Sjögren’s syndrome and therapeutic radiation for head and neck cancers result in atrophy of the salivary glands and consequent xerostomia (dry mouth). To clarify the mechanisms of salivary gland dysfunction, we have established a system for the primary culture of parotid acinar cells. We found that the expression patterns of claudins are remarkably changed during culture. These changes are considered a process of dedifferentiation since the expression levels of other differentiation markers also alter. Acinar markers, such as amylase and aquaporin-5, are decreased and, in contrast, duct markers such as claudin-4 and cytokeratin-14 are increased. In the late phase of culture, mesenchymal markers begin to be expressed. These results suggest that acinar cells transiently change to duct-like cells during epithelial mesenchymal transition. Inhibitors of Src and p38 MAP kinases suppress these changes and simultaneously increase the expression of acinar marker proteins. Activation of p38 MAP kinase occurs during cell isolation from the parotid glands, which is suppressed by Src kinase inhibitors. Therefore, cellular stresses caused by cell isolation trigger the signal that induces the transition to duct-like cells and dedifferentiation. There is a possibility that salivary acinar cells have differentiation plasticity, which is regulated by the Src-p38 MAP kinase signaling pathway.

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Regeneration of periodontal tissues, lost as a result of periodontal disease, is a key objective of periodontal treatment. Although several periodontal regeneration therapies have been devised, the origin of the undifferentiated cells that regenerate periodontal tissues remains unknown. Therefore, in the present study, to clarify the existence of osteoblast progenitor cells in the periodontal ligament, as well as to investigate the mechanism of alveolar bone regeneration without any effects from the original bone, we evaluated osteoblast differentiation induced by transplantation of GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Ten days after transplantation, initial alveolar bone was formed apart from the cementum in the bifurcation region. After 20 days, this bone tissue had expanded to almost all of the bifurcation. GFP localization showed that the osteoblasts were derived from the transplant. Alpha-SMA- and BMP4-positive cells were observed near the root surface at 5 days after transplantation. With the progress of alveolar bone regeneration, osteoblasts expressing Runx2 and Osterix appeared in the bone-forming region. These results indicate that periodontal ligament tissue remaining on the root surface after a tooth extraction contains undifferentiated cells that have the ability to regenerate alveolar bone. The process of osteoblast differentiation in this model might be similar to that for normal alveolar bone formation. Thus, periodontal ligament cells might be useful for the regeneration of alveolar bone in tissue engineering applications.

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The submandibular gland (SMG), like teeth and other glandular tissues, develops under epithelial-mesenchymal interaction. Its process is regulated by various signaling molecules, including growth/differentiation factors, which are synthesized as inactive precursors and activated via limited proteolysis. At their processing site, multi-basic amino acid sequences, such as Arg-X-Lys/Arg/X-Arg, commonly exist. It is revealed that subtilisin-like proprotein convertases (SPCs) catalyze such processing; however, little is known about the role of SPCs in SMG development. This study therefore focused on the physiological role of an SPC member, PACE4 (SPC4), in SMG development. In the organ culture system of rat embryonic SMG rudiments, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK), a potent inhibitor of SPCs, inhibited the branching morphogenesis (BM) of SMG and the expression of AQP5, an exocrine-type water channel. In contrast, other inhibitors of trypsin-like serine proteases, including leupeptin and other peptidyl-CMKs, affected neither BM nor AQP5 expression. Dec-RVKR-CMK also suppressed the expression of PACE4, but not another SPC, furin. Additionally, the PACE4-specific antibody suppressed BM and AQP5 expression similarly. A rescue experiment to find the key molecule, which was recovered from Dec-RVKR-CMK-induced suppression of BM and AQP5 expression, revealed that signaling by BMP2, whose precursor is a candidate physiological substrate of PACE4, is involved in the BM and AQP5 expression. Further, the transcriptional silencing of PACE4 by PACE4-specific siRNAs caused the suppression of BM and AQP5 expression in the present organ culture system. These observations strongly suggest that PACE4 plays an important role in SMG development.

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Mineralization of bone matrix starts with the formation of matrix vesicles secreted by osteoblasts, which subsequently grow into mineralized nodules. These nodules are globular structures formed by mineral crystals assembled in a radial fashion, and appear to be the final inducers of collagen mineralization. Despite its apparent simplicity, the course of bone mineralization is tightly controlled by many factors in the microenvironment. In this review, we will present our recent experiments on the general processes of bone mineralization. First, we will discuss the ultrastructural alteration of mineral crystals in the context of magnesium (Mg) insufficiency, which causes an elevation of calcium (Ca) concentrations in bone and of the pure form of hydroxyapatite. Such elevated Ca concentrations also induced premature collagen mineralization without the mediation of mineralized nodules. This could mean that the availability of elements, such as Mg, in the bone microenvironment is important for the in vivo generation of the pure form of hydroxyapatite. Next, we will examine collagen mineralization in the context of ascorbic acid insufficiency. Ascorbic acid is essential for collagen formation and, when it is not available, the typical, stout collagen microfibrils with striations cannot be identified. In their place, filamentous fibrils of collagen without striations, i. e., immature collagen microfibrils, are observed. In the osteoid, however, many mineralized nodules feature some sort of “mineral extensions” towards the immature microfibrils. It seems that collagen microfibrils, even if immature, serve as a scaffold for the mineralization of collagen fibrils. These findings reaffirm that a sophisticated set of regulatory steps is necessary for proper mineralization in bone.

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Kampo medicines are clinically used for the treatment of various diseases, and their use for oral diseases is desired; however, only a few kampo medicines are adaptable for diseases of the oral region. Against this background, we carried out an investigation into the application of preexisting kampo medicines to diseases of the oral region. Here, we indicate the effects of kampo medicines on periodontal disease, drug-induced gingival overgrowth, and xerostomia using in vitro or animal models. (i) Shosaikoto has an anti-inflammatory effect and is used for the treatment of pneumonia and bronchitis. Because shosaikoto decreases lipopolysaccharide (LPS)-induced prostaglandin E₂ production by gingival fibroblasts, it may be effective to improve inflammation in periodontal tissue. (ii) Saireito has an inhibitory effect on cell proliferation and is used for the treatment of idiopathic retroperitoneal fibrosis. Because saireito suppresses nifedipine-induced gingival fibroblast proliferation, it may be effective for the treatment of drug-induced gingival overgrowth. (iii) Byakkokaninjinto and goreisan are used for the treatment of xerostomia. Because diabetes is accompanied by xerostomia, the effects of these two medicines on salivary secretion have been examined using streptozotocin-induced diabetic mice. Both medicines recover the blood glucose level and secretory rate of saliva to almost normal levels. These results suggest that kampo medicines may be adaptable for periodontal disease, drug-induced gingival overgrowth, and diabetes-mediated xerostomia.

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The resorption of alveolar bone in periodontitis and/or following tooth extraction results in a significant problem in an individual’s oral health-related quality of life. Strategies employing growth factor proteins and/or stem cell-based therapy have been successfully used to regenerate alveolar bone augmentation/regeneration; however, cost-effectiveness issues illustrate the need for developing alternative strategies. One promising approach is the development of conventional chemical and biological therapeutics to strongly stimulate endogenous cells to regenerate. Small-molecule compounds can have a variety of biological functions, serving as cell signaling molecules, as tools in molecular biology, and as drugs in medicine. Therefore, the discovery of natural and novel small-molecule synthetic regulators of differentiation and maturation of osteoblasts and/or osteoclasts would accelerate the development of bone regenerative medicine. Recently, “chemical biology” has started to play an increasingly important role in both drug discovery and elucidating the mechanism of biological phenomena. This review first addresses the current needs and strategies for alveolar bone regeneration, followed by the potential small molecules associated with bone regeneration, with a concluding section on chemical biology-based high throughput screening approaches for identifying small molecules targeting osteoblast/osteoclast differentiation.

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Non-steroidal anti-inflammatory drugs (NSAIDs) act against not only inflammation but also down-regulation of bone remodeling. The inhibitory mechanisms of their action on bone remodeling are still unclear. We hypothesized that an NSAID, diclofenac, down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFκB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrate-resistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner, leading to the abolition of osteoclastic bone resorption. Levels of cathepsin K transcripts, an osteoclastic resorption marker, were down regulated. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFκB and phosphorylated NFκB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFκB reduction, which regulates osteoclast differentiation and activation.

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Meckel’s cartilage, discovered by German anatomist J. F. Meckel (1781-1833), is hyaline cartilage formed in the mandibular process of the first branchial arch of vertebrate embryos. An intermediate portion of Meckel’s cartilage is absorbed by multinucleated cells and disappears during the following developmental stages in mammals. The process of Meckel’s cartilage disappearance is not accompanied by the apoptosis of chondrocytes. The most posterior portion of the cartilage is changed to auditory ossicles by endochondral ossification, and the rest of the posterior portion transdifferentiates into sphenomandibular ligament. Meckel’s cartilage is not considered an anlage of the mandible, since bony tissue of the mandible is independently formed by membranous ossification. Chondrocytes are differentiated from mesodermal cells in general, whereas cells forming Meckel’s cartilage are differentiated from ectodermal mesenchymal cells of neural crest origin. In reptiles, Meckel’s cartilage ossifies and forms bone in the lower jaws, and cretaceous mammals had calcified Meckel’s cartilage in addition to a mandible. During the evolution from reptile to mammal, the mode of mastication changed, and the origin of bone in the lower jaw may have been gradually transferred from Meckel’s cartilage to the mandible.

