Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

TGF-ß1 stimulates cyclooxygenase-2 expression and PGE2 production of human dental pulp cells: Role of ALK5/Smad2 and MEK/ERK signal transduction pathways.

BACKGROUND/PURPOSES: TGF-ß1 is an important growth factor that may influence the odontoblast differentiation and matrix deposition in the reactionary/reparative dentinogenesis to dental caries or other tooth injuries. TGF-ß1 exerts its effects through various signaling pathways, such as Smads and MAPKs. Cyclooxygenase-2 (COX-2) is a membrane-associated enzyme that produces prostaglandin E2 (PGE2) at sites of pulpal injury and inflammation, which leads to tissue swelling, redness and pain. The purposes of this study were to investigate the differential signal transduction pathways of TGF-ß1 that mediate COX-2 stimulation and PGE2 production in dental pulp cells. METHODS: Pulp cells were exposed to TGF-ß1 with/without SB431542 (an ALK5/Smad2 inhibitor) and U0126 (a MEK/ERK inhibitor). MTT assay was used to estimate cell viability. Enzyme-linked immunosorbent assay (ELISA) was used for measurement of PGE2 levels. RT-PCR and western blot were used to determined COX-2 mRNA and protein, respectively. RESULTS: Exposure to TGF-ß1 (1-10 ng/ml) increased the COX-2 mRNA and protein level of cultured pulp cells. Exposure to TGF-ß1 (0.1-10 ng/mL) significantly stimulated PGE2 production of dental pulp cells. Under the pretreatment of SB431542, the stimulatory effect of TGF-ß1 on COX-2 level of pulp cells was inhibited. Similarly, U0126 also partly inhibited the TGF-ß1-induced COX-2 expression. CONCLUSION: TGF-ß1 increased the COX-2 and PGE2 level of cultured pulp cells. The effect of TGF-ß1 on COX-2 protein expression was associated with ALK5/Smad2/3 and MEK/ERK pathways. These events are important in the early inflammation, repair and regeneration of dental pulp in response to injury.

bFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration. OBJECTIVES: The purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling. METHODS: SCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining. RESULTS: SCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05). CONCLUSION: bFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.

Clinical and Radiographic Characteristics of Vertical Root Fractures in Endodontically and Nonendodontically Treated Teeth.

INTRODUCTION: A vertical root fracture (VRF) is a root fracture extending along the longitudinal axis of roots and is often noted in endodontically treated teeth. However, the clinical and radiographic characteristics of VRFs are not completely known. METHODS: A total of 65 teeth with 68 vertical fractured roots in 58 Chinese patients were investigated. The clinical examination records and radiographic images were reviewed in detail. RESULTS: A total of 24 male (41.38%) and 34 female (58.62%) patients aged 25-90 years (average = 57 years) were included; 51 (87.93%) and 7 (12.07%) patients exhibited 1 tooth and 2 teeth with VRFs, respectively, in the dentition. VRFs occurred mainly in the mesial root (20 roots, 57.14%) of the mandibular molars (29 teeth, 44.62%). Clinically, teeth with VRFs usually presented a periodontal probing depth >5 mm (44 teeth, 91.67%, P < .001) with a prosthesis (55 teeth, 84.62%, P < .001) and a relatively intact dentition (42 patients exhibited <4 missing teeth in the dentition, 77.78%, P < .001). Most of the nonendodontically treated VRFs exhibited attrited occlusal surfaces. Radiographic characteristics of the teeth with VRFs were typically associated with prior root canal treatment (56 teeth, 86.15%, P < .001), periodontal bone loss (62 teeth, 95.38%, P < .001), apical bone loss (52 teeth, 80.00%, P < .001), and periodontal ligament widening (61 teeth, 93.85%, P < .001). The mesial roots of the mandibular molars were most susceptible to VRFs in both endodontically and nonendodontically treated teeth. CONCLUSIONS: These results elucidated some clinical and radiographic and diagnostic features that facilitate VRF identification.

Basic Fibroblast Growth Factor Regulates Gene and Protein Expression Related to Proliferation, Differentiation, and Matrix Production of Human Dental Pulp Cells.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays differential effects on the proliferation, differentiation, and extracellular matrix turnover in various tissues. However, limited information is known about the effect of bFGF on dental pulp cells. The purposes of this study were to investigate whether bFGF influences the cell differentiation and extracellular matrix turnover of human dental pulp cells (HDPCs) and the related gene and protein expression as well as the role of the mitogen-activated protein kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling pathway. The expression of fibroblast growth factor receptors (FGFRs) in HDPCs was also studied. METHODS: The expression of FGFR1 and FGFR2 in HDPCs was investigated by reverse-transcription polymerase chain reaction. HDPCs were treated with different concentrations of bFGF. Cell proliferation was evaluated using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell differentiation was evaluated using alkaline phosphatase (ALP) staining. Changes in messenger expression of cyclin B1 and tissue inhibitor of metalloproteinase (TIMP) 1 were determined by reverse-transcription polymerase chain reaction. Changes in protein expression of cdc2, TIMP-1, TIMP-2, and collagen I were determined by Western blotting. U0126 was used to clarify the role of MEK/ERK signaling. RESULTS: HDPCs expressed both FGFR1 and FGFR2. Cell viability was stimulated by 50-250 ng/mL bFGF. The expression and enzyme activities of ALP were inhibited by 10-500 ng/mL bFGF. At similar concentrations, bFGF stimulates cdc2, cyclin B1, and TIMP-1 messenger RNA and protein expression. bFGF showed little effect on TIMP-2 and partly inhibited collagen I expression of pulp cells. U0126 (a MEK/ERK inhibitor) attenuated the bFGF-induced increase of cyclin B1, cdc2, and TIMP-1. CONCLUSIONS: bFGF may be involved in pulpal repair and regeneration by activation of FGFRs to regulate cell growth; stimulate cdc2, cyclin B1, and TIMP-1 expression; and inhibit ALP. These events are partly associated with MEK/ERK signaling.

Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1-modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth.

INTRODUCTION: Transforming growth factor ß1 (TGF-ß1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-ß1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2. METHODS: SCAPs were exposed to TGF-ß1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho-enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA). RESULTS: TGF-ß1 stimulated the growth and collagen content of cultured SCAPs. TGF-ß1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-ß1-induced cell growth and collagen content in SCAPs. TGF-ß1 stimulated ALP activity at lower concentrations (0.1-1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-ß1-induced ALP activity but showed little effect on 10 ng/mL TGF-ß1-induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-ß1. CONCLUSIONS: TGF-ß1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-ß1 and SCAP for engineering of pulpal regeneration and apexogenesis.