Protein Adsorption Force Regulates The Responses Of Stem Cell To Substrates Tensile Strain

Protein adsorption on the surface of biomaterials is the first event happened when biomaterials are implanted into the human body,so that the adsorbed protein layer acts as a bridge between cells and biomaterials.After cells become specifically adhered onto the biomaterials,cell traction force（CTF）will be generated and then transmitted to biomaterials in a top-down mode of cell-protein interface→protein layer→protein-material interface→biomaterial,by which cells can sense and measure the mechanical properties of biomaterials.On the other hand,biomaterials are constantly experiencing various mechanical stretch strains in vivo and the mechanical stretch strain will be transmitted to cells in a bottom-up mode of materials→material-protein interface→protein layer→protein-cell interface to regulate cells behavior.The material-protein interface is a key mediator for CTF and stretch strain transmission in both two modes.We believe that the strength of the protein-material interface,that is,protein adsorption force(F_ad),should have vital roles in CTF and mechanical strain transmission.Although many studies have shown that F_ad can regulate the transmission of CTF and thus the response of cells to the mechanical properties of the substrates,there is a lack of evidence about the importance of F_ad in the transmission of substrate stretch strain and the regulation of cell response to substrate stretch strain.Herein,fibronectin（FN）as a protein model and rat mesenchymal stem cells（rMSCs）as a cell model were used in our study to investigate the regulation of F_ad to the transmission of substrate stretch strain and subsequent cell response to substrate stretch strain.The main works and conclusions are summarized as follows:（1）The regulation and quantification of F_ad:Self-assembled monolayers（SAMs）were used to construct-CH₃,-OH and-NH₂ SAMs on the surface of rigid poly（dimethyl siloxane）（PDMS）elastic membranes.The results from X-ray photoelectron spectroscopy（XPS）and static water contact angles verify the successful construction of various SAMs.Then the F_ad values of FN on various SAMs were detected by using a parallel plate flow chamber/microsphere technique and the results show that surface chemistry could regulate F_ad effectively,presenting a trend of-NH₂(recorded as F_max)>-CH₃(recorded as F_med)>>-OH(recorded as F_min).Furthermore,FN molecules were covalently bound to PDMS to simulate an extra-maximum F_ad of FN（recorded as Fmax⁺）.（2）The effect of F_ad on the transmission of CTF and stretch strain:In order to evaluate the effect of F_ad on CTF and stretch-strain transmission,immunofluorescence technique was used to observe reorganization or desorption of the adsorbed FN on various substrates when FN was subjected to pure CTF（static group）,pure mechanical stretching（cell-free group,0.2 Hz 10%24 h cyclic uniaxial stretching）,and both of CTF and mechanical stretching（cells+mechanical stretch）.Pure CTF led to obvious desorption of the adsorbed FN layer on the F_minsubstrate,which may prevent the transmission of CTF to the substrate.In contrast,the FN layers on F⁺_max,F_maxand F_medsubstrates experienced different degrees of reorganization,which could promise the transmission of CTF to the substrate.In the absence of cells,the pure mechanical stretch did not damage any substrate-FN interface so as to ensure the transmission of stretch strain to cells.When both CTF and mechanical stretch existed,the mechanical stretch exacerbated the desorption of the FN layer on F_minsubstrate,which should prevent both CTF and stretch strain from being transmitted to the substrate or cells,while the F⁺_max,F_max,and F_med surfaces could guarantee the effective transmission of CTF and stretch strain.（3）Mediation of F_ad to rMSCs orientation and osteogenic differentiation in response to stretch strain:On F_min substrates,the cells demonstrated random orientation and low osteogenic differentiation due to the inability of cells to sense the stretch strain.However,cells seeded on high F_ad surfaces(F⁺_max,F_max and F_medsurfaces)can sense the stretch strain so as to take a parallel（5%strain）or a perpendicular（10%strain）orientation with the stretch direction,and the osteogenic differentiation was stronger than on F_min surfaces.In summary,this study confirms the important role of F_ad in the transmission of CTF and mechanical stretch strain as well as the regulation of cell fate.The F_adregulates the biological effects of rMSCs by affecting the transmission of CTF and stretch strain.This work provides a new perspective to understand the mechanism that mechanical stretch stimuli regulate cell behaviors and may help to guide the design of biomaterials and bioreactors.