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The imbalance between bone resorption and bone formation in favor of resorption results in bone loss and deterioration of bone architecture. Osteoblast differentiation is a sequential event accompanying biogenesis of matrix vesicles and mineralization of collagen matrix with hydroxyapatite crystals. Considerable efforts have been made in developing naturally-occurring plant compounds, preventing bone pathologies, or enhancing bone regeneration. Coumarin aesculetin inhibits osteoporosis through hampering the ruffled border formation of mature osteoclasts. However, little is known regarding the effects of aesculetin on the impairment of matrix vesicle biogenesis. MC3T3-E1 cells were cultured in differentiation media with 1-10 μM aesculetin for up to 21 days. Aesculetin boosted the bone morphogenetic protein-2 expression, and alkaline phosphatase activation of differentiating MC3T3-E1 cells. The presence of aesculetin strengthened the expression of collagen type 1 and osteoprotegerin and transcription of Runt-related transcription factor 2 in differentiating osteoblasts for 9 days. When ≥1-5 μM aesculetin was added to differentiating cells for 15-18 days, the induction of non-collagenous proteins of bone sialoprotein II, osteopontin, osteocalcin, and osteonectin was markedly enhanced, facilitating the formation of hydroxyapatite crystals and mineralized collagen matrix. The induction of annexin V and PHOSPHO 1 was further augmented in ≥5 μM aesculetin-treated differentiating osteoblasts for 21 days. In addition, the levels of tissue-nonspecific alkaline phosphatase and collagen type 1 were further enhanced within the extracellular space and on matrix vesicles of mature osteoblasts treated with aesculetin, indicating matrix vesicle-mediated bone mineralization. Finally, aesculetin markedly accelerated the production of thrombospondin-1 and tenascin C in mature osteoblasts, leading to their adhesion to preformed collagen matrix. Therefore, aesculetin enhanced osteoblast differentiation, and matrix vesicle biogenesis and mineralization. These findings suggest that aesculetin may be a potential osteo-inductive agent preventing bone pathologies or enhancing bone regeneration.

Background: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes., Methods: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence., Results: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway., Conclusions: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells.

Cirsium setidens (Dunn) Nakai, commonly known as gondre, is a perennial herb that grows predominantly in South Korea. It contains several bioactive phytochemicals with antioxidant, anti‑cancer, anti‑tumor and anti‑inflammatory properties. The present study aimed to investigate the effects of methanolic extracts of gondre on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). As characterized by nuclear magnetic resonance spectroscopy and matrix‑assisted laser deposition/ionization (time‑of‑flight) mass spectrometry, the methanol extract of gondre was found to be enriched with pectolinarin. After 48 h, enhanced viability of hPDLSCs was observed in the presence of gondre compared with under control conditions, suggesting the biocompatibility of gondre. Notably, biocompatibility was markedly affected by gondre concentration in cultured media. Relatively high cell viability was observed in medium containing 0.05% gondre. Furthermore, mineralization was significantly higher in hPDLSCs in the presence of gondre compared with that in control cells, indicating their mineralization potential. Increased expression of various transcription markers, such as collagen 1, runt‑related transcription factor 2, bone sialoprotein and alkaline phosphatase, was also detected when hPDLSCs were stimulated with gondre compared with in the control groups, further confirming the superior osteogenic potential of gondre extract for tissue engineering applications, particularly in bone tissues.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 μl of 1,25(OH) 2 D 3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 μl of extract/L containing 0.4 μg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 μg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 μg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals., (© 2020 Blackwell Verlag GmbH.)

Strategies aimed at delaying the onset of bone tissue degeneration and the resulting skeletal fragility are key to decrease the risk of bone fracture correlated to ageing. The therapeutic properties of sulfurous thermal waters (STWs), rich in hydrogen sulfide (H 2 S), have been claimed for centuries. However, the direct regulation of bone cells by STWs has not been investigated yet. Here we aimed at analyzing the effect of STWs on cultured human mesenchymal stromal cells (hMSCs) derived from bone tissue. Two concentrations of STWs from 2 health spa centers in Italy (here named STW-1 and STW-2) containing, respectively, high and moderate quantities of H 2 S, were added to the culture media. Cytotoxicity and osteogenic differentiation were evaluated. We provided first evidence that treatment of hMSCs with STWs results in a sharp increase in intracellular H 2 S content, coherent with the different concentrations of H 2 S, thereby reveling that STWs-released H 2 S is internalized by cells. STWs treatment significantly induced osteogenic differentiation of hMSCs. In particular, mineral apposition was increased with a similar pattern by the two STWs, while mRNA expression of osteogenic markers (BSP, OC, RUNX-2, OPN) was differently affected. Only STW-2 induced a significant, dose-dependent increase in these gene expression. These findings support the rationale for the use of STWs as a complementary treatment of bone wasting diseases., (Copyright © 2020. Published by Elsevier Masson SAS.)

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 ( ID2 ) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2 , Osterix , and IBSP , which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature.

Previous studies have suggested that platycodin D is implicated in bone biology and ameliorates osteoporosis development. Platycodin D repressed the osteoclast activity and enhanced bone mineral density in the mouse model. However, the effects of platycodin D on osteoblast differentiation have not been elucidated yet. In C3H10T1/2 cells, platycodin D upregulated osteogenic markers including alkaline phosphatase (ALP), bone sialoprotein, and collagen type 1 alpha 1, and transcription factors, such as Runx2 and osterix, subsequently enhancing the bone mineralization. In a molecular mechanism study, platycodin D induced β-catenin nuclear accumulation by upregulating GSK3β phosphorylation. Furthermore, platycodin D upregulated the ALP activity and enhanced the mineralization process in osteoblast cells via the sirtuin 1/β-catenin pathways. Taken together, these results suggested that platycodin D could be an effective therapeutic compound against osteoporosis because of its regulatory effects during the osteoblast differentiation., (© 2019 Wiley Periodicals, Inc.)

The main objective of this study was to enhance the biological performance of resorbable polymeric scaffolds for bone tissue engineering. Specifically, we focused on both microstructure and surface modification of the scaffolds to augment adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC). Moreover, a new cell seeding method assuring 90% seeding efficiency on the scaffolds was developed. Poly(l‑lactide‑co‑glycolide) (PLGA) scaffolds with monomodal and bimodal pore distribution were produced by solvent casting/phase separation followed by porogen leaching and modified with artificial extracellular matrices (aECM) consisting of collagen type I and high sulphated hyaluronan (sHya). The application of two porogens resulted in bimodal pore distribution within the PLGA scaffolds as shown by scanning electron microscopy and microcomputer tomography. Two types of pores with diameters 400-600 μm and 2-20 μm were obtained. The scaffolds were successfully coated with a homogenous layer of aECM as shown by Sirius red and toluidine blue staining. In vitro study showed that presence of bimodal pore distribution in combination with collagen/sHya did not significantly influence hMSC proliferation and early osteogenic differentiation compared to scaffolds with monomodal pore distribution. However, it enhanced mineralization as well as the expression of Runt-related transcription factor 2, osteopontin and bone sialoprotein II. As a result PLGA scaffolds with bimodal pore distribution modified with collagen/sHya can be considered as prospective material promoting bone regeneration., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Insufficient bone volume remains a key issue when using dental implants. Adipose tissue‑derived stem cells (ADSCs) can accelerate bone healing when combined with dental implants. To improve the application of ADSCs for dental uses, the present study aimed to identify optimal implantation conditions. Mesenchymal stem cell‑derived exosomes can induce naïve stem cells to differentiate through the osteogenic lineage. In the present study, exosomes derived from 3T3L1 preadipocytes (3T3L1‑exo) were purified and characterized. The effects and potential mechanisms of 3T3L1‑exo on 3T3L1 cell ossification were examined by reverse transcription‑quantitative polymerase chain reaction, western blotting, electron microscopy, RNA sequencing and histological analysis. The current study confirmed that 3T3L1‑exo enhanced 3T3L1 preadipocyte osteogenic differentiation, as revealed by upregulation of osteogenic differentiation‑associated genes and increased Alizarin Red staining. Furthermore, the microRNA (miR) expression profiles of 3T3L1‑exo and 3T3L1 preadipocytes were sequenced and compared. The results of a further analysis demonstrated that miR‑223 expression was reduced in 3T3L1 preadipocytes stimulated by 3T3L1‑exo compared with in unstimulated cells. This finding suggested that 3T3L1‑exo promoted 3T3L1 bone formation by decreasing miR‑223 through a competitive mechanism, another miRNA, or another factor. The mechanism by which miR‑223 is decreased warrants further investigation. In conclusion, the application of 3T3L1‑exo may be useful for investigating preadipocyte‑induced bone regeneration.

Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.

Objective: The role of Hertwig's epithelial root sheath (HERS) cells in periodontal formation has been controversial. This study aimed to further clarify whether HERS cells participate in formation of the periodontium, and the necessity of HERS cells in differentiation of dental follicle cells (DFCs) for periodontal regeneration., Design: HERS cells and DFCs were isolated and identified from post-natal 7-day Sprauge-Dawley rats. In vitro, direct co-culture of HERS cells and DFCs as well as the individual culture of HERS and DFCs were performed and followed by alizarin red staining and the quantitative real-time polymerase chain reaction analysis. For in vivo evaluation, the inactivated dentin matrix (iTDM) was fabricated. HERS cells and DFCs were seeded in combination or alone on iTDM and then transplanted into the rat omentum. Scanning electron microscope and further histological analysis were carried out., Results: In vitro, mineral-like nodules were found in the culture of HERS cells alone or HERS + DFCs either by alizarin red staining or scanning electronic microscope. The mineralization and fiber-forming relevant mRNA expressions, such as bone sialoprotein, osteopontin, collagen I and collagen III in HERS + DFCs were significantly higher than that of the HERS or DFCs alone group. After transplantation in vivo, cementum and periodontal ligament-like tissues were formed in groups of HERS + DFCs and HERS alone, while no evident hard tissues and attached fibers were found in DFCs alone., Conclusions: Hertwig's epithelial root sheath cells directly participate in the formation of the periodontium, and they are essential for the differentiation of dental follicle cells to form periodontal structures. The combination use of Hertwig's epithelial root sheath cells and dental follicle cells is a promising approach for periodontal regeneration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background and Objective: Induced pluripotent stem cells (iPSCs) are a candidate cell source in periodontal regenerative therapy. Enamel matrix derivative (EMD) has been shown to regenerate periodontal tissues, and atelocollagen sponge (ACS) is considered a suitable scaffold or carrier for growth factors. This study aimed to investigate the effect of combined use of EMD and an ACS scaffold on cell behaviors and differentiation of mouse iPSCs (miPSCs) in vitro., Material and Methods: Following embryonic body formation from miPSCs, dissociated cells (miPS-EB-derived cells) were seeded onto ACS with or without EMD, and cultured in osteoblast differentiation medium. Scanning electron microscopy and histological analyses were used to assess cell morphology and infiltration within the ACS. Cell viability (metabolism) was determined using an MTS assay, and expression of mRNA of osteoblastic differentiation markers was assessed by quantitative RT -PCR. Alkaline phosphatase (ALP) staining intensity and activity were evaluated. Mineralization was assessed by von Kossa staining, and calcium content was quantitated using the methylxylenol blue method., Results: By 24 hours after seeding, miPS-EB-derived cells in both the EMD and control groups had attached to and infiltrated the ACS scaffold. Scanning electron microscopy images revealed that by day 14, many cytoplasmic protrusions and extracellular deposits, suggestive of calcified matrix, were present in the EMD group. There was a time-dependent increase in cell viability up to day 3, but no difference between groups was observed at any time point. The levels expressed of ALP and osterix genes were significantly higher in the EMD group than in the control group. Expression of runt-related transcription factor 2 was increased in the EMD group compared with the control group on day 7. EMD upregulated the expression of bone sialoprotein and osteopontin on day 14, whereas expression of osteocalcin was lower at all time points. The staining intensity and activity of ALP were higher in the EMD group than in the control group. Mineralization levels and calcium contents were significantly higher in the EMD group throughout the observation period., Conclusion: These data suggest that combining ACS with EMD increases levels of osteoblastic differentiation and mineralization in miPS-EB-derived cells, compared with ACS used alone., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In situ tissue engineering has become a novel strategy to repair periodontal/bone tissue defects. The choice of cytokines that promote the recruitment and proliferation, and potentiate and maintain the osteogenic differentiation ability of mesenchymal stem cells (MSCs) is the key point in this technique. Stromal cell‑derived factor‑1 (SDF‑1) and basic fibroblast growth factor (bFGF) have the ability to promote the recruitment, and proliferation of MSCs; however, the differential effect of SDF‑1 and bFGF pretreatment on MSC osteogenic differentiation potency remains to be explored. The present study comparatively observed osteogenic differentiation of bone morrow MSCs (BMMSCs) pretreated by bFGF or SDF‑1 in vitro. The gene and protein expression levels of alkaline phosphatase (ALP), runt related transcription factor 2 (Runx‑2) and bone sialoprotein (BSP) were detected using reverse transcription‑quantitative polymerase chain reaction and western blotting. The results showed that the expression of ALP mRNA on day 3, and BSP and Runx‑2 mRNA on day 7 in the bFGF pretreatment group was significantly higher than those in SDF‑1 pretreatment group. Expression levels of Runx‑2 mRNA, and ALP and Runx‑2 protein on day 3 in the SDF‑1 pretreatment group were higher than those in the bFGF pretreatment group. However, there was no significant difference in osteogenic differentiation ability on day 14 and 28 between the bFGF‑ or SDF‑1‑pretreatment groups and the control. In conclusion, bFGF and SDF‑1 pretreatment inhibits osteogenic differentiation of BMMSCs at the early stage, promotes it in the medium phase, and maintains it in the later stage during osteogenic induction, particularly at the mRNA level. Out of the two cytokines, bFGF appeared to have a greater effect on osteogenic differentiation.

Aim: To analyze the effect of three mitogen-activated protein kinase (MAPK) inhibitors, namely, SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor) in Dex-stimulated MDPC-23 cell differentiation and mineralization., Methods: Experiment was divided into five groups, control (cells without Dex and inhibitors treatment), Dex (cells with Dex treatment but without inhibitors), Dex + SB202190, Dex + SP600125, and Dex + PD98059. Cell differentiation was assessed by alkaline phosphatase (ALP) activity assay and real time RT-PCR. Cell mineralization was investigated by alizarin red staining., Results: Exposure to SB202190 (20  μ M) significantly decreased the mineral deposition in Dex-treated cells as demonstrated by alizarin red staining. Treatment of SP600125 (20  μ M) attenuated the mineralization as well, albeit at a lower degree as compared to SB202190 (20  μ M). Similarly, SB202190 (20  μ M) completely abrogated the ALP activity stimulated by Dex at six days in culture, while no changes were observed with regard to ALP activity in SP600125 (20  μ M) and PD98059 (20  μ M) treated cells. The upregulation of bone sialoprotein (BSP), ALP, and osteopontin (OPN) in Dex challenged cells was completely inhibited by SB202190., Conclusion: Blockade of p38-MAPK signaling pathway resulted in significant inhibition of ALP activity, mineralization, and downregulation of osteogenic markers. The data implicated that p38 signaling pathway plays a critical role in the regulation of MDPC-23 cells differentiation and mineralization.

