the cell / material interface 
 has proven crucial to influencing cellular 
 behavior , with substrate topography , accessible 
 area / shape , stiffness , dimensionality , and protein 
 tethering known to be some of the physical 
 stimuli perceivable to cells . this ability to control cell fate , without , 
 or with , minimal use of soluble factors , such as growth factors ( e.g. , 
 bmp-2 ) , is highly sought after in the 
 field of tissue engineering ; however , a complete understanding of 
 the mechanisms used by cells to distinguish these physical stimuli 
 is not yet fully understood . there 
 is emerging evidence that the mobility of the material surface 
 alters cell behavior . different systems have been developed that make use of hydrated 
 mobility , which is the motion of a hydrophilic material interface 
 as a consequence of the interaction with the surrounding water . for 
 example , increasing the tether length of synthetic adhesive peptides 
 to the underlying substrate enhances cell adhesion , cell spreading , 
 and the formation of focal adhesions . peg - based block copolymers with modulated chain mobility alter fibroblast 
 adhesion and morphology . moreover , model chemistries , 
 tethered to glass with dynamic properties , obtained by controlling 
 side - chain length ( number of c ) , have been shown to influence mesenchymal 
 stem cell ( msc ) phenotype . these cell / material 
 interactions involve fluctuating picoscale forces , exerted by cells 
 upon the surface , which can trigger the movement of the polymer surface . it must be noted that the concept of surface 
 mobility is different from surface stiffness . the latter is generally 
 sensed by cells after assembling focal adhesions and probing the surface 
 with nanoscale forces ( 5.5 nn/m ) , while surface mobility involves single receptor interactions 
 with much lower and fluctuating forces ( picoscale ) . polymers , 
 the chains of which are mobile entities with dynamics 
 directly linked to temperature , are extensively used as biomaterials . 
 below the glass - transition temperature ( tg ) , the glassy state , polymer chains are almost frozen and movements 
 are mainly restricted to the side groups of the chains at the sub - nanometer 
 level . above tg , a liquid - like state , 
 the free volume drastically increases and polymer chains are highly 
 mobile within distances of tens of nanometers . this is demonstrated conceptually , in the context of this 
 work , in figure 1 . direct evidence of the scale of these movements can be obtained from 
 experiments in thin polymer films . varying 
 polymer thickness from 100 to 20 nm leads to monotonically diminished tg , which demonstrates that both film thickness 
 and the movement of polymer chains share the same nanometer range . conceptual figure of how polymer 
 side - chain length affects the mobility by increasing free volume ( represented 
 by the white area ) and its effect on the fibronectin layer ( monomer 
 shown above ) . pma(x = 1 ) leads to adsorption of globular 
 fibronectin , with pea(x = 2 ) , pba(x = 4 ) , and pha(x = 6 ) all leading to the formation 
 of fibronectin nanonetworks . the fact that the same organization of 
 the protein occurs on these three polymers allows discussion of the 
 effect of surface mobility on protein layers with the same initial 
 conformation / distribution and assessment of the effect of mobility , 
 disregarding major conformational effects of the proteins induced 
 by surface chemistry . cells interact with surfaces , polymer or otherwise , through 
 a layer 
 of adsorbed proteins using transmembrane integrins , which assemble 
 into large multiprotein complexes called focal adhesions . these allow cells to indirectly detect surface 
 properties by the latter s ability to affect the conformation 
 and flexibility of the extracellular matrix ( ecm ) proteins , consequently 
 exposing key cell - binding residues on these matrix proteins . a prime 
 example of these types of proteins is fibronectin ( fn ) , a 440 
 kda dimer protein , which binds predominantly to 51 integrins through the rgd and phrsn ( synergy ) 
 domains located in repeats iii10 and iii9 , respectively ( shown schematically in figure 1 ) . physiologically , it maintains a globular 
 conformation , but via cellular stimuli it can unfold into an extended 
 conformation , exposing domains responsible for lateral assembly and 
 network formation , thus forming an integral part of the ecm . previous work has demonstrated that surface 
 chemistry can alter the amount and conformation of fn adsorbed onto 
 materials , determining its bioactivity : garcia et al . , using model 
 surface chemistry , showed that the integrin binding domain of fn can 
 be presented to cells with different biological activity depending 
 on the hydrophilic / hydrophobic balance of the surface . changes in the protein orientation / conformation , 
 as a consequence of its conjugation to a surface , also translate into 
 an altered activity . because cells 
 only can respond to the surface mobility indirectly , 
 via the adsorbed protein layer , it is the aim of this work to observe 
 how surface mobility translates into interfacial 
 mobility of the protein layer . among the broad range of available 
 polymers , this work has selected a family of poly(alkyl acrylates ) , 
 with tg well below 37 c ( with a 
 vinyl backbone and side groups coo(ch2)xh , where x = 1 , 2 , 4 , and 6 for 
 poly - methyl , ethyl , butyl , and hexyl acrylates , respectively ) , which interact strongly with fn and 
 on which fn self - assembles into a network of nanofibrils ( for x  2 ) , as shown 
 in figure 1 . thus , 
 interface mobility of the protein layer is expected to be directly 
 linked to the mobility of the underlying polymer surface . this work 
 therefore shows that the mobility of hydrophobic polymers ( hydration - independent ) 
 is a fundamental , dynamic property . this can then be translated into 
 the interfacial layer of adsorbed fn , and this subsequently plays 
 a role in cell adhesion , reorganization , and differentiation . 1 mg / ml fibronectin from human 
 plasma ( sigma - aldrich ) was labeled using the fluorotag fitc conjugation 
 kit ( sigma - aldrich ) . the protocol provided with the kit was adapted 
 for fibronectin labeling ( by adjusting the fn / fitc labeling ratio ) . 
