Assessment of Reference Genes Stability in Cortical Bone of Obese and Diabetic Mice.

Introduction: Bone, a pivotal structural organ, is susceptible to disorders with profound health implications. The investigation of gene expression in bone tissue is imperative, particularly within the context of metabolic diseases such as obesity and diabetes that augment the susceptibility to bone fractures. The objective of this study is to identify a set of internal control genes for the analysis of gene expression. Methods: This study employs reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) to assess gene expression in bone tissue. We selected fourteen housekeeping genes and assessed their stability in the cortical bone of mouse models for obesity and diabetes using four well-established algorithms (GeNorm, BestKeeper, NormFinder, and the comparative Delta Ct method). Results and Conclusion: We identified Rpl13a as the mostly stably expressed reference gene in cortical bone tissue from mouse models of obesity and diabetes (db/db), while Gapdh was found to be the most stable reference gene in another diabetes model, KKAy mice. Additionally, Ef1a, Ppia, Rplp0, and Rpl22 were identified as alternative genes suitable for normalizing gene expression in cortical bone from obesity and diabetes mouse models. These findings enhance RT-qPCR accuracy and reliability, offering a strategic guide to select reference gene for studying bone tissue gene expression in metabolic disorders.

Comparison of the biomechanical differences in the occlusal movement of wild-type and BMP9 knockout mice with apical periodontitis.

Apical periodontitis is a common clinical disease caused by bacteria; bacterial metabolites can cause an imbalance in bone homeostasis, bone mass reduction, and tooth loss. Bone resorption in apical periodontitis causes a concentration of stress in the tooth and periodontal tissues during occlusion, which aggravates the disease. Emerging evidence indicates that bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), may play an important role in tooth and dentoalveolar development. Herein, we investigated the role of BMP9 in the development of apical periodontitis and its effects on the biomechanics of dentoalveolar bone. Apical periodontitis models were established in five BMP9 knockout (KO) mice and five C57BL/6 WT (wild-type) mice. At baseline and 14, 28, and 42 days after modeling, in vivo micro-computed tomography analysis and three-dimensional (3D) reconstruction were performed to evaluate the apical lesion in each mouse, and confirm that the animal models were successfully established. Finite element analysis (FEA) was performed to study the stress and strain at the alveolar fossa of each mouse under the same vertical and lateral stress. FEA revealed that the stress and strain at the alveolar fossa of each mouse gradually concentrated on the tooth cervix. The stress and strain at the tooth cervix gradually increased with time but were decreased at day 42. Under the same lingual loading, the maximum differences of the stress and strain at the tooth root in KO mice were greater than those in WT mice. Thus, these findings demonstrate that BMP9 could affect the biomechanical response of the alveolar fossa at the tooth root in mice with apical periodontitis. Moreover, the effects of BMP9 on the biomechanical response of the alveolar bone may be site-dependent. Overall, this work contributes to an improved understanding of the pathogenesis of apical periodontitis and may inform the development of new treatment strategies for apical periodontitis.

BMP4-regulated human dental pulp stromal cells promote pulp-like tissue regeneration in a decellularized dental pulp matrix scaffold.

Pulp regeneration with stem cells is a promising alternative for treating periapical and pulp diseases of young permanent teeth. The aim of this study was to characterize decellularized dental pulp extracellular matrix (dECM) and investigate whether bone morphogenetic protein 4 (BMP4) regulates dental pulp stromal cells (DPSC)-mediated pulp regeneration combined with dECM. Dental pulp isolated from healthy third molars was decellularized with 10% sodium dodecyl sulfate (SDS) and Triton X-100. H&E staining, DAPI staining and electron microscopy were used to observe the dECM structure. The Cell Counting Kit-8 assay was used to analyse cell proliferation. Recombinant adenovirus was used to overexpress BMP4 in DPSCs. The cells were cultured in dECM and dECM + three-dimensional (3D) Vitrogel systems, and bone/dentin/angiogenesis marker expression was evaluated by real-time polymerase chain reaction (RT-PCR) and ALP staining. DPSCs mixed with dECM and BMP4 were transplanted into nude mice, and pulp-like tissue formation was evaluated. The expression of osteogenic and angioblastic genes was increased, and pulp-like tissue formed in vivo. Thus, dECM promotes DPSC proliferation, BMP4 and dECM together accelerate pulp-like tissue formation by DPSCs in vitro.

Matrigel Scaffolding Enhances BMP9-induced Bone Formation in Dental Follicle Stem/Precursor Cells.

