Early Osteoblast Differentiation Research Articles

LncPVT1 regulates osteogenic differentiation of human periodontal ligament cells via miR-10a-5p/brain-derived neurotrophic factor.

Identifying the factors affecting osteoblast differentiation of periodontalligament cells(PDLCs) can help enhance the regeneration of periodontal tissue. LncRNA plasmacytoma variant translocation 1 (lncPVT1) is an important regulatory factor involved in many biological processes, but its role in osteogenesis remains unclear. Expressions of osteogenic markers were detected by quantitative reverse transcription polymerase chain reaction and Western blot analysis. Alkaline phosphatase staining was conducted for early osteoblast differentiation and alizarin red S staining was used for mineral deposition. RNA sequencing was used to identify the miRNAs regulated by lncPVT1 during osteogenesis. Cell transfection was used to overexpress or knockdown lncPVT1 and miR-10a-5p. Dual luciferase reporter assays were conducted to analyze the binding of miR-10a-5p to brain-derived neurotrophic factor (BDNF). LncPVT1 was significantly increased during osteogenic induction of PDLCs. Overexpression of lncPVT1 promoted osteogenesis, whereas lncPVT1 knockdown inhibited this process. RNA sequencing showed that miR-10a-5p expression was significantly increased after lncPVT1 knockdown. RNA immunoprecipitation assay further demonstrated the binding potential of lncPVT1 and miR-10a-5p. MiR-10a-5p inhibited the osteogenesis of PDLCs, and partially reversed the stimulatory effects of lncPVT1. Subsequently, we identified a predicted binding site for miR-10a-5p on BDNF and confirmed it using dual luciferase reporter assays. Moreover, lncPVT1 upregulated the expression of BDNF, whereas miR-10a-5p downregulated BDNF expression. BDNF promoted osteogenesis and partially rescued the si-lncPVT1-mediated inhibition of PDLCs osteogenic differentiation. LncPVT1 positively regulated the osteogenic differentiation of PDLCs via miR-10a-5p and BDNF. Our results provide a promising target for enhancing the osteogenic potential of PDLCs.

Possibility of RANKL as a pharmacological target

In bone tissue, RANKL acts as a positive regulator of bone turnover through both RANKL forward signaling, which induces osteoclast maturation, and RANKL reverse signaling, which promotes early osteoblast differentiation. Inhibition of osteoclast maturation by blocking RANKL forward signaling inevitably reduces the supply of osteoclast-derived coupling factors, leading to the suppression of bone formation. If we can design an agent that blocks RANKL forward signaling while simultaneously activating RANKL reverse signaling, the influence of reduced supply of osteoclast-derived coupling factors can be mitigated, which may be useful in the treatment of bone destructive diseases. Our previous study has shown that the cross-linking of RANKL molecules on the cell surface triggers the activation reverse signaling. Therefore, we screened various structures of IgG Fc-fusion protein constructs with multivalent binding sites for RANKL by arranging anti-RANKL single-chain antibody variable regions (scFv). Consequently, it was found that a structure in which the domain order inside the scFv was VH-VL order was advantageous for its ability to activate RANKL reverse signaling. Finally, the optimal structure was selected, and its pharmacological activity was evaluated using the ovariectomized mouse model. Results confirmed that the activation of RANKL reverse signaling could mitigate the decrease in bone formation associated with the suppression of osteoclast maturation.

Fabrication of Hydroxyapatite with Bioglass Nanocomposite for Human Wharton's-Jelly-Derived Mesenchymal Stem Cell Growing Substrate.

