Film-forming polymer solutions containing cholesterol myristate and berberine mediate pressure ulcer repair via the Wnt/ß-catenin pathway.

Pressure ulcer (PU) is a worldwide problem that is difficult to address because of the related inflammatory response, local hypoxia, and repeated ischaemia/reperfusion, causing great suffering and financial burden to patients. Traditional Chinese medicine turtle plate powder can treat skin trauma, but its composition is complex and inconvenient to use. Here, we combined cholesterol myristate (S8) with berberine (BBR), with anti-inflammatory and antibacterial effects, as a drug and used hydroxypropyl methylcellulose and polyvinylpyrrolidone K30 as carriers to construct a novel film-forming polymeric solution (S8 + BBR FFPS), comprehensively study its reparative effect on PU and explore the potential mechanism in rat PU models. The results showed that S8 + BBR FFPS inhibits excessive inflammatory response, promotes re-epithelialization, and promotes hair follicle growth during the healing process of PU, which may be related to the activation of the Wnt/ß-catenin signalling pathway by S8 + BBR FFPS to mediate hair follicle stem cell proliferation and maintain skin homeostasis. Therefore, S8 + BBR FFPS may be a potential candidate for the treatment of chronic skin injury, and its association with the Wnt/ß-catenin signalling pathway may provide new ideas to guide the design of biomaterial-based wound dressings for chronic wound repair.

Alendronate-induced Perturbation of the Bone Proteome and Microenvironmental Pathophysiology.

Objectives: Bisphosphonates (BPs) are powerful inhibitors of osteoclastogenesis and are used to prevent osteoporotic bone loss and reduce the risk of osteoporotic fracture in patients suffering from postmenopausal osteoporosis. Patients with breast cancer or gynecological malignancies being treated with BPs or those receiving bone-targeted therapy for metastatic prostate cancer are at increased risk of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Although BPs markedly ameliorate osteoporosis, their adverse effects largely limit the clinical application of these drugs. This study focused on providing a deeper understanding of one of the most popular BPs, the alendronate (ALN)-induced perturbation of the bone proteome and microenvironmental pathophysiology. Methods: To understand the molecular mechanisms underlying ALN-induced side-effects, an unbiased and global proteomics approach combined with big data bioinformatics was applied. This was followed by biochemical and functional analyses to determine the clinicopathological mechanisms affected by ALN. Results: The findings from this proteomics study suggest that the RIPK3/Wnt/GSK3/ß-catenin signaling pathway is significantly perturbed upon ALN treatment, resulting in abnormal angiogenesis, inflammation, anabolism, remodeling, and mineralization in bone cells in an in vitro cell culture system. Conclusion: Our investigation into potential key signaling mechanisms in response to ALN provides a rational basis for suppressing BP-induced adverse effect and presents various therapeutic strategies.

The Delivery of a Wnt Pathway Inhibitor Toward CSCs Requires Stable Liposome Encapsulation and Delayed Drug Release in Tumor Tissues.

The Wnt signaling pathway is involved in tumorigenesis and various stages of tumor progression, including the epithelial-mesenchymal transition, metastasis, and drug resistance. Many efforts have been made to develop drugs targeting this pathway. CGX1321 is a porcupine inhibitor that can effectively block Wnt ligand synthesis and is currently undergoing clinical trials. However, drugs targeting the Wnt pathway may frequently cause adverse events in normal tissues, such as the intestine and skin. Formulation of the drug inside liposomes could enable preferential drug delivery to solid tumor tissues and limit drug exposure in normal organs. We developed a strategy to stably encapsulate CGX1321 inside liposomes with minimal drug releases in circulation. The liposomal drugs were shown to interfere with the aberrant Wnt signaling specifically in tumor tissues, resulting in focused effects on LGR5+ CSCs (cancer stem cells), while sparing other cells from significant cytotoxicity. We showed it is feasible to use such a CSC elimination approach to treat malignant cancers prone to rapid progression using a LoVo tumor model as well as a GA007 patient derived xenograft (PDX) model. Nano drug delivery systems may be required for precision medicine in cancer therapy.

