Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. MATERIAL AND METHODS: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. CONCLUSION: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.

Effects of EDTA on the hydration mechanism of mineral trioxide aggregate.

Ethylenediaminetetraacetic acid (EDTA) is commonly used during the preparation of obstructed root canals that face a high risk of root perforation. Such perforations may be repaired with mineral trioxide aggregate (MTA). Due to EDTA's ability to chelate calcium ions, we hypothesized that EDTA may disrupt the hydration of MTA. Using scanning electron microscopy and energy-dispersive x-ray spectroscopy, we found that MTA specimens stored in an EDTA solution had no crystalline structure and a Ca/Si molar ratio considerably lower than those obtained for specimens stored in distilled water and normal saline. Poor cell adhesion in EDTA-treated MTA was also noted. X-ray diffraction indicated that the peak corresponding to portlandite, which is normally present in hydrated MTA, was not shown in the EDTA group. The microhardness of EDTA-treated specimens was also significantly reduced (p < 0.0001). These findings suggest that EDTA interferes with the hydration of MTA, resulting in decreased hardness and poor biocompatibility.

Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro.

Transforming growth factor-beta (TGF-beta) is important in regulating the repair and regeneration of damaged dental pulp. For further elucidating the roles of different isoforms of TGF-beta in the healing and inflammatory processes of human dental pulp, we found that TGF-beta1, TGF-beta2 and TGF-beta3 inhibited the growth of two human dental pulp cell strains in vitro by 19-29, 18-25 and 23-26%, respectively, at a concentration of 0.5 ng/ml. TGF-beta also differentially stimulated the collagen synthesis of pulp cells. Collagen synthesis increased by 1 ng/ml of TGF-beta1 and TGF-beta2 by 42 and 51%, respectively. TGF-beta3 (0.1-1 ng/ml) lacked of stimulatory effect on collagen synthesis of pulp cells. Pulp cells have the intrinsic capacity to contract collagen lattice, leading to decreasing of lattice diameter. An 8 h exposure to TGF-beta1 and TGF-beta2 enhanced the pulp cell-populated collagen lattice contraction at concentrations ranging from 0.2 to 3 ng/ml. At similar concentrations, TGF-beta3 lacked of this stimulatory effect. When collagen lattice were detached after 24 h of exposure, TGF-beta1 and TGF-beta2 (0.6-3 ng/ml) induced the pulp cells-populated collagen lattice contraction within 4-8h of gel detachment. These results indicate that TGF-beta-induced collagen lattice contraction is a late cellular event. These in vitro results indicate that effects of TGF-beta isoforms on the growth, collagen synthesis and collagen lattice contraction of pulp cells may play crucial roles in the pathobiological processes of dental pulp.

Treatment of tooth fracture by medium-energy CO2 laser and DP-bioactive glass paste: the interaction of enamel and DP-bioactive glass paste during irradiation by CO2 laser.

Acute trauma or trauma associated with occlusal disturbance can produce tooth crack or fracture. Although several methods are proposed to treat the defect, however, the prognosis is generally poor. If the fusion of a tooth fracture by laser is possible, it will offer an alternative to extraction or at least serve as an adjunctive treatment in the reconstruction. We have tried to use a continuous-wave CO2 laser and a newly developed DP-bioactive glass paste (DPGP) to fuse or bridge tooth crack or fracture lines. Both the DP-bioactive glass paste and tooth enamel have strong absorption bands at the wavelength of 10.6 microm. Therefore, under CO2 laser, DPGP and enamel should have an effective absorption and melt together. The interface between DPGP and enamel could be regarded as a mixture of DPGP and enamel (DPG-E). The study focused on the phase transformation, microstructure, functional group and thermal behavior of DPG-E with or without CO2 laser irradiation, by the analytical techniques of XRD, FTIR, DTA/TGA, and SEM. The results of XRD showed that the main crystal phase in the DPG-E was dicalcium phosphate dihydrate (CaHPO4.2H2O). It changed into CaHPO4, gamma-Ca2P2O7, beta-Ca2P2O7 and finally alpha-Ca2P2O7 with increasing temperature. In the FTIR analysis, the 720 cm(-1) absorption band ascribed to the P-O-P linkage in pyrophosphate rose up and the intensities of the OH- bands reduced after laser irradiation. In regard to the results of DTA/TGA after irradiation, the weight loss decreased due to the removal of part of absorption water and crystallization water by the CO2 laser. SEM micrographs revealed that the melted masses and the plate-like crystals formed a tight chemical bond between the enamel and DPGP. We expect that DPGP with the help of CO2 laser can be an alternative to the treatment of tooth crack or fracture.

