BACKGROUND: In applications on dental/orthopedic implants and bone regeneration, biomaterials contained magnesium have been widely used. However, the mechanism underlying the biologic effects is still largely unknown. In addition, previous reports of osteogenic effect of magnesium mainly relied on studies using ATCC osteosarcoma cell lines but not normal human osteoblasts. OBJECTIVE: This study was designed to test the effect of magnesium on osteogenic phenotypic behaviors of normal human osteoblasts. METHODS: Normal human osteoblasts derived from human alveolar bone were cultured in triplicate in growth media with varies concentrations of supplemental magnesium: 0.5mM, 1mM, 2mM, 4mM, 8mM and 16mM as the study groups and 0mM as a control group for the time intervals of 7 days, 10 days, 14 days and 21days. Cell proliferation was measured by crystal violet dye staining. Expression of osteocalcin was measured by Quantikine Elisa and mineralization of cultures was measured by Alizarin Red staining. The data were normalized per cell basis. Statistical analysis was done using ANOVA and Student’s t test. Results: Osteocalcin expression was upregulated in groups with supplemented magnesium at 0.5mM (1.16folds, p<0.01 ), 1.0mM (1.22folds, p<0.01 ), 2.0mM (1.37folds, p<0.01 ) at day 21 compared to control, while at 4mM ( p<0.01 ) and above showed down-regulation. Alizarin Red stained cultures showed higher degree of mineralization at 1mM ( p=0.0228 ) and 2mM ( p=0.0142) compared to control. Groups with 4mM and above showed less calcium deposition. Similar results have been gained also on day 10 and day 14 for both assays. CONCLUSION: Osteogenesis of normal human osteoblasts could be significantly upregulated by 2mM supplemental magnesium. These data are important for manufacturing magnesium-containing biomaterials for bone tissue regeneration and implants.

Statins, hydroxymethylglutaryl-coenzyme-A reductase inhibitors (HMG-Co-A), are known to reduce plasma cholesterol levels. Interestingly, Simvastatin was previously reported to have a positive effect on the proliferation and odontoblastic differentiation of human dental pulp cells. However, the biocompatibility of Simvastatin has not been studied thoroughly. The purpose of this study was to further compare the effectiveness of different concentrations of Simvastatin on the attachment, proliferation, differentiation, toxicity, mineralization, and flow cytometry of human dental pulp cells (HDPCs) and osteoblasts. HDPCs and osteoblasts were cultured with Simvastatin at various concentrations of 1, 10, 25, 50, 75, 100 μmol/L, and 0 μmol/L was used as a control. The cell attachment was evaluated at 16 hours for HDPCs and 9 hours for osteoblasts. The proliferation rate, differentiation, cytotoxicity, and mineralization were investigated at 7, 14 and 21 days. Cell cycle and apoptosis were assessed at 1 and 3 days. Statistical analysis was performed using ANOVA. P-values ≤0.05 were considered statistically significant. The results showed that 25 μmol/L demonstrated the highest cell attachment efficiency when compared to the control in HDPCs (P<0.05). There was no statistical significance (P>0.05) amongst the groups in the cell attachment efficiency in osteoblasts. All tested concentrations showed a significant decrease in the proliferation rate and mineralization (P<0.001) and an increase in cytotoxicity and cytostasis (P<0.001) in both cell types. ALP levels increased in HDPCs and osteoblasts (P<0.001). DSP and RUNX2 levels decreased in HDPCs (P<0.001). OSC levels were increased in osteoblasts, but RUNX2 was decreased (P<0.001). Cell cycle and apoptosis significantly increased as time increased (P<0.001) in both cell types. In conclusion, the present findings showed that Simvastatin adversely affects the proliferation, cell viability of HDPCs and osteoblasts by inducing apoptosis, which were confirmed by flow cytometry results. There was an increase in the odontogenic and osteogenic markers hinting at early differentiation, which decreased as time increased. / 2025-07-10T00:00:00Z

No description available.

No description available.

Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. To determine the role of OA in skeletal homeostasis in vivo. we utilized a mouse model with a natural mutation in the osteoactivin gene. This mutation is due to a premature stop codon, which results in the generation of a truncated 150 amino acid OA protein. This animal, which we will refer to as OA mutant, was shown by ìCT and histomorphometric analysis to have increased bone volume, trabecular thickness, and trabecular number compared to wild-type (WT) mice at 4 weeks of age, which is a time at which bone formation is most active. Histological analysis of long bones stained with TRAP (tartrate resistant acid phosphatase) and colorimetric analysis of serum TRAP 5b levels indicated that the numbers of osteoclasts are significantly increased in OA mutant samples. Interestingly, although the numbers of osteoclasts as compared to WT were higher in OA mutant mice, serum levels of C-telopeptide of type I collagen (CTX) and osteocalcin, biomarkers for bone resorption and bone formation respectively, were significantly decreased. These data suggested that in mice the presence of truncated OA protein results in increased osteoclast number, but that they are inefficient in resorbing bone and may in part contribute to the increase in bone volume in OA mutant mice in vivo. To further investigate the role of OA in osteoclast differentiation, osteoclasts were differentiated from hematopoietic stem cell progenitors ex vivo. HSCs were cultured in the presence of 50 ng/ml of M-CSF for two days and then with M-CSF and 100 ng/ml of RANKL in the presence or absence of 50 ng/ml recombinant OA. We observed a dramatic increase in multinucleated TRAP-positive osteoclasts and the number of nuclei per osteoclast in OA-treated cultures compared to control. Additionally, analysis of HSCs showed increased cell proliferation in response to exogenous OA treatment. When osteoclasts were differentiated in ex vivo cultures derived from OA mutant and WT mice, we observed decreased osteoclast number, size, and function in OA mutant compared to WT cultures. This decrease was abrogated when cultures were treated exogenously with recombinant OA. Quantitative PCR analysis of RNA isolated during osteoclast differentiation from WT and OA mutant mice reveal decreased gene expression of critical osteoclast differentiation and functional markers, which explains the osteoclast defect observed ex vivo. To investigate the role of OA in osteoblast differentiation, primary osteoblasts were derived from mesenchymal progenitors isolated from calvariae of WT and OA mutant neonatal pups. OA mutant osteoblasts were found to have decreased alkaline phosphatase (ALP) staining and activity at day 14 in culture. Furthermore when cultures were differentiated to 21 days to simulate matrix mineralization in vitro, OA mutant osteoblasts exhibited decreased Alizarin Red and Von Kossa staining. Quantitative measurement of calcium also showed decreased mineral deposition in OA mutant mice compared to WT. Electron microscopic and protein studies were able to eliminate the notion of ER stress or cell toxicity as a result of ER stress playing a role in the delayed osteoblast differentiation observed in OA mutant osteoblasts. Furthermore, OA mutant osteoblasts exhibited decreased proliferation and survival ex vivo. These data reveal an effect of osteoactivin in osteoblasts ex vivo. This study provided an in vivo tool to study the role of osteoactivin in bone cells and the regulation of bone formation and bone resorption by this molecule. Taken together, these findings suggest that the presence of truncated OA leads to increased bone volume due to defective interplay between bone-resorbing osteoclasts and bone-forming osteoblasts. Data presented here support the notion of osteoactivin as a novel molecule in modulating skeletal homeostasis in vivo. / Cell Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Elimination of bacteria and active infection within an infected root canal system is one of the primary objectives of nonsurgical root canal treatment. One of the measures of successful root canal treatment is subsequent bone healing of periapical lesions caused by previous infection. A previous study by Yumoto et al. showed that electromagnetic wave stimulation can increase proliferation of osteoblastic cells with no cytotoxicity, and it can also up-regulate growth factors such as vascular endothelial growth factor and platelet-derived growth factor.18 They also showed increased proliferation of an immortalized osteoblastic MC3T3-E1 cell line 3 days following electromagnetic stimulation (EMS).18 Previously, Pauly et al. found increased alkaline phosphatase (ALP) activity with 10 mA EMS application to primary murine calvaria-derived osteoblastic cells with 5 pulses at 1 second