Protein Layer And Protein-material Interfacial Force And Their Regulation To Force Transmission And Stem Cells Behaviors In Response To The Mechanical Properties Of Material

Extracellular matrix（ECM）coordinates and directs various activities of cells,tissues,organs,or even the whole human body.Abnormal ECM is closely related to the occurrence and development of diseases.Accordingly,regulation of ECM is a new strategy for disease treatment including the repair of defected tissues.To realize this strategy,an in-depth understanding of the mechanism for cell-ECM interactions is the precondition.When a biomaterial is implanted into human body,the proteins in interstitial fluid or blood will immediately adsorb onto the material surface,forming a new artificial ECM of protein-material composite.Subsequently,cells adhere on the protein layer to generate cell traction force（CTF）.Through the generation/transmission of CTF,cells try to sense and respond to the mechanical properties of materials.“Cell-protein layer-material”acts as the common force transmission system for CTF and the mechanical properties of materials.We recognize that the protein layer is essentially‘a polymer membrane’.Therefore,we hypothesize that its spacial organization and mechanical properties are key factors to regulate CTF transmission.Besides,CTF transmission must pass protein-substrate interface,so that we suppose that the protein-substrate interfacial force(F_ad)may cooperate with the protein layer to regulate CTF transmission as well.Unfortunately,most of the existing reports focus on the biological aspects of the protein layer and pay little attention to its material attributes.The effects of the protein layer and F_ad on CTF transmission and further on cell behavior have received even less attention.Clarifying the roles of the protein layer and F_ad is critically important for a more in-depth interpretation of the cell-ECM interactions and for guidance of the design and fabrication of biomaterials.In this work,the organization of protein layer and F_ad were carefully investigated by using fibronectin as model protein.Thereafter,a multifunctional platform capable of controlling the organization of FN layer,F_ad and the mechanical properties of the substrate and quantifying the CTF transmitted to substrate was established.By using this platform,the roles of FN layer and F_ad to regulate CTF generation/transmission and subsequent cell behavior responding to the mechanical properties of the substrate were examined by using rat mesenchymal stem cells as model cells.The main works and conclusions are summarized as follows:（1）Investigation of the FN organization and F_adVarious polydimethylsiloxane（PDMS）substrates with pendent-OH,-NH₂ and-CH₃ groups were constructed by using surface modification technologies,and the adsorption of FN was investigated.It was found that the F_ad followed an order as PDMS-OH（4.91±1.19 p N/FN）<PDMS-NH₂（28.27±7.55 p N/FN）<PDMS-CH₃（71.22±11.94 p N/FN）.The protein amount and adsorption rate of FN took similar order to F_ad.Regarding the spacial organization of FN layer,the FN molecules on hydrophilic PDMS-OH demonstrated a more natural compact structure and took a“flat flying”state,in which the FN-FN interaction was weak and the horizontal rigidity is higher than the vertical one.On PDMS-NH₂ surface,the electrostatic interaction between PDMS-NH₂ and FN molecules obviously changed the FN conformation and the extended FN molecules took a“side lying”state to form network,in which the FN-FN interaction was strong and the average rigidity of the FN layer was relatively high.On PDMS-CH₃,the strong hydrophobicity significantly destroyed FN conformation and some large hydrophobic cavities existed in the FN layer,which decreased the volumetric density and the average rigidity of the FN layer.These results indicate that the surface chemistries can regulate the organization of protein and F_ad.（2）Construction of a multifunctional platform that can simultaneously modulate FN organization,F_ad and substrate mechanical properties and quantify CTF on the substrateBy using the calculation results from the Euler Bernoulli beam theory,PDMS with different Young’s modulus and micropost height was selected to regulate the rigidity and stress relaxation.Consequently,three pure elastic substrates,i.e.PDMS-High（E～2126.35 k Pa）,PDMS-Medium(E_eff～20.92 k Pa)and PDMS-Low(E_eff～7.77 k Pa),and one substrate with similar rigidity to PDMS-Low yet obvious stress relaxation,i.e.PDMS-Low+(E_eff～5.86 k Pa)were constructed.By modifying the microposts surfaces with-OH and-NH₂,the FN organization and F_ad could be effectively regulated.Moreover,according to the deformation of microposts,the values of CTF transmitted to the substrate could be quantified.