牙骨质基质胍提取物促牙龈成纤维细胞、成骨细胞粘附研究

目的 :鉴定牙骨质基质胍提取物内牙骨质蛋白的生物活性。方法 :制取牙骨质基质胍提取物 ,检测人牙龈成纤维细胞、MC3T3 E1成骨细胞两种细胞在含牙骨质基质提取物浓度分别为 2 .5、5、10、2 0μg/ml的DMEM培养液中孵化 1h的贴壁率并以加牛血清蛋白 1mg/ml为空白对照组。检测两种细胞在含牙骨质基质提取物浓度 10 μg/ml的DMEM培养液中孵化 30、60、90、12 0min贴壁率。 结果 :不同浓度的牙骨质基质提取物均可促进牙龈成纤维细胞和成骨细胞的粘附 ,并呈浓度依赖性 ,尤以 10 μg/ml的牙骨质基质提取物的浓度为较佳浓度。细胞的贴壁率随牙骨质基质提取物作用时间的延长逐步升高 ,并以 90min为较理想的作用时间。结论 :人牙骨质基质胍提取物含有可促进牙龈成纤维细胞和成骨细胞粘附的牙骨质活性蛋白     

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts

Objective: Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts.

Materials and methods: Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E₂, interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays.

Results: PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24?h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p?p?2, IL-6, IL-8 and MMP-1. Treatment of IL-1β stimulated fibroblasts in combination with PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE₂, IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE₂, IL-6, IL-8 or MMP-1 by gingival fibroblasts.

Conclusions: Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.     

Interleukin-6 production by human gingival fibroblasts

The ability of human gingival fibroblasts to synthesize interleukin-6 (IL-6) was studied using in vitro and immunohistochemical techniques. Culture supernatants of human gingival fibroblasts contained significant quantities of IL-6 activity which could be stimulated by fetal calf serum, recombinant interleukin-1 beta and lipopolysaccharide. The activity in the supernatants was specifically attributed to IL-6 since up to 97% of the activity could be inhibited by an anti-IL-6 antibody. Immunohistochemical studies on low-density human gingival fibroblast cultures indicated that the cells were associated with material reactive to the anti-IL-6 antibody. This localization was seen on the cell surface and in the cytoplasm of the cells. Immunoreactivity towards IL-6 was also noted in sections of human gingivae. Moderate staining was seen in the connective tissues and lower portions of the gingival epithelium, while intense staining was seen at foci of inflammation. The identification of IL-6 with human gingival tissues and cells implicates this lymphokine in the molecular events associated with the inflammatory periodontal diseases.     

Enhancement by extracts of mineralized tissues of protein production by human gingival fibroblasts in vitro

Non-confluent cell cultures were exposed to both guanidine and guanidine-EDTA extracts of cementum, dentine and alveolar bone, at concentrations from 2 to 50 μg/ml for 48 h. The cells were radioactively labelled during the last 24 h. Total protein production was measured via incorporation of radioactive proline; collagen production was estimated by digestion of the radioactive protein mixture with bacterial collagenase. All guanidine-EDTA extracts elicited statistically-significant increases in total protein production when compared to controls. At 50 μg/ml of extract, the increase in protein production was 340, 143 and 338 per cent for bone, cementum and dentine, respectively. Similar results were obtained for collagen production. Guanidine-EDTA extracts also stimulated an increase in the production of specific proteins, as ascertained by gel electrophoresis. In contrast, the guanidine extracts had no effect on either protein or collagen production. Thus the functions of gingival fibroblasts can be altered by proteins from associated mineralized tissues. Identification of such proteins and their biological functions would enhance knowledge of the mechanisms that regulate connective-tissue regeneration.     

