Kazuhiko Nakata

The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential.

Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation.

Age-dependent decline in dental pulp regeneration after pulpectomy in dogs.

The age-associated decline in the regenerative abilities of mesenchymal stem cells (MSCs) may be due to age-related changes in reduction in number, intrinsic properties of MSCs and extrinsic factors of the extracellular environment (the stem cell niche). The effect of age on the efficacy of MSC transplantation on regeneration, however, has not been clearly demonstrated due to variable methods of isolation of MSCs and variations in stem cell populations. In this study, dental pulp stem cell (DPSC) subsets were isolated from young and aged dog teeth based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs). In order to study the age-associated changes, their biological properties and stability were compared and the regenerative potential was examined in a pulpectomized tooth model in aged dogs. MDPSCs from aged dogs were efficiently enriched in stem cells, expressing trophic factors with high proliferation, migration and anti-apoptotic effects as in MDPSCs from young dogs. However, pulp regeneration was retarded 120 days after autologous transplantation of aged MDPSCs. We further demonstrated that isolated periodontal ligament stem cells (PDLSCs) from aged dogs, representative of migrating stem cells from outside of the tooth compartment to regenerate pulp tissue, had lower proliferation, migration and anti-apoptotic abilities. These results therefore provide a better understanding of the mechanisms involved in the age-dependent decline in pulp regeneration, which are attributed to a decrease in the regenerative potential of resident stem cells.

Mouse-induced pluripotent stem cells differentiate into odontoblast-like cells with induction of altered adhesive and migratory phenotype of integrin.

Methods for differentiating induced pluripotent stem (iPS) cells into odontoblasts generally require epithelial–mesenchymal interactions. Here, we sought to characterize the cells produced by a ‘hanging drop’ technique for differentiating mouse iPS cells into odontoblast-like cells that requires no such interaction. Cells were cultured by the hanging drop method on a collagen type-I (Col-I) scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4) without an epithelial–mesenchymal interaction. We evaluated the expression of odontoblast-related mRNA and protein, and the proliferation rate of these cells using reverse-transcription polymerase chain reaction, immunofluorescence staining, and BrdU cell proliferation enzyme-linked immunosorbent assay, respectively. The differentiated cells strongly expressed the mRNA for dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (Dmp-1), which are markers of mature odontoblasts. Osteopontin and osteocalcin were not expressed in the differentiated cells, demonstrating that the differentiated iPS cells bore little resemblance to osteoblasts. Instead, they acquired odontoblast-specific properties, including the adoption of an odontoblastic phenotype, typified by high alkaline phosphatase (ALP) activity and calcification capacity. The cell-surface expression of proteins such as integrins α2, α6, αV and αVβ3 was rapidly up-regulated. Interestingly, antibodies and siRNAs against integrin α2 suppressed the expression of DSPP and Dmp-1, reduced the activity of ALP and blocked calcification, suggesting that integrin α2 in iPS cells mediates their differentiation into odontoblast-like cells. The adhesion of these cells to fibronectin and Col-I, and their migration on these substrata, was significantly increased following differentiation into odontoblast-like cells. Thus, we have demonstrated that integrin α2 is involved in the differentiation of mouse iPS cells into odontoblast-like cells using the hanging drop culture method, and that these cells have the appropriate physiological and functional characteristics to act as odontoblasts in tissue engineering and regenerative therapies for the treatment of dentin and/or dental pulp damage.

Lack of correlation between the amount of eugenol released from zinc oxide-eugenol sealer and cytotoxicity of the sealer.

The purpose of this study was to examine a possible correlation between the eugenol released from a zinc oxide-eugenol sealer (Canals) and the degree of cytotoxicity. The cytotoxicity and eugenol release from root canal filling material containing eugenol was examined for test solutions at several experimental periods. No positive correlation was found between eugenol release and cytotoxicity of the root canal filling material.

A Survey of the Incidence of Single-Visit Endodontics

This survey was conducted to obtain answers to some basic questions regarding the timing of root canal obturation. A questionnaire was sent via e-mail to 738 randomly chosen United States endodontists listed in the 1998 to 1999 membership roster of the American Association of Endodontists. One hundred fifty-six replies were received. In pulpectomy cases, root canal obturation at the first visit was carried out by 55.8% of the responding endodontists; in infected root canal cases, the percentage was 34.4%. Of the responding endodontists, 34.2% indicated that their patients had experienced some trouble after root canal obturation at the first visit.

Anaerobic Bacterial Extracts Influence Production of Matrix Metalloproteinases and Their Inhibitors by Human Dental Pulp Cells

The role of human dental pulp (HDP) cells in extracellular matrix degradation in pulpitis is still unclear. In this study, the effects of sonicated bacterial extracts (SBEs) from Prevotella intermedia, Fusobacterium nucleatum, Porphyromonas endodontalis, and Porphyromonas gingivalis on the balance between the production of matrix metalloproteinases (MMPs) and that of their inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] by HDP cells were examined. HDP cells were treated with SBEs, and their culture media were later harvested. MMP activities and TIMP concentrations were determined by use of independent measurement strategies and sensitive ELISAs. The production of MMP-1 and MMP-2 was accelerated by all SBE. On the other hand, TIMP-1 production was slightly elevated; and TIMP-2 production was markedly inhibited by all of the extracts. SBEs derived from these anaerobic bacteria seemed to affect the acceleration of extracellular matrix degradation activity by HDP cells. These findings suggest that HDP cells stimulated by bacterial byproducts may be involved in the pathogenesis of pulpitis.

