Quantification of Single Human Dermal Fibroblast Contraction.

¹Tim R. Fray, MSc., ¹Justin E. Molloy, DPhil., ²Mark P. Armitage, PhD., ¹John C. Sparrow, DPhil. Tissue Engineering (1998) In Press 1 Department of Biology, University of York, PO Box 373, York, YO1 5YW. UK.
2 Smith and Nephew Group Research Centre, Heslington, York, YO1 5DF.

Contraction forces produced by single, human dermal fibroblasts, cultured on deformable silicone membranes, have been quantified using video microscopy and image analysis. Cell contraction causes a deformation of the substrate, which appears as a series of surface wrinkles perpendicular to the long axis of the cell. Local surface deformation was measured from the two dimensional displacement of small latex beads embedded in the surface layer to which the fibroblasts adhere. From the motion of the latex beads, the contractile forces of the cells were calculated.²

Fibroblast Contraction This animation demonstrates the procedure for measuring the contraction and shows the sequence of events before and after the addition of SDS to the culture media. The SDS initiates disintegration of cell integrity and as the cell relaxes, its grip on the surface is lost. The latex beads embedded in the surface then return to their original resting positions. The animation is analysed frame by frame and the displacement of the beads is calculated using image software. A calibrated glass micro-needle was used to measure the force required to stretch the surface by a known amount (the surface stiffness)¹ which then allows us to convert the distance the beads have moved into the force required to do so. A series of vector forces are calculated and the overall vector giving the overall contraction force of the cell.

In vivo, such forces are thought to cause contraction of the skin’s dermal layer and promote wound closure. Normal contraction is vital to prevent infection and water loss. However, aberrant cellular behaviour is thought to be responsible for a variety of wound pathologies, such as hypertrophic and keloid scarring.

We have found that contractile forces of 2.65 µN/cell were produced. This is similar to those produced by single smooth muscle cells,³ approximately ten times greater than the forces measured for keratocytes, and 3 orders of magnitude greater than previously published values for fibroblasts that had been cultured in a collagen gel.⁴ Our goal is to understand the mechanisms that determine the polarity and maximum force of contraction and also to study differences in the behaviour of fibroblasts and myofibroblasts.

1. Burton, K., and Taylor, D.L.
Traction forces of cytokinesis measured with optically modified elastic substrata.
Nature 385, 450, 1997.

2. Lee, J., Leonard, M., Oliver, T., Ishihara, A., and Jacobson, K.
Traction forces generated by locomoting keratocytes.
J. Cell Biol. 127, 1957, 1994.

3. Warshaw, D.M.
Force:velocity relationship in single isolated toad stomach smooth muscle cells.
J. Gen. Physiol. 89, 771, 1987.

4. Eastwood, M., McGrouther, D.A., and Brown, R.A.
A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling.
Biochim. Biophys. Acta 120, 186, 1994.