Effect of Cells Derived from Periodontal Ligament Tissue on Bone Formation.

BACKGROUND/AIM: Recent reports indicate that sclerostin is secreted by periodontal ligament tissue-derived (PDL) cells during orthodontic force loading and that the secreted sclerostin contributes to bone metabolism. However, the detailed mechanism is poorly understood. The aim of this study was to determine how PDL cells affect bone formation. MATERIALS AND METHODS: Rat periodontal ligament tissue was immunohistochemically stained for sclerostin. Cultured primary PDL cells, osteoblasts, and skin fibroblasts (Sfbs) isolated from rat periodontal ligament tissue, calvaria, and skin, respectively, were examined. Osteoblasts were cultured with control conditioned medium (Cont-CDM) and PDL cell culture conditioned medium (PDL-CDM) for up to 21 days. Cultured osteoblasts were then stained with alkaline phosphatase and von Kossa stain. Osteoblasts cultured in each conditioned medium were analyzed by real-time quantitative PCR for bone Gla protein (Bgp), Axin2, and Ki67 expression. PDL cells used to obtain conditioned medium were analyzed for Sost, Ectodin and Wnt1 expression and compared with expression in Sfbs. RESULTS: Expression of sclerostin was observed in periodontal ligament tissue by immunohistochemical staining. The formation of mineralization nodules was inhibited in PDL-CDM compared with Cont-CDM in osteoblast culture. In PDL-CDM, the expression levels of Bgp and Axin2 in osteoblasts were decreased compared with Cont-CDM. In PDL cells, expression levels of Sost and Ectodin were much higher than in Sfbs; however, expression of Wnt1 was lower in PDL cells compared with Sfbs. CONCLUSION: PDL cells secrete various proteins, including sclerostin and suppress osteogenesis in osteoblasts through the canonical Wnt pathway.

Effects of Irradiation by Carbon Dioxide Laser Equipped With a Water Spray Function on Bone Formation in Rat Tibiae.

BACKGROUND/AIM: Irradiation of tissue with carbon dioxide (CO2) laser shows a characteristic thermal effect that causes vaporization of tissue in the target region. However, the thermal effect in places other than the target region induces tissue damage. Two methods are used: high reactive-level laser therapy (HLLT), aimed at surgical treatment, and low reactive-level laser therapy (LLLT), aimed at cell and tissue activation. In both, vaporization of tissue is induced by thermal damage. A water spray function may ameliorate thermal damage from CO2 laser irradiation. In this study, we irradiated CO2 laser on rat tibiae with or without a water spray function and examined the effects of this technique on bone metabolism. MATERIALS AND METHODS: Bone defects were created in rat tibiae by dental bur in a Bur group and by laser in laser irradiation groups with (Spray group) and without (Air group) water spray function. At 1 week postoperatively, histological analyses of tibiae were performed using hematoxylin and eosin staining, immunohistochemical staining (IHC) with anti-sclerostin antibody, and 3-dimensional (3D) observation using micro-computed tomography. RESULTS: Histological findings and 3D observation confirmed induction of new bone formation following laser irradiation in both the Air and Spray groups. No bone formation was seen in the Bur group. IHC revealed that the activity of osteocytes in the region of irradiated cortical bone was markedly impaired in the Air group, but osteocyte impairment was ameliorated in the Spray group and absent in the Bur group. CONCLUSION: The water spray function appears effective in reducing thermal damage to tissues irradiated by CO2 laser. CO2 lasers with water spray function may be useful in bone regeneration therapy.

Oxytocin Facilitates Dentinogenesis of Rat Dental Pulp Cells.

INTRODUCTION: Oxytocin (OT) is a neurohypophysial hormone that plays a role in lactation and parturition and exerts diverse biological actions via the OT receptor. Recently, several studies have reported that OT stimulates bone formation by osteoblasts in osteoporosis. We focused on OT and hypothesized that OT can stimulate the differentiation of odontoblasts as well as osteoblasts. The aim of this study was to verify whether OT is an essential factor in dentinogenesis; we examined the effects of OT on dentinogenesis using a long-term culture system of rat dental pulp cells. METHODS: Using a culture system of rat dental pulp cells with Otr knocked out by CRISPR-Cas9 genome editing, we examined the effects of OT on odontoblastlike cell differentiation as reflected by dentin formation. RESULTS: We confirmed that OT stimulated mineralized nodule formation and the expression of both dentin sialoprotein and bone Gla protein messenger RNAs (mRNAs) in the culture system. Interestingly, the cultured cells treated with OT also exhibited an increase of both Wnt10a and Lef-1 mRNA. The Otr knockout cells showed inhibition of nodule formation and mRNA expression, and these phenomena remained despite OT treatment. These results indicate the following: OT regulates odontoblastlike cell differentiation via the OT receptor, it stimulates dentin formation, and the Wnt canonical pathway is closely related to these effects. CONCLUSIONS: The present results suggest that OT can promote odontoblastlike cell differentiation, resulting in increased dentin formation, and that OT could be an important factor for dentinogenesis.

