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This study aimed to investigate the effects of phosphate concentration on osteoblastic differentiation in the murine preosteoblastic cell line MC3T3-E1 within 3D collagen gel culture system. To examine the impact of phosphate concentrations on cellular behavior, we constructed the 3D collagen gel culture system. The 3D culture system was used to observe morphological changes and to detect the expression of osteogenic markers (Runx2 and Dmp1). High (50 mM) concentrations of inorganic phosphate (Pi), a phosphate donor, reduce the cellular matrix production and proliferation compared with the control concentration (10 mM) during the experimental period. Immunohistochemical analysis revealed that Runx2 expression at high phosphate concentration was increased compared with the control concentration, while after 2 weeks, Runx2 expression at high phosphate concentration was decreased compared with the control concentration. Dmp1 expression was increased in high phosphate concentration from 1 week throughout the experimental period compared with the control concentration. Our findings suggest that phosphate concentration plays a crucial role in modulating osteoblastic differentiation in 3D cell culture. A High phosphate concentration appears to accelerate Runx2 expression at initial stage, and subsequently suppress it, while maintaining sustained Dmp1 expression. Our results suggests that the influence of phosphate on osteoblastic differentiation within 3D cell culture may contribute to elucidation of the mechanisms underlying bone formation and mineralization related to skeletal development and bone disease pathogenesis. [ABSTRACT FROM AUTHOR]

Adenosquamous carcinoma (ASC) is a rare variant of squamous cell carcinoma (SCC). It is thought to be more aggressive with poorer prognosis than conventional SCCs. Therefore, accurate preoperative diagnosis is crucial for adequate treatment strategy. In this report, we will present a case of ASC arising in left mandibular gingiva of 76-year-old male. Histological examination revealed both SCC component and adenocarcinoma (AdC) component. Also, transition from AdC to short spindle-shaped and pleomorphic cells, which is known as sarcomatoid change, was observed. Immunohistochemical study revealed that SCC component was positive for p40 and negative for Cytokeratin 7 (CK7), whereas, AdC component was negative for p40 and positive for CK7. In area with sarcomatoid change, immunohistochemical analysis revealed that negative expression of both CK7 and E-cadherin but positive expression of Vimentin and nuclear Snail/Slug, indicating the possible role of epithelial-mesenchymal transition (EMT) in AdC to sarcomatoid transition. [ABSTRACT FROM AUTHOR]

Diagnostic methods of oral squamous cell carcinoma (SCC) using artificial intelligence (AI) and digital-histopathologic images have been developed. However, previous AI training methods have focused on the cellular atypia given by the training of high-magnification images, and little attention has been paid to structural atypia provided by low-power wide fields. Since oral SCC has histopathologic types with bland cytology, both cellular atypia and structural atypia must be considered as histopathologic features. This study aimed to investigate AI ability to judge oral SCC in a novel training method considering cellular and structural atypia and their suitability. We examined digitized histological whole-slide images from 90 randomly selected patients with tongue SCC who attended a dental hospital. Image patches of 1000 × 1000 pixels were cut from whole-slide images at 0.3125-, 1.25-, 5-, and 20-fold magnification, and 90,059 image patches were used for training and evaluation. These image patches were resized into 224 × 224, 384 × 384, 512 × 512, and 768 × 768 pixels, and the differences in input size were analyzed. EfficientNet B0 was utilized as the convolutional neural network model. Gradient-weighted class activation mapping (Grad-CAM) was used to elucidate its validity. The proposed method achieved a peak accuracy of 99.65% with an input size of 512 × 512 pixels. Grad-CAM suggested that AI focused on both cellular and structural atypia of SCC, and tended to focus on the region surrounding the basal layer. Training AI regarding both cellular and structural atypia using various magnification images simultaneously may be suitable for the diagnosis of oral SCC. [ABSTRACT FROM AUTHOR]

Using a study on mice, the roles of transcription factors, Runx2 and Cbfbeta, in embryonic skeletal development are analyzed.

