Inducing cathepsin L expression/production, lysosomal activation, and autophagy of human dental pulp cells by dentin bonding agents, camphorquinone and BisGMA and the related mechanisms.

Camphorquinone (CQ) and resin monomers are included in dentin bonding agents (DBAs) and composite resin to restore tooth defects due to abrasion, crown fracture, or dental caries. DBAs, CQ, and bisphenol A-glycidyl methacrylate (BisGMA) applications influence the biological activities of the dental pulp. The current investigation aimed to delineate the effect of DBAs, CQ, and BisGMA on cathepsin L production/expression, lysosomal activity, and autophagy induction in human dental pulp cells (HDPCs). HDPCs were exposed to DBAs, CQ, or BisGMA with/without inhibitors for 24 h. Enzyme-linked immunosorbent assay was employed to determine the cathepsin L level in culture medium. The cell layer was utilized to measure cell viability by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) assay. Real-time PCR was used to evaluate the mRNA expression. Western blotting or immunofluorescent staining was used to study protein expression. Lysosomal density was evaluated by lysotracker red staining. We found that DBAs, CQ, and BisGMA stimulated cathepsin L mRNA, protein expression, and production in HDPCs. In addition, CQ and BisGMA induced lysosomal activity, Beclin1, ATG12, LC3B, Bax, and p53 expression in HDPCs, indicating the stimulation of autophagy. Glutathione (GSH) prevented CQ- and BisGMA-induced cytotoxicity. Moreover, E64d, cathepsin L inhibitor (two cathepsin inhibitors), and Pifithrin-α (a p53 inhibitor) showed little preventive effect toward CQ- and BisGMA-induced cytotoxicity. Autophagy inhibitors (NH4Cl, Lys05) mildly enhanced the CQ- and BisGMA-induced cytotoxicity. These results indicate that DBAs stimulated cathepsin L, possibly due to their content of CQ and BisGMA that may induce cathepsin L in HDPCs. CQ and BisGMA stimulated lysosomal activity, autophagy, and apoptosis, possibly via induction of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. In addition, CQ and BisGMA cytotoxicity was related to redox change and autophagy. These events are important role in pulpal changes after the restoration of tooth decay using CQ- and BisGMA-containing DBAs and resin composite.

Stimulation phosphatidylinositol 3-kinase/protein kinase B signaling by Porphyromonas gingivalis lipopolysacch aride mediates interleukin-6 and interleukin-8 mRNA/protein expression in pulpal inflammation.

BACKGROUND/PURPOSE: The signaling mechanisms for Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammation in human dental pulp cells are not fully clarified. This in vitro study aimed to evaluate the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in PgLPS-induced pulpal inflammation. METHODS: Human dental pulp cells (HDPCs) were challenged with PgLPS with or without pretreatment and coincubation with a PI3K/Akt inhibitor (LY294002). The gene or protein levels of PI3K, Akt, interleukin (IL)-6, IL-8, alkaline phosphatase (ALP), osteocalcin and osteonectin were analyzed by reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and immunofluorescent staining. In addition, an enzyme-linked immunosorbent assay was used to analyze IL-6 and IL-8 levels in culture medium. RESULTS: In response to 5 µg/ml PgLPS, IL-6, IL-8, and PI3K, but not Akt mRNA expression of HDPCs, was upregulated. IL-6, IL-8, PI3K, and p-Akt protein levels were stimulated by 10-50 µg/ml of PgLPS in HDPCs. PgLPS also induced IL-6 and IL-8 secretion at concentrations higher than 5 µg/ml. Pretreatment and co-incubation by LY294002 attenuated PgLPS-induced IL-6 and IL-8 mRNA expression in HDPCs. The mRNA expression of ALP, but not osteocalcin and osteonectin, was inhibited by higher concentrations of PgLPS in HDPCs. CONCLUSION: P. gingivalis contributes to pulpal inflammation in HDPCs by dysregulating PI3K/Akt signaling pathway to stimulate IL-6 and IL-8 mRNA/protein expression and secretion. These results are useful for understanding the pulpal inflammation and possible biomarkers of inflamed pulp diagnosis and treatment.

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.

Horizontal root fracture in posterior teeth without dental trauma: A diseased condition with special characteristics.

