Osteoblast Differentiation Markers Research Articles

Anthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3β and subsequently activating β-catenin

BackgroundThe bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. PurposeThis study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. Study design and methodsCell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. ResultsAHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3β (GSK-3β); particularly, isovitexin-4′-O-glucoside most strongly binds to the peptide backbone of GSK-3β at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3β at SER9 (an inactive form) and released β-catenin into the nucleus. Pretreatment with FH535, a Wnt/β-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. ConclusionAHs stimulate osteogenic activities through the inhibition of GSK-3β and subsequent activation of β-catenin, leading to anti-osteoporosis effects.

FAM20C plays a critical role in the development of mouse vertebra

Background ContextFamily with sequence similarity 20-member C (FAM20C) is a protein kinase that is responsible for the phosphorylation of many secretory proteins; however, its roles in spine or vertebra development have not be studied. PurposeThe aim of this investigation is to analyze the roles of FAM20C in vertebra development. Study Design/SettingA mouse study of the Fam20c gene using conditional knockout to assess the effects of its inactivation on vertebra development. MethodsBy breeding Sox2-Cre mice with Fam20cflox/flox mice, Sox2-Cre;Fam20cflox/flox mice (abbreviated as cKO mice) are created. X-ray radiography, resin-casted scanning electron microscopy, Hematoxylin and Eosin staining, safranin O staining, Goldner's Masson trichrome staining, Von Kossa staining, tartrate-resistant alkaline phosphatase staining, immunohistochemistry staining, Western Immunoblotting and real-time PCR were employed to characterize the vertebrae of cKO mice compared to the normal control mice. ResultsInactivation of Fam20c in mice results in remarkable spine deformity, severe morphology and mineralization defects, altered levels of osteoblast differentiation markers, reduction of activity of the Wnt/β-catenin signaling pathway and reduced level of osteoclastogenesis in the vertebrae. ConclusionsFAM20C plays an essential role in vertebral development; it may regulate vertebral formation through the Wnt/β-catenin signaling pathway. Clinical significanceMutations in the human FAM20C gene are associated with Raine syndrome. The findings of this study provide valuable clues for the clinical management of Raine syndrome regarding spine manifestations in patients.

CD14, A toll-like receptor co-factor, influences osteoclast differentiation and activity, but does not alter osteoblastic potential of bone-marrow preursors in mice

