Spreading Of Human Fibroblasts Research Articles

Hydrogels with Dual Gradients of Mechanical and Biochemical Cues for Deciphering Cell-Niche Interactions.

Cell niche is a multifactorial environment containing complex interactions between biochemical and physical cues. Although extensive studies have examined the effects of biochemical or physical cues alone on cell fate, how biochemical and mechanical signals interact to influence cell fates remains largely unknown. To address this challenge, here we report a polyethylene glycol-based gradient hydrogel platform as biomimetic cell niche containing independently tunable matrix stiffness and biochemical ligand density. The versatility of this platform is demonstrated by fabricating and characterizing single gradient or orthogonally aligned dual gradient hydrogels. These gradients result in differential elongation and spreading of human fibroblasts. Both hydrogel stiffness and biochemical ligand density are independently tunable by sequential photopolymerization. By controlling light exposure, a broad range of hydrogel stiffness and different types/doses of biochemical ligands can be incorporated. Such tunability facilitates customization of this platform for investigating complex cell-niche interactions associated with various cell types, such as stem cells and cancer cells. The outcomes of such studies may help identify optimal niche cues to promote desiralbe stem fates and tissue regeneration or inhibit diseases progression.

Modification of Bacterial Cellulose with Organosilanes to Improve Attachment and Spreading of Human Fibroblasts.

Bacterial Cellulose (BC) synthesized by Acetobacter xylinum has been a promising candidate for medical applications. Modifying BC to possess the properties needed for specific applications has been reported. In this study, BCs functionalized by organosilanes were hypothesized to improve the attachment and spreading of Normal Human Dermal Fibroblast (NHDF). The BC gels obtained from biosynthesis were dried by either ambient-air drying or freeze drying. The surfaces of those dried BCs were chemically modified by grafting methyl terminated octadecyltrichlorosilane (OTS) or amine terminated 3-aminopropyltriethoxysilane (APTES) to expectedly increase hydrophobic or electrostatic interactions with NHDF cells, respectively. NHDF cells improved their attachment and spreading on the majority of APTES-modified BCs (∼70-80% of area coverage by cells) with more rapid growth (∼2.6-2.8× after incubations from 24 to 48h) than on tissue culture polystyrene (∼2×); while the inverse results (< 5% of area coverage and stationary growth) were observed on the OTS-modified BCs. For organosilane modified BCs, the drying method had no effect on in vitro cell attachment/spreading behaviors.

Relationship between surface properties (roughness, wettability) of titanium and titanium alloys and cell behaviour

Cell attachment and spreading to titanium-based alloy surfaces is a major parameter in implant technology. In this paper, substratum surface hydrophobicity, surface free energy, interfacial free energy and surface roughness were investigated to ascertain which of these parameters is predominant in human fibroblast spreading. Two methods for contact angle measurement were compared: the sessile drop method and the captive bubble two-probe method. The relationship between surface roughness and the sessile drop contact angles of various engineered titanium surfaces such as commercial pure titanium (cp-Ti), titanium–aluminium–vanadium alloy (Ti–6Al–4V), and titanium–nickel (NiTi), was shown. Surface free energy (SFE) calculations were performed from contact angles obtained on smooth samples based on the same alloys in order to eliminate the roughness effect. SFE of the surfaces have been calculated using the Owens–Wendt (OW) and Van Oss (VO) approaches with the sessile drop method. The OW calculations are used to obtain the dispersive ( γ d) and polar ( γ p) component of SFE, and the VO approach allows to reach the apolar ( γ LW) and the polar acid–base component ( γ ab) of the surface. From captive bubble contact angle experiments (air or octane bubble under water), the interfacial free energy of the different surfaces in water was obtained. A relationship between cell spreading and the polar component of SFE was found. Interfacial free energy values were low for all the investigated surfaces indicating good biocompatibility for such alloys.

Protein adsorption and fibroblast adhesion on irradiated polysiloxane surfaces.

A very peculiar case of differential cell response towards polysiloxane surfaces of very similar composition is investigated. Poly(hydroxymethylsiloxane) (PHMS) surfaces treated either by O(2)-plasma or 6 keV Ar(+)-beams have been used to test the adhesion, proliferation and spreading of human fibroblasts. The surface chemical structure and nanomorphology were investigated by means of X-ray photoelectron spectroscopy (XPS), surface free energy measurements and atomic force microscopy (AFM). In spite of the close compositional and morphological similarity of the modified surfaces, the viability of the adhered cells, evaluated by means of optical microscopy and epifluorescence microscopy, was found to be very different in the two cases. The study of the features of the adsorbed protein adlayer on the two types of surfaces was performed by XPS and AFM and indicated that the overall cell behavior is connected to a quite different protein aggregation process, occurring respectively on the plasma- and Ar(+)-modified polysiloxane surfaces. It is suggested that the specific biological response of the modified surfaces is determined by the chemical structure at the nanometric level.

The influence of substratum surface free energy on growth and spreading of human fibroblasts in the presence and absence of serum proteins.

To determine whether the surface free energy of polymer materials influences the spreading of 13 polymers and glass were related to spreading and growth of human skin fibroblasts. Experiments were performed in both the presence and absence of serum proteins. We calculated the surface free energy from contact angles of phosphate-buffered saline (PBS), n-propanol/PBS mixtures, and alpha-bromonaphthalene on the polymers, using the concept of polar and dispersion components accounting for spreading pressures. Cell spreading and substratum surface free energy (gamma s) showed a characteristic sigmoid relationship both in the presence and in the absence of serum proteins; good spreading only occurred when gamma s was higher than approximately 57 erg . cm-2. In the presence of serum proteins, cell spreading is similar on most materials; only few materials show relatively high cell spreading. Cell growth in the presence of serum proteins did not differ significantly on the various polymers with reference to their gamma s values. In contrast, two groups of polymers could be distinguished in the absence of serum with respect to cell growth. The first group showed increasing cell growth with increasing gamma s, whereas the second group showed consistently low cell growth. The results demonstrate the complex relationship between cell spreading and substratum surface free energy as well as the role of serum proteins in modifying the surface characteristics of polymers in relation to cell spreading and growth.

Qualitative and quantitative differences in spreading of human fibroblasts on various protein coats. Modulation by treatment of the cells with amines.

In contrast to established cell lines, normal human skin fibroblasts spread on their own fibronectin. The present investigation has examined whether human fibroblasts, like established cell lines, would be capable of spreading on substrata coated with proteins with different reactivity towards the cell surface. Coverslips were coated with human serum, fibronectin, alpha 2 macroglobulin-trypsin, a polyspecific anti-fibroblast antibody and a polyspecific anti-calf-serum antibody. The attachment of the cells to these substrate was of the same extent. Spreading was examined qualitatively using phase, interference contrast and reflection contrast optics on live cells, as well as scanning electron microscopy on fixed cells. To quantitate the maximum degree of cell spreading a semi-automated system was used, which measured the cell perimeter on a large number of cells. The distributions of the degree of cell spreading on the five substrata were compared statistically. The qualitative and quantitative differences observed on the various substrata could be further differentiated by adding various amines to the cells during 60 min spreading or during a 30 min preincubation before spreading. No strict correlation could be found between the effect of the amines on attachment or on spreading and their presumed effects on cellular transglutaminases. The results clearly indicate that the spreading of human fibroblasts can be modulated by the nature of the substratum and that, by using quantitative methods, these differences in behaviour can be measured accurately.

Role of the a2M-receptor in attachment and spreading of human fibroblasts

Surface molecules are thought to be involved in the attachment and spreading of cells on a substratum coated with specific ligands (Rauvala et al., 1981; Carter et al., 1981; Michl et al., 1979). Since human fibroblasts carry a surface receptor for a 2Macroglobulin-protease complexes (Van Leuven et al., 1979), we examined whether these cells would attach and spread selectively on a substratum coated with the specific ligand. The presence of the receptor on dissociated cells was demonstrated, as well as the stability of the protein coat on the substratum. Moreover, we determined that the a 2M-T coat expressed the antigen observed only on molecules which interact selectively with the cells. Cell attachment occurred rapidly on complexed but at the same rate as on uncomplexed a 2M and on a 2M derivatized with mono-dansylcadaverine ( a 2M-DC); the latter was shown not to interact specifically with the cells (Van Leuven et al., 1979). Cell spreading on a 2M-T was limited and had the same extent as on a 2M-DC. The cells remained capable of ingesting a 2M-trypsin complexes during attachment, they did not ingest the a 2M-trypsin substratum and their attachment or spreading was not inhibited by the addition of an excess a 2M-trypsin in the attachment medium. Our results do suggest that the a 2M receptor is not involved in attachment or spreading of fibroblasts on an a 2M-coat or that its involvement does not result in facilitated spreading of the cells.

Initial adhesion of human fibroblasts in serum-free medium: Possible role of secreted fibronectin

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secreted and adsorbed on the substratum surface. Human skin fibroblasts attached and spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblast attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2–8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kidney cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells onto the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-insoluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.