Horizontal root fractures (HRF) were observed mostly in the anterior teeth of young adults due to dental injury. However, HRFs in posterior teeth (PHRF) without dental trauma cannot be neglected. The etiology and risk factors of PHRF were unclear. Lower premolars and palatal root of maxillary molars were particularly affected, indicating the specificity of this diseased entity. PHRF were mainly reported in Asian population, suggesting possible racial difference. Whereas most PHRF teeth showed symptoms mimicking endodontic and periodontal lesions, some affected teeth were asymptomatic. Periodontal pocket, soft tissue swelling, chronic pain or discomfort during mastication were commonly noted. Diagnosis of PHRF depended on thorough clinical examination, radiographic images or exploratory surgery. Intracanal bleeding and electronic apex locator confirmation during endodontic treatment were also useful for diagnosis. Flexible splinting, endodontic/periodontal treatment or root amputation were treatment strategies to preserve the fractured teeth. The aim of this narrative review is to summarize the demography, tooth and root distribution, diagnostic methods, etiology and possible related factors, clinical features, radiographic characteristics, and the treatment schemes of PHRF without dental trauma. A better understanding and identification of this particular root fracture could be achieved. The diagnostic tools and practical management are useful for clinical guides.

Surface Modified ß-Tricalcium phosphate enhanced stem cell osteogenic differentiation in vitro and bone regeneration in vivo.

A major number of studies have demonstrated Beta-tricalcium phosphate (ß-TCP) biocompatibility, bioactivity, and osteoconductivity characteristics in bone regeneration. The aim of this research was to enhance ß-TCP's biocompatibility, and evaluate its physicochemical properties by argon glow discharge plasma (GDP) plasma surface treatment without modifying its surface. Treated ß-TCP was analyzed by scanning electron microscopy (SEM), energy-dispersive spectrometry, X-ray photoelectron spectroscopy (XPS), X-ray diffraction analysis, and Fourier transform infrared spectroscopy characterization. To evaluate treated ß-TCP biocompatibility and osteoblastic differentiation, water-soluble tetrazolium salts-1 (WST-1), immunofluorescence, alkaline phosphatase (ALP) assay, and quantitative real-time polymerase chain reaction (QPCR) were done using human mesenchymal stem cells (hMSCs). The results indicated a slight enhancement of the ß-TCP by GDP sputtering, which resulted in a higher Ca/P ratio (2.05) than the control. Furthermore, when compared with control ß-TCP, we observed an improvement of WST-1 on all days (p < 0.05) as well as of ALP activity (day 7, p < 0.05), with up-regulation of ALP, osteocalcin, and Osteoprotegerin osteogenic genes in cells cultured with the treated ß-TCP. XPS and SEM results indicated that treated ß-TCP's surface was not modified. In vivo, micro-computed tomography and histomorphometric analysis indicated that the ß-TCP test managed to regenerate more new bone than the untreated ß-TCP and control defects at 8 weeks (p < 0.05). Argon GDP treatment is a viable method for removing macro and micro particles of < 7 µm in size from ß-TCP bigger particles surfaces and therefore improving its biocompatibility with slight surface roughness modification, enhancing hMSCs proliferation, osteoblastic differentiation, and stimulating more new bone formation.

Genetic Susceptibility and Protein Expression of Extracellular Matrix Turnover-Related Genes in Oral Submucous Fibrosis.

Betel quid (BQ) chewing increased the risk of oral cancer and oral submucous fibrosis (OSMF), an oral premalignant disorder (OPMD) with malignant transformation potential. BQ components such as areca nut (AN), trauma by coarse AN fiber, catechin, copper, alkaloids, stimulated reactive oxygen species (ROS), inflammation and cytotoxicity are suggested to be the contributing factors. They may induce tissue inflammation, proliferation of fibroblasts and collagen deposition, myofibroblast differentiation and contraction, collagen cross-links and inhibit collagen phagocytosis, finally leading to the development of OSMF and oral cancer. These events are mediated by BQ components-induced changes of extracellular matrix (ECM) turnover via regulation of TGF-ß1, plasminogen activator inhibitor-1 (PAI-1), cystatin, lysyl oxidase (LOX) and tissue inhibitors of metalloproteinases (TIMPs) and metalloproteinases (MMPs). Genetic susceptibility is also involved in these disease processes. Further understanding the molecular mechanisms of BQ-induced OSMF and oral cancer can be helpful for future disease prevention and treatment.

Glow Discharge Plasma Treatment on Zirconia Surface to Enhance Osteoblastic-Like Cell Differentiation and Antimicrobial Effects.