Mast cells are multifunctional immune cells that participate in many important processes such as defense against pathogens, allergic reactions, and tissue repair. These cells perform their functions through the release of a wide variety of mediators. This release occurs mainly through cross-linking IgE (immunoglobulin E) bound to high affinity IgE receptors by multivalent antigens. The abundance of mast cells in connective tissue, surrounding blood vessels, and their involvement in the early stages of bone repair support the possibility of physiological and pathological interactions between mast cells and osteoblasts. However, the participation of mast cell mediators in osteogenesis is not fully understood. Therefore, the objective of this work was to investigate the role of mast cell mediators in the acquisition of the osteogenic phenotype in vitro. The results show that pooled mast cell mediators can affect proliferation, morphology, and cytoskeleton of osteoblastic cells, and impair the activity and expression of alkaline phosphatase as well as the expression of bone sialoprotein. Also, mast cell mediators inhibit the expression of mRNA for those proteins and inhibit the formation and maturation of calcium nodules and consequently inhibit mineralization. Therefore, mast cell mediators can modulate osteogenesis and are potential therapeutic targets for treatments of bone disorders.

RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase-dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2-binding regions of Bglap2 and Ibsp. Growth of MC3T3-E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin-bound P-ERK, P-RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di-methylation, another gene activation-associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono-, di-, and tri-methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation-dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2-deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non-phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin-associated P-ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P-RUNX2-dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427-2435, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objective: To investigate the effect of Wnt3a on osteogenic differentiation of human dental pulp stem cells (DPSC). Methods: DPSCs were subjected to different concentrations of Wnt3a (0, 5, 20, 50 and 100 μg/L) and at seven days after culture the alkaline phosphatase (ALP) activity was tested. Mineralized nodule formation was examined by alizarin red staining. Osteogenic-related gene expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type Ⅰ (COL-Ⅰ), Runt-related transcription factor-2 (RUNX2) was examined by quantitative real-time PCR (qPCR). Results: After seven days of induction by DPSC, Wnt3a protein could inhibit the ALP activity (concentration 0: 1.076±0.203, 5 μg/L: 0.828±0.118, 20 μg/L: 0.505±0.044, 50 μg/L: 0.499±0.038, 100 μg/L: 0.483±0.060). The expression of OCN in 5 μg/L Wnt3a group (0.092±0.005) was lower than that in culture medium (0.858±0.190)( P <0.05). Alizarin red staining showed that 5 μg/L Wnt3a had no mineralization induction effect on DPSC. Conclusions: Wnt3a could inhibit osteogenic differentiation of dental pulp stem cells.

Objective: Vascular calcification is associated with increased risk of myocardial infarction and stroke. The objective of this work was to examine the ability of 17β-estradiol (E2) to stimulate calcification of vascular smooth muscle cells (VSMC) in vivo, using aged apolipoprotein E-null mice with advanced atherosclerotic lesions, and subsequently to explore underlying mechanisms in vitro., Approach and Results: Silastic E2 capsules were implanted into male and female apolipoprotein E-null mice aged 34 weeks. Plaque and calcified area were measured in the aortic sinus and innominate artery after 8 weeks. Immunohistochemical analysis examined expression of the estrogen receptors (estrogen receptor alpha and estrogen receptor beta [ERβ]). VSMC expression of osteogenic markers was examined using digital polymerase chain reaction. Advanced atherosclerotic lesions were present in all mice at the end of 8 weeks. In both male and female mice, E2 increased calcified area in a site-specific manner in the aortic sinus independently of plaque growth or lipid levels and occurred in association with a site-specific decrease in the proportion of ERβ-positive intimal cells. Calcified lesions expressed collagen I and bone sialoprotein, with decreased matrix Gla protein. In vitro, E2 suppressed ERβ expression and increased VSMC mineralization, demonstrating increased collagen I and II, osteocalcin and bone sialoprotein, and reduced matrix Gla protein and osteopontin. Antagonism or RNA silencing of estrogen receptor alpha, ERβ, or both further increased VSMC mineralization., Conclusions: We have demonstrated that E2 can drive calcification in advanced atherosclerotic lesions by promoting the differentiation of VSMC to osteoblast-like cells, a process which is augmented by inhibition of estrogen receptor alpha or ERβ activity., (© 2017 American Heart Association, Inc.)

Objective: To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages, and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity, thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs., Methods: IPSCs derived from human gingival fibroblasts at passages 5, 10, 15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine, then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining, including bone related markers: osteocalcin (OCN), bone sialoprotein (BSP); periodontal ligament related markers: periostin, vimentin; and cementum related markers: cementum attachment protein (CAP), cementum protein 1 (CEMP1). The untreated spontaneous differentiation groups were set as negative controls respectively., Results: iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindle-like shape similar to fibroblasts upon periodontal specific differentiation. All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5: t=2.125, P=0.003; P10: t=2.246, P=0.021; P15: t=3.754, P=0.004; P20: t=3.933, P=0.002), but no significant difference in their calcium deposition were detected within passages 5, 10, 15 and 20 (periodontal differentiation: F=2.365, P=0.109; spontaneously differentiation: F=2.901, P=0.067). Periodontal tissue related marker expressions of iPSCs at all passages had also been significantly upgraded under the treatment of GDF-5 (P<0.05), but still, no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP: F=0.926 7, P=0.450; vimentin: F=0.917 1, P=0.455; CEMP1: F=2.129, P=0.1367)., Conclusion: Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs, as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging. Therefore, iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.

Objectives: Mesenchymal stem cells (MSCs) offer a promising therapy in dentistry because of their multipotent properties. Selecting donor MSCs is crucial because Beagle dogs (canines) commonly used in preclinical studies have shown variable outcomes, and it is unclear whether canine MSCs (cMSCs) are skeletal site specific. This study tested whether jaw and long bone cMSCs have disparate in vitro and in vivo multilineage differentiation capabilities., Study Design: Primary cMSCs were isolated from the mandible (M-cMSCs) and femur (F-cMSCs) of four healthy Beagle dogs. The femur served as the non-oral control. Clonogenic and proliferative abilities were assessed. In vitro osteogenic, chondrogenic, adipogenic, and neural multilineage differentiation were correlated with in vivo bone regeneration and potential for clinical applications., Results: M-cMSCs displayed two-fold increase in clonogenic and proliferative capacities relative to F-cMSCs (P = .006). M-cMSCs in vitro osteogenesis based on alkaline phosphatase (P = .04), bone sialoprotein (P = .05), and osteocalcin (P = .03), as well as adipogenesis (P = .007) and chondrogenesis (P = .009), were relatively higher and correlated with enhanced M-cMSC bone regenerative capacity. Neural expression markers, nestin and βIII-tubulin, were not significantly different., Conclusions: The enhanced differentiation and bone regenerative capacity of mandible MSCs may make them favorable donor graft materials for site-specific jaw bone regeneration., (Copyright © 2016. Published by Elsevier Inc.)

