Junko Hatakeyama

Amelogenin-mediated Regulation of Osteoclastogenesis, and Periodontal Cell Proliferation and Migration

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

A Method for Rapid Demineralization of Teeth and Bones

Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

The Effect of Valproic Acid on Mesenchymal Pluripotent Cell Proliferation and Differentiation in Extracellular Matrices

Valproic acid (2-n-propylpentanoic acid, VPA) is a widely used antiepileptic and anticonvulsant drug. Previous studies have reported that VPA effects osteogenesis in vivo and in vitro, yet it remains unclear whether VPA promotes cell differentiation of osteoblasts derived from mesenchymal cells. The purpose of this study was to clarify the effect of VPA on undifferentiated pluripotent mesenchymal cell proliferation and differentiation into osteoblasts while analyzing the impact of the absence or presence of extracellular matrices (ECMs). Mouse mesenchymal cells were cultured on non-coated plastic, type I collagen-coated, and fibronectin-coated plates in the absence or presence of VPA. A cell proliferation assay was performed in which modified formazan dye content was analyzed and proliferation nuclear antigen (PCNA)-positive cells were counted at various concentrations of VPA. A high concentration of VPA did not clearly alter cell morphology, but large numbers of stress fibers were observed in these cells and the cell proliferation ratio was decreased with positive PCNA counts. In the presence of matrices, the cell proliferation ratio decreased at low VPA concentrations compared with the ratio obtained in the absence of these ECMs. On the other hand, VPA promoted osteoblastic differentiation in the presence of type I collagen. These findings indicate that for undifferentiated mesenchymal cells, VPA promotes a decrease in the cell proliferation rate in the presence of ECMs and promotes osteoblastic differentiation, both of which could provide insight into additional mechanisms of osteoblastic cell differentiation caused by VPA.

Amelogenins: Multi-Functional Enamel Matrix Proteins and Their Binding Partners

Amelogenins are the most abundant extracellular matrix proteins secreted by ameloblasts during tooth development and are important for enamel formation. Recently, amelogenins have been detected not only in ameloblasts, which are differentiated from the epithelial cell lineage, but also in other tissues, including mesenchymal tissues at low levels, suggesting that amelogenins possess other functions in these tissues. The therapeutic application of an enamel matrix derivative rich in amelogenins resulted in the regeneration of cementum, alveolar bone, and periodontal ligament (PDL) in the treatment of experimental or human periodontitis, indicating the attractive potential of amelogenin in hard tissue formation. In addition, a full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) regulate cementoblast/PDL cell proliferation and migration in vitro. Interestingly, amelogenin null mice show increased osteoclastogenesis and root resorption in periodontal tissues. Recombinant amelogenin proteins suppress osteoclastogenesis in vivo and in vitro, suggesting that amelogenin is involved in preventing idiopathic root resorption. Amelogenins are implicated in tissue-specific epithelial-mesenchymal or mesenchymal-mesenchymal signaling; however, the precise molecular mechanism has not been characterized. In this review, we first discuss the emerging evidence for the additional roles of M180 and LRAP as signaling molecules in mesenchymal cells. Next, we show the results of a yeast two-hybrid assay aimed at identifying protein-binding partners for LRAP. We believe that gaining further insights into the signaling pathway modulated by the multifunctional amelogenin proteins will lead to the development of new therapeutic approaches for treating dental diseases and disorders.

Amelogenin splice isoforms stimulate chondrogenic differentiation of ATDC5 cells

OBJECTIVE Amelogenins are the most abundant matrix proteins in enamel. Among the amelogenin isoforms, full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) are expressed in various tissues and are implicated as signalling molecules in mesenchymal cells. Here, we examined the effects of M180 and LRAP on a chondrogenic cell line, ATDC5, to investigate the role of amelogenins in chondrogenesis. MATERIALS AND METHODS Recombinant mouse M180- or LRAP-protein-containing medium or control medium was mixed with a chondrogenesis-stimulating medium, and changes in the phenotype, gene expression levels and cell proliferation of cultured ATDC5 cells were analysed. RESULTS The addition of amelogenins increased alkaline phosphatase activity and glycosaminoglycan secretion at 14 and 21 days of culture, respectively, as compared with the control. Quantitative PCR (Q-PCR) analysis revealed that LRAP increased the gene expression levels of Runx2, Col2a1 and Aggrecan at 7 days of differentiation. Moreover, both M180 and LRAP significantly increased the gene expression levels of ALP, Aggrecan, Col10a1 and osteopontin at 28 days of culture. Bromodeoxyuridine assay and Q-PCR analysis for Wnt signalling indicated that both M180 and LRAP reduced proliferation, but induced the cell differentiation possibly through altered non-canonical Wnt signalling. CONCLUSION M180 and LRAP accelerate chondrogenic differentiation and maturation of ATDC5 cells.

