Protein-material Interfacial Force And Its Regulation To Cell Behavior In Response To Mechanical Stimuli

Seed cells and scaffolds are key elements for artificial organs construction and tissue engineering.The mechanical microenvironment provided by the mechanical properties and mechanical stretch of scaffold materials plays a pivotal role in regulating cell behavior and tissue/organ formation.After a cell becomes specifically adhered on material,it may produce cell traction force?CTF?.Through the transmission of CTF,cell can sense and gauge the mechanical properties of material.In addition,mechanical stretch results in material strain.Through the transmission of material strain,cell regulates itself to adapt the mechanical stretch.Therefore,the transmission of CTF and material strain are essentially important for cells to sense and respond the mechanical stimuli.It is well known that protein adsorption onto material surface is the initial step before cell adheres on biomaterial.The adsorbed protein layer acts as a bridge between cell and material.We suppose that cell-protein interface,protein layer and protein-material interface form a complete system for force transmission.Though intensive attention has been paid to cell-protein interface for its contributions to force transmission and cell behavior,rare attention has been paid to the protein-material interfacial force(F_ad).Clarifying the roles of F_ad is essentially important for a more in-depth interpretation of the responding mechanism of cells to mechanical microenvironment and for an optimal design of scaffolds as well.In this study,two platforms,one for quantifying and the other for regulating F_ad were established and the effectiveness of F_ad in modulating cell behavior was examined by using fibronectin?FN?as model protein and osteoblasts as model cells.Thereafter,the regulation of F_ad to transmission of CTF and stretch strain and further to cell responses were thoroughly studied by using rat mesenchymal stem cells?rMSCs?as model cells.The main works and conclusions are summarized as follows:?1?Establishing the platform for F_ad quantification by using a parallel plate flow chamber/microsphere techniqueFN and albumin bovine?BSA?were covalently bound to NH₂-functionalized microsphere by using sulfo-SMPB as a coupling agent,forming FN-or BSA-conjugated microspheres.The protein-conjugated microspheres were then adsorbed to the bottom of parallel plate flow chamber and then subjected to flow shear stress.The percentage of remained protein-conjugated microspheres on the surface was plotted as a function of the shear stress,within which the applied shear stress capable of removing 50%of the initially adsorbed microspheres was denoted by?_50%as a critical shear stress.By using a sphere/plane adhesion model,F_ad per protein molecular was calculated from?_50%.The obtained F_ad values were similar to the data from previous reports,which indicates that the established platform based on the parallel plate flow chamber/microsphere technique is effective in quantifying F_ad.?2?Establishing the platform for regulating the magnitude of F_adVarious self-assembled monolayer?SAMs?with surface-OH,-CH₃ or-NH₂ were prepared on glass coverslips by using silanization reaction.Static water contact angles and X-ray photoelectron spectroscopy?XPS?results verified the successful preparation of SAMs.The F_ad values of FN and BSA on various SAMs was quantified by the parallel plate flow chamber/microsphere technique.The results from both FN and BSA indicate an obvious chemistry dependence of F_ad as NH₂-SAMs?25.6±7.5 p N?>CH₃-SAMs?14.1±4.1 pN?>>OH-SAMs?1.5±0.4 pN?and NH₂-SAMs?6.5±4.9pN?>CH₃-SAMs?4.7±3.6 pN?>>OH-SAMs?1.1±0.8 p N?,which suggests that the SAMs-based platform can be used to regulate the magnitude of F_ad.?3?Validating the regulation of F_ad to cell behaviorIn view of the fact that surface chemistries can affect the conformation?active sites?of adsorbed protein and further the cell behavior,both the active sites and F_ad were investigated about their effects on cell behavior by using FN as a model protein and osteoblasts as a model cell.FN monoclonal antibody?HFN 7.1?was used to detect the active binding domains of FN and the expression and organization of focal adhesions were detected to indicate the magnitude of CTF.It is demonstrated that the initial osteoblast adhesion and CTF?2 hours?were mainly governed by the active binding domains of FN exposed on SAMs,yet the late cell adhesion and CTF?12 hours?were