Treatment of tooth fracture by medium energy CO2 laser and DP-bioactive glass paste: compositional, structural, and phase changes of DP-bioglass paste after irradiation by CO2 laser.

Nowadays, fractured teeth are difficult to treat effectively. Currently, root fractures are usually treated by root amputation, hemisection or tooth extraction. If the fusion of tooth fracture by laser were possible, it would offer a different therapy to repair fracture teeth. We tried to use a developed DP-bioactive glass paste to fuse or bridge the tooth crack line by a medium energy continuous-wave CO2 laser. The study is divided into three parts: (1) The compositional and structure changes in tooth enamel and dentin after laser treatment; (2) The phase transformation and recrystallization of DP-bioactive paste during exposure to the CO2 laser; (3) The thermal interactions and bridge mechanism between DP-bioactive glass paste and enamel/dentin when they are subjected to CO2 laser. The present report will focus on the second part that will examine the changes of laser-exposed DP-bioactive glass paste by means of X-ray diffractometer (XRD), Fourier transforming infrared spectroscopy (FTIR), differential thermal analysis/thermogravimetric analysis (DTA/TGA), and scanning electron microscopy (SEM). From the study, we could find that the temperature increase due to laser irradiation is greater than 900 degrees C and that the DP-bioactive glass paste could be melted in a short period of time after irradiation. In the study, we successfully developed a DP-bioactive glass paste which could form a melting glass within seconds after exposure to a medium energy density continuous-wave CO2 laser. The paste will be used in the near future to bridge the enamel or dentin surface crack by the continuous-wave CO2 laser.

Comparison of Nd:YAG laser versus scaling and root planing in periodontal therapy.

BACKGROUND: The Nd:YAG laser has recently been used in the treatment of periodontal disease. However, although a clinical reduction of probing depth and gingival inflammation to this new approach has been reported, it has not been fully evaluated. Interleukin-1 beta (IL- 1beta), a potent stimulator of bone resorption, has been identified in gingival crevicular fluid (GCF), which is closely associated with periodontal destruction. The aim of this study was to compare the effects of Nd:YAG laser treatment versus scaling/root planing (SRP) treatment on crevicular IL-1beta levels in 52 sampled sites obtained from 8 periodontitis patients. METHODS: One or 2 periodontitis-affected sites with a 4 to 6 mm probing depth and horizontal bone loss from 3 adjacent single-root teeth in each of 4 separate quadrants were selected from patients for clinical documentation and IL-1beta assay. Sampling site(s) from each diseased quadrant was randomly assigned to one of the following groups: 1) subgingival laser treatment (20 pps, 150 mJ) only; 2) SRP only; 3) laser treatment first, followed by SRP 6 weeks later; or 4) SRP first, followed by laser therapy 6 weeks later. The GCF was collected and the amount of IL-1beta was assayed by enzyme-linked immunosorbent assay (ELISA). Clinical parameters and GCF were measured at baseline and biweekly after therapy for 12 weeks. RESULTS: An obvious clinical improvement (marked decrease in the number of diseased sites with gingival index > or =2) and reduction of crevicular IL- 1beta were found in all groups. The level of IL- 1beta was significantly lower in the SRP group (P = 0.035) than in the laser therapy group for the duration of the 12 weeks. The laser combined SRP therapy group showed a further reduction of IL- 1beta (6 to 12 weeks after treatment) than either laser therapy alone or SRP combined laser therapy. CONCLUSIONS: Our data suggest that laser therapy appeared to be less effective than traditional SRP treatment. Of the 4 treatment modalities, inclusion of SRP was found to have a superior IL- 1beta response, when compared to other therapies without it. In addition, no additional benefit was found when laser treatment was used secondary to traditional SRP therapy.

