Fu-Mei Huang

Platelet-rich Fibrin Increases Proliferation and Differentiation of Human Dental Pulp Cells

INTRODUCTION Platelet-rich fibrin (PRF) by Choukrouns technique is derived from an autogenous preparation of concentrated platelets without any manipulation. When delicately pressed between 2 gauzes, the PRF clot becomes a strong membrane with high potential in clinical application. However, the effect of PRF on dental pulp cells (DPCs) remains to be elucidated. This study was to determine the biological effects of PRF on DPCs. METHODS PRF samples were obtained from 6 healthy volunteers. Human DPCs were derived from healthy individuals undergoing extraction for third molars. Cell proliferation resulting from PRF was evaluated by colorimetric assay. Western blot was used to evaluate the expression of osteoprotegerin (OPG). Alkaline phosphatase (ALP) activity was examined by substrate assay. RESULTS PRF did not interfere with cell viability of DPCs (P > .05). DPCs were observed to attach at the edges of PRF by phase-contrast microscopy. PRF was found to increase DPC proliferation during 5-day incubation period (P < .05). PRF was found to increase OPG expression in a time-dependent manner (P < .05). ALP activity was also significantly up-regulated by PRF (P < .05). CONCLUSIONS PRF was demonstrated to stimulate cell proliferation and differentiation of DPCs by up-regulating OPG and ALP expression. These findings might serve as a basis for preclinical studies that address the role of PRF in reparative dentin formation.

Cytotoxic and nongenotoxic effects of phenolic compounds in human pulp cell cultures

Phenolic compounds are widely used in clinical dentistry as sedatives for the dental pulp, as disinfectants for caries, and as root canal medications. The pathobiological effects of various phenolic compounds on human dental pulp fibroblasts were investigated with Hoechst 33258 fluorescence assay and DNA precipitation assay. All phenolic compounds showed cytotoxicity in Hoechst 33258 fluorescence assay by inhibiting cellular DNA in a concentration-dependent manner. The 50% inhibition concentrations required to decrease the cellular DNA contents by guaiacol, phenol, eugenol, and thymol were 9.8, 4.5, 0.9, and 0.5 mM, respectively. However these phenolic compounds did not cause DNA single-strand breaks in cultured human pulp fibroblasts. These results indicate that phenolic compounds are cytotoxic agents but are without genotoxic effects on human pulp fibroblasts in vitro. However care should be taken to reduce the possibility of pulpal as well as periapical irritations from inadvertent extrusion of these substances in clinical usage.

Induction of Vascular Endothelial Growth Factor Gene Expression by Proinflammatory Cytokines in Human Pulp and Gingival Fibroblasts

Vascular endothelial growth factor (VEGF) enhances the permeability of blood vessels, which is an important vascular change observed during inflammatory processes. The purpose of this in vitro study was to investigate the effect of proinflammatory cytokines on the expression of VEGF mRNA gene in human pulp and gingival fibroblasts. Interlukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were used to evaluate VEGF mRNA gene expression in human pulp and gingival fibroblasts. The levels of mRNAs were measured by quantitative reverse-transcriptase polymerase chain reaction analysis. Both IL-1alpha and TNF-alpha induced significantly high levels of VEGF mRNA gene expression in human pulp and gingival fibroblasts (p < 0.05). In addition, TNF-alpha was found to be more effective in the induction of VEGF mRNA gene expression in pulp than gingival fibroblasts (p < 0.05). Moreover, IL-1alpha was found to be more effective in the induction of VEGF mRNA gene expression than TNF-alpha in gingival fibroblast cultures (p < 0.05). These results indicate that proinflammatory cytokines can induce VEGF mRNA gene expression, and such an effect may partially contribute to the destruction of pulpal and periapical tissues through promoting expansion of the vascular network coincident to progression of the inflammation.