Expression of MMP-8 and MMP-13 in the development of periradicular lesions

AIM To elucidate the expressions of MMP-8 and MMP-13 in experimentally induced rat periradicular lesions by means of the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining. METHODOLOGY Thirty rats were used and periradicular lesions in mandibular first molar teeth were established following pulp exposure. The animals were sacrificed at 0 (no exposure control), 1, 2, 3, 4 and 6 weeks after pulp exposure. The right molars were used for RT-PCR analysis of MMP-8 and MMP-13. The left molars were subjected to immunohistochemical staining with both MMPs. The areas of these lesions were measured histometrically, and the numbers of both reactive cells in the periapical portion were counted per unit area. Significant differences were analysed by the Mann-Whitney U-test. RESULTS MMP-8 gene expression gradually increased from 2 to 4 weeks, but slightly decreased at 6 weeks. MMP-13 gene expression gradually increased from 1 to 3 weeks. At 4 and 6 weeks, the level of expression was as high as that at 3 weeks. Immunohistochemically, MMP-8 was first detected at 2 weeks and gradually increased until 4 weeks. MMP-13 gradually increased from 1 to 4 weeks. Both MMPs decreased at 6 weeks. The area of the periradicular lesions gradually increased from 1 to 4 weeks, showing a large increase in week 2 and 3 in particular, but then decreased in week 6. MMP-13-expressing cells were significantly greater than MMP-8-positive cells at week 1 and 2. CONCLUSIONS These findings indicate that MMP-8 and MMP-13 were related to the development of periradicular lesions. It is suggested that MMP-13 increased from an early stage during their development and that MMP-8 is involved in the progression of tissue destruction including bone resorption.

Evaluation of Correspondence of Dental Computed Tomography Imaging to Anatomic Observation of External Root Resorption

Radiography using a compact dental CT, the 3D Accuitomo XYZ Slice View Tomograph (3D Accuitomo), was performed for examining the root resorption of a mandibular premolar in a patient who needed endodontic therapy. The images obtained by the 3D Accuitomo clearly showed the apparent presence of external root resorption of the tooth. As the root resorption had progressed seriously, the diagnosis was that it was impossible to save the tooth. After having obtained informed consent from the patient, the tooth was extracted. The surface of the root of the extracted tooth was examined visually without magnification, and the observations were compared with the findings made by 3D Accuitomo imaging for evaluating the reliability of the dental CT for diagnostic imaging of root resorption. The radiographic and visual examination results corresponded completely. Thus the 3D Accuitomo depicted the aspects of root resorption accurately; and its application for clinical use should be very effective for examining and diagnosing root resorption.

Effects of lipopolysaccharides on human dental pulp cells

Human dental pulp cells were treated with 1, 10, and 100 micrograms/ml of lipopolysaccharide (LPS). The effects of treatment were examined by measurement of the DNA content, protein content, and alkaline phosphatase activity of the cells. LPS samples were purified from Porphyromonas gingivalis, Porphyromonas endodontalis, and Fusobacterium nucleatum isolated from root canals, and Escherichia coli 0111:B4 LPS was used as a positive control. At a concentration of 1 microgram/ml, none of the LPSs caused any change in the production of DNA or protein, whereas the amount of DNA was increased at 10 micrograms/ml and inhibited at 100 micrograms/ml. Protein synthesis was decreased by LPSs at both 10 and 100 micrograms/ml. Alkaline phosphatase activity was not changed at any concentration of LPS tested.

Similar in vitro effects and pulp regeneration in ectopic tooth transplantation by basic fibroblast growth factor and granulocyte-colony stimulating factor.

OBJECTIVES Granulocyte-colony stimulating factor (G-CSF) has been shown to have combinatorial trophic effects with dental pulp stem cells for pulp regeneration. The aim of this investigation is to examine the effects of basic fibroblast growth factor (bFGF) in vitro and in vivo compared with those of G-CSF and to assess the potential utility of bFGF as an alternative to G-CSF for pulp regeneration. MATERIALS AND METHODS Five different types of cells were examined in the in vitro effects of bFGF on cell migration, proliferation, anti-apoptosis, neurite outgrowth, angiogenesis, and odontogenesis compared with those of G-CSF. The in vivo regenerative potential of pulp tissue including vasculogenesis and odontoblastic differentiation was also compared using an ectopic tooth transplantation model. RESULTS Basic fibroblast growth factor was similar to G-CSF in high migration, proliferation and anti-apoptotic effects and angiogenic and neurite outgrowth stimulatory activities in vitro. There was no significant difference between bFGF and G-CSF in the regenerative potential in vivo. CONCLUSIONS The potential utility of bFGF for pulp regeneration is demonstrated as a homing/migration factor similar to the influence of G-CSF.