 in brief , 250 l of 1 mg / ml fibronectin was incubated with fitc 
 in a fluorescent molecule to protein ratio of 125:1 for 2 h. the labeled 
 fibronectin was then separated from unconjugated molecules via a g-25 
 sephadex column . the success of the conjugation procedure was determined 
 by measuring the absorbance of the retrieved fractions at 280 ( protein ) 
 and 495 nm ( fitc ) and calculated using equations provided . polymers 
 were synthesized by radical polymerization of acrylate monomers using 
 1 wt % benzoin . polyacrylate solutions were prepared by dissolving 
 bulk polymers in toluene , with a 4% w / v solution for pma and pea and 
 a 6% w / v solution for pba and pha . 12 mm diameter glass coverslips 
 were cleaned by sonication in ethanol and dried at 60 c . 100 
 l of polymer solution was added to the surface and spin - coated 
 for 30 s at 3000 rpm . residual solvent was removed by drying at 60 
 c in vacuum for 1 h. polymer surfaces were coated with a 20 
 g / ml fibronectin solution in dpbs for 10 min ( for afm studies ) 
 or 1 h ( for domain availability , mobility measurement , and cell culture ) . they were then washed in dpbs and milli - q water and in the case of 
 afm studies dried with n2 . this difference in time is to 
 aid in the imaging of the network via afm , which can reduce in clarity 
 at higher time points due to adsorption of more protein . phase images were obtained 
 for coated and uncoated polymer surfaces via afm in ac mode ( nanowizard 
 3 bioscience afm , jpk ) . a pyramidal silicon nitride tip , with a cantilever 
 spring constant of 3 n / m and a resonance frequency of 75 khz 
 ( mpp-21120 , bruker ) , was used . fractal dimension analysis was carried 
 out on the images of fn - coated samples using the imagej fractal box 
 count analysis tool , using box sizes of 2 , 3 , 4 , 6 , 8 , 12 , 16 , 32 , 
 and 64 pixels . force spectroscopy curves were obtained , after calibration 
 of tip sensitivity and spring constant , with a set - point of 10 nn , 
 a zeta length of 10 m , a constant duration of 1 s , and at room 
 temperature . analysis was performed using the jpk processing software 
 ( v4.3.21 ) , and force curves were fitted with a hertz model at 50 nm 
 indentation . the water contact angle of the adsorbed protein surfaces 
 was measured both statically and dynamically , with the latter providing 
 the hysteresis angle of the surface ( optical tensiometer theta , biolin 
 scientific ) . advancing angle was measured by adding water 
 to the original static volume and receding by removing it at a rate 
 of 0.1 l / s . photobleaching 
 was performed 
 on the fluorescent , protein - coated surfaces , with the four polymers 
 and glass as a control on an olympus fv1000 . the samples were stored 
 at 37 c ( under cell culture conditions ) , and the fluorescence 
 signal was measured over a period of 120 h using a zeiss observer 
 z.1 widefield microscope . a 20  50 m area 
 was selected manually across the bleach border region ( figure 6a ) . using imagej , a surface 
 intensity profile was obtained , where each column of pixels ( for one 
 value of x ) was averaged , yielding a line graph ( figure 6b ) across the edge 
 of the bleached area . to quantify the changes , using originpro 8 , 
 the center of the linear region was taken as the central point of 
 the selection area ( x = 10 m ) , and a linear 
 fit over 0.5 m ( 1 m total ) on either side of this point 
 was used to determine the linear gradient for each time point ( figure 6b , red line ) . for 
 each time point , the gradient of the fluorescence intensity across 
 the border was obtained ( figure 6c ) , which was then fitted with an exponential growth 
 ( over time ) , shown in eq 1.1where y0 is the y value at time zero , k is the rate constant , and t is time . the availability of 
 fn adsorbed on polymer surfaces as well as that of the rgd and synergy 
 domains was determined by elisa . samples were blocked for 30 min with 
 1% w / v bsa ( sigma - aldrich ) . to determine the availability of fn , we 
 incubated rabbit polyclonal anti - fn antibody ( sigma - aldrich , 1:10  000 ) 
