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![Cell adhesion in response to homo and porcine plasma fibronectin. (A) U2OS cells were plated on coverslips coated with the indicated fibronectin concentration (μg/ml) for 30 min and then the images were taken by phase contrast microscopy. Bar, 100 μm. (B) The plot shows the area of cells spreading on coverslips coated with the indicated fibronectin concentration (μg/ml). Data are mean ± s.e.m. (n = 80 cells in every condition). ***p < 0.001. (C) U2OS cells were plated on the indicated concentration of fibronectin for 30 min and then their cell attachment was measured. Fold of cells remaining attached on the indicated concentration of fibronectin relative to that on 0 μg/ml fibronectin. Data are mean ± s.e.m. (n = 8 independent experiments). *p < 0.05; **p<0.01; ***p < 0.001. (D) TIRFM images of U2OS cells that had been plated for 1.5 h on coverslips coated with the indicated fibronectin concentration and immunostained with paxillin. Bar, 10 μm. (E) Plot shows the sum of the total area of paxillin-marked focal adhesions within a cell versus the fibronectin concentration. Data are mean ± s.e.m. [homo: n =15 cells (0 μg/ml), 20 cells (0.5 μg/ml), 13 cells (1 μg/ml), 20 cells (2 μg/ml), 18 cells (5 μg/ml), 15 cells (10 μg/ml); porcine: n= 9 cells (0 μg/ml), 14 cells (0.5 μg/ml), 11 cells (1 μg/ml), 9 cells (2 μg/ml), 11 cells (5 μg/ml), 10 cells (10 μg/ml)]. *p < 0.05; **p<0.01; ***p < 0.001. (F) Confocal images of U2OS cells that were plated for 1.5 h on coverslips coated with the indicated fibronectin concentration (μg/ml). Bar, 10 μm. (Bottom) Relative fluorescence intensity taken along the line highlighted in the confocal image with the edge being marked with arrows and the distance.](profile/Jean-Cheng-Kuo/publication/318968740/figure/fig2/AS:614013620731908@1523403663697/Cell-adhesion-in-response-to-homo-and-porcine-plasma-fibronectin-A-U2OS-cells-were.png)
Cell adhesion in response to homo and porcine plasma fibronectin. (A) U2OS cells were plated on coverslips coated with the indicated fibronectin concentration (μg/ml) for 30 min and then the images were taken by phase contrast microscopy. Bar, 100 μm. (B) The plot shows the area of cells spreading on coverslips coated with the indicated fibronectin concentration (μg/ml). Data are mean ± s.e.m. (n = 80 cells in every condition). ***p < 0.001. (C) U2OS cells were plated on the indicated concentration of fibronectin for 30 min and then their cell attachment was measured. Fold of cells remaining attached on the indicated concentration of fibronectin relative to that on 0 μg/ml fibronectin. Data are mean ± s.e.m. (n = 8 independent experiments). *p < 0.05; **p<0.01; ***p < 0.001. (D) TIRFM images of U2OS cells that had been plated for 1.5 h on coverslips coated with the indicated fibronectin concentration and immunostained with paxillin. Bar, 10 μm. (E) Plot shows the sum of the total area of paxillin-marked focal adhesions within a cell versus the fibronectin concentration. Data are mean ± s.e.m. [homo: n =15 cells (0 μg/ml), 20 cells (0.5 μg/ml), 13 cells (1 μg/ml), 20 cells (2 μg/ml), 18 cells (5 μg/ml), 15 cells (10 μg/ml); porcine: n= 9 cells (0 μg/ml), 14 cells (0.5 μg/ml), 11 cells (1 μg/ml), 9 cells (2 μg/ml), 11 cells (5 μg/ml), 10 cells (10 μg/ml)]. *p < 0.05; **p<0.01; ***p < 0.001. (F) Confocal images of U2OS cells that were plated for 1.5 h on coverslips coated with the indicated fibronectin concentration (μg/ml). Bar, 10 μm. (Bottom) Relative fluorescence intensity taken along the line highlighted in the confocal image with the edge being marked with arrows and the distance.