Immunohistochemical Study of Amelogenin and Lysosome-Associate Membrane Proteins (LAMPs) in Cartilage

La amelogenina es una de las proteinas de la matriz del esmalte secretadas por ameloblastos durante la formacion del esmalte en el desarrollo dentario. Estudios recientes demuestran que la amelogenina se expresa en los condrocitos. Las proteinas de membrana asociadas a lisosomas (LAMPs) se han identificado como proteinas de union asociadas a la amelogenina; se ha sugerido que actuan como receptores de senalizacion de la amelogenina. El proposito de este estudio fue aclarar la localizacion de la amelogenina y las LAMPs en el cartilago de crecimiento y nodulos cartilaginosos en cultivos de micromasa. Articulaciones de la rodilla del raton, que incluian la placa de crecimiento tibial de 4 semanas de edad y cultivos de micromasa de celulas mesenquimales del brote del miembro despues de 2 semanas se fijaron en paraformaldehido y procesaron rutinariamente. Los cortes fueron sometidos a inmunotincion con amelogenina, colageno tipo II y X, LAMP-1 y LAMP-3 . Se observo inmunorreaccion positiva de amelogenina tanto en la zona proliferacion e hipertrofica del cartilago de crecimiento despues del pretratamiento enzimatico. Ademas, los nodulos cartilaginosos en el cultivo de micromasa eran inmunopositivos para la amelogenina. Los condrocitos en la zona de proliferacion de la placa de crecimiento fueron immunopositivos a LAMP-1, mientras que los condrocitos de la zona hipertrofica se tineron debilmente. Estas observaciones indican que la amelogenina puede estar presente en la matriz del cartilago producida tanto in vivo e in vitro, ademas la amelogenina puede estar implicada en la formacion de cartilago mediante la via de senalizacion de LAMP-1.

Growth Differentiation Factor 5 (GDF-5) Induces Matrix Metalloproteinase 2 (MMP-2) Expression in Periodontal Ligament Cells and Modulates MMP-2 and MMP-13 Activity in Osteoblasts

Growth differentiation factor 5 (GDF-5), a member of the transforming growth factor beta (TGF-) superfamily expressed in periodontal tissues, promotes extracellular matrix (ECM) in periodontal ligament (PDL) cells. Matrix metalloproteinases (MMP) are proteolytic enzymes that degrade ECM and are expressed in PDL cells. To date, little is known about the regulation of MMP synthesis and secretion in PDL cells. The aim of this study is to examine the effects of GDF-5 on MMP production and activity in PDL cells. GDF-5 increased both collagen type I alpha 2 (Col I2) and MMP-2 gene expression in cells derived from mouse PDL tissues after 3 days of culture. Because PDL cells represent a heterogeneous population, we examined gelatinolytic activity and gene expression profiles in an osteoblast cell line. After 6, 12, 24, and 48 hours of culture, GDF-5 increased both Col I2 and MMP gene expression in osteoblasts. GDF-5 also promoted MMP-2 activity as revealed by gelatin zymography after 7 days of culture. In the presence of the p38 MAP kinase inhibitor SB202190, on the other hand, MMP-2 activity was blocked. Taken together, these results indicate that GDF-5 may increase simultaneously the gene expression of type I collagen and MMP-2 in the osteoblast-like cells among the PDL cells and p38 MAP kinase pathway in osteoblast could involve in the regulation of MMP-2 enzyme activity induced by GDF-5.

Histologic evaluation of the effects of Emdogain gel on injured root apex in rats.

INTRODUCTION This study investigated the effects of Emdogain gel (EMD) on the injured open apex within periapical lesions. METHODS Periapical lesions were induced in rats by opening the pulp chambers of the mandibular first molars and filing the apical foramen through the distal root canal with #25 K-files to make an open apex. The teeth were exposed to the oral environment for 7 days. Then we irrigated the distal root canals and divided them into EMD-treated and propylene glycol alginate-treated groups. The rats were killed 7, 14, and 28 days after treatment and examined histochemically. RESULTS In the EMD-treated rats, more cells expressed transforming growth factor-β1 or bone morphogenetic protein-2 at 7 days after treatment, and the regeneration of cementum and bone was observed around the root apex at 14 days after treatment. Conversely, in the propylene glycol alginate-treated group, few cells expressed transforming growth factor-β1 or bone morphogenetic protein-2, and apical periodontal tissue recovery was rarely seen within the periapical lesions throughout the experiment. CONCLUSIONS These results suggest that EMD does not irritate injured periapical tissue and may create a favorable environment that promotes the healing of destroyed periapical tissues.

Immunopathology of Apical Periodontitis and Refractory Cases

Apical periodontitis is a relatively frequently encountered disease in clinical dentistry; however, its pathogenesis and etiology are not easily elucidated. Therefore, it is not always cured, even when carefully following the highest standards of treatment and intractable apical periodontitis may occur. In addition, in long-term root canal treatment of difficult cases with intractable pain, there may be misunderstandings between the dentist and patient. While acute pain is an indispensable symptom in detecting lesions and disease, sustained chronic pain can decrease an individual’s quality of life with various negative outcomes, including decreased motivation to work. Therefore, endodontic treatments and pain control measures for a diseased tooth in intractable apical periodontitis must be developed. This review outlines the progression from the onset of the lesion and examines the immunology of apical periodontitis based on studies of model animals, indicating that interleukin-1β is a key factor in elucidating the disease state and is expected to lead to the development of an effective treatment for refractory cases.