 for 1 h , followed by a 1 h incubation with biotinylated horse antirabbit 
 secondary antibody ( vectorlabs , 1:10  000 ) , both at room temperature 
 ( rt ) . samples were then incubated with hrp - streptavidin ( r&d systems ) 
 for 20 min , washed , and incubated with hrp substrate ( r&d systems ) 
 for 20 min . after stopping the reaction , using the stop solution , absorbance was measured at 450 ( maximal absorbance of the tag ) and 
 540 nm ( blank control , to determine background absorbance ) . to assess 
 the availability of cell - binding domains , we incubated samples at 
 rt with monoclonal mouse primary antibody ( mab1937 , 1:20,000 in 1% 
 bsa or hfn 7.1 antibody for the synergy or rgd domain , respectively ) . the samples were then washed with 0.5% tween 20 and incubated with 
 goat antimouse hrp - tagged secondary antibody ( 1:10  000 in 1% 
 bsa solution ) for 1 h ( rt ) . after washing with 0.5% tween 20 , samples 
 were incubated with hrp substrate solution in the absence of light 
 for 20 min . the reaction was terminated with stop solution ( r&d 
 systems ) and absorbance was measured . l929 
 fibroblasts and c2c12 myoblasts were 
 cultured in dmem ( 1 ) dulbecco s modified eagle medium 
 ( + 4.5 g / l d - glucose , + l - glutamine , gibco ) , containing 
 1% antibiotic mix of penicillin and streptomycin and 10% ( l929 ) or 
 20% ( c2c12 ) fbs ( gibco ) . to observe cell - mediated protein reorganization , 
 we seeded cells on fibronectin - coated coverslips , within a 24-well 
 plate , on glass and polymer surfaces at a density of 5000 
 cells / cm in the presence of 10% fbs . after incubating 
 for 3 h cells they were blocked with 1% bsa for 20 min then stained 
 for actin for 1 h with rhodamine phallotoxin r415 ( life technologies , 
 1:40 ) . actin ( stained with r415 ) was used as a mask for the reorganization 
 of the underlying fn layer within the cell areas and determining the 
 morphological characteristics of the cell . for the studies of the 
 differentiation of c2c12 , cells were diluted in differentiation medium 
 ( dmem 41965 + 1% ps + 1% its - x ) and seeding was performed on coated 
 surfaces at 18  500 cells / cm . after 3 h the media 
 was refreshed with differentiation medium and blebbistatin was added 
 to experimental samples at 10 m . after 4 days samples were 
 washed and fixed ( 20:2:1 etoh 70%/formaldehyde 37%/acetic acid ) for 
 10 min ( 4 c ) . cells were then incubated with 5% goat serum for 
 1 h ( rt ) . subsequently , cells were incubated with antisarcomeric myosin 
 antibody ( 1:250 ) , followed by cy3 antimouse antibody ( 1:200 ) , both 
 for 1 h at 37 c , in the absence of light . in all instances , where relevant , one - way anova tests 
 were performed to determine any statistically significant differences : 
 * p  0.05 , * * p  0.01 , 
 * * * p  0.001 , and * * * * p  
 0.0001 ( graphpad software , la jolla , ca ) . the linear gradient of bleaching 
 analysis was determined in originpro 8 ( orginlab , northampton , ma ) , 
 with this subsequently being transferred to and analyzed in graphpad 
 prism 6 . in 
 this study four polymers with similar physicochemical properties , 
 each comprising a vinyl backbone with a side group  coo(ch2)xh ( x = 1 , x = 2 , x = 4 , and x = 
 6 ) , were used . the spin - coated surfaces were found to be smooth , with 
 similar root - mean - square ( rms ) roughness of 37.5  5.5 
  10 pm and thickness of 7.4  2.3  10 nm , regardless of composition . furthermore , afm nanoindentation 
 determined that all surfaces had young s moduli 1 mpa , 
 greater than the 40 kpa stiffness threshold that cells can detect ( figure 2 ) . ( b ) force curve from 
 measurements , showing an example of the initial 50 nm indentation 
 of a polymer surface (  ) , on which the hertz model was applied 
 to calculate the young s moduli . ( c ) young s moduli 
 of polymer films ; each point on the graph is an average derived from 
 64 measurements ( per sample ) . measurement of the water contact angle of these polymer surfaces , 
 with and without fn coating , as well as comparing the labeled and 