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Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate...
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... determine whether homo and porcine fibronectins are comparable when regulating adhesion strength and the organization of F-actin, we initially compared the effect of homo and porcine fibronectin on cell spreading and adhesion. The area of cell spreading was measured after 30 min using U2OS cells that had been seeded onto plates coated with increasing concentrations of homo or porcine fibronectin (Figure 2A). The results revealed that the area of cell spreading increased as the concentration of fibronectin increased for both fibronectins ( Figure 2B). Cell adhesive capacity was also quantified and this revealed that increasing concentrations of homo or porcine fibronectin promoted cell adherence to fibronectin ( Figure 2C). Next, we immunolabelled and visualized the cellular pattern of the FA marker paxillin ( Figure 2D) using cells seeded on increasing concentration of homo or porcine fibronectin for 1.5 h. The results showed an increased area of adhesion www.impactjournals.com/oncotarget fibronectin from homo and porcine plasma. In step 1, cleared plasma was passed through a pre-column of Sepharose CL-4B in order to collect high molecular weight proteins. In step 2, the flow-through materials obtained from Sepharose CL-4B was loaded on a pre-column of gelatin-Sepharose Fast Flow 4B. After removing unbound proteins by sequentially washed with TBS-EDTA, 1 M NaCl and 0.2 M Arginine (Arg), the fibronectin was eluted with 1 M Arg and then dialyzed against TBS for 48 h at 4°C. In step 3, dialyzed material was applied to an Arg-Sepharose Fast Flow 4B column. After washing the column with TBS-EDTA, the fibronectin was eluted from the gel using 0.3 M NaCl/TBS-EDTA, and then dialyzed against TBS for 24 h at 4°C. Finally each of the fibronectins were concentrated using a Vivaspin 20 centrifugal concentrator (Molecular Weight Cut Off: 100 kDa). (B) The eluted fractions obtained from the Arg-Sepharose Fast Flow 4B column were analyzed by Western blotting using antibodies against fibronectin (FN) and Coomassie blue staining. (C) HFF1, Hela and U2OS cells plated on 6-well plates coated with 0 and 10 μg/ml homo or porcine plasma fibronectin for 16 h were assayed for wound- healing migration, which was monitored by time-lapse microscopy. The still images were obtained at the indicated times after wounding. The dotted lines mark the edge of the wound at the 0-h, 6-h and 12-h time points of wound-healing migration. Bar, 200 μm. Bottom: the percentage of wound closure was calculated using Metamorph software. Data are mean ± s.e.m. (n = 5 independent ...
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... determine whether homo and porcine fibronectins are comparable when regulating adhesion strength and the organization of F-actin, we initially compared the effect of homo and porcine fibronectin on cell spreading and adhesion. The area of cell spreading was measured after 30 min using U2OS cells that had been seeded onto plates coated with increasing concentrations of homo or porcine fibronectin (Figure 2A). The results revealed that the area of cell spreading increased as the concentration of fibronectin increased for both fibronectins ( Figure 2B). Cell adhesive capacity was also quantified and this revealed that increasing concentrations of homo or porcine fibronectin promoted cell adherence to fibronectin ( Figure 2C). Next, we immunolabelled and visualized the cellular pattern of the FA marker paxillin ( Figure 2D) using cells seeded on increasing concentration of homo or porcine fibronectin for 1.5 h. The results showed an increased area of adhesion www.impactjournals.com/oncotarget fibronectin from homo and porcine plasma. In step 1, cleared plasma was passed through a pre-column of Sepharose CL-4B in order to collect high molecular weight proteins. In step 2, the flow-through materials obtained from Sepharose CL-4B was loaded on a pre-column of gelatin-Sepharose Fast Flow 4B. After removing unbound proteins by sequentially washed with TBS-EDTA, 1 M NaCl and 0.2 M Arginine (Arg), the fibronectin was eluted with 1 M Arg and then dialyzed against TBS for 48 h at 4°C. In step 3, dialyzed material was applied to an Arg-Sepharose Fast Flow 4B column. After washing the column with TBS-EDTA, the fibronectin was eluted from the gel using 0.3 M NaCl/TBS-EDTA, and then dialyzed against TBS for 24 h at 4°C. Finally each of the fibronectins were concentrated using a Vivaspin 20 centrifugal concentrator (Molecular Weight Cut Off: 100 kDa). (B) The eluted fractions obtained from the Arg-Sepharose Fast Flow 4B column were analyzed by Western blotting using antibodies against fibronectin (FN) and Coomassie blue staining. (C) HFF1, Hela and U2OS cells plated on 6-well plates coated with 0 and 10 μg/ml homo or porcine plasma fibronectin for 16 h were assayed for wound- healing migration, which was monitored by time-lapse microscopy. The still images were obtained at the indicated times after wounding. The dotted lines mark the edge of the wound at the 0-h, 6-h and 12-h time points of wound-healing migration. Bar, 200 μm. Bottom: the percentage of wound closure was calculated using Metamorph software. Data are mean ± s.e.m. (n = 5 independent ...
