Osteoblast Marker Genes Research Articles

Astragalin Promotes Osteoblastic Differentiation in MC3T3-E1 Cells and Bone Formation in vivo.

Astragalin (AG) is a biologically active flavonoid compound that can be extracted from a number of medicinal plants. However, the effects of AG on osteoblastic differentiation in mouse MC3T3-E1 cells and on bone formation in vivo have not been studied fully. In this study, we found that the activities of alkaline phosphatase (ALP) and mineralized nodules in MC3T3-E1 cells were both significantly increased after treatment with AG (5, 10, and 20 μM). Meanwhile, the mRNA and protein levels of osteoblastic marker genes in MC3T3-E1 cells after AG treatment were markedly increased compared with a control group. In addition, the levels of BMP-2, p-Smad1/5/9, and Runx2 were significantly elevated in AG-treated MC3T3-E1 cells. Moreover, we found that the protein levels of Erk1/2, p-Erk1/2, p38, p-p38, and p-JNK were also significantly increased in AG-treated MC3T3-E1 cells compared to those in the control group. Finally, in vivo experiments demonstrated that AG significantly promoted bone formation in an ovariectomized (OVX)-induced osteoporotic mouse model. This was evidenced by significant increases in the values of osteoblast-related parameters (BFR/BS, MAR, Ob.S/BS, and Ob.N/B.Pm) and bone histomorphometric parameters (BMD, BV/TV, Tb.Th, and Tb.N.) in OVX mice after AG treatment (5, 10, and 20 mg/kg). Collectively, these results demonstrated that AG may promote osteoblastic differentiation in MC3T3-E1 cells via the activation of the BMP and MAPK pathways and promote bone formation in vivo. These novel findings indicated that AG may be a useful bone anabolic agent for the prevention and treatment of osteoporosis.

FGF adsorbed mesoporous bioactive glass with larger pores in enhancing bone tissue engineering.

Mesoporous bioactive glass (MBG) is performed as a bone tissue engineering material because of its good bioactivity, biocompatibility and osteoinducion characteristics. Here, we propose MBG with larger pores (MBG-L) adsorbed fibroblast growth factor (FGF) to facilitate osteoblast differentiation and matrix mineralization. Specifically, we observed that MBG-L promotes calcium deposit precipitation in vitro. In addition, adhesion, proliferation, differentiation and matrix mineralization were promoted after osteoblast cultured on MBG-L/FGF. Interestingly, we found that the transcriptional activity of the critical transcription factor Runx2 was increased through MAPK pathway after osteoblast cultured on MBG-L/FGF. Support for this result, we found that the expression of osteoblastic marker genes, Osteocalcin (Ocn), Osteopontin (Opn), and Runx2 were increased. Thus, our findings provided that MBG-L/FGF could be a promising new material in bone tissue engineering.

ELP2 negatively regulates osteoblastic differentiation impaired by tumor necrosis factorα in MC3T3-E1 cells through STAT3 activation.

Tumor necrosis factor‐α (TNF‐α) is a pluripotent signaling molecule. The biological effect of TNF‐α includes slowing down osteogenic differentiation, which can lead to bone dysplasia in long‐term inflammatory microenvironments. Signal transducer and activator of transcription 3 (STAT3)‐interacting protein 1 (StIP1, also known as elongator complex protein 2, ELP2) play a role in inhibiting TNF‐α‐induced osteoblast differentiation. In the present study, we investigated whether and how ELP2 activation mediates the effects of TNF‐α on osteoblastic differentiation. Using in vitro cell cultures of preosteoblastic MC3T3‐E1 cells, we found that TNF‐α inhibited osteoblastic differentiation accompanied by an increase in ELP2 expression and STAT3 activation. Forced ELP2 expression inhibited osteogenic differentiation of MC3T3‐E1 cells, with a decrease in the expression of osteoblast marker genes, alkaline phosphatase activity, and matrix mineralization capacity. In contrast, ELP2 silencing ameliorated osteogenic differentiation in MC3T3‐E1 cells, even after TNF‐α stimulation. The TNF‐α‐induced inhibitory effect on osteoblastic differentiation was therefore mediated by ELP2, which was associated with Janus kinase 2 (JAK2)/STAT3 activation. These results suggest that ELP2 is upregulated at the differentiation of MC3T3‐E1 cells into osteoblasts and inhibits osteogenic differentiation in response to TNF‐α through STAT3 activation.

Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.

KLF2 regulates osteoblast differentiation by targeting of Runx2

Osteoblast differentiation plays a critical role in bone formation and maintaining balance in bone remodeling. Runt-related transcription factor 2 (Runx2) is a central transcription factor regulating osteoblast differentiation and promoting bone mineralization. Until now, the molecular regulatory basis and especially the gene regulatory network of osteogenic differentiation have been unclear. Krüppel-like factor 2 (KLF2) is a zinc finger structure and DNA-binding transcription factor. The current study aimed to investigate the physiological function of KLF2 in osteoblast differentiation. Our results indicate that KLF2 is expressed in pre-osteoblast MC3T3-E1 cells and primary osteoblasts. Interestingly, KLF2 expression is increased in osteoblasts during the osteoblastic differentiation process. Overexpression of KLF2 in MC3T3-E1 cells promoted the expression of the osteoblastic differentiation marker genes Alp, Osx, and Ocn, and stimulated mineralization by increasing Runx2 expression at both the mRNA and protein levels. In contrast, knockdown of KLF2 produced the opposite effects. Importantly, we found that KLF2 could physically interact with Runx2. KLF2 promoted osteoblast differentiation by regulating Runx2 and physically interacting with Runx2. Taken together, the findings of this study identify KLF2 as a novel regulator of osteoblast differentiation. Our findings suggest that KLF2 might be a new therapeutic target for bone disease.Krüppel-like factor 2 (KLF2) is expressed in primary osteoblasts and is upregulated during osteoblast differentiation. The authors show that KLF2 promotes osteoblast differentiation and mineralization by increasing expression of the transcription factor Runx2, which is necessary for osteoblast function. Therefore, KLF2 might be a novel therapeutic target for bone disease.

Ethyl acetate and n-butanol fraction of Cissus quadrangularis promotes the mineralization potential of murine pre-osteoblast cell line MC3T3-E1 (sub-clone 4).

In a sequel to investigate osteogenic potential of ethanolic extract of Cissus quadrangularis (CQ), the present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ-EA) and butanol (CQ-B) extracts of CQ on mouse pre-osteoblast cell line MC3T3-E1 (sub-clone 4) with an objective to isolate an antiosteoporotic compound. Growth curve, proliferation, and viability assays showed that both the extracts were nontoxic to the cells even at high concentration (100 µg/ml). The cell proliferation was enhanced at low concentrations (0.1 µg/ml and 1 µg/ml) of both the extracts. They also upregulated the osteoblast differentiation and mineralization processes in MC3T3-E1 cells as reflected by expression profile of osteoblast marker genes such as RUNX2, Osterix, Collagen (COL1A1), Alkaline Phosphatase (ALP), Integrin-related Bone Sialoprotein (IBSP), Osteopontin (OPN), and Osteocalcin (OCN). CQ-EA treatment resulted in early differentiation and mineralization as compared with the CQ-B treatment. These findings suggest that low concentrations of CQ-EA and CQ-B have proliferative and osteogenic properties. CQ-EA, however, is more potent osteogenic than CQ-B.

Metformin promotes differentiation of human bone marrow derived mesenchymal stem cells into osteoblast via GSK3β inhibition.

