Synergic Effects Of PDMS Substrate Stiffness And Topography On Osteogenic Differentiation Of Rat Bone Mesenchymal Stem Cells

Objective:In this study, we aim to investigate the synergic effects of polydimethylsiloxane (PDMS) substrate stiffness and topography on the morphology, proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs), and to make sure whether the substrate stiffness and topography can synergistically contribute to osteogenic differentiation, and to find the ideal range of stiffness and topography. To lay a theoretical foundation for the research and development of bone tissue engineering materials.Methods:1. Characterization of the stiffness and topography of human fenur bone and cell interface. The stiffness and topography of bone and cell interface were measured by atomic force microscope and scanning electron microscope, as a basis for the preparation of different stiffness and surface topography of PDMS substrate.2, Preparation of biomimetic PDMS substrate. Fabrication of silicon wafers with different ridges and grooves structure using electron beam lithography and plasma etching technique. PDMS substrates with different stiffness and topography were obtained by molding. Groups:group A hardness is 3.5 MPa, no ridges and grooves; group B hardness is 3.5 MPa, ridges and grooves width of 300 nm; group C hardness is 3.5 MPa, ridges and grooves width of 1800nm; group D hardness is 0.27 MPa, no ridges and grooves; Group E hardness is 0.27 MPa, ridges and grooves width of 300nm; Group F hardness is 0.27 MPa, ridges and grooves width of 1800 nm.3. The morphology, proliferation and osteogenic differentiation of rBMSCs were detected on PDMS substrates with different stiffness and topography. rBMSCs were cultured on the basis of the above groups PDMS substrates. Inverted fluorescence microscope was used to observe the morphology of rBMSCs. CCK-8 reagent was used to detect the proliferation of rBMSCs. Alkaline phosphatase (ALP) kit was used to detect the ALP activity of rBMSCs. Immunofluorescence technique was used to detect the expression of osteocalcin (OCN) and collagen I (COLI). QRT-PCR technique was used to detect the expression of Runx2 mRNA.Results:1. The stiffness and topography of the bone and cell interface, which is the stiffness and topography of the extracellular matrix. The diameter of collagen fibers that constitute the major components of the extracellular matrix was found to range from dozens of nanometers to several hundred nanometers. The average diameter of the three samples of collagen fibers was 289±118 nm,365±140 nm,198±98 nm respectively. The average hardness value of the three samples of trabecular bone was 0.93±0.09 MPa,3.58±0.14 MPa,2.44±0.20 MPa.2. The stiffness of the PDMS substrate was 3.5±0.16 MPa and 0.27±0.015 MPa, with a mass ratio of 5:1 and 33:1 mixed 184 pre polymer and 184 curing agent. Scanning electron microscopy was used to measure the diameter of PDMS substrate surface ridges and grooves structure, which were 300 nm and 1800nm, respectively.3. rBMSCs appeared to have little stress fibers on the 0.27 MPa PDMS substrates, apparent stress fibers form when cells were cultured on the 3.5 MPa PDMS substrates. Representative images of cells spreading on different substrate show that those cells on soft substrates spreading very little compared with the extent of spreading on stiff substrates. Under the condition of the same stiffness, rBMSCs on planar surfaces and 300nm ridge width,300nm groove width substrate exhibited a flattened and polygonal morphology as well as the largest average cell area compared to 1800nm ridge width,1800 nm groove width substrate, however we found that rBMSCs were elongated and aligned along patterns of grooves and ridges on 1800nm width substrate, which were spindly. At the same time point, when the substrate topography was identical, the proliferation rate of rBMSCs on the stiff substrate (3.5 MPa) was significantly higher than that of the soft substrate (0.27 MPa). However when the substrate stiffness was identical, the proliferation rate of rBMSCs have no significant difference on the different topography of the substrate. When the substrate stiffness was identical, the number of osteocalcin and collagen I secretion, ALP activity, Runx2 mRNA expression of rBMSCs on 300nm ridge width,300nm groove width substrate was significantly more than that of the planar surfaces and 1800nm ridge width,1800nm groove width substrate groups, when the substrate topography was identical, the number of osteocalcin and collagen I secretion, ALP activity, Runx2 mRNA expression of rBMSCs on the hard substrate (3.5 MPa) was higher than that of the soft substrate(0.27 MPa). rBMSCs cultured on substrate with stiffness of 3.5 MPa, 300nm ridge width,300 nm groove width showed greater proliferation, spreading, cytoskeleton arrangement, and OCN and COLI secretion, ALP activity, Runx2 mRNA expression were significantly increased as compared to cells cultured on other groups.Conclusions:1. Stiffness and topography of the substrate can affect the morphology of rBMSCs.2. Substrate stiffness promotes rBMSCs proliferation more obviously.3. Substrate stiffness and topography can synergistically promote osteogenic differentiation of rBMSCs, substrate with stiffness of 3.5 MPa,300 nm ridge width, 300 nm groove width promote the osteogenic differentiation trend more pronounced.4. The research findings not only help to understand the physical factors in the pathogenesis of certain diseases (such as osteoporosis), but also provide a theoretical basis for the development of new materials for bone tissue engineering.