Drug Repurposing in Dentistry; towards Application of Small Molecules in Dentin Repair.

One of the main goals of dentistry is the natural preservation of the tooth structure following damage. This is particularly implicated in deep dental cavities affecting dentin and pulp, where odontoblast survival is jeopardized. This activates pulp stem cells and differentiation of new odontoblast-like cells, accompanied by increased Wnt signaling. Our group has shown that delivery of small molecule inhibitors of GSK3 stimulates Wnt/ß-catenin signaling in the tooth cavity with pulp exposure and results in effective promotion of dentin repair. Small molecules are a good therapeutic option due to their ability to pass across cell membranes and reach target. Here, we investigate a range of non-GSK3 target small molecules that are currently used for treatment of various medical conditions based on other kinase inhibitory properties. We analyzed the ability of these drugs to stimulate Wnt signaling activity by off-target inhibition of GSK3. Our results show that a c-Met inhibitor, has the ability to stimulate Wnt/ß-catenin pathway in dental pulp cells in vitro at low concentrations. This work is an example of drug repurposing for dentistry and suggests a candidate drug to be tested in vivo for natural dentin repair. This approach bypasses the high level of economical and time investment that are usually required in novel drug discoveries.

Promoting osteogenic differentiation of human-induced pluripotent stem cells by releasing Wnt/ß-catenin signaling activator from the nanofibers.

Implants that can enhance the stem cells differentiation in the absence of the chemical osteogenic growth factors will attract the great interest of orthopedic scientists. Inorganic polyphosphate (poly-P), as a ubiquitous biological polymer, is one of the factors that can be an alternative for osteogenic growth factors via activating Wnt/ß-catenin signaling. In this study, poly-P was incorporated at the blend of polycaprolactone (PCL)/poly (l-lactic acid) (PLLA) electrospun nanofibers and then osteogenic differentiation potential of human-induced pluripotent stem cells (iPSCs) was investigated by the important bone markers. 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) and scanning electron microscopy results confirmed the biocompatibility of the fabricated nanofibers, while higher proliferation rate of iPSCs was detected in PCL-PLLA(poly-P) group compared with the PCL-PLLA and tissue culture plate groups. Alkaline phosphatase activity, calcium content, and gene expression results demonstrated that osteogenic differentiation of iPSCs was increased when cultured on PCL-PLLA(poly-P) in comparison with other groups. According to the results, PCL-PLLA(poly-P) could be considered as a promising candidate for use as bone implants.

A synthetic DNA-binding inhibitor of SOX2 guides human induced pluripotent stem cells to differentiate into mesoderm.

Targeted differentiation of human induced pluripotent stem cells (hiPSCs) using only chemicals would have value-added clinical potential in the regeneration of complex cell types including cardiomyocytes. Despite the availability of several chemical inhibitors targeting proteins involved in signaling pathways, no bioactive synthetic DNA-binding inhibitors, targeting key cell fate-controlling genes such as SOX2, are yet available. Here, we demonstrate a novel DNA-based chemical approach to guide the differentiation of hiPSCs using pyrrole-imidazole polyamides (PIPs), which are sequence-selective DNA-binding synthetic molecules. Harnessing knowledge about key transcriptional changes during the induction of cardiomyocyte, we developed a DNA-binding inhibitor termed PIP-S2, targeting the 5'-CTTTGTT-3' and demonstrated that inhibition of SOX2-DNA interaction by PIP-S2 triggers the mesoderm induction in hiPSCs. Genome-wide gene expression analyses revealed that PIP-S2 induced mesoderm by targeted alterations in SOX2-associated gene regulatory networks. Also, employment of PIP-S2 along with a Wnt/ß-catenin inhibitor successfully generated spontaneously contracting cardiomyocytes, validating our concept that DNA-binding inhibitors could drive the directed differentiation of hiPSCs. Because PIPs can be fine-tuned to target specific DNA sequences, our DNA-based approach could be expanded to target and regulate key transcription factors specifically associated with desired cell types.

Regeneration of large bone defects using mesoporous silica coated magnetic nanoparticles during distraction osteogenesis.