Metformin, a common and first-line drug for diabetes mellitus, is widely used in the world. Recently, many studies have documented that osteogenesis could be mediated by metformin. However, the specific mechanism by which metformin affects osteogenesis has not been clearly identified. Therefore, the aim of this study is to evaluate the role of GSK3β in metformin-induced osteogenic differentiation of mesenchymal stem cells (MSCs). Osteoblast-marker genes, including Col-1, OCN, and RUNX2, were measured by RT-PCR in differentiated MSCs treated with Metformin. Osteogenic differentiation viability was measured by Alkaline phosphatase (ALP) assays and Alizarin Red Staining. The expression of GSK3β, β-catenin and AMPK were measured by Western blotting in MSCs treated with metformin. We found that metformin at 100 μM significantly promoted osteogenic differentiation of human mesenchymal stem cells (hBMSCs). Next, we showed that GSK3β and Wnt signaling pathway are involved in metformin-induced osteogenic differentiation of hBMSCs. Furthermore, osteogenic differentiation of hBMSCs induced by metformin could be eliminated by inhibiting phosphorylation of GSK3β. The data suggested that metformin promoted the osteoblast differentiation of MSCs by, at least partly, inhibiting GSK3β activity. Additionally, we also found that AMPK plays an essential role in the inhibition of GSK3β by metformin.

Activation of TGR5 promotes osteoblastic cell differentiation and mineralization

Impairment of normal osteoblast differentiation has been associated with bone loss-related disorders, such as osteoporosis. Takeda G-protein coupled receptor 5 (TGR5) has been identified as an important modulator of bile acid and energy homeostasis. Little information regarding the effects of TGR5 on osteoblastic bone formation and matrix mineralization has been reported before. In the current study, we found that TGR5 was expressed in osteoblast-like cell line MC3T3-E1 cells. Osteogenic medium (OM) stimulation promoted the expression of TGR5 in a dose-dependent manner. Notably, treatment with the specific TGR5 agonist GPBARA increased ALP activity, matrix mineralization, and expressions of osteoblastic differentiation marker genes, such as ALP, OCN, and Osx, by promoting the expression of Runx-2. Silencing of TGR5 by transfection with TGR5 siRNA abolished these effects. Also, we found that the AMPK/eNOS pathway was involved in this process. Blockage of AMPK activation using its specific inhibitor compound C abolished the effect of GPBARA-induced increase in ALP activity, matrix mineralization, and expressions of osteoblastic differentiation marker genes. The obtained results provide a new insight into the physiological function of TGR5 in bone formation and suggest that TGR5 might be a novel therapeutic target for bone diseases.

A Novel Rhein Derivative Modulates Bone Formation and Resorption and Ameliorates Estrogen-Dependent Bone Loss.

Osteoporosis, an osteolytic disease that affects millions of people worldwide, features a bone remodeling imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Identifying dual target-directed agents that inhibit excessive bone resorption and increase bone formation is considered an efficient strategy for developing new osteoporosis treatments. Rhein, a natural anthraquinone, can be isolated from various Asian herbal medicines. Rhein and its derivatives have been reported to have various beneficial pharmacological effects, especially their bone-targeting ability and anti-osteoclastogenesis activity. Moreover, hydrogen sulfide (H2 S) was reported to prevent ovariectomy- (OVX-) induced bone loss by enhancing bone formation, and sulfur replacement therapy has been considered a novel and plausible therapeutic option. Based on this information, we synthesized a rhein-derived thioamide (RT) and investigated its effects on bone resorption and bone formation in vitro and in vivo. It has been found that the RT-inhibited receptor activator of the nuclear factor-κB (NF-κB) ligand- (RANKL-) induced osteoclastogenesis and bone resorption in a dose-dependent manner. The expression of osteoclast marker genes was also suppressed by RT treatment. Furthermore, exploration of signal transduction pathways indicated that RT markedly blocked RANKL-induced osteoclastogenesis by attenuating MAPK pathways. However, RT treatment in an osteoblastic cell line, MC3TE-E1, indicated that RT led to an increase in the deposition of minerals and the expression of osteoblast marker genes, as demonstrated by Alizarin Red staining and alkaline phosphatase activity. Importantly, an OVX mouse model showed that RT could attenuate the bone loss in estrogen deficiency-induced osteoporosis in vivo with a smart H2 S-releasing property and that there was a considerable improvement in the biomechanical properties of bone. Accordingly, our current work highlights the dual regulation of bone remodeling by the rhein-derived molecule RT. This may be a highly promising approach for a new type of anti-osteoporosis agent. © 2018 American Society for Bone and Mineral Research.

Cellular Response to 3-D Printed Bioactive Silicate and Borosilicate Glass Scaffolds.

