Tenomodulin expression in the periodontal ligament enhances cellular adhesion.

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain

<i>Gli1</i> Haploinsufficiency Leads to Decreased Bone Mass with an Uncoupling of Bone Metabolism in Adult Mice

<div><p>Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of <i>Gli1</i>, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of <i>Gli1</i>^+/− adult mice revealed that <i>Gli1</i> haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of <i>Gli1</i>^+/− precursors, and the impairment was rescued by <i>Gli1</i> expression via adenoviral transduction. In addition, <i>Gli1</i>^+/− precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and <i>Gli1</i>^+/− adult mice, we found that the <i>Gli1</i>^+/− mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that <i>Gli1</i>, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.</p></div

Osteoblast differentiation in cultures of precursor cells from WT and <i>Gli1</i>^+/− mice.

<p>(<b>A</b>) mRNA expression of osteoblast marker genes in 14-day osteogenic cultures of BMSCs in the presence of the Smoothened agonist. mRNA expression was analyzed by real-time RT-PCR. (<b>B</b>) ALP and von Kossa stainings in 14-day osteogenic cultures of BMSCs in the presence of the Smoothened agonist. (<b>C</b>) mRNA expression of osteoblast marker genes in 7-day osteogenic cultures of osteoblast precursors (OPs) isolated from neonatal calvarias, in the presence of the Smoothened agonist. (<b>D</b>) ALP and von Kossa staining in 7-day osteogenic cultures of OPs in the presence of the Smoothened agonist. (<b>E</b>) Rescue of the expression levels of osteoblast marker genes in <i>Gli1</i>^+/− OPs by the adenoviral overexpression of <i>Gli1</i> in the presence of the Smoothened agonist. WT or <i>Gli1</i>^+/− OPs were infected with Ax-GFP (−) or Ax-Gli1 (+) at MOI 10.</p

Comparison of bone fracture healing between 8-week-old WT and <i>Gli1</i>^+/− mice.

<p>(<b>A</b>) Soft X-ray pictures of the fracture sites of all WT (n = 8) and <i>Gli1</i>^+/− (n = 8) male mice tested at 2 weeks after the fracture. (<b>B</b>) Representative micro-CT images of the callus in WT and <i>Gli1</i>^+/− male mice. Bar, 1 mm. (<b>C</b>) The areas of horizontal cross-sections at fracture lines (left) and the volume of the calluses (right) of WT and <i>Gli1</i>^+/− mice, calculated using 3D-micro-CT data. Data are means ± SDs of eight mice per genotype. *p<0.05 vs. WT. (<b>D</b>) H&E and alcian blue double staining of the calluses 2 weeks after the fracture. Bar, 200 µm.</p

Survival rate of <i>Gli1</i> mutant mice during postnatal 10 days.

<p>Percentages are in parentheses.</p><p>Survival rate of <i>Gli1</i> mutant mice during postnatal 10 days.</p

Radiological findings of long bones in wild-type (WT) and <i>Gli1</i>^+/− mice.

<p>(<b>A</b>) Three-dimensional micro-computed tomography (3D-micro-CT) images of the distal femurs of representative 8-week-old WT and <i>Gli1</i>^+/− male mice. Sagittal sections, transverse sections, and 3D reconstruction images of the primary spongiosa are shown for each genotype. Bar, 1 mm. (<b>B</b>) Histomorphometric analyses of 3D-micro-CT data. BMD, bone mineral density; BV/TV, bone volume per tissue volume; Tb.Th, trabecular thickness; Tb.N trabecular number parameters. Data are means ± SDs of eight male mice per genotype. *p<0.05 vs. WT.</p

Osteoclast differentiation in cultures of bone marrow cells from WT and <i>Gli1</i>^+/− mice.

