Role of serum vitronectin and fibronectin in adhesion of fibroblasts following seeding onto tissue culture polystyrene

Abstract

The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in tissue culture. In this study the contribution that adsorption of serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of fibroblast cells during the first 90 min following seeding was determined for two modified tissue culture polystyrenes, as model biomaterial surfaces. The amount of serum Vn and Fn which adsorbed onto tissue culture grade polystyrene (TCP) from different serum concentrations over the range of 0.1-30% (v/v) were determined and compared to attachment of cells of the BHK-21 and HT1080 fibroblast lines. There was no simple correlation between the amount of Fn or the amount of Vn adsorbed and cell attachment and spreading. The requirement for Fn or Vn for attachment and spreading of BHK-21 or HT1080 cells onto modified polystyrene (either TCP or to Primaria) during the first 90 min of cell culture was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of BHK-21 or HT1080 cells onto TCP or Primaria surfaces were reduced in a concentration-dependent manner when the cells were seeded in medium containing 2% (v/v) or higher concentrations of Vn-depleted serum. BHK-21 cells or HT1080 cells seeded in medium containing Fn-depleted serum (which contained Vn) attached and spread onto TCP or Primaria. Both BHK-21 cells and HT1080 cells failed to attach to TCP or Primaria when seeded in medium containing serum depleted of both Vn and Fn. The requirement for serum Vn or Fn for fibroblast attachment to TCP was also tested using cells of a human dermal fibroblast strain. The attachment of the dermal fibroblasts to TCP during the first 90 min of culture was not decreased by depletion of Vn from the 15% (v/v) serum, but there was a reduction in the proportion of the attached cells which had spread. Selective depletion of serum Fn did not have any effect on either cell attachment or spreading. Our results show that for fibroblast cells, particularly with cell lines such as BHK-21 or HT1080 but also with cell strains, the first binding of cells onto tissue culture polystyrene when plated in medium containing serum is a result of adsorption onto the surface of serum Vn. The adsorption of serum Vn onto the surface overcomes the effect of serum components which tend to decrease cell attachment.

Proteins at Interfaces

“…The differential affinity model is supported by studies that showed large differences in the adsorption of fibronectin onto various surfaces (59)(60)(61)(62). In addition, the initial spreading of 3T3 cells on HEMA-EMA copolymers is linearly correlated with the amount of fibronectin adsorbed to the surfaces from serum (61).…”

“…Similarly, plasma deposited polymers which enhanced 3T3 and muscle myoblast cell growth also enhanced fibronectin adsorption from serum in comparison to untreated surfaces (63). Depletion studies, in which the fibronectin or vitronectin are selectively removed from the serum, have shown that vitronectin, rather than fibronectin, appears to be the primary adhesion factor for certain cells and surfaces (62,(64)(65)(66). However, the most recent studies of this type show that the enhanced affinity of fibronectin for the nitrogen rich Primaria tissue culture surface in comparison to ordinary oxygen rich tissue culture surfaces is sufficient to provide for the attachment of human vein endothelial cells to Primaria from serum depleted of vitronectin.…”

Mammalian Target of Rapamycin Positively Regulates Collagen Type I Production via a Phosphatidylinositol 3-Kinase-independent Pathway

“…For example, recent studies linked mTOR to the signaling pathway of the ␣ V ␤ 3 integrin (64). ␣ V ␤ 3 integrin, a receptor for vitronectin, is the primary molecule required for attachment and spreading of fibroblasts on cell culture dishes (65). It is well documented that collagen message stability decreases after seeding fibroblasts in collagen matrices, whereas plating cells on plastic increases collagen synthesis (66).…”

The influence of nanoscale topographical cues on initial osteoblast morphology and migration

“…integrins) are involved in the process of pattern recognition (Bershadsky et al, 2006). Immediately after contact of a substrate with cell culture medium, proteins (most importantly fibronectin and vitronectin in serum) start to adhere to the surface (Steele et al, 1992;Garcia and Boettiger, 1999). Specific binding sites in these proteins (the best known being the RGD peptide sequence) are recognized by integrin heterodimers on the cell membrane.…”