Distraction osteogenesis (DO) represents an effective but undesirably lengthy treatment for large bone defects. Both magnetic nanoparticles and silicon have been shown to induce osteogenic differentiation of mesenchymal stem cells (MSCs), the key participant in bone regeneration. We herein synthesized mesoporous silica coated magnetic (Fe3O4) nanoparticles (M-MSNs) and evaluated its potential for acceleration of bone regeneration in a rat DO model. The M-MSNs exhibited good biocompatibility and remarkable capability in promoting the osteogenic differentiation of MSCs via the canonical Wnt/ß-catenin pathway in vitro. More importantly, local injection of M-MSNs dramatically accelerated bone regeneration in a rat DO model according to the results of X-ray imaging, micro-CT, mechanical testing, histological examination, and immunochemical analysis. This study demonstrates the notable potential of M-MSNs in promoting bone regeneration during DO by enhancing the osteogenic differentiation of MSCs, paving the way for clinical translation of M-MSNs in DO to repair large bone defects.

Intermittent compressive force induces human mandibular-derived osteoblast differentiation via WNT/ß-catenin signaling.

Mechanical force induces an efflux of ATP that regulates osteoblast differentiation. However, the effect of mechanical force-induced ATP efflux on WNT/ß-catenin signaling remains unclarified. The aim of this study was to investigate the effect of intermittent compressive force (ICF) and ICF-induced extracellular ATP on osteoblast differentiation via WNT/ß-catenin signaling in human mandibular-derived osteoblast precursors (hMOBPs). The hMOBPs were subjected to ICF (1.5 g/cm2 , 0.3 Hz) for 20 h. To investigate the role of ATP, Apyrase (0.5 units/mL), an enzyme that hydrolyzes ATP, was added 30 min before ICF was applied. The extracellular ATP levels were measured immediately after ICF was removed. The mRNA expression of osteogenic related genes, including WNT was evaluated via quantitative real time polymerase chain reaction. In vitro mineralization was determined by Alizarin Red S staining. The localization of ß-catenin was detected using immunofluorescence staining and lentiviral-TOP-dGFP reporter assay. The results demonstrated that ICF increased ATP efflux and in vitro mineralization by hMOBPs. In addition, OSX, ALP, and WNT3A mRNA expression and ß-catenin nuclear translocation increased when ICF was applied. The upregulation of these genes was reduced by Apyrase, suggesting the role of ICF-induced ATP on osteoblast differentiation. Notably, ICF altered the mRNA expression of purinergic 2X receptors (P2XRs). A P2X1R antagonist (NF449) downregulated ICF-induced WNT3A, OSX, and ALP mRNA expression. Moreover, when 25 µM α, ß-meATP, a P2X1R agonist, was added, WNT3A, and OSX expression increased. In conclusion, our results demonstrate that ICF-induced ATP enhanced hMOBP differentiation. This enhancement was associated with WNT/ß-catenin signaling and P2X1R activation.

Nicotine inhibits osteogenic differentiation of human periodontal ligament cells under cyclic tensile stress through canonical Wnt pathway and α7 nicotinic acetylcholine receptor.

BACKGROUND AND OBJECTIVES: Nicotine, the main psychoactive component of tobacco, affects cell metabolism, proliferation, adhesion and, importantly, the osteogenic differentiation of fibroblasts. Approximately 15% of all orthodontic patients are adults among who one-fifth are smokers. Hence, it is necessary to have insight into the effects of nicotine on the osteogenic differentiation of hPDLCs during orthodontic tooth movement. This study aimed to investigate the effects and mechanisms of nicotine on the osteogenic differentiation of human periodontal ligament cells (hPDLCs) under the application of cyclic tensile stress. MATERIAL AND METHODS: hPDLCs were obtained from donor third molars. The hPDLCs were treated with nicotine and/or cyclic tensile stress that was applied with a cell stress plus unit. The effect of nicotine on cell viability was analyzed using the MTT assay. The osteogenic differentiation of hPDLCs was detected by alkaline phosphatase staining, Alizarin Red S staining, quantitative real-time polymerase chain reaction and western blotting. RESULTS: In combination with cyclic tensile stress, nicotine prevented the tensile stress-induced increase in alkaline phosphatase activity, formation of mineralization nodules and the upregulation of mRNA and protein expression of Runt-related transcription factor 2, transcription factor Sp7 and collagen type I; however, canonical Wnt pathway was activated. Furthermore, the addition of Dickkopf-related protein 1 and α-bungarotoxin counteracted the negative effect of nicotine and rescued the osteogenic differentiation of hPDLCs, respectively. CONCLUSION: These results indicate that nicotine prevents the increased osteogenic potential of hPDLCs induced by cyclic tensile stress by binding to an α7 nicotinic acetylcholine receptor and activating the canonical Wnt pathway.

