Fibroblast contractile force is independent of the stiffness which resists the contraction.

Using a device named the cell force monitor, the contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-glycosaminoglycan (GAG) matrices over the first 24 h following cell attachment. In this paper, the effect of a variation in the stiffness that resists matrix contraction by cells on the contractile force generated by the cells was determined. Data from these experiments revealed that the contractile force generated by the fibroblasts was independent of the stiffness of the resistance within the range tested (0.7-10.7 N/m). These results suggest that during the time when fibroblasts are attaching to and spreading on collagen-GAG matrices the contractile forces they generate are force limited, not displacement limited. Therefore, the cytoskeletal mechanism of force generation, corresponding with cell elongation, is capable of increasing the displacement of adhesion sites in order to develop the same level of force. Although a detailed understanding of how the passive mechanical signals provided by substrate materials affect cell processes is still unavailable, in vitro modeling of cell-mediated contraction continues to provide useful information.

Growth factor regulation of smooth muscle actin expression and contraction of human articular chondrocytes and meniscal cells in a collagen-GAG matrix.

Recent work has demonstrated that human articular chondrocytes and meniscus cells can express the gene for a contractile actin isoform, alpha-smooth muscle actin (SMA), in vivo. The objective of the present study was to evaluate the effects of two growth factors, transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF)-BB, on the SMA content of these cells and their contraction of a collagen-glycosaminoglycan (GAG) analog of extracellular matrix in vitro. TGF-beta1 was found to markedly increase SMA content of the cells and PDGF-BB decreased SMA expression, with both findings achieving statistical significance. A notable finding was the increased contraction of the collagen-GAG matrix induced by TGF-beta1 and the decrease in contraction resulting from PDGF-BB treatment, indicating a causal relationship between expression of SMA and the contractility of the cells. A novel cell force monitor, employed to estimate the force exerted per cell, demonstrated a higher force exerted by the TGF-beta1-treated cells. The findings demonstrate that the expression of SMA by articular chondrocytes and meniscal cells and their associated contractile behavior can be regulated by selected growth factors. This work provides a foundation for the rational investigation of the mechanisms underlying SMA-enabled contraction of these cell types and the control of this behavior in tissue engineering.

Fibroblast contraction of a collagen-GAG matrix.

Contractile cells, found in wounded or diseased tissues, are associated with the formation of scar tissue. The complexity of contraction in vivo has led to the development of models of contraction by cells in vitro. In this work, a device was developed which quantitatively measured the contractile force developed by fibroblasts seeded into a collagen-glycosaminoglycan porous matrix in vitro. This device differed from most of those previously described in that it directly transferred cellular contractile force to the force transducer (a cantilever beam) and that it used a porous matrix rather than a collagen gel. The data for the increase in contractile force with time were fit to a mathematical equation using two fitting parameters. Data were then compared using the fitting parameters and the cell density. A study of the effect of cell density on the contractile force resulted in a linearly proportional relationship. Subsequent normalization of force by cell density or number resulted in a value of contractile force per cell, 1 nN, that was independent of cell density. The time for the contractile force to develop was also independent of cell density. These results suggest that, in this system, cells develop contractile force individually, irrespective of the force generated by surrounding cells.

Micromechanics of fibroblast contraction of a collagen-GAG matrix.

The contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-GAG matrices over time. We have identified the microscopic deformations developed by individual fibroblasts which lead to the observed macroscopic matrix contraction. Observation of live cells attached to the matrix revealed that matrix deformation occurred as a result of cell elongation. The time dependence of the increase in average fibroblast aspect ratio over time corresponded with macroscopic matrix contraction, further linking cell elongation and matrix contraction. The time dependence of average fibroblast aspect ratio and macroscopic matrix contraction was found to be the result of the stochastic nature of cell elongation initiation and of the time required for cells to reach a final morphology (2-4 h). The proposed micromechanics associated with observed buckling or bending of individual struts of the matrix by cells may, in part, explain the observation of a force plateau during macroscopic contraction. These findings indicate that the macroscopic matrix contraction measured immediately following cell attachment is related to the extracellular force necessary to support cell elongation, and that macroscopic time dependence is not directly related to microscopic deformation events.

Tendon cell contraction of collagen-GAG matrices in vitro: effect of cross-linking.

The contraction of connective tissue cells can play important roles in wound healing and pathological contractures. The effects of this contractile behavior on cell-seeded constructs for tissue engineering have not yet been investigated. The goal of this work was to investigate in vitro tendon cell-mediated contraction of collagen-glycosaminoglycan (GAG) matrices cross-linked using selected methods. Highly porous collagen-GAG sponges were seeded with calf tendon cells and the projected area and DNA content of the sponges measured at 3, 7, 14, and 21 days post-seeding. Immunohistochemistry was performed to determine if alpha-smooth muscle actin (SMA) was associated with the cell contraction of the matrices. Dehydrothermal (DHT) treatment alone was not sufficient to resist contraction by the seeded tendon fibroblasts. Cross-linking of the collagen-GAG sponges to the extent that the modulus was three times that of sponges treated by DHT alone was necessary to resist contraction. SMA was seen in the cytoplasm of most cells in all sponges at all time periods. The results provide a rational basis for the determination of the mechanical properties of collagen matrices required for engineering certain connective tissues.