Recently, composite scaffolding has found many applications in hard tissue engineering due to a number of desirable features. In this present study, hydroxyapatite/bioglass (HAp/BG) nanocomposite scaffolds were prepared in different ratios using a hydrothermal approach. The aim of this research was to evaluate the adhesion, growth, viability, and osteoblast differentiation behavior of human Wharton’s-jelly-derived mesenchymal stem cells (hWJMSCs) on HAp/BG in vitro as a scaffold for application in bone tissue engineering. Particle size and morphology were investigated by TEM and bioactivity was assessed and proven using SEM analysis with hWJMSCs in contact with the HAp/BG nanocomposite. Viability was evaluated using PrestoBlueTM assay and early osteoblast differentiation and mineralization behaviors were investigated by ALP activity and EDX analysis simultaneously. TEM results showed that the prepared HAp/BG nanocomposite had dimensions of less than 40 nm. The morphology of hWJMSCs showed a fibroblast-like shape, with a clear filopodia structure. The viability of hWJMSCs was highest for the HAp/BG nanocomposite with a 70:30 ratio of HAp to BG (HAp70/BG30). The in vitro biological results confirmed that HAp/BG composite was not cytotoxic. It was also observed that the biological performance of HAp70/BG30 was higher than HAp scaffold alone. In summary, HAp/BG scaffold combined with mesenchymal stem cells showed significant potential for bone repair applications in tissue engineering.

Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

Free Transplantation of a Tissue Engineered Bone Graft into an Irradiated, Critical-Size Femoral Defect in Rats.

Healing of large bone defects remains a challenge in reconstructive surgery, especially with impaired healing potential due to severe trauma, infection or irradiation. In vivo studies are often performed in healthy animals, which might not accurately reflect the situation in clinical cases. In the present study, we successfully combined a critical-sized femoral defect model with an ionizing radiation protocol in rats. To support bone healing, tissue-engineered constructs were transferred into the defect after ectopic preossification and prevascularization. The combination of SiHA, MSCs and BMP-2 resulted in the significant ectopic formation of bone tissue, which can easily be transferred by means of our custom-made titanium chamber. Implanted osteogenic MSCs survived in vivo for a total of 18 weeks. The use of SiHA alone did not lead to bone formation after ectopic implantation. Analysis of gene expression showed early osteoblast differentiation and a hypoxic and inflammatory environment in implanted constructs. Irradiation led to impaired bone healing, decreased vascularization and lower short-term survival of implanted cells. We conclude that our model is highly valuable for the investigation of bone healing and tissue engineering in pre-damaged tissue and that healing of bone defects can be substantially supported by combining SiHA, MSCs and BMP-2.

Biological Mechanisms of Paeonoside in the Differentiation of Pre-Osteoblasts and the Formation of Mineralized Nodules.

Paeonia suffruticosa is a magnificent and long-lived woody plant that has traditionally been used to treat various diseases including inflammatory, neurological, cancer, and cardiovascular diseases. In the present study, we demonstrated the biological mechanisms of paeonoside (PASI) isolated from the dried roots of P. suffruticosa in pre-osteoblasts. Herein, we found that PASI has no cytotoxic effects on pre-osteoblasts. Migration assay showed that PASI promoted wound healing and transmigration in osteoblast differentiation. PASI increased early osteoblast differentiation and mineralized nodule formation. In addition, PASI enhanced the expression of Wnt3a and bone morphogenetic protein 2 (BMP2) and activated their downstream molecules, Smad1/5/8 and β-catenin, leading to increases in runt-related transcription factor 2 (RUNX2) expression during osteoblast differentiation. Furthermore, PASI-mediated osteoblast differentiation was attenuated by inhibiting the BMP2 and Wnt3a pathways, which was accompanied by reduction in the expression of RUNX2 in the nucleus. Taken together, our findings provide evidence that PASI enhances osteoblast differentiation and mineralized nodules by regulating RUNX2 expression through the BMP2 and Wnt3a pathways, suggesting a potential role for PASI targeting osteoblasts to treat bone diseases including osteoporosis and periodontitis.

Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells.

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 μM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 μM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 μM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)–Smad1/5/8 and Wnt–β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.

Scleraxis upregulated by transforming growth factor-β1 signaling inhibits tension-induced osteoblast differentiation of priodontal ligament cells via ephrin A2.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-β1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-β receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-β1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.

Vitamin A and Bone Health: A Review on Current Evidence.

