S. Balaji1, N. Hann1, R. Ranjan1, C. Moles1, A. F. Shaaban1, T. M. Crombleholme1,3, P. Bollyky2, S. G. Keswani1 1Cincinnati Children’s Hospital Medical Center,Pediatric General Thoracic And Fetal Surgery,Cincinnati, OH, USA 2Stanford School Of Medicine,Infectious Diseases, Department Of Medicine,Palo Alto, CA, USA 3Children’s Hospital Colorado,Center For Children’s Surgery,Aurora, CO, USA
Introduction: Mid gestation fetal skin heals without scar and is characterized by negligible resting tension and distinct extracellular matrix (ECM) with elevated levels of hyaluronan (HA) produced by fetal fibroblasts. In contrast, adult skin is characterized by relatively low levels of HA, and much higher resting tension and scar formation. Of note, wounds of a critical size even in the fetus heal with a scar suggesting a role for biomechanical forces in the regulation of the fetal regenerative phenotype. There is little data that examined the interaction of mechanical stress and regulation of the ECM, specifically HA synthesis. Taken together, we hypothesize that higher mechanical tension may alter the fibroblast regulation of HA synthases (HAS1-3).
Methods: Primary murine adult fibroblasts (AFb) and fetal fibroblasts (FFb) were cultured with +/- mechanical stretch for 24 hours. Static tension at 58 KPa (resting tension of adult mouse skin) was applied using flexcell apparatus to induce tonic stretch. Stretch induced phenotypic changes in FFb and AFb were assessed by changes in PCM (particle exclusion assay), HAS1-3 synthases (qPCR) and cell morphology and cytoskeleton changes (a-SMA Immunohistochemistry). Data presented as average+/-SD, n=4/group, p values by t-Test or ANOVA.
Results:AFb and FFb demonstrate differential cell responses to biomechanical stretch. Under static condition, AFb had significantly lower HAS1 (AFb 1.4+/-0.4 vs FFB 4.6+/-1.1, p<0.01) and HAS2 (AFb 1.2+/-0.25 vs FFB 2.7+/-0.8, p<0.01) gene expression that encodes for fetal-like high molecular weight hyaluronan, and smaller PCM (AFb 1.84+/-0.08 vs FFb 2.78+/-0.14; p<0.001). AFb also demonstrated thicker a-SMA fibers compared to FFb, but the fiber orientation appeared random in both cell types. 24 hours of mechanical stretching resulted in the loss of FFb phenotype with a significant decrease in HAS1 (FFb+stretch 1.0+/-0.35 vs FFb 4.6+/-1.1, p<0.01) and HAS2 (FFb+stretch 1.0+/-.2 vs FFb 2.7+/-.8, p<0.01) gene expression and a significant reduction in PCM formation (FFb+stretch 1.98+/-.15 vs FFb 2.78+/-.14, p<0.01) in FFb, to levels similar to the AFb under static condition. Stretching increased HAS1 expression in AFb (AFb+stretch 3.0+/-.6 vs AFb 1.4+/-0.4, p<.05), but had no effect on HAS2. Interestingly a-SMA staining demonstrated that stretching resulted in thickening of the stress fibers in FFb, but not AFb, and a trend toward reorientation of actin fibers in the strained cells perpendicular to the direction of stretch in both AFb and FFb.
Conclusion:Our data suggest that biomechanical forces may have a significant role in influencing the dermal fibroblasts’ morphology and their regulation of the ECM in the fetal and adult wound healing phenotype. Understanding the contribution of mechanical environment via morphological and phenotypic alterations may yield novel therapeutic targets in recapitulating fetal regenerative healing in postnatal tissues.