Reconstruction of rabbit mandibular bone defects using carbonate apatite honeycomb blocks with an interconnected porous structure.

Carbonate apatite (CO3Ap) granules are useful as a bone substitute because they can be remodeled to new natural bone in a manner that conforms to the bone remodeling process. However, reconstructing large bone defects using CO3Ap granules is difficult because of their granular shape. Therefore, we fabricated CO3Ap honeycomb blocks (HCBs) with continuous unidirectional pores. We aimed to elucidate the tissue response and availability of CO3Ap HCBs in the reconstruction of rabbit mandibular bone defects after marginal mandibulectomy. The percentages of the remaining CO3Ap area and calcified bone area (newly formed bone) were estimated from the histological images. CO3Ap area was 49.1 ± 4.9%, 30.3 ± 3.5%, and 25.5 ± 8.8%, whereas newly formed bone area was 3.0 ± 0.6%, 24.3 ± 3.3%, and 34.7 ± 4.8% at 4, 8, and 12 weeks, respectively, after implantation. Thus, CO3Ap HCBs were gradually resorbed and replaced by new bone. The newly formed bone penetrated most of the pores in the CO3Ap HCBs at 12 weeks after implantation. By contrast, the granulation tissue scarcely invaded the CO3Ap HCBs. Some osteoclasts invaded the wall of CO3Ap HCBs, making resorption pits. Furthermore, many osteoblasts were found on the newly formed bone, indicating ongoing bone remodeling. Blood vessels were also formed inside most of the pores in the CO3Ap HCBs. These findings suggest that CO3Ap HCBs have good osteoconductivity and can be used for the reconstruction of large mandibular bone defects. The CO3Ap HCB were gradually resorbed and replaced by newly formed bone.

Expression and Role of IL-1ß Signaling in Chondrocytes Associated with Retinoid Signaling during Fracture Healing.

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1ß (IL-1ß) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1ß and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1ß in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1ß and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1ß decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1ß-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1ß treatment, and the IL-1ß-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1ß-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1ß via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process.

The Roles of Indian Hedgehog Signaling in TMJ Formation.

The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes. Uniquely, progenitor cells reside near the articular surface, proliferate, undergo chondrogenesis, and mature into hypertrophic chondrocytes. During the past decades, there has been a growing interest in the molecular mechanisms by which the TMJ develops and acquires its unique structural and functional features. Indian hedgehog (Ihh), which regulates skeletal development including synovial joint formation, also plays pivotal roles in TMJ development and postnatal maintenance. This review provides a description of the many important recent advances in Hedgehog (Hh) signaling in TMJ biology. These include studies that used conventional approaches and those that analyzed the phenotype of tissue-specific mouse mutants lacking Ihh or associated molecules. The recent advances in understanding the molecular mechanism regulating TMJ development are impressive and these findings will have major implications for future translational medicine tools to repair and regenerate TMJ congenital anomalies and acquired diseases, such as degenerative damage in TMJ osteoarthritic conditions.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling.

Analysis of the sublingual artery using contrast-enhanced computed tomography.

INTRODUCTION: This in vivo study aimed to clarify the position of the sublingual artery (SLA) relative to the mandibular bone and to infer the potential risk for injury during dental implant surgery. METHODS: Contrast-enhanced computed tomography images of the mouth of 50 edentulous patients (100 sides) treated at Tokushima University Hospital were reviewed. Curved planar reconstructed images perpendicular to the alveolar ridge were processed and classified into molar, premolar, canine, and incisor regions. The SLA and its branches were identified, and the distance from the mandible to the SLA was measured. RESULTS: The SLA was located close to the mandible (<2 mm) in the molar, premolar, canine, and incisor segments in 12.0% (95% confidence interval 5.6%-18.4%), 20.6% (12.6%-28.7%), 30.5% (21.3%-39.8%), and 41.8% (28.8%-54.9%) cases, respectively. The SLA was located within ±3 mm craniocaudally to the upper wall of the mandibular canal in the molar and premolar regions in 50% of cases and within ±5 mm craniocaudally to the mylohyoid ridge in the canine and incisor regions in the other cases, with no sex or age-related differences. The vertical distance from the alveolar ridge to the SLA was influenced by sex and age owing to alveolar resorption, indicating that the alveolar ridge is not a reliable reference for predicting SLA position. CONCLUSIONS: As the risk of SLA injury always exist during dental implant placement and there is no way to confirm the SLA pathways in a patient, clinicians must avoid injuring the sublingual soft tissue.

