Characterization of mesenchymal stem cells from human normal and hyperplastic gingiva.

Human gingiva plays an important role in the maintenance of oral health and shows unique fetal-like scarless healing process after wounding. Here we isolate and characterize mesenchymal stem cells from human normal and hyperplastic gingival tissues (N-GMSC and H-GMSC, respectively). Immunocytochemical staining indicated that gingival lamina propria contained Stro-1 and SSEA-4 positive cells, implying existence of putative gingival MSC. Under attachment-based isolating and culturing condition, gingival MSC displayed highly clonogenic and long-term proliferative capability. By using single colony isolation and expansion approaches, we found both N-GMSC and H-GMSC possessed self-renewal and multipotent differentiation properties. N-GMSC and H-GMSC showed distinct immunoregulatory functions in a murine skin allograft setting via up-regulation of putative systemic regulatory T cells (Tregs). N-GMSC and H-GMSC were capable of regenerating collagenous tissue following in vivo transplantation, in which H-GMSC exhibited more robust regenerative capability. These findings suggest that gingival tissue contains tissue-specific mesenchymal stem cell population and is an ideal resource for immunoregulatory therapy due to its substantial availability and accessibility. In addition, gingival MSC over-activation may contribute to gingival hyperplastic phenotype.

Immunohistochemical expression of mast cell tryptase in giant cell fibroma and inflammatory fibrous hyperplasia of the oral mucosa.

This study analysed the immunohistochemical expression of mast cell tryptase in giant cell fibromas (GCFs). In addition, the possible interaction of mast cells with stellate giant cells, as well as their role in fibrosis and tumour progression, was investigated. For this purpose, the results were compared with cases of inflammatory fibrous hyperplasia (IFH) and normal oral mucosa. Thirty cases of GCF, 30 cases of IFH and 10 normal mucosa specimens used as control were selected. Immunoreactivity of mast cells to the anti-tryptase antibody was analysed quantitatively in the lining epithelium and in connective tissue. In the epithelial component (p=0.250) and connective tissue (p=0.001), the largest mean number of mast cells was observed in IFHs and the smallest mean number in GCFs. In connective tissue, the mean percentage of degranulated mast cells was higher in GCFs than in IFHs and normal mucosa specimens (p<0.001). Analysis of the percentage of degranulated mast cells in areas of fibrosis and at the periphery of blood vessels also showed a larger mean number in GCFs compared to IFHs and normal mucosa specimens (p<0.001). The percent interaction between mast cells and stellate giant cells in GCFs was 59.62%. In conclusion, although mast cells were less numerous in GCFs, the cells exhibited a significant interaction with stellate giant cells present in these tumours. In addition, the results suggest the involvement of mast cells in the induction of fibrosis and modulation of endothelial cell function in GCFs.

Upregulation of heme oxygenase-1 expression in gingiva after cyclosporin A treatment.

BACKGROUND: Heme oxygenase (HO)-1 is a stress-inducible protein that confers cytoprotection, but its role in gingiva during cyclosporin A (CsA) therapy is unknown. We used in vivo and in vitro models to investigate the expression of mRNA and protein for HO-1 in gingiva upon CsA treatment. METHODS: Twenty-six male Sprague-Dawley rats were assigned to two groups after the establishment of edentulous ridges. Rats in the CsA group received CsA, 30 mg/kg/day for 4 weeks, whereas control rats received mineral oil only. All rats were killed after 4 weeks, and the edentulous gingivae were excised. mRNA and protein expression of HO-1 in gingivae were determined using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively. For the in vitro study, cultured human gingival fibroblasts were harvested after treatment with various concentrations of CsA, and HO-1 mRNA and protein expression were determined using RT-PCR and Western blotting, respectively. RESULTS: Mean gingival HO-1 mRNA expression was greater in the CsA group than in the control animals (P = 0.076). IHC staining for HO-1 protein was significantly greater in the gingivae of CsA-treated rats than in those of the control group. In fibroblast cultures, expression of HO-1 mRNA and protein also increased significantly after CsA treatment. CONCLUSION: CsA upregulates the gingival expression of HO-1, which may exert a cytoprotective effect.

Expression of peroxiredoxin I in plasma cells of oral inflammatory diseases.

