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Separate different tags with a comma. To include a comma in your tag, surround the tag with double quotes. Please enable cookies in your browser to get the full Trove experience. Skip to content Skip to search. Published Leiden : Brill, Language English View all editions Prev Next edition 6 of 6. Mittal, K. Subjects Cell adhesion molecules. Cell adhesion.

ISBN 13: 9789004190788

Surface active agents. Cellular control mechanisms. Notes Formerly CIP.

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Includes bibliographical references. View online Borrow Buy Freely available Show 0 more links Error bars represent the standard deviation of three independent experiments. Similar levels of MMP2 are found on the different surfaces. By contrast, MMP9 and Runx2 expressions are highly dependent on surface chemistry and with enhanced level on the hydrophilic surfaces. The intensity of each band was referred to the level of Gapdh on the same sample. There is a lack of understanding of the cell-material interaction from an integrated point of view that includes the amount and state of the adsorbed layer of proteins on the material surface, cell adhesion - including integrin expression and focal adhesion formation - cell signaling, matrix reorganization, secretion and degradation, i.

Some efforts have been devoted in the literature to correlate the material surface properties, especially surface chemistry, to protein adsorption and cell adhesion [37] — [40]. From a mechanistic point of view, it is known that the influence of surface chemistry on cell behavior is a consequence of the intermediate layer of proteins adsorbed on the material surface.

That is to say, cells interact with synthetic material surfaces via the previously deposited layer of FN. The sequence of events would be the following: FN is a macromolecule that display a globular conformation in solution; upon adsorption on a particular surface chemistry, interactions between the chemical groups of the surface and the FN domains triggers changes in the conformation of the protein that might lead to complete unfolding and exposure of groups that were hidden in solution. Consequently, the effect of the material surface chemistry is indirectly received by cells via the adsorbed layer of FN.

This is in agreement with results obtained on this family of SAMs by radiolabeling the protein [41]. That is to say, it is known that FN is adsorbed in higher amount on hydrophobic CH 3 surfaces than hydrophilic ones OH [23]. Our results established the existence of a linear correlation between surface wettability Figure 1a and the density of adsorbed FN Figure 1b for this family of mixed SAMs.

It is known that FN has a compact folded structure in physiological buffer that is stabilized through ionic interactions between arms [43]. FN interactions with chemical groups of the substrate CH 3 give rise to conformational changes in the molecule that must lead to the occlusion of the cell binding domains III 9— It is likely that FN orients at the CH 3 surface, so that its hydrophobic segments interact with the methyl groups in PEA, maybe throughout the heparin-binding fragment [44].

Different supramolecular organization of the protein at the material interface is also reflected in protein distribution on the material surface, as directly observed with AFM images in Figure 2 and Figures S1 , S2 , S3 : globular aggregates on the hydrophilic surfaces and fibrillar-like structures on the methyl terminated SAMs. Differences in the availability of FN adhesion domains on the different SAMs influence the initial cell-material interaction, as determined by focal adhesion formation and F-actin cytoskeleton development Figure 3.

The influence of surface chemistry on FN conformation and cell adhesion has been established for SAMs based on different chemical groups. In particular, differences in integrin binding and focal adhesion assembly between OH and CH 3 SAMs most likely resulted from surface chemistry dependent differences in the functional presentation of adsorbed FN, whose major integrin-binding RGD domain is particularly sensitive to the underlying chemistry [41] , [45]. Phosphorylation of FAK has been shown to be sensitive to surface chemistry [45]. In our case, increasing the fraction of hydroxyl groups on the sample leads to similar FAK levels both for gene and protein expression, Figure 4 but with higher and higher levels of phosphorylation of Y, the autophosphorylation site in FAK and a binding site for Src and PI-3 kinases [47] , which suggests a stepwise activation of signaling cascades as a function of hydroxyl groups on the surface increases.

That is to say, activation of signaling pathways is directly related to integrin binding and focal adhesion formation, which are regulated by the availability of binding domains in FN upon adsorption on different chemistries Figures 1 , 2 , 3. It has been demonstrated that FAK regulates cell adhesion strengthening via integrin activation and binding [48]. Moreover, our results are consistent with the role Y autophosphorylation site plays in adhesion strengthening and integrin binding rate.

Mutation or blocking of the Y autophosphorylation site blocked FAK-mediated adhesive responses, cell migration and spreading [48] — [51]. After initial cell adhesion, cells tend to reorganize the adsorbed layer of proteins at the material interface before secreting their own matrix. In this way, FN synthesized by cells assembles into a network of fibrils. During this assembly, however, FN needs to undergo distinct conformational changes, which on adsorption to the substrate can be limited. This may explain why materials surfaces affect FN matrix formation [52] , [53].

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After 2. It has been suggested that the ability of cells to reorganize the adsorbed layer of proteins at the material interface must be a consequence of the strength of interaction between the ECM proteins and the material surface, e. However, additional reasons must be considered when seeking the molecular origin of this fact, which must also be a consequence of the following sequence of events: i the availability of cell adhesion domains after FN adsorption on the SAM surface is higher in the samples with higher OH content Figure 1 ; ii integrin expression and focal adhesion formation is enhanced on the more hydrophilic surfaces Figure 3 , Figure S4 ; iii phosphorylation of FAK is enhanced on the SAMs with higher OH contents Figure 4.

To reorganize the adsorbed layers of proteins, cells must develop mechanical forces on the substrate through a contractile mechanism. Contractility results from dynamic interactions between actin filaments and myosin, which are regulated via phosphorylation of myosin light chain MLC. Rho GTPases control the formation of stress fibers and focal adhesion assembly by modulating MLC phosphorylation and generating actin-myosin contractility [56].

It is well known that inhibitors of contractility also down-regulated tyrosine phosphorylation of FAK [57] — [59] ; more recently it has been shown that contractility-mediated cell forces also require FAK phosphorylation [60] , a fact that supports our reorganization patterns in dependence of the fraction of OH groups: FN is better reorganized on those substrates on which FAK phosphorylation occurs more efficiently Figures 4 , 5. The dynamics of FN secretion and formation of a fibrillar matrix late matrix occurs preferentially on the samples with the higher contents of OH groups Figure S5 ; see e.

These results support the hypothesis that late matrix formation is in need not only of cell adhesion on the substrate, but some cell movements, in the range of the size of the focal adhesion plaques, must take place so matrix deposition occurs normally [61]. Late matrix formation has been related to the ability of cells to rearrange the initially adsorbed protein layer, especially when comparing cell adhesion on hydrophilic and hydrophobic substrates [52] — [54]. Except organization, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling.

Matrix remodeling is a subject of an extensive biomedical research, but how it relates to the biocompatibility of materials remains unclear. The importance of the proteolytic activity of cells has been already considered in the design of biomaterials by incorporating MMP sensitive sequences, which have shown to be mandatory in tissue regeneration in 3D, including cell proliferation, migration and angiogenesis [62] — [65].

Nevertheless, the effect of material chemistry on the proteolytic activity of cells has not been addressed so far. Our results show that the activation of proteolytic routes in these cells is an MMP-dependent phenomenon sensitive to surface chemistry. MMP2 has FN type II repeats inserted into the catalytic domain [67] and it has been found to cleavage FN and vitronectin into small fragments in vivo , which leads to increased cell adhesion and migration [67] , [68].

In this sense, MMP2 expression was constant on every FN-coated surface, regardless the underlying chemistry Figure 6 , 7. By contrast, MMP9 expression increases as the fraction of OH groups in the sample does Figures 6 , 7 , which suggests a direct relationship between FN activity at the cell-material interface and MMP9 expression, as a consequence of a sequence of events that include integrin expression Figure S4 , focal adhesion formation Figure 3 , matrix reorganization Figure 5 and FAK phosphorylation Figure 4.

Runx2 is a key transcription factor in regulation of bone development and osteoblast differentiation.

Surface Tension of Water, Capillary Action, Cohesive and Adhesive Forces - Work & Potential Energy

The consequence of interfering with endogenous Runx2 is a defect in normal osteoblast development or function [73]. It has been reported a direct relationship between MMP activity and osteblasts markers [74]. In this sense, MMP9 is a direct target of Runx2 in bone tissue, suggesting a regulatory link between Runx2, the expression of MMP9, and cell migration [75] , [76].

Figures 6 and 7 also suggest a correlation between Runx2 and MMP9 activation on every surface chemistry.


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That is to say, Figures 6 and 7 show that both protein and gene expression levels of Runx2 and MMP9 are directly correlated, with low values on the CH 3 -rich SAMs, that increases as the OH content in the surface does. This result supports the idea that surface chemistry-mediated activation of MMP9 occurs in a physiological-like way, as its activation at the cell-material interface involves also the upregulation of its direct target Runx2, as occurs in vivo.

Overall, surface chemistry modulates FN dynamics at the cell-material interface. Further, our results demonstrate that surface chemistry is an external parameter able to trigger proteolytic routes in cells in an MMP-dependent manner. Our results demonstrate the ability of synthetic biomaterials as new tools to direct matrix degradation, which must provide the field with new strategies to investigate fundamental aspects of the phenomenon, as well as the inclusion of parameters to take into account during the design of scaffolds for regenerative medicine, aiming at controlling matrix protein dynamics at the cell-material interface.

Surfaces were validated by water contact angle measurements Dataphysics OCA. Si-cantilevers from Veeco Manchester, UK were used with force constant of 2. Drive amplitude was mV and the amplitude setpoint A sp was 1. After adsorption, samples were rinsed in PBS to eliminate the non-adsorbed protein. AFM was performed in the tapping mode immediately after sample preparation. The enhanced chemiluminescence detection system GE Healthcare was used prior to exposing the blot to X-ray.

Image analysis of the western bands was done using in house software [26]. Primary monoclonal antibody HFN7. After washing 0. Each experiment was performed in triplicate. Samples were then rinsed in 0.


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A Leica DMB fluorescent microscope was used for cellular imaging. The ability of cells to reorganize adsorbed FN i.

The role of adhesion energy in controlling cell–cell contacts

The evolution of FN in the ECM was followed by immunofluorescence after different culture times and following the same procedure as described before. The oligonucleotides sequence used for PCR reactions are listed in Table 1. All reactions were done at least per triplicate and RNA template was obtained from independent experiments. All experiments were performed at least three times in triplicate unless otherwise noted.

Surface and Interfacial Aspects of Cell Adhesion

If treatment level differences were determined to be significant, pair-wise comparisons were performed using a Tukey post hoc test. Fibronectin distribution on the different substrates as observed by the phase magnitude in AFM at different magnifications. Cellular reorganization of adsorbed FN and synthesized FN fibrils on the different surfaces after 2.

The technique employed in these figures is immunofluorescence with anti-FN antibody. It is shown the adsorbed FN on the material surface red bottom and the way cells rearrange this layer of FN resulting in black-dark areas as well as enhanced intensity of the fluorescence as a consequence of the formation of FN fibrils by cells. It is shown a broad cell population 20—30 cells per image after different culture times, so that not only FN reorganization is observed but also FN secretion can be accounted for.

Fluorescence distribution and intensity is in agreement with protein expression displayed in Figure 6. The corresponding image for F-actin is also included for the sake of cell identification. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Background The cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Introduction The interaction of cells with foreign materials takes place via the adsorbed layer of proteins such as fibronectin FN , vitronectin, and fibrinogen, representing the soluble matrix proteins in the biological fluids [1].

Download: PPT. Figure 1. Figure 2. Cell adhesion and signaling The organization of proteins involved in the formation of focal adhesion complexes provides an opportunity to learn more about the effectiveness of cell-to-substrate interactions. Figure 3. Figure 4. Fibronectin reorganization and secretion Figure 5 shows the cellular reorganization of adsorbed FN after 2.

Figure 5. Matrix degradation The ability of cells to degrade ECM was investigated by characterizing the expression of two different matrix metalloproteinases MMPs and correlated with Runx2 expression. Figure 6. Figure 7. Discussion There is a lack of understanding of the cell-material interaction from an integrated point of view that includes the amount and state of the adsorbed layer of proteins on the material surface, cell adhesion - including integrin expression and focal adhesion formation - cell signaling, matrix reorganization, secretion and degradation, i.

Statistical analysis All experiments were performed at least three times in triplicate unless otherwise noted.

Cell adhesion

Supporting Information. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5.


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Figure S6. References 1. Grinnell F Focal adhesion sites and the removal of substratum-bound fibronectin. J Cell Biol — View Article Google Scholar 2. Hynes RO Integrins: bidirectional, allosteric signaling machines. Cell — View Article Google Scholar 3. Biomaterials — View Article Google Scholar 4. Nature Reviews Molecular Cell Biology 5: 56— View Article Google Scholar 5. Spie J Tissue engineering and reparative medicine. Ann NY Acad Sci 1—9. View Article Google Scholar 6. Science — View Article Google Scholar 7. Altankov G, Groth T Reorganization of substratum-bound fibronectin on hydrophilic and hydrophobic materials is related to biocompatibility.

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