Vitamin A is a fat-soluble micronutrient essential for growth, immunity, and good vision. The preformed retinol is commonly found in food of animal origin whereas provitamin A is derived from food of plant origin. This review summarises the current evidence from animal, human and cell-culture studies on the effects of vitamin A towards bone health. Animal studies showed that the negative effects of retinol on the skeleton were observed at higher concentrations, especially on the cortical bone. In humans, the direct relationship between vitamin A and poor bone health was more pronounced in individuals with obesity or vitamin D deficiency. Mechanistically, vitamin A differentially influenced the stages of osteogenesis by enhancing early osteoblastic differentiation and inhibiting bone mineralisation via retinoic acid receptor (RAR) signalling and modulation of osteocyte/osteoblast-related bone peptides. However, adequate vitamin A intake through food or supplements was shown to maintain healthy bones. Meanwhile, provitamin A (carotene and β-cryptoxanthin) may also protect bone. In vitro evidence showed that carotene and β-cryptoxanthin may serve as precursors for retinoids, specifically all-trans-retinoic acid, which serve as ligand for RARs to promote osteogenesis and suppressed nuclear factor-kappa B activation to inhibit the differentiation and maturation of osteoclasts. In conclusion, we suggest that both vitamin A and provitamin A may be potential bone-protecting agents, and more studies are warranted to support this hypothesis.

A Novel Long Noncoding RNA, Lnc-OAD, Is Required for Bone Morphogenetic Protein 2- (BMP-2-) Induced Osteoblast Differentiation.

Long noncoding RNAs (lncRNAs) play very important roles in cell differentiation. Our recent study has demonstrated that a novel lncRNA named lnc-OAD modulated 3T3-L1 adipocyte differentiation. In the present study, we examined the roles of lnc-OAD in bone morphogenetic protein 2- (BMP-2-) induced osteoblast differentiation. Lnc-OAD expression was increased during BMP-2-induced osteoblast differentiation in C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblast cells. Knockdown of lnc-OAD expression by specific siRNA remarkably decreased early osteoblast differentiation. In addition, stable knockdown of lnc-OAD by lentivirus vector also significantly inhibited late osteoblast differentiation and matrix mineralization in vitro. Conversely, stably overexpressed lnc-OAD with lentiviral vector accelerated osteoblast differentiation. Mechanistically, knockdown of lnc-OAD reduced significantly the phosphorylation of AKT and the expression of Osterix induced by BMP-2, while overexpression of lnc-OAD enhanced the phosphorylation of AKT and the expression of Osterix. Taken together, our study suggests that lnc-OAD plays an important role in modulating BMP-2-induced osteoblast differentiation via, at least in part, regulating the AKT-Osterix signaling axis.

Resveratrol protects MC3T3-E1 cells against cadmium-induced suppression of osteogenic differentiation by modulating the ERK1/2 and JNK pathways

Resveratrol (RES) is a natural polyphenolic compound with a broad range of physiological and pharmacological properties. Previous studies have shown that RES also plays an important role in protecting and promoting early bone metabolism and differentiation. The accumulation of cadmium (Cd), one of the world’s most poisonous substances, can inhibit skeletal growth and bone maturation, thus causing osteoporosis. However, whether RES can prevent the Cd-induced inhibition of osteogenic differentiation remains unknown. In this study, we found that RES promoted the early maturity of osteoblastic MC3T3-E1 cells, as demonstrated by the significantly increased mRNA and protein expression of a range of differentiation markers, including alkaline phosphatase (ALP), collagen 1 (COL1), bone morphogenetic protein-2 (BMP-2), and runt-related transcription factor 2 (RUNX2). In contrast, we found that cadmium chloride (CdCl2) inhibited the viability and osteogenic maturity of MC3T3-E1 cells. We also demonstrated that RES pretreatment for 30 min provided significant protection against Cd-induced apoptosis and attenuated the inhibition of osteogenic differentiation induced by Cd by modulating ERK1/2 and JNK signaling. In conclusion, our results indicate that RES is a potential femoral protectant that not only enhance the viability and early differentiation of osteoblasts, but also protect osteoblasts from cadmium damage.