The participation of reactive oxygen species (ROS) in the immune response, both as pathogen killers and as mediators of signaling pathways, is well established. However, little is known about the enzymes responsible for ROS elimination in immune cells. Peroxiredoxin I (PrdxI) is a multifunctional enzyme that exhibits thioredoxin-dependent peroxidase activity. It has been described as a major hydrogen peroxide (H(2)O(2))-inducible protein in mouse peritoneal macrophages. In order to characterize its participation in the antioxidant defense of inflammatory/immune cells in greater detail, we evaluated its expression at sites of the oral cavity affected by inflammatory disorders induced by different agents (infectious, chemical, mechanical or tumor). In this study we demonstrated, by immunohistochemistry, that PrdxI is expressed in plasma cells, but not in B lymphocytes, regardless of the inflammation-inducing agent. We suggest that PrdxI induction could be considered a crucial part of the cellular adaptive response to the B-cell differentiation process to cope with the additional H(2)O(2) associated with massive disulfide bond formation during immunoglobulin folding in the endoplasmic reticulum of plasma cells. PrdxI could diminish the tissue damage that accompanies inflammation.

The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts.

BACKGROUND: The immunosuppressive agent cyclosporin is associated with a number of major side-effects including the development of gingival overgrowth. Although the pathogenesis of cyclosporin-induced gingival overgrowth remains unclear, it has been suggested that the finely regulated balance between extracellular matrix synthesis and degradation may be disturbed, resulting in an accumulation of excess connective tissue components within the gingival tissue. The aim of this study was to investigate the effect of cyclosporin on matrix metalloproteinases (MMP)-1 and tissue inhibitors of MMP (TIMP)-1 expression at the mRNA, protein, and enzyme activity levels. METHODS: Gingival fibroblasts were grown to confluence and then cultured in serum-free medium supplemented with cyclosporin over the concentration range of 0 to 2000 ng/ml. MMP-1 and TIMP-1 mRNA levels in cultures were determined by reverse transcription polymerase chain reaction (RT-PCR), protein levels in whole conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA), and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. Tissue mRNA levels in normal and overgrown gingiva were also determined by RT-PCR. RESULTS: Results indicated that cyclosporin inhibited MMP-1 expression at both the mRNA and protein level in a dose- and time-dependent fashion. The effects on TIMP-1 expression were less clear, cyclosporin inhibiting mRNA expression, but having no effect on TIMP-1 protein levels at any concentration studied. Addition of the drug resulted in reduced levels of collagenolytic activity in the culture medium. MMP-1 mRNA expression was significantly reduced in overgrown compared to normal tissue. CONCLUSIONS: These results add support to the hypothesis that the accumulation of collagen seen in gingival overgrowth can be explained by a cyclosporin-induced inhibition of collagenolytic activity within the gingival tissues.

C-1 esterase inhibitor dysfunction localised to the periodontal tissues: clues to the role of stress in the pathogenesis of chronic periodontitis?

BACKGROUND: C1-esterase inhibitor (C1eIn) is an important modulator of complement activation via the classical pathway. Deficiencies or dysfunction involving this inhibitor underlie the condition of angioneurotic oedema. AIM: The purpose of this report is to describe a female patient who presented at the age of 24 years with an apparently aggressive form of periodontitis and severe oedema, localised to the free gingival tissues. After 21 years of repeated surgical reduction of the gingiva, a diagnosis of C1eIn dysfunction was made. METHODS: Exhaustive serological investigations were performed along with histopathology. RESULTS: All investigations were unremarkable, until the function of the C1eIn molecule was investigated. These demonstrated a functional activity of only 29% and a raised C1q at 157 mg/l. Subsequent repeated investigation with careful specimen handling demonstrated undetectable levels of C1eIn and normal C1q. A diagnosis of C1eIn dysfunction was made, although at present it is unclear whether this represents an unusual variant of hereditary dysfunctional C1eIn deficiency. The patient was managed by various means, including steriodal and non-steroidal drugs, the latter forming part of her maintenance regime. CONCLUSIONS: To our knowledge, this is the first case of angio-oedema localised to the free gingiva. The role of stress in the acute exacerbations of oedema and bone loss is discussed along with the diagnostic pitfalls associated with this case.

Matrix metalloproteinases (MMP-8 and -9) and neutrophil elastase in gingival crevicular fluid of cyclosporin-treated patients.

