Expression of Wnt3a, Wnt10b, ß-catenin and DKK1 in periodontium during orthodontic tooth movement in rats.

OBJECTIVE: To investigate the expression of Wnt3a, Wnt10b, ß-catenin and DKK1 in the periodontal ligament (PDL) during orthodontic tooth movement (OTM) in rats. MATERIALS AND METHODS: Nickel-titanium closed-coil springs were used to deliver an initial 50 g mesial force to the left maxillary first molars in 30 rats. The force was kept constant for 1, 3, 5, 7, 10 and 14 days until the animals were sacrificed. The right maxillary molars without force application served as control. Paraffin-embedded sections of the upper jaws were prepared for histological and immunohistochemical analyses to detect Wnt3a, Wnt10b, ß-catenin and DKK1 expression in PDL. RESULTS: Wnt3a, Wnt10b, ß-catenin and DKK1 were expressed on both the ipsilateral and contralateral sides of PDL in each group. After the application of orthodontic force, the expression of ß-catenin and DKK1 was initially increased and then decreased on both sides, with maximal levels of expression at day 7 and day 10, respectively. On the compression side, Wnt3a and Wnt10b levels started to increase at day 5, while on the tension side, these two molecules began to increase at day 1. Furthermore, the expression levels of Wnt3a, Wnt10b, and ß-catenin were much stronger on the tension side than on the compression side at any of the observation points, while DKK1 level was much higher on the compression side. CONCLUSION: Wnt3a, Wnt10b, ß-catenin and DKK1 expression may be related to the periodontal tissue remodeling following the application of an orthodontic force in rats. These observations suggest that the Wnt/ß-catenin signaling pathway may play a crucial role in periodontal tissue remodeling during OTM.

Downregulation of Wnt causes root resorption.

INTRODUCTION: There are multiple causes of external root resorption, but absent a disease state, it is most often observed when excessive physical force is used during orthodontic treatment. Even without mechanical stimulation, however, root resorption can still occur. The purpose of this study was to test whether Wnt signaling plays a role in pathologic root resorption, by conditionally deleting Wntless (Wls) from odontoblasts and osteoblasts and then evaluating the phenotypic effects on the maintenance of the root surface. METHODS: Ten (age, 1 month) and 20 (age, 3 months) OCN-Cre;Wls(fl/fl) mice and their wild-type littermates were evaluated using microcomputed tomography, histology, and immunohistochemistry. Phenotypic alterations in the alveolar bone, dentin, and cementum were characterized and quantified. RESULTS: In a genetic model of reduced Wnt signaling, we found that RANKL expression is upregulated, and osteoprotegerin expression is downregulated. This molecular disruption results in an increase in osteoclast activity, a decrease in osteoblast activity, and extensive, spontaneous root resorption. A genetic strain of mice in which Wnt signaling is elevated exhibits thicker cementum, whereas, even in the perinatal period, OCN-Cre;Wls(fl/fl) mice exhibit thinner cementum. CONCLUSIONS: Taken together, these data demonstrate that Wnts regulate cementum homeostasis, and that idiopathic cases of root resorption might have as their etiology a reduction in endogenous Wnt signaling.

Wntless regulates dentin apposition and root elongation in the mandibular molar.

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;Wls(CO/CO) mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, ß-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.

Wnt5a/Ror2 mediates temporomandibular joint subchondral bone remodeling.

Increased subchondral trabecular bone turnover due to imbalanced bone-resorbing and bone-forming activities is a hallmark of osteoarthritis (OA). Wnt5a/Ror2 signaling, which can derive from bone marrow stromal cells (BMSCs), takes a role in modulating osteoblast and osteoclast formation. We showed previously that experimentally unilateral anterior crossbites (UACs) elicited OA-like lesions in mice temporomandibular joints (TMJs), displaying as subchondral trabecular bone loss. Herein, we tested the role of BMSC-derived Wnt5a/Ror2 signaling in regulating osteoclast precursor migration and differentiation in this process. The data confirmed the decreased bone mass, increased tartrate-resistant acid phosphatase (TRAP)-positive cell number, and enhanced osteoclast activity in TMJ subchondral trabecular bone of UAC-treated rats. Interestingly, the osteoblast activity in the tissue of TMJ subchondral trabecular bone of these UAC-treated rats was also enhanced, displaying as upregulated expressions of osteoblast markers and increased proliferation, migration, and differentiation capabilities of the locally isolated BMSCs. These BMSCs showed an increased CXCL12 protein expression level and upregulated messenger RNA expressions of Rankl, Wnt5a, and Ror2. Ex vivo data showed that their capacities of inducing migration and differentiation of osteoclast precursors were enhanced, and these enhanced capabilities were restrained after blocking their Ror2 signaling using small interfering RNA (siRNA) assays. Reducing Ror2 expression in the BMSC cell line by siRNA or blocking the downstream signalings with specific inhibitors also demonstrated a suppression of the capacity of the BMSC cell line to promote Wnt5a-dependent migration (including SP600125 and cyclosporine A) and differentiation (cyclosporine A only) of osteoclast precursors. These findings support the idea that Wnt5a/Ror2 signaling in TMJ subchondral BMSCs enhanced by UAC promoted BMSCs to increase Cxcl12 and Rankl expression, in which JNK and/or Ca(2+)/NFAT pathways were involved and therefore were engaged in enhancing the migration and differentiation of osteoclast precursors, leading to increased osteoclast activity and an overall TMJ subchondral trabecular bone loss in the UAC-treated rats.