Enamel Matrix Derivative in Diffusion Chamber Implanted Subcutaneously in Rat Induces Formation of Fibrous Connective Tissue Containing Abundant Blood Vessels.

BACKGROUND: Enamel matrix derivative (EMD) is widely used for regeneration therapy in dental clinical situations, but the mechanism of EMD bioactivity remains obscure. To clarify this mechanism, we focused on the formation of connective tissue and blood vessels. The aim of this study was to confirm whether EMD induces the formation of connective tissue and blood vessels by using the diffusion chamber (DC) technique. MATERIALS AND METHODS: Individual DCs containing EMD (DC-EMD) or propylene glycol alginate (PGA) were implanted subcutaneously in rat dorsum. At 4 weeks after the implantation, histological analysis of DCs was performed using azan staining. RESULTS: DC-EMD induced the formation of much larger amounts of connective tissue containing abundant blood vessels than did DC-PGA. CONCLUSION: The results indicated that EMD can induce the formation of both connective tissue and blood vessels. This bioactivity may contribute to the mechanism whereby EMD induces tissue regeneration.

Lymphocyte enhancer-binding factor 1: an essential factor in odontoblastic differentiation of dental pulp cells enzymatically isolated from rat incisors.

Lymphocyte enhancer-binding factor 1 (Lef1), a HMG-domain protein, is thought to play important roles in inductive tissue interaction during tooth development. Lef1 knockdown in mice causes arrest at the bud stage in tooth development. As this gene participates in the regulation of a large and diverse set of peptide growth factors in ectomesenchymal cell differentiation of dental papilla, Lef1 appears to be a key factor in odontoblast differentiation. However, the relationship between Lef1 and odontoblast differentiation is still unclear. To analyze the biological roles of Lef1 in regulating odontoblast differentiation, we transiently overexpressed or suppressed Lef1 in cultured dental pulp cells. Lef1-overexpressing cells expressed higher levels of dentin sialoprotein (DSPP), osteocalcin and alkaline phosphatase (ALP) mRNA and formed larger numbers of mineralized nodules compared to control cells. However, Msx-1 expression or cell proliferation was unaffected by overexpression of Lef1. To further examine the role of Lef1 in dental pulp cells, we knocked down Lef1 expression in dental pulp cells using short interfering RNA (siRNA). Transient expression of siRNA against Lef1 markedly reduced Lef1 mRNA levels, and Lef1-suppressed cells expressed lower levels of DSPP, osteocalcin and ALP mRNA compared to control cells. Furthermore, the formation of mineralized nodules was inhibited by siRNA against Lef1; however, neither Msx-1 expression or cell proliferation was inhibited by siRNA against Lef1. These results outline the role of Lef1 in accelerating odontoblast differentiation by regulating DSP and osteocalcin mRNA expression in dental pulp cells, confirming that Lef1 is a key factor for odontoblast differentiation.

Effect of glypican-1 gene on the pulp cells during the reparative dentine process.

GPC-1 (glypican-1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors and members of the TGF-ß (transforming growth factor beta-1) family. The function of cell-surface proteoglycans in the reparative dentine process has been under investigation. Gpc-1 was detected with similar frequency as tgf-ß1 in the cDNA library using mRNA from the odontoblast-like cell-enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc-1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc-1 on odontoblast-like cell differentiation using siRNA (small interfering RNA) to down-regulate gpc-1 expression. Immunohistological examination showed that GPC-1 was expressed in pulp cells entrapped by fibrodentine and odontoblast-like cells as well as TGF-ß1. The mRNAs for gpc-1, -3 and -4, except for gpc-2, were expressed during odontoblast-like cell differentiation in pulp cells. The relative levels of gpc-1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down-regulation of gpc-1 expression resulted in a 3.9-fold increase in tgf-ß1 expression in pulp cells and a 0.3-fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc-1 and tgfß-1 expression are necessary for the onset of differentiation, but should be down-regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc-1 expression in odontoblast-like cells is associated with the early differentiation but not with the formation of reparative dentine.

Effect of Small-molecule GSK3 Antagonist on Differentiation of Rat Dental Pulp Cells into Odontoblasts.