l-Quebrachitol Promotes the Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells: Involvement of the BMP-2/Runx2/MAPK/Wnt/ß-Catenin Signaling Pathway.

Osteoporosis is widely recognized as a major health problem caused by an inappropriate rate of bone resorption compared to bone formation. Previously we showed that d-pinitol inhibits osteoclastogenesis but has no effect on osteoblastogenesis. However, the effect on osteoblast differentiation of its isomer, l-quebrachitol, has not yet been reported. The purpose of this study was, therefore, to investigate whether l-quebrachitol promotes the osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. Moreover, the molecular mechanism of action of l-quebrachitol was further explored. Here, it is shown for the first time that l-quebrachitol significantly promotes proliferation and cell DNA synthesis. It also enhances mineralization accompanied by increases in mRNA expression of bone matrix proteins including alkaline phosphatase (ALP), collagen type I (ColI), osteocalcin (OCN), and osteopontin (OPN). In addition, l-quebrachitol upregulates the mRNA and protein expression of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), while down-regulating the receptor activator of the nuclear factor-κB ligand (RANKL) mRNA level. Moreover, the expression of regulatory genes associated with the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/ß-catenin signaling pathways are also upregulated. These findings indicate that l-quebrachitol may promote osteoblastogenesis by triggering the BMP-2-response as well as the Runx2, MAPK, and Wnt/ß-catenin signaling pathway.

Wnt/ß-Catenin Signaling Determines the Vasculogenic Fate of Postnatal Mesenchymal Stem Cells.

Vasculogenesis is the process of de novo blood vessel formation observed primarily during embryonic development. Emerging evidence suggest that postnatal mesenchymal stem cells are capable of recapitulating vasculogenesis when these cells are engaged in tissue regeneration. However, the mechanisms underlining the vasculogenic differentiation of mesenchymal stem cells remain unclear. Here, we used stem cells from human permanent teeth (dental pulp stem cells [DPSC]) or deciduous teeth (stem cells from human exfoliated deciduous teeth [SHED]) as models of postnatal primary human mesenchymal stem cells to understand mechanisms regulating their vasculogenic fate. GFP-tagged mesenchymal stem cells seeded in human tooth slice/scaffolds and transplanted into immunodeficient mice differentiate into human blood vessels that anastomize with the mouse vasculature. In vitro, vascular endothelial growth factor (VEGF) induced the vasculogenic differentiation of DPSC and SHED via potent activation of Wnt/ß-catenin signaling. Further, activation of Wnt signaling is sufficient to induce the vasculogenic differentiation of postnatal mesenchymal stem cells, while Wnt inhibition blocked this process. Notably, ß-catenin-silenced DPSC no longer differentiate into endothelial cells in vitro, and showed impaired vasculogenesis in vivo. Collectively, these data demonstrate that VEGF signaling through the canonical Wnt/ß-catenin pathway defines the vasculogenic fate of postnatal mesenchymal stem cells. Stem Cells 2016;34:1576-1587.

Notch/Wnt cross-signalling regulates stemness of dental pulp stem cells through expression of neural crest and core pluripotency factors.