Anti-tumor effect in human breast cancer by TAE226, a dual inhibitor for FAK and IGF-IR in vitro and in vivo.

Focal adhesion kinase (FAK) is a 125-kDa non-receptor type tyrosine kinase that localizes to focal adhesions. FAK overexpression is frequently found in invasive and metastatic cancers of the breast, colon, thyroid, and prostate, but its role in osteolytic metastasis is not well understood. In this study, we have analyzed anti-tumor effects of the novel FAK Tyr(397) inhibitor TAE226 against bone metastasis in breast cancer by using TAE226. Oral administration of TAE226 in mice significantly decreased bone metastasis and osteoclasts involved which were induced by MDA-MB-231 breast cancer cells and increased the survival rate of the mouse models of bone metastasis. TAE226 also suppressed the growth of subcutaneous tumors in vivo and the proliferation and migration of MDA-MB-231 cells in vitro. Significantly, TAE226 inhibited the osteoclast formation in murine pre-osteoclastic RAW264.7 cells, and actin ring and pit formation in mature osteoclasts. Moreover, TAE226 inhibited the receptor activator for nuclear factor κ B Ligand (RANKL) gene expression induced by parathyroid hormone-related protein (PTHrP) in bone stromal ST2 cells and blood free calcium concentration induced by PTHrP administration in vivo. These findings suggest that FAK was critically involved in osteolytic metastasis and activated in tumors, pre-osteoclasts, mature osteoclasts, and bone stromal cells and TAE226 can be effectively used for the treatment of cancer induced bone metastasis and other bone diseases.

The Soluble Factor from Oral Cancer Cell Lines Inhibits Interferon-γ Production by OK-432 via the CD40/CD40 Ligand Pathway.

(1) Background: OK-432 is a penicillin-killed, lyophilized formulation of a low-toxicity strain (Su) of Streptococcus pyogenes (Group A). It is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously showed that (i) OK-432 treatment induces a high amount of IFN-? production from peripheral blood mononuclear cells (PBMCs), and (ii) conditioned medium (CM) from oral cancer cells suppresses both the IFN-? production and cytotoxic activity of PBMCs driven by OK-432. The aim of this study was to determine the inhibitory mechanism of OK-432-induced IFN-? production from PBMCs by CM. (2) Methods: We performed cDNA microarray analysis, quantitative RT-PCR, and ELISA to reveal the inhibitory mechanism of CM. (3) Results: We found that CD40 plays a key role in IFN-? production via IL-12 production. Although OK-432 treatment upregulated the expression levels of the IL-12p40, p35, and CD40 genes, CM from oral cancer cells downregulate these genes. The amount of IFN-? production by OK-432 treatment was decreased by an anti-CD40 neutralizing antibody. (4) Conclusions: Our study suggests that uncertain soluble factor(s) produced from oral cancer cells may inhibit IFN-? production from PBMCs via suppressing the CD40/CD40L-IL-12 axis.

Histological comparison of three apatitic bone substitutes with different carbonate contents in alveolar bone defects in a beagle mandible with simultaneous implant installation.

Since bone apatite is a carbonate apatite containing carbonate in an apatitic structure, carbonate content may be one of the factors governing the osteoconductivity of apatitic bone substitutes. The aim of this study was to evaluate the effects of carbonate content on the osteoconductivity of apatitic bone substitutes using three commercially available bone substitutes for the reconstruction of alveolar bone defects of a beagle mandible with simultaneous dental implant installation. NEOBONE, Bio-Oss, and Cytrans that contain 0.1, 5.5, and 12.0 mass% of carbonate, respectively, were used in this study. The amount of newly formed bone in the upper portion of the alveolar bone defect of the beagle's mandible was 0.7, 6.6, and 39.4% at 4 weeks after surgery and 4.7, 39.5, and 75.2% at 12 weeks after surgery for NEOBONE, Bio-Oss, and Cytrans, respectively. The results indicate that bone-to-implant contact ratio was the largest for Cytrans. Additionally, the continuity of the alveolar ridge was restored in the case of Cytrans, whereas the continuity of the alveolar ridge was not sufficient when using NEOBONE and Bio-Oss. Both Cytrans and Bio-Oss that have a relatively larger carbonate content in their apatitic structure was resorbed with time. We concluded that carbonate content is one of important factors governing the osteoconductivity of apatitic bone substitutes.