BACKGROUND: Gingival overgrowth (GO) is one of the most important side effects of cyclosporin A (CsA) medication, but its pathogenesis is not completely understood. The aim of this study was to identify and compare collagenase-2 (MMP-8), gelatinase-B (MMP-9), and neutrophil (PMN)-elastase levels in gingival crevicular fluid (GCF) from 15 renal transplant patients receiving CsA therapy and exhibiting CsA GO, 14 patients with gingivitis, and 10 periodontally healthy subjects. METHODS: Clinical data were obtained on plaque index, papilla bleeding index, and hyperplastic index from each site studied. GCF samples and clinical data were collected from: 2 sites exhibiting CsA GO (CsA GO+) and 2 sites not exhibiting CsA GO (CsA GO-) in each CsA-treated patient; 2 diseased sites in each patient with gingivitis; and 2 healthy sites in each subject with clinically healthy periodontium. CsA GO+ and CsA GO- sites were divided into 2 subgroups as clinically not inflamed (PBI = 0) and inflamed (PBI > or =1). GCF MMP-8, MMP-9, and PMN-elastase levels were analyzed by immunofluorometric assay. RESULTS: GCF MMP-8 and -9 levels and clinical degrees of gingival inflammation in CsA GO+ sites were similar to those in diseased sites. However, GCF elastase levels were significantly lower in CsA GO+ sites compared to those in diseased sites. GCF MMP-8, -9 and PMN-elastase levels were not different between CsA GO- sites and healthy sites. Additionally, GCF MMP-8 and -9 levels in inflamed CsA GO+ sites were higher but not statistically significantly than those in diseased sites. In contrast, GCF PMN-elastase levels in inflamed CsA GO+ sites were significantly lower than the levels in diseased sites. CONCLUSIONS: These results show that CsA therapy does not have a significant effect on GCF MMP-8 and MMP-9 levels, but the gingival inflammation seems to be the main reason for their elevations. However, low GCF PMN-elastase levels can be an important factor in the pathogenesis of CsA-induced gingival overgrowth. CsA therapy does not eliminate the potential use of GCF MMP-8 and -9 as future diagnostic markers of gingival inflammation.

Phenytoin and cyclosporin A suppress the expression of MMP-1, TIMP-1, and cathepsin L, but not cathepsin B in cultured gingival fibroblasts.

BACKGROUND: Fibroblasts are known not only to synthesize and secrete extracellular matrix proteins, but also to degrade them for connective tissue remodeling. Drug-induced gingival overgrowth is characterized by a massive accumulation of extracellular matrix components in gingival connective tissues. Although some previous reports suggested that causative drugs stimulated the fibroblast proliferation, the results are not conclusive yet. In this study, we hypothesized that drug-induced gingival overgrowth could be a consequence of impaired ability of matrix degradation rather than an enhanced proliferation of gingival fibroblasts induced by these drugs. METHODS: Normal human gingival fibroblasts were cultured with or without either 20 microg/ml of phenytoin or 200 ng/ml of cyclosporin A. Total RNA and cellular proteins were collected every day for RT-PCR analyses and for measuring lysosomal enzyme activity. In addition, an immunohistochemical study was performed to detect lysosomal enzymes in cells from enlarged gingiva of the patients with phenytoin-induced gingival overgrowth. RESULTS: RT-PCR analyses revealed that these drugs suppressed the expression of MMP-1, TIMP-1, and cathepsin L, but not that of cathepsin B in a time-dependent manner. Then, we measured the activity of lysosomal enzymes and cathepsin B and L. The results indicated that although cathepsin B activity was not observed to be impaired, regardless of the drugs used in these cells, both total and active forms of combined activity of cathepsins B and L were suppressed in a time-dependent manner. CONCLUSIONS: The results indicate that, besides suggested effects of these drugs on gingival fibroblasts and/or on accumulated cells in the gingival tissues, extracellular matrix-degrading ability, particularly that by cathepsin L, is also suppressed by cyclosporin A and phenytoin in gingival fibroblasts, and that lysosomal enzyme plays an important role in the pathogenesis of drug-induced gingival hyperplasia.

Induction of superoxide dismutase isozymes by tumor necrosis factor-alpha and lipopolysaccharide in cultured normal and hyperplastic gingival fibroblasts.

To determine whether lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) are involved in the induction of superoxide dismutase (SOD) in gingival tissue, we examined their effect on induction of SOD isozymes in cultured normal (NGF) and phenytoin-induced hyperplastic (PHF) gingival fibroblasts. Treatment of both NGFs and PHFs with 10 to 50 ng/mL TNF-alpha for 24 hours increased the level of manganese SOD (MnSOD) to as much as four times the level of untreated cultures. PHFs, but not NGFs, were shown to be responsive to TNF-alpha in eliciting a significant increase in copper-zinc SOD (Cu/ZnSOD), albeit in a lesser amount than MnSOD. Additionally, treatment of both types of cells with 5 to 50 mg/mL of LPS for 24 hours also elicited an increase in the levels of MnSOD. Again, an LPS-induced increase in Cu/ZnSOD levels could only be demonstrated in PHFs, but not in NGFs. These observations were further confirmed by comparing the achromatic bands associated with SOD isozymes exhibited in the electrophoretogram using a nondenaturing polyacrylamide electrophoresis technique. These results indicate that TNF-alpha and LPS were capable of inducing both MnSOD and Cu/ZnSOD simultaneously in PHF fibroblasts. PHFs may be inherently more capable than NGFs in combating oxidative stress.