Dental pulp stem cells (DPSCs) from adult teeth express neural crest (NC) markers together with core transcriptional factors associated with stem cell pluripotency, such as Oct4a, Sox2, c-Myc, Rex1, Stella/Dppa3, Ssea1/Fut4, Lin28 and Nanog. The possibility to boost the natural stemness features of DPSCs by mild methods, that do not involve gene and/or chromatin modification or gene transfection, is highly desirable for cell therapy. Canonical Wnt and Notch are two highly conserved developmental signalling pathways that are involved in NC emergence and stem cell self-renewal. We determined that both pathways coordinate to regulate the expression of core pluripotency and NC factors in DPSCs. Pharmacological inhibition of the Notch pathway for 48 h, by the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), abolished the expression of NC and core factors. In addition, it induced a silencing of the canonical Wnt signalling and a clear reduction in the stemness potential of DPSCs, as shown by a reduced ability to generate mature, fully differentiated osteoblasts and adipocytes. Conversely, pharmacological activation of the Wnt pathway for 48 h, by either the glycogen synthase kinase 3 beta (GSK3-ß) inhibitor 6-bromoindirubin-3´-oxime (BIO) or the human recombinant protein Wnt-3a, not only largely increased the expression of NC and core factors, but also increased the efficiency of DPSCs to differentiate into mature osteoblasts and adipocytes. These results showed that a short preconditioning activation of Wnt/Notch signalling by small molecules and/or recombinant proteins enhanced the stemness and potency of DPSCs in culture, which could be useful for optimising the therapeutic use of these and other tissue-specific stem cells.

Porphyromonas gingivalis lipopolysaccharides affect gingival stem/progenitor cells attributes through NF-κB, but not Wnt/ß-catenin, pathway.

AIM: This study investigates for the first time the effect of Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) on proliferative/regenerative aptitudes of gingival stem/progenitor cells (G-MSCs). MATERIAL AND METHODS: G-MSCs (n = 5) were treated by 0, 10 ng/ml, 100 ng/ml, 1 µg/ml or 10 µg/ml Pg-LPS. At 1 hour, Toll-like receptor 4 (TLR-4) expression and NF-κB and Wnt/ß-catenin signalling pathways were examined. Colony-forming unit assay was conducted at day 12. At 24 and 48 hours, MTT test, ALP activity, mRNA for tumour necrosis factor-α (TNF-α), interleukin-6, collagen-I (Col-I), collagen-III, RUNX-2, alkaline phosphatase (ALP), osteonectin and protein expression of interleukin-6 and TNF-α were analysed. RESULTS: With increasing Pg-LPS, TLR-4 was upregulated, pNF-κB-p65 rose from median (Q25/Q75) 6.56% (4.19/7.90) to 13.02% (8.90/16.50; p = 0.002) and pNF-κB-p65/tNF-κB-p65 from 0.14(0.10/0.17) to 0.30(0.21/0.42; p = 0.002). pß-Catenin, tß-catenin and pß-catenin/tß-catenin showed no differences. Increasing Pg-LPS concentration increased cell numbers from 288.00(72.98/484.32) to 861.39 (540.41/1599.94; p = 0.002), ALP mRNA from 0.00(0.00/0.01) to 0.56(0.00/1.90; p = 0.004) and TNF-α from 32.47(12.11/38.57) to 45.32(28.68/48.65; p = 0.036). Over time, ALP activity increased from 0.89(0.78/0.95) to 1.90(1.83/2.09; p < 0.001), mRNA for TNF-α from 0.00(0.00/0.12) to 0.01(0.00/0.06; p = 0.007), mRNA for Col-I from 82.70(0.03/171.50) to 124.00(52.85/232.50; p = 0.019), while mRNA for RUNX-2 decreased from 1.73(0.92/3.20) to 0.84(0.48/1.47; p = 0.005). CONCLUSIONS: Pg-LPS upregulated G-MSCs' proliferation, without attenuation of their regenerative potential. The effects were NF-κB, but not Wnt/ß-catenin, pathway dependent.

Pamidronate Disodium Leads to Bone Necrosis via Suppression of Wnt/ß-Catenin Signaling in Human Bone Marrow Mesenchymal Stem Cells In Vitro.