Effects of butyrate and propionate on the adhesion, growth, cell cycle kinetics, and protein synthesis of cultured human gingival fibroblasts.

BACKGROUND: Various periodontal and root canal pathogens, such as the Bacteroides species, can produce significant amounts of short chain fatty acids (SCFA). The roles of SCFA in the pathogenesis of periodontal disease are still not fully understood. METHODS: We therefore investigated 2 main SCFA, butyrate and propionate, on the functional behavior of cultured human gingival fibroblasts (GF) such as cell growth, protein synthesis, cell adhesion capacity, and cell cycle progression. RESULTS: Butyrate and propionate inhibited the growth of healthy (HGF) and inflamed gingival fibroblasts (IGF) in a dose dependent manner. At concentrations of 4, 8, and 16 mM, butyrate suppressed the cell growth by 11 to 58%, 16 to 60%, and 50 to 71%, respectively. The response of cultured gingival fibroblasts to SCFA showed individual differences. Morphologically, GF became larger and more flattened in appearance following exposure to butyrate (>8 mM) and propionate (>24 mM) for 5 days. Inhibitory effects of butyrate (>2 mM) and propionate (>8 mM) on the growth of GF were due possibly to their inhibition of cell-cycle progression. At concentrations of 2 and 8 mM, butyrate led to G0/G1 arrest. Elevation of the exposure concentration to 8 to 24 mM further result in G2/M phase arrest of GF. On the other hand, propionate, at concentrations ranging from 4 to 24 mM, led to G0/G1 arrest. Butyrate (>2 mM) inhibited the proline-rich protein synthesis of GF. At concentrations of 4, 8, 16, and 24 mM, butyrate inhibited the protein synthesis of HGF-1 by 42%, 43%, 51%, and 54%, respectively. In all strains of cultured GF, the suppressive effect of propionate is less than that of butyrate. At concentration range of 4 to 24 mM, propionate suppressed the protein synthesis of HGF-1 by 23 to 43%. However, both butyrate and propionate (4 to 48 mM) exerted little effects on the adhesion of GF to type I collagen within 3 hours of incubation. CONCLUSIONS: These results suggested that SCFA released by pathogenic microorganisms can contribute to the gingival tissue dysfunction and breakdown through their actions on specific biological functions of GF.

Thrombin-stimulated growth, clustering, and collagen lattice contraction of human gingival fibroblasts is associated with its protease activity.

BACKGROUND: Thrombin is a serine protease produced following gingival tissue injury or inflammation. It regulates the functional behavior of injury-neighboring cells via the activation of specific protease-activated receptors (PAR). Thrombin's role in gingival tissue healing and inflammatory response processes is not yet well understood. METHODS: We investigated the effects of thrombin on gingival fibroblast (GF) growth [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay], collagen lattice contraction, and associated morphological changes. RESULTS: Thrombin (>1 U/ml), but not thrombin receptor (PAR-1) agonist peptide (SFLLRN, single letter amino acid code, abbreviated as TRAP, 1 to 50 microg/ml), stimulated the growth and clustering of cultured human GF in vitro. Growth-stimulatory effects of thrombin were inhibited by D-Phe-Pro-ArgCH2Cl (PPACK), a serine protease inhibitor. By contrast, trypsin (>10 microg/ml), a PAR-2 activator, suppressed the growth of GF. Thrombin (>0.2 U/ml) and TRAP (10 to 25 microg/ml), but not trypsin, prostaglandin E2 (0.01 to 0.5 microg/ml), or bovine serum albumin (BSA) (1 to 80 microg/ml), induced the GF-populated collagen lattice contraction within 30 to 60 minutes of exposure. The thrombin-induced collagen lattice contraction was inhibited by PPACK (20 microg/ml) and an actin filament polymerization inhibitor, cytochalasin B (1 microg/ml). The collagen lattice contraction induced by TRAP was also inhibited by cytochalasin B, but not by PPACK. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), the expression of PAR-1, and to a lesser extent PAR-3, was observed for human GF, although little PAR-2 and PAR-4 expression was noted. CONCLUSIONS: These results indicate that thrombin is important in periodontal wound healing and inflammatory processes by promoting the growth and contraction of GF. The stimulatory effects of thrombin are associated with its protease activation of thrombin receptors.

