Regulation of fibrotic changes by the synergistic effects of cytokines, dimensionality and matrix: Towards the development of an in vitro human dermal hypertrophic scar model

Abstract

Current therapeutic strategies to reduce scarring in full thickness skin defect offer limited success due to poor understanding of scar tissue formation and the underlying signaling pathways. There is an urgent need to develop human cell based in vitro scar tissue models as animal testing is associated with ethical and logistic complications and inter-species variations. Pro-inflammatory cytokines play critical role in regulating scar development through complex interplay and interaction with the ECM and corresponding signaling pathways. In this context, we assessed the responses of cultured fibroblasts with respect to their differentiation into myofibroblasts using optimised cytokines (TGF-β1, IL-6 and IL-8) for scar formation in 2D (tissue culture plate, collagen type I coated plate) vs 3D collagen type I gel based constructs. We attempted to deduce the role of dimensionality of cell culture matrix in modulating differentiation, function and phenotype of cultured fibroblasts. Validation of the developed model showed similarity to etiology and pathophysiology of in vivo hypertrophic scar with respect to several features: 1) transition of fibroblasts to myofibroblasts with convincing expression of α-SMA stress fibers; 2) contraction; 3) excessive collagen and fibronectin secretion; 4) expression of fibrotic ECM proteins (SPARC and Tenascin); 5) low MMP secretion. Most importantly, we elucidated the involvement of TGF-β/SMAD and Wnt/β-catenin pathways in developing in vitro dermal scar. Hence, this relatively simple in vitro human scar tissue equivalent may serve as an alternative for testing and designing of novel therapeutics and help in extending our understanding of the complex interplay of cytokines and related dermal scar specific signaling. Statement of SignificanceScarring of the skin affects almost millions of people per year in the developed world alone, nevertheless the complex pathophysiology and the precise signaling mechanisms responsible for this phenomenon of skin scarring are still unknown. A number of anti-scar drugs are being developed and being tested on animals and monolayer models. However, testing the efficacy of these drugs on lab based 3D in vitro models may prove extremely useful in recapitulating the 3D microenvironment of the native scar tissue. In that context in this study we have demonstrated the development of 3D in vitro dermal scar model, by optimizing a constellation of factors, such as combination of cytokines (TGF-β1,IL-6,IL-8) and cellular dimensionality in inducing the differentiation of dermal fibroblasts to myofibroblasts. This in vitro scar model was successful in replicating hallmark features of hypertrophic scar such as excessive synthesis of fibrotic extracellular matrix, perturbed matrix homeostasis, contraction, diminished MMP synthesis. The study also highlighted significant involvement of TGF-β/SMAD and Wnt/β-catenin signaling pathways in in vitro scar formation.