Correlation Between Different Tissue Expansion Protocols And Changes In Gene Expression
Joanna K. Ledwon1, Jolanta M. Topczewska2, Chiang C. Huang3, Arun K. Gosain1.
1Northwestern University Feinberg School of Medicine, Department of Surgery Plastic Division, Stanley Manne Children’s Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA, 2Northwestern University Feinberg School of Medicine, Department of Pediatrics, Stanley Manne Children’s Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA, 3University of Wisconsin, Joseph J Zilber School of Public Health, Milwaukee, Milwaukee, IL, USA.
Purpose. Tissue expansion (TE) relies on skin growth in response to mechanical forces. Although TE is widely used in reconstructive surgery, there has been minimal investigation into the transcriptional activity in the skin under stretch. The present study evaluates the molecular response of skin to TE to identify pathways that could potentially be manipulated to improve the efficacy of TE and investigate the effect of eight TE protocols on expression of mechanosensitive genes.
Methods. Using a porcine TE model, the transcriptome was mapped (RNA-seq analysis) in the skin samples that were stretched by inflation of expanders with 30ml or 60ml saline for 1 hour to 14 days. Contralateral unexpanded sites served as controls. Differentially expressed genes were detected with NOIseq (FC>2 and probability>0.8). qRT-PCR analysis was performed to verify results obtained by RNA-seq. Statistical analysis was performed using GraphPad Prism 7 software. The significance of obtained results was calculated using the unpaired student’s t-test. P-values≤0.05 were considered significant.
Results. Differential analysis revealed significant changes in the expression of 220 genes (P<0.005), representing a diverse response pattern (Fig. 1). qPCR analysis on biopsies collected from apical (a), middle (m) and periphery (p) of expanders showed that expression of mechanosensitive genes depends on magnitude of mechanical forces (Fig. 2A). The highest increase of MMP1 expression (FC=41-787, P>0.001) was detected in the apical biopsies, exposed on the highest pressure. In biopsies “m” the increase in MMP1 expression was 4-8 times smaller than in biopsies “a”, but still significantly higher than in control (FC=52-175, P>0.001). In biopsies “p” the MMP1 expression was not significantly changed. The evaluation of SFRP2 expression at 3 days after fill with 30ml saline revealed 5 times higher response (P=0.001) when additional injection with the same volume was performed 7 days earlier, suggesting that multiple injections stimulate the molecular response of skin in TE more effectively. The opposite effect was observed for double injection with 60ml saline, indicating that when pressure is too high this can negatively affect the molecular response and limit skin growth and regeneration (Fig. 2B).
Conclusions. RNA-seq analysis revealed numerous genes responding to tissue expansions that are involved in a variety of biological processes including homeostasis, proliferation, differentiation, and inflammation. To achieve the most effective stimulation of skin growth, real-time evaluation of pressure within the expanders will be incorporated in subsequent studies. Understanding the correlation between time of expansion, magnitude of pressure and molecular response of skin in TE may help to optimize clinical outcomes, leading to the most efficient recruitment of new skin and make expansion feasible even in compromised tissue beds.
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