Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth, which remains a significant problem. The risk factors appraised include the duration of treatment. However, there are no stereological and biochemical studies exploring the effects of long-term CsA therapy on gingival tissue. The purpose of the present study was to investigate the level of TGF-beta1 in saliva and describe the densities of fibroblasts and collagen fibers in the gingival tissue of rats treated with CsA for long periods. Rats were treated for 60, 120, 180 and 240 days with a daily subcutaneous injection of 10 mg/kg of body weight of CsA. At the end of the experimental periods, saliva was collected for the determination of TGF-beta1 levels. After histological processing, the oral epithelium and the connective tissue area were measured as well as the volume densities of fibroblasts (Vf) and collagen fibers (Vcf). After 60 and 120 days of CsA treatment, there was a significant increase in Vf and Vcf as well as a significant increase in TGF-beta1 levels. After 180 and 240 days, reduction in the gingival overgrowth associated with significant decreases in the level of TGF-beta1, and also decreased Vf and Vcf, were observed. The data presented here suggest that after long-term therapy, a decrease in TGF-beta1 levels occurs, which might contribute to an increase in the proteolytic activity of fibroblasts in the gingiva, favoring the normality of extracellular matrix synthesis.

Human periodontal ligament stem cells (PDLSCs) have been studied as a promising strategy in regenerative approaches. The protease-activated receptor 1 (PAR 1 ) plays a key role in osteogenesis and has been shown to induce osteogenesis and increase bone formation in PDLSCs. However, little is known about its effects when activated in PDLSCs as a cell sheet construct and how it would impact bone formation as a graft in vivo. Here, PDLSCs were obtained from 3 patients. Groups were divided into control, osteogenic medium and osteogenic medium + PAR 1 activation by TFLLR-NH2 peptide. Cell phenotype was determined by flow cytometry and immunofluorescence. Calcium deposition was quantified by Alizarin Red Staining. Cell sheet microstructure was analyzed through light, scanning electron microscopy and histology and transplanted to Balb/c nude mice. Immunohistochemistry for bone sialoprotein (BSP), integrin β1 and collagen type 1 and histological stains (H&E, Van Giesson, Masson’s Trichrome and Von Kossa) were performed on the ex-vivo mineralized tissue after 60 days of implantation in vivo . Ectopic bone formation was evaluated through micro-CT. PAR 1 activation increased calcium deposition in vitro as well as BSP, collagen type 1 and integrin β1 protein expression and higher ectopic bone formation (micro-CT) in vivo.

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