（3）Regulation of FN organization and F_ad to the generation and transmission of CTFThe generation and transmission of CTF were investigated by using immunofluorescence,Western blot and scanning electron microscopy.It was found that the magnitude of CTF initially generated by r MSCs primarily depended on the number of the exposed adhesive sites（RGD sequences）on the surface of FN layer,and the CTF followed an order as PDMS-OH>PDMS-NH₂.When CTF was transmitted to FN layer,the weak F_ad and FN-FN interaction on PDMS-OH led to desorption or rupture of the FN layer and the consequent decrease in the generated CTF.On the other hand,the strong F_ad and FN-FN interaction on PDMS-NH₂ promoted the reorganization of FN molecules,leading to the dynamic increase of the generated CTF.When CTF was transmitted to the substrates,the magnitude of CTF transmitted to per unit area of substrate did not change with adhesion time and almost kept constant on PDMS-OH（4.784 n N/μm²）and PDMS-NH₂（4.045 n N/μm²）.These results suggest that the FN organization and F_ad dynamically regulate the magnitude of the generated CTF through regulating the transmission of CTF along the FN layer and FN-substrate interface.（4）Regulation of FN organization and F_ad to the perception and responses of r MSCs to the substrate mechanical propertiesFinally,the cell spreading,locomotion and differentiation of r MSCs were examined by using immunofluorescence staining,inverted microscope,q RT-PCR,etc.On substrates with identical FN organization and F_ad,the spreading areas of r MSCs always increased with increasing rigidity of the pure elastic substrate,and the stress relaxation of substrates promoted cell spreading.The cell areas took an order as High≈Low+>Medium>Low.On the other hand,the cell locomotion decreased with increasing rigidity,and stress relaxation inhibited cell locomotion,taking an order as High≈Low+<Medium<Low.As expected,high rigidity together stress relaxation promoted osteogenic differentiation（High≈Low+>Medium>Low）whereas low rigidity promoted adipogenic differentiation（High≈Low+<Medium<Low）.These results verified that the rigidity and stress relaxation regulate the spreading,locomotion and differentiation of stem cells.On substrates with identical mechanical properties,the cell behavior highly depended on the FN organization and F_ad.Specifically,on PDMS-OH,the desorption or rupture of the FN layer due to the weak F_ad and FN-FN interaction led to formation of more nascent adhesions,resulting in decreased spreading,increased cell polarity and locomotion,and more adipogenic differentiation.Contrarily,on PDMS-NH₂,the effective reorganization by CTF due to the strong F_ad and FN-FN interaction led to formation of more mature focal adhesions,resulting in increased spreading,decreased cell polarity and locomotion,and more osteogenic differentiation.These results indicate that FN organization and F_ad regulate the formation of nascent adhesions or mature focal adhesions and the dynamic generation and transmission of CTF and further regulate cell spreading,locomotion and differentiation.In summary,we have established a multifunctional platform integrating the abilities to regulate the spacial organization of protein layer,F_ad and the mechanical properties of substrates and quantifying the transmitted CTF.By using this platform,it is verified that the FN organization and F_ad control the dynamic generation and transmission of CTF,which further regulate the perception and biological responses of cells to the mechanical properties of substrates.The investigation on FN organization from the perspective of material science and the introduction of both FN organization and F_ad not only add new contents to the researches on protein adsorption and biological interfaces,but also help to gain more comprehensive and in-depth understanding on cell-material interactions.The obtained mechanism may guide the design and fabrication of artificial ECM to facilitate tissue repair.