| Deficiency of the posterior maxillary teeth and maxillary bone in the maxillary posterior region can severely affect facial contours,as well as physiological functions such as speech,chewing,and swallowing.When the bone height in the posterior maxillary region is extremely insufficient,the difficulty of implant restoration is greatly increased.In the reconstruction of the maxillary bone in the posterior maxillary region,the maxillary sinus floor elevation bone grafting technique is usually used to meet the requirements of implantation and tooth crown restoration,but the osteogenesis efficiency in this area is a challenging issue.Clinically used artificial bone graft materials have a certain bone guidance function,but they have limitations in mobilizing the in-situ mesenchymal stem cells(MSCs)for osteogenic transformation,resulting in a low autogenous bone replacement rate,significant differences between new bone and natural autogenous bone,and a long bone formation cycle.An efficient in-situ autogenous bone regeneration therapy strategy needs to be designed based on the in-situ osteogenic mechanism of the maxillary sinus area.Recent studies have shown that the sinus floor bone wall is not the sole source of bone regeneration.The maxillary sinus membrane,known as the Schneiderian membrane(SM),also exhibits certain osteogenic potential.MSCs derived from the SM(SMMSCs)have been extracted and identified.However,the exact hierarchical location of SMMSCs in the SM and whether different types of SMMSCs exist within the SM are not yet recognized.SMMSCs derived from different tissue structures may exhibit different cellular biological behaviors and corresponding osteogenic characteristics.Obtaining a defined source of a single type of MSCs from human SM can help develop new therapeutic strategies for maxillary sinus bone regeneration,as well as serve as seed cells for preclinical research tools and bone tissue engineering biomaterials.Designing osteogenic materials that adapt to the specific characteristics of different SMMSCs will be a significant breakthrough in addressing the challenge of bone formation speed in the maxillary sinus region in the future.This study aims to explore whether there are multiple types of MSCs in the complete human SM,such as lamina propria layer-derived MSCs(LMSCs)and periosteum layer-derived MSCs(PMSCs),and to investigate whether various SMMSCs can be clearly separated by cell surface markers,so that the specific types of SMMSCs have potential application value for stem cell therapy.Meanwhile,whether different SMMSCs have different osteogenic properties during in vitro culture,and whether there are cell-cell interactions were evaluated to reveal whether there are cell communication-related mechanisms among various SMMSCs that are positioned adjacently in vivo to regulate each other’s osteogenic behaviors.In order to further explore the role of SMMSCs in the osteogenic microenvironment of the maxillary sinus and guide the improvement of the osteogenic potential of SM from the perspective of MSCs,this study plans to prepare specific site micron-sized spherical biomimetic mineralized light-induced double cross-linked methacrylated gelatin-fibrinogen hydrogel microspheres,fully simulating the extracellular matrix structure with collagen protein components,non-collagen protein components,and mineral deposition in early bone formation,thus deeply restoring the changes in osteogenic properties of SMMSCs on the mineralized matrix surface,as well as the cell communication between different SMMSCs.The scaffold material has a multifunctional cell contact surface for cell recruitment,proliferation,and osteogenic differentiation,and can provide sufficient space for cell migration and moderate material degradation rate,serving as an effective research tool for SMMSCs.Finally,this study aims to propose potential mechanisms for maxillary sinus in situ osteogenesis mediated by SMMSCs and explore whether SMMSCs can act as chemotactic factor delivery tools to promote the migration of surrounding environmental cells into the osteogenic space,thereby enhancing sinus bone regeneration levels,accelerating maxillary sinus osteogenesis process,and improving bone quality.The specific experiments are divided into four parts:In the first part of the study,MSCs derived from the lamina propria layer(LMSCs)and periosteum layer(PMSCs)of the human SM were isolated,extracted,and cultured in vitro,and standardized in vitro and in vivo identification of MSCs were performed,simultaneously conducting RNA sequencing analysis to discover the differences in gene expression levels between the two cells and further distinguish the two cells.In the second part of the study,the differential characteristics of the osteogenic direction of the two SMMSCs were evaluated in detail,according to the expression of osteogenic proteins of the two cells at different time points,and the osteogenic abilities of the two cells were analyzed.The development of a biomineralized extracellular matrix with rich collagen and non-collagenous protein components was designed to highly simulate the osteogenic microenvironment,and then the osteogenic characteristics of the two cells in this biomineralized matrix microenvironment were comprehensively analyzed in vitro and in vivo.In the third part of the study,the interaction between PMSCs and LMSCs was analyzed,specifically the cell recruitment and osteogenesis between the two types of stem cells.And comprehensive analysis of changes in cell recruitment and osteogenesis between the two types of SMMSCs in the biomineralized matrix microenvironment was performed in vitro and in vivo.RNA sequencing analysis was used to evaluate the specific mechanism pathway enrichment of this cell interaction,and western blot was used to validate the expression of the corresponding pathways,proposing the interaction mechanism pathway of PMSCs and LMSCs in the biomineralized matrix.In the fourth part of the study,an early bone formation animal model after MSFE of rabbits was designed using a biomineralized matrix,and the migration of PMSCs and LMSCs on the biomineralized matrix was evaluated in vivo,and the bone formation situation in the maxillary sinus at different time points was analyzed.The following conclusions were drawn from the above experiments:1.There are two types of MSCs in the SM of the human maxillary sinus,namely,LMSCs with CD171 positive and PMSCs with CD171 negative.2.LMSCs and PMSCs have different osteogenic potentials.Under two-dimensional culture,the osteogenic ability of PMSCs is significantly stronger than that of LMSCs.In the three-dimensional culture of biomimetic mineralized matrix,the osteogenic ability of the two types of cells was obvious,but the osteogenic ability of PMSCs was stronger than that of LMSCs.3.PMSCs have the recruiting ability for LMSCs.PMSCs attached to the surface of the biomineralized matrix further enhance the recruiting ability of LMSCs and promote the osteogenic action of LMSCs.This result is closely related to the enhancement of the PKC/FAK/PI3K/Akt pathway in both types of cells.The key factor for PMSCs to promote the biological function of LMSCs is SDF-1.4.In the maxillary sinus elevation bone formation model,it was found that there is synergistic osteogenesis mediated by intercellular recruitment between the two types of MSCs,thereby accelerating bone regeneration in the maxillary sinus region. |
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