Temperature elevation on the root surface during Nd:YAG laser irradiation in the root canal.

Nd:YAG lasers have been suggested as a potential tool in endodontic therapy because of their sterilizing and sealing effects on the dentinal tubules. However, the generation of heat in the root canal by laser irradiation may produce potentially harmful effects on adjacent tissues. The purpose of this study was to evaluate the temperature elevation on the root surface when Nd:YAG laser was irradiated in the root canal. The apical third area of 90 single-rooted teeth were irradiated with normal pulsed Nd:YAG laser (50, 80, 100, 150, and 200 mJ/pulse; 20, 25, and 30 pulses/s). The temperature elevation was measured and recorded on the root surface simultaneously. The temperature elevation did not exceed 10 degrees C only when the laser energy output was below 100 mJ/pulse and under 20 pulses/s.

Prevalence and distribution of cervical dentin hypersensitivity in a population in Taipei, Taiwan.

The prevalence, distribution, and possible causal factors of cervical dentin hypersensitivity were studied in a population attending the Health Examination Center of National Taiwan University Hospital. A total of 780 patients were examined for the presence of cervical dentin hypersensitivity by means of a questionnaire and intraoral tests. There were 253 patients (32%) who claimed to have hypersensitive teeth at present and 90 patients (12%) who reported a history of hypersensitive teeth. The intraoral distribution of hypersensitivity showed that premolars and molars were the most common teeth sensitive to the air and probe stimuli, while the incisors were the least sensitive ones. The presence and history of dentin hypersensitivity were positively correlated with previous tooth-brushing and periodontal disease. Only a few of the patients who claimed to have dentin hypersensitivity had tried treatment with desensitizing tooth-pastes (11%) or sought professional help (5%).

The combined occluding effect of sodium fluoride varnish and Nd:YAG laser irradiation on human dentinal tubules.

Various methods and materials used in the treatment of dentin hypersensitivity are thought to achieve a therapeutic benefit by tubule occlusion. The aim of the present study was to evaluate the combined occluding effect of sodium fluoride varnish and Nd:YAG laser irradiation on human dentinal tubules. Thirty-six dentin specimens with exposed dentinal tubule orifices were used in this study. The samples were randomly divided into four groups. Groups A, B, and C were varnished by sodium fluoride, whereas group D served as a control. Then, group C was lased by 30 mJ of Nd:YAG laser, 10 pulses/s for 2 min by light painting. Three hours later, groups B and C were brushed by an electrical toothbrush for 30 min. Under SEM observation, the control group showed numerous exposed dentinal tubule orifices, and the sodium fluoride varnished specimens showed closure of exposed dentinal tubule orifices. After electrical toothbrushing, most of the sodium fluoride varnish was brushed away, except in the specimens that were irradiated by Nd:YAG laser. Over 90% of the dentinal tubule orifices were occluded by sodium fluoride varnish combined with Nd:YAG laser irradiation.

Sealing depth of Nd:YAG laser on human dentinal tubules.

Dentin permeability and hypersensitivity are both reduced when the dentinal tubules are occluded. Previous scanning electron microscopic studies showed that Nd:YAG laser could cause melting of dentin and closure of exposed dentinal tubules without dentin surface cracking. Therefore, the aim of the present study was to evaluate the sealing depth of Nd:YAG laser on human dentinal tubules. Thirty-six dentin specimens with exposed dentinal tubule orifices were used in this study. Samples were randomly divided into three groups. Groups A and B were lased by Nd:YAG laser at energy of 30 mJ with 10 pulses/s for a stroke along the dentin surface. Group C was not lased and served as a control. Subsequently, group B was frozen in liquid nitrogen and split by a sharp chisel. Under SEM observation, nonlased specimens showed numerous exposed dentinal tubule orifices, and lased specimens showed melting of dentin and closure of exposed dentinal tubule orifices. The sealing depth of Nd:YAG laser on human dentinal tubules was approximately 4 microns.

A comparison of the morphological changes after Nd-YAG and CO2 laser irradiation of dentin surfaces.