Purpose: Changes in subchondral bone structure occur in osteoarthritis (OA), and are visible by imaging as subchondral sclerosis on radiographs, and bone marrow lesions (BMLs) by MRI. These changes are the result of active bone remodeling that occurs in OA. BMLs in particular are correlated with pain in OA patients. We previously demonstrated that bone remodeling was significantly reduced after joint injury in mice deficient in CD14. CD14 enhances Toll-like receptor (TLR) mediated inflammation by acting as a co-receptor for multiple TLRs, increasing TLR sensitivity to damage associated molecular patterns (DAMPS) to activate proinflammatory signaling pathways. Soluble CD14, TLRs, and DAMPs (TLR ligands) are all elevated in OA patients, and sCD14 has been implicated as a biomarker of symptom and structural severity in OA. Increasing evidence suggestions CD14/TLR signaling influences bone remodeling as well, through activation of osteoclast precursor cells. Here, we investigate the potential mechanism by which CD14 influences bone remodeling through in vitro analysis of osteoclastogenic and osteoblastic potential and function. Methods: Bone marrow (BM) hematopoietic and mesenchymal precursors were isolated from long bones of 12-week-old male wild-type C57BL/6 (WT) and CD14-deficient (CD14-/-) mice. Briefly, BM was flushed from the femurs and tibia, and cells were cultured for 24 hours to separate non-adherent from adherent population. For osteoclastic differentiation, the non-adherent cells were seeded at a density of 26,000 cells per cm2 and were cultured in presence of monocyte/macrophage colony-stimulating factor (M-CSF; 35 ng/ml) and receptor activator of nuclear factor kβ ligand (RANKL; 100 ng/ml) for 9 days. Osteoblastic differentiation was accomplished by seeding the adherent BM cells at 30,000 cells per cm2 and culturing in the presence of 10 mM β-glycerophosphate ,50 μM ascorbic acid, and 100 nM Dexamethasone for up to 19 days. Osteoclasts were identified as multinucleated cells with positive TRAP staining. The resorptive ability of osteoclasts was assayed by quantifying resorption pits formed on bone slices. For osteoblast differentiation, Alizarin red was used to detect mineralized bone matrix. At the completion of each assay RNA was isolated from cultures and Real-time quantitative PCR analysis was performed to detect changes in mRNA levels of osteoclast (i.e. calcitonin receptor/CTR and cathepsin K/Ctsk) and osteoblast (i.e. osteopontin/OPN and osteocalcin/BLAP) differentiation markers. Results: Cells from CD14-/- mice demonstrated decreased percentages of TRAP positive cells and multi-nucleated cells (p value = 0.01; Fig. 1 A-D) and reduced Ctsk mRNA (p value = 0.03; Fig. 1 E&F), compared to WT controls. CD14-/- cells showed a 50% reduction in resorption pits and area compared to WT (Fig. 1 G&I). No significant differences were observed between strains in osteoblastic potential measured by mineralization and gene expression of osteoblastic markers (Fig. 2). Conclusions: CD14-/- BM precursor cells have reduced capacity to differentiate into functional osteoclasts, while osteoblastic potential is retained. This suggests that the reduced capacity of CD14-deficient osteoclastic cells may lead to the reduced bone remodeling in response to joint injury seen in the DMM model, by disrupting the balance between resorption and formation. Future experiments will determine the influence of DAMPs on osteoclast differentiation and activity, and the density of osteoclasts in SCB after DMM injury. These experiments will lead to a better understanding of the role of CD14 and TLR pathways on bone remodeling in response to joint injury and OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to alkalinization-induced vascular calcification in vitro.

ObjectiveIn order to find new strategies for the prevention of vascular calcification in uremic individuals especially treated by dialysis and develop novel therapeutic targets in vascular calcification, we explore the role of KCa3.1 in alkalinization‐induced VSMCs calcification in vitro.MethodRat VSMCs calcification model was established by beta‐glycerophosphate (β‐GP, 10 mM) induction. The pH of Dulbecco's modified Eagle's medium (DMEM) was adjusted every 24 h with 10 mM HCl or 10 mM NaHCO3. The mineralization was measured by Alizarin Red staining and O‐cresolphthalein complex one method. mRNA and protein expression were detected by RT‐PCR and Western blot or immunofluorescence. Ca2+ influx was measured by Elisa.ResultThe results indicated that alkalization induced an increase in Ca2+ influx to enhance VSMCs calcification. Furthermore, the increase of calcification was associated with the expression of KCa3.1 via advanced expression of osteoblastic differentiation markers alkaline phosphatase (ALP) and Runt‐related transcription factor 2 (Runx2). Blocking KCa3.1 with TRAM‐34 or shRNA vector can significantly lowered the effects of calcification in the activity of ALP and Runx2 expression.ConclusionTogether all, our studies suggested that alkalinization can promote vascular calcification by upregulating KCa3.1 channel and enhancing osteogenic/chondrogenic differentiation by upregulating Runx2. The specific inhibitor TRAM‐34 and KCa3.1‐shRNA ameliorated VSMCs calcification by downregulating KCa3.1.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

LGR5 enhances the osteoblastic differentiation of MC3T3‑E1 cells through the Wnt/β‑catenin pathway

