Tissue spreading on implantable substrates is a competitive outcome of cell-cell vs. cell-substratum adhesivity

Abstract

While the interactions of cells with polymeric substrata are widely studied, the influence of cell-cell cohesiveness on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or "tug-of-war" between cell-cell and cell-substratum adhesions. We have generated both a "library" of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesiveness. Cell-substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell-cell cohesiveness was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell-cell cohesiveness in a series of copolymeric substrata having different levels of cell-substratum adhesivity. In these experiments, cell-cell cohesiveness was measured by tissue surface tensiometry, and cell-substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell-cell cohesiveness dramatically slowed the spreading rate of cell aggregates.

Figure 1

Variations in the molar fraction f of PEG present in copolymers of DTE and PEG make it possible to modulate the adhesivity of the polymer surface to cells by changing the amount and conformation of serum proteins adsorbed onto the polymer surface. Molecular weight of PEG: 1,000 g/mole; molecular weight of the copolymers varied from about 70,000 to 100,000 g/mole. With increasing f, the copolymer surface becomes more hydrophilic, as indicated by a decrease in the air-water contact angle (2).

Figure 2

Quantitative analysis of single cell attachment on poly(DTE-co-PEG carbonate) substrata shows that in all L cell types, rate of attachment markedly declined with increasing PEG content. Fluorescence-labeled (calcein-AM dye) single cell suspensions of low, intermediate, and high cohesivity were allowed to attach for 24 h on polymeric substrata containing 0–5% PEG. Rate of cell attachment was measured as fluorescence intensity at each time point. Data points represent averages of measurements made in duplicate and repeated at least three times.

Figure 3

At a constant level of cell–cell adhesion, cell population spreading on poly(DTE-co-PEG carbonate) substrates depends on the adhesivity of the substratum. Single cell suspensions of all L cell types, exemplified here by LN5 (a–d) and LR1 (e–h) were allowed to attach for 24 h to polymeric substrata containing 0–5% PEG. Note cadherin-specific differences in culture morphology. In all cases, cell population spreading was markedly reduced by increased PEG content of the substratum (top to bottom). Cells seeded on the substratum of highest PEG content tended to aggregate on the substratum, from which the aggregates were easily removed by gentle agitation. (Scale bar = 100 μm.)

Figure 4

Matrix of results showing the interplay between cell–cell cohesivity and cell–substratum adhesivity. Increasing cell–cell cohesivity decreases aggregate spreading rate whereas increasing cell–substratum adhesivity increases aggregate spreading rate. Small, fluorescence-labeled cell aggregates of low, intermediate, and high cohesivity (top to bottom) were allowed to spread for 24 h on polymeric substrata containing 0%, 3%, and 5% PEG (left to right). The most cohesive aggregates hardly spread at all on even the most adhesive substratum (G) and even the least cohesive aggregates were unable to spread significantly on the least adhesive substratum (C). (Scale bar = 100 μm.)

Figure 5

Serum-derived proteins mediate cell aggregate spreading. PKH2 fluorescence-labeled cell aggregates of low cohesivity (LN2 cells, 1.9 dyne/cm) were deposited and cultured for 24 h on a poly(DTE-carbonate)-coated surface in medium containing 10% FBS (Left) or TCM (TM) serum substitute (Right). Cells emigrated rapidly in the presence of serum but not at all in its absence.