We report a case of a fibrous odontogenic lesion occurring in the maxilla of a man in his 60s. The patient presented with an asymptomatic radiolucent area related to the upper left premolar on radiography. Histological examination of the lesion obtained through an incisional biopsy revealed dense fibrous connective tissue containing small, rounded islands of odontogenic epithelium. The differential diagnoses included central odontogenic fibroma and sclerosing odontogenic carcinoma. Despite being completely different entities, the histological similarities between the two tumors raise diagnostic challenges, with fibrous connective tissue and odontogenic epithelium being the common features. Owing to the finding of unclear small or thin epithelial nests with a high nuclear-cytoplasmic ratio and despite its small size, the lesion was suspected of being malignant, and a maxillary resection was performed. Sections from the resected specimen displayed a small or tiny odontogenic epithelium with cellular atypia and invasion by epithelial cells in and around the nerve fasciculi; therefore, a final diagnosis of sclerosing odontogenic carcinoma was made. Nonetheless, the presence of an odontogenic epithelium in or around the nerve fascicles is not always an evidence of malignancy in the jaw. Caution should be exercised when differentiating invasive epithelia from epithelial residues to provide appropriate treatment. [ABSTRACT FROM AUTHOR]

The main purpose of cytological examination in the oral region is to screen for squamous cell carcinoma or intraepithelial neoplasms; thus, the background tends to be considered a deterrent for microscopy. From this perspective, liquid-based cytology (LBC) is favorable for preparing clear samples with few backgrounds. However, background hemocytes are sometimes of critical importance in the diagnosis. We report two cases of oral malignant lymphoma, plasmablastic lymphoma, and anaplastic large cell lymphoma in which careful observation of the background in scraping LBC sample contributed to the early diagnosis. Atypical lymphoid cells were observed only in a very small part of the LBC samples from the presented patients; however, cytological findings, such as large lymphoid cells with outstanding nucleoli, large mitotic cells, or intermediate-to-large lymphoid cells with pleomorphic nuclei were sufficient for obtaining a cytological diagnosis of malignant lymphoma. Although the number and cell size of leukocytes in LBC with Papanicolaou staining were significantly different from those in air-dried conventional smears with Romanovsky staining, which are commonly preferred for the discrimination of hemocytes, the corresponding cytological features could be observed. Therefore, attention should be paid to the background as well as squamous epithelium to prepare for such unexpected cases. The LBC examination with Papanicolaou staining alone can suggest the possibility of malignant lymphoma. • Case presentation of oral malignant lymphoma in scraping liquid-based cytology (LBC) • Attention should be paid to the background as well as squamous epithelium. • Cytological features of malignant lymphoma could be observed in LBC. [ABSTRACT FROM AUTHOR]

Leiomyosarcoma of the oral cavity is a very rare tumor type associated with aggressive clinical behavior and a low survival rate. However, this tumor may also be encountered as a slow-growing, non-ulcerated, painless mass in its early stage. We report a case of leiomyosarcoma, affecting the gingival mucosa of a 24-year-old male. The tumor mimicked an epulis, because it was encountered as a slow-growing, discreet, firm, non-ulcerated, painless mass without any evidence of resorption of the maxillary bone. Excision of the "epulis" resulted in a histological diagnosis of leiomyosarcoma. The histological findings of a wider excision did not indicate the presence of any tumor cells. The patient has since remained tumor-free for 3 years. A literature review, related to the gingiva of the maxilla or mandible, indicated only a few cases wherein the tumor behavior was similarly mild and benign as it was in this case. Thus, when the tumor is diagnosed at an early stage, while it is small and non-aggressive, successful treatment can lead to a good prognosis. [ABSTRACT FROM AUTHOR]

Multiple endocrine neoplasia (MEN) type 2B is the rarest and most aggressive form of MEN syndrome. MEN 2B patients manifest characteristic oral and facial features besides the neural crest cell–derived tumors, including medullary carcinoma, pheochromocytoma, mucosal neuroma, and ganglioneuromatosis of the gut. We report a case of MEN 2B diagnosed on the basis of the warning signs of mucosal neuroma and multiple neural hyperplasias in the maxillary bone resected during orthognathic surgery. A subsequent systemic examination under the pathologic diagnosis of neural lesions revealed medullary thyroid carcinoma, megacolon, thickened corneal nerves, and RET gene mutation, thus verifying the diagnosis of MEN 2B. An immunohistochemical study revealed an increased number of unmyelinated Schwann cells in the hyperplastic nerves. We suggest that intraosseous neural hyperplasia is a specific finding of the MEN 2B syndrome in addition to the known oral and facial manifestations. [ABSTRACT FROM AUTHOR]