BACKGROUND: It has been reported that glycogen synthase kinase 3 (GSK3) antagonist promoted the reparative formation of dentin. The aim of the present study was to evaluate whether treatment schedule of Tidegrusib® (TG), a small-molecule GSK3 antagonist, affected in vitro differentiation of dental pulp cells toward odontoblast-like cells. MATERIALS AND METHODS: Pulp cells isolated from rat incisors were repeatedly exposed to TG for the first 6 h (intermittent exposure) or the full 48 h (continuous exposure) of each 48-h incubation cycle. Histological analysis of alkaline phosphatase and von Kossa staining were performed. The expression of dentin sialophosphoprotein (Dspp) and osteocalcin (Ocn) mRNA were examined by real-time polymerase chain reaction. Western blotting assays were used to monitor the expression of ß-catenin and its phosphorylated form. RESULTS: When pulp cells were intermittently exposed to TG for only the first 6 h of each incubation cycle, pulp cells differentiated into odontoblast-like cells, characterized by an increase in alkaline phosphatase activity, nodule formation, and mRNA expression of Dspp. and Ocn; this did not occur under the continuous exposure. Phosphorylation of ß-catenin was enhanced by continuous exposure to TG compared with intermittent exposure. CONCLUSION: These results suggest that the TG-induced odontoblast-like cell differentiation reflects in vivo reparative dentin formation and depends on the exposure time.

Biological Properties of the Aggregated Form of Chitosan Magnetic Nanoparticle.

BACKGROUND/AIM: Chitosan-coated iron oxide nanoparticles (Chi-NP) have gained attention because of their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. In this study, we investigated various biological properties of Chi-NP. MATERIALS AND METHODS: Chi-NP was prepared by mixing magnetic NP with chitosan FL-80. Particle size was determined by scanning and transmission electron microscopes, cell viability by MTT assay, cell cycle distribution by cell sorter, synergism with anticancer drugs by combination index, PGE2 production in human gingival fibroblast was assayed by ELISA. RESULTS: The synthetic process of Chi-NP from FL-80 and magnetic NP increased the affinity to cells, up to the level attained by nanofibers. Upon contact with the culture medium, Chi-NP instantly formed aggregates and interfered with intracellular uptake. Aggregated Chi-NP did not show cytotoxicity, synergism with anticancer drugs, induce apoptosis (accumulation of subG1 cell population), protect the cells from X-ray-induced damage, nor affected both basal and IL-1ß-induced PGE2 production. CONCLUSION: Chi-NP is biologically inert and shows high affinity to cells, further confirming its superiority as a scaffold for drug delivery.

Photodynamic Therapy with Pyoktanin Blue and Diode Laser for Elimination of Enterococcus faecalis.

BACKGROUND/AIM: Enterococcus faecalis is responsible for most cases of endodontic treatment failure. Despite various conventional disinfection methods, root canals are not completely free of microorganisms. Photodynamic therapy (PDT) is a new antimicrobial strategy that involves the use of a non-toxic photosensitizer (PS) and a light source. The aim of this study was to evaluate the antimicrobial effect of PDT using diode laser and pyoktanin blue (PB) and confirm the nontoxicity of PB as a PS. MATERIALS AND METHODS: Laser irradiation with an output power of 3 W was performed with PB as the PS to a bacterial solution containing E. faecalis. Then, the number of colony-forming units was counted. PB cytotoxicity was also assessed by the MTT assay. RESULTS: E. faecalis counts were reduced after laser irradiation, laser irradiation with PB, or the combination thereof compared to the control, non-irradiation or water. The 50% cytotoxic concentration value for adult human dermal fibroblasts incubated with PB for 1 min was 108 µg/ml. CONCLUSION: Diode laser irradiation in combination with PB as the PS is efficacious for the elimination of E. faecalis without toxic effects to human dermal fibroblasts. This strategy might be useful for root canal irrigants.

Effects of surface microtopography of titanium disks on cell proliferation and differentiation of osteoblast-like cells isolated from rat calvariae.

The surface topography of implant fixture is an important factor affecting the osseointegration. We herein demonstrated the effects of surface microtopography of titanium disks on proliferation and differentiation of osteoblast-like cells isolated from rat calvariae. Titanium disks with machine surface (MS), rough surface (R1) and rough surface combined with small cavities (R2) were used in an in vitro culture system. Rough surfaces (R1 and R2 disks) induced stronger osteoblast proliferation and differentiation (BGP and sclerostin mRNA expressions and calcium content) than the smooth surface (MS disk). Furthermore, surface microtopography of R2 disk, which was rough with small cavities, more strongly induced cell proliferation and mineralized bone matrix production than R1 disk. Our results suggest that surface microtopography influences osteoblast proliferation and differentiation. R2 disk, which is rough with small cavities, may be used in implant fixtures to increase osseointegration.