PURPOSE: Pamidronate disodium-associated bone necrosis is poorly understood at the cellular and molecular levels. This study proposes a pathway leading to the pamidronate disodium-mediated inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMMSCs) derived from the mandible in vitro. MATERIALS AND METHODS: Primary human BMMSCs were isolated from the mandible and marrow tissue. A proliferation assay was performed to determine the experimental concentration of pamidronate disodium. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin red S (ARS) staining were assessed after treatment with pamidronate disodium (0, 0.1, 0.5, 1, 5, 10 µg/mL). Quantitative real-time polymerase chain reaction and western blotting specific for Wnt and ß-catenin signaling genes or proteins were performed after treatment with pamidronate disodium 0.5 µg/mL. Wnt3a was used to observe the osteogenic differentiation of BMMSCs during treatment with pamidronate disodium 0.5 µg/mL. RESULTS: As expected, pamidronate disodium 1, 5, and 10 µg/ml were unfavorable for BMMSC growth (P < .05), whereas 0.1 and 0.5 µg/mL did not affect BMMSC growth (P ≥ .05). BMMSCs treated with pamidronate disodium 0.5 µg/mL had lower ALP activity, ALP staining, and ARS staining (P < .05), and BMMSCs treated with low concentrations (<0.5 µg/mL) of pamidronate disodium had the same levels of ALP activity, ALP staining, and ARS staining as the control (0 µg/mL). Pamidronate disodium 0.5 µg/mL decreased the expression of genes and proteins involved in Wnt and ß-catenin signaling. BMMSCs with Wnt3a and pamidronate disodium 0.5 µg/mL had higher ALP activity, ALP staining, and ARS staining (P < .05). CONCLUSIONS: Pamidronate disodium inhibited Wnt and ß-catenin signaling, which controls osteogenic differentiation in BMMSCs. Wnt3a, a Wnt and ß-catenin signaling activator, reversed the negative effects caused by pamidronate disodium to salvage the osteogenic defect in BMMSCs.

TNF-α was involved in calcium hydroxide-promoted osteogenic differentiation of human DPSCs through NF-κB/p38MAPK/Wnt pathway.

It has been reported that calcium hydroxide can induce proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying molecular mechanisms are still unclear. In this study, we sought to explore the role of calcium hydroxide in the cell proliferation and directional differentiation of DPSCs and to study the regulatory effect of NF-κB, p38MAPK, and Wnt signaling on differentiation of DPSCs. CCK8 cell assay, Wound Healing Assay, and Alkaline Phosphatase Staining Assay were respectively used to determine the proliferation rate, migration and ALP expression of DPSCs. Alizarin Red Staining Assay was used to observe the mineralization of DPSCs. RT-PCR analysis and Western Blot Analysis displayed the expression of related fators at mRNA and protein level, respectively. In the present study, we found that NF-κB, p38MAPK, and Wnt signaling could abolish calcium hydroxide-induced proliferation of DPSCs. The inhibition of NF-κB, p38MAPK, and Wnt signaling suppressed the migration, ALP expression, and mineralization of DPSCs. NF-κB, p38MAPK, and Wnt signaling involved in directional differentiation of DPSCs. Moverover, calcium hydroxide could activate NF-κB, p38MAPK, and Wnt pathway by regulating TNF-α. Our study showed that NF-κB, p38MAPK, and Wnt signaling pathway were involved in calcium hydroxide-induced proliferation, migration, mineralization, and osteogenic differentiation in DPSCs. Calcium hydroxide affected NF-κB, p38MAPK, and Wnt pathway by regulating TNF-α.

Chitosan scaffolds induce human dental pulp stem cells to neural differentiation: potential roles for spinal cord injury therapy.