Objectives: Low concentrations of tumour necrosis factor-alpha (TNF-α) have been reported to promote osteogenic differentiation. In this study, a series of in vitro experiments was performed to investigate underlying molecular mechanisms involved., Materials and Methods: MC3T3-E1 murine preosteoblasts were treated with TNF-α at doses of 0, 0.1 or 1 ng/mL. The ephrinB2-EphB4 signalling pathway was activated using ephrinB2-fc, or inhibited using lentiviruses encoding siRNAs specifically targeting EphB4. Cell proliferation/survival was evaluated using the Cell Counting Kit-8 (CCK-8) assay, and expression levels of Runx2, BSP, ephrinB2 and EphB4 were determined using RT-PCR and Western blotting. ALP activity in these cells was also determined, and mineral nodule formation was evaluated with alizarin red S staining., Results: Low concentrations of TNF-α had no influence on cell proliferation/survival. However, expression levels of Runx2, BSP, ephrinB2 and EphB4, as well as ALP activity and mineral nodule formation, were significantly enhanced in MC3T3-E1 cells treated with low concentrations of TNF-α. Moreover, activation of the ephrinB2-EphB4 signalling pathway by ephrinB2-fc enhanced TNF-α-induced osteogenic differentiation, while down-regulation of EphB4 level reversed the positive effect of TNF-α., Conclusions: Low concentrations of TNF-α promoted osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway., (© 2016 John Wiley & Sons Ltd.)

Purpose: Collagen barrier membranes were first introduced to regenerative periodontal and oral surgery to prevent fast ingrowing soft tissues (ie, epithelium and connective tissue) into the defect space. More recent attempts have aimed at combining collagen membranes with various biologics/growth factors to speed up the healing process and improve the quality of regenerated tissues. Recently, a new formulation of enamel matrix derivative in a liquid carrier system (Osteogain) has demonstrated improved physico-chemical properties for the adsorption of enamel matrix derivative to facilitate protein adsorption to biomaterials. The aim of this pioneering study was to investigate the use of enamel matrix derivative in a liquid carrier system in combination with collagen barrier membranes for its ability to promote osteoblast cell behavior in vitro., Materials and Methods: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto porcine-derived collagen membranes alone (control) or porcine membranes + enamel matrix derivative in a liquid carrier system. Control and enamel matrix derivative-coated membranes were compared for cell recruitment and cell adhesion at 8 hours; cell proliferation at 1, 3, and 5 days; and real-time polymerase chain reaction (PCR) at 3 and 14 days for genes encoding Runx2, collagen1alpha2, alkaline phosphatase, and bone sialoprotein. Furthermore, alizarin red staining was used to investigate mineralization., Results: A significant increase in cell adhesion was observed at 8 hours for barrier membranes coated with enamel matrix derivative in a liquid carrier system, whereas no significant difference could be observed for cell proliferation or cell recruitment. Enamel matrix derivative in a liquid carrier system significantly increased alkaline phosphatase mRNA levels 2.5-fold and collagen1alpha2 levels 1.7-fold at 3 days, as well as bone sialoprotein levels twofold at 14 days postseeding. Furthermore, collagen membranes coated with enamel matrix derivative in a liquid carrier system demonstrated a sixfold increase in alizarin red staining at 14 days when compared with collagen membrane alone., Conclusion: The combination of enamel matrix derivative in a liquid carrier system with a barrier membrane significantly increased cell attachment, differentiation, and mineralization of osteoblasts in vitro. Future animal testing is required to fully characterize the additional benefits of combining enamel matrix derivative in a liquid carrier system with a barrier membrane for guided bone or tissue regeneration.

Tumor necrosis factor-α (TNF-α) stimulates osteoclast differentiation and suppresses osteoblast differentiation, leading to bone loss and decreased bone mass in local inflammation areas in patients with rheumatoid arthritis. The growth factor progranulin (PGRN) is expressed in various types of cells and play crucial roles in the pathogenesis of atherosclerosis and arthritis by blocking TNF-α. Here, we investigated the role of PGRN in blocking TNF-α-mediated inhibition of osteoblast differentiation and the regulatory mechanism. C2C12 stem cell was induced by bone morphogenetic protein-2 (BMP-2) for osteoblast differentiation. A significant increase in ALP activity (P < 0.001), as well as the expression of ColI, Ocn, and Bsp in the induced cells (P < 0.01 and P < 0.001) were observed; the marker gene expression and ALP activity were inhibited by TNF-α (P < 0.01 and P < 0.001). PGRN significantly blocks the TNF-α-mediated inhibition of osteoblast differentiation, evidenced by the ALP activity (P < 0.05 and P < 0.01), Alizarin red staining, the expression of ColI, Ocn, and Bsp (P < 0.05 and P < 0.01) and the osteoblast key transcription factor gene Runx2 (P < 0.01), Osx (P < 0.05), and ATF4 (P < 0.05). Mechanical study indicated that PGRN significantly blocks the TNF-α-mediated stimulation of NF-kB signaling (P < 0.01 and P < 0.001). PGRN exerts a protective effect on osteoblast differentiation in an inflammatory environment. Thus, we concluded that the treatment of osteoblasts with PGRN could be used in the future to prevent or treat rheumatoid arthritis-associated bone loss., Competing Interests: The authors declare no conflict of interest.

Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs) with desired water dispersibility were achieved with the regulation of poly (acrylic acid). Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of -22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS) solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP) activity assays together with the osteocalcin (OCN) and bone sialoprotein (BSP) expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration.

Bone morphogenetic protein 7 (BMP-7) is a multifunctional differentiation factor that belongs to the transforming growth factor superfamily. BMP-7 induces gene expression of protein tyrosine phosphatase-like, member A/cementum attachment protein (PTPLA/CAP) and cementum protein 1 (CEMP1), both of which are markers of cementoblasts and cementocytes. In the previous study, we reported that BMP-7 treatment enhanced PTPLA/CAP and CEMP1 expression in both normal and immortal human periodontal ligament (PDL) cells. To elucidate the molecular mechanisms of the gene expression of these molecules, in this study, we identified a functional transcription activator binding region in the promoter region of PTPLA/CAP and CEMP1 that is responsive to BMP signals. Here, we report that some short motifs termed GC-rich Smad-binding elements (GC-SBEs) that are located in the human PTPLA/CAP promoter and CEMP1 promoter are BMP-7 responsive as analyzed with luciferase promoter assays. On the other hand, we found that transcription of Sp7/Osterix and PTPLA/CAP was up-regulated after 1 week of BMP-7 treatment on purified normal human PDL cells as a result of gene expression microarray analysis. Furthermore, transcription of Sp7/Osterix, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP) was up-regulated after 2 weeks of BMP-7 treatment, whereas gene expression of osteo/odontogenic markers such as integrin-binding sialoprotein (IBSP), collagen, type I, alpha 1 (COL1A1), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP) was not up-regulated in purified normal or immortal human PDL cells as a result of qRT-PCR. The results suggest that BMP-7 mediates cementogenesis via GC-SBEs in human PDL cells and that its molecular mechanism is different from that for osteo/odontogenesis.

Epithelial-mesenchymal interaction occurs during development of various tissues, including teeth and bone. Recently, a preameloblast-conditioned medium (PA-CM) from mouse apical bud cells (ABCs), a type of dental epithelial cell, was found to induce odontogenic differentiation of dental pulp stem cells and promote dentin formation. The aims of the present study were to investigate the effects of PA-CM on human bone marrow mesenchymal stem cells (hBMSCs) in vitro, and to investigate the bone regenerative capacity in vivo through epithelial-mesenchymal interactions of developmental osteogenesis. Coculturing with ABCs and PA-CM treatment upregulated osteoblast differentiation markers of hBMSCs compared to cells cultured alone. PA-CM accelerated mineralized nodule formation and also increased bone sialoprotein promoter activity in hBMSCs. PA-CM facilitated the migration of hBMSCs, but did not significantly influence proliferation. PA-CM promoted bone formation of hBMSCs in vivo. Radiographic and histologic findings showed that PA-CM induced the bony regeneration at calvarial defects in rat. Taken together, these data show that PA-CM enhances the migration and osteogenic differentiation of hBMSCs in vitro and induces bone formation in vivo.