In vitro evaluation of the cytotoxicity and genotoxicity of root canal medicines on human pulp fibroblasts

An intracanal medicine is often required because microorganisms in the dentinal tubes may be difficult to eliminate completely by instrumentation. Phenolic compounds are widely used in dental treatment as sedatives for the dental pulp or as disinfectants for caries and the root canal. In this study, propidium iodide fluorescence and DNA precipitation assay were used to investigated the cytotoxic and genotoxic effects of camphorated phenol and camphorated parachlorophenol on cultured human pulp fibroblasts in vitro. Both medicines reduced the content of double-stranded polynucleic acid of fibroblasts over a 24-h culture period in a concentration-dependent manner. Camphorated parachlorophenol was more cytotoxic than camphorated phenol. But, both medicines did not cause genotoxicity on pulp cells. The advantage of these experimental methods are simplicity and rapidity. Furthermore, this experimental system may be useful for preliminary cytotoxicity and genotoxicity screening of various dental medicines in vitro.

Proinflammatory cytokines induce cyclooxygenase-2 mRNA and protein expression in human pulp cell cultures.

The increased release of prostaglandins (PG) within pulpal tissues is considered to play a pathogenic role during pulpal disease progression. The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX). COX-2 is an inducible enzyme believed to be responsible for PG synthesis at site of inflammation. The effect of proinflammatory cytokines on human pulp cells with special reference to COX-2 expression has not been reported earlier. The aim of the present study was to investigate the effects of interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha) on the expression of COX-2 mRNA gene and protein in cultured human pulp cells. Investigations of the time dependence of COX-2 mRNA expression in proinflammatory cytokines-treated human pulp cells revealed a rapid accumulation of the transcript, a significant signal first detectable 1 h after exposure. In addition, both IL-1alpha and TNF-alpha up-regulated COX-2 protein expression by human pulp cells. The kinetics of this response showed that COX-2 was detectable in cell lysates as early as 2 h post proinflammatory cytokines challenge and remained elevated throughout the 24-h incubation period. This suggests that one of the pathogenic mechanisms of pulpal inflammation in vivo may be the synthesis of COX-2 by resident cells in response to a proinflammatory cytokines challenge. COX-2 may play an important role in the regulation of prostanoid formation in the pathogenesis of pulpal inflammation. Taken together, we propose that the use of selective COX-2 inhibitors might provide a valuable tool in the control of pulpal inflammation.

Cytotoxic evaluation of root canal filling materials on primary human oral fibroblast cultures and a permanent hamster cell line.

To date there has been very little data on the cytotoxic response of different cell lines to root canal sealers. The objective of this study was to determine the cytocompatibility of three different extracts of root canal sealers and to compare the cytotoxic response of these materials on two different primary human oral fibroblasts (derived from gingiva and buccal mucosa) and one permanent hamster cell line (V79 cells). Cytotoxicity was judged using an assay of tetrazolium bromide reduction. The results showed that root canal sealers (AH plus, Canals, and N2) were cytotoxic to primary human oral fibroblast cultures and V79 cells. It was found that N2 was the most toxic root canal sealer among those tested in all cultures. The toxicity decreased in an order of N2 > AH plus approximately = Canals. The sensitivity of toxicity depended on the materials tested and the cell culture system used.

Induction of Tissue Plasminogen Activator Gene Expression by Proinflammatory Cytokines in Human Pulp and Gingival Fibroblasts

Plasminogen activator converts plasminogen to plasmin, and plasmin activates the latent matrix metalloproteinases. Tissue plasminogen activator (t-PA) is one of the important proteolysis factors present in human inflamed tissues. However, few studies reported on the mechanisms of tissue destruction via a t-PA proteolysis pathway in pulpal and periapical diseases. The subsequent reactions leading to pulpal and periapical injury after the induction of proinflammatory cytokines remains to be elucidated. The aim of this study was to investigate the effects of interleukin-1alpha and tumor necrosis factor-alpha on the expression of t-PA mRNA gene in cultured human pulp and gingival fibroblasts. The mRNAs for t-PA were measured by reverse transcription-polymerase chain reaction at 2, 6, and 24 h. The results show that both cytokines induced significantly high levels of t-PA mRNA gene expression in human pulp fibroblasts. The peak of t-PA mRNA levels induced by both proinflammatory cytokines was at the 6-h incubation period. Interleukin-1alpha was found to be more effective in induction of t-PA gene expression than tumor necrosis factor-alpha. In addition, a similar induction pattern was also found in human gingival fibroblasts. These results indicate that proinflammatory cytokines can induce t-PA gene expression and such an effect may partially contribute to the destruction of pulpal and periapical tissues through dysregulated pericellular proteolysis. An understanding of the mechanism could not only further define the role of immune events in pulpal and periapical diseases but also have important implication for pharmacological intervention.

