The effect of root detoxification on human gingival fibroblasts

Comparative growth or inhibition of growth of human gingival fibroblasts on experimental root surfaces was used to assess the efficacy in vitro of the Cavitron and Prophy-Jet. Six teeth with a hopeless periodontal prognosis, slated for prosthetic replacement, were extracted and sectioned into 12 specimens. Experimental specimens were instrumented with the Cavitron or Cavitron and Prophy-Jet. Four calculus-covered control specimens were not treated. Eight root specimens in fibroblast tissue culture were stained with 1.0% toluidine blue for determination of fibroblast viability after 48 hours. No fibroblast growth took place on calculus control specimens. Cavitron specimens showed light fibroblast growth and viability. Cavitron/Prophy-Jet specimens showed superior growth and vitality of fibroblasts.     

Adhesion and growth of cultured human gingival fibroblasts on periodontally involved root surfaces treated by Er:YAG laser

BACKGROUND: The application of Er:YAG laser irradiation, approved in 1997 to be used on dental hard tissues, has been investigated for periodontal therapy. The aim of this study was to analyze the biocompatibility of root surfaces treated by Er:YAG laser. METHODS: Adhesion and growth of cultured human gingival fibroblasts on root surfaces treated by either irradiation with Er:YAG laser or curet were compared. Thirty single-rooted teeth extracted because of periodontal disease were used. Calculus deposits on all experimental surfaces were removed, and the teeth were divided into three groups according to the applied treatment: group A, root planing with Gracey curet no. 3/4; group B, two irradiations with laser (60 mJ/pulse, 10 Hz; 10" each with 10-second interval, 3 J/cm2); group C, two irradiations with laser (100 mJ/pulse, 10 Hz; 10" each with 10-second interval, 5 J/cm2). Fragments (5 mm x 6 mm) were obtained from the experimental surfaces. Then, 1 x 10(3) cells were seeded on the top of each fragment. One, 2, and 3 days after seeding the specimens were prepared for scanning electron microscopy analysis, and the cells on the electronmicrographs were counted. The data obtained in triplicate were statistically compared by the Kruskall-Wallis test complemented by the Dunn test (P < or = 0.05). RESULTS: Human gingival fibroblasts adhered to and grew on all treated surfaces. Group B presented a significantly higher cell count than did the other two groups at days 1 and 2. Three days after seeding the cultured fibroblasts of groups A and B reached total confluence. The cell count of group B was significantly higher than that of group C. CONCLUSION: The surfaces treated with 60 mJ/pulse Er:YAG laser irradiation promoted faster adhesion and growth than surfaces treated with either root planing or 100 mJ/pulse Er:YAG laser irradiation.     

The effect of smoking on epithelial proliferation in healthy and periodontally diseased marginal gingival epithelium

BACKGROUND: Smoking causes an increase in the thickness of gingival epithelium, which is the outcome of increased keratinocyte proliferation or loss. Smoking-related changes in the proliferative activity of the gingival epithelium are largely uncharacterized for periodontal diseases. The aim of the present study was to determine the effects of smoking on the proliferation of the epithelium in periodontally diseased marginal gingiva by comparing the expression patterns of two different proliferation markers. METHODS: Gingival biopsies (N=60) were obtained from smokers who had clinically healthy gingiva (n=10), smokers with gingivitis (n=10), smokers with periodontitis (n=10), non-smokers with clinically healthy gingiva (n=10), non-smokers with gingivitis (n=10), and non-smokers with periodontitis (n=10). The quantitative measurement of maximum epithelial thickness was performed on hematoxylin and eosin-stained sections. The expression patterns for proliferating cell nuclear antigen (PCNA) and Ki67 were evaluated immunohistochemically. RESULTS: The percentage of PCNA-positive cells was higher than the percentage of Ki67-positive cells in all groups (P<0.001). When the mean values of PCNA and Ki67 were compared in each group, a statistically significant difference was observed only in the healthy smoker group (P=0.003). Significant differences in PCNA proliferation indices were only found between the smoker group and the non-smoker healthy group (P=0.015). CONCLUSIONS: Smoking had an affect on the proliferation of cells in the oral gingival epithelium, regardless of periodontal status. The increase in thickness of the epithelium was not associated with smoking; periodontal status and inflammation seemed to be more important factors. Smoking induced the replication activity of gingival epithelium and induced DNA repair.     