DNA content and alkaline phosphatase expression in cells of different gingival overgrowths.

The present study compared the alkaline phosphatase (ALPase) expression and DNA content at specific periods in cultured cells derived from non-inflamed enlarged gingivae of idiopathic gingivofibromatosis (IGF) and phenytoin-induced hyperplasia (PHG). Cultured cells from healthy gingiva or periodontal ligament (PDL) were used as controls. The DNA assay, ALPase assay and cytochemical staining for ALPase in cultured cells were performed at four, seven, and nine days. The presence of intense ALPase activity was a prominent feature in cultured IGF cells, whereas very low ALPase activity was detected in PHG cells. The cell lines tested showed no significant differences in DNA content. The expression of ALPase in these cells was population density-dependent. The observation that cells isolated from both types of gingival overgrowth exhibited a different ALPase profile at variance with normal gingival fibroblasts suggested that a distinct pathogenic mechanism may be involved in each type of gingival overgrowth.

Alkaline phosphatase activity in cultured cells of human healthy and hyperplastic gingivae: a preliminary study.

The present study compared the alkaline phosphatase (ALP) expression in cultured cells derived from different types of gingival hyperplasia. Tissue biopsies were obtained from patients with clinically non-inflamed, enlarged gingivae of idiopathic gingivofibromatosis (IGF) and phenytoin-induced hyperplasia (PHG). Fibroblasts obtained from the gingivae or periodontal ligaments (PDL) of the same or different healthy subjects were used as controls. Fibroblasts were plated and the ALP activity and total amount of DNA per well were studied in each isolated cell type at 4, 7 and 9 days of culture. The results revealed that fibroblasts derived from IGF tissue synthesized a large amount of ALP by day 7 and day 9 of culture. The amount of ALP activity was cell density-dependent and the strongest expression of enzyme activity was noted after cell confluence. In contrast, only very low ALP activity was detected in cells derived from normal gingivae and PHG. Cytochemical examination of these cells revealed similar observations. The fact that fibroblasts derived from PHG tissue did not exhibit ALP in vitro suggests that there was a shift of cell phenotype in this tissue. The increased expression of ALP in IGF cells may imply a developmental trend toward osteoblastic phenotypes.

[Essential and iatrogenic gingival hyperplasia. Its morphology and significance]. / Les hyperplasies gingivales essentielles et iatrogéniques. Morphologie et signification.

11 specimens were carried out in patients with idiopathic gingival hyperplasia (3 cases), gravidic hyperplasia (1 case), iatrogenic hyperplasia (5 cases after cyclosporin A administrated in patients with cardiac grafts, 2 cases after treatment by Adalat). By optic microscopy, the deep collagen base is thickened, associated sometimes to an inflammatory process. By histoenzymology, the fibroblasts have high activities of their oxidative enzymes and also of the enzymes of protein synthesis. The electron microscopy corroborates the numerous globular fibroblasts with well-developed rough endoplasmic reticulum. These results prove the main role of fibroblasts in these lesions and the etiopathogenesis of this hyperplasia is discussed.

Immunohistochemical identification of alpha-1-antitrypsin, alpha-1-antichymotrypsin, and lysozyme in focal hyperplastic gingivitis.

Tissues from twenty-four patients with focal hyperplastic gingival lesions containing calcification were stained for lysozyme (muramidase), alpha-1-antitrypsin, and alpha-1-antichymotrypsin. In eighteen of the twenty-four cases the tissues stained positively for lysozyme, and in all instances the tissues stained positively for alpha-1-antitrypsin and alpha-1-antichymotrypsin. These data suggest that the fibrous components of these lesions are derived from tissue histiocytes.

Gingival tissue reactions to orthodontic closure of extraction sites. Histologic and histochemical studies.

The formation of a hyperplastic gingival tissue with invagination was observed in the extraction areas in seven patients aged 12 to 17 years who, after extraction of the maxillary first premolars, were undergoing orthodontic treatment with an edgewise appliance. This tissue was excised when about 2 mm. of the space remained to be closed. Histologic and histochemical analyses of the biopsy areas demonstrated hyperplasia and increased metabolism in the invaginated epithelium as well as increased production of glucose aminoglycans in the surrounding connective tissue. Loss of collagen was noted in the same regions. There was evidence of bone remodeling rather than only bone resorption in the biopsy area. It was concluded that stimulation from the orthodontic forces was responsible for the hyperplastic tissue reaction and that the increased amounts of glucose aminoglycans may be responsible for possible relapses.