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a Wnt-associated gene that contributes to cell proliferation and self-renewal in various organs. LGR5 is expressed in Ewing sarcoma, and LGR5-overexpressing mesenchymal stem cells promote fracture healing. However, the effects of LGR5 on osteoblastic differentiation remain unclear. The aim of the present study was to explore the function of LGR5 in osteoblastic differentiation. LGR5 was overexpressed or knocked down in the MC3T3-E1 pre-osteoblastic cell line via lentiviral transfection and its function in osteoblastic differentiation was investigated. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), osteocalcin and collagen type I a1 were determined, and ALP and Alizarin red staining were performed. In addition, the effects of LGR5 modulation on β-catenin and the expression of target genes in the Wnt pathway were investigated. The results revealed that the overexpression of LGR5 promoted osteoblastic differentiation. This was associated with enhancement of the stability of β-catenin and its levels in the cell nucleus, which enabled it to activate Wnt signaling. By contrast, the inhibition of LGR5 decreased the osteogenic capacity of MC3T3-E1 cells. These results indicate that LGR5 is a positive regulator of osteoblastic differentiation, whose effects are mediated through the Wnt/β-catenin signaling pathway. This suggests suggesting that the regulation of LGR5/Wnt/β-catenin signaling has potential as a therapy for osteoporosis.

Ferulic acid-Cu(II) and Zn(II) complexes promote bone formation

The current study aims to synthesize and investigate the effects of Ferulic acid-copper(II) (FA-Cu) and Ferulic acid-Zinc(II) (FA-Zn) complexes on bone formation. The initial biocompatibility test and ALP activity assay showed that the FA-Cu and FA-Zn complexes have reached the optimum concentrations of 40 μM. An in-vitro, cellular level evaluation showed that these complexes promote the ALP activity and calcium deposition in osteoblasts. At the molecular level analysis, these complexes increased the osteoblast specific markers, Runx2 and type 1 collagen mRNAs and secretory protein levels of osteocalcin (OC) and osteonectin (ON). Also, the expression of the co-activator of osteoblast differentiation, TAZ increased and the effect of these complexes decreased the co-suppressor HDAC4. We tested the bone forming effect of these complexes in (1) zebrafish embryo bone development, (2) osteoblast distribution in scale, and (3) tail fracture models. FA-Cu and FA-Zn complex exposures have enhanced bone mineralization in zebrafish embryos' development, increased osteoblast distribution in adult zebrafish scales, and reinforced bone healing by increasing ossification at the injury site. In summary, our findings have shown that FA stimulates bone formation, and the complexes of FA-Cu and FA-Zn have further improved it.

Inflammatory Cytokine TNF-a Controls Mesenchymal Stem Fate by Regulating JMJD3 Expression

Introduction: Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate in to osteoblast, adipocytes and chondrocytes. Lineage specification of MSC is governed by various systemic hormones, systemic and local growth factors and cytokines. TNF-α is an inflammatory cytokine produced at the site of tissue injuries and known to regulates MSC migration and differentiation. However, its role on lineage specification and differentiation of MSCs remain complex and elusive. In this study we explored the same utilizing human bone marrow and adipocyte derived MSCs. Experimental Methods: Human MSCs derived from bone marrow and adipocytes were differentiated in to osteoblast and adipocytes in the presence or absence of TNF-α. Expressions of osteoblast and adipocyte differentiation markers were assessed by qRT-PCR. The key epigenetic factor of lineage specification JMJD3 was depleted in MSCs utilizing lentiviral ShRNA. Results: TNF-α promoted the osteoblastic and inhibited the adipogenic differentiation of MSC as assessed by Alizarin and oil red O staining, respectively. Consistently, while inducing the key osteogenic factors, TNF- α repressed the adipogenic markers in MSCs. Mechanistically, TNF-α regulates MSC fate by inducing lysine-specific demethylase JMJD3/KDM6B, which is a key epigenetic factor that determines mesenchymal stem cell lineage specification. ShRNA mediated knockdown of JMD3 in MSCs inhibited TNF- α mediated activation and inhibition of osteogenic and adipogenic differentiation, respectively. Conclusion: Our study uncovers the novel mechanisms of TNF-α mediated MSC lineage commitment and differentiation and thus highlight JMJD3 as mediator of TNF-α actions in MSCs.

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.