Bone tissue engineering requires a combination of cells, efficient biochemical and physicochemical factors, and biocompatible scaffolds. In this study, we evaluated the potential use of injectable Matrigel as a scaffold for the delivery of rat dental follicle stem/precursor cells (rDFSCs) transduced by bone morphogenetic protein (BMP) 9 to enhance osteogenic differentiation in vitro and promote ectopic bone formation in vivo. Recombinant adenovirus was used to overexpress BMP9 in rDFSCs. Alkaline phosphatase activity was measured using a histochemical staining assay and a chemiluminescence assay kit. Quantitative real-time polymerase chain reaction was used to determine mRNA expression levels of bone-related genes including distal-less homeobox 5 (DLX5), osteopontin (OPN), osterix (Osx), and runt-related transcription factor 2 (Runx2). Matrix mineralization was examined by Alizarin Red S staining. rDFSCs proliferation was analyzed using the Cell Counting Kit-8 assay. Subcutaneous implantation of rDFSCs-containing Matrigel scaffolds was used, and micro-computed tomography analysis, histological evaluation, and trichrome staining of implants extracted at 6 weeks were performed. We found that BMP9 enhanced alkaline phosphatase activity and mineralization in rDFSCs. The expression of bone-related genes (DLX5, OPN, Osx, and Runx2) was also increased as a result of BMP9 stimulation. Micro-computed tomography analysis and histological evaluation revealed that the bone masses retrieved from BMP9-overexpressing rDFSCs were significantly more pronounced in those with than in those without Matrigel. Our results suggest that BMP9 effectively promote osteogenic differentiation of rDFSCs, and Matrigel facilitate BMP9-induced osteogenesis of rDFSCs in vivo.

BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple lineages including osteoblastic lineage. Osteogenic differentiation of MSCs is a cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development, which is regulated by numerous signaling pathways including bone morphogenetic proteins (BMPs). Through a comprehensive analysis of the osteogenic activity, we previously demonstrated that BMP9 is the most potent BMP for inducing bone formation from MSCs both in vitro and in vivo. However, as one of the least studied BMPs, the essential mediators of BMP9-induced osteogenic signaling remain elusive. Here we show that BMP9-induced osteogenic signaling in MSCs requires intact Notch signaling. While the expression of Notch receptors and ligands are readily detectable in MSCs, Notch inhibitor and dominant-negative Notch1 effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo. Genetic disruption of Notch pathway severely impairs BMP9-induced osteogenic differentiation and ectopic bone formation from MSCs. Furthermore, while BMP9-induced expression of early-responsive genes is not affected by defective Notch signaling, BMP9 upregulates the expression of Notch receptors and ligands at the intermediate stage of osteogenic differentiation. Taken together, these results demonstrate that Notch signaling may play an essential role in coordinating BMP9-induced osteogenic differentiation of MSCs.

Label-free electrochemical sensor to investigate the effect of tocopherol on generation of superoxide ions following UV irradiation.

BACKGROUND: Generation of reactive oxygen species (ROS), triggered by ultraviolet radiation (UVR), is associated with carcinogenesis of the skin. UV irradiation induced superoxide anion (O2•-) is the key ROS involved in the cellular damage. The cytoprotective efficacy of an unknown anti-oxidant compound can be evaluated by analyzing the production of O2•- from treated cells. METHODS: In this study, a glass carbon electrode functionalized with nanotube@DNA-Mn3(PO4)2 composite was applied to quantitative determination of generation of highly unstable O2•- from the melanoma A375 cell line following UVR(UV, UVA and UVB). In addition, the cytoprotective efficacy of anti-oxidant α-tocopherol was evaluated by quantifying the production of O2•-. RESULTS: The results showed that, UVR triggers generation of O2•- in melanoma A375 cells, and α-tocopherol is effective in diminishing the production of O2•- following UV irradiation. By comparing the conventional cell-survival assays results, we found that our simple and quick electrochemical sensing method can quantify O2•- generation through the biological activity of an anti-oxidant compound (α-tocopherol). CONCLUSION: Our label-free electrochemical quantification method for ROS (O2•- major) in cells facing UVR stress demonstrates its potential application for high-throughput screening of anti-oxidation compounds.

Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

PURPOSE: The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. METHODS: Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. RESULTS: Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. CONCLUSIONS: Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

Telomerase reverse transcriptase mediates EMT through NF-κB signaling in tongue squamous cell carcinoma.

Locoregional lymph nodes metastasis in oral tongue squamous cell carcinoma represents one of important and common prognostic factors for poor clinical outcome. The human Telomerase Reverse Transcriptase (hTERT) is one of key players in cancer metastasis and stemness, but its exact function in tongue squamous cell carcinoma remains unknown. Here, we aim to understand the role of hTERT by utilizing the CRISPR/Cas9 gene editing system to deplete hTERT in the SCC-15 cell line. Functional comparison of SCC-15 control and knockout cells (hTERT-/-) showed that loss of hTERT suppressed cell proliferation and migration/invasion. Furthermore, hTERT depletion significantly decreased tumorigenesis, including alterations in cellular morphology that areindicative for epithelial-mesenchymal transition (EMT). Mechanistically we demonstrated that the hTERT knockout attenuates NF-κB signaling via a negative feedback regulation in tumorprogression. From these results we propose a novel molecular mechanism of hTERT to promote SCC-15 invasion and metastasis via NF-κB activation. We conclude that targeting hTERT may represent a new therapeutic strategy to improve therapy and survival of tongue squamous cell carcinoma patients.

Comparative analysis of prophages in Streptococcus mutans genomes.

Prophages have been considered genetic units that have an intimate association with novel phenotypic properties of bacterial hosts, such as pathogenicity and genomic variation. Little is known about the genetic information of prophages in the genome of Streptococcus mutans, a major pathogen of human dental caries. In this study, we identified 35 prophage-like elements in S. mutans genomes and performed a comparative genomic analysis. Comparative genomic and phylogenetic analyses of prophage sequences revealed that the prophages could be classified into three main large clusters: Cluster A, Cluster B, and Cluster C. The S. mutans prophages in each cluster were compared. The genomic sequences of phismuN66-1, phismuNLML9-1, and phismu24-1 all shared similarities with the previously reported S. mutans phages M102, M102AD, and ϕAPCM01. The genomes were organized into seven major gene clusters according to the putative functions of the predicted open reading frames: packaging and structural modules, integrase, host lysis modules, DNA replication/recombination modules, transcriptional regulatory modules, other protein modules, and hypothetical protein modules. Moreover, an integrase gene was only identified in phismuNLML9-1 prophages.

The healing of alveolar bone defects with novel bio-implants composed of Ad-BMP9-transfected rDFCs and CHA scaffolds.

Cells, scaffolds, and growth factors play important roles in bone regeneration. Bone morphogenetic protein 9 (BMP9), a member of BMP family, could facilitate osteogenesis by regulating growth factors and promoting angiogenesis. Similar to other stem cells, rat dental follicle stem cells (rDFCs), the precursor cells of cementoblasts, osteoblasts and periodontal ligament cells, can self-renew and exhibit multipotential capacity. Coralline hydroxyapatite (CHA) has good biocompatibility and conductivity required for bone tissue engineering. Here, we reported that BMP9 could enhance the osteogenic differentiation of rDFCs in cell culture. Moreover, our results suggested that BMP9 acted through the Smad1/5/8 signaling pathway. We also produced a novel scaffold that encompasses bio-degradable CHA seeded with recombinant adenoviruses expressing BMP9-transfected rDFCs (Ad-BMP9-transfected rDFCs). With this implant, we achieved more alveolar bone regeneration in the alveolar bone defect compared to blank group, CHA group and rDFCs group. Our results provided a novel bio-implants composed of Ad-BMP9-transfected rDFCs and CHA scaffolds and its mechanism is regarding the activation of Smad1/5/8 signaling pathway in BMP9-induced rDFCs osteogenesis.

Anthelmintic mebendazole enhances cisplatin's effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is one of the most common and aggressive types of human cancers worldwide. Nearly a half of HNSCC patients experience recurrence within five years of treatment and develop resistance to chemotherapy. Thus, there is an urgent clinical need to develop safe and novel anticancer therapies for HNSCC. Here, we investigate the possibility of repurposing the anthelmintic drug mebendazole (MBZ) as an anti-HNSCC agent. Using the two commonly-used human HNSCC lines CAL27 and SCC15, we demonstrate MBZ exerts more potent anti-proliferation activity than cisplatin in human HNSCC cells. MBZ effectively inhibits cell proliferation, cell cycle progression and cell migration, and induces apoptosis of HNSCC cells. Mechanistically, MBZ can modulate the cancer-associated pathways including ELK1/SRF, AP1, STAT1/2, MYC/MAX, although the regulatory outcomes are context-dependent. MBZ also synergizes with cisplatin in suppressing cell proliferation and inducing apoptosis of human HNSCC cells. Furthermore, MBZ is shown to promote the terminal differentiation of CAL27 cells and keratinization of CAL27-derived xenograft tumors. Our results are the first to demonstrate that MBZ may exert its anticancer activity by inhibiting proliferation while promoting differentiation of certain HNSCC cancer cells. It's conceivable the anthelmintic drug MBZ can be repurposed as a safe and effective agent used in combination with other frontline chemotherapy drugs such as cisplatin in HNSCC treatment.