The effect of EDTA on the attachment and growth of cultured human gingival fibroblasts in periodontitis-affected root surface

The purpose of the present study was to analyze the effects of 5% and 24% EDTA on the attachment of gingival fibroblasts to periodontally diseased root surfaces. A flat root surface was created on human teeth that were extracted due to severe periodontitis. The teeth were etched with the following concentrations of etylediaminetetraacetic acid (EDTA) for two minutes: 5% (group I) and 24% (group II). Group III was soaked in saline and served as a control. The specimens and fibroblasts were incubated in a culture medium for 24 hours each day for one and two weeks and photographed using scanning electron microscopy. Each specimen was examined for the migration of cells into the etched and non-etched root surface. No fibroblasts could be detected on the saline groups. More fibroblasts could attach to the surface treated with 24% EDTA than with 5% EDTA. It was concluded that supersaturated EDTA at 24% enhances the attachment of gingival fibroblasts to the root surface.     

Histological assessment of root cementum at periodontally healthy and diseased human teeth

This study aimed to investigate and compare the lipid and polysaccharide content of the cemental surfaces of healthy and periodontally-involved teeth. Thirty periodontally-involved single-rooted teeth from fifteen patients with localized juvenile, adult and rapidly progressive periodontitis were included in the experimental group and 5 healthy teeth were assessed in the control group. Frozen serial sections were obtained and stained with hematoxylin-eosin for morphological assessment. Oil-Red-O and Alcian Blue-Periodic Acid Schiff stains were used to evaluate the presence of lipids, neutral and acidic polysaccharides using light microscopy. It was found that with hematoxylin-eosin staining in the experimental group, both the involved and uninvolved cementum surfaces of teeth, which belong to all periodontitis groups, showed generally irregular surfaces that contain some resorption areas. Alcian Blue-Periodic Acid Schiff positive staining was observed only superficially and at the areas associated with microbial dental plaque. However, Oil-Red-O staining was positive only superficially at 5 teeth that belonged to localized juvenile and rapidly progressive periodontitis groups. Apparent lipopolysaccharide staining into cementum was not seen in any of the diseased teeth. The results presented here suggest that endotoxin was only localized in superficial layers and associated with only microbial colonization.     

Topographic distribution of subgingival plaque along root surfaces of human periodontally diseased teeth

Abstract The purpose of this study was to characterize the topographical distribution and organization of subgingival plaque in periodontally diseased teeth. 26 extracted teeth were fixed and processed for undecaicified histological evaluation. The sections were cut perpendicular to the long axis of the tooth and analyzed by phase-contrast microscopy. The coronal portion of the analyzed roots showed a dense accumulation of filamentous forms, fusiform rods, coccoid forms and loosely aggregated spirochetes. The middle and apical portions showed a non-uniform distribution of the microflora, with microorganisms representing all the known morphotypes. Furthermore, plaque was detected below undisturbed periodontal fibers, indicating that plaque not only forms apically, but also in a lateral direction, penetrating and colonizing below areas where periodontal fibers are inserted into the root surface.     

Anti-bacterial effect of citric acid treatment of periodontally diseased root surfaces In vitro

Abstract This investigation examined whether citric acid may exert an anti-bacterial effect against plaque deposits present on root surfaces in vitro. Aerobic and anaerobic blood-agar plate cultures were prepared from plaque samples obtained from the proximal root surfaces of 20 periodontally diseased human teeth following extraction. Ten teeth were exposed to saturated citric acid (pH 1) for 3 min, followed by rinsing in sterile 0.85% saline and plaque samples were then obtained immediately adjacent to those sites sampled initially. Controls consisted of using sterile water instead of citric acid on a further five teeth. The numbers of colonies present on pre- and post-treatment culture plates were counted al 24 h. The results indicated that citric acid application reduced, in all instances, the numbers of colonies grown from post-exposure plaque samples as compared to pre-exposure samples. No colonies were detected in 55% of aerobic and 30% of anaerobic cultures of acid-treated root surface samples. For aerobic cultures, citric acid exposure reduced the number of colonies grown from >10⁴ to <100 in 95% of the root surfaces sampled, while for anaerobic cultures, reduction from >10⁴ to <100 was found in 80% of surfaces sampled. The findings indicate that citric acid exerts anti-bacterial activity against microbial plaque deposits present on periodontally diseased root surfaces in vitro.     

Fatty acids of healthy and periodontally diseased root substance in human teeth.