Synthesis and characterization of magnesium diboride nanosheets in alginate/polyvinyl alcohol scaffolds for bone tissue engineering

Boride, which belongs to the distinct category of ceramic materials, has attracted significant attention in tissue engineering applications. Magnesium diboride (MgB2) consists of a plane of magnesium atoms sandwiched between the layers of boron. Even though MgB2 showed its role in various applications, its effect on osteogenesis has not yet been investigated. In this study, we synthesized MgB2 nanosheets (MgB2NS), a new class of 2D-nanoscale structures, by the ultrasonication exfoliation method and incorporated them into a polymeric mixture of alginate (Alg) and polyvinyl alcohol (PVA) by the freeze-drying procedure. The synthesized scaffolds (Alg/PVA/MgB2NS) were characterized by SEM, XRD, FT-IR, protein adsorption, swelling, degradation, and biomineralization studies. These scaffolds were non-toxic to mouse mesenchymal stem cells (mMSCs). MgB2NS in the scaffolds enhanced osteoblast differentiation of mMSCs at the molecular level by the expression of Runx2 and osteoblast differentiation marker genes and at the cellular level by alkaline phosphatase, alizarin Red and von Kossa staining. Overall, our results showed that MgB2NS in Alg/PVA scaffolds have osteogenic potential, suggesting their possible use in bone tissue engineering applications.

The Myokine Irisin Promotes Osteogenic Differentiation of Dental Bud-Derived MSCs.

Simple SummaryIrisin is a recently discovered protein, mainly produced in the muscle tissue, whose action is proving effective in many other tissues. The crosstalk between muscle and bone has been long since demonstrated, and physical activity has shown to have an impressive positive effect in both tissues. Irisin production increases with exercising and drops with sedentariness and aging, indicating that the molecule is involved in sarcopenia and in bone mass reduction. Although skeleton is target of irisin, its mechanism of action on bone cells has not yet been completely elucidated. The aim of this work is to analyze the effect of irisin on osteoblast differentiation; to this purpose, we used a stem cell model reproducing the osteoblastogenesis and the bone-forming processes. We performed an in vitro study exploring the main osteoblast markers in the presence of irisin. We found that irisin has an impressive effect on the most peculiar osteoblast feature: the bone mineral matrix secretion process. Moreover, irisin demonstrated an inductive effect on osteoblast osteocalcin production. Both results suggest a stimulating effect of irisin in bone formation. The association we observed between irisin addition and osteoblast osteocalcin production should be further investigated.The myokine irisin, well known for its anabolic effect on bone tissue, has been demonstrated to positively act on osteoblastic differentiation processes in vitro. Mesenchymal stem cells (MSCs) have captured great attention in precision medicine and translational research for several decades due to their differentiation capacity, potent immunomodulatory properties, and their ability to be easily cultured and manipulated. Dental bud stem cells (DBSCs) are MSCs, isolated from dental tissues, that can effectively undergo osteoblastic differentiation. In this study, we analyzed, for the first time, the effects of irisin on DBSC osteogenic differentiation in vitro. Our results indicated that DBSCs were responsive to irisin, showed an enhanced expression of osteocalcin (OCN), a late marker of osteoblast differentiation, and displayed a greater mineral matrix deposition. These findings lead to deepening the mechanism of action of this promising molecule, as part of osteoblastogenesis process. Considering the in vivo studies of the effects of irisin on skeleton, irisin could improve bone tissue metabolism in MSC regenerative procedures.

BMP9 Can Induce Schwann Cells Expressing Simian Virus 40 T Antigen to Differentiate into Fat and Bone In Vivo and In Vitro.

