins was detected by immunofluorescence staining and Western Blot.3.In vivo study on the promotion of osteogenesis in maxillary sinus by NELL-1protein.A rabbit maxillary sinus floor elevation model was constructed and different grafts were implanted in the maxillary sinus,and the specimens were taken at 8 and12 w after surgery.HE staining and Masson staining were used to evaluate the osteogenic effect of the implants in different groups and their biological safety.Results and Conclusion:1.Human MSMSCs were obtained by enzymatic digestion combined with tissue block apposition method,and the purified MSMSCs were mostly long shuttle-shaped,closely arranged with each other in a swirling and fish swarm shape,positive for stem cell surface markers CD90,CD73 and CD105,and negative for CD34,CD45 and HLA-DR,with osteogenic,lipogenic and chondrogenic differentiation potential.2.In vitro experiments showed that NELL-1 protein had a pro-proliferative effect on human MSMSCs;NELL-1 protein promoted the ALP activity and increased the expression of m RNA and protein such as β-catenin,Runx-2 and OCN in human MSMSCs;immunofluorescence and Western Blot results showed that after the effect of antagonist Dkk1 The immunofluorescence and Western Blot results showed that the expression of β-catenin was significantly reduced in both NELL-1 protein group and control cells after the effect of antagonist Dkk1.The above results confirmed that NELL-1 protein could improve the osteogenic differentiation of human MSMSCs in vitro,which might be achieved by upregulating the expression of Wnt/βcatenin signaling pathway.3.In vivo experiments demonstrated that in the rabbit maxillary sinus floor elevation model,Micro-CT results showed higher bone volume and bone density of new bone in the experimental group,and HE and Masson staining results showed that the bone tissue in this group was more mature and had good biosafety.The above results confirmed that NELL-1 protein could significantly promote bone regeneration in rabbit maxillary sinus floor. |
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