Peri-implantitis is the pathological condition of connective tissue inflammation and the progressive loss of supporting bone around dental implants. One of the primary causes of peri mucositis evolving into peri-implantitis is bacterial infection, including infection from Porphyromonas gingivalis. Enhancing the surface smoothness of implants helps to prevent P. gingivalis adhesion to the implant's surface. Interaction analyses between bacteria and the surface roughness of zirconia (Zr) discs subjected to a glow discharge plasma (GDP) treatment compared with non-plasma-treated autoclaved control Zr discs were done. Examinations of the material prosperities revealed that the GDP-treated Zr group had a smoother surface for a better wettability. The GDP-treated Zr discs improved the proliferation of the osteoblast-like cells MG-63, and the osteoblastic differentiation was assessed through alkaline phosphatase detection and marker gene bone sialoprotein (Bsp) and osteocalcin (OC) induction. Scanning electron microscopy demonstrated a relatively low P. gingivalis adhesion on GDP-treated Zr disks, as well as lower colonization of P. gingivalis compared with the control. Our findings confirmed that the GDP treatment of Zr discs resulted in a significant reduction of P. gingivalis adhesion and growth, demonstrating a positive correlation between surface roughness and bacteria adhesion. Therefore, the GDP treatment of Zr dental implants can provide a method for reducing the risk of peri-implantitis.

Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-ß/Smad2-dependent and -independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin.

BACKGROUND: There are 200-600 million betel quid (BQ) chewers in the world. BQ increases oral cancer risk. Matrix metalloproteinase-9 (MMP-9) is responsible for matrix degradation, cancer invasion and metastasis. Whether areca nut extract (ANE), a BQ component, stimulates MMP-9 secretion, and the related signaling pathways awaits investigation. RESULTS: ANE (but not arecoline) stimulated MMP-9 production of gingival keratinocytes and SAS cancer epithelial cells. ANE stimulated TGF-ß1, p-Smad2, and p-TAK1 protein expression. ANE-induced MMP-9 production/expression in SAS cells can be attenuated by SB431542 (ALK5/Smad2 inhibitor), 5Z-7-Oxozeaenol (TAK1 inhibitor), catalase, PD153035 (EGFR tyrosine kinase inhibitor), AG490 (JAK inhibitor), U0126 (MEK/ERK inhibitor), LY294002 (PI3K/Akt inhibitor), betel leaf (PBL) extract, and hydroxychavicol (HC, a PBL component), and melatonin, but not by aspirin. CONCLUSIONS: AN components contribute to oral carcinogenesis by stimulating MMP-9 secretion, thus enhancing tumor invasion/metastasis. These events are related to reactive oxygen species, TGF-ß1, Smad2-dependent and -independent signaling, but not COX. These signaling molecules can be biomarkers of BQ carcinogenesis. PBL, HC and melatonin and other targeting therapy can be used for oral cancer treatment. METHODS: ANE-induced MMP-9 expression/secretion of oral epithelial cells and related TGF-ß1, Smad-dependent and -independent signaling were studied by MTT assay, RT-PCR, western blotting, immunofluorescent staining, and ELISA.

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.

IL-1ß induced IL-8 and uPA expression/production of dental pulp cells: Role of TAK1 and MEK/ERK signaling.

BACKGROUND/PURPOSE: Interleukin 1 beta (IL-1ß) is a pro-inflammatory cytokine involved in the inflammatory processes of dental pulp. IL-8 and urokinase plasminogen activator (uPA) are two inflammatory mediators. However, the role of transforming growth factor beta-activated kinase-1 (TAK1) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways in responsible for the effects of IL-1ß on IL-8 and uPA expression/secretion of dental pulp cells are not clear. METHODS: Human dental pulp cells were exposed to IL-1ß with/without pretreatment with 5z-7-oxozeaneaeol (a TAK1 inhibitor) or U0126 (a MEK/ERK inhibitor). TAK1 activation was determined by immunofluorescent staining. The protein expression of IL-8 was tested by western blot. The expression of IL-8 and uPA mRNA was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). The secretion of IL-8 and uPA was measured by enzyme-linked immunosorbent assay. RESULTS: Exposure of dental pulp cells to IL-1ß (0.1-10 ng/ml) stimulated IL-8 and uPA expression. IL-1ß also induced IL-8 and uPA secretion of dental pulp cells. IL-1ß stimulated p-TAK1 activation of pulp cells. Pretreatment and co-incubation of pulp cells by 5z-7oxozeaenol (1 and 2.5 µM) and U0126 (10 and 20 µM) prevented the IL-1ß-induced IL-8 and uPA expression. 5z-7oxozeaenol and U0126 also attenuated the IL-1ß-induced IL-8 and uPA secretion. CONCLUSION: IL-1ß is important in the pathogenesis of pulpal inflammatory diseases and repair via stimulation of IL-8 and uPA expression and secretion. These events are associated with TAK1 and MEK/ERK signaling. Blocking of TAK1 and MEK/ERK signaling has potential to control inflammation of dental pulp.