Gene Expression Analysis of Cultured Rat-Endothelial Cells after Nd:YAG Laser Irradiation by Affymetrix GeneChip Array.

Endothelial cells and dental pulp cells enhance osteo-/odontogenic and angiogenic differentiation. In our previous study, rat pulp cells migrated to Nd:YAG laser-irradiated endothelial cells in an insert cell culture system. The purpose of this study was to examine the possible changes in the gene expression of cultured rat aortic endothelial cells after Nd:YAG laser irradiation using affymetrix GeneChip Array. Total RNA was extracted from the cells at 5 h after laser irradiation. Gene expressions were evaluated by DNA array chip. Up-regulated genes were related to cell migration and cell structure (membrane stretch, actin regulation and junctional complexes), neurotransmission and inflammation. Heat-shock 70 kDa protein (Hsp70) was related to the development of tooth germ. This study offers candidate genes for understanding the relationship between the laser-stimulated endothelial cells and dental pulp cells.

Induction of Apoptosis in Human Oral Keratinocyte by Doxorubicin.

BACKGROUND/AIM: We have previously reported that doxorubicin (DXR) showed much higher cytotoxicity against human oral squamous cell carcinoma cell lines compared to normal human mesenchymal normal oral cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell), yielding high tumor-specificity. However, we unexpectedly found that doxorubicin showed potent cytotoxicity against human normal oral keratinocytes and primary gingival epithelial cells. In the present study, we investigated the reproducibility, underlining mechanisms and generality of this unexpected finding. MATERIALS AND METHODS: Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, fine cell structure by transmission electron microscopy and apoptosis induction by western blot analysis. RESULTS: Doxorubicin induced keratinocyte toxicity, regardless of cell density and concentration of FBS in the culture medium. Doxorubicin induced apoptosis (characterized by the loss of cell surface microvilli, chromatin condensation, nuclear fragmentation and caspase-3 activation) in keratinocytes. A total of 11 anticancer drugs showed similar keratinocyte toxicity. Alkaline extract of the leaves of Sasa senanensis Rehder partially alleviated the DXR-induced keratinocyte cytotoxicity by promoting cell growth. CONCLUSION: The present study suggested that oral keratinocyte toxicity is a novel adverse effect of most anticancer agents.

Spatiotemporal expression of sclerostin in odontoblasts during embryonic mouse tooth morphogenesis.

INTRODUCTION: Sclerostin is the product of the SOST gene. Loss-of-function mutations in the SOST gene result in a high bone mass phenotype, thus confirming that sclerostin is a negative regulator of bone mass. SOST knockdown in humans also causes oral and dental malformations. However, the relationship between sclerostin and tooth development is unclear. METHODS: Using immunohistochemical techniques, we investigated sclerostin expression during fetal mouse tooth development and adult mouse tooth morphogenesis. RESULTS: Sclerostin was expressed in the secretory odontoblasts located along the ameloblasts of fetal mouse tooth germ and adult incisor. Sclerostin expression was also observed in the fetal and adult osteocytes in the jaw bone. CONCLUSION: These results suggest that sclerostin, one of the important regulatory factors of differentiated odontoblast function, may usable in vital pulp therapy.

Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells.

Platelet-derived growth factor (PDGF) has recently been demonstrated to control the expression of alkaline phosphatase and proteoglycan synthesis of odontoblastic cells in dental pulp tissues. Although PDGF appears to be closely related to dentinogenesis, much about the mode of action of PDGF on odontoblast differentiation remains unclear. In this study, we examined the effects of three PDGF dimers (PDGF AA, AB, and BB) on odontoblastic differentiation of dental pulp cells in long-term mineralized cultures. Dental pulp cells isolated from rat lower incisors were continuously treated with each of PDGF AA, AB, and BB in separate cultures for 20 days. The three PDGF dimers suppressed alkaline phosphatase activity, osteocalcin and calcium content, and the formation of dentin-like nodules. The expression of mRNA for dentin sialoprotein (DSP) in the cells was inhibited by PDGF AA treatment, whereas PDGF AB and BB treatment stimulated the expression of DSP, even though the dentin-like nodule formation was inhibited. Although the effects of PDGF on odontoblastic differentiation varied among the dimers, the cells expressed both PDGF alpha and beta receptors, whose quantities were similar. These results suggest that PDGF exerts diverse effects on odontoblastic differentiation depending on its dimeric form. These in vitro findings explain, at least in part, the in vivo action of PDGF in dentinogenesis during the repair process of damaged dental pulp.