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... determine whether homo and porcine fibronectins are comparable when regulating adhesion strength and the organization of F-actin, we initially compared the effect of homo and porcine fibronectin on cell spreading and adhesion. The area of cell spreading was measured after 30 min using U2OS cells that had been seeded onto plates coated with increasing concentrations of homo or porcine fibronectin (Figure 2A). The results revealed that the area of cell spreading increased as the concentration of fibronectin increased for both fibronectins ( Figure 2B). Cell adhesive capacity was also quantified and this revealed that increasing concentrations of homo or porcine fibronectin promoted cell adherence to fibronectin ( Figure 2C). Next, we immunolabelled and visualized the cellular pattern of the FA marker paxillin ( Figure 2D) using cells seeded on increasing concentration of homo or porcine fibronectin for 1.5 h. The results showed an increased area of adhesion www.impactjournals.com/oncotarget fibronectin from homo and porcine plasma. In step 1, cleared plasma was passed through a pre-column of Sepharose CL-4B in order to collect high molecular weight proteins. In step 2, the flow-through materials obtained from Sepharose CL-4B was loaded on a pre-column of gelatin-Sepharose Fast Flow 4B. After removing unbound proteins by sequentially washed with TBS-EDTA, 1 M NaCl and 0.2 M Arginine (Arg), the fibronectin was eluted with 1 M Arg and then dialyzed against TBS for 48 h at 4°C. In step 3, dialyzed material was applied to an Arg-Sepharose Fast Flow 4B column. After washing the column with TBS-EDTA, the fibronectin was eluted from the gel using 0.3 M NaCl/TBS-EDTA, and then dialyzed against TBS for 24 h at 4°C. Finally each of the fibronectins were concentrated using a Vivaspin 20 centrifugal concentrator (Molecular Weight Cut Off: 100 kDa). (B) The eluted fractions obtained from the Arg-Sepharose Fast Flow 4B column were analyzed by Western blotting using antibodies against fibronectin (FN) and Coomassie blue staining. (C) HFF1, Hela and U2OS cells plated on 6-well plates coated with 0 and 10 μg/ml homo or porcine plasma fibronectin for 16 h were assayed for wound- healing migration, which was monitored by time-lapse microscopy. The still images were obtained at the indicated times after wounding. The dotted lines mark the edge of the wound at the 0-h, 6-h and 12-h time points of wound-healing migration. Bar, 200 μm. Bottom: the percentage of wound closure was calculated using Metamorph software. Data are mean ± s.e.m. (n = 5 independent ...