A pilot trial on the molecular pathophysiology of traumatic temporomandibular joint bony ankylosis in a sheep model. Part II: The differential gene expression among fibrous ankylosis, bony ankylosis and condylar fracture.

OBJECTIVE: The purpose of the study was to preliminarily explore the differential expressions of a series of genes regulating bone formation in temporomandibular joint (TMJ) fibrous ankylosis, bony ankylosis and condylar fracture healing. METHODS: The cDNA from either the bony ankylosed callus or fracture callus of the 6 sheep, as described in the part I, were both used in the study. The differences of gene expressions between bony ankylosis and condylar fracture at 1, 3, and 6 months postoperatively were measured by real-time PCR, with 2 samples at each time point. In addition, another 2 sheep were added to have fibrous ankylosis induced on the right TMJ, and 1 sheep was sacrificed at 3 and 6 months after surgery, respectively. The differences of gene expressions between fibrous and bony ankylosis at 3 and 6 months postoperatively were measured by real-time PCR. RESULTS: Bony ankylosis showed higher mRNA expression trends in Wnt2b, Wnt5a, ß-Catenin, Lef1, CyclinD1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn, Bmp2, and Bmp7 compared to fibrous ankylosis, although no statistical analysis was performed due to the very small sample size. Whereas bony ankylosis showed a significant lower expression of Wnt5a, ß-Catenin, Lef1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn and Bmp4 compared to condylar fracture at several time points (P < 0.05). CONCLUSION: Our data provided a preliminary molecular evidence for the hypothesis that the development of traumatic TMJ bony ankylosis was the course of delayed bone healing or hypertrophic nonunion, and deserved to be further studied.

A pilot trial on the molecular pathophysiology of traumatic temporomandibular joint bony ankylosis in a sheep model. Part I: Expression of Wnt signaling.

OBJECTIVE: To preliminarily investigate the temporal patterns of the endogenous mRNA expression for members of the Wnt signaling and a series of genes regulating bone formation during the development of traumatic temporomandibular joint (TMJ) bony ankylosis in a sheep model. METHODS: Six sheep were used for the induction of bony ankylosis of TMJ. We performed a condylar fracture, excision of the lateral 2/3 disc and serious injury to the glenoid fossa to induce bony ankylosis on the right TMJ. An isolated condylar fracture was performed on the left side. Two sheep were sacrificed at 1 month, 3 months, and 6 months after surgery, respectively. The specimens from the ankylosed joint and the condylar fracture were harvested for RNA extraction respectively. In this report (Part I), only the bony ankylosed samples were used for analysis of gene expressions. The specimens 1 month postoperatively were taken as the control, and the changes of expression of target genes over time were examined by real-time PCR. RESULTS: mRNA expression of Wnt1, Wnt2b, Wnt3a, ß-catenin, Sfrp1, Lrp6, Lef1, CyclinD1, and Runx2 was up-regulated at 3 and 6 months compared with 1 month. The expression of Wnt5a, Sox9, and Osterix was up-regulated with a peak at 3 months, and then fell back to the basal levels at 6 months. The expression of Ocn began to up-regulate until 6 month postoperatively. CONCLUSION: Our findings suggested that Wnt signaling was involved in the formation of traumatic TMJ bony ankylosis and thus may be a potential therapeutic target for the treatment of the disease in the future.

Effects of WNT10A on proliferation and differentiation of human dental pulp cells.