In this comparative study, samples of root substance from healthy and periodontally diseased human teeth were analyzed for fatty acids by means of a gas-chromatographic technique. The sample preparation procedure allowed specimens from individual teeth to be analyzed. The content of the fatty acids C16:0, C16:1, C18:0, and C18:1 in the superficial layer of the periodontally diseased teeth was significantly higher than that of healthy teeth. In the inner layer, there was no such difference. Four different 3-hydroxy fatty acids (3-OH C14:0, 3-OH C15:0, 3-OH C16:0, and 3-OH C17:0) were identified by combined gas-chromatography/mass-spectrometry in the tooth substance of periodontally diseased teeth. This indicates the presence of lipid A, which is the toxic component of bacterial lipopolysaccharides.     

Scanning electron microscopy in vitro study on the effect of Carisolv application on periodontally diseased root surfaces

The purpose of this study was to evaluate the characteristics of diseased root surfaces treated by the association of scaling and the application of Carisolv. Twenty-four uniradicular periodontally involved teeth were used in this study. The teeth were divided randomly into three groups: eight teeth were scaled and root planed until there was a complete visible removal of calculus (group 1), Carisolv was applied on the root surfaces of eight teeth twice for 30 seconds before scaling with a sharp curette (group 2), and eight teeth received the same treatment as in group 2 but with a blunt curette (group 3). Specimens were examined using scanning electron microscopy. The superficial aspect of the roots from group 1 presented scratches that mirrored the curette cutting edge, and the smear layer completely covered the surface. Root surfaces from groups 2 and 3 also presented a smear layer that covered the surface completely, but it was somewhat smoother than group 1. The use of Carisolv as an adjunct to scaling and root planing presented no advantage for smear layer removal over scaling alone, suggesting that no benefit is obtained by the use of Carisolv during periodontal mechanical treatment.     

The distribution and quantitation of cementum-bound lipopolysaccharide on periodontally diseased root surfaces of human teeth

The aim of this study was to estimate the concentration of cementum-bound LPS on a group of 12 teeth that had been extracted because of periodontitis. LPS on scaled root surfaces was labelled by immunogold/silver staining. The concentrations of LPS were estimated by quantifying the amount of bound silver label, using X-ray microanalysis in areas free of plaque or calculus. These were compared against standards of known LPS concentration, which were separately prepared for each sample. Cementum-bound LPS was detected at concentrations of up to 2 EU/mm2 of affected root surface. However, most of the root surfaces had considerably lower concentrations than this, the mean of all samples never exceeding 0.7 EU/mm2. LPS concentrations were highest on cementum towards the apical regions of the affected pocket. These findings confirm that cementum-bound LPS is only present in low concentrations on affected teeth, and suggest that the clinical significance of cementum-associated LPS may have been over-estimated in the past. The demonstration of LPS appears to be more important as an indicator of retained bacteria and calculus than of cementum-bound LPS per se.     

Inhibitory effects of human gingival extracts on bone resorption in vitro

The effects of human gingival extracts on in vitro , bone resorption were examined and related to the amount of plaque, gingival inflammation and bone resorption present at the time of sampling. Twenty patients with varying degrees of periodontal disease were used in this study. The plaque index and bleeding index were recorded. the marginal gingiva removing surgically and immediately freeze dried. Alveolar bone resorption was then measured from the cemento enamel junction (CFI) to the alveolar bone crest directly during surgical exposure or on radiographs. The bone resorting activity of extracts from freeze dried gingiva was then tested in an in vitro bone resorption assay. No bone resorting activity was noted in 17 of the 18 extracts tested and thus no correlation could be found with the clinical indices. Mean values of the T/C ratios for 18 extracts tested indicate a biphasic suppression of control resorption by the extracts. The extracts suppressed resorption stimulated by known bone resorbing agents. H [eating of the extract prior to testing eliminated this suppressive effect. It is concluded that inhibitors of bone resorption are present in human gingival extracts. Their presence may explain our inability to demonstrate bone resorhing activity in human gingiva.     

Attachment, growth and synthesis by human gingival fibroblasts on demineralized or fibronectin-treated normal and diseased tooth roots

Regeneration of a fibrous attachment to tooth roots previously exposed to periodontal pockets requires interaction between the root surface and fibroblasts. Experiments were designed to assess whether or not fibroblasts will attach, grow and synthesize normally on tooth roots and determine the efficacy of various treatments of diseased root surfaces on these activities. Extracted teeth were collected, washed and stored frozen until used. The roots were sectioned and the surface area determined. All roots were thoroughly planed to remove most of the cementum and some were then treated with fibronectin or citric acid. Each root was placed in a Linbro well and a suspension of fibroblasts in Dulbecco Vogt medium with 10% fetal calf serum added. After cell attachment, each root was transferred to a fresh well and incubated. Some roots were examined by scanning electron microscopy. Attachment and growth were assessed by harvesting and counting cells after various incubation times. The pattern of protein synthesis was determined by preparing fluorographs from cultures pulse labeled with radioactive S35-methionine. Cells attach and grow on planed root surfaces. The number that attach to normal and diseased roots is greatly enhanced by pretreatment with fibronectin or citric acid. By day three, the cells become confluent at approximately 20,000/cm2. The pattern of proteins produced is comparable to that seen in cultures maintained in petri dishes. After root planing, cells attach, grow and synthesize equally well on diseased and normal roots.     

