Topography On PDMS Affects β-catenin To Promote Osteogenesis Through Autophagy

Objective:Class II malocclusion is a common type of orthodontic malocclusion in clinical practice.The main manifestations are that the molars of the patients are distally related,the anterior teeth are deeply overlaid,and the mandible is retracted.Because the sagittal relationship of such patients is not adjusted,severe cases can lead to temporomandibular joint osteoarthritis(TMJ OA).Condylar joint reconstruction is a commonly used surgical method for patients with severe TMJ OA.For the healing of condyle bone defect,one of the treatment methods in bone tissue engineering is the replacement of artificial bone materials.Polydimethylsiloxane(PDMS)is a polymer material that can well mimic the extracellular matrix.Its topography drives the change of cell morphology,which greatly affects the cell biology process,especially in cell differentiation.It is known that the different dimensional surface topography arranged on the PDMS substrate will affect the osteogenic differentiation ability,but the osteogenic signal transduction related to the surface topography is not fully understood.The topography of the PDMS substrate surface is related to the function of β-cateninguided osteogenic transcription factors in the nucleus and autophagy in the cytoplasm.Autophagy is an important physiological mechanism for achieving the self-stability of osteoblasts.Recent studies have shown that autophagy has a significant regulatory role in osteoblast self-renewal and differentiation.However,the role of autophagy in the effect of different dimensional topography of PDMS substrate surface on the osteogenic differentiation ability of osteoblast is still unclear.This study explores the effect of PDMS substrate surface topography on the autophagy and osteogenic differentiation of osteoblasts and explores the response of osteoblast to topography and the underlying mechanism.Seeking to reveal the signal transduction in the regulation of osteogenesis by the topological topography of different dimensions on the surface,laying a theoretical foundation for material interface modification and the development of new bone tissue engineering materials.Methods:In this study,we mainly explored the role of autophagy in the influence of different topologies of PDMS substrate surface on osteogenic differentiation.Plasma etching technology is used to fabricate silicon wafer templates with different dimensions,and the PDMS substrate surface of different dimensions is re-engraved.The different dimensions of the prepared PDMS substrate are examined by atomic force microscope,and the osteoblast precursor cells MC3T3-E1 are cultured on the substrate surface of different dimensions.The morphology of MC3T3-E1 cultured on different topologies was observed by an inverted microscope,and the proliferation of MC3T3-E1 cultured on different topologies was detected by the CCK-8 kit.Detection of osteogenic differentiation ability of MC3T3-E1 on PDMS with different dimensions by immunofluorescence staining,alizarin red staining,q RT-PCR,Western blot and other methods;by constructing Super8 x TOPFlash plasmid and Super8 x FOPFlash plasmid,immunofluorescence staining,Western blot and other methods were used to detect the expression changes of β-catenin in MC3T3-E1;the protein expression of autophagy-related genes p62 and LC3II/I were detected by Western blot;transfection by si RNA technology and other methods analyze the correlation between autophagy and β-catenin and then prove its relationship with osteogenic differentiation.Results:1.Atomic force microscopy detects the topological morphology of the prepared PMDS substrate surface,and proves that the topological morphology of different dimensions is prepared: Flat,W0.5,W3,W10,W27(Wavelength W: μm)2.The adhesion and proliferation of MC3T3-E1 cells were confirmed to be affected by the topography of the PDMS substrate surface in different dimensions.Doublelabelled fluorescent staining of actin(cytoskeleton)and nucleus showed that MC3T3-E1 adhered better to W3.CCK-8 analysis proved that the substrates with different dimensions of topological morphology still support cell proliferation after 72 hours of culture,especially the number of cells on the surface of W10 is more than that on other surfaces.3.Different dimensions of PDMS substrate surface have different effects on the osteogenic differentiation of MC3T3-E1 cells.The results of ALP immunofluorescence staining and RUNX2,OSX m RNA and protein expression indicate that MC3T3-E1 cells on W27 substrate have the weakest osteogenic differentiation ability.4.The osteogenic differentiation of MC3T3-E1 cells is related to β-catenin entering the nucleus and activating Wnt signalling.Luciferase gene report analysis showed that β-catenin transcription activity increased.Western blot and immunofluorescence staining showed that the W3 basal group had the highest content of β-catenin into the nucleus.5.Different dimensions of PDMS substrate surface have different effects on the autophagy of MC3T3-E1 cells.Western blot and GFP-LC3 showed that the level of autophagy in the W3 basal group was higher.6.MC3T3-E1 cell autophagy affects osteogenic differentiation by regulating β-catenin.When the W3 basal group was added with the autophagy inhibitor CQ,the β-catenin non-silent group had a lower nuclear content,but it was higher than the β-catenin silent group,and the osteogenic differentiation ability was reduced.After the agonist RAPA was added,the β-catenin gene was not silenced and the nuclear content increased,and the osteogenic differentiation ability increased.Conclusions:This study determined that the topological morphology of different dimensions has an impact on the osteogenic differentiation of osteoblasts.In particular,W3 promotes β-catenin to enter the nucleus and thus affects the osteogenic differentiation of osteoblasts.W3-induced autophagy is an important reason for promoting β-catenin to enter the nucleus.This study reveals the potential mechanism of osteoblast differentiation mediated by topological morphology and provides ideas for improving the surface modification of biomaterials.These insights help us to improve our understanding of the interaction between cell osteogenic differentiation and topography,and provide new theories for the application of bone tissue engineering and regenerative medicine.