 unlabeled protein , allowed for a further understanding of the physical 
 characteristics ( figure 3 ) . there was a minimal change in the contact - angle hysteresis ( hysteresis 
 = advancing angle  receding angle , figure 3 ) and in the static contact angle ( sca ) ( supplementary figure s1 ) , with increasing polymer 
 chain length in the absence of fibronectin ( figure 3a ) ; however , both advancing and receding 
 angles were seen to increase , with increasing side chain length ( figure 3b ) . coating the polymers 
 with fn led to a decrease in the receding angles , with a concomitant 
 increase in the advancing angles ( figure 3c ) . furthermore , there was no change in the 
 wettability properties when labeled fn was used compared with that 
 of the unlabeled protein ( figure 3a ) . phase imaging of tapping mode afm , with fractal 
 dimension analysis , 
 showed that fitc labeling of the fn protein did not affect the distribution 
 and conformation adopted by fn upon adsorption on the different surfaces 
 ( figure 4 ) . with or 
 without the fitc label , it was noted that network formation did not 
 occur on pma ( x = 1 ) , where globular aggregates were 
 observed , while pea ( x = 2 ) , pba ( x = 4 ) , and pha ( x = 6 ) surfaces supported the formation 
 of fibrillar protein networks ; with longer 
 adsorption times , the network structure is maintained and becomes 
 denser ( supplementary figure s2 ) . this 
 indicates that fitc is an appropriate means through which to analyze 
 the interfacial mobility of fn and how this property relates to domain 
 availability and cell - mediated reorganization . dynamic water contact angles were measured 
 on the polymer surfaces before and after coating with a fn solution 
 of concentration 20 g / ml . ( b ) 
 advancing ( aca ) and receding ( rca ) angles of the uncoated polymer 
 surface and ( c ) of the surfaces after coating with fn and labeled 
 fn . the surface density 
 of fn , as quantified by the bca assay , shows similar adsorbed levels 
 on each surface ( supplementary figure s3 ) . the adsorbed fn ( regions of interest shown schematically in figure 5a ) was further characterized 
 in terms of its availability on the material surface , considering 
 both general availability of the protein and the specific availability 
 of key domains for cell binding.figure 5b shows that the 
 availability of the adsorbed fn , determined via polyclonal antibody 
 binding , decreases on the more mobile surfaces . importantly , in contrast 
 with this , the availability of the cell - binding domains , the rgd ( figure 5c ) and phsrn ( synergy ) 
 sites ( figure 5d ) , 
 as indicated by the arrows in figure 5a , was observed to increase on the more mobile , network - forming , 
 polymers . fibronectin distribution on the polymer 
 surfaces by afm ( ac mode ) after adsorption from a solution of concentration 
 20 g / ml . fn is organized into nanonetworks on pea ( x = 2 ) , pba ( x = 4 ) , and pha ( x = 6 ) . these 1 m  1 m phase images demonstrate 
 the similarities in protein distribution in the labeled and unlabeled 
 forms . the values shown underneath the network forming polymers relate 
 to the fractal dimension ( d ) , a descriptor of the complexity of a 
 pattern that accounts for the network connectivity . polymer surfaces coated with fitc - labeled fn were 
 used to measure 
 the interfacial mobility of the protein layer adsorbed onto this family 
 of polymers ( figure 6 ) . specifically , by bleaching large areas 
 of each of the protein - coated polymer surfaces , the long - term change 
 in the surface fluorescence intensity profile was measured . fn mobility 
 is linked to the fluorescence recovery into the bleached area , together 
 with movement of  dark  species into the untreated area , 
 resulting in a local change in the fluorescence signal . the focus 
 here was upon the edge of the bleached region due to the small length 
 scale of surface mobility limiting the effects to this boundary region 
 ( figure 6a ) . the linear 
 fit of the central region , as defined by the 1 m region spanning 
 the central point of the selection area ( x = 10 m , 