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... determine whether homo and porcine fibronectins are comparable when regulating adhesion strength and the organization of F-actin, we initially compared the effect of homo and porcine fibronectin on cell spreading and adhesion. The area of cell spreading was measured after 30 min using U2OS cells that had been seeded onto plates coated with increasing concentrations of homo or porcine fibronectin (Figure 2A). The results revealed that the area of cell spreading increased as the concentration of fibronectin increased for both fibronectins ( Figure 2B). Cell adhesive capacity was also quantified and this revealed that increasing concentrations of homo or porcine fibronectin promoted cell adherence to fibronectin ( Figure 2C). Next, we immunolabelled and visualized the cellular pattern of the FA marker paxillin ( Figure 2D) using cells seeded on increasing concentration of homo or porcine fibronectin for 1.5 h. The results showed an increased area of adhesion www.impactjournals.com/oncotarget fibronectin from homo and porcine plasma. In step 1, cleared plasma was passed through a pre-column of Sepharose CL-4B in order to collect high molecular weight proteins. In step 2, the flow-through materials obtained from Sepharose CL-4B was loaded on a pre-column of gelatin-Sepharose Fast Flow 4B. After removing unbound proteins by sequentially washed with TBS-EDTA, 1 M NaCl and 0.2 M Arginine (Arg), the fibronectin was eluted with 1 M Arg and then dialyzed against TBS for 48 h at 4°C. In step 3, dialyzed material was applied to an Arg-Sepharose Fast Flow 4B column. After washing the column with TBS-EDTA, the fibronectin was eluted from the gel using 0.3 M NaCl/TBS-EDTA, and then dialyzed against TBS for 24 h at 4°C. Finally each of the fibronectins were concentrated using a Vivaspin 20 centrifugal concentrator (Molecular Weight Cut Off: 100 kDa). (B) The eluted fractions obtained from the Arg-Sepharose Fast Flow 4B column were analyzed by Western blotting using antibodies against fibronectin (FN) and Coomassie blue staining. (C) HFF1, Hela and U2OS cells plated on 6-well plates coated with 0 and 10 μg/ml homo or porcine plasma fibronectin for 16 h were assayed for wound- healing migration, which was monitored by time-lapse microscopy. The still images were obtained at the indicated times after wounding. The dotted lines mark the edge of the wound at the 0-h, 6-h and 12-h time points of wound-healing migration. Bar, 200 μm. Bottom: the percentage of wound closure was calculated using Metamorph software. Data are mean ± s.e.m. (n = 5 independent ...
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... 2 ) as the concentration of each fibronectin increased; the quantification was in terms of the area of paxillin-marked adhesion ( Figure 2E). To further determine whether there were fibronectin-dependent changes in adhesion strength that affected actin cytoskeleton organization, U2OS cells were plated onto plates coated with increasing concentration of homo or porcine fibronectin for 1.5 h; these were then immunolabelled for F-actin. The results revealed a dense network of F-actin covering the peripheral region of cells for both fibronectins, and the organization of the actin cytoskeleton spread to entire cells as the size of the cells increased ( Figure 2F). Similar results were observed using Hela cells (Supplementary Figure 2) and human foreskin fibroblasts (HFF1) cells (Supplementary Figure 3). The two isolated fibronectins have been shown to be able to geometrically patterned and used as substrates to control the microenvironment of individual mesenchymal stem cells; this in turn modulated the decision of cells to adhere and spread [23][24][25]. We found that patterned substrates coated with increasing concentrations of either homo or porcine fibronectin had similar effects in terms of controlling cell shape, modifying the degree of cell spreading on micro- patterned areas and affecting the organization of paxillin- marked focal adhesions and F-actin (Supplementary Figure 4). Thus it would seem that homo and porcine fibronectins are equally capable of promoting cell adhesion strength, modifying actin cytoskeleton organization and affecting cell migration and that these changes occur in a fibronectin concentration-dependent ...