influenced by F_ad.F_ad regulated the reorganization or desorption of FN by early CTF,which further influenced the late cell adhesion and the consequent fibrillogenesis of endogenous FN,verifying that the controlled F_ad by SAMs can influence cell behavior and thus can be employed in the study of force transmission.?4?Investigating the effect of F_ad on the transmission of CTF and subsequent stem cell response to mechanical properties of substratesFirst,a platform capable of simultaneously modulate substrate mechanical properties and F_ad was established as follows.Stiff?E²200 kPa,with negligible stress relaxation?and soft?E¹ kPa,with obvious stress relaxation?poly?dimethyl siloxane??PDMS?substrates were prepared and modified with surface-OH and-NH₂ groups.It was found that the magnitude of F_ad had nothing to do with the mechanical properties of PDMS yet depended on the surface chemisty:NH₂-SAMs?35±11 p N and 31±10pN?>>OH-SAMs?3.5±1.1 pN and 3.0±1.0 pN?.The F_ad on OH-SAMs and NH₂-SAMs was recorded as F_min and F_max respectively.Extremely,FN moleculars were covalently bound to PDMS to represent a maximum F_ad(F_max).Then the influences of F_ad on the transmission of CTF and cell responses were studied by using rMSCs as model cells.The reorganization/desorption of adsorbed FN and the deformation of substrates were detected to indicate the transmission of CTF to FN layer and substrates respectively.It was found that FN on F_min substrates could not resist CTF so as to significantly desorb from the substrates by CTF,which prevents CTF from being transmitted to substrates.As a result,what rMSCs can perceive is the weak mechanical feedback from the proteins rather than the mechanical properties of substrates,leading to more adipogenic differentiation on both stiff and soft substrates.On the other hand,CTF could be well transmitted to F_med and F_max substrates so that the mechanical properties of substrates can be perceived by r MSCs,inducing more osteogenic differentiation on both stiff and soft PDMS.More osteogenic differentiation even on soft PDMS might result from the stress relaxation of soft PDMS,which,however,should be tested in future work.These results verify that F_ad regulates the transmission of CTF to substrates and further determines the fate of stem cells.?5?Investigating the effect of F_ad on the transmission of stretch strain and subsequent stem cell responseStiff PDMS substrates?E²200 kPa,no obvious stress relaxation?with various F_ad(F_min,F_med and F_max)were seeded with rMSCs for 12 hours and then subjected to 0.2Hz,10%strain of cyclic uniaxial stretch for 24 hours by using a commercial stretch device.As a control,the substrates without seeded cells were subjected to similar stretch strain in order to see the effect of stretch strain alone on the adsorbed FN.It is found that substrate strain alone had no significant effect on the adsorbed FN regardless of the magnitude of F_ad.On the other hand,for substrates with seeded cells,the FN was reorganized or desorbed and rMSCs aligned perpendicular to stretch axis depending on the magnitude of F_ad.Specifically,FN on F_min substrates was obviously desorbed and the F-actin and focal adhesions were not well organized.Stretch strain further promoted the desorption of FN by CTF.As a result,the stretch strain could not be transmitted to rMSCs and thus the trend of rMSCs realignment was weak on F_min substrates.On F_med and F_max substrates,the FN can resist CTF and the F-actin and focal adhesions were well organized.As a result,the stretch strain was well transmitted to rMSCs,resulting in perpendicular alignment of to rMSCs to the stretch axis.These results verify that F_ad regulatesthetransmissionofstretchstrainthroughmedicatingthe reorganization/desorption of FN by CTF and further regulates the response of cells to stretch strain.In summary,we have established a platform for quantifying F_ad and a platform for regulating F_ad.By using these two platforms,it is verified that F_ad regulates the transmissionofCTFandsubstratestrainthroughmodulatingthe reorganization/desorption of the adsorbed protein by CTF,which further determines the perception and response of cells to the mechanical properties and stretch strain of substrates.The introduction of F_ad not only provides a new perspective for understanding cell-protein-material interactions but also broadens the scope of biomechanics.In addition,the obtained mechanism involving F_ad may guide the optimal design of scaffolds for tissue regeneration.