 detailed further in methods ) , provided a gradient , plotted for each 
 time point ( figure 6b ) . an exponential growth ( eq 1 ) to the variation of this gradient value over time was fitted 
 and used as an indicator of the mobility of the protein layer ( figure 6c ) . exposure of epitopes of fn when adsorbed 
 on polymer surfaces from a solution of concentration 20 g / ml . ( a ) fniii710 model with arrows pointing to binding 
 sites for mab1937 and hfn 7.1 . ( c ) rgd cell - binding site exposure ( hfn 7.1 
 ab ) . measuring the rate of change in fluorescence 
 intensity profile as an indicator of protein mobility . ( b ) linear 
 gradient value of the bleach border used to determine any changes 
 of the intensity profile at each time point . ( c ) comparison of the 
 gradient value at each time point ( n = 10 ) for each 
 of the polymer surface . this is then used to determine the rate constant 
 ( k value ) of the exponential growth equation . ( d ) 
 gradient of the linear region of the bleach border changes with respect 
 to time for each of the polymers . ( e ) ascertained k values compared with the glass - transition temperature , tg , of the polymers . a nonmonotonic dependence of the interfacial mobility of 
 the fibronectin 
 ( as indicated by k , the rate constant ) adsorbed onto 
 the surface , with respect to the mobility of the polymer ( represented 
 by their glass - transition temperatures ) , was observed ( figure 6d , e ) . upon increasing polymer 
 mobility , at 37 c , from pma ( tg  
 10 c ) to pea ( tg  20 
 c ) , a reduction in fn interfacial mobility was observed . upon 
 further increasing polymer mobility to pba ( tg  50 c ) , the fn mobility returns to 
 be similar to that seen on pma , and for pha ( tg  70 c ) a significant increase in fn 
 mobility was observed . cells were well spread on the protein - coated polymers and the glass 
 control after 3 h. well - developed cytoskeletons were observed in all 
 cases , and no significant differences in cell size and shape were 
 noted ( supplementary figure s4 ) . the use 
 of fitc - labeled fibronectin permitted the observation of the extent 
 of cell - mediated reorganization of the adsorbed protein layer without 
 the complication of cell - secreted fibronectin being taken into account . figure 7a shows the difference 
 in the relative intensity of fibronectin underneath the cell area 
 as compared with the area outside the cell . indeed , this difference 
 in the intensity of fluorescence coming from fn beneath or outside 
 the cell area can readily be observed on all of the protein - coated 
 surfaces ( figure 7b ) . 
 not only can reorganization be seen but also the labeled fn indicates 
 where the cell is exerting the greatest amount of force on the surfaces , 
 indicated by the brighter and darker areas , where fn is accumulated 
 or taken from , respectively . this semiquantitative approach allowed 
 for the determination of a trend in the extent of cell - mediated protein 
 reorganization on each of the surfaces . pma ( x = 
 1 ) and pea ( x = 2 ) showed the smallest extent of 
 cell - mediated reorganization , with fn reorganization increasing with 
 the mobility of the protein layer . cells on pha allowed for the highest 
 extent of fn reorganization , thus replicating the trend of the mobility 
 of the protein layer ( figure 6c ) . ( a ) relative differences in the fluorescence 
 intensity within the cell area ( as defined by the stained actin ) compared 
 with outside the cell area . ( b ) cell ( actin , red ) with the corresponding 
 fibronectin layer ( green ) below for glass , pma ( x = 1 ) , pea(x = 2 ) , pba(x = 4 ) , 
 and pha(x = 6 ) , respectively ( scale bar = 25 m ) . cell differentiation . differentiation of c2c12 
 cells cultured on 
 fn coated collagen i , pma , pea , pba , and pha ( a ) without and ( b ) with 
 a contractility inhibitor ( blebbistatin ) . coli is the differentiation 
 control , and the percentage of differentiation is measured as the 
 ratio of sarcomeric myosin - positive cells . ( c ) representative pictures : 
 nuclei ( blue ) and sarcomeric myosin ( red ) for each of the surfaces . 