The repair and regeneration of loaded segmental bone defects is a challenge for both materials and biomedical science communities. Our recent work demonstrated the capability of bioactive glass in supporting bone healing and defect bridging using a rabbit femur segmental defect model without growth factors or bone marrow stromal cells (BMSCs). Here in the current work, a comprehensive in vitro evaluation of bioactive silicate (13-93) and borosilicate (2B6Sr) glass scaffolds was conducted to provide further understanding of their biological performances and to establish a correlation between in vitro and in vivo behaviors. Our in vitro evaluation using a murine MC3T3-E1 cell line confirmed the capability of both scaffolds to support cell attachment, vascular endothelial growth factor (VEGF) formation, and to stimulate mineral deposition and osteoblast marker gene expression. In particular, borosilicate (2B6Sr) glass showed a better capability in supporting the mineralization and gene expression than silicate (13-93) glass, consistent with a faster bone healing ability in vivo. The current in vitro results, combined with our previous in vivo findings, provide a strong basis for the further translational evaluation of bioactive glass scaffolds and for potential preclinical practice. © 2018 Wiley Periodicals, Inc. J. Biomed. Mater. Res. Part B, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 818-824, 2019.

Tricin enhances osteoblastogenesis through the regulation of Wnt/β-catenin signaling in human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stromal cells that are capable of differentiate into multilineage cell types including bone in vitro and in vivo. Aim of the study: They have been widely developed as a therapeutic approach for bone tissue repair and regeneration. However, the efficiency of lineage specific differentiation still needs improvement. We investigated the effect of Tricin on the proliferation of human adult MSCs by alamar blue assay, the mineralization of MSCs by staining of calcium deposition, and the expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs. Moreover, we also checked the effects of Tricin on the expression of key genes in Wnt/β-catenin signaling pathway. We found Tricin could promote proliferation of MSCs, as well as their mineralization. Tricin could also enhance the expression of osteogenesis marker genes including Bone sialoprotein, osteocalcin, Alkaline phosphatase, and RUNX2. Furthermore, we found out that neither siβ-catenin nor GSK-3β had effect on Tricin treatment, but siWnt3α could neutralize the effect of Tricin. Tricin can enhance osteoblastogenesis through the regulation of Wnt/β-catenin signaling pathway in human adult MSCs in vitro.

Lumichrome inhibits osteoclastogenesis and bone resorption through suppressing RANKL-induced NFAT activation and calcium signaling.

The dynamic balance between bone resorption and bone formation is crucial to maintain bone mass. Osteoclasts are key cells that perform bone resorption while osteoblasts and osteocytes function in bone formation. Osteoporosis, a bone metabolism disease characterized by bone loss and degradation of bone microstructure, occurs when osteoclastic bone resorption outstrips osteoblastic bone synthesis. The interaction between receptor activator of nuclear factor κB ligand (RANKL) and RANK on the surface of bone marrow macrophages promotes osteoclast differentiation and activation. In this study, we found that lumichrome, a photodegradation product of riboflavin, inhibits RANKL-induced osteoclastogenesis and bone resorption as determined by tartrate-resistant acid phosphatase staining, immunofluorescence, reverse transcription-polymerase chain reaction, and western blot. Our results showed that lumichrome represses the expression of osteoclast marker genes, including cathepsin K (Ctsk) and Nfatc1. In addition, lumichrome suppressed RANKL-induced calcium oscillations, NFATc1, NF-κB, and MAPK signaling activation. Moreover, lumichrome promoted osteoblast differentiation at an early stage, as demonstrated by upregulated expression of osteoblast marker genes Alp, Runx2, and Col1a1. We also found that lumichrome reduces bone loss in ovariectomized mice by inhibiting osteoclastogenesis. In summary, our data suggest the potential of lumichrome as a therapeutic drug for osteolytic diseases.

Synthesis and characterization of silibinin/phenanthroline/neocuproine copper(II) complexes for augmenting bone tissue regeneration: an in vitro analysis.