Ameloblastin (AMBN) is an enamel sheath protein that presumably has a role in determining the prismatic structure of growing enamel crystals. To investigate the relationship between the molecular evolution of the AMBN gene and development of enamel prismatic structures, it is considered to be of great significance in the identification of homologues of the AMBN genes in nonmammals whose teeth lack an enamel prismatic structure. Several clones containing AMBN cDNA were isolated from an African clawed toad tooth cDNA library by screening with a polymerase chain reaction (PCR) method. Sequence analysis of the clones revealed that they were derived from different genes (toad-A and toad-B), which were found to contain ORFs encoding 408- and 352-amino-acid proteins, respectively. The N-terminal part of the toad AMBN proteins and the phosphorylation motif for casein kinase II, as well as several features, were found to be highly conserved throughout the evolution of tetrapods. Exon–intron boundaries were shared by toad and caiman genes with the exception of exons 6, 7 and 10 while human and caiman genes shared them exclusive of exons 8 and 9 which have been found only in the human. As for exon 7, it was absent in both toad genes. Moreover, the AMBN genes were transcribed only in the upper jaw, presumably in teeth. These results may provide useful information for investigation of the evolution of enamel. [Copyright &y& Elsevier]

Ameloblastin (AMBN) is one of the enamel sheath proteins which presumably has a role in determining the prismatic structure of growing enamel crystals. There may therefore be a relationship between the molecular evolution of the AMBN gene and the development of enamel prismatic structures. To investigate whether such a relationship exists, it was necessary to identify the homologues of the AMBN gene in a reptile whose teeth lack an enamel prismatic structure. To this end, several clones containing AMBN cDNA were isolated from caiman jaws using the reverse transcription–polymerase chain reaction (RT–PCR) method. Sequence analysis of the AMBN cDNA revealed an open reading frame of 1221 bp encoding a 407-amino-acid protein. Translation of the caiman cDNA starts at the methionine corresponding to the second of two putative start codons conserved in mammalian AMBN genes. The N-terminal part of the caiman AMBN shows high amino acid sequence similarities to human, pig, cattle, rat and mouse AMBN sequences, as well as several other features that have been conserved throughout the evolution of reptiles and mammals. Unexpectedly, the nucleotide sequences of the 3′ untranslated region (UTR) are also conserved, not only within mammalian genes but also between reptilian and mammalian genes. The caiman AMBN gene is a single-copy gene, transcribed only in the jaws, presumably in teeth. [Copyright &y& Elsevier]

Abstract: Dentin matrix protein 1 (DMP1), a noncollagenous bone matrix protein produced by osteocytes, regulates matrix mineralization and phosphate homeostasis. The lack of a precise assay for circulating DMP1 levels impairs further investigation of the protein's biological significance. Because full-length precursor DMP1 is cleaved into NH2- and COOH-terminal fragments during the secretory process, we developed two new sandwich ELISAs for the NH2- and COOH-terminal fragments of rat DMP1. One of these ELISAs, ELISA 1–2, is based on two affinity-purified polyclonal antibodies against the DMP1-1 and DMP1-2 peptides of the NH2-terminal fragment, whereas the other, ELISA 4–3, is based on two affinity-purified polyclonal antibodies against the DMP1-3 and DMP1-4 peptides of the COOH-terminal fragment. The polyclonal antibodies were characterized in immunohistochemical and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) studies. Immunohistochemical analyses of rat bone using these polyclonal antibodies revealed DMP1 immunoreactivity in osteocytes and pericanalicular matrix, consistent with the previously reported osteocyte-specific expression of DMP1. LC–MS/MS analyses of rat plasma-derived immunoreactive products affinity-extracted with these antibodies revealed the presence of DMP1 in circulating blood. The ELISAs established with these antibodies met accepted standards for reproducibility, repeatability, precision, and accuracy. Circulating DMP1 and levels of other biochemical markers (osteocalcin, Trap5b, Dkk-1, and SOST) were measured in 2-, 4-, 8-, 12-, 18-, 24-, 72-, and 96-week-old Wistar male rats. Circulating DMP1 levels determined by ELISAs 1–2 and 4–3 significantly decreased with age. During rapid skeletal growth (2–12weeks), DMP1 levels measured by ELISA 4–3 were over three times higher than those measured by ELISA 1–2; however, DMP1 levels in old animals (72 and 96weeks) were almost the same when measured by either ELISA. DMP1 levels determined by both ELISAs were most highly positively correlated with the level of Dkk-1, second most highly correlated with the level of osteocalcin, and less highly correlated with the levels of Trap5b and SOST. These novel sandwich ELISAs for rat DMP1 are highly specific and allow precise measurements of circulating DMP1, which may be a new biochemical marker for osteocyte-mediated bone turnover. [Copyright &y& Elsevier]