Mitogenic activity of cementum components to gingival fibroblasts

Cementum forms the interface between root dentin and periodontal ligament through which periodontal connective tissue is attached to root surfaces. We have examined how cementum components influence the biological activities of gingival fibroblasts. Cementum was harvested from freshly extracted human teeth and extracted sequentially with 0.5 mol/L acetic acid, 4 mol/L guanidine-0.5 mol/L EDTA, and bacterial collagenase. The extracts were concentrated and analyzed for mitogenic activity to human gingival fibroblasts. DNA synthesis was assayed by measurement of [3H]thymidine incorporation by quiescent fibroblasts activated to divide, and cell growth was determined by the counting of cells over a 10-day period. Results showed that extracts of cementum stimulated quiescent gingival fibroblasts to synthesize DNA and grow. The stimulation was dose-dependent, and most of the stimulatory activity was extracted by acid. Addition of small quantities of serum potentiated the mitogenic activity to levels greater than those of control cultures containing 10% fetal calf serum. The mitogenic activity was heat-stable, but it was destroyed by trypsin. Neither platelet-derived growth factor (PDGF) nor epidermal growth factor (EGF) was detectable in the cementum extract, and extracts of human dentin and skin contained very little mitogenic activity. We conclude that cementum contains substances capable of regulating the growth of gingival fibroblasts, and that these substances may play an important role in gingival connective tissue formation and regeneration.     

Biological effects of cementum and bone extracts on human periodontal fibroblasts

BACKGROUND: Non-collagenous proteins of mineralized tissues play important roles in bone induction during mineralization and in regulating the activity of many types of mesenchymal cells. This study was conducted to determine the effects of acetic acid extracts of bone and cementum on alkaline phosphatase (ALPase) activity and in vitro mineralization of cultured human periodontal fibroblasts (hPF). METHODS: Alveolar bone and cementum obtained from clinically healthy subjects were extracted by a solution containing 0.5 M acetic acid and enzyme inhibitors. Osteoblastic phenotypes of hPF were assayed by ALPase activity, gene expression of bone marker proteins, and the ability to produce in vitro mineralization in culture media containing 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate, and 10(-7) M dexamethasone. The effects of cementum and bone extracts on the expression of osteoblastic phenotypes in hPF were also determined. RESULTS: Many protein components, varying in molecular weight from 10 to 14 to 120 kDa, were detectable in 10% SDS-PAGE of both cementum and alveolar bone extracts. The hPF cells were found to exhibit a moderate ALPase activity when compared with rat osteosarcoma (ROS) 17/2.8 cells under the same experimental conditions. Gene expression for ALPase, osteocalcin bone sialoprotein, osteopontin, and BMP-7 at mRNA message was detected by RT-PCR in hPF and ROS 17/2.8 cells. The confluent hPF and ROS 17/2.8 cells showed evidence of calcium deposition in the extracellular milieu at 30 and 15 to 30 days' cultures, respectively, under a mineralization medium. The hPF appeared to form mineralized foci with morphological characteristics different from the mineralized nodules produced by ROS 17/2.8 cells. The addition of low concentrations (5 microg/ml) of either cementum or bone extract produced an increase in the size and number of mineralization spots, as well as greater ALPase activity in both hPF and ROS 17/2.8 cultures during the observation periods. CONCLUSIONS: These results suggest that hPF possess certain mineralizing phenotypes, and that acetic acid extracts of bone and cementum contain components capable of stimulating osteogenic differentiation of hPF.     

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.     

Intracellular interleukin-1 alpha production in human gingival fibroblasts is differentially regulated by various cytokines.

Interleukin-1 (IL-1) may play a critical role in immune and inflammatory responses in inflamed gingiva, and it is synthesized by a wide variety of host cells. In this study, we examined the regulatory effects of various cytokines on bioactive membrane IL-1 and intracellular IL-1 alpha production in cultured human gingival fibroblasts (HGF). Recombinant human (rh) IL-1 beta stimulated membrane IL-1 activity, which was mainly attributed to IL-1 alpha. rhIL-1 beta and rh tumor necrosis factor (TNF)-alpha stimulated HGF to produce intracellular IL-1 alpha, whereas rh interleukin-6 (IL-6), rh interleukin-4 (IL-4), and rh interferon (IFN)-gamma did not do so. Intracellular IL-1 alpha production induced by rhIL-1 beta or rhTNF-alpha may be partially related to protein kinase C (PKC) activation, because rhIL-1 beta or rhTNF-alpha-induced intracellular IL-1 alpha production was stimulated by pre-treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, but was suppressed by the pre-treatment with 1-(5-isoquinoline-sulfonyl) -2-methylpiperazine dihydrochloride (H-7), which is a PKC inhibitor. rhIL-4 inhibited rhIL-1 beta- or rhTNF-alpha-induced intracellular IL-1 alpha production, but rhIL-6 had no effect on this production. Pre-treatment with rh IFN-gamma remarkably enhanced intracellular IL-1 alpha production induced by subsequent treatment with rhIL-1 beta or rhTNF-alpha. Simultaneous treatment with rhIFN-gamma and rhIL-1 beta inhibited rhIL-1 beta-induced intracellular IL-1 alpha production, but co-treatment with rhIFN-gamma and rhTNF-alpha enhanced rhTNF-alpha-induced intracellular IL-1 alpha production. These results suggest that in inflamed gingiva, pro-inflammatory cytokines such as IL-1 beta and TNF-alpha may induce bioactive intracellular IL-1 alpha production in human gingival fibroblasts and that this production can be differentially modulated by T-cell-derived cytokines such as IFN-gamma or IL4.     

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.     

Thymosin beta4 is cytoprotective in human gingival fibroblasts

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.     

Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts.

To understand orthodontic tooth movement and determine optimal orthodontic force from a biological viewpoint, nitric oxide production in cultured human periodontal ligament fibroblasts was measured at varying levels of hydraulic pressure. The fibroblasts in a culture flask were exposed to the controlled change in hydraulic pressure, and intracellular nitric oxide levels were measured in real time by a nitric oxide-binding fluorescent dye, diaminofluorescein-2. The fibroblasts produced a significantly larger amount of nitric oxide at the pressure of 75 and 100 mmHg, compared with the pressure of 0, 25, and 50 mmHg (P <.0001, one-way ANOVA, and P <.05, Tukey-Kramer test). Immunohistochemically, the cultured fibroblasts expressed brain nitric oxide synthase. The pressure level to enhance nitric oxide production was comparable to the magnitude of clinically used orthodontic force (80 g/cm(2)). Nitric oxide might be a key regulator in orthodontic tooth movement.     

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。     

Prostaglandin production by human gingival fibroblasts inhibited by triclosan in the presence of cetylpyridinium chloride

BACKGROUND: The effect of triclosan plus the cationic detergent cetylpyridinium chloride (CPC) was evaluated for prostaglandin inhibition in human gingival fibroblasts. Since triclosan has previously been shown to inhibit proinflammatory cytokine induced prostaglandin E2 (PGE2) production, we wanted to determine if triclosan, in the presence of CPC, could enhance these effects. METHODS: Initial studies determined that both triclosan and CPC were cytotoxic to human gingival fibroblasts in concentrations exceeding 1.0 microg/ml for either agent longer than 24 hours in a tissue culture. Therefore, subsequent studies measuring prostaglandin biosynthesis and cyclooxygenase (COX)-1 and COX-2 mRNA expression were performed in concentrations and times that did not significantly affect cell viability. RESULTS: PGE2 biosynthesis was dose dependently inhibited by both triclosan and triclosan and CPC when challenged by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta. At pharmacologically relevant concentrations, triclosan and CPC inhibited IL-1beta-induced PGE2 production to a greater extent than triclosan alone (P = 0.02). Moreover, enhanced COX-2 mRNA repression was observed with triclosan and CPC in comparison to triclosan alone in IL-1beta and TNF-alpha stimulated cells. No effect on COX-1 gene expression was observed. Further analysis of cell signaling mechanisms of triclosan and CPC indicates that nuclear factor-kappa B (NF-kappaB) and not p38 mitogen-activated protein kinase (MAPK) signaling may be impaired in the presence of triclosan and CPC. CONCLUSION: This study indicates that triclosan and CPC are more effective at inhibiting PGE2 at the level of COX-2 gene regulation, and this combination may offer a potentially better anti-inflammatory agent in the treatment of inflammatory lesions in the oral cavity.     

