Human Gingival Fibroblasts Display a Non-Fibrotic Phenotype Distinct from Skin Fibroblasts in Three-Dimensional Cultures

Abstract

Scar formation following skin injury can be a major psychosocial and physiological problem. However, the mechanisms of scar formation are still not completely understood. Previous studies have shown that wound healing in oral mucosa is faster, associates with a reduced inflammatory response and results to significantly reduced scar formation compared with skin wounds. In the present study, we hypothesized that oral mucosal fibroblasts from human gingiva are inherently distinct from fibroblasts from breast and abdominal skin, two areas prone to excessive scar formation, which may contribute to the preferential wound healing outcome in gingiva. To this end, we compared the phenotype of human gingival and skin fibroblasts cultured in in vivo-like three-dimensional (3D) cultures that mimic the cells' natural extracellular matrix (ECM) niche. To establish 3D cultures, five parallel fibroblast lines from human gingiva (GFBLs) and breast skin (SFBLs) were seeded in high density, and cultured for up to 21 days in serum and ascorbic acid containing medium to induce expression of wound-healing transcriptome and ECM deposition. Cell proliferation, morphology, phenotype and expression of wound healing and scar related genes were analyzed by real-time RT-PCR, Western blotting and immunocytochemical methods. The expression of a set of genes was also studied in three parallel lines of human abdominal SFBLs. Findings showed that GFBLs displayed morphologically distinct organization of the 3D cultures and proliferated faster than SFBLs. GFBLs expressed elevated levels of molecules involved in regulation of inflammation and ECM remodeling (MMPs) while SFBLs showed significantly higher expression of TGF-β signaling, ECM and myofibroblast and cell contractility-related genes. Thus, GFBLs display an inherent phenotype conducive for fast resolution of inflammation and ECM remodeling, characteristic for scar-free wound healing, while SFBLs have a profibrotic, scar-prone phenotype.

Distinct phenotype and therapeutic potential of gingival fibroblasts

“…They also contain elevated levels of immunomodulatory and anti-fibrotic TGF-b3 relative to pro-fibrotic TGF-b1 as compared with similar skin wounds (13,36,39,40,49,50). Interestingly, accumulating evidence has suggested that the gingival wound-healing response may also depend on a distinct phenotype of gingival fibroblasts (11,13,38,51). Are gingival fibroblasts different from other oral fibroblasts?…”

“…The findings showed that gingival fibroblasts are programmed for fast resolution of inflammation, ECM remodeling and angiogenesis conducive for fast and scarless wound healing. In contrast, skin fibroblasts are prone to abundant ECM deposition, active TGF-b signaling and contractility, characteristics of fibrosis and scar formation (51). Thus, gingival fibroblasts may provide an advantage over skin fibroblasts in therapeutic modalities aiming to promote fast and scarless wound healing.…”

“…This may, at least in part, depend on the distinct phenotype of gingival cells (Table II). Gingival fibroblasts secrete proangiogenic vascular endothelial growth factor (VEGF), CXCL12 (stromal cellederived factor-1a), CXCL8 (IL-8), CCL2 (monocyte chemoattractant protein-1), and CXCR1, and promote endothelial cell growth in vitro (51,94,95), but the relevance of this for angiogenesis in vivo or in in vitro models is not known. Gingival fibroblasts also produce abundantly factors that promote re-epithelialization, including keratinocyte growth factor (FGF7) and hepatocyte growth factor/scatter factor (HGF/SF) (96).…”

“…For example, gingival fibroblasts produce more osteoprotegerin and less osteoclastogenic factors as compared with skin fibroblasts, resulting in decreased osteoclastogenic induction in vitro (103). They also produce high levels of certain matrix metalloproteinases (51), which activate or inactivate inflammatory mediators and their inhibitors (104). In addition, they are less responsive to stimulation of IL-1b expression by bacterial lipopolysaccharide (105), whereas they show a pronounced growth inhibition by prostaglandin E2 (PGE2) caused by elevated expression of PGE2 receptor EP2 as compared with skin fibroblasts (106).…”

Gingival Wound Healing

“…A recent study has shown that decreased TGF-β signaling and consequent reduced levels of connective tissue growth factor (CTGF) may be responsible for the progressive loss of collagen in the aged skin (Quan et al 2010). Although TGF-β regulation may differ in skin versus gingival cells (Mah et al 2014), it is plausible that gingival cells may experience changes in their response to TGF-β during aging. Collagen fibers are significantly modified and reorganized during the slow tissue-remodeling process in which molecules change their general orientation.…”