 in panel b there was significant differences between coli and polymer 
 surfaces with 1,2 ( p = * * * * ) and 3 ( p = * ) carbons in the side chain . finally , c2c12 cells ( mouse myoblast cells capable of differentiating 
 into mature myotubes in vitro ) were used to assess the ability of 
 these polymer  protein interfaces to induce cell differentiation 
 ( figure 8) , with col 
 i used a standard control for differentiation . the highest levels 
 of differentiation were seen on pea ( x = 2 ) and pba 
 ( x = 4 ) , with the level of differentiation reducing 
 on pha ( x = 6 ) ( figure 8a ) . inhibition of cell contractility using 
 blebbistatin , a myosin inhibitor , adversely affected c2c12 cell morphology 
 and differentiation ( figure 8b ) . interestingly , inhibiting the cells ability to 
 exert force produced a trend opposite to that of the protein mobility . previous work has shown that polymer surface 
 mobility affects the 
 cellular response in a nonmonotonic fashion . it has been shown by a number of groups 
 that surface mobility , in various forms , can have a profound effect 
 on cellular attachment , morphology , and response . this new work 
 has sought to directly relate how the observed protein mobility and 
 organization at the cell / material interface , determined by the polymer 
 surface mobility , influences the cellular response . the set of polymers 
 presented here differ only slightly in their side - chain chemistry 
 by the sequential addition of methyl groups . as previously mentioned , 
 all surfaces were above the detectable stiffness threshold of the 
 cells , despite variation ( figure 2 ) . the large variation in young s modulus seen 
 in pha ( x = 6 ) can be attributed to the adhesive 
 nature of this polymer . minimal differences in wettability of the 
 untreated polymer surfaces , by observation of the static contact angles 
 ( supplementary figure s1 ) , were noted . contact - angle hysteresis was also similar on all surfaces and increased 
 after fn coating ( figure 3a ) . the advancing angle of the protein - coated surface was 
 seen to increase with increasing side chain length and mobility of 
 the polymer , while the opposite was seen to be true in the case of 
 the receding angle ( figure 3c ) . these differences in dynamic contact - angle measurements 
 reveal a different state of the adsorbed protein , suggesting an increase 
 in protein surface coverage from pma ( x = 1 ) to the 
 rest of the polymers ( compatible with the unfolding of the protein 
 and the formation of fibrils , figure 4 ) . on the more mobile , protein network - forming polymers 
 ( x  2 ) , the increase in contact angles hysteresis 
 indicates an enhanced ability of the adsorbed protein to undergo molecular 
 rearrangement at the water / air interface , which is compatible with 
 an increased mobility of the protein layer . this increase in the hysteresis 
 was noted in both polymers coated with labeled and unlabeled fn ( figure 3a ) , indicating that 
 both have similar physical properties . this is supported by 
 data shown in figure 4 , which shows that the conformation of the adsorbed protein changes 
 drastically between pma ( x = 1 ) and pea ( x = 2 ) ( and the rest of the more mobile 
 polymers ) . this conformational change , from globular to fibrillar , 
 is believed to be driven by the orientation of key hydrophobic residues 
 to interact with the polymer backbone . this leads to the exposure of fn - fn binding sites , which in turn 
 drives further conformational changes through electrostatic and entropic 
 factors . of key importance is the fact 
 that there appears to be minimal differences between the labeled and 
 unlabeled fn after adsorption on material surfaces , which would preclude 
 its use in these studies . furthermore , measurement of the fractal 
 dimension of the network - forming surfaces ( x  
 2 ) , as seen in figure 4 , shows minimal differences in conformation and complexity of the 
 protein networks . this implies that the contribution of protein conformation 
 in the bioactivity of these protein networks is not likely to be a 
 significant factor . while previous work has shown that the stiffness 
 of a substrate can affect cellular behavior , 
 this can be discounted 
 here in favor of surface mobility . for the elasticity / stiffness of 
 the ecm to play a role in cell response the range of moduli previously reported is within 
 the range 1 to 40 kpa , which is 
 within the range of stress that cells are able to exert on a surface ; 
 the maximum force is reported to be between 1 and 5 nn m ( kpa ) . the stiffness 
 of these surfaces , as determined by nanoindentation , is shown to be 
 in the megapascal range ( figure 2 ) ; hence cells should not be able to deform the underlying 
 substrate . further evidence of this lies in the lack of difference 
 in cell 
 morphology ( specifically size , supplementary figure s4 ) , which has previously been observed to change upon detection 
 of substrates with different mechanical properties . furthermore , the cytoskeleton was not seen to be arranged 