In the recent decades, flavonoid metal complexes have been widely investigated for their multifaceted role in enabling osteoblast differentiation and bone formation. Silibinin complexed with copper(II) ion has been synthesized along with two mixed ligand complexes, namely copper(II) silibinin-phenanthroline and neocuproine as co-ligands, and their positive role in promoting neovacularization and osteoblast differentiation was investigated. Silibinin mono complex [Cu(sil)(H2O)2] and [Cu(sil)(phen)] showed similar UV-visible absorption in the region of 315 and 222nm, whereas Cu(silibinin)(neocuproine) [Cu(sil)(neo)] showed a blueshift in the 320nm transition. The involvement of carbonyl group present in the C-ring in metal ion chelation was identified by FT-IR analysis. Thermal gravimetric analysis (TGA) depicted that [Cu(sil)(neo)] has higher thermal stability when compared with the control silibinin and Cu-silibinin mono, and phen complexes. Cu-silibinin complexes were found to be non-toxic to human MG-63 cells and mouse mesenchymal stem cells (MSCs). Our investigations identified the positive role of these complexes in promoting osteoblast differentiation by enhancing calcium deposition and alkaline phosphatase (ALP) activity at the cellular level and stimulation of osteoblastic marker genes such as Runx2, ALP, type 1 collagen, and OCN mRNAs expression at the molecular level. These complexes also supported angiogenesis by upregulation of VEGF and Ang 1 expression in mouse MSCs. Hence, our results suggest that the potential of these metal complexes along with mixed ligand complexes promoted osteoblast differentiation, thus warranting its candidature for bone tissue regeneration application.

Asiaticoside induces osteogenic differentiation of human periodontal ligament cells through the Wnt pathway.

Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside-induced osteogenic differentiation of hPDL cells. hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real-time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of β-catenin was analyzed by both immunofluorescence and western blot. The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real-time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose-dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of β-catenin as evaluated by immunofluorescence staining and western blot analysis. Pre-treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside-induced β-catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside-induced DMP1 protein expression. The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/β-catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.

CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal (BMS) cells derived from Chip−/− mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip−/− mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

Evaluation of osteoblast differentiation and function when cultured on mesoporous bioactive glass adsorbed with testosterone.

Mesoporous bioactive glass (MBG), a kind of porous materials with great osteoconductive and osteoinductive ability, shows promising application in bone tissue engineering due to its high specific surface area, orderly channel structure, and large pore volume. Here we reported that the proliferation, differentiation, and mineralization were promoted in MC3T3-E1 cells cultured on MBG which adsorbed with testosterone (MBG/T). We found that transcriptional activity of Runx2 which is a critical transcription factor is increased in MC3T3-E1 cells cultured on MBG/T. Intriguingly, we observed that ERK phosphorylation was enhanced in MC3T3-E1 cells cultured on MBG/T. We showed that activated Runx2 in MC3T3-E1 cells cultured on MBG/T is through Erk1/2 phosphorylation. Consistent with this result, we also found that the expression of osteoblastic marker genes were increased. Therefore, we concluded that osteoblast differentiation and mineralization was enhanced after cells cultured on MBG/T through Erk1/2-activated Runx2 pathway. Our findings provided that MBG/T is a potential material in the process of bone repair.

Rescue of a cherubism bone marrow stromal culture phenotype by reducing TGFβ signaling.