Transforming growth factor-beta stimulates interleukin-11 production by human periodontal ligament and gingival fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta is a potent multifunctional polypeptide, abundant in the bone matrix. Interleukin (IL)-11 is a pleiotropic cytokine with effects on multiple cell types. The present study was performed to evaluate the regulatory effects of TGF-beta on IL-11 production by human periodontal ligament cells (PDL) and human gingival fibroblasts (HGF). MATERIAL AND METHODS: The expression of TGF-beta receptor in PDL and HGF were observed using flow cytometry. PDL and HGF were stimulated with TGF-beta with or without protein kinase C (PKC) inhibitors and activator. IL-11, bone morphogenetic protein-2 (BMP-2) and TGF-beta mRNA expression was quantified by real-time polymerase chain reaction (PCR). IL-11 production was measured using enzyme-linked immunosorbent assay. RESULTS: PDL and HGF expressed both TGF-beta receptor I and TGF-beta receptor II on the cell surfaces. IL-11 mRNA expression and IL-11 production were augmented by TGF-beta in both PDL and HGF, with higher values in PDL. PKC inhibitors partially suppressed TGF-beta-induced IL-11 production in PDL and HGF, whereas activator enhanced it. TGF-beta mRNA and BMP-2 mRNA expression were up-regulated by TGF-beta in PDL. CONCLUSION: These results suggest that PDL produce IL-11 in response to TGF-beta.     

Interleukin-1 stimulates proteoglycan and hyaluronic acid production by human gingival fibroblasts in vitro

The effect of recombinant interleukin 1β [IL-1β] on proteoglycan and hyaluronic acid synthesis by human gingival fibroblasts has been investigated. It was found to stimulate gingival fibroblast proliferation in a dose dependent fashion with the midpoint of this response being in the 10⁻¹¹ mol/L range. At a concentration of 10⁻¹¹ mol/L, IL-1β stimulated proteoglycan synthesis by 40 per cent. Although IL-1β can stimulate cell proliferation and prostaglandin synthesis, its effect on proteoglycan synthesis was independent of these parameters. The kinetics of proteoglycan degradation in the presence or absence of IL-1β was monitored by pulse chase experiments and were found not to differ between treated and untreated cultures. The molecular size and carbohydrate composition of the proteoglycans was not affected by IL-1β. Additional studies revealed the synthesis of hyaluronic acid was also stimulated by IL-1β. As for the proteoglycans, inhibition of cell proliferation did not affect the stimulatory effect of IL-1β. However, blockage of prostaglandin synthesis abolished the stimulatory effect of IL-1β on hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis was found to be related to elevated levels of the enzyme hyaluronate synthetase. Molecular size analysis of newly synthesized hyaluronic acid revealed that cells treated with IL-1β synthesized more large molecular mass hyaluronic acid. Taken together, these findings are considered to reflect the ability of gingival fibroblasts to respond to inflammatory mediators in a manner indicative of early tissue repair.     

Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts

Lin S‐J, Lu H‐K, Lee H‐W, Chen Y‐C, Li C‐L, Wang L‐F. Nitric oxide inhibits androgen receptor‐mediated collagen production in human gingival fibroblasts. J Periodont Res 2012; 47: 701–710. © 2012 John Wiley & Sons A/S Background and Objective: In our previous study, we found that flutamide [an androgen receptor (AR) antagonist] inhibited the up‐regulation of collagen induced by interleukin (IL)‐1β and/or nifedipine in gingival fibroblasts. The present study attempted to verify the role of nitric oxide (NO) in the IL‐1β/nifedipine‐AR pathway in gingival overgrowth. Material and Methods: Confluent gingival fibroblasts derived from healthy individuals (n = 4) and those with dihydropyridine‐induced gingival overgrowth (DIGO) (n = 6) were stimulated for 48 h with IL‐1β (10 ng/mL), nifedipine (0.34 μm ) or IL‐1β + nifedipine. Gene and protein expression were analyzed with real‐time RT‐PCR and western blot analyses, respectively. Meanwhile, Sircol dye‐binding and the Griess reagent were, respectively, used to detect the concentrations of total soluble collagen and nitrite in the medium. Results: IL‐1β and nifedipine simultaneously up‐regulated the expression of the AR and type‐I collagen α1 [Colα1(I)] genes and the total collagen concentration in DIGO cells (p < 0.05). IL‐1β strongly increased the expression of inducible nitric oxide synthase (iNOS) mRNA and the nitrite concentration in both healthy and DIGO cells (p < 0.05). However, co‐administration of IL‐1β and nifedipine largely abrogated the expression of iNOS mRNA and the nitrite concentration with the same treatment. Spearman’s correlation coefficients revealed a positive correlation between the AR and total collagen (p < 0.001), but they both showed a negative correlation with iNOS expression and the NO concentration (p < 0.001). The iNOS inhibitor, 1400W, enhanced IL‐1β‐induced AR expression; furthermore, the NO donor, NONOate, diminished the expression of the AR to a similar extent in gingival fibroblasts derived from both healthy patients and DIGO patients (p < 0.05). Conclusion: IL‐1β‐induced NO attenuated AR‐mediated collagen production in human gingival fibroblasts. The iNOS/NO system down‐regulated the axis of AR/Colα1(I) mRNA expression and the production of AR/total collagen proteins by DIGO cells.     

Inhibitory effect of periodontally diseased root extracts on the growth of human gingival fibroblasts

Cementum shavings obtained from periodontally diseased and nondiseased areas of 100 removed, single-rooted teeth were extracted with either pyrogen-free water (PFW) for 5 minutes, 1 M citric acid for 5 minutes or 45% phenol-PFW for 90 minutes at 65 degrees C. The extracts were membrane-filtered, dialyzed exhaustively versus PFW, lyophilized, weighed and then dissolved in complete growth medium. The phenol-water or citric acid extracts of cementum shavings from periodontally diseased roots were positive for endotoxin by the limulus lysate assay (LLA). Pyrogen-free water extracts of diseased or phenol-water extracts of nondiseased cementum shavings were negative, or only slightly positive, respectively, for endotoxin by LLA. Media containing the various extracts were added to logarithmically growing cultures of human gingival fibroblasts (HGF). Separate cultures of HGF were exposed to Escherichia coli endotoxin at concentrations of 50, 100, 250 and 500 micrograms/ml to determine the growth-inhibitory effects of a known endotoxin. Cell growth was analyzed by measuring the incorporation of tritiated thymidine into cells. Suppression of HGF growth from 30 to 49% by E. coli endotoxin was concentration-dependent and linear over the concentration range of endotoxin tested. Pyrogen-free water extracts of diseased (endotoxin negative) or phenol-water extracts of nondiseased cementum shavings (slightly endotoxin positive) did not effect HGF growth. However, citric acid or phenol-water extracts of diseased cementum shavings (highly endotoxin positive) significantly suppressed HGF growth 58% and 61%, respectively. These results indicate that citric acid is effective in removing cytotoxic substances, presumably endotoxin, from cementum shavings and suggest that citric acid treatment is effective clinically in detoxifying periodontally diseased root surfaces.