The purpose of this study was to compare the morphological changes after Nd-YAG and CO2 laser irradiation on dentin surfaces with or without the smear layer. Eighty-one 3-mm-thick dentin specimens collected from the middle third of molar crowns were used. The dentin surfaces were ground to #320, #400, and #600 grit in series to create a smear layer. Half of the specimens were treated with 14% EDTA for 2 min to remove the smear layers. The lasers were applied on each specimen perpendicularly with 1-mm focus distance to the dentin surface for 4 s. The parameters for the Nd-YAG laser were 50 mJ, 100 mJ, and 150 mJ at 10 pps, 20 pps, and 30 pps, and for the CO2 laser were 2 W, 3 W, and 4 W at 5 ms x 20 pps, 10 ms x 10 pps, 20 ms x 20 pps, 50 ms x 2 pps, 100 ms x 2 pps, and 200 ms x 2 pps. The results showed that the Nd-YAG laser caused crater and melting of the dentin surface, especially in dentin specimens with smear layers. The CO2 laser produced extensive cracking lines on dentin surfaces with a smear layer, whereas surface erosion and crater formation were found on specimens without a smear layer. In conclusion, both the laser types and smear layer have a significant influence on the morphological changes of dentin surfaces irradiated by lasers.

The quality of ultrasonic root-end preparation: a quantitative study.

Ultrasonic root-end preparation techniques have recently been introduced and revolutionized the field of endodontic surgery. The purpose of this study was to compare quantitatively the quality of root-end preparation techniques prepared by a specially designed ultrasonic retrotip with those prepared in a traditional manner by a microhandpiece bur. Twenty roots with two canals and an isthmus from extracted maxillary human molars were selected for this study. After instrumentation, obturation, and root-end resection, root-end preparations were made using either an ultrasonic retrotip or a conventional microhandpiece bur. With the aid of the image processing and analysis system, the specimens were inspected under a stereomicroscope for further evaluation of the quality of the shape and size of preparation. The results of this investigation showed that the ultrasonic root-end preparations produced more conservative and less perforated cavities than those made with conventional microhandpiece bur preparations.

Root deformation during root-end preparation.

Ultrasonic root-end preparation techniques have recently been introduced and revolutionized the field of endodontic surgery. However, several reports claimed that there was an increasing incidence of crack formation after ultrasonic root-end preparation. As yet, little work has focused on the root deformation during root-end preparation. Thus, the purpose of this investigation was to measure the amount of root deformation during root-end preparation with the use of microhandpiece and ultrasonic systems by using strain gauge methods, and simultaneously to detect any cracks with the aid of the stereomicroscope, stain, and an image processing system. The results demonstrated the ultrasonic instrumentation produced significantly greater strain on average than that generated with the microhandpiece system. From the viewpoint of fracture, any technique that could diminish the strain on the root would decrease the likelihood of fracture; however, no crack was observed on any resected surface of roots in this study.

Phase, compositional, and morphological changes of human dentin after Nd:YAG laser treatment.

Although techniques for repairing root fracture have been proposed, the prognosis is generally poor. If the fusion of a root fracture by laser is possible, it will offer an alternative to extraction. Our group has attempted to use lasers to fuse a low melting-point bioactive glass to fractured dentin. This report is focused on the phase, compositional, and morphological changes observed by means of X-ray diffractometer, Fourier transforming infrared spectroscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy in human dentin after exposure to Nd:YAG laser. The irradiation energies were from 150 mJ/ pulse-10 pps-4 s to 150 mJ/pulse-30 pps-4 s. After exposure to Nd:YAG laser, dentin showed four peaks on the X-ray diffractometer that corresponding to a-tricalcium phosphate (TCP) and beta-TCP at 20 = 30.78 degrees/34.21 degrees and 32.47 degrees/33.05 degrees, respectively. The peaks of a-TCP and beta-TCP gradually increased in intensity with the elevation of irradiation energy. In Fourier transforming infrared analysis, two absorption bands at 2200 cm(-1) and 2015 cm(-1) could be traced on dentin treated by Nd:YAG laser with the irradiation energies beyond 150 mJ/pulse-10 pps-4 s. The energy dispersive X-ray results showed that the calcium/phosphorus ratios of the irradiated area proportionally increased with the elevation of irradiation energy. The laser energies of 150 mJ/ pulse-30 pps-4 s and 150 mJ/pulse-20 pps-4 s could result in the a-TCP formation and collagen breakdown. However, the formation of glass-like melted substances without a-TCP at the irradiated site was induced by the energy output of 150 mJ/ pulse-10 pps-4 s. Scanning electron micrographs also revealed that the laser energy of 150 mJ/ pulse-10 pps-4 s was sufficient to prompt melting and recrystallization of dentin crystals without cracking. Therefore, we suggest that the irradiation energy of Nd:YAG laser used to fuse a low melting-point bioactive glass to dentin is 150 mJ/ pulse-10 pps-4 s.