 differently , which is also expected if the cell can detect the mechanical 
 properties of the surface . finally , the 
 traction forces exerted by a cell on the surface are dependent on 
 the stiffness of a surface . these traction 
 forces change in relation to the size of focal adhesions , which is 
 also not seen to change here ( supplementary figure s5 ) . further factors , such as 
 surface roughness , have been shown to have implications in cell behavior ; this , too , can be discounted because this parameter is similar ( rms 
 roughness 37.5  5.5  10 pm ) on all 
 surfaces . the fact that these surfaces ( pea ( x = 
 2 ) , pba ( x = 4 ) , pha ( x = 6 ) ) have 
 similar physical and chemical properties allows the attribution of 
 any differences in cell behavior to subtle changes in surface mobility 
 of the polymers . the overall 
 availability of 
 fn and its domains ( shown schematically in figure 5a ) on the polymer 
 surfaces after adsorption changes for the different surfaces , decreasing 
 with increasing side - chain length ( figure 5b ) . this may be related to the unfolding 
 of fn , meaning that single molecules occupy a greater surface area . in contrast , the availability of the adhesion tripeptide , rgd , was 
 seen to increase on the network - forming surfaces ( pea ( x = 1 ) , pba ( x = 4 ) , pha ( x = 6 ) ) 
 ( figure 5c ) . this demonstrates 
 that the unfolding of fn , due to surface interactions , should positively 
 alter cell adhesion . the availability of the synergy site ( phsrn ) 
 was seen to be significantly elevated on network - forming surfaces , 
 with no significant differences between pea ( x = 
 2 ) , pba ( x = 4 ) , and pha ( x = 6 ) 
 ( figure 5d ) . as previously alluded to , it has been established , in previous work , 
 that the conformation of the protein layer can be affected by surface 
 chemistry . furthermore , it has been shown 
 that the orientation of the fibronectin molecules attached to the 
 surface can have an effect on their bioactivity . the conformational change of fn upon adsorption onto pma ( x = 1 ) versus the rest of the polymers ( x  2 ) observed via afm ( figure 4 ) is reflected on the different domain availability 
 measured by elisa ( figure 5 ) and leads to a different bioactivity of the protein layer . on the more mobile polymers ( x  2 ) , the 
 conformation and domain availability of fn upon adsorption is seen 
 to be comparable ( figure 5 ) . this suggests that whereas conformation plays a role in 
 the change of fn activity between pma and pea , the mobility is the 
 dominant factor behind the differences in cellular response on the 
 protein network - forming surfaces ( figures 7 and 8) . it is 
 well - established that cells interact with surfaces through an interfacial 
 layer of extracellular matrix proteins ( e.g. , fn ) . furthermore , it has been hypothesized that polymer surface 
 mobility can be translated through this protein layer , subsequently 
 being detected by cells . determination 
 of the mobility of the adsorbed protein on the polymer surface , via 
 fluorescence recovery of the bleach border region , has provided an 
 unexpected result , where the interfacial mobility does not correlate 
 directly with glass - transition temperature ( polymer mobility ) . previous 
 work has shown that the mobility of polymer chains at specific temperatures 
 increases as the glass - transition temperature , tg , decreases . therefore , at 37 
 c , polymer surface mobility will increase with direct proportionality 
 to side - chain length , that is , mobility increasing from pma(x = 1 ) < pea(x = 2 ) < pba(x = 4 ) < pha(x = 6 ) . one would thus 
 expect that when the interaction of the protein with the underlying 
 polymer surface is strong enough , as is the case of this family of 
 polymers , the mobility of the protein layer would be a reflection 
 of that of the material surface ; however , a nonmonotonic dependence 
 of protein mobility on polymer mobility was noted . it is believed 
 that the initial decrease in fn mobility between pma ( x = 1 ) and pea ( x = 2 ) is due to the observed formation 
 of a fn nanonetwork on the pea surface , compared with the globular 
 conformation on pma surfaces . the network is likely formed through 
 interaction with the pea ( and pba and pha surfaces ) providing access 
 to the 70 kda amino - terminal fragment i15 , which 
 is vital to matrix formation . it can 
 be postulated that network formation may restrict the freedom of motion 
 of the protein , thus reducing mobility . upon further increase in the 
 polymer mobility ( pba ( x = 4 ) and pha ( x = 6 ) ) , fn mobility is also seen to increase in direct proportionality 
 to the increased mobility of the underlying polymer surface . these 
 data , together with the afm ( figure 4 ) and domain availability ( figure 5 ) results , indicate that the increasing mobility 
 of the polymer surfaces ( from x = 2 to 6 ) compensates 
 for the restricted motion within the fn network , without significantly 
 altering the conformation of the protein . with cells responding 
 to substrate - defined factors , like stiffness and topography , it therefore stands to reason that this change in mobility , mediated 