Bone regeneration using a porcine bone substitute collagen composite in vitro and in vivo.

The biocharacteristics of xenogeneic grafts make them a possible substitute for autogenous bone grafts in dental bone graft procedures. This study aimed to develop a novel porcine graft with collagen capable of generating new bone in bone defects via osteoconduction over 8 weeks of healing and to compare it with a porcine graft. The porcine collagen graft was made to undergo a cell viability test (MTT) and alkaline phosphatase assay (ALP). The surgical procedure was performed in 20 male adult New Zealand white rabbits. Four calvarial critical-size defects of 6 mm in diameter were prepared in each rabbit. The upper left defect was filled with a porcine graft of 500-1000 µm, the upper right with a porcine collagen graft, the lower left with hydroxyapatite/beta-tricalcium phosphate and the lower right served as the control without any filling material. The rabbits were divided and sacrificed at 2, 4, 6 and 8 weeks after surgery. Histological and micro-CT scan results showed that the performance of the porcine collagen graft is superior for regenerating new bone. Porcine collagen graft showed cell viability and osteoblast-like cell differentiation in vitro. The results indicate that porcine collagen graft is a potential bone substitute for clinical application.

In Vitro and In Vivo Study of a Novel Porcine Collagen Membrane for Guided Bone Regeneration.

For years, in order to improve bone regeneration and prevent the need of a second stage surgery to remove non-resorbable membranes, biological absorbable membranes have gradually been developed and applied in guided tissue regeneration (GTR). The present study's main objective was to achieve space maintenance and bone regeneration using a new freeze-dried developed porcine collagen membrane, and compare it with an already commercial collagen membrane, when both were used with a bovine xenograft in prepared alveolar ridge bone defects. Prior to surgery, the membrane's vitality analysis showed statistically significant higher cell proliferation in the test membrane over the commercial one. In six beagle dogs, commercial bone xenograft was packed in lateral ridge bone defects prepared in the left and right side and then covered with test porcine collagen membrane or commercial collagen membrane. Alveolar height changes were measured. Histomorphometric results, in vitro and in vivo properties indicated that the new porcine collagen membrane is biocompatible, enhances bone xenograft osteoconduction, and reduces the alveolar ridge height reabsorption rate.

Development and Testing of X-Ray Imaging-Enhanced Poly-L-Lactide Bone Screws.

Nanosized iron oxide particles exhibit osteogenic and radiopaque properties. Thus, iron oxide (Fe3O4) nanoparticles were incorporated into a biodegradable polymer (poly-L-lactic acid, PLLA) to fabricate a composite bone screw. This multifunctional, 3D printable bone screw was detectable on X-ray examination. In this study, mechanical tests including three-point bending and ultimate tensile strength were conducted to evaluate the optimal ratio of iron oxide nanoparticles in the PLLA composite. Both injection molding and 3D printing techniques were used to fabricate the PLLA bone screws with and without the iron oxide nanoparticles. The fabricated screws were implanted into the femoral condyles of New Zealand White rabbits. Bone blocks containing the PLLA screws were resected 2 and 4 weeks after surgery. Histologic examination of the surrounding bone and the radiopacity of the iron-oxide-containing PLLA screws were evaluated. Our results indicated that addition of iron oxide nanoparticles at 30% significantly decreased the ultimate tensile stress properties of the PLLA screws. The screws with 20% iron oxide exhibited strong radiopacity compared to the screws fabricated without the iron oxide nanoparticles. Four weeks after surgery, the average bone volume of the iron oxide PLLA composite screws was significantly greater than that of PLLA screws without iron oxide. These findings suggested that biodegradable and X-ray detectable PLLA bone screws can be produced by incorporation of 20% iron oxide nanoparticles. Furthermore, these screws had significantly greater osteogenic capability than the PLLA screws without iron oxide.