Abstract: In an attempt to identify the histological properties of the klotho-deficient (kl/kl) bone matrix, bone mineralization and the localization of Ca2+-binding bone matrix proteins – osteocalcin, dentin matrix protein-1 (DMP-1) and matrix Gla protein (MGP) – were examined in kl/kl tibiae. While a widespread osteocalcin staining could be verified in the wild-type bone matrix, localization of the same protein in the kl/kl tibiae seemed rather restricted to osteocytes with only a faint staining of the whole bone matrix. In wild-type mice, MGP immunoreactivity was present at the junction between the epiphyseal bone and cartilage, and at the insertion of the cruciate ligaments. In kl/kl mice, however, MGP was seen around the cartilaginous cores of the metaphyseal trabeculae and in the periphery of some cells of the bone surface. DMP-1 was identified in the osteocytic canalicular system of wild-type tibiae, but in the kl/kl tibiae this protein was mostly found in the osteocytic lacunae and in the periphery of some cells of the bone surface. Mineralization of the kl/kl bone seemed somewhat defective, with broad unmineralized areas within its matrix. In these areas, mineralized osteocytes along with their lacunae and osteocytic cytoplasmic processes were found to have intense osteocalcin and DMP-1 staining. Taken together, it might be that the excessive production of Ca2+-binding molecules such as osteocalcin and DMP-1 by osteocytes concentrates mineralization around such cells, disturbing the completeness of mineralization in the kl/kl bone matrix. [Copyright &y& Elsevier]

Abstract: Adipose tissue-derived adipokines play important roles as regulators of skeletal growth and development. CTRP1, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the CTRP1 in chondrocyte proliferation and maturation using a mouse chondrocytic cell line, N1511. The CTRP1 protein was strongly expressed and predominantly distributed in the reserve and proliferative chondrocytes in the fetal growth plate and its mRNA decreased during the maturation of N1511 chondrocytes. Recombinant CTRP1 promoted proliferation of immature proliferating N1511 chondrocytes in a dose-dependent manner, whereas it inhibited maturation of maturing N1511 chondrocytes. The stimulatory effect of CTRP1 on chondrocyte proliferation was associated with activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway. On the other hand, the inhibitory effect of CTRP1 on chondrocyte maturation is associated with suppression of the ERK1/2 pathway. These results suggest a novel physiological role for CTRP1 in endochondral ossification. [Copyright &y& Elsevier]

Abstract: A case of multifocal nodular oncocytic hyperplasia (MNOH) of the bilateral parotid gland is presented. An 80-year-old woman was admitted to hospital because of painless swellings in bilateral parotid regions. Histologically, the nodular lesion had incomplete capsules and engulfed the surrounding parotid gland at the periphery. The lesions were mostly composed of clear cells, while the peripheries of the lesions had typical oncocytic cells with abundant fine granules. The histological existence of the clear cell component in the lesions led to misdiagnoses of other clear cell neoplasms. However, this case had multiple nodules in bilateral glands. No evidence of malignant histological findings was found. Moreover, the clear cells, as well as the oncocytic cells, were demonstrated to have mitochondria and glycogen in their cytoplasm using special staining. Based on these findings, the diagnosis of this case was MNOH in the parotid gland. We also discuss the differential diagnosis for clear cell lesions. [Copyright &y& Elsevier]

Abstract: Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods. [Copyright &y& Elsevier]

Periodontal ligament-associated protein-1 (PLAP-1)/asporin is a recently identified novel member of the small leucine-rich repeat proteoglycan family. PLAP-1/asporin is involved in chondrogenesis, and its involvement in the pathogenesis of osteoarthritis has been suggested. We report that PLAP-1/asporin is also expressed specifically and predominantly in the periodontal ligament (PDL) and that it negatively regulates the mineralization of PDL cells. In situ hybridization analysis revealed that PLAP-1/asporin was expressed specifically not only in the PDL of an erupted tooth but also in the dental follicle, which is the progenitor tissue of the PDL during tooth development. Overexpression of PLAP-1/asporin in mouse PDL-derived clone cells interfered with both naturally and bone morphogenetic protein 2 (BMP-2)-induced mineralization of the PDL cells. On the other hand, knockdown of PLAP-1/asporin transcript levels by RNA interference enhanced BMP-2-induced differentiation of PDL cells. Furthermore co-immuno-precipitation assays showed a direct interaction between PLAP-1/asporin and BMP-2 in vitro, and immunohistochemistry staining revealed the co-localization of PLAP-1/asporin and BMP-2 at the cellular level. These results suggest that PLAP-1/ asporin plays a specific role(s) in the periodontal ligament as a negative regulator of cytodifferentiation and mineralization probably by regulating BMP-2 activity to prevent the periodontal ligament from developing non-physiological mineralization such as ankylosis. [ABSTRACT FROM AUTHOR]