Effects of acidic calcium phosphate concentration on setting reaction and tissue response to ß-tricalcium phosphate granular cement.

Beta-tricalcium phosphate granular cement (ß-TCP GC), consisting of ß-TCP granules and an acidic calcium phosphate (Ca-P) solution, shows promise in the reconstruction of bone defects as it sets to form interconnected porous structures, that is, ß-TCP granules are bridged with dicalcium phosphate dihydrate (DCPD) crystals. In this study, the effects of acidic Ca-P solution concentration (0-600 mmol/L) on the setting reaction and tissue response to ß-TCP GC were investigated. The ß-TCP GC set upon mixing with its liquid phase, based on the formation of DCPD crystals, which bridged ß-TCP granules to one another. Diametral tensile strength of the set ß-TCP GC was relatively the same, at ∼0.6 MPa, when the Ca-P concentration was 20-600 mmol/L. Due to the setting ability, reconstruction of the rat's calvarial bone defect using ß-TCP GC with 20, 200, and 600 mmol/L Ca-P solution was much easier compared to that with ß-TCP granules without setting ability. Four weeks after the reconstruction, the amount of new bone was the same, ∼17% in both ß-TCP GC and ß-TCP granules groups. Cellular response to ß-TCP granules and ß-TCP GC using the 20 mmol/L acidic Ca-P solution was almost the same. However, ß-TCP GC using the 200 and 600 mmol/L acidic Ca-P solution showed a more severe inflammatory reaction. It is concluded, therefore, that ß-TCP GC, using the 20 mmol/L acidic Ca-P solution, is recommended as this concentration allows surgical techniques to be performed easily and provides good mechanical strength, and the similar cellular response to ß-TCP granules. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:22-29, 2020.

Fabrication of porous carbonate apatite granules using microfiber and its histological evaluations in rabbit calvarial bone defects.

Carbonate apatite (CO3 Ap) granules are known to show good osteoconductivity and replaced to new bone. On the other hand, it is well known that a porous structure allows bone tissue to penetrate its pores, and the optimal pore size for bone ingrowth is dependent on the composition and structure of the scaffold material. Therefore, the aim of this study was to fabricate various porous CO3 Ap granules through a two-step dissolution-precipitation reaction using CaSO4 as a precursor and 30-, 50-, 120-, and 205-µm diameter microfibers as porogen and to find the optimal pore size of CO3 Ap. Porous CO3 Ap granules were successfully fabricated with pore size 8.2-18.7% smaller than the size of the original fiber porogen. Two weeks after the reconstruction of rabbit calvarial bone defects using porous CO3 Ap granules, the largest amount of mature bone was seen to be formed inside the pores of CO3 Ap (120) [porous CO3 Ap granules made using 120-µm microfiber] followed by CO3 Ap (50) and CO3 Ap (30). At 4 and 8 weeks, no statistically significant difference was observed based on the pore size, even though largest amount of mature bone was formed in case of CO3 Ap (120). It is concluded, therefore, that the optimal pore size of the CO3 Ap is that of CO3 Ap (120), which is 85 µm.

Retinoic Receptor Signaling Regulates Hypertrophic Chondrocyte-specific Gene Expression.

BACKGROUND/AIM: Retinoid signaling is important for the maturation of growth-plate chondrocytes. The effect of retinoid receptor gamma (RARγ) signaling on the expression of genes in hypertrophic chondrocytes is unclear. This study investigated the role of RARγ signaling in regulation of hypertrophic chondrocyte-specific genes. MATERIALS AND METHODS: The gene expression in mouse E17.5 tibial cartilage was examined by in situ hybridization analysis. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting were used for analysis of mRNA and phosphorylated mitogen-activated protein kinase (MAPK). RESULTS: mRNA expression of Rarg and connective tissue growth factor (Ccn2) was detected in maturing chondrocytes throughout the cartilaginous skeletal elements. In chondrogenic ATDC5 cells, an RARγ agonist induced the gene expression of type-X collagen (Col10A1), transglutaminase-2 (Tg2), matrix metalloproteinase-13 (Mmp13), and Ccn2 mRNA, whereas a retinoic acid pan-agonist suppressed RARγ agonist-stimulated gene expression. Phosphorylated extracellular signal regulated-kinases (pERK1/2), p-p38, and phosphorylated c-Jun N-terminal kinase (pJNK) MAPK were time-dependently increased by RARγ agonist treatment. Experimental p38 inhibition led to a severe drop in the RARγ agonist-stimulated expressions of Col10A1, Tg2, Mmp13, and Ccn2 mRNA. CONCLUSION: RARγ signaling is required for the differentiation of hypertrophic chondrocytes, with differential cooperation with p38 MAPK.

Clinical significance and pathogenic function of connective tissue growth factor (CTGF/CCN2) in osteolytic mandibular squamous cell carcinoma.

BACKGROUND: Mandibular bone destruction is a frequent occurrence in oral squamous cell carcinoma. However, the relationship between the bone destruction and associated factors is unclear. Here, the role and diagnostic utility of connective tissue growth factor (CCN2) in bone destruction of the mandible was investigated. PATIENTS AND METHODS: The production of CCN2 was explored by using immunohistochemistry on paraffin-embedded tissues from 20 cases of mandibular squamous cell carcinoma. The effect of CCN2 on osteoclastogenesis was examined in vitro by using total bone marrow cell populations from male mice. RESULTS: Immunohistochemical analysis showed that CCN2-positive signals were closely associated with destructive invasion of the mandible by oral squamous cell carcinomas. Consistent with these results, recombinant human CCN2 (rCCN2) stimulated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cell formation in vitro. CONCLUSION: CCN2 can be considered a diagnostic marker and target for treatment in oral osteolytic mandibular squamous cell carcinoma.

Roles of Ihh signaling in chondroprogenitor function in postnatal condylar cartilage.

Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1 month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3 months, articular cartilage consisted of a 2-3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72 h, respectively. Notably, EdU injection in 3 month-old mice labeled both progenitors and maturing chondrocytes by 96 h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings indicate that the chondroprogenitor layer is continuously engaged in condylar growth postnatally and its organization and functioning depend on hedgehog signaling.

Oral Squamous Cell Carcinoma-derived Sonic Hedgehog Promotes Angiogenesis.

BACKGROUND: Sonic hedgehog (SHH) signaling is related to the pathogenesis of oral squamous cell carcinoma (OSCC), but its role in OSCC is not yet well understood. In this study, we analyzed the role of SHH signaling in OSCC. MATERIALS AND METHODS: We examined the expression pattern of SHH and its signal proteins in clinically resected OSCC samples by immunohistochemistry. We also evaluated the function of SHH signaling using the hedgehog signaling inhibitor cyclopamine in vivo and in vitro by proliferation, migration and angiogenesis analyses. RESULTS: We found that SHH was highly expressed in human tongue OSCC, whereas patched (PTCH1), glioma-associated oncogene 1 (GLI1) and GLI2 proteins were expressed in the microvascular cells in the tumor invasive front. Administration of cyclopamine to mice suppressed the growth and angiogenesis of OSCC xenografts in vivo. Moreover, cyclopamine inhibited endothelial cell proliferation and migration, and reduced aorta vascular length in the rat. CONCLUSION: These findings suggest that OSCC-derived SHH stimulates angiogenesis at the tumor invasive front.

Expression and Role of Sonic Hedgehog in the Process of Fracture Healing with Aging.