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Meckel’s cartilage (MC), which develops as a template of the first skeleton of the mandible, undergoes site-specific fates. The anterior portion contributes to the formation of the rostral part of the mandible by endochondral ossification, while the posterior portion is involved in the formation of the ossicle bones. In contrast, the posterior part of the midportion (PM portion; located in the soft tissue) forms the sphenomandibular ligament, but its formation mechanism remains unclear. We found that chondrocytes in the PM portion have the potential to transform into other phenotypic cells under in vivo and in vitro conditions. In the present review, we focus on the results of cellular transformation based on our investigations. It has been clarified that MC chondrocytes in the PM portion transform into fibroblastic cells by phenotypic switching of the chondrocytes themselves in response to epidermal growth factor. In contrast to their conversion to fibroblastic cells in vivo, MC chondrocytes subjected to long-term culture or transplanted into ectopic sites, such as the spleen, expressed bone-type proteins accompanied by transformation into osteocytic cells. Since phenotypic transformation did not occur in mesoderm-derived costal chondrocytes, the findings suggest that neural crest-derived MC chondrocytes have high potential for transformation in response to altered stimuli.

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Meckel’s cartilage is a temporary supporting tissue that forms during the embryonic period. Unlike the distal (anterior), posterior, and proximal portions, the middle portion degenerates with the death of chondrocytes as well as resorption of the cartilaginous matrix by chondroclasts without giving rise to ossification. Perichondral cells initially differentiate into osteoblasts and subsequently form periosteal bone on the lateral surface of Meckel’s cartilage closest to the incisor teeth, and then chondrocytes become hypertrophied in the restricted position of Meckel’s cartilage. Thereafter, the calcified periosteal bone and cartilage are resorbed by osteoclasts and chondroclasts; however, little is known regarding the mechanisms by which the middle portion is disintegrated during the embryonic period. Based on immunohistochemical and in situ zymographic findings in mice, we discuss the role of matrix metalloproteinases in the disintegration of the middle portion of Meckel’s cartilage.

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Meckel’s cartilage is a supporting tissue in the embryonic mandible that disappears during development. The fate of mammalian Meckel’s cartilage differ according to three (proximal, middle and distal) regions. The middle region of the cartilage forms fibrous tissue and we focused on the middle region of Meckel’s cartilage located within the mandible. During development, macrophages penetrate the perichondium at 16 days of gestation (E16), and then chondrocytes start to express interleukin-1β and matrix metalloproteinase-9, degrade glycoconjugates in the matrix, synthesize type I collagen, and decrease the expression of Type II collagen. Chondrocytes constitutively express monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α) and interleukin-1 receptors. Organ culture study also shows the disappearance of Meckel’s cartilage at E17 but not E14. These results indicate the dynamic matrix changes of Meckel’s cartilage during development and suggest that the functional changes of chondrocytes for disappearance might be induced by interleukin-1β secreted by penetrating macrophages.

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A set of Hox genes expressed during the embryonic process presents a crucial system to set up the body plan in animal phyla. Current systematic approaches, such as bio-imaging, epigenetic regulation and phylogenetic genome comparison, illuminated new comprehensive insights into Hox genes as an evolutionary constraint of the vertebrate body plan, which provides a novel counter-view to give further insights into the evolution and development of the craniofacial and dental systems as non-Hox systems.

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Calretinin is a 29 kDa calcium-binding protein, which is widely expressed in the central and peripheral neural tissue. It has also been demonstrated in odontogenic epithelium during odontogenesis and in neoplastic odontogenic tissues. The lining epithelium of eight cases of unicystic ameloblastoma, six cases of dentigerous cyst, six cases of odontogenic keratocyst, reclassified as keratocystic odontogenic tumor (KCOT), and four cases of solid/multicystic ameloblastoma was examined for the expression of calretinin. No positive staining was observed in any of the dentigerous cysts and keratocystic odontogenic tumor linings. In comparison, coarse dark brown staining was seen in the stellate reticulum of solid multicystic ameloblastoma and more superficial epithelial layers of unicystic ameloblastoma. In conclusion, we have highlighted calretinin to be a specific immunohistochemical marker for neoplastic ameloblastic tissue that can be used as an important diagnostic aid in the differential diagnosis of unicystic ameloblastoma and cystic odontogenic lesions.