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats.

BACKGROUND: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti-inflammatory effect. However, there are few reports about its anti-inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell-based therapy for treating periodontitis. METHODS: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1ß, IL-6, and IL-8 were determined. The anti-inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. RESULTS: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS-stimulated hPDLCs via nuclear factor (NF)-kappa B, p38/extracellular signal-regulated kinase, and mitogen-activated protein kinase/activator protein-1 pathways. Moreover, in the LPS and ligature-induced periodontitis rat model, BTZ suppressed expression of TNF-α, IL-1ß, IL-6, and IL-8, reduced the ratio of receptor activator of NF-κB ligand/osteoprotegerin, and prevented alveolar bone absorption. CONCLUSION: These findings demonstrate the anti-inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.

Hypoxia pathway and hypoxia-mediated extensive extramedullary hematopoiesis are involved in ursolic acid's anti-metastatic effect in 4T1 tumor bearing mice.

Hypoxic in the tumor mass is leading to the myeloproliferative-like disease (leukemoid reaction) and anemia of body, which characterized by strong extensive extramedullary hematopoiesis (EMH) in spleen. As the key transcription factor of hypoxia, hypoxia-inducible factor-1 (HIF-1) activates the expression of genes essential for EMH processes including enhanced blood cell production and angiogenesis. We found ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, inhibited growth of breast cancer both in vivo and in vitro. The suppression was mediated through the inhibition of multiple cell pathways linked to inflammation, proliferation, angiogenesis, and metastasis. UA also suppressed the leukemoid reaction and the EMH phenomenon of the tumor bearing mice without any significant suppression on body weight (i.p. by 20 mg/kg for 28 days). This is associated with the significant decrease in white blood cells (WBC), platelets (PLT) and spleen weight. During this process, we also detected the down-regulation of cell proliferative genes (PCNA, and ß-catenin), and metastatic genes (VEGF, and HIF-1α), as well as the depression of nuclear protein intensity of HIF-1α. Furthermore, the expression of E2F1, p53 and MDM2 genes were increased in UA group when the VEGF and HIF-1α was over-expressed. Cancer cells were sensitive to UA treating after the silencing of HIF-1α and the response of Hypoxic pathway reporter to UA was suppressed when HIF-1α was over expressed. Overall, our results from experimental and predictive studies suggest that the anticancer activity of UA may be at least in part caused by suppressing the cancer hypoxia and hypoxia-mediated EMH.

In Vitro Biocompability/Osteogenesis and In Vivo Bone Formation Evalution of Peptide-Decorated Apatite Nanocomposites Assisted via Polydopamine.

Enhancing the biocompatibility and osteogenic activity of nano-apatite for applications in bone graft substitutes and bone tissue engineering have been the current challenge in regeneration of lost bone. Inspired by mussels, here we have developed facile biomimetic approaches for preparation of two types of peptide-conjugated apatite nanocompsoties assisted by polydopamine (pDA). We exploited polydopamine chemistry for the modification of nano-apatite crystals: polydopamine coated apatite (HA-c-pDA) and polydopamine template-mediated apatite (HA-t-pDA), on which bone forming peptide was subsequently immobilized under weakly basic conditions to obtain peptide-conjugated apatite nanocomposites (HA-c-pep and HA-t-pep, respectively). TEM images revealed that HA-c-pDA displayed typically rod-like morphology, while HA-t-pDA was sponge-like structure where pDA sheets were decorated by needle-like apatite crystals with low degree of crystallinity. In the cell culture experiments, HA-t-pep nanocomposite exhibited higher cell proliferation, spreading, and alkaline phosphatase activity as well as calcium nodule-formation, compared with pristine nano-HA and HA-c-pep nanocomposite. We then implanted the peptide-decorated apatite into rabbit calvarial defects and analyzed bone formation after 2 months. The data revealed that HA-t-pep group exhibited remarkably enhanced bioactivity and bone formation in vivo. Based on these results, our biomimetic approach could be a promising tool to develop peptide-conjugated apatites for bone regeneration. Meanwhile, the excellent biocompatibility and high osteogenesis of the peptide-conjugated apatite nanocomposite might confer its great potentials in bone repair, bone augmentation, as well as coating of biomedical implants.

Wnt and BMP Signaling Crosstalk in Regulating Dental Stem Cells: Implications in Dental Tissue Engineering.