Thrombin-induced DNA synthesis of cultured human dental pulp cells is dependent on its proteolytic activity and modulated by prostaglandin E2.

To clarify the roles of alpha-thrombin and prostaglandin E2 (PGE2) in the healing and inflammatory processes of dental pulp, their effects on the DNA synthesis of human pulp cells were investigated by measurement of [3H]thymidine incorporation. At a concentration range of 1 to 25 units/ml, alpha-thrombin stimulated DNA synthesis of the pulp cells by 1.5 to 2.6-fold. On the contrary, PGE2 (> 0.05 microgram/ml) suppressed DNA synthesis by 24 to 39%. Using reverse transcriptase-polymerase chain reaction, thrombin receptor mRNA expression was identified in the pulp cells. Furthermore, alpha-thrombin-induced DNA synthesis could be inhibited by antithrombin III (2 units/ml) with heparin (2 units/ml) or D-Phe-Pro-ArgCH2Cl (50 micrograms/ml). PGE2 (0.1 to 0.5 microgram/ml) also inhibited the thrombin-induced DNA synthesis by 39 to 64%. These results imply that pulp cells express the thrombin receptor that is activated by the serine protease activity of thrombin. Interactions of thrombin and PGE2 are important in modulating the inflammatory and healing processes of the pulp.

Thrombin activates the growth, cell-cycle kinetics, and clustering of human dental pulp cells.

Thrombin is activated during vascular injury and inflammation of the dental pulp. In the present study, we found that thrombin can stimulate the proliferation of pulp cells in a dose-dependent manner as analyzed by modified MTT assay. The cell number increased by 1.6, 1.77, and 2.14-fold over that of control after exposure to 5, 10, and 20 units/ml of thrombin for 5 days. Flow cytometry studies also found that thrombin (10 units/ml) can induce the cell cycle progression of pulp cells after 24 h of incubation, as revealed by increasing the proportion of cells in the S phase and the G2/M phase from 29 to 72%. Moreover, exposure to thrombin (> 5 units/ml) for 3 days led to marked clustering of pulp cells. We concluded that thrombin can regulate the growth, cell cycle progression, and functional reorganization of the pulp tissue during pulp healing and inflammatory processes.

Induction of dental pulp fibroblast matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression by interleukin-1alpha and tumor necrosis factor-alpha through a prostaglandin-dependent pathway.

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.

Collagen gene expression in human dental pulp cell cultures.

Pulp cells from human permanent molars were isolated and established in culture; 40% showed positive alkaline phosphatase staining. When incubated with 50 micrograms/ml of ascorbic acid and 10 mM of beta-glycerophosphate, the cells formed a mineralized extracellular matrix; they could thus have the potential to differentiate into odontoblast-like cells in vitro. Collagen synthesis was analysed by SDS interrupted gel electrophoresis, Northern blot and slot blot: the cells produced predominantly (approximately 99%) type I collagen and only trace amount of type III collagen. The ratio of alpha 1 (I) to alpha 2(I) procollagen chains was about 68:32, indicating that no significant amount of collagen type I trimer was synthesized in this system. The ratios of alpha 1(I), alpha 2(I) and alpha 1(III) procollagen mRNAs were about 61:25:1; these were compatible with the ratios of corresponding procollagen alpha chains. In addition, a novel 5.8 kb pro alpha 1(III) mRNA was detected. These observations indicate that collagen synthesis in these cultured pulp cells was regulated at the transcriptional level.