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... 2 ) as the concentration of each fibronectin increased; the quantification was in terms of the area of paxillin-marked adhesion ( Figure 2E). To further determine whether there were fibronectin-dependent changes in adhesion strength that affected actin cytoskeleton organization, U2OS cells were plated onto plates coated with increasing concentration of homo or porcine fibronectin for 1.5 h; these were then immunolabelled for F-actin. The results revealed a dense network of F-actin covering the peripheral region of cells for both fibronectins, and the organization of the actin cytoskeleton spread to entire cells as the size of the cells increased ( Figure 2F). Similar results were observed using Hela cells (Supplementary Figure 2) and human foreskin fibroblasts (HFF1) cells (Supplementary Figure 3). The two isolated fibronectins have been shown to be able to geometrically patterned and used as substrates to control the microenvironment of individual mesenchymal stem cells; this in turn modulated the decision of cells to adhere and spread [23][24][25]. We found that patterned substrates coated with increasing concentrations of either homo or porcine fibronectin had similar effects in terms of controlling cell shape, modifying the degree of cell spreading on micro- patterned areas and affecting the organization of paxillin- marked focal adhesions and F-actin (Supplementary Figure 4). Thus it would seem that homo and porcine fibronectins are equally capable of promoting cell adhesion strength, modifying actin cytoskeleton organization and affecting cell migration and that these changes occur in a fibronectin concentration-dependent ...
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... 2 ) as the concentration of each fibronectin increased; the quantification was in terms of the area of paxillin-marked adhesion ( Figure 2E). To further determine whether there were fibronectin-dependent changes in adhesion strength that affected actin cytoskeleton organization, U2OS cells were plated onto plates coated with increasing concentration of homo or porcine fibronectin for 1.5 h; these were then immunolabelled for F-actin. The results revealed a dense network of F-actin covering the peripheral region of cells for both fibronectins, and the organization of the actin cytoskeleton spread to entire cells as the size of the cells increased ( Figure 2F). Similar results were observed using Hela cells (Supplementary Figure 2) and human foreskin fibroblasts (HFF1) cells (Supplementary Figure 3). The two isolated fibronectins have been shown to be able to geometrically patterned and used as substrates to control the microenvironment of individual mesenchymal stem cells; this in turn modulated the decision of cells to adhere and spread [23][24][25]. We found that patterned substrates coated with increasing concentrations of either homo or porcine fibronectin had similar effects in terms of controlling cell shape, modifying the degree of cell spreading on micro- patterned areas and affecting the organization of paxillin- marked focal adhesions and F-actin (Supplementary Figure 4). Thus it would seem that homo and porcine fibronectins are equally capable of promoting cell adhesion strength, modifying actin cytoskeleton organization and affecting cell migration and that these changes occur in a fibronectin concentration-dependent ...
Citations
... The sorting of fibronectin into exosomes depends on binding to integrins [104]. N-glycans on fibronectin have a role in the positive regulation of cell adhesion and directed cell migration via integrin-mediated signals [105]. ...
Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available. In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided. In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.
... In vitro studies have demonstrated that FN is important for cell migration and attachment to the ECM, as well as cell-to-ECM communication, a process known as dynamic reciprocity. 15,20 Such interactions may at least partially explain the robust evidence of host cell infiltration, neovascularization, and tissue remodelling observed in vivo. Thus, AL-only, AL-CL, or AL-CL-AL grafts that have been decellularized may lose up to 80% of the FN available in FTPM, as well as up to 50% of other important proteins, such as bFGF, IL-1RA, and TIMP-2, among others not tested here. ...
Allografts derived from live‐birth tissue obtained with donor consent have emerged as an important treatment option for wound and soft tissue repairs. Placental membrane derived from the amniotic sac consists of the amnion and chorion, the latter of which contains the trophoblast layer. For ease of cleaning and processing, these layers are often separated with or without re‐lamination and the trophoblast layer is typically discarded, both of which can negatively affect the abundance of native biological factors and make the grafts difficult to handle. Thus, a full‐thickness placental membrane that includes a fully‐intact decellularized trophoblast layer was developed for homologous clinical use as a protective barrier and scaffold in soft tissue repairs. Here, we demonstrate that this full‐thickness placental membrane is effectively decellularized while retaining native extracellular matrix (ECM) scaffold and biological factors, including the full trophoblast layer. Following processing, it is porous, biocompatible, supports cell proliferation in vitro, and retains its biomechanical strength and the ability to pass through a cannula without visible evidence of movement or damage. Finally, it was accepted as a natural scaffold in vivo with evidence of host‐cell infiltration, angiogenesis, tissue remodelling, and structural layer retention for up to 10 weeks in a murine subcutaneous implant model.
... Collagen is the main structural protein of the extracellular matrix and promotes cell adhesion to substrates [26]. Fibronectin is another extracellular matrix protein and plays a major role in cell adhesion through the generation of focal adhesions [27]. Adhesion of colorectal cancer cells HCT116 to the hydrophilic zone with and without biochemical functionalization was investigated. ...
In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell–material and cell–cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.
... Numerous studies have employed FN coating on platforms to study cell attachment and viability, given the diverse cell-binding properties of FN, its ability to enhance cell spreading and migration, and its role in essential biological processes [18]. FN interacts with and activates cell surface integrin receptor, which then connects with the actin cytoskeleton in cells and forms focal adhesions (FAs) to regulate cell migration [24]. ...
... Both NP460 and NPC43 cells had a higher traversing probability of 30.0% and 91.9%, respectively. The FN coating on the trench sidewalls of the top and bottom layers attracted more cells into the narrow trenches by promoting the assembly of FAs, which play a role in force transmission and regulate migration [24]. The hydrophobic ridges of the top layers hindered the cells from returning to the top layer ridges. ...
The extracellular matrix (ECM) serves as a complex scaffold with diverse physical dimensions and surface properties influencing NPC cell migration. Polydimethylsiloxane (PDMS), a widely used biocompatible material, is hydrophobic and undesirable for cell seeding. Thus, the establishment of a biomimetic model with varied topographies and surface properties is essential for effective NPC43 cell separation from NP460 cells. This study explored how ECM surface properties influence NP460 and NPC43 cell behaviors via plasma treatments and chemical modifications to alter the platform surface. In addition to the conventional oxygen/nitrogen (O2/N2) plasma treatment, O2 and argon plasma treatments were utilized to modify the platform surface, which increased the hydrophilicity of the PDMS platforms, resulting in enhanced cell adhesion. (3-aminopropyl)triethoxysilane and fibronectin (FN) were used to coat the PDMS platforms uniformly and selectively. The chemical coatings significantly affected cell motility and spreading, as cells exhibited faster migration, elongated cell shapes, and larger spreading areas on FN-coated surfaces. Furthermore, narrower top layer trenches with 5 µm width and a lower concentration of 10 µg/mL FN were coated selectively on the platforms to limit NP460 cell movements and enhance NPC43 cell separation efficiency. A significantly high separation efficiency of 99.4% was achieved on the two-layer scaffold platform with 20/5 µm wide ridge/trench (R/T) as the top layer and 40/10 µm wide R/T as the bottom layer, coupling with 10 µg/mL FN selectively coated on the sidewalls of the top and bottom layers. This work demonstrated an innovative application of selective FN coating to direct cell behavior, offering a new perspective to probe into the subtleties of NPC cell separation efficiency. Moreover, this cost-effective and compact microsystem sets a new benchmark for separating cancer cells.
... Fibronectin possesses binding sites for heparin, fibrin, and collagen [18], as well as several integrin binding sites which may be recognized by different cell lines [19][20][21][22]. Fibronectin is found to favor cell adhesion [23,24] and spreading [25,26]. Hence, coating the surface of biomaterials with fibronectin has been a viable strategy for enhancing cellular response to the biomaterials. ...