 through the protein layer , would affect cell behavior . figure 7 shows how these factors can 
 affect cell - mediated reorganization of the protein interface . the 
 synthesis and reorganization of the ecm in vivo it is 
 observed here that increased protein mobility allows cells to organize 
 the protein network with greater ease in a similar to trend to that 
 observed for the protein mobility despite minimal change in morphological 
 cues ( supplementary figure s4 ) . previous 
 work has also observed increased cell - mediated organization with increased 
 protein mobility . here increased protein 
 mobility was achieved by reduction in adsorbed fn density as well 
 as the interspersing of vitronectin with the system , both of which 
 enhanced reorganization . it has also been observed that this interfacial 
 mobility does not transfer through differential recruitment of integrins 
 on each of these polymers ( supplementary figure s5 ) . maximal differentiation is observed 
 on pea ( x = 1 ) and pba ( x = 4 ) surfaces , 
 with significant increase noted compared with collagen ( typical control 
 material for myotube formation ) , pma ( x = 1 ) , and 
 pha ( x = 6 ) . the difference in differentiation potential 
 on these surfaces is accentuated by use of the myosin ii inhibitor , 
 blebbistatin . with minimal changes in 
 the nature of the protein network indicated in figures 4 and 5 as well as 
 the minimal changes in morphological cues observed on l929 cells ( supplementary figure s4 ) , these 
 data can infer , not only , that the interfacial mobility of fn on the 
 material surfaces increases the ability of cells to adhere and reorganize 
 fn , but also that activation of cell contractility is required for 
 mobility - dependent myotube formation . when cell contractility is inhibited , 
 the less mobile surfaces produced greater levels of differentiation . this dependence of cell differentiation on surface mobility is in 
 line with previous observations in mesenchymal stem cells on these 
 polymers . this , in conjunction with the 
 minimal morphological differences of l929 cells , helps to demonstrate 
 the effect of surface mobility on cell behavior . it has been shown that fluorescence labeling of fn allows direct 
 quantification of its mobility at the cell / material interface . a family 
 of polyalkyl acrylates has been used , upon which fn is organized into 
 nanonetworks ( at x  2 ) , so that there are 
 no major differences in the initial state of the protein layer adsorbed 
 on material surfaces . this work establishes that polymer mobility 
 is translated into interfacial mobility of the adsorbed protein layer 
 and that this , in turn , affects cell response . indeed , the ability 
 of cells to reorganize the protein layer is enhanced on the more mobile 
 surfaces . it has been further shown that the sensing of mobility is 
 related to cell contractility ; this influences the ability of cells 
 to differentiate on substrates of different interfacial mobility , 
 altering the cell differentiation response .
cells , by interacting with surfaces 
 indirectly through a layer 
 of extracellular matrix proteins , can respond to a variety of physical 
 properties , such as topography or stiffness . 
 polymer surface mobility 
 is another physical property that is less well understood but has 
 been indicated to hold the potential to modulate cell behavior . 
 polymer 
 mobility is related to the glass - transition temperature ( tg ) of the system , the point at which a polymer transitions 
 from an amorphous solid to a more liquid - like state . 
 this work shows 
 that changes in polymer mobility translate to interfacial mobility 
 of extracellular matrix proteins adsorbed on the material surface . 
 this study has utilized a family of polyalkyl acrylates with similar 
 chemistry but different degrees of mobility , obtained through increasing 
 length of the side chain . 
 these materials are used , in conjunction 
 with fluorescent fibronectin , to determine the mobility of this interfacial 
 layer of protein that constitutes the initial cell  material 
 interface . 
 furthermore , the extent of fibronectin domain availability 
 ( iii9 , iii10 , - the integrin binding site ) , 
 cell - mediated reorganization , and cell differentiation was also determined . 
 a nonmonotonic dependence of fibronectin mobility on polymer surface 
 mobility was observed , with a similar trend noted in cell - mediated 
 reorganization of the protein layer by l929 fibroblasts . 
 the availability 
 of the integrin - binding site was higher on the more mobile surfaces , 
 where a similar organization of the protein into networks at the material 
 interface was observed . 
 finally , differentiation of c2c12 myoblasts 
 was seen to be highly sensitive to surface mobility upon inhibition 
 of cell contractility . 
 altogether , these findings show that polymer 
 mobility is a subtle influence that translates to the cell / material 
 interface through the protein layer to alter the biological activity 
 of the surface .