Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway

Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.     

Promoting osteogenic differentiation of human-induced pluripotent stem cells by releasing Wnt/β-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/β-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.     

Stimulatory effect of 17β-estradiol on osteogenic differentiation potential of rat adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ADSCs) are considered as a potential cell source for regenerative medicine and tissue engineering. Although ADSCs have greater proliferation capacity than bone marrow stem cells (BMSCs), lower differentiation ability of these cells limits their utility in experimental and clinical studies. The purpose of this study was to investigate whether 17β-estradiol (E(2)) has a stimulatory effect on osteogenic differentiation potential of ADSCs in vitro. ADSCs were isolated from visceral adipose tissues of rats and treated with different concentrations of E(2) in osteogenic medium (OM) for 21 days. The differences in osteogenic differentiation potential of the cultures were assessed by von Kossa staining, measurement of alkaline phosphatase (ALP) activity and calcium levels. ADSCs cultured in OM supplemented with E(2) showed greater bone-like nodule formation and mineral deposition in comparing with the cells grown in OM. In addition, ALP activity and calcium levels also were significantly higher in the cultures exposed to E(2) than the cells treated only with OM (p < 0.005, n = 5). Our results suggest that E(2) may stimulate the osteogenic differentiation of ADSCs and therefore, can be used as an inducing agent to improve the efficiency of these cells in in vitro and in vivo studies.     

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.     

RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1–10 ng/ml) of TNF-α and decreased in high concentration (50–100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.     

Novel mini β-TCP 3D perfusion bioreactor for proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Applications of bioreactors in combination with scaffolding materials are promising in tissue-engineering fields. To rapidly produce bone mesenchymal stem cells (MSCs) suitable for osteogenic differentiation (OSD), we developed a novel technique for β-TCP (β-tricalcium phosphate) scaffolds preparation and employed these scaffolds to build a new type of mini three dimensional (3D) perfusion bioreactor. Compared to the 2D static culture, MSCs acquired much higher amplification rate and alkaline phosphatase (ALP) activity over a 24-day culture period. Interestingly, the Specific ALP activity was independent of the growth rate under adequate osteogenic inducement, suggesting there may be an OSD bottleneck at a single-cell level. Furthermore, cells on scaffolds exhibited gradually reduced total migration rate (MR) and relatively constant local MR, both ideal for bone regeneration. Excellent adhesion of MSCs to the smooth scaffolds surface, especially the layer structures, was seen on scanning electron microscopy images, demonstrating fine compatibility between scaffold and cells. Our results indicate this simplified, integrated and potentially modularizable 3D bioreactor could enable the osteocytes producing from MSCs for expected applications.     

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.     

Adenosine triphosphate enhances osteoblast differentiation of rat dental pulp stem cells via the PLC–IP3 pathway and intracellular Ca2+ signaling

Intracellular Ca²⁺ signals are essential for stem cell function and play a significant role in the differentiation process. Dental pulp stem cells (DPSCs) are a potential source of stem cells; however, the mechanisms controlling cell differentiation remain largely unknown. Utilizing rat DPSCs, we examined the effect of adenosine triphosphate (ATP) on osteoblast differentiation and characterized its mechanism of action using real-time Ca ²⁺ imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca ²⁺ deposition in the extracellular matrix via Alizarin Red S staining. This was consistent with upregulation of osteoblast genes BMP2, Mmp13, Col3a1, Ctsk, Flt1, and Bgn. Stimulation of DPSCs with ATP (1–300 µM) increased intracellular Ca ²⁺ signals in a concentration-dependent manner, whereas histamine, acetylcholine, arginine vasopressin, carbachol, and stromal-cell-derived factor-1α failed to do so. Depletion of intracellular Ca ²⁺ stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca ²⁺ free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP ₃) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca ²⁺ signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca ²⁺-dependent mechanism via the PLC-IP ₃ pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.     

Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-β activation

Transforming growth factor-β (TGF-β) is a critical regulator of bone development and remodeling. TGF-β must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-β activation and TSP1 control of TGF-β activation is critical for regulation of TGF-β activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-β inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-β activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-β. Cultured MSCs express TSP1 and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. TSP1 and active TGF-β block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-β, since a peptide which blocks TSP1 TGF-β activation reduced TGF-β activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-β activity is a critical determinant of osteoblast differentiation.     

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.     

Human dental pulp stem cells expressing transforming growth factor β3 transgene for cartilage-like tissue engineering

Background aimsThe aim of this study was to engineer sizable three-dimensional cartilage-like constructs using stem cells isolated from human dental pulp stem cells (DPSCs).MethodsHuman DPSCs were isolated from teeth extracted for orthodontic treatment and enriched further using immuno-magnetic bead selection for stem cell marker CD146. Chondrogenic lineage differentiation of DPSCs induced using recombinant transforming growth factor β3 (TGFβ3) was verified by pellet culture. Because the use of recombinant proteins is associated with rapid degradation and difficult in vivo administration, we constructed the recombinant adeno-associated viral vector encoding human TGFβ3 and determined the best multiplicity of infection for DPSCs. Transduced DPSCs were seeded on poly-l-lactic acid/polyethylene glycol (PLLA/PEG) electrospun fiber scaffolds demonstrating proper attachment, proliferation and viability as shown by scanning electron microscopy micrographs and CCK-8 cell counting kit. Scaffolds seeded with DPSCs were implanted in the back of nude mice.ResultsTransduced DPSCs highly expressed human TGFβ3 for up to 48 days and expressed chondrogenic markers collagen IIa1, Sox9 and aggrecan, as verified by immunohistochemistry and messenger RNA (mRNA). Immunohistochemistry for TGFβ3/DPSC constructs (n = 5/group) showed cartilage-like matrix formation with glycosaminoglycans. In vivo constructs with TGFβ3/DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSC constructs (n = 5/group). Western blot analysis confirmed this expression pattern on the protein level (n = 3/group).ConclusionsImmuno-selected DPSCs can be successfully differentiated toward chondrogenic lineage, while expressing the chondrogenic inducing factor. Seeded on PLLA/PEG electrospun scaffold, human DPSCs formed three-dimensional cartilage constructs that could prove useful in future treatment of cartilage defects.     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.     

Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.     

Effects of Wnt/β-catenin signalling on proliferation and differentiation of apical papilla stem cells

Objectives: The Wnt signalling pathway has been shown to play an important role in tooth development, however its effects with stem cells from the apical papilla (SCAP) have remained unclear. The purpose of this study was to determine effects of Wnt/β‐catenin on proliferation and differentiation of SCAP in vitro. Materials and methods: SCAP were obtained, identified and cultured. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of mineralization‐related genes and mineralized nodule formation were measured in presence or absence of various concentrations of lithium chloride. Results: MTT assay and flow cytometry demonstrated that Wnt/β‐catenin activity could promote proliferation of SCAP. Real‐time PCR analysis found that Wnt/β‐catenin strongly upregulated expression of dentine sialophosphoprotein, osteocalcin and ALP in SCAP after incubation with mineralization induction medium, while ALP and alizarin red staining indicated that Wnt/β‐catenin enhanced ALP activity and formation of mineralized nodules. Conclusion: Our results suggest that canonical Wnt/β‐catenin signalling promotes proliferation and odonto/osteogenic differentiation of SCAP.