Limonoid Triterpene, Obacunone Increases Runt-Related Transcription Factor 2 to Promote Osteoblast Differentiation and Function.

Root bark of Dictamnus dasycarpus Turcz. has been widely used as a traditional medicine and is a well-known anti-inflammatory agent. We isolated limonoid triterpene, obacunone (Obac) from the dried root bark of D. dasycarpus. Obac has been reported to exhibit varieties of biological activities including anti-inflammatory, anti-cancer, and anti-oxidant effects. This study aimed to investigate the beneficial effects and biological mechanisms of Obac in osteoblast differentiation and bone matrix mineralization. In the present study, Obac at concentrations ranging from 1 to 100 μM showed no proliferation effects in MC3T3-E1. The treatment of Obac (1 and 10 μM) increased wound healing and migration rates in a dose-dependent manner. Alkaline phosphatase (ALP) staining and activity showed that Obac (1 and 10 μM) enhanced early osteoblast differentiation in a dose-dependent manner. Obac also increased late osteoblast differentiation in a dose-dependent manner, as indicated by the mineralized nodule formation of ARS staining. The effects of Obac on osteoblast differentiation was validated by the levels of mRNAs encoding the bone differentiation markers, including Alp, bone sialoprotein (Bsp), osteopontin (Opn), and osteocalcin (Ocn). Obac increased the expression of bone morphogenetic protein (BMP), and the phosphorylation of smad1/5/8, and the expression of runt-related transcription factor 2 (RUNX2); Obac also inhibited GSK3β and upregulated the protein level of β-catenin in a dose-dependent manner during osteoblast differentiation. Obac-mediated osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin and a Wnt/β-catenin inhibitor, Dickkopf-1 (Dkk1) with the abolishment of RUNX2 expression and nuclear accumulation by Obac. Taken together, the findings of this study demonstrate that Obac has pharmacological and biological activates to promote osteoblast differentiation and bone mineralization through BMP2, β-catenin, and RUNX2 pathways, and suggest that Obac might be a therapeutic effect for the treatment and prevention of bone diseases such as osteoporosis and periodontitis.

Early B-cell Factor1 (Ebf1) promotes early osteoblast differentiation but suppresses osteoblast function.

Early B cell factor 1 (Ebf1) is a transcription factor that regulates B cell, neuronal cell and adipocyte differentiation. We and others have shown that Ebf1 is expressed in osteoblasts and that global deletion of Ebf1 results in increased bone formation in vivo. However, as Ebf1 is expressed in multiple tissues and cell types, it has remained unclear, which of the phenotypic changes in bone are derived from bone cells. The aim of this study was to determine the cell-autonomous and differentiation stage-specific roles of Ebf1 in osteoblasts. In vitro, haploinsufficient Ebf1+/- calvarial cells showed impaired osteoblastic differentiation indicated by lower alkaline phosphatase (ALP) activity and reduced mRNA expression of osteoblastic genes, while overexpression of Ebf1 in wild type mouse calvarial cells led to enhanced osteoblast differentiation with increased expression of Osterix (Osx). We identified a putative Ebf1 binding site in the Osterix promoter by ChIP assay in MC3T3-E1 osteoblasts and showed that Ebf1 was able to activate Osx-luc reporter construct that included this Ebf1 binding site, suggesting that Ebf1 indeed regulates osteoblast differentiation by inducing Osterix expression. To reconcile our previous data and that of others with our novel findings, we hypothesized that Ebf1 could have a dual role in osteoblast differentiation promoting early but inhibiting late stages of differentiation and osteoblast function. To test this hypothesis in vivo, we generated conditional Ebf1 knockout mice, in which Ebf1 deletion was targeted to early or late osteoblasts by crossing Ebf1fl/fl mice with Osx- or Osteocalcin (hOC)-Cre mouse lines, respectively. Deletion of Ebf1 in early Ebf1Osx-/- osteoblasts resulted in significantly increased bone volume and trabecular number at 12weeks by μCT analysis, while Ebf1hOC-/- mice did not have a bone phenotype. To conclude, our data demonstrate that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. However, Ebf1 inhibits bone accrual in the Osterix expressing osteoblasts in vivo but it is redundant in the maintenance of mature osteoblast function.

