Initial assessment of gingival biotype as a potential source of variability in the migration, contraction and gene expression of fibroblasts.

OBJECTIVE: Gingival biotype refers to the clinical classification of gingiva based on the thickness of the tissue, with thick gingival tissues more resistant to trauma and recession than the thin variant. However, to date there has never been an analysis of whether fibroblasts isolated from different biotypes possess inherent phenotypic differences. We hypothesized that gingival fibroblasts from thick and thin biotype would exhibit differences in migration, contraction and gene expression in vitro in the presence of either transforming growth factor beta one (TGF-ß1) or tumor necrosis factor alpha (TNFα), two major cytokines involved in wound repair. DESIGN: Migration was quantified using closure of scratch wound assays, contraction was assessed using attached and detached collagen lattices and extracellular matrix related gene expression using Taqman Realtime polymerase chain reaction. RESULTS: Human gingival fibroblasts isolated from both biotypes showed similar rates of closure of scratch wounds, which was not influenced by the addition of TGF-ß1 or TNFα. Fibroblasts from both biotypes contracted detached, but not attached, collagen gels to 50 % of their original weight although this contraction was not associated with incorporation of α-smooth muscle actin into stressfibres under any tested culture condition. Analysis of gene expression showed that POSTN, and ACTA2 mRNA levels did not significantly change, but CCN2 and COL1A2 mRNA levels were significantly higher in thick compared to thin fibroblasts in response to TGF-ß1. CONCLUSION: While supra-cellular factors influence the healing, esthetic outcomes and recession in thin gingival biotypes, differences in gingival fibroblast gene expression in response to growth factors may also play a role and warrants further investigation.

Fibronectin synthesis, but not α-smooth muscle expression, is regulated by periostin in gingival healing through FAK/JNK signaling.

During skin healing, periostin facilitates myofibroblast differentiation through a ß1 integrin/FAK dependent mechanism and continued expression is associated with scarring. In contrast to skin, gingival tissue does not typically scar upon injury, but the role of periostin in gingival healing has never been investigated. Using a rat gingivectomy model, we show that the gingival architecture is re-established within 14 days of wounding. Periostin mRNA levels peak at day 7 post-wounding, with persistence of periostin protein in the connective tissue through day 14. Collagen type I and lysyl oxidase mRNA levels peak at day 7 post wounding, which corresponded with the peak of fibroblast proliferation. Although α-smooth muscle actin mRNA levels increased 200-fold in the tissue, no myofibroblasts were detected in the regenerating tissue. In vitro, human gingival fibroblast adhesion on periostin, but not collagen, was inhibited by blocking ß1 integrins. Fibroblasts cultured on periostin exhibited similar rates of proliferation and myofibroblast differentiation to cells cultured on collagen only. However, human gingival fibroblasts cultured in the presence of periostin exhibited significantly increased fibronectin and collagen mRNA levels. Increases in fibronectin production were attenuated by pharmacological inhibition of FAK and JNK signaling in human gingival fibroblasts. In vivo, mRNA levels for fibronectin peaked at day 3 and 7 post wounding, with protein immunoreactivity highest at day 7, suggesting periostin is a modulator of fibronectin production during gingival healing.

Periostin and CCN2 Scaffolds Promote the Wound Healing Response in the Skin of Diabetic Mice.

IMPACT STATEMENT: Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.

Nifedipine and phenytoin induce matrix synthesis, but not proliferation, in intact human gingival connective tissue ex vivo.

Drug-induced gingival enlargement (DIGE) is a fibrotic condition that can be caused by the antihypertensive drug nifedipine and the anti-seizure drug phenytoin, but the molecular etiology of this type of fibrosis is not well understood and the role of confounding factors such as inflammation remains to be fully investigated. The aim of this study was to develop an ex vivo gingival explant system to allow investigation of the effects of nifedipine and phenytoin alone on human gingival tissue. Comparisons were made to the histology of human DIGE tissue retrieved from individuals with DIGE. Increased collagen, fibronectin, and proliferating fibroblasts were evident, but myofibroblasts were not detected in DIGE samples caused by nifedipine and phenytoin. In healthy gingiva cultured in nifedipine or phenytoin-containing media, the number of cells positive for p-SMAD2/3 increased, concomitant with increased CCN2 and periostin immunoreactivity compared to untreated explants. Collagen content assessed through hydroxyproline assays was significantly higher in tissues cultured with either drug compared to control tissues, which was confirmed histologically. Matrix fibronectin levels were also qualitatively greater in tissues treated with either drug. No significant differences in proliferating cells were observed between any of the conditions. Our study demonstrates that nifedipine and phenytoin activate canonical transforming growth factor-beta signaling, CCN2 and periostin expression, as well as increase collagen density, but do not influence cell proliferation or induce myofibroblast differentiation. We conclude that in the absence of confounding variables, nifedipine and phenytoin alter matrix homeostasis in gingival tissue explants ex vivo, and drug administration is a significant factor influencing ECM accumulation in gingival enlargement.