Titanium (Ti-6Al-4V) substrates were functionalized through the covalent binding of fibronectin, and the effect of the existence of this extracellular matrix protein on the surface of the material was assessed by employing mesenchymal stem cell (MSC) cultures. The functionalization process comprised the usage of the activation vapor silanization (AVS) technique to deposit a thin film with a high surface density of amine groups on the material, followed by the covalent binding of fibronectin to the amine groups using the N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The biological effect of the fibronectin on murine MSCs was assessed in vitro. It was found that functionalized samples not only showed enhanced initial cell adhesion compared with bare titanium, but also a three-fold increase in the cell area, reaching values comparable to those found on the polystyrene controls. These results provide compelling evidence of the potential to modulate the response of the organism to an implant through the covalent binding of extracellular matrix proteins on the prosthesis.
... Fibronectin possesses binding sites for heparin, fibrin and collagen [18], as well as several integrin binding sites which may be recognized by different cell lines [19][20][21][22]. Fibronectin is found to favor cell adhesion [23,24] and spreading [25,26]. Hence, coating the surface of biomaterials with fibronectin has been a viable strategy for enhancing cellular response to the biomaterials. ...
Titanium (Ti-6Al-4V) substrates were functionalized through the covalent binding of fibronectin, and the effect of the presence of this extracellular matrix protein on the surface of the material was assessed employing mesenchymal stem cell (MSC) cultures. The functionalization process com-prised the usage of the activation vapor silanization (AVS) technique to deposit a thin film with a high surface density of amine groups on the material, followed by the covalent binding of fi-bronectin to the amine groups using the N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hy-drochloride / N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The biological effect of the fibronectin on murine MSCs was assessed in vitro. It was found that functionalized samples not only showed enhanced initial cell adhesion compared with bare titanium, but also a three-fold increase in the cell area, reaching values comparable to those found on the polystyrene controls. These results represent a clear indication of the potential of modulating the response of the or-ganism to an implant through the covalent binding of extracellular matrix proteins on the pros-thesis.
... Previous research demonstrated that collagen was responsible for promoting hMSC adhesion and proliferation when compared to polystyrene coated plates (Somaiah et al. 2015;Salzig et al. 2016). Other studies have also evaluated the impact of molecules such as fibronectin, which was previously reported to enhance cell attachment and modulate cell differentiation (Veevers-Lowe et al. 2011;Somaiah et al. 2015;Salzig et al. 2016;Hsiao et al. 2017). While fibronectin is involved in cell adhesion and cell migration processes, collagen has a critical role to ensure a regulated functioning of the connective tissues throughout the body. ...
... Additionally, the authors have reported that collagen substrates may be responsible for enhancing hMSCs' osteogenic ability, which may benefit bone-related clinical applications (Somaiah et al. 2015). Similarly, fibronectin has been demonstrated to play a major role in cell adhesion and migration (Ruoslahti 1984;Ramos et al. 2016;Hsiao et al. 2017). Previous studies have reported that hMSC migration is also regulated by fibronectin (Veevers-Lowe et al. 2011). ...
Bioactive materials interact with cells and modulate their characteristics which enable the generation of cell-based products with desired specifications. However, their evaluation and impact are often overlooked when establishing a cell therapy manufacturing process. In this study, we investigated the role of different surfaces for tissue culture including, untreated polystyrene surface, uncoated Cyclic Olefin Polymer (COP) and COP coated with collagen and recombinant fibronectin. It was observed that human mesenchymal stromal cells (hMSCs) expanded on COP-coated plates with different bioactive materials resulted in improved cell growth kinetics compared to traditional polystyrene plates and non-coated COP plates. The doubling time obtained was 2.78 and 3.02 days for hMSC seeded in COP plates coated with collagen type I and recombinant fibronectin respectively, and 4.64 days for cells plated in standard polystyrene treated plates. Metabolite analysis reinforced the findings of the growth kinetic studies, specifically that cells cultured on COP plates coated with collagen I and fibronectin exhibited improved growth as evidenced by a higher lactate production rate (9.38 × 10⁵ and 9.67 × 10⁵ pmol/cell/day, respectively) compared to cells from the polystyrene group (5.86 × 10⁵ pmol/cell/day). This study demonstrated that COP is an effective alternative to polystyrene-treated plates when coated with bioactive materials such as collagen and fibronectin, however COP-treated plates without additional coatings were found not to be sufficient to support cell growth. These findings demonstrate the key role biomaterials play in the cell manufacturing process and the importance of optimising this selection.
