Teeth develop from reciprocal interactions between mesenchyme cells and epithelium, where the epithelium provides the instructive information for initiation. Based on these initial tissue interactions, we have replaced the mesenchyme cells with mesenchyme created by aggregation of cultured non-dental stem cells in mice. Recombinations between non-dental cell-derived mesenchyme and embryonic oral epithelium stimulate an odontogenic response in the stem cells. Embryonic stem cells, neural stem cells, and adult bone-marrow-derived cells all responded by expressing odontogenic genes. Transfer of recombinations into adult renal capsules resulted in the development of tooth structures and associated bone. Moreover, transfer of embryonic tooth primordia into the adult jaw resulted in development of tooth structures, showing that an embryonic primordium can develop in its adult environment. These results thus provide a significant advance toward the creation of artificial embryonic tooth primordia from cultured cells that can be used to replace missing teeth following transplantation into the adult mouth.

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.

We present direct evidence of an activator-inhibitor system in the generation of the regularly spaced transverse ridges of the palate. We show that new ridges, or rugae, marked by stripes of Sonic hedgehog (Shh) expression, appear at two growth zones where the space between previously laid-down rugae increases. However, inter-rugal growth is not absolutely required: new stripes still appear when growth is inhibited. Furthermore, when a ruga is excised new Shh expression appears, not at the cut edge but as bifurcating stripes branching from the neighbouring Shh stripe, diagnostic of a Turing-type reaction-diffusion mechanism. Genetic and inhibitor experiments identify Fibroblast Growth Factor (FGF) and Shh as an activator-inhibitor pair in this system. These findings demonstrate a reaction-diffusion mechanism likely to be widely relevant in vertebrate development.

BackgroundMatricellular proteins, including periostin, are important for tissue regeneration.Methods and FindingsPresently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient (−/−) mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin γ2. Periostin was associated with laminin γ2, and this association enhanced the proteolytic cleavage of the laminin γ2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin−/− mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin−/− mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-κB.ConclusionThese results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing.

Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a cilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of the embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1 mutants also had ectopic diastema teeth and accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.

Previous studies have provided the biological basis for the therapeutic use of enamel matrix derivative (EMD) at sites of periodontal regeneration. A purpose of this study is to determine effects of EMD on cell growth, osteoblastic differentiation and insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) production in human periodontal ligament cells (HPLC). We also examined participation of endogenous IGF-I and TGF-beta 1 with EMD-stimulated cell growth in these cells. HPLCs used in this study were treated with EMD alone or in combination with antihuman IGF-I antibody (anti-hIGF-I) or anti-hTGF-beta 1, recombinant human bone morphogenetic protein-2 (rhBMP-2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], rhTGF-beta 1 or rhIGF-I. After each treatment, cell growth, the production of IGF-I and TGF-beta 1 and the expression of osteoblastic phenotypes were evaluated. EMD stimulated cell growth in dose-dependent and time-dependent manners. EMD was also stimulated to express IGF-I and TGF-beta 1 at protein and mRNA levels. The EMD-stimulated cell growth was partially suppressed by cotreatment with anti-hIGF-I or anti-hTGF-beta 1, and cell growth was also stimulated by treatment with rhIGF-I or rhTGF-beta 1. rhBMP-2 stimulated alkaline phosphatase (ALPase) activity and ALPase mRNA expression, and 1,25(OH)2D3 stimulated ALPase and osteocalcin mRNA expression. However, EMD showed no effect on the osteoblastic phenotypes expression. These results demonstrated that EMD has no appreciable effect on osteoblastic differentiation, however it stimulates cell growth and IGF-I and TGF-beta 1 production in HPLC, and that these endogenous growth factors partially relate to the EMD-stimulated cell growth in HPLC.

DPLC retain the capability to differentiate into an osteoblast lineage and may act in the regeneration of periodontal ligament with new cementum formation, whereas these cells may have a limited influence on alveolar bone formation during the course of periodontal regeneration.

Podoplanin is a type-I transmembrane sialomucinlike protein, which is expressed in a wide range of cell types and is involved in platelet aggregation and tumor metastasis. Here, we investigated the function, regulation, and expression of podoplanin in osteosarcoma. Podoplanin expression was observed in three osteosarcoma cell lines (MG-63, HOS, and U-2 OS) with platelet aggregation-inducing ability, which was blocked by podoplanin small-interfering RNA or a neutralizing antibody. Overexpression of podoplanin in nonmetastatic Dunn osteosarcoma cells promoted cell migration without attenuating cell proliferation. Both podoplanin and TGF-␤1 were up-regulated by c-Fos induction in MC3T3-E1 osteoblastic cells, and were highly expressed in c-Fos transgenic mouse osteosarcomas and c-Fos-transformed osteosarcoma cell lines. Immunohistochemistry of human osteosarcoma tissue microarrays (n ‫؍‬ 133) showed staining of tumor cells embedded in an excess of irregular neoplastic bone matrix in 100% of tumors undergoing so-called "normalization/maturation." Podoplanin was also expressed in osteosarcoma subtypes, with 65% of osteoblastic, 100% of chondroblastic, and 79% of fibroblastic tumors. CD44 and pERM immunohistochemistry showed coexpression with podoplanin in both mouse and human osteosarcoma. Podoplanin expression was significantly higher in metastatic osteosarcomas (n ‫؍‬ 6) than in primary osteosarcomas (n ‫؍‬ 10). Our data suggest that podoplanin, which is not expressed in normal osteoblasts but in osteocytes, is aberrantly expressed in transformed osteoblasts and in osteosarcoma, and is under AP-1 transcriptional control. Thus podoplanin is a candidate molecule for ther- Osteosarcoma (OS) is the most common primary malignant bone tumor, with a high tendency to metastasize to the lung. Despite recent advances in modern chemotherapy, the average survival after a recurrence in distant organs is less than 1 year.1 In contrast, of patients who present with no metastasis, approximately 70% will be long-term survivors.2 Therefore, there is a strong necessity to better understand the molecular mechanisms of metastasis to deliver innovative life-saving and life-enhancing therapies to patients.Platelet aggregation is one of the crucial steps involved during the sequential tumor metastasis process to escape from the host immune system and form tumor emboli in distant organs. Several earlier studies have shown that platelet aggregating capability of tumor cells from colon cancer and melanoma is correlated with their metastatic potential in vivo.3-5 Podoplanin, a type-I transmembrane sialomucin-like glycoprotein, is a platelet aggregation-inducing factor 6 that is expressed in a wide range of tumors 7-9 and has been reported to be associated with poor outcome of oral and esophageal squamous cell carcinomas.10,11 Podoplanin promotes cell migration and cell invasion, 9,12 and also plays a key role in epithelial-mesenchymal transition in Madin-Darby canine kidney (MDCK) type-II cells.