Cytotoxicity of the bacterium Actinobacillus actinomycetemcomitans extracts in human gingival fibroblasts

Filter-sterilized sonic extracts (SE) of strains of Actinobacillus actinomycetemcomitans were shown to inhibit the proliferation of human gingival fibroblasts in vitro. The inhibition was dose-dependent: a 50 per cent inhibitory dose of 2 micrograms protein/ml was found for A. actinomycetemcomitans strain Y4. The inhibitory activity could be neutralized by homologous antiserum and was heat inactivated by temperatures of 80 degrees C or greater. The fibroblast-inhibitory activity was present in SEs of both leukotoxic-producing and non-leukotoxic strains of A. actinomycetemcomitans, suggesting that a separate agent is responsible for leukotoxicity and fibroblast inhibition. A short (10 min) exposure of the fibroblasts to the A. actinomycetemcomitans SE was sufficient to inhibit irreversibly cell proliferation, provided that serum was present at the time that the cells were exposed to the SE. SE-challenged fibroblasts exhibited a marked decrease in the rate of DNA synthesis, but no inhibition of RNA or protein synthesis. Although the SE-treated cells did not proliferate, they appeared to remain intact and viable; and displayed no gross morphological alterations.     

The effect of chlorhexidine treatment of root surfaces on the attachment of human gingival fibroblasts in vitro

Chlorhexidine mouthrinse is a widely used adjunct in periodontal therapy due to its bactericidal effects. The effect of this agent on chronic gingivitis and wound healing following surgical therapy in animals and humans has been favorable. The re-establishment of lost connective tissue attachment to the root surface following periodontal therapy is a desirable goal in which the ability of periodontal ligament fibroblasts to reattach to root surfaces of periodontally involved teeth is a critical event. Understanding the effect of chlorhexidine on fibroblast attachment will provide the rationale for its use during the healing phase of periodontal surgery. For this study, impacted third molars were sectioned into 4 pieces. Groups of 10 root pieces were exposed to 0.12% chlorhexidine or saline for 3 minutes followed by a distilled water rinse. The root pieces were incubated with human gingival fibroblasts (HGF) using standard tissue culture techniques for 1, 2, 4, 6, and 8 hours. HGF were prelabeled with 3H-thymidine to a standard specific activity. The surface area of each root piece was determined and the attached cells quantified by using scintillation spectroscopy. The number of cells per unit area was then calculated and the data expressed as cells/mm2. The repeated measures design was statistically analyzed by repeated measures analysis of variance. There was a significant difference between the number of attached cells in the chlorhexidine and the control groups (P less than 0.001). Exposure of root surfaces to chlorhexidine significantly inhibits subsequent fibroblast attachment which may interfere with regeneration of the periodontium. Hence, the data suggest that efforts should be made to minimize chlorhexidine contact with the root surface with physical barriers.     

Expression of CDIa on monocytes cultured with supernatants from periodontally diseased gingival epithelial cells

OBJECTIVE: Langerhans cells are believed to originate from the monocyte lineage and have been reported to increase in number with plaque accumulation and gingival inflammation. The aim of this study was to investigate the effects of local gingival epithelial factors on the induction of CDla, a Langerhans cell phenotype, on monocyte rich populations.
MATERIALS AND METHODS: Peripheral blood monocyte rich populations from healthy subjects were cultured for 24 h with either healthy gingival or periodontally diseased gingival epithelial supernatants. Additionally, the monocyte rich populations were cultured with cytokines IL-Iα, IL-Iβ, IL-6 and TNF-α which are known to be produced by epithelial cells or co-cultured with autologous epithelial cells. The percent CDIa positive cells was determined using FACS analysis.
RESULTS: Healthy gingival supernatants did not induce CDIa expression in monocyte rich populations, however, a significant increase in per cent CDla⁺ cells for monocyte rich populations cultured with five (P < 0.01) of six periodontal gingival epithelial supernatants was found. IL-lα or TNF-α (10ng/well) resulted in a significant increase in the per cent CDla⁺ cells (P < 0.01). Depletion of CDla⁺ Langerhans cells from healthy gingival epithelium did not enhance induction of CDIa expression in monocyte rich populations. Monocyte rich populations cultured together with non-depleted epithelial cultures resulted in a decreased percent of CDla⁺ cells.
CONCLUSION: These findings indicated that epithelial factor/s associated with periodontally involved epithelia, may be involved in inducing a Langerhans cell phenotype in monocyte rich populations. The data also provide indirect evidence for a role of Langerhans cells in inhibiting induction of CDIa in healthy epithelium.     

The presence of nicotine on root surfaces of periodontally diseased teeth in smokers

There is a growing body of scientific evidence to support the concept that the use of tobacco products significantly contributes to the progression of periodontal disease or is detrimental to healing following periodontal therapy. Several studies have shown toxic effects of nicotine on peripheral circulation and the immune response. The purpose of the present study was to identify and compare the quantity of nicotine present on root planed and nonroot planed surfaces of teeth from smokers. Twenty-nine single-rooted teeth from 11 smokers were extracted, brushed clean, and the roots sectioned longitudinally. The respective halves were either left untreated (Group A) or thoroughly root planed (Group B). Pulpal tissue was removed and the individual root sections weighed. Each half was extracted for nicotine using a methylene chloride technique. Quantification was performed using high pressure liquid chromatography (HPLC) and the sections compared on a nicotine per root weight basis. Results showed a greater amount of nicotine present on non-root planed sections than on treated sections, although some treated specimens revealed small amounts of the substance. These findings suggest that nicotine is present on the root surface but is largely removed by thorough root planing. Its presence is not surprising in light of the recent finding that nicotine and cotinine, the major metabolite of nicotine, are found in gingival crevicular fluid. Recent studies have shown a particularly harmful effect of nicotine on fibroblasts. Its presence on root surfaces may, therefore, impair wound healing and alter the host response in periodontal disease. The use of tobacco products in conjunction with periodontal therapy may interfere with optimal healing and/or lead to further periodontal breakdown.     

Cytotoxicity of a new root canal filling material on human gingival fibroblasts

This study evaluated the cytotoxicity of the root canal sealing materials Resilon and Epiphany versus gutta-percha, Grossman's sealer, Thermaseal, and Sealapex. Using human gingival fibroblasts the fibroblasts cultures were incubated for either 1 or 24 h to test the cytotoxicity after freshly mixing or after 24 h of setting. Fibroblasts were then stained with trypan blue, to determine number of dead cells. Data were analyzed using ANOVA and t tests. Resilon was similar to gutta-percha and the control. Epiphany was less cytotoxic than Grossman's sealer at both the 1 and 24 h time periods. Epiphany was more cytotoxic than Sealapex at the 1-h time period but less cytotoxic at the 24 h time period. These results indicated that Resilon had a lower cytotoxicity and that Epiphany was more cytotoxic than conventional materials.     

牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞生物学性能的影响

目的:观察牙齿外伤钛固定夹板(titanium trauma splint,TTS)浸提液对人牙龈成纤维细胞(human gingival fibroblasts,HGF)生物学性能的影响,为临床应用提供理论依据。方法:分离、培养鉴定人牙龈成纤维细胞,将牙齿外伤钛固定夹板浸提液加入人牙龈成纤维细胞培养基中,通过倒置显微镜观察细胞形态学改变、MTT法细胞毒性实验和细胞凋亡实验,观察牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞生物学性能的影响。结果:牙齿外伤钛固定夹板浸提液对人牙龈成纤维细胞的形态无明显改变,其增殖和凋亡的影响较正常人牙龈成纤维细胞相比无明显细胞毒性,不引起细胞凋亡。结论:牙齿外伤钛固定夹板具有良好的生物安全性,有一定的临床应用前景。     

Attachment of human gingival fibroblasts to periodontally involved root surface following scaling and/or etching procedures: a scanning electron microscopy study

This study evaluated, in vitro, fibroblast attachment to periodontally involved root surfaces which were either root planed or acid/chelated by different agents. Specimens were divided into 3 groups of 12 specimens each. The root surfaces were root planed with a Gracey 7/8 curette, an EMS or an Amdent piezo-electric scaler and treated with saline, citric acid, tetracycline hydrochloride or EDTA to produce different surface textures. They were then cultured with fibroblasts for 72 h and examined by scanning electron microscopy. There was a significantly greater number of fibroblasts attached to specimens treated with citric acid, tetracycline and EDTA than to those root planed only. Furthermore, fibroblasts were more likely to attach to rough-surfaced than to smooth-surfaced specimens.