INTRODUCTION: Wingless-type MMTV integration site family, member 10A (WNT10A) plays crucial roles in odontogenesis. The aim of this study was to investigate the effects of WNT10A on human dental pulp cells (DPCs), which contain a mixed population of cells, including stem and progenitor cells, and participate in dentin repair or dentin-pulp regeneration. METHODS: Healthy human premolars extracted for orthodontic reasons were used as a study model. The expression of WNT10A protein in dental pulp was determined by immunohistochemistry. The messenger RNA expression of WNT10A and Wnt-related genes was analyzed by semiquantitative reverse-transcription polymerase chain reaction. DPCs were enzymatically separated from pulp tissues, cultured, and passaged. The biological effects of WNT10A on DPCs were investigated using recombinant lentivirus encoding WNT10A complementary DNA. WNT10A-induced changes in DPC proliferation were assessed by methyltetrazolium assay and flow cytometry. In order to determine the effects of WNT10A on DPC differentiation, the activity of alkaline phosphatase (ALP), an early marker of odontoblastic differentiation, was assessed using an ALP activity assay kit, and the expression levels of odontoblast-specific genes, including DSPP, DMP1, ALP, and COL1A1, were detected by quantitative polymerase chain reaction and Western blot. RESULTS: WNT10A protein was clearly identified in the cytoplasm of DPCs. Semiquantitative reverse-transcription polymerase chain reaction indicated the expression of WNT10A and Wnt-related genes in pulp tissues as well as in passaging DPCs. Lentiviral overexpression of WNT10A enhanced proliferation of DPCs and down-regulated ALP activity and the expression of odontoblast-specific genes. CONCLUSIONS: WNT10A promotes the proliferation of DPCs and negatively regulates their odontoblastic differentiation.

YAP overexpression affects tooth morphogenesis and enamel knot patterning.

Teeth develop through distinct morphological stages. At the cap stage, a compactly clustered and concentrically arranged cell mass, the enamel knot, appears at the tip of the enamel organ. Cells in this knot express sets of key molecules, and as such have been proposed to act as a signaling center directing tooth morphogenesis and tooth cusp formation. YAP is a transcriptional co-activator of the Hippo signaling pathway that is essential for the proper regulation of organ growth. In this study, we analyzed the tooth phenotype in transgenic mice that overexpressed a constitutively active form of YAP in the dental epithelium. We found that overexpression of YAP resulted in deformed tooth morphogenesis with widened dental lamina. In addition, the enamel knot was mislocated to the upper portion of the enamel organ, where it remained devoid of proliferating cells and contained apoptotic cells with intense Edar transcripts and reduced E-cadherin expression. Interestingly, some signaling molecules, such as Shh, Fgf4, and Wnt10a, were not expressed in this mislocated enamel knot, but remained at the tip of the enamel organ. Analysis of these data suggests that the signaling center is induced by reciprocal epithelial-mesenchymal interactions, and its induction may be independent of the enamel knot.

A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.

Localization of Bmp-4, Shh and Wnt-5a transcripts during early mice tooth development by in situ hybridization.

A comparative nonisotopic in situ hybridization (ISH) analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.

Presence of ghost cells and the Wnt signaling pathway in odontomas.

BACKGROUND: Although it has been reported that ghost cells are present in odontomas, the generation mechanism of these cells is unclear. To evaluate the presence of ghost cells and involvement of the Wnt signaling pathway, we examined the expression of hard keratins, beta-catenin and Lef-1 in odontomas. METHODS: Sixty-nine cases of odontoma were examined immunohistochemically with the use of antibodies against human hair proteins, beta-catenin and Lef-1. RESULTS: Expression of hard keratins was found only in the cytoplasm of ghost cells in 46 (66.7%) of the 69 odontomas. Compound odontomas (78.8%) showed a higher incidence of ghost cells than complex odontomas (29.4%). Histopathologically, ghost cells were found within odontogenic epithelium adjacent to immature enamel and in the centre of Liesegang-ring-like calcified materials. Expression of beta-catenin and Lef-1 was observed in the cytoplasm and nucleus of odontogenic epithelial cells adjacent to the ghost cells in immature odontomas. CONCLUSION: These findings suggest that odontoma is a hard keratin-expressing tumor-like lesion, and that the Wnt signaling pathway may be involved in the formation of ghost cells in odontomas.

Localization of Bmp-4, Shh and Wnt-5a transcripts during early mice tooth development by in situ hybridization

A comparative nonisotopic in situ hybridization (ISH) analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.

No presente trabalho, realizou-se uma análise comparativa não isotópica por hibridização in situ a fim de se detectar a presença de transcritos de Bmp-4, Shh e Wnt-5a durante as fases iniciais da odontogênese em camundongos, desde a iniciação até o estágio de capuz. No estágio de iniciação, observou-se expressão precoce de Bmp-4 no epitélio e no mesênquima subjacente, enquanto que a expressão de Shh ocorreu durante o estágio de capuz, na região de espessamento do revestimento epitelial odontogênico, tornando-se mais intensa na área de nó do esmalte. Os transcritos de Wnt-5a foram expressos somente no mesênquima durante os estágios de iniciação, botão e capuz, com intenso sinal na região no mesênquima na fase de botão. Estes resultados mostraram que Bmp-4, Shh e Wnt-5a são expressos desde os estágios mais precoces do desenvolvimento dentário, sugerindo que o gene Wnt-5a seja expresso em populações celulares distintas daquelas que expressam Bmp-4 e Shh.