<p>(<b>A</b>) Formation of TRAP-positive multinucleated osteoclasts by the co-culture of BMSCs and BMMΦ derived from WT or <i>Gli1</i>^+/− mice. (<b>B</b>) The numbers of osteoclasts expressed as means ± SDs of 4 wells per group in (A). *p<0.05 vs. the control group (WT BMSC × WT BMMΦ). (<b>C</b>) mRNA expression of <i>Rankl</i>, <i>Opg</i>, <i>Dmp1</i>, and <i>Sost</i> in 14-day osteogenic cultures of BMSCs. The mRNA expression was analyzed by real-time RT-PCR. <i>Rankl</i>, receptor activator of nuclear factor-κB ligand; <i>Opg</i>, osteoprotegerin; <i>Dmp1</i>, dentin matrix acidic phosphoprotein 1; <i>Sost</i>, sclerostin. *p<0.05 vs. WT. (<b>D</b>) Formation of TRAP-positive multinucleated osteoclasts in 5-day cultures of RAW cells in the presence or absence of sonic hedgehog (Shh) and cyclopamine (Cyc). (<b>E</b>) The numbers of osteoclasts in (D) expressed as means ± SDs of 4 wells per group. (<b>F</b>) mRNA expression of <i>Ptch1</i> in cultured RAW cells in the presence or absence of Shh and cyclopamine (Cyc). Data are means ± SDs of 4 wells per group. (<b>G</b>) mRNA expression of cathepsin K (<i>Ctsk</i>) and <i>NFATc1</i> in osteoclasts derived from WT or <i>Gli1</i>^+/− BMMΦ. Cells were cultured with recombinant M-CSF (10 ng/mL), RANKL (100 ng/mL), and Shh (25 nM). The mRNA expression was analyzed by real-time RT-PCR.</p

Histological findings of adult WT and <i>Gli1</i>^+/− mice.

<p>(<b>A</b>) von Kossa staining and toluidine blue staining of the distal femur sections of representative 8-week-old WT and <i>Gli1</i>^+/− male mice. Bar, 100 µm. (<b>B</b>) Histomorphometric analyses of bone volume and bone formation parameters in distal femurs from 8-week-old WT and <i>Gli1</i>^+/− male mice. OS/BS, osteoid surface per bone surface; MAR, mineral apposition rate; BFR, bone formation rate per bone surface; dLS/BS, double-labeled surface per bone surface; sLS/BS, single-labeled surface per bone surface. Data are means ± SDs of five male mice per genotype. *p<0.05 vs. WT. (<b>C</b>) TRAP staining of the distal femur sections of representative 8-week-old WT and <i>Gli1</i>^+/− male mice. Bar, 100 µm. (<b>D</b>) Histomorphometric analyses of bone resorption parameters in the distal femurs of 8-week-old WT and Gli1^+/− male mice. ES/BS, eroded surface per bone surface; N. Oc/B. Pm, number of osteoclasts per 100 mm of bone perimeter; Oc. S/BS, osteoclast surface per bone surface. In (<b>B</b>) and (<b>D</b>), data are means ± SDs of five mice per genotype. *p<0.05 vs. WT.</p

Primary syphilis with a tongue ulcer mimicking tongue cancer: a case report

The main symptom in primary syphilis is a small, painless, sore or ulcer called a chancre on the penis, vagina, or around the anus, although chancres can sometimes appear in the mouth or on the lips, fingers, or buttocks. We present the case of a man in his early 60 s with a chief complaint of a painful tongue ulcer. An ulcerated, indurated, and hemorrhagic lesion (23 × 14 mm) was found on the ventral tongue surface, near the oral floor. Palpation identified several swollen, mobile, elastic cervical lymph nodes, with no tenderness. We initially diagnosed tongue cancer; however, during a subsequent detailed examination for a malignant tumor, including biopsy and obtaining additional history, his disease was finally identified as primary syphilis with multiple swollen cervical lymph nodes. Oral amoxicillin and probenecid were started, and after 14 days, there was partial reduction in the size of the submandibular lymph nodes and the ulcer on the left tongue margin. The number of patients with syphilis in Japan increased by eight times from 2012 to 2018. We suggest that dentists consider primary syphilis as a differential diagnosis for oral refractory ulcer with induration and obtain a detailed patient history

Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration