Effect Of Substrate Stiffness On The Morphology And Biological Behaviors Of Maxillary Schneiderian Sinus Membrane-Derived Stem Cells

Objectives:Maxillary sinus floor elevation is often used to solve the problem of insufficient bone mass in the posterior maxillary area during implant surgery.The procedure is briefly summarized as elevating the maxillary sinus membrane and implanting biomaterials between the membrane and the bony wall of sinus floor as needed.However,the principle of the technology remains controversial.At present,some scholars have proposed that there are stem cells in maxillary sinus membrane,which have the potential to induce osteogenic differentiation.Therefore,new bone during maxillary Schneiderian sinus elevation may be generated by MSSM-derived stem cells（maxillary Schneiderian sinus membranederived stem cells）.How MSSM-derived stem cells respond to the biomechanical properties of the environment is worthy of further exploration.Among various biophysical signals,matrix stiffness has been proved to affect the biological behavior and function of various cells through mechanical interactions,and the effects are different between different cells.At present,there is still a lack of relevant studies on the effect of stiffness on MSSM-derived stem cells.In this study,we intended to isolate and culture MSSM-derived stem cells and further explore the effects of stiffness on their morphology,adhesion,proliferation and osteogenic differentiation.Studying the biological behavior of MSSMderived stem cells and exploring how to enhance the osteogenic ability of these cells can provide a theoretical basis for clinical operation of maxillary sinus elevation and selection of proper biomaterials.Methods:1.Isolation,culture and identification of MSSM-derived stem cellsAfter isolating maxillary sinus membrane from Japanese white rabbits,the tissues were separated into single cells by enzyme digestion method and primary cell culture was performed.After the cells were cultured to 2-4 generations,the self-renewal ability was detected by cell sphere-forming experiment.Flow cytometry was used to identify surface markers.The osteogenic,adipogenic and chondrogenic differentiation abilities were evaluated by alizarin red,oil red and alcian blue staining.2.Effects of stiffness on morphology,adhesion and proliferation of MSSM-derived stem cellsThe cells were cultured on surfaces with different matrix stiffness,and their morphology was observed after fluorescence staining with 4’,6-diamidino-2-phenylindole（DAPI）and Fluorescein Isothiocyanate（FITC）labeled phalloidin.The adhesion behaviors of cells were observed after fluorescence staining of vinculin.CCK-8 was used to evaluate the proliferation of cells cultured on the different substrate at several time points.3.Effect of stiffness on osteogenic ability of MSSM-derived stem cells in vitroAfter the cells were cultured on the substrates of different stiffness for osteogenic induction,m RNA expression of alkaline phosphatase（ALP）,osteopontin（OPN）,runt-related transcription factor-2（RUNX-2）,bone morphogenetic protein-2（BMP-2）and collagen,type I,alpha 1（COL1A1）and OPN protein expression were detected by real-time quantitative polymerase chain reaction（q RT-PCR）and immunofluorescence at several time points.Results:1.The MSSM-derived cells were successfully isolated and cultured,which had the ability of self-renewal and the potential of osteogenic,adipogenic and chondrogenic differentiation.Mesenchymal associated antigens of cell surface,CD29,CD90 and CD44 were positive,while hematopoietic related markers CD34 and CD45 were negative.2.The cells were cultured on three substrates with stiffness of 13-16,48-53 and 62-68 k Pa respectively.The higher the stiffness,the more conducive to the extension of cells and the stronger the fluorescent expression of vinculin,and more conducive to cell proliferation.3.After the cells were cultured on three kinds of stiffness substrates for osteogenic induction and differentiation,the m RNA expression levels of ALP,OPN,RUNX-2,BMP-2 and COL1A1,and OPN protein expression increased with increase of stiffness Conclusions:The MSSM-derived cells extracted in this experiment were stem cells.In the stiffness range of 13 to 68 k Pa,the higher the stiffness,the more conducive to cell extension,adhesion and proliferation.In addition,stiffer substrates also promoted the osteogenic differentiation ability of MSSM-derived stem cells.