In our previous study, we constructed Schwann cells (SCs) that stably express Simian virus 40 T antigen (SV40T-SCs). SV40T-SCs functions and markers are similar to those of neural crest cells. There we used bone morphogenetic protein 9 (BMP9) to induce SV40T-SCs differentiation in vitro and in vivo and study possible related mechanism. SV40T-SCs differentiation was induced by BMP9 conditioned medium. The lipogenic differentiation of SV40T-SCs was assessed by Oil Red O staining. Alizarin red and Alcian blue staining, and alkaline phosphatase (ALP) assays were used to evaluate the SV40T-SCs osteogenic differentiation. The expression of adipocyte differentiation (c/EBPα and c/EBPβ) and osteoblast differentiation markers (OSX and RUNX2) were detected by quantitative polymerase chain reaction (qPCR). To study possible mechanism related to SV40T-SCs differentiation, the P53 and E2F1 activity were assessed by luciferase reporter plasmid, and Slug and E-cadherin expression by qPCR. In vivo, SV40T-SCs infected by Ad-BMP9 or Ad-GFP were injected under the skin of nude mice. After 4-6 W, the mice were euthanized and subcutaneously mass formed at injecting sites was collected for pathological analysis. After SV40T-SCs were cultured in BMP9 conditioned medium, lipid droplets were formed in the cytoplasm of these cells. Alizarin red and Alcian blue staining were positive, and ALP activity of SV40T-SCs increased significantly. The expression of adipocyte differentiation (c/EBPα and c/EBPβ) and osteoblast differentiation markers (OSX and RUNX2) in SV40T-SCs was upregulated by BMP9. SV40T significantly increased Slug expression and decreased E-cadherin expression. SV40T-SCs infected with Ad-BMP9 were able to differentiate into adipose tissue and form a small bone matrix under the nude mice skin. SV40T-SCs have the ability to differentiate into adipocytes and osteoblasts in vivo and in vitro. SV40T can upregulate the Slug expression and downregulate the E-cadherin expression to produce endothelial-to-mesenchymal transition (EMT). The multidirectional differentiation ability of SV40T-SCs may be related to EMT.

Osteolineage depletion of mitofusin2 enhances cortical bone formation in female mice.

Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of β-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.

Palmul-Tang, a Korean Medicine, Promotes Bone Formation via BMP-2 Pathway in Osteoporosis.

Osteoporosis is a common skeletal disease in post-menopausal women. Palmul-tang, an herbal medicine, has been treated for gynecological disease such as anemia, anorexia, anti-fatigue, unspecified menstruation and female infertility in East Asia. In this study, ameliorative effects of Palmul-tang soft extracts (PMT), a Korean Medicine, on osteoporosis were investigated. Ovariectomized (OVX) osteoporotic ICR mice were intragastrically administrated PMT for 4 weeks. The level of bone mineral density (BMD) was analyzed in bone tissues by dual X-ray absorptiometry. The bone medullary cavity and deposition of collagen were investigated by histological analysis. In addition, the BMP-2 signaling-related molecules, osteoblastic differentiation and formation markers, were determined in femoral tissues. The levels of BMD and bone mineral content were significantly increased in tibia, femurs and LV by treatment of PMT. PMT replenished bone marrow cavity and increased collagen deposition in bone marrow cells of femur. In addition, administration of PMT recovered serum ALP, bALP, osteocalcin and calcium levels in osteoporotic mice. Moreover, PMT treatment up-regulated the expressions of BMP-2, RUNX2 and OSX with its downstream factors, ALP, OPN and BSP-1, in the femoral tissues. Taken together, PMT restored the bone minerals and improvement of bone integrity by bone-forming BMP-2 signaling pathway. These results demonstrate that PMT could be an ameliorative agent for osteoporosis.

The TrkA agonist gambogic amide augments skeletal adaptation to mechanical loading.

The periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant increase in the fold change of Ngf, Wnt7b, and Axin2 mRNA expression as compared to vehicle (control). To test the effect of GA on osteoblasts directly, we cultured MC3T3-E1 cells for up to 21days in osteogenic differentiation media containing NGF, GA, or vehicle (control). Media containing GA induced the significant upregulation of the osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in a dose-dependent manner, whereas treatment with NGF was not associated with any significant increases in these markers. Furthermore, consistent with our in vivo findings, we observed that administration of 50nM of GA upregulated expression of Ngf at both Day 3 and Day 7. However, cells treated with the highest dose of GA (500nM) had significantly increased apoptosis and impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia.