Thrombin inhibitor argatroban modulates bone marrow stromal cells behaviors and promotes osteogenesis through canonical Wnt signaling

AimsCoagulation is a common event that play a double-edged role in physiological and pathological process. Anti-coagulation methods were applied in joint surgery or scaffolds implantation to encourage new vascular formation and avoid coagulation block. However, whether anti-coagulation drug perform regulatory roles in bone structure is unknown. This study aims to explore a direct thrombin inhibitor, argatroban, effects on bone marrow stromal cells (BMSCs) and decipher the underlying mechanisms. Materials and methodsArgatroban effects on BMSCs were investigated in vivo and in vitro. The drug was applied in periodontal disease model mice and bone loss was evaluated by μCT and histology. BMSCs were treated with different doses argatroban or vehicle. Cellular reactions were analyzed using wound healing assay, qRT-PCR, Alizarin Red S staining and western blotting. Key findingsWe demonstrated that local injection of argatroban can rescue bone loss in periodontal disease in vivo. To explore the underlying mechanism, we examined that cell proliferation and differentiation capability. Proliferation and migration of BMSCs were both inhibited by applying lower dose of argatroban. Interestingly, without affecting osteoclastogenesis, osteogenic differentiation was significantly induced by argatroban, which were shown by extracellular mineralization and upregulation of early osteoblastic differentiation markers, alkaline phosphatase, Osteocalcin, transcription factors RUNX2 and Osterix. In addition, molecular analysis revealed that argatroban promoted β-catenin nuclear translocation and led to an increase of osteogenesis through activating canonical Wnt signaling. SignificanceTaken together, our results show the novel application of the anti-coagulation compound argatroban in the commitment of BMSCs-based alveolar bone regeneration and remodeling.

Leukemia inhibitory factor treatment attenuates the detrimental effects of glucocorticoids on bone in mice.

Glucocorticoids (GCs) are widely used drugs for the treatment of inflammatory and autoimmune diseases. However, a severe side effect induced by long-term GC therapy is osteoporosis. Leukemia inhibitory factor (LIF) - a glycoprotein 130 (gp130) dependent cytokine and member of the interleukin-6 cytokine family - is an activator protein 1 (AP-1) target gene that may be involved in one of the mechanisms underlying GC-induced bone loss. Indeed, we previously reported that the mRNA expression level of LIF was enhanced upon osteogenic differentiation, but was significantly decreased in GC-treated osteoblasts. In this study, we show that in vitro LIF treatment rescues the decreased early osteogenic differentiation and mineralization of GC-treated osteoblasts. Furthermore, we also demonstrate that in vivo LIF treatment protects against GC-mediated trabecular bone loss by decreasing the loss of both trabecular bone formation and osteoblast numbers. This protection appears to be conferred by LIF rescuing GC decreased activity of Stat3, MAPK, and Akt signaling pathways. Thus, the specific targeting of LIF signaling may represent a new therapeutic strategy to prevent GC-induced trabecular bone loss.

M2-like macrophage infiltration and transforming growth factor-β secretion during socket healing process in mice

ObjectiveMacrophages are involved in tissue inflammation and repair through cytokine secretion. However, the contribution of macrophages to healing and osteogenesis after tooth extraction remains unclear. Therefore, we investigated the distribution of osteoblastic cells and macrophages in the early healing process after tooth extraction. MethodsThe maxillary first molars of 6-week-old male mice were extracted. The maxilla was collected 1, 3, and 7 days after extraction. The states of socket healing, localization of osteoblastic markers, and macrophage infiltration were sequentially observed by micro-CT imaging and immunohistochemistry. ResultsOn day 3 after tooth extraction, α-smooth muscle actin (SMA)-positive cells, osteoprogenitor cells at fracture healing, were observed in the socket. Several α-SMA-positive cells also expressed Runx2, the early osteoblast differentiation marker. The infiltration of F4/80-positive, mature macrophages and CD206-positive, M2-like macrophages was noted in the socket. However, CD169-positive macrophages (Osteomac), which are involved in fracture healing, were not detected in the socket. F4/80-positive and CD206-positive macrophages also showed the localization of transforming growth factor-β (TGF-β), which promotes osteoprogenitor cell proliferation and early differentiation. Phosphorylated Smad3, a downstream mediator of the signal activity of TGF-β, was detected in α-SMA-positive cells. On day 7, the extracted socket contained a large amount of new bone. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were detected on bone surfaces. ConclusionOur data indicate that M2-like macrophages regulate the proliferation and differentiation of α-SMA-positive cells by secreting TGF-β at the early stage of socket healing, and also suggest the importance of macrophages in healing and bone formation after tooth extraction.