Abstract: Matrix metalloproteinase-20 (MMP20; enamelysin) is important for proteolytic processing of extracellular matrix (ECM) proteins during the formation of enamel and plays a critical role in proteolytic processing of amelogenin (AMEL), the most abundant enamel ECM protein. MMP20 might have played a role in the emergence of teeth, because jawless vertebrates with primordial teeth on their external skeletons may have possessed the MMP20 gene, and MMP20 and enamel ECM proteins are thought to have evolved together in a special relationship over time. Thus, an understanding of the molecular evolution of the MMP20 gene is important for elucidating the evolution of enamel and it is necessary to identify the orthologs of the MMP20 gene in non-mammals, as it has been identified in mammals. In the present study, orthologs of the MMP20 genes from a reptile (caiman) and an amphibian (African clawed toad) were cloned and characterized. Comparisons of the orthologs revealed that the MMP20 proteins were highly conserved throughout the evolution of tetrapods. Further, the caiman, toad, and mammalian MMP20 shared several unique features specific for MMP20, but not for other matrix metalloproteinases. In addition, the toad MMP20 gene was transcribed only in the upper jaw, presumably in teeth. These results suggest that MMP20 in a common ancestor of tetrapods might have been recruited for the processing of AMEL and conserved over 350 million years of evolution. [Copyright &y& Elsevier]

Fam20C, which phosphorylates many secretory proteins with S-x-E/pS motifs, is highly expressed in bone and tooth tissues, implying that Fam20C-mediated phosphorylation is critical for regulation of these mineralized tissues. Previous studies of Fam20C-deficient mice revealed that Fam20C plays important roles in bone formation and mineralization. However, Fam20C-deficient mice develop hypophosphatemia, a systemic factor that masks the local effect of Fam20C in the bone tissue; consequently, the local role of Fam20C remains unknown. To elucidate the local function of Fam20C in bone tissue, we studied osteoblast-specific Fam20C transgenic (Fam20C-Tg) mice, which have no alteration in serum calcium and phosphate levels. Fam20C-Tg mice had more highly phosphorylated proteins in bone tissue than wild-type mice. In cortical bone of Fam20C-Tg mice, bone volume, mineralization surface (MS/BS), and mineral apposition rate (MAR) were elevated; in addition, the transgenic mice had an elevated number of vascular canals, resulting in an increased cortical porosity. Osteocyte number was elevated in the transgenics, but osteoblast number was unchanged. The microstructure of bone matrix characterized by the preferential orientation of collagen and apatite, was degraded and thus the mechanical function of bone material was deteriorated. In trabecular bone of Fam20C-Tg mice, bone volume was reduced, whereas MS/BS and MAR were unchanged. Osteoclast number was elevated and eroded surface area was non-significantly elevated with an increased serum CTX-I level, whereas osteoblast number was unchanged. These findings indicated that Fam20C overexpression in osteoblasts promotes cortical bone formation by increasing MS/BS and MAR and promoting osteocyte differentiation, but does not affect trabecular bone formation. Furthermore, Fam20C overexpression indirectly promotes osteoclastic bone resorption in cortical and trabecular bones. Our findings show that osteoblastic Fam20C-mediated phosphorylation in bone tissue regulates bone formation and resorption, and bone material quality. • Osteoblast-specific Fam20C transgenic (Tg) mice have no alteration in serum Ca and P. • Tg mice have more highly-phosphorylated proteins in bone than wild-type mice. • Osteoblastic Fam20C promotes cortical bone formation by an enhanced mineralization. • Osteoblastic Fam20C induces transition from osteoblast into osteocyte. • Osteoblastic Fam20C indirectly promotes osteoclastic bone resorption. [ABSTRACT FROM AUTHOR]