Cell-based transplantation strategies hold great potential for spinal cord injury (SCI) repair. Chitosan scaffolds have therapeutic benefits for spinal cord regeneration. Human dental pulp stem cells (DPSCs) are abundant available stem cells with low immunological incompatibility and can be considered for cell replacement therapy. The purpose of this study is to investigate the role of chitosan scaffolds in the neural differentiation of DPSCs in vitro and to assess the supportive effects of chitosan scaffolds in an animal model of SCI. DPSCs were incubated with chitosan scaffolds. Cell viability and the secretion of neurotrophic factors were analyzed. DPSCs incubated with chitosan scaffolds were treated with neural differentiation medium for 14 days and then neural genes and protein markers were analyzed by Western blot and reverse transcription plus the polymerase chain reaction. Our study revealed a higher cell viability and neural differentiation in the DPSC/chitosan-scaffold group. Compared with the control group, the levels of BDNF, GDNF, b-NGF, and NT-3 were significantly increased in the DPSC/chitosan-scaffold group. The Wnt/ß-catenin signaling pathway played a key role in the neural differentiation of DPSCs combined with chitosan scaffolds. Transplantation of DPSCs together with chitosan scaffolds into an SCI rat model resulted in the marked recovery of hind limb locomotor functions. Thus, chitosan scaffolds were non-cytotoxic and provided a conducive and favorable microenvironment for the survival and neural differentiation of DPSCs. Transplantation of DPSCs might therefore be a suitable candidate for treating SCI and other neuronal degenerative diseases.

Mesenchymal Wnt/ß-Catenin Signaling Controls Epithelial Stem Cell Homeostasis in Teeth by Inhibiting the Antiapoptotic Effect of Fgf10.

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/ß-catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/ß-catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no ß-catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO-treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/ß-catenin. Stem Cells 2015;33:1670-1681.

Gene-expression analysis of cementoblasts and osteoblasts.

BACKGROUND AND OBJECTIVE: Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice. MATERIAL AND METHODS: OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization. RESULTS: Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts. CONCLUSION: The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.

Lithium chloride promotes the odontoblast differentiation of hair follicle neural crest cells by activating Wnt/ß-catenin signaling.

The Wnt/ß-catenin signalling pathway contributes to the maintenance of pluripotency and partial reprogramming of stem cells. Postnatal neural crest cells (NCCs) can differentiate into odontoblast-like cells due to their multi-potential property, but further endeavors need to be made to promote odontogenic differentiation of hair follicle neural crest cells (hfNCCs). This study investigated whether the Wnt pathway activator lithium chloride (LiCl) promotes odontoblast differentiation of hfNCCs. Change of proliferation, ß-catenin and pluripotency markers of hfNCCs were examined after treatment with LiCl. An in vitro odontoblast differentiation model of hfNCCs was built using dental cell conditioned media (DC-CM). The effects of LiCl on odontoblast differentiation of hfNCCs showed that proliferation and expression of ß-catenin in the cytosolic and nuclear compartments were increased in the LiCl-treated hfNCCs, and the pluripotency marks, Oct4, Klf4, Sox2 and Nanog, were more highly expressed in the LiCl-treated group than in the control group. The odontoblast markers such as DSP, DMP1 and Runx2, could be detected in hfNCCs induced by DC-CM, but in LiCl -treated group all three markers had stronger expression. Expression of ß-catenin in the nuclear of LiCl-treated hfNCCs induced by DC-CM was higher than in the other groups. The data indicate that the Wnt pathway activator LiCl can promote proliferation and odontoblast differentiation of hfNCCs, and chemical approaches are of benefit in obtaining more desirable seed cell types for cell-based therapies.

Effects of Er-Zhi-Wan on microarchitecture and regulation of Wnt/ß-catenin signaling pathway in alveolar bone of ovariectomized rats.

Recent studies have shown that Er-Zhi-Wan (EZW), a traditional Chinese medicine consisting of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL), had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan (EZW) on the microarchitecture and the regulation of Wnt/ß-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups: sham operation group (sham, n=10), ovariectomy (OVX) group (n=10), and OVX with EZW treatment group (EZW group, n=10). From one week after ovariectomy, EZW (100 mg/mL) or vehicle (distilled water) was fed (1 mL/100 g) once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchitecture, serum levels of E2, bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b (TRAP5b), as well as mandibular mRNA expression of Wnt/ß-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), ß-catenin and dickkopf homolog 1 (DKK1). The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Furthermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and ß-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/ß-catenin signaling pathway.