Ziyuglycoside I Upregulates RUNX2 through ERK1/2 in Promoting Osteoblast Differentiation and Bone Mineralization.

Sanguisorba officinalis L. (Rosaceae) is a perennial herbaceous plant and its roots have been used as an important traditional medicine for over 2000 years. Ziyuglycoside I (Ziyu), an active compound isolated from the roots of S. officinalis L., has shown biological effects such as anti-oxidant, antiviral, and antiwrinkle activities. This study aimed to elucidate the underlying mechanisms of action of Ziyu on cytotoxicity, migration, and differentiation of pre-osteoblasts. Herein, at concentrations ranging from 1 to 100 [Formula: see text]M, Ziyu was not cytotoxic against pre-osteoblasts. Alkaline phosphatase activity assay and staining, and migration assay showed that Ziyu increased cell migration and promoted early osteoblast differentiation, followed by the enhancement of mineralized nodule formation in a dose-dependent manner, as indicated by Alizarin Red S staining. In addition, Ziyu increased the protein levels of runt-related transcription factor 2 (RUNX2) during osteoblast differentiation, whereas it did not affect the phosphorylation of Smad1/5/8 and GSK3b and expression of [Formula: see text]-catenin. Ziyu also activated ERK1/2 and mitogen-activated protein kinase during osteoblast differentiation, and ERK1/2 inhibitor attenuated Ziyu-mediated RUNX2 expression and nuclear accumulation. Furthermore, Ziyu-mediated early and late osteoblast differentiation was significantly suppressed by the inhibition of ERK1/2, which was accompanied by attenuation in the mRNA levels of osteoblast-related genes including bone sialoprotein, osteopontin, and osteocalcin. Taken together, the findings of this study provide evidence that Ziyu promotes cell migration, osteoblast differentiation, and bone mineralization and suggest a potential role for Ziyu in the treatment of bone diseases.

Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor.

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 μM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 μM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 μM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased β-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Surface modification of titanium with collagen/hyaluronic acid and bone morphogenetic protein 2/7 heterodimer promotes osteoblastic differentiation.

The aim of this study was to evaluate the effects of a collagen/hyaluronic acid coating without or with incorporated heterodimeric bone morphogenetic protein 2/7 (BMP2/7) on in-vitro osteoblastic differentiation on titanium discs. The multilayer collagen/hyaluronic acid coatings without or without incorporated BMP2/7 were deposited on titanium discs via a layer-by-layer technique. The effects of the coatings were evaluated by assessing the alkaline phosphatase (ALP) activity (an early osteoblastic differentiation marker) and the osteocalcin expression (a late osteoblastic differentiation marker). The expression levels of the osteoblastic genes, such as alkaline phosphatase 2 (AKP2) and osteocalcin (OC) were detected using real-time RT-PCR. ALP activity and OC expression were significantly increased when cells were cultured with collagen/hyaluronic acid+BMP2/7 heterodimer (p<0.05). The same result was found in cells with the expression of a BMP2/7 fusion gene, OC and AKP2. These results indicated that collagen/hyaluronic acid+BMP2/7 heterodimer-coated discs might have the potential to greatly enhance osseointegration than a either BMP2 or BMP7 solution or a mixture of BMP2 and BMP7 BMP2/7.