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1 Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, USA
2 Department of Biomedical Engineering, University of Boston, Boston, MA, USA
* To whom correspondence should be addressed. E-mail: russell{at}uic.edu.
Cardiac myocyte cultures usually require pharmacological intervention to prevent over-proliferation of contaminating non-myocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10µm vertical projections that we term "pegs", to which over 80% of rat neonatal cardiac fibroblasts attach within 48 of hours after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes (p<0.001) and a concomitant 60% decrease (p<0.01) in cyclin D1 protein levels suggesting a G1/S1 cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5µM or 20µM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% (p<0.001), suggesting that this signalling pathway plays an important role in the process. Using mobile and immobile 10µm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation since mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces (p<0.01). This suggests an increase in the proportion of myocytes on the pegged surfaces. Connexin-43 protein expression was also increased suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo.
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