Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA-treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase-polymerase chain reaction revealed that the CsA-treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA-treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% +/- 2.7% and 24.3% +/- 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% +/- 5.3% and 81.3% +/- 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen-coated latex beads. Fibroblasts isolated from CsA-treated gingiva on day 8 and day 30 contained 5.7% +/- 0.6% and 9.9% +/- 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% +/- 5.5% and 33.3% +/- 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA-treated rat gingiva.

Osteopontin (OPN) is a major glycosylated phosphoprotein in bone matrix and is produced by several cells including osteoblasts, osteoclasts and macrophages. OPN levels increase in active sites of bone metabolism. Recently, several bone-related proteins were identified in gingival crevicular fluid (GCF) to seek markers of alveolar bone resorption in periodontal disease. In this study, we investigated the existence of OPN in GCF and the correlation between OPN level in GCF and probing depth (PD) of sampling sites in 98 periodontitis patients and 35 healthy subjects. An immunoblotting analysis using 10% polyacrylamide gel showed that two forms of OPN with molecular masses of 54 and 66 kDa and several degraded fragments were detected in most GCF samples from diseased sites (PD > 4 mm). In GCF samples from healthy sites (PD < or = 3 mm), only one form (54 kDa) was observed, but any degraded fragments were not detected. When OPN amounts in GCF samples were determined by ELISA, a weak. but significant correlation was observed between OPN amount in GCF and PD (r=0.32, p=0.0013). These results demonstrate that OPN exists in GCF and that OPN level in GCF increases with the progression of periodontal disease.

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA-treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase-polymerase chain reaction revealed that the CsA-treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA-treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% +/- 2.7% and 24.3% +/- 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% +/- 5.3% and 81.3% +/- 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen-coated latex beads. Fibroblasts isolated from CsA-treated gingiva on day 8 and day 30 contained 5.7% +/- 0.6% and 9.9% +/- 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% +/- 5.5% and 33.3% +/- 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA-treated rat gingiva.

These findings suggest that the decrease in collagen degradation due to lower phagocytosis is closely associated with the increase in Type I collagen accumulation in nifedipine-treated rat gingiva.

It has been shown that mirrorimage duplications of the zeugopodia and digits are formed when MRC-5 fibroblasts producing hepatocyte growth factor (HGF) are applied to the anterior region of the chick limb bud (Yonei et al.[1993] Dev. Biol. 16024C253). To evaluate the role of HGF in limb development, we observed the expression pattern of the HGF gene using in situ hybridization. The HGF gene was expressed in the mesoderm of the limb bud and in the central core region of mandibular arch and maxillary processes at stages 17 to 24. When both wing and leg buds begin to extend distally, the HGF gene is expressed in the mesenchymal cells, but not in the ectodermal cells and somites. Concomitant with establishment of the apical ectodermal ridge, distal mesenchymal cells of the limb bud express the HGF gene intensely with a gradient higher in the distal region. The HGF expression is later confined to the ventral and subapical mesenchyme of the limb bud, although no signal is detectable in the apical and non-ridge ectoderm. However, signal for the c-met proto-oncogene encoding the HGF receptor is not detectable in the limb bud at stages 17 to 24. These results suggest that HGF produced in the limb mesoderm may be involved in initial induction and maintenance of the apical ectoderm during limb development.