We utilized a bone marrow stromal culture system to investigate changes in TGFβ signaling in a mouse model for cherubism (Sh3bp2KI/KI). Interestingly, bone marrow cultures derived from cherubism mice not only displayed impaired osteoblast differentiation, but also had spontaneous osteoclast formation. PAI1, a target gene of TGFβ signaling, was elevated 2-fold in cherubism CD11b-,CD45- cells compared to wild type cells, while the expression of BAMBI, an inhibitor of TGFβ signaling, was down-regulated. We also discovered that treatment of cherubism cultures with antagonists of the TGFβ signaling pathway could largely rescue osteoblast differentiation and markedly reduce spontaneous osteoclast formation. Treatment with the type I TGFβ receptor small molecule inhibitor SB505124 increased osteoblast reporter gene Col1a1-2.3 expression 24-fold and increased the expression of osteoblast gene markers Osterix (Sp7) 25-fold, Bone Sialoprotein (BSP) 7-fold, Osteocalcin (Bglap1) 100-fold, and Dentin Matrix Protein 1 (DMP1) 35-fold. In contrast, SB505124 treatment resulted in a significant reductions in osteoclast number and size. Gene expression analyses for RANKL, a positive regulator of osteoclast formation was 2.5-fold higher in osteoblast cultures derived from Sh3bp2KI/KI mice compared to wild type cultures, whereas OPG, an inhibitor of RANKL was 5-fold lower. However, SB505124 treatment reduced RANKL almost back down to wild type levels, while increasing OPG expression. Our studies also implicate a role for TGFβ ligands in the etiology of cherubism. Blocking of TGFβ ligands with the monoclonal antibody 1D11 increased Col1a1-2.3 reporter expression 4-fold and 13-fold in cultures derived from Sh3bp2KI/+ and Sh3bp2KI/KI mice, respectively. Serum levels of latent TGFβ1 were also 2-fold higher in SH3BP2KI/KI mice compared to wild type littermates. Taken together, these studies provide evidence that elevated levels of TGFβ signaling may contribute to the disease phenotype of cherubism and a reduction in pathway activity may be an effective therapeutic approach to treat this rare disease.

The Role of Mitochondria in Methamphetamine-induced inhibitory effects on osteogenesis of Mesenchymal Stem Cells

Methamphetamine (METH) abuse causes significant physical, psychological, and social concerns. Therefore, in this study, we investigated its effects on osteogenic differentiation of mesenchymal stem cells (MSCs). We found that METH dose-dependently affected MSCs viability. Upon osteogenic induction, the 3 and 30 µmol/l METH dosages without deleterious effects on MSCs viability resulted in the down-regulation of osteoblastic marker genes (Alp, Bglap, and Runx2), suppression of the protein expression of RUNX2, and decreased ALP activity and mineralization ability. Mitochondria are essential during osteogenesis of MSCs. Our analysis on mitochondrial function revealed that METH decreased ATP production, suppressed the oxygen consumption rate, and depolarized the mitochondrial membrane potential, but it had no significant effects on the protein expression of the five complexes on the respiratory chain. Additionally, METH could impair mitochondrial biogenesis, as demonstrated by decreased mtDNA and down-regulated biogenesis factors. Mitochondrial fusion regulators were also decreased at the mRNA and protein levels. However, mitochondrial fission and mitophagy were not affected. In conclusion, our study revealed that exposure to METH could result in decreased mitochondrial biogenesis and fusion as well as mitochondrial dysfunction, and thus it suppressed the osteogenesis of MSCs.

Fabrication of calcium phosphate-loaded carboxymethyl cellulose non-woven sheets for bone regeneration

Calcium phosphate-loaded carboxymethyl cellulose non-woven sheets (CMC/CaP sheet) were fabricated and their potential to induce in vitro osteoblast differentiation and in vivo bone regeneration were investigated. The CMC/CaP sheets were prepared by alternately soaking protonated-CMC non-woven sheets in CaCl2 and Na2HPO4 aqueous solutions. Because of its slow water uptake rate, the protonated-CMC was successfully loaded with a mixed phase of brushite and hydroxyapatite. In vitro, the CMC/CaP sheet induced osteoblast differentiation of human mesenchymal stromal cells (hMSCs), as shown by calcification and the upregulation of osteoblast marker genes. In absence of CaP, hMSCs on the CMC sheet had enhanced expression of alkaline phosphatase (ALP) only, indicative of early osteoblast differentiation. Finally, bone regeneration by the CMC/CaP sheet was demonstrated in a mouse calvarial defect model, based on micro-computed tomography (micro-CT), Masson’s trichrome staining, and immunostaining for osteoblast markers. Cells expressing the transcription factor Sp7/Osterix, which is essential for osteoblast differentiation, were detected around the new bone. The combined effect of CMC and CaP enhanced osteoblast differentiation and the CMC/CaP non-woven sheet was found to be an easy-to-handle and flexible scaffold for bone regeneration.

A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system

ObjectiveThree-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids. DesignhDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components. ResultsCompared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions. ConclusionsThe present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods.