Variables affecting integrin expression of cells grown on substrates in vitro Several studies have investigated the factors which affect integrin expression by

cells cultured on different substrates. It was reported that differences were seen depending on the type of cells used, and the substrates on which the cells were grown (Hormia and Kononen, 1994; Sinha et al., 1994; Dean et al, 1995; Gronowicz and McCarthy, 1996; Sinha and Tuan, 1996).

1.6.4.1 Integrin expression is affected by both substrate composition and topography

Sinha and Tuan (1996) cultured primary human OB cells on Ti and cobalt- chromium (Co-Cr) discs, and control tissue culture polystyrene. They used immunohistochemistry to identify the integrins expressed. Significant differences in integrin expression were noted. These were tabulated as follows, where (+) indicates greater than 50% of cells cultured on a particular surface demonstrated positive staining.

Table 1.3 Summary of the integrin subunits expressed by primary human cells cultured on substrates of different surface roughness

OB

Substrate «2 tt3 « 4 « 5 a^ a y _{Pi P3}

Polished Ti + + 4- + + +

Rough Ti -h + 4- + +

Polished Co-Cr + + 4- -h 4-

Rough Co-Cr -I- -h -k +

Polystyrene + + 4-

The results show that except for as and as, all integrin subunits were expressed by cells grown on the control tissue culture polystyrene surface. Regarding the metal substrates, as and ae subunits were only noted on cells cultured on polished Ti, while Ps was detected on all surfaces, except on rough Co-Cr. as was not detected on any of the metal surfaces. This suggests that the Fn receptor was not used by the cells to attach to the metals, and this may indicate that Fn may not be easily adsorbed on these surfaces. The suggestion that Fn was not used for the attachment of OB cells to metallic substrates is in agreement with the results of Schneider and Burridge (1994a), where it was shown that serum Vn was used for the adhesion of OB cells on Ti and glass. Only when plated on Fn coated surfaces, did the OB cells concentrate Pi integrins within their focal adhesions, indicating the presence of the major Fn receptor, agpi.

By selective removal of serum Vn and Fn from the culture medium, Steele et al.

(1992) showed that the initial attachment and spreading of fibroblasts and endothelial cells onto synthetic polymeric surfaces, such as tissue culture polystyrene, were also dependent upon the serum Vn component, and not on the Fn component. In another study, Howlett et al. (1994) showed that the attachment of human bone derived cells plated in medium during the first 90 minutes of culture was also primarily a result of adsorption of serum Vn onto the surface of tissue culture polystyrene, cpTi, stainless steel and alumina. Selective removal of Vn from the tissue culture medium resulted in 70% loss of cell adhesion and spreading on these materials, compared to only a reduction of <5% when Fn was depleted from the medium.

Sinha and Tuan (1996) also detected positive staining for az, a4, and pi on all the substrates, although previous studies on primary human bone integrin expression quoted earlier had found relatively low amounts of a2 and no tt4. az is a known collagen receptor, and this suggests that OB cells may bind to collagen through an integrin mediated interaction on the substrate where az was expressed. This is true for all bone cells, since 90% of the protein expressed is collagen. However, as was only seen on polished Ti and not on rough, and also not on Co-Cr regardless of the texture. This may reflect differences in the ECM ligand synthesis or serum protein ligand adsorption due to the different surface roughness, although the chemical composition of the substrates may be the same. Nevertheless, as pi is a multifunctional receptor which can bind to Fn, collagen and laminin, and this study suggests that Fn may also be used by the OB cells to attach to polished Ti.

Sinha and Tuan (1996) also observed an association between integrin expression and cell morphology. They noted that OB cells on polished Ti surfaces were larger and more elongated, compared to cells on polished Co-Cr. Both as and ae were expressed on polished Ti, but not on polished Co-Cr. They also noted that the cells on rough Ti were more rounded than those on rough Co-Cr. The only difference in integrin expression on these substrates was the absence of Ps on rough Ti. The authors therefore suggested that as, ae and Ps may play significant roles in the cell spreading process, since in an earlier experiment, Sinha et al. (1994) found that cytoskeletal organisation and stability were enhanced on Ti, compared to Co-Cr and PS.

The suggestion that cell shape may be related to integrin expression agrees with that of Chen et al. (1992), who found that integrin levels were up-regulated in cells which had been kept in suspension for some time (up to 24 h), before being allowed to adhere to substrates. However, if under adherent conditions the cells did not express a particular integrin subunit, then leaving the cells in suspension or in a rounded condition would not lead to induction of that subunit.

In another experiment, Gronowicz and McCarthy (1996) cultured SaOS-2 cells on discs made of 'Tivanium' (a Ti alloy), ’Zimaloy' (a Co-Cr alloy), 'plastic' and glass.

The metals were prepared similarly, i.e. both were sandblasted with 600 grit AI2O3, and then blasted with 100 mesh glass beads. The authors used Western blot analysis to identify and compare the integrins detected just prior to inoculation onto the substrates, and then 24 h after plating. The experiments were carried out in serum-ffee medium (SFM), and in some instances the cells were treated with cycloheximide to prevent synthesis of endogenous proteins. The integrins expressed are shown in Table 1.4. Changes in the integrin expression are indicated by arrows showing the magnitude of reduction or increased expression at the end of 24 h after cell inoculation.

Table 1.4 Summary of the integrin subunits expressed by SaOS-2 cells cultured in SFM on different substrates with similar surface topography

Substrate _«1 «2 « 5 ttv _Pi Ps

Tivanium i 2.7X Î iJxT 4.9xt I

Zimaloy i 1.8xt 1.7xt 3.8xt :

Plastic 2.5xt t 2x f 1.9xt 9.5XÎ small Î

Glass i small f 6.4xt i

The main finding in this study was that cells attached to the metals even when protein synthesis was inhibited. The authors concluded that protein synthesis was not needed for cell attachment, and that there was direct integrin binding to the metals, since ROD peptides in the medium inhibited cell attachment to Tivanium and Zimaloy by 28% and 40%, respectively. However, the mechanisms by which integrins may bind directly to the metals are unknown. When cells were pre-treated with polyclonal antibodies to Fn prior to inoculation on the discs, binding to Tivanium and Zimaloy was inhibited by 63% and 49%, respectively. However, when cells were pre-treated with antibody to the Vn receptor, no significant effect on cell adhesion to the metals was noted. Integrin expression after 24 h of attachment showed a great increase in the Fn receptor (aspi) but not in the Vn receptor (avPs or avPs). This confirms the attachment inhibition results, which show that the majority of the OB cells in this study attached to metals via the Fn receptor, and not via the Vn receptor.

4 h on glass, smooth Ti or etched Ti surfaces expressed localised avPs focal contacts, but only a diffuse avPi distribution. Cells grown on rough sandblasted Ti expressed a diffuse distribution of both ayp3 and aypi. Vinculin-containing focal contacts were seen in cells grown on glass and the smoother Ti, but not on the cells grown on sandblasted Ti surfaces. This finding suggests that fibroblasts bind to Vn through local focal contacts on smooth Ti, and bind to Fn through a diffused ECM contact association (Chen and Singer, 1982). However, as the study was carried out in complete medium, it is not certain if the attachment proteins were incorporated from the serum in the culture medium, or were cell derived, or both, as Fn and Vn are present in serum, and both promote cell adhesion (Fath et al., 1989).

The results of the above studies indicate that different cultured cells use different integrins to attach to different substrates, and that the profile of integrin expression may be altered by both substrate composition and topography.

1.6.4.2 Different cell types exhibit different preferences fo r the same ECM molecules

Dean et al. (1995) used gingival cells (fibroblasts and epithelial cells) to determine if laminin and Fn could influence cell attachment to implant surfaces in vitro.

They showed that coating of implant surfaces with Fn resulted in two to three times enhancement of gingival fibroblast binding on all the implant surfaces tested, with a lesser effect on epithelial cells. On the other hand, coating of the implant surfaces with laminin resulted in three to four times enhancement of gingival epithelial cell binding on all implant surfaces, with a lesser effect on the fibroblasts. The surface roughness of the substrates on which the proteins were laid down had little influence on the results.

1.6.4.3 Patterns o f focal contact localisation differ with substrate material

By vinculin immunostaining, Sinha et al. (1994) showed that focal contact localisation was different on various substrates as a function of time, even though the number of cells forming focal contacts on the substrates was the same. On plastic surfaces, focal contacts were initially formed at the cell periphery but became completely redistributed throughout the cell by 12 h. However, on Ti and Co-Cr

substrates, redistribution of focal contacts was delayed until 24 h. The authors suggested that peripheral contact formation may be adequate to anchor the cells to metal substrates, while attachment to plastic required many focal contacts throughout the cell surface. These results suggest that cell adherence was stronger and more stable on metals than on plastic surfaces, and that stable adhesion occurs if enough bonds/attachment sites are formed.