The present study aimed to investigate the effects of simvastatin on the bone differentiation capacity and immunological characteristics of bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and cultured in medium containing 1.0 µmol/ml simvastatin. The alkaline phosphatase activity, mRNA expression levels of osteocalcin and bone sialoprotein, and calcium nodule formation were assessed to determine the osteogenic differentiation capability of BMSCs. To investigate alterations in the immunological properties of simvastation‑treated BMSCs, the immunogenicity of these cells and the effect of BMSCs on phytohemagglutinin‑stimulated lymphocyte proliferation were also assessed. Following treatment with simvastatin, the alkaline phosphatase activity, and mRNA expression levels of osteocalcin and bone sialoprotein were increased significantly in the BMSCs. In addition, von Kossa staining revealed a brown calcium‑positive reaction zone in simvastatin‑treated cells. Simvastatin‑induced BMSCs revealed no affect on the proliferation of allogeneic lymphocytes, however, inhibited phytohemagglutinin‑induced lymphocyte proliferation. Collectively, simvastatin promoted the osteogenic differentiation of BMSCs significantly without affecting their immunosuppressive properties.

Osteoporosis (OP) is considered a complex disease with a strong genetic impact, mainly affecting post-menopausal women and is also a common cause of fracture. Elucidating the molecular mechanisms that regulate the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is crucial to developing treatment strategies to combat OP. In the present study, we found that ectopic viral integration site‑1 (Evi1) was highly expressed during the process of adipogenesis of rat BMSCs. Notably, Evi1 levels markedly increased on day 3 of adipogenic differentiation following the addition of adipogenic induction supplements. In addition, we interfered with the expression of the Evi1 gene in the adipogenesis of BMSCs by supplementing adenoviral plasmids and measured the expression levels of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), peroxisome proliferator‑activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) by RT-qPCR and western blot analysis. The mRNA and protein levels of osteogenic and adipogenic markers in the BMSCs were up‑ and downregulated, respectively following the silencing of siEvi1. Our experimental results substantiate that the suppression of Evi1 in BMSCs by RNA interference inhibits adipogenic differentiation, while it promotes osteogenic differentiation. The results from our study demonstrated that the Evi1 gene may be targeted as a therapeutic strategy for promoting bone formation.

The differentiation of pluripotent mesenchymal stem cells to mature osteoblasts is crucial for the maintenance of the adult skeleton. In rheumatic arthritis, osteoblast differentiation is impaired by the overproduction of cytokine tumor necrosis factor (TNF)‑α. It has been demonstrated that TNF-α is able to inhibit osteoblast differentiation through the activation of nuclear factor (NF)-κB signaling. As a result of the critical role of TNF-α and NF-κB in the pathogenesis of bone-loss associated diseases, these factors are regarded as key targets for the development of therapeutic agents. In the current study, the role of the NF-κB inhibitor gliotoxin (GTX) in the regulation of osteoblast differentiation was evaluated. The non-toxic GTX doses were determined to be ≤ 3 µg/ml. It was revealed that GTX was able to block TNF-α-induced inhibition of osteoblast differentiation, as indicated by alkaline phosphatase (ALP) activity and ALP staining assays, as well as the expression levels of osteoblast-associated genes Col I, Ocn, Bsp, Runx2, Osx and ATF4. Additionally, it was identified that gliotoxin directly promoted bone morphogenetic protein-2-induced osteoblast differentiation. GTX was found to inhibit the accumulation of NF-κB protein p65 in the nucleus and reduce NF-κB transcriptional activity, suggesting that GTX potentiated osteoblast differentiation via the suppression of NF-κB signaling.

Polyelectrolyte multilayer (PEMU) coatings built layer by layer with alternating pairs of polyelectrolytes can be tuned to improve cell interactions with surfaces and may be useful as biocompatible coatings to improve fixation between implants and tissues. Here, we show that human mesenchymal stromal cells (hMSCs) induced with bone differentiation medium (BDM) to become osteoblasts biomineralize crosslinked PEMUs built with the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly(acrylic acid) (PAA). Degrees of hMSC osteoblast differentiation and surface biomineralization on the smooth PAH-terminated PEMUs (PAH-PEMUs) and microstructured PAA-terminated PEMUs (PAA-PEMUs) reflect differences in cell-deposited extracellular matrix (ECM). BDM-induced hMSCs expressed higher levels of the early osteoblast differentiation marker alkaline phosphatase and collagen 1 (COL1) sooner on PAA-PEMUs than on PAH-PEMUs. Cells on both types of PEMUs proceeded to express the later stage osteoblast differentiation marker bone sialoprotein (BSP), but the BDM-induced cells organized a more amorphous Collagen I and denser BSP localization on PAA-PEMUs than on PAH-PEMUs. These ECM properties correlated with greater biomineralization on the PAA-PEMUs than on PAH-PEMUs. Together, these results confirm the suitability of PAH/PAA PEMUs as a substrate for hMSC osteogenesis and highlight the importance of substrate effects on ECM organization and BSP presentation on biomineralization., (© 2014 Wiley Periodicals, Inc.)

Calcium phosphate (CaP) scaffolds have been widely and successfully used with osteoblast cells for bone tissue regeneration. However, it is necessary to investigate the effects of these scaffolds on odontoblast cells' proliferation and differentiation for dentin tissue regeneration. In this study, three different hydroxyapatite (HA) to beta tricalcium phosphate (β-TCP) ratios of biphasic calcium phosphate (BCP) scaffolds, BCP20, BCP50, and BCP80, with a mean pore size of 300μm and 65% porosity were prepared from phosphoric acid (H2PO4) and calcium carbonate (CaCO3) sintered at 1000°C for 2h. The extracts of these scaffolds were assessed with regard to cell viability and differentiation of odontoblasts. The high alkalinity, more calcium, and phosphate ions released that were exhibited by BCP20 decreased the viability of human dental pulp cells (HDPCs) as compared to BCP50 and BCP80. However, the cells cultured with BCP20 extract expressed high alkaline phosphatase activity and high expression level of bone sialoprotein (BSP), dental matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP) genes as compared to that cultured with BCP50 and BCP80 extracts. The results highlighted the effect of different scaffold ratios on the cell microenvironment and demonstrated that BCP20 scaffold can support HDPC differentiation for dentin tissue regeneration., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Osteogenic behaviour of osteoblasts from trabecular, cortical and subchondral bone were examined to determine any bone type-selective differences in samples from both osteoarthritic (OA) and osteoporotic (OP) patients. Cell growth, differentiation; alkaline phosphatase (TNAP) mRNA and activity, Runt-related transcription factor-2 (RUNX2), SP7-transcription factor (SP7), bone sialoprotein-II (BSP-II), osteocalcin/bone gamma-carboxyglutamate (BGLAP), osteoprotegerin (OPG, TNFRSF11B), receptor activator of nuclear factor-κβ ligand (RANKL, TNFSF11) mRNA levels and proangiogenic vascular endothelial growth factor-A (VEGF-A) mRNA and protein release were assessed in osteoblasts from paired humeral head samples from age-matched, human OA/OP (n = 5/4) patients. Initial outgrowth and increase in cell number were significantly faster (p < 0.01) in subchondral and cortical than trabecular osteoblasts, in OA and OP, and this bone type-related differences were conserved despite consistently faster growth in OA. RUNX2/SP7 levels and TNAP mRNA and protein activity were, however, greater in trabecular than subchondral and cortical osteoblasts in OA and OP. BSP-II levels were significantly greater in trabecular and lowest in cortical osteoblasts in both OA and OP. In contrast, BGLAP levels showed divergent bone type-selective behaviour; highest in osteoblasts from subchondral origins in OA and trabecular origins in OP. We found virtually identical bone type-related differences, however, in TNFRSF11B:TNFSF11 in OA and OP, consistent with greater potential for paracrine effects on osteoclasts in trabecular osteoblasts. Subchondral osteoblasts (OA) exhibited highest VEGF-A mRNA levels and release. Our data indicate that human osteoblasts in trabecular, subchondral and cortical bone have inherent, programmed diversity, with specific bone type-related differences in growth, differentiation and pro-angiogenic potential in vitro.