Aging is one of the risk factors for delayed fracture healing. Sonic hedgehog (SHH) protein, an inducer of embryonic development, has been demonstrated to be activated in osteoblasts at the dynamic remodeling site of a bone fracture. Herein, we compared and examined the distribution patterns of SHH and the functional effect of SHH signaling on osteogenesis and osteoclastogenesis between young (5-week-old) and aged (60-week-old) mice during fracture healing. We found that SHH was expressed in bone marrow cells from the fractured site of the rib of young mice on day 5, but was barely detectable in the corresponding cells from the rib of aged mice. SHH was also detected in osteoblasts and bone marrow cells at the callus remodeling stage on days 14 and 28 in both young and aged mice. The number of alkaline phosphatase (ALP)-positive osteoblasts was significantly higher in young mice on days 5 and 14, whereas the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was significantly higher in aged mice. SHH stimulated significantly more osteoblast formation in the young compared to old mice. SHH stimulated the osteoclast formation directly in the aged mice and suppressed the formation indirectly through osteoprotegerin expression in the young mice. Results indicate that an aged-related delay of fracture healing may contribute to the unbalanced bone formation and resorption, regulated by hedgehog signaling.

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling.

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.

Novel Midkine Inhibitor iMDK Inhibits Tumor Growth and Angiogenesis in Oral Squamous Cell Carcinoma.

Midkine is a heparin-binding growth factor highly expressed in various human malignant tumors. However, its role in the growth of oral squamous cell carcinoma is not well understood. In this study, we analyzed the antitumor effect of a novel midkine inhibitor (iMDK) against oral squamous cell carcinoma. Administration of iMDK induced a robust antitumor response and suppressed cluster of differentiation 31 (CD31) expression in oral squamous cell carcinoma HSC-2 cells and SAS cells xenograft models. iMDK inhibited the proliferation of these cells dose-dependently, as well as the expression of midkine and phospho-extracellular signal-regulated kinase in HSC-2 and SAS cells. Moreover, iMDK significantly inhibited vascular endothelial growth factor and induced tube growth of human umbilical vein endothelial cells in a dose-dependent fashion. These findings suggest that midkine is critically involved in oral squamous cell carcinoma and iMDK can be effectively used for the treatment of oral squamous cell carcinoma.

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction.

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.

Expression and roles of CCN2 in dental epithelial cells.

Connective tissue growth factor (CCN2) regulates diverse cellular functions, including tooth development. In order to delineate the precise role of CCN2 in the epithelium during odontogenesis, we investigated how it is expressed and what roles it may have in primary cultures of epithelial cells derived from developing tooth germ of the bovine fetus. Ccn2 mRNA and protein were strongly expressed in the inner dental epithelium, which is consistent with the expression of transforming growth factor-ß2 mRNA and proliferating cell nuclear antigen. Bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) were also expressed in the inner dental epithelium, indicating that CCN2 functionally interacts with these factors in the epithelium. The stimulatory effects of FGF2 on cell proliferation and BMP4 on cell differentiation were additively up-regulated by CCN2 in a newly-established dental epithelium cell culture. Taken together, our data provide clear evidence that CCN2 is synthesized by inner dental epithelial cells, and appears to act as an autocrine factor, which regulates dental epithelial cell proliferation and differentiation in concert with growth factors.

A case of adenoid cystic carcinoma associated with IgG4-related disease.

INTRODUCTION: Immunoglobulin G4-related disease (IgG4-RD) is an inflammatory condition associated with elevated serum IgG4 levels and tissue infiltration by IgG4-expressing plasma cells. We present a case of adenoid cystic carcinoma (ACC) of the submandibular gland with possible involvement of IgG4-RD. PRESENTATION OF CASE: The patient was a 59-year-old man presenting with a swollen right submandibular gland. Laboratory tests revealed IgG4 levels of 176mg/dl (reference range: 4.8-105). An initial open biopsy for histological diagnosis showed chronic sialadenitis. The region was monitored on an outpatient basis, and finally the right submandibular was totally resected because malignant tumor could not be excluded. Histological examination of the submandibular gland showed an ACC with lymphocytic infiltration containing many IgG4-positive plasma cells in the tumor stroma. DISCUSSION: We have described a case that indicated a possible involvement of ACC with IgG4-RD. This allows us to speculate that longstanding IgG4-RD may progress to malignancy or infiltration of IgG4-positive plasma cells through the signals of tumor stimuli. Further investigations are required to determine the potential pathogenic mechanism underlying this unique tumor. CONCLUSION: This case underscores that caution is needed in the diagnosis of masses with high serum IgG4 levels, as the differential diagnosis includes malignancy.