Adsorption Forces Of Various Proteins On Substrates And Its Effect On Stem Cell Differentiation

Seed cells,scaffold materials,and biologically active molecules are three basic elements required in tissue engineering.The scaffold materials provide physical support for the seed cells and form local microenvironments.It is well known that,upon implantation of scaffolds in body,protein adsorption onto the scaffold materials is the first event before cell adhesion.Therefore,the adsorbed protein layer is an important link between cells and materials.Once cells become specifically adhered,cell traction force（CTF）would be generated via the formation of focal adhesions and cytoskeletons.Cells sense and respond to the mechanical porperties of underlying materials through the transmission of CTF from cells to materials.It is reported that the adsorption force(F_ad)of fibronectin on the surfaces of materials can regulate the transmission of cell traction force（CTF）and further the responses of stem cells to the mechanical properties of materials.Based on the fact that there are various types of proteins in body fluid,we speculate that the type of proteins should affect F_advalues and thus the F_ad-mediated CTF transmission and stem cell differentiation.Therefore,in this present work,bovine serum albumin（BSA）,the most abundant protein in blood and two important extracellular matrix proteins,type collagen I（COL I）and fibronectin（FN）were selected as model proteins,and their F_advalues were quantitatively detected by using a constructed platform capable of regulating and detecting F_advalues.Finally,the effects of the F_adof COL I on CTF transmission of rat mesenchymal stem cells（r MSCs）and further on the responses of r MSCs to the mechanical properties of substrates were investigated and compared with those for the F_adof FN from previous studies.The main works and conclusions are summarized as follows:（1）Stiff polydimethylsiloxane（PDMS）without stress relaxation（CH₃-10）and soft PDMS with stress relaxation（CH₃-100）were first prepared.Then self-assembled monolayers（SAMs）with hydroxyl（OH）and amino（NH₂）were constructed on CH₃-10and CH₃-100 surfaces by using surface plasma treatment and（3-Aminopropyl）triethoxy silane（APTES）modification of the obtained OH,respectively.The results from water contact angle,X-ray Photoelectric Spectrometer and Atomic Force Microscope detection verify the successful preparation of CH₃-,OH-,and NH₂-SAMs,which provides a platform to regulate the F_advalue of BSA,COL I and FN in the following study.（2）The F_advalue of BSA,COL I and FN on the surface of OH-,NH₂-and CH₃-SAMs were quantitatively detected by using a parallel plate flow chamber combined with a spherical/surface model.The results indicate that the F_advalues presented a trend as-OH<-NH₂<-CH₃for BSA and FN,and a trend of-OH<-CH₃<-NH₂for COL I,suggesting that the surface chemistry of substrates can regulate F_ad.More importantly,F_advalues demonstrate an obvious dependence on the type of protein,following a trend of BSA<<FN<COL I.（3）The regulation of the F_adof COL I to CTF transmission of r MSCs and further on cell behavior was investigated by immunofluorescence staining,observation of substrate deformation under phase contrast microscope,alkaline phosphatase staining,and Oil Red O staining.The high F_advalues of COL I on both OH and NH₂SAMs enable the successful transmission of CTF to substrates.As a result,r MSCs can sense the high Young’s modulus of stiff PDMS substrate and the stress relaxation of soft PDMS substrates and thus show strong osteogenetic differentiation.This is obviously different from the differentiation of r MSCs on FN-adsorbed OH substrates reported in previous studies:r MSCs on both soft and stiff OH substrates demonstrate more adipogenetic differentiation because of the low F_adon OH substrates and the resulting failure of CTF transmission and perception of mechanical properties of substrates.In conclusion,we found that F_adis affected by the type of protein,and the different F_addue to protein types may result in the difference in CTF transmission and thus the responses of stem cells to the mechanical properties of materials.These findings add new evidences to show the importance of F_adand provide a new perspective to understand the mechanism for cell-materials interaction and a new strategy to design biomaterials for tissue engineering.