Effects of Black Jade on Osteogenic Differentiation of Adipose Derived Stem Cells under Benzopyrene

Jade, a popular gemstone symbolizing beauty, grace, and longevity, is known to improve blood circulation; however, scientific research evidence is still lacking. The effect of black jade extract on the expression levels of apoptotic and osteogenic genes was validated using qPCR and flow cytometry. In combination with the use of a fluorescence microscope, osteogenic differentiation and the stained osteocytes count were analyzed. Under the pressure of benzo(a)pyrene, dermal cell apoptosis was accelerated and the osteogenic differentiation of adipose-derived stem cells (ASCs) was suppressed; but black jade extract counteracted the effects. Through an anti-apoptotic mechanism, the extract suppressed the expression of apoptotic proteins Bax and cytochrome C to 9 and 4.8 times, respectively, compared to that in dermal cells exposed to benzo(a)pyrene. During osteogenic differentiation of ASCs, the extract enhanced their differentiation despite being exposed to benzo(a)pyrene, and the relative levels of the osteoblast differentiation markers osteoponin, osteocalcin, and sclerostin were 1.87, 2.54, and 3.9 times higher, respectively, than those in the conditioned medium by benzo(a)pyrene. These effects of the extract indicate that black jade extract is very useful when applied as a functional biomaterial.

Asarylaldehyde enhances osteogenic differentiation of human periodontal ligament stem cells through the ERK/p38 MAPK signaling pathway

Periodontitis is an inflammatory disease that affects tooth-supporting tissues. Chronic inflammation can progress to periodontitis, which results in loss of alveolar bone. Asarylaldehyde is a potential substance for bone metabolism present in natural compounds. Here, we propose the application of asarylaldehyde in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to prevent bone loss. We investigated the effect of asarylaldehyde on hPDLSCs together with bone differentiation media in vitro. The osteogenic differentiation effect was observed after treatment of hPDLSCs with several concentrations of asarylaldehyde. After 21 days, osteogenic cells were identified by mineralization. We also observed that asarylaldehyde increased the mRNA expression of osteoblast-specific markers in hPDLSCs. Interestingly, asarylaldehyde regulated the levels of alkaline phosphatase (ALP) transcriptional activity through the p38/extracellular-signal-regulated kinase (ERK) signaling pathway. Notably, asarylaldehyde induced hPDLSCs to promote osteogenic differentiation. These results suggest that asarylaldehyde plays a key role in the osteogenic differentiation of hPDLSCs. Asarylaldehyde may be a good candidate for the application of natural compounds in future in periodontal regeneration.

Primary Cilia as a Biomarker in Mesenchymal Stem Cells Senescence: Influencing Osteoblastic Differentiation Potency Associated with Hedgehog Signaling Regulation.

Bone tissue engineering-based therapy for bone lesions requires the expansion of seeding cells, such as autologous mesenchymal stem cells (MSCs). A major obstacle to this process is the loss of the phenotype and differentiation capacity of MSCs subjected to passage. Recent studies have suggested that primary cilia, primordial organelles that transduce multiple signals, particularly hedgehog signals, play a role in senescence. Therefore, we explored the relationships among senescence, primary cilia, and hedgehog signaling in MSCs. Ageing of MSCs by expansion in vitro was accompanied by increased cell doubling time. The osteogenic capacity of aged MSCs at passage 4 was compromised compared to that of primary cells. P4 MSCs exhibited reductions in the frequency and length of primary cilia associated with decreased intensity of Arl13b staining on cilia. Senescence also resulted in downregulation of the expression of hedgehog components and CDKN2A. Suppression of ciliogenesis reduced the gene expression of both Gli1, a key molecule in the hedgehog signaling pathway and ALP, a marker of osteoblastic differentiation. This study demonstrated that the senescence of MSCs induced the loss of osteoblastic differentiation potency and inactivated hedgehog signaling associated with attenuated ciliogenesis, indicating that primary cilia play a mediating role in and are biomarkers of MSC senescence; thus, future antisenescence strategies involving manipulation of primary cilia could be developed.

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.

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.