Periodontal ligament-associated protein-1 (PLAP-1) is a newly discovered member of the extracellular matrix family of proteins known as proteoglycans and is a negative regulator that plays a crucial role in the homeostasis of periodontal tissues. It can protect the periodontal ligament from excessive osteogenesis. However, the molecular mechanisms of PLAP-1 during osteogenic differentiation and osteogenesis remain unclear. In this study, we constructed a PLAP-1 recombinant retroviral plasmid vector named pBABE-hygro-PLAP-1. We transfected this plasmid into rat bone marrow stromal cells (rBMSCs) to obtain a stable cell line with overexpression of PLAP-1 to verify whether PLAP-1 also acts as an inhibitory factor in rBMSCs during bone mineralization. A rBMSC line stably overexpressing PLAP-1 was established successfully as determined by the mRNA levels of PLAP-1, which were measured by real time-qPCR (RT-qPCR), and protein expression, which was measured by immunocytochemistry and western blot analysis. At the same time, a Cell Counting Kit-8 assay did not reveal any statistically significant changes in the transfected cells (P > 0.05). Then, mineral-inducing cultures were performed, and mineralized nodules were observed at weeks 2, 3 and 4 under a microscope. Alizarin Red (Sigma) staining was performed at 4 week to illustrate calcium accumulation. The mineralized nodules in the PLAP-1-transfected rBMSC group were fewer than those in the control groups. The time span of the formation of the mineralized nodules was prolonged. Meanwhile, osteogenic genes were also detected in the mineral-inducing cells by RT-qPCR. An RT-qPCR analysis demonstrated that the levels of the osteoblast markers of rBMSCs that were transfected with pBABE-hygro-PLAP-1, including Runx2, Osterix, alkaline phosphatase, bone sialoprotein and osteocalcin, were lower than those in the non-transfected rBMSCs and rBMSCs that were transfected with empty vector (P < 0.01). These results suggest that PLAP-1 has an inhibitory function in rBMSCs when they differentiate into osteoblast-like cells.

Recent studies of new surface modifications that superimpose well-defined nanostructures on microrough implants, thereby mimicking the hierarchical complexity of native bone, report synergistically enhanced osteoblast maturation and local factor production at the protein level compared to growth on surfaces that are smooth, nanorough, or microrough. Whether the complex micro/nanorough surfaces enhance the osteogenic response by triggering similar patterns of integrin receptors and their associated signaling pathways as with well-established microrough surfaces, is not well understood. Human osteoblasts (hOBs) were cultured until confluent for gene expression studies on tissue culture polystyrene (TCPS) or on titanium alloy (Ti6Al4V) disks with different surface topographies: smooth, nanorough, microrough, and micro/nanorough surfaces. mRNA expression of osteogenesis-related markers such as osteocalcin (BGLAP) and bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2), BMP4, noggin (NOG) and gremlin 1 (GREM1) were all higher on microrough and micro/nanorough surfaces, with few differences between them, compared to smooth and nanorough groups. Interestingly, expression of integrins α1 and α2, which interact primarily with collagens and laminin and have been commonly associated with osteoblast differentiation on microrough Ti and Ti6Al4V, were expressed at lower levels on micro/nanorough surfaces compared to microrough ones. Conversely, the αv subunit, which binds ligands such as vitronectin, osteopontin, and bone sialoprotein among others, had higher expression on micro/nanorough surfaces concomitantly with regulation of the β3 mRNA levels on nanomodified surfaces. These results suggest that the maturation of osteoblasts on micro/nanorough surfaces may be occurring through different integrin engagement than those established for microrough-only surfaces.

Aloe vera is a traditional wound healing medicine. We hypothesized acemannan, a polysaccharide extracted from Aloe vera gel, could affect bone formation. Primary rat bone marrow stromal cells (BMSCs) were treated with various concentrations of acemannan. New DNA synthesis, VEGF, BMP-2, alkaline phosphatase activity, bone sialoprotein, osteopontin expression, and mineralization were determined by [(3)H] thymidine incorporation assay, ELISA, biochemical assay, western blotting, and Alizarin Red staining, respectively. In an animal study, mandibular right incisors of male Sprague-Dawley rats were extracted and an acemannan treated sponge was placed in the socket. After 1, 2, and 4 weeks, the mandibles were dissected. Bone formation was evaluated by dual-energy X-ray absorptiometry and histopathological examination. The in vitro results revealed acemannan significantly increased BMSC proliferation, VEGF, BMP-2, alkaline phosphatase activity, bone sialoprotein and osteopontin expression, and mineralization. In-vivo results showed acemannan-treated groups had higher bone mineral density and faster bone healing compared with untreated controls. A substantial ingrowth of bone trabeculae was observed in acemannan-treated groups. These data suggest acemannan could function as a bioactive molecule inducing bone formation by stimulating BMSCs proliferation, differentiation into osteoblasts, and extracellular matrix synthesis. Acemannan could be a candidate natural biomaterial for bone regeneration.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is guided by various physical and biochemical factors. Among these factors, modulus (i.e., rigidiy) of the ECM has gained significant attention as a physical osteoinductive signal that can contribute to endochondral ossification of a cartilaginous skeletal template. However, MSCs also participate in intramembranous bone formation, which occurs de novo from within or on a more compliant tissue environment. To further understand the role of the matrix interactions in this process, we evaluated osteogenic differentiation of hMSCs cultured on low moduli (102, 390 or 970 Pa) poly(N-isopropylacrylamide) (p(NIPAAm)) based semi-interpenetrating networks (sIPN) modified with the integrin engaging peptide bsp-RGD(15) (0, 105 or 210 µM). Cell adhesion, proliferation, and osteogenic differentiation of hMSCs, as measured by alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), bone sialoprotein-2 (iBSP), and osteocalcien (OCN) protein expression, was highest on substrates with the highest modulus and peptide concentrations. However, within this range of substrate stiffness, many osteogenic cellular functions were enhanced by increasing either the modulus or the peptide density. These findings suggest that within a compliant and low modulus substrate, a high affinity adhesive ligand serves as a substitute for a rigid matrix to foster osteogenic differentiation.