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Recently, magnesium (Mg) deficiency has been identified as a possible risk factor for osteoporosis. This study investigated the effect of Mg deficiency on the morphology of the periodontal ligament (PDL) and alveolar bone in rats. Wistar rats (8 weeks old) were fed either a normal control diet (n=24) or Mg-deficient diet (n=24) for 28 days. Fluorescent labels, tetracycline or calcein, were administrated intraperitoneally each week during the experimental period. Serum Mg, calcium (Ca), phosphorus (P) and alkaline phosphatase (ALP) were measured at the end of the experiment. The maxilla, including the first molar and the femurs, were collected to detect the fluorescent labels, for histological observation, histochemical localization of ALP, and X-ray microanalysis. In rats fed the Mg-deficient diet, serum Mg, P, and ALP were significantly decreased, but serum Ca was increased, while tetracycline and calcein labeling showed partly retarded new bone formation in femoral midshafts; however, new bone formation on the periosteal side of the alveolar crest area of the maxilla was not affected by Mg deficiency over the experimental period. Histological observations demonstrated a widening of the periodontal ligament space, disarray of the periodontal fibers, and an increase in fibroblasts with collagen-containing vesicles in their cytoplasm. Color mapping showed that Mg content was significantly decreased in bone formed in the alveolar crest area. In summary, Mg deficiency causes disarray of the periodontal fibers and influences collagen fiber metabolism in growing rats. While bone growth of the alveolar bone on the periosteal side was not affected by Mg deficiency over the experimental period, no new bone formation on the periodontal side was observed.

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one of the major concerns of denture wearers is adaptation to new or replacement dentures, regardless of their experience of wearing dentures. The purpose of this study was to investigate the changes in oral stereognosis ability (OSA) that occur with the insertion of a new denture in experienced and non-experienced complete denture wearers. Age and gender-matched 8 experienced and 8 non-experienced complete denture wearers were tested. OSA was assessed by measuring the accuracy of solid object size perception at 3 stages, namely, just before (pre-treatment), 30 min after (30 min post-treatment) and one month after (1 month post-treatment) the insertion of new dentures. Two-way ANOVA with post-hoc Tukey’s test was used to analyze the experience factor (experienced vs. non-experienced) and test interval (pre-treatment, 30 min post-treatment and 1 month post-treatment). OSA was influenced by the test interval (F_2,23=4.3, p=0.04). OSA was significantly increased 1 month post-treatment compared to 30 min post-treatment (Tukey’s test p<0.05). There was no significant difference in OSA between experienced and non-experienced groups (F_1,13=0.76, p=0.41). There was a significant interaction of experience by test interval (F_2,23=69.44, p<0.001). The findings suggested that there was a significant improvement in the OSA of both experienced and non-experienced denture wearers after wearing new dentures for 1 month, irrespective of the previous experience of denture wearing.

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Sterilized particles of anorganic bovine bone matrix (ABBM: Bio-Oss^®), widely used for sinus floor augmentation, are characterized by high bone conductivity and long persistence due to slow resorption. Here we report the histological and histochemical features of ABBM particles applied for bone augmentation in 5 adult patients with special reference to the mode of osteoclastic resorption of xenogenic material. Bone cores retrieved at 6 months after surgery contained densely packed ABBM particles fully or partially encapsulated by the newly induced bone and showed immunoreactivity for osteopontin (OPN) at the bone/ABBM boundary. Multinuclear cells showing enzymatic reactions for tartrate-resistant acid phosphatase (TRAP) were in contact with 48.26±9.43% of the ABBM surface but only 2.13±0.84% of that of induced bone. TRAP-positive multinuclear cells attached to the ABBM particles were enriched with mitochondria but, with a few exceptions, lacked a ruffled border and immunoreactivity for cathepsin K. These data indicate that a predominantly large proportion of TRAP-positive cells at the graft site are hypofunctional osteoclasts or osteoclastic cells at 6 months after surgery, and explain the long persistence of grafted ABBM particles despite the abundance of TRAP-positive multinuclear cells. Graft samples retrieved at 2 weeks from the extraction socket suggested the role of tiny ABBM fragments for the induction of TRAP-positive multinuclear cells in the early postoperative phase. Due to the limited availability of samples, how grafted xenogenic material is involved in bone remodeling in later time periods is yet to be determined.

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