Akihiro Hosoya

Hard Tissue Formation in Subcutaneously Transplanted Rat Dental Pulp

While dental pulp appears to be able to form mineralized matrices that do not always resemble dentin, the precise characteristics of the hard tissue and the mechanism of its induction remain unknown. Therefore, we evaluated hard tissue induced by transplantation of pulp into subcutaneous tissue. Seven days after transplantation, initial hard tissue was formed at the inner periphery of the pulp. After 14 days, this hard tissue expanded inwardly. Mineralized matrix was immunopositive for osteocalcin, osteopontin, and bone sialoprotein, but negative for dentin sialoprotein. Transplantation of GFP-labeled pulp into wild-type rats showed these formative cells to have been derived from the transplant. TEM observation revealed apatite crystals within necrotic cells and matrix vesicles at the initial stage of calcification. These results indicate that pulp cells possess the ability to form a bone- or cementum-like matrix. Calcification of the matrix may occur in necrotic cells and matrix vesicles, followed by collagenous calcification.

Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections

To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following or bypassing fixation in 4% paraformaldehyde, these sections were decalcified in 5% EDTA for 0-5xa0min. Before decalcification, marked radiopacity of bone matrix was observed in contact microradiography (CMR) images, and electron probe microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In fixed and unfixed sections without decalcification, immunolocalization of bone matrix proteins were almost restricted to osteoid. After 1xa0min of decalcification, reduced radiopacity was apparent in the CMR images, and less phosphorus and calcium was observed by EPMA, which completely disappeared by 5xa0min decalcification. After 3-5xa0min of decalcification, unfixed sections showed that these proteins were immunolocalized in bone matrix, but were not detectable in osteoid. However, fixed sections demonstrated that these were found in both bone matrix and osteoid. The present findings suggest that bone matrix proteins are embedded in calcified matrix which is separated from the aqueous environment and that they hardly move, probably due to firm bonding with each other. In contrast, matrix proteins in osteoid are subject to loss after decalcification because they may be bound to scattered apatite crystals, not to each other.

Ripply2 is essential for precise somite formation during mouse early development

The regions of expression of Ripply1 and Ripply2, presumptive transcriptional corepressors, overlap at the presomitic mesoderm during somitogenesis in mouse and zebrafish. Ripply1 is required for somite segmentation in zebrafish, but the developmental role of Ripply2 remains unclear in both species. Here, we generated Ripply2 knock‐out mice to investigate the role of Ripply2. Defects in segmentation of the axial skeleton were observed in the homozygous mutant mice. Moreover, somite segmentation and expression of Notch2 and Uncx4.1 were disrupted. These findings indicate that Ripply2 is involved in somite segmentation and establishment of rostrocaudal polarity.

Immunohistochemical Localization of α-Smooth Muscle Actin During Rat Molar Tooth Development

The dental follicle contains mesenchymal cells that differentiate into osteoblasts, cementoblasts, and fibroblasts. However, the characteristics of these mesenchymal cells are still unknown. α-Smooth muscle actin (α-SMA) is known to localize in stem cells and precursor cells of various tissues. In the present study, to characterize the undifferentiated cells in the dental follicle, immunohistochemical localization of α-SMA was examined during rat molar tooth development. Rat mandibles were collected at embryonic days (E) 15-20 and postnatal days (P) 7-28. Immunohistochemical stainings for α-SMA, periostin, Runt-related transcription factor-2 (Runx2), tissue nonspecific alkaline phosphatase (TNAP), and bone sialoprotein (BSP) were carried out using paraffin-embedded sections. α-SMA localization was hardly detected in the bud and cap stages. At the early bell stage, α-SMA-positive cells were visible in the dental follicle around the cervical loop. At the late bell to early root formation stage (P14), these cells were detected throughout the dental follicle, but they were confined to the apical root area at P28. Double immunostaining for α-SMA and periostin demonstrated that α-SMA-positive cells localized to the outer side of periostin-positive area. Runx2-positive cells were visible in the α-SMA-positive region. TNAP-positive cells in the dental follicle localized nearer to alveolar bone than Runx2-positive cells. BSP was detected in osteoblasts as well as in alveolar bone matrix. These results demonstrate that α-SMA-positive cells localize on the alveolar bone side of the dental follicle and may play a role in alveolar bone formation.

Involvement of cell-cell and cell-matrix interactions in bone destruction induced by metastatic MDA-MB-231 human breast cancer cells in nude mice

To clarify the mechanisms of bone destruction associated with bone metastases, we studied an animal model in which inoculation of MDA-MB-231 human breast cancer cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone using morphological techniques. On the bone surfaces facing the metastatic tumor cells, there existed many tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. TRAP-positive mononuclear osteoclast precursor cells were also observed in the tumor nests. Immunohistochemical studies showed that the cancer cells produced parathyroid hormone-related protein (PTHrP) but not receptor activator of NF-κB ligand (RANKL). Histochemical and immunohistochemical examinations demonstrated that alkaline phosphatase and RANKL-positive stromal cells were frequently adjacent to TRAP-positive osteoclast-like cells. Immunoelectron microscopic observation revealed that osteoclast-like cells were in contact with RANKL-positive stromal cells. MDA-MB-231 cells and osteoclastlike cells in the tumor nests showed CD44-positive reactivity on their plasma membranes. Hyaluronan (HA) and osteopontin (OPN), the ligands for CD44, were occasionally colocalized with CD44. These results suggest that tumorproducing osteoclastogenic factors, including PTHrP, upregulate RANKL expression in bone marrow stromal cells, which in turn stimulates the differentiation and activation of osteoclasts, leading to the progression of bone destruction in the bone metastases of MDA-MB-231 cells. Because the interactions between CD44 and its ligands, HA and OPN, have been shown to upregulate osteoclast differentiation and function, in addition to the cell-cell interactions mediated by RANK and RANKL, the cell-matrix interactions mediated by these molecules may also contribute to the progression of osteoclastic bone destruction.