Induction of cyclooxygenase-2 mRNA and protein expression by epoxy resin and zinc oxide-eugenol based root canal sealers in human osteoblastic cells

An ideal root canal sealer should be nonirritating to the surrounding tissues. Unfortunately, all histological investigation demonstrated that all types of root canal sealer can induce mild to severe inflammatory alternations. However, there is little information on the precise mechanisms about root canal sealers-induced inflammatory reaction. Cyclooxygenase-2 (COX-2) is an inducible enzyme believed to be responsible for prostaglandin synthesis at site of inflammation. The aim of the present study was to investigate the effects of epoxy resin (AH26) and zinc oxide-eugenol based (Endomethansone and N2) root canal sealers on the expression of COX-2 mRNA gene and protein in cultured human osteoblastic cells. Investigations of the time dependence of COX-2 mRNA expression in root canal sealer-treated human osteoblastic cells revealed a rapid accumulation of the transcript, a significant signal first detectable within 2h and diminished to control level after 24h. In addition, all root canal sealers also induced COX-2 protein expression in human osteoblastic cells. Furthermore, to elucidate whether induction of COX-2 is associated with cytotoxicity, NS-398 (a selective COX-2 inhibitor), was added to test its protective effects. NS-398 at non-cytotoxic dose is not able to prevent root canal sealers-induced cytotoxicity. Taken together, the activation of COX-2 expression may be one of the pathogenesis of root canal sealers-induced periapical inflammation. In addition, root canal sealers-induced cytotoxicity is not directly via the induction of COX-2 expression.

Induction of Cyclooxygenase-2 mRNA and Protein Expression in Human Pulp Cells Stimulated with Black-pigmented Bacteroides

Cyclooxygenase-2 (COX-2) is induced after the activation of cells by a variety of proinflammatory agents. Recently, evidence has shown that COX-2 may play a role in the pathogenesis of pulpal inflammation. However, little is known regarding the mechanism of pulpal inflammation at the site of bacterial infection. The purpose of this study was to determine the effects of the supernatants from black-pigmented Bacteroides (Porphyromonas endodontalis, Porphyromonas gingivalis, and Prevotella intermedia) on the COX-2 expression in primary human pulp cells in vitro. Investigations of the time dependence of COX-2 mRNA expression in black-pigmented Bacteroides-treated human pulp cells revealed a rapid accumulation of the transcript, a significant signal first detectable after 1 h of exposure. In addition, black-pigmented Bacteroides also up-regulated COX-2 protein expression in human pulp cells. Data from our in vitro experiment showed that black-pigmented Bacteroides were capable of stimulating COX-2 expression in human pulp cells. These results indicate that black-pigmented Bacteroides species may play an important role in the pathogenesis of pulpal inflammation. The activation of COX-2 may be one of the distinct host degradative pathways in the pathogenesis of microbial-induced pulpal/periapical inflammation.

Resinous Perforation-Repair Materials Inhibit the Growth, Attachment, and Proliferation of Human Gingival Fibroblasts

The choice of repair material is one of the important factors in the prognosis of the endodontically treated tooth with a perforation defect. The cytotoxicity of perforation-repair materials must be investigated to ensure a safe biological response. The aim of this in vitro study was to evaluate the effect of resin-modified, glass-ionomer cement, compomer, and resin on human-gingival fibroblasts. Human gingival fibroblasts from crown lengthening surgery were cultured by using an explant technique with the consent of the patient. Cytotoxicity was judged by using an assay of tetrazolium bromide reduction. The results showed that resin-modified, glass-ionomer cement Fuji II LC, compomer Compoglass, and resin SpectrumTPH (TPH) were cytotoxic to primary human gingival fibroblast cultures by inhibiting cell growth and proliferation. TPH alone had an effect on cell attachment. It was found that TPH was the most cytotoxic repair material among those tested in all cultures. The toxicity decreased in the order of TPH>FLC>CG.