Regulation Of Protein-materials Interfacial Force (F_ad) To Cells’ Utilization On Adsorbed Fibronectin:Remodeling,Degradation And Endocytosis

When biomaterials are implanted,extracullar（ECM）proteins will quickly adsorb onto material surface and then cells become adhereed through the adsorbed protein layer.Adhered cells may generate cell traction force（CTF）and via the transmission of CTF,cells sense and respond to the properties of material.In the force transmission system of cell-ECM protein-material,the interfacial force between ECM protein and material(F_ad)plays an important role.It is reported that,in order to adapt the properties of materials,cells may utilize and modify extracellular proteins through reorganization/desorption,endocytosis or degradation,yet rare studies pay attention to the contribution of F_ad.Probing the influence of F_ad on the utilization of adsorbed ECM proteins should help to provide an in-depth understanding on the mechanism by which cells respond to the mechanical microenvironment and induce new design ideas for tissue regeneration scaffolds.In this work,the effects of F_ad on the utilization（reorganization/desorption,endocytosis,degradation）of adsorbed ECM protein was explored by using fibronectin（FN）as model protein and rat mesenchymal stem cells（r MSCs）as model cell.The main research contents and conclusions are as follows:（1）Construction of substrates with tunable F_ad and measurement of F_adThe surface of polydimethylene siloxane（PDMS）was modified with-OH,-NH₂,and-CH₃ by means of self-assemble monolayer（SAMs）technology.Then the F_advalues between adsorbed FN and various PDMS were quantified by using the parallel plate flow chamber/microsphere technique.The F_ad values are 3.71±0.78 p N on OH-PDMS,24.02±8.28 p N on NH₂-PDMS and 81.80±5.70 p N on CH₃-PDMS,verifying that surface chemistries can regulate F_ad.（2）Regulation of F_ad on reorganization/desorption of adsorbed proteinsAfter r MSCs were seeded on various PDMS preadsorbed with FITC-FN（FN labeled with fluorescein isothiocyanate）for 2 h,4 h,and 6 h,respectively,FITC-FN and cells were observed by immunofluorescence staining.It was found that low F_ad on-OH surface could not resist CTF and obvious reorganization,aggregation,and desorption of FN were observed.A higher F_ad on-NH₂ surface could resist CTF and thus produce slight FN reorganization and good cell adhesion/spreading.However,on surfaces with extremely high F_ad,no reorganaization or desorption of FN was observed.These results reveal that F_ad can regulate the reorganization/desorption of the adsorbed protein layer.（3）Regulation of F_ad on degradation of adsorbed proteinsProteolytic degradation is an important approach to remove ECM.In this study,the expression of matrix metalloproteinase-2（MMP-2）was detected by Zymography after r MSCs were cultured for 4 h,12 h and 24 h and the degradation of FITC-FN was further semi-quantified.We found that,when F_ad is strong enough to retard normal reorganization of FITC-FN,cells may secret MMP-2 to degrade FN with an attempt to obtain a comfortable microenvironment.These results indicate that F_ad regulates the degradation of ECM proteins.（4）Regulation of F_ad to endocytosis of adsorbed proteinEndocytosis is highly dependent on clathrin.To investigate the regulation of F_ad to endocytosis of adsorbed protein,the colocalization of intracellular clathrin and adsorbed FN was observed by using immunofluorescence staining,and the endocytosis intensity was detected by flow cytometry.It was found that,at early cell adhesion（2 h）,more FN and larger aggregates of FN were endocytosized by cells on low F_ad surface.After cell adhesion of 4 h and 6 h,however,more endocytosis happened on-NH₂ surfaces rather than on-CH₃ or covalently immobilized FN surfaces with extremely strong F_ad.Further observation of Rhodamine-FN and GFP r MSCs by using a living cell workstation indicated that,endocytosis happened even after cell adhesion of 10 min on all F_adsurfaces,therafter（cell adhesion of 0.5 h）FN aggregation and desorption occurred on low F_ad surface yet only tiny dotted fluorescent aggregation without FN desorption was observed on high F_ad surfaces.These results demonstrate that F_ad regulates the endocysis of ECM proteins and endocytosis might happen before protein aggregation/desorption.In summary,we have successfully established substrates with tunable F_ad,and verified that F_ad is an important factor to regulate the utilization of adsorbed ECM protein by cells.This work investigates cell-material interaction from the viewpoint of interfacial force(F_ad)and provides an insight into the modes and temporal sequence of cell-ECM protein interactions.