... Fibronectin induces strong adhesion in many cell types. [52,53] Therefore, high concentrations make cells so adherent that their cell speed decreases, while integrins are less engaged at lower concentrations of fibronectin, resulting in lower adhesion and freer movement. ...
Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). The present work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses to macrophages in 2D environments. Laminin 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared to cells on fibronectin. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. The present study also demonstrates that laminin 111 exerts a suppressive effect toward fibronectin, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated in the cell downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory modes based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in 3D and in vivo contexts warrants further study.
... Some studies have already shown that poloxamer can induce osteogenic and adipogenic differentiation in mesenchymal stem cells, then poloxamer may also induce osteogenic differentiation of pre-osteoblast cells [55,56]. Regarding bioactive glasses, their ionic dissolution products, such as calcium and phosphate ions, can interact with undifferentiated cells and induce their osteogenic differentiation [57][58][59]. Considering the PL-BG5Ho formulation, even though the poloxamer and the bioactive glass do not show a synergic effect on an early stage of differentiation, we must consider both formulation components as a mineralizing agent role. ...
Bisphosphonates are a class of drugs that induce bone cancer cell death and favor bone regeneration, making them suitable for bone cancer treatment. However, when combined with bioactive glasses to enhance bone regeneration, a chemical bond between biphosphonates and the glass surface inactivates their mechanism of action. A new colloidal hydrogel-based drug delivery system could overcome that limitation once bisphosphonates, such as zoledronic acid (ZA), are incorporated into hydrogel micelles, avoiding their interaction with the glass surface. In this work, we proposed formulations based on a poloxamer 407 thermo-responsive hydrogel matrix containing holmium-doped bioactive glass nanoparticles and different concentrations (0.05 and 5 mg/mL) of ZA. We characterized the influence of the glass and the ZA on the hydrogel properties. In addition, a drug concentration screening was performed, and biological characterizations evaluated the best result. The biological characterization consisted of evaluating cytotoxicity and in vitro bone regeneration ability through cell migration and quantification of genes related to osteogeneses through RT-PCR. The results suggest that the addition of glasses and ZA to the poloxamer did not significantly influence the sol-gel transition of the hydrogels (around 13 °C) regardless of the ZA content. However, the ZA at high concentration (PL-ZA100) decreased the enthalpy of gel formation from 68 to 43 kJ.mol-1 when compared with the pure hydrogel formulation (PL), suggesting a water structurer role of ZA, which is withdrawn when glass particles are added to the system (PL-BG5Ho-ZA100). Solid-state 31P nuclear resonance spectroscopy results showed that part of the ZA is chemically bonded to the glass surface, which explains the withdrawal in the water structurer role of ZA when the glasses were incorporated into the hydrogel. Besides, based on the drug release results, we proposed a model where part of the ZA is "free," encapsulated in the hydrogel matrix, while another part of the ZA is bonded to the glass surface. Finally, considering the in vitro results and our proposed model, the ratio between "free" and "bonded" ZA in our drug delivery systems showed in vitro evidence of a cancer treatment that selectively kills osteosarcoma cells while still favoring an osteogenic microenvironment. By overcoming the limitation of combining bisphosphonates with bioactive glasses, hydrogel-based drug delivery systems can be a solution for the development of new formulations proposed for bone cancer treatment in conjunction with bone regeneration.
... This finding could be attributed to that the continued release of calcium ions during setting time of MTA material (29), although it is not component of MTA (30), has the ability to stimulate several signaling molecules, such as transforming growth factor-β (TGF-β), macrophage colony-stimulating factor (MCSF). Calcium ions also can form crystals that attract fibronectin, which controls cellular adhesion and differentiation (31). In addition, MTA can uncouple and activate growth factors nested in the proximal dentin (32) and also increase the secretion of TGF-β1 from pulp cells (33). ...