Objective: To get the stem cells from the young permanent tooth apical papillae, and observe the osteogenic differentiation of the cells after cultured with acellular dermal matrix (ADM)., Methods: Young permanent tooth apical papillae were obtained by the oral surgeon. The cells from the apical papillae were isolated, cultured and analyzed through a flow cytometer. The cells in the experimental group were induced both osteogenic and adipogenic differentiation. The cells were not induced in the control group.Both groups were evaluated by staining and real-time polymerase chain reaction (real-time PCR) to examine the quantity of RNAs in the experimental group. The cells from apical papillae were also cultured with ADM. These cells were also induced both osteogenic and adipogenic differentiation in the experimental group, and not induced in the control group. The measures of staining and real-time PCR were also carried out., Results: The cells from the apical papillae proliferated in a rapid rate. Of which 70.3% in cultures were positive for Stro-1, and 96% positive for CD105 according to flow cytometric analysis. After induction, the RNA level related to osteogenic and adipogenic differentiation expressed higher in the experimental group than those of the control group without induction obviously, such as osteocalcin (OCN), bone sialoprotein (BSP), liver X-recepter α (LXRα), lipoprotein lipase(LPL), peroxisome proliferator activated receptor γ (PPAR-γ), and scavenger receptor class B type 1(SR-B1). The cells cultured with ADM also had a fast proliferation, and grew attached to ADM. After induction, the RNA level of OCN and BSP had a higher expression than the control group (P > 0.05), and LPL also expressed higher (P < 0.05)., Conclusion: The study approved that there were a big amount of stem cells in the young permanent tooth apical papilla obtained by oral surgery, which had significant osteogenic potential. The cells still proliferated well when they were cultured with ADM as a kind of collagen skelecton. The results showed that ADM could be performed as a base to support the stem cells to survive the environment, and it also could play a role in osteogenic differentiation of stem cells from apical papilla.

In the present study, the in vitro effects of novel strontium-modified calcium phosphate bone cements (SrCPCs), prepared using two different approaches on human-bone-marrow-derived mesenchymal stem cells (hMSCs), were evaluated. Strontium ions, known to stimulate bone formation and therefore already used in systemic osteoporosis therapy, were incorporated into a hydroxyapatite-forming calcium phosphate bone cement via two simple approaches: incorporation of strontium carbonate crystals and substitution of Ca(2+) by Sr(2+) ions during cement setting. All modified cements released 0.03-0.07 mM Sr(2+) under in vitro conditions, concentrations that were shown not to impair the proliferation or osteogenic differentiation of hMSCs. Furthermore, strontium modification led to a reduced medium acidification and Ca(2+) depletion in comparison to the standard calcium phosphate cement. In indirect and direct cell culture experiments with the novel SrCPCs significantly enhanced cell proliferation and differentiation were observed. In conclusion, the SrCPCs described here could be beneficial for the local treatment of defects, especially in the osteoporotic bone., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on osteogenic differentiation of mouse osteoblast cell line MC3T3-E1., Methods: PgW83 was cultured under standard anaerobic conditions and extracted by sonication. Mouse osteoblast cell line MC3T3-E1 was cultured with various concentrations of the extraction (0, 10, 100, 1000 mg/L). Western blotting was applied to investigate the expression of osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN) and osteonectin (ON). The activity of alkaline phosphatase (ALP) was detected by microplate reader after 14 days. Mineralization nodule formation was measured by alizarin red staining after 21 days., Results: Compared with the control group, the extracts of Pg decreased OC and ON expression in a dose-dependent manner (OC relative expression:1.000 ± 0.000,0.852 ± 0.110,0.625 ± 0.451,0.213 ± 0.053), (ON relative expression: 1.000 ± 0.000, 1.035 ± 0.133,0.141 ± 0.023,0.020 ± 0.003) (P < 0.05). The expression of OPN was down-regulated significantly in MC3T3-E1 treated with 1000 mg/L extraction (0.572 ± 0.162) compared with control group, 10 and 100 mg/L (1.000 ± 0.000, 1.029 ± 0.135, 1.199 ± 0.337) (P < 0.05). The expression of BSP remained unchanged when the cells were cultured with or without extraction (BSP relative expression:1.000 ± 0.000,0.831 ± 0.182,0.897 ± 0.115,0.778 ± 0.235) (P > 0.05). Meanwhile, the extracts of Pg decreased ALP activity [control group:(0.0275 ± 0.0014) U/gprot, 10 mg/L: (0.0140 ± 0.0011) U/gprot, 100 mg/L: (0.0057 ± 0.0013) U/gprot, 1000 mg/L: (0.0020 ± 0.0008) U/gprot] (P < 0.05) and reduced mineralization nodule formation., Conclusions: The results suggest that Pg may inhibit osteoblasts'osteogenic function by down-regulation of osteogenic differentiation related proteins.

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation., (Copyright © 2012 Wiley Periodicals, Inc.)

Objectives: We prepared 3D poly (ε-caprolactone) (PCL) nanofibre scaffolds and tested their use for seeding, proliferation, differentiation and migration of mesenchymal stem cell (MSCs)., Materials and Methods: 3D nanofibres were prepared using a special collector for common electrospinning; simultaneously, a 2D PCL nanofibre layer was prepared using a classic plain collector. Both scaffolds were seeded with MSCs and biologically tested. MSC adhesion, migration, proliferation and osteogenic differentiation were investigated., Results: The 3D PCL scaffold was characterized by having better biomechanical properties, namely greater elasticity and resistance against stress and strain, thus this scaffold will be able to find broad applications in tissue engineering. Clearly, while nanofibre layers of the 2D scaffold prevented MSCs from migrating through the conformation, cells infiltrated freely through the 3D scaffold. MSC adhesion to the 3D nanofibre PCL layer was also statistically more common than to the 2D scaffold (P < 0.05), and proliferation and viability of MSCs 2 or 3 weeks post-seeding, were also greater on the 3D scaffold. In addition, the 3D PCL scaffold was also characterized by displaying enhanced MSC osteogenic differentiation., Conclusions: We draw the conclusion that all positive effects observed using the 3D PCL nanofibre scaffold are related to the larger fibre surface area available to the cells. Thus, the proposed 3D structure of the nanofibre layer will find a wide array of applications in tissue engineering and regenerative medicine., (© 2012 Blackwell Publishing Ltd.)

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation., (Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix (ECM) involved in different steps of the regulation of cellular differentiation. In this study artificial extracellular matrices (aECM) consisting of collagen (Col) I and different GAG derivatives were used as a substrate for human mesenchymal stromal cells (hMSC) to study osteogenic differentiation in vitro. hMSC were cultured on aECM containing col and hyaluronan sulfates (HyaS) with increasing degrees of sulfation (DS(S)) and were compared with aECM containing col and the natural GAG hyaluronan or chondroitin 4-sulfate. hMSC were analyzed for osteogenic differentiation markers such as calcium phosphate deposition, tissue non-specific alkaline phosphatase (TNAP) and expression of runt-related transcription factor 2 (runx2), osteocalcin (ocn) and bone sialoprotein II (bspII). Compared with aECM containing Col and natural GAG all Col/HyaS-containing aECM induced an increase in calcium phosphate deposition, TNAP activity and tnap expression. These effects were also seen in the absence of dexamethasone (an established osteogenic supplement). The expression of runx2 and ocn was not altered and the expression of bspII was diminished on the col/HyaS-containing aECM. The impact of the Col/HyaS-containing aECM on hMSC differentiation was independent of the DS(S) of the HyaS derivatives, indicating the importance of the primary (C-6) hydroxyl group of N-acetylglucosamine. These results suggest that Col/HyaS-containing aECM are able to stimulate hMSC to undergo osteogenic differentiation even in the absence of dexamethasone, which makes these matrices an interesting tool for hMSC-based tissue engineering applications and biomaterial functionalizations to enhance bone formation., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tumor necrosis factor-alpha (TNF-α) is one major inflammatory factor peaking at 24 h after bone fracture in response to injury; its role in bone healing is controversial. The aims of this study were to investigate whether the duration of exposure to TNF-α is crucial for the initiation of bone regeneration and to determine its underlying mechanism(s). We demonstrated that 24 h of TNF-α treatment significantly abrogated osteocalcin gene expression by human primary osteoblasts (HOBs). However, when TNF-α was withdrawn after 24 h, bone sialoprotein and osteocalcin gene expression levels in HOBs at day 7 were significantly up-regulated compared with the HOBs without TNF-α treatment. In contrast, continuous TNF-α treatment down-regulated bone sialoprotein and osteocalcin gene expression. In addition, in an indirect co-culture system, HOBs pretreated with TNF-α for 24 h induced significantly greater osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) than the HOBs without TNF-α treatment. TNF-α treatment also promoted endogenous bone morphogenetic protein 2 (BMP-2) production in HOBs, while blocking the BMP-2 signaling pathway with Noggin inhibited osteogenic differentiation of ASCs in the co-culture system. Furthermore, activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after TNF-α treatment occurred earlier than BMP-2 protein expression. BMP-2 production by HOBs and osteogenic differentiation of ASCs in the co-culture system with HOBs was significantly decreased when HOBs were pretreated with TNF-α in combination with the p38 MAPK-specific inhibitor (SB203580). Taken together, we provide evidence that exposure duration is a critical element in determining TNF-α's effects on bone regeneration. We also demonstrate that the p38 MAPK signaling pathway regulates the expression of BMP-2 in osteoblasts, which then acts through a paracrine loop, to direct the osteoblast lineage commitment of mesenchymal stem cells.

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling., (Copyright © 2011 Wiley Periodicals, Inc.)

Objective: To investigate the effects of emdogain, enamel matrix derivative (EMD), on the proliferation, cell cycle, mineralization and ultrastructure of human periodontal ligament (PDL) cells in vitro., Methods: The influence of cell growth on PDL cells was evaluated by Cell Counting Kit-8 (CCK-8) in the presence and absence of emdogain, after 1, 3, and 5 d of culture. DNA synthesis and ultrastructure of PDL cells were observed by flow cytometry(FCM) and transmission electron microscopy (TEM) in the presence and absence of emdogain after 3 d of culture. The increasing of osteogenic capacity was verified by the expression changes of osteogenic differentiation markers of bone sialoprotein (BSP) and osteopontin (OPN) in emdogain-treated PDL cells by immunohistochemicl staining., Results: Incubation of PDL cells with emdogain after 3 d significantly stimulated cell growth and DNA synthesis. Emdogain enhanced the osteogenic potential of PDL cells by high expression of osteogenic differentiation markers of BSP and OPN., Conclusion: The data indicate that Emdogain enhances cell proliferation and promotes differentiation of PDL cells, which contributes to periodontal tissue regeneration .

PDL (periodontal ligament) is a source of multi-potent stem cells in humans and their differentiation potential to a cementoblast and osteoblast phenotypes has been shown. Tissue construction from PDL-derived cells could be considered as a valuable technique for periodontal regenerative medicine. On these basis, we determined the role of CEMP1 (cementum protein 1) as a factor to induce differentiation of human PDL cells in a 3D (three-dimensional) fashion. Human PDL cells were grown in an RCCS (rotary cell culture system) D-410 RWV (rotating wall vessel) bioreactor, and maintained in either experimental (CEMP1 2.5 μg/ml) or control media during 4 weeks. Cell proliferation in the presence of CEMP1 was determined. The tissue-like structure formed was analysed histologically, stained with Alizarin Red and Alcian Blue. ALP (alkaline phosphatase)-specific activity, immunostaining, RT-PCR (reverse transcription-PCR) and Western blotting were performed to determine the expression of BSP (bone sialoprotein), enamel [AMBN (ameloblastin) and AMEL (amelogenin)], cementum [CAP (cementum attachment protein) and CEMP1] and cartilage-related proteins (Sox9, aggrecan, types II and X collagens). Our results show that hrCEMP1 (human recombinant CEMP1) promoted cell proliferation by human PDL cells in 3D cultures and induced the formation of a tissue-like structure resembling bone and/or cementum and material similar to cartilage. The addition of hrCEMP1 to the 3D human PDL cell cultures increased ALP-specific activity by 2.0-fold and induced the expression of markers for the osteogenic, cementogenic and chondrogenic phenotypes at the mRNA and protein levels. Our data show that human PDL cells in 3D cultures with the addition of CEMP1 has the potential to be used for the bioengineering reconstruction of periodontal tissues and cartilage since our results suggest that CEMP1 stimulates human PDL cells to differentiate towards different phenotypes.

Postnatal dental pulp stem cells (DPSCs) represent a unique precursor population in the dental pulp, which have multipotential and harbor great potential for tissue engineering purposes. However, for therapy applications, transplanted cells are often exposed to unfavorable conditions such as cytokines released from necrotic or inflammatory cells in injured tissues. It is not clear how stem cells exposed to these conditions changes in their characteristics. In this study, the effects of pro-inflammatory cytokines, such as IL-1 and TNF, on DPSCs were investigated. Cells were treated with IL-1, TNF, or both for 3, 7, and 12 days. The cultures were evaluated for cell proliferation, ALP activity, and real-time PCR. We found that a short treatment (3 days) of pro-inflammatory cytokines induced the odontogenic differentiation of DPSCs. Furthermore, post 3 days treatment with pro-inflammatory cytokines, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis demonstrated that the cultures gave obviously mineralized tissue formation, especially for both IL-1 and TNF applied. These data suggest that IL-1 and TNF produced in the early inflammatory reaction may induce the mineralization of DPSCs., (Copyright © 2011 Wiley Periodicals, Inc.)

Adipose-derived stromal cells (ADSCs) can be differentiated in vitro into several mesenchyme-derived cell types. We had previously described depot-specific differences in the adipocyte differentiation of ADSCs, and consequently we hypothesized that there may also be depot-specific differences in osteoblast differentiation of ADSCs. For this study, the osteoblast differentiation potential of rat subcutaneous ADSCs (scADSCs) and perirenal visceral ADSCs (pvADSCs) was compared. Osteoblast differentiation media (OM) induced markers of the osteoblastic phenotype in scADSCs, but not in pvADSCs. ADSCs harvested from rats with diet-induced visceral obesity (DIO) exhibited reduced osteoinduction, compared to lean controls, but adipocyte differentiation was not affected. Expression of the pro-osteogenic transcription factor Msx2 was significantly higher in naïve scADSCs from lean and DIO rats than in pvADSCs. Our findings indicate that ADSCs from different anatomical sites are uniquely pre-programmed in vivo in a depot-specific manner, and that diet-induced metabolic disturbances translate into reduced osteoblast differentiation of ADSCs., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Mitogen-activated protein kinase (MAPK) pathways are involved in stem cell differentiation. However, the odontoblastic differentiation-inducing effects by mineral trioxide aggregate (MTA) via MAPK pathways have not been clarified in human dental pulp stem cells (DPSCs). In this study we investigated the effects of MTA on cell viability and production of differentiation markers, and the involvement of MAPK signaling pathways in cultured human DPSCs. Cells were cultured with MTA, and the viability and differentiation productions of the cells were determined using the MTT assay and real-time PCR analysis, respectively. MAPK activation was measured by western blotting. MTA at concentrations of 20 and 10 mg/ml was toxic for human DPSCs. MTA significantly increased the expression of alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), type I collagen (COLI), osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs and induced the phosphorylation of p42 and p44 (p42/44), p38 and c-Jun N-terminal kinases 1 and 2 (JNK1/2) MAPK. Furthermore, the inhibitor of p42/44 MAPK attenuated the MTA-induced odontoblastic differentiation. These data indicated that MTA-induced odontoblastic differentiation of human DPSCs was via MAPK pathways, which may play a key role in the repair responses of dentin-pulp-like complexes.