Tooth is a complex hard tissue organ and consists of multiple cell types that are regulated by important signaling pathways such as Wnt and BMP signaling. Serious injuries and/or loss of tooth or periodontal tissues may significantly impact aesthetic appearance, essential oral functions and the quality of life. Regenerative dentistry holds great promise in treating oral/dental disorders. The past decade has witnessed a rapid expansion of our understanding of the biological features of dental stem cells, along with the signaling mechanisms governing stem cell self-renewal and differentiation. In this review, we first summarize the biological characteristics of seven types of dental stem cells, including dental pulp stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, dental follicle precursor cells, periodontal ligament stem cells, alveolar bone-derived mesenchymal stem cells (MSCs), and MSCs from gingiva. We then focus on how these stem cells are regulated by bone morphogenetic protein (BMP) and/or Wnt signaling by examining the interplays between these pathways. Lastly, we analyze the current status of dental tissue engineering strategies that utilize oral/dental stem cells by harnessing the interplays between BMP and Wnt pathways. We also highlight the challenges that must be addressed before the dental stem cells may reach any clinical applications. Thus, we can expect to witness significant progresses to be made in regenerative dentistry in the coming decade.

Osteoinductive peptide-functionalized nanofibers with highly ordered structure as biomimetic scaffolds for bone tissue engineering.

The construction of functional biomimetic scaffolds that recapitulate the topographical and biochemical features of bone tissue extracellular matrix is now of topical interest in bone tissue engineering. In this study, a novel surface-functionalized electrospun polycaprolactone (PCL) nanofiber scaffold with highly ordered structure was developed to simulate the critical features of native bone tissue via a single step of catechol chemistry. Specially, under slightly alkaline aqueous solution, polydopamine (pDA) was coated on the surface of aligned PCL nanofibers after electrospinning, followed by covalent immobilization of bone morphogenetic protein-7-derived peptides onto the pDA-coated nanofiber surface. Contact angle measurement, Raman spectroscopy, and X-ray photoelectron spectroscopy confirmed the presence of pDA and peptides on PCL nanofiber surface. Our results demonstrated that surface modification with osteoinductive peptides could improve cytocompatibility of nanofibers in terms of cell adhesion, spreading, and proliferation. Most importantly, Alizarin Red S staining, quantitative real-time polymerase chain reaction, immunostaining, and Western blot revealed that human mesenchymal stem cells cultured on aligned nanofibers with osteoinductive peptides exhibited enhanced osteogenic differentiation potential than cells on randomly oriented nanofibers. Furthermore, the aligned nanofibers with osteoinductive peptides could direct osteogenic differentiation of human mesenchymal stem cells even in the absence of osteoinducting factors, suggesting superior osteogenic efficacy of biomimetic design that combines the advantages of osteoinductive peptide signal and highly ordered nanofibers on cell fate decision. The presented peptide-decorated bone-mimic nanofiber scaffolds hold a promising potential in the context of bone tissue engineering.

Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs).

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. Here, we investigate if BMP9 and Wnt/ß-catenin act synergistically on odontogenic differentiation. Using the immortalized SCAPs (iSCAPs) isolated from mouse apical papilla tissue, we demonstrate that Wnt3A effectively induces early osteogenic marker alkaline phosphatase (ALP) in iSCAPs, which is reduced by ß-catenin knockdown. While Wnt3A and BMP9 enhance each other's ability to induce ALP activity in iSCAPs, silencing ß-catenin significantly diminishes BMP9-induced osteo/odontogenic differentiation. Furthermore, silencing ß-catenin reduces BMP9-induced expression of osteocalcin and osteopontin and in vitro matrix mineralization of iSCAPs. In vivo stem cell implantation assay reveals that while BMP9-transduced iSCAPs induce robust ectopic bone formation, iSCAPs stimulated with both BMP9 and Wnt3A exhibit more mature and highly mineralized trabecular bone formation. However, knockdown of ß-catenin in iSCAPs significantly diminishes BMP9 or BMP9/Wnt3A-induced ectopic bone formation in vivo. Thus, our results strongly suggest that ß-catenin may play an important role in BMP9-induced osteo/ondontogenic signaling and that BMP9 and Wnt3A may act synergistically to induce osteo/odontoblastic differentiation of iSCAPs. It's conceivable that BMP9 and/or Wnt3A may be explored as efficacious biofactors for odontogenic regeneration and tooth engineering.

Bone morphogenetic protein-9 effectively induces osteo/odontoblastic differentiation of the reversibly immortalized stem cells of dental apical papilla.

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.

Crosstalk between Wnt/ß-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells.

Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/ß-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on ß-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERß expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERß expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.