Generation of an in vitro 3D cell sheet-based model of scar-like tissue

Considering the prevalent misunderstanding in both the mechanisms and pathology underlying skin deformities, scarring still presents itself as one of the unsolved issues in the clinics. Key players like fibroblasts and the localized biochemical role of transforming growth factor beta 1 (TGF1) have been tightly associated to both its development and progression. TGF1 is responsible for the phenotypical switch of resident fibroblasts into myofibroblasts which in turn leads to the enhancement of immature extracellular matrix (ECM) synthesis and generation of tensional forces that then primes ECM reorganization required for wound remodeling. However, a deregulation of this process leads to myofibroblast persistence in the wound site and an inflammatory loop and the appearance of certain skin pathologies (e.g hypertrophic scars).
Benchtop models of hypertrophic scars rely on scarce human ex vivo samples or standard 2D cultures of fibroblasts isolated from hypertrophic scar tissue. We therefore propose the use of human dermal fibroblast cell sheets (hDFbsCS) as the first step to attain cell sheets with a myofibroblast-like phenotype to generate cohesive in vitro 3D scar-like tissues.
hDFbsCS were produced as previously described1, and stimulated with TGF1 up to 21 days. Following phenotype and ECM characterization, 3 hDFbsCS were stacked to obtain a 3D structure. Gene (q-PCR) and protein (Western blot and immunocytochemistry) analysis showed that hDFbs cell sheets, when stimulated with TGF-β1 present an increased expression of α-SMA, fibronectin (FN) ED-A and FN ED-B, characteristic of a myofibroblast-like phenotype. When looking into the expression of scar ECM-associated proteins, hDFbs cell sheets obtained in the presence of TGF- β1 produced higher amounts of fibronectin and collagen I. Stable 3D constructs with a noticeable level of integration after a total of 21 days of culture, were formed upon stacking of the cell sheets obtained after 7days of culture in the presence of TGF- β1. The secretome analysis of scar related soluble factors, such as MMP-1, MMP-2 and IL6 showed similar expression patterns to published data.
Therefore, our results suggest that it is possible to create cohesive 3D scar-like tissue structures from hDFbsCS driving the possibility to develop accurate in vitro 3D scar models to study wound healing deregulation.
1M.T. Cerqueira (2014) Acta Biomaterialia 10(7).
1M.G. Rohami (2016) Matrix Biology 44-46.