Association of TIMP-2 with extracellular matrix exposed to mechanical stress and its co-distribution with periostin during mouse mandible development.

Matrix remodeling is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Periostin, originally identified in a mouse osteoblastic library, plays a role in cell adhesion and migration and in mechanical stress-induced matrix remodeling. In this study, we analyzed and compared the distribution patterns of TIMP-2 and periostin during mouse mandible development. Immunohistochemical staining for TIMP-2 and periostin was carried out on serial cryosections obtained from mice at embryonic days 13–16, postnatal day 2 (P2), P35, and 12xa0weeks of age. TIMP-2 and periostin exhibited a strikingly similar protein distribution during mandible development. From bud to early bell stages of molars, TIMP-2 and periostin were highly expressed on the lingual and anterior sides of the basement membrane and on the adjacent jaw mesenchyme. In pre- and postnatal incisors, the basement membrane of the apical loop and dental follicle was immunostained for TIMP-2 and periostin. At postnatal stages, TIMP-2 and periostin were prominently confined to the extracellular matrix (ECM) of gingival tissues, periodontal ligaments, and tendons (all recipients of mechanical strain). However, periostin was solely detected in the lower portion of the inner root sheath of hair follicles. Gingiva of P2 cultured in anti-TIMP-2 antibody-conditioned medium showed markedly reduced staining of periostin. We suggest that TIMP-2 and periostin are co-distributed on ECM exposed to mechanical forces and coordinately function as ECM modulators.

VDR in Osteoblast-Lineage Cells Primarily Mediates Vitamin D Treatment-Induced Increase in Bone Mass by Suppressing Bone Resorption

Long‐term treatment with active vitamin D [1α,25(OH)2D3] and its derivatives is effective for increasing bone mass in patients with primary and secondary osteoporosis. Derivatives of 1α,25(OH)2D3, including eldecalcitol (ELD), exert their actions through the vitamin D receptor (VDR). ELD is more resistant to metabolic degradation than 1α,25(OH)2D3. It is reported that ELD treatment causes a net increase in bone mass by suppressing bone resorption rather than by increasing bone formation in animals and humans. VDR in bone and extraskeletal tissues regulates bone mass and secretion of osteotropic hormones. Therefore, it is unclear what types of cells expressing VDR preferentially regulate the vitamin D–induced increase in bone mass. Here, we examined the effects of 4‐week treatment with ELD (50u2009ng/kg/day) on bone using osteoblast lineage‐specific VDR conditional knockout (Ob‐VDR‐cKO) and osteoclast‐specific VDR cKO (Ocl‐VDR‐cKO) male mice aged 10 weeks. Immunohistochemically, VDR in bone was detected preferentially in osteoblasts and osteocytes. Ob‐VDR‐cKO mice showed normal bone phenotypes, despite no appreciable immunostaining of VDR in bone. Ob‐VDR‐cKO mice failed to increase bone mass in response to ELD treatment. Ocl‐VDR‐cKO mice also exhibited normal bone phenotypes, but normally responded to ELD. ELD‐induced FGF23 production in bone was regulated by VDR in osteoblast‐lineage cells. These findings suggest that the vitamin D treatment‐induced increase in bone mass is mediated by suppressing bone resorption through VDR in osteoblast‐lineage cells.

Differential regulation of TIMP-1, -2, and -3 mRNA and protein expressions during mouse incisor development

Tissue inhibitors of metalloproteinases (TIMPs) possess multiple functions, in addition to their matrix metalloproteinase (MMP) inhibitory activity. The continuously growing incisor of mouse possesses a stem cell compartment at the apical end of the epithelium (the apical loop) and thus provides an excellent tool to analyze the mechanisms of organogenesis and cytodifferentiation. To understand the functions of TIMPs in tooth development, we have analyzed the gene expression and protein localization of TIMP-1, -2, and -3 during mouse incisor development, from embryonic day 13 (E13) to postnatal day 3 (P3). TIMP-1 was present on the basement membrane during early developmental stages. At P2, TIMP-1 was strongly detected along the apical loop, transiently disappeared from the basement membrane in the cytodifferentiation zone, and later reappeared at the distal end of functional ameloblasts. Expression of TIMP-2 protein was restricted to the outer part of the apical loop throughout the examined stages. At P2, TIMP-2 was present on the basement membrane at the outer part of the apical loop. The dental follicle also expressed Timp-2, and the corresponding protein was abundant within the extracellular matrix. Timp-3 mRNA was highly expressed in the mesenchyme surrounding the apical loop. During matrix formation, Timp-3 was expressed by subodontoblasts, and the protein was detected in this layer and between odontoblasts. Distinct temporal and spatial expression patterns of TIMPs suggest divergent functions of these factors in incisor organogenesis.

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.

Immunohistochemical Study of Osteodentin in the Unerupted Rat Incisor

To characterize osteodentin in the pre-functional rat incisor, we performed histological and histochemical evaluation of the anterior apex of the incisor in 3-day-old rats. The unerupted incisor was composed of osteodentin, with numerous cells present in the anterior apex. The osteodentin was immunopositive for osteocalcin, bone sialoprotein, and dentin sialoprotein, with an immunolocalization pattern similar to that of dentin. Back-scattered electron microanalysis (BSE) and electron probe microanalysis (EPMA) showed that osteodentin was not uniformly calcified. These results indicate that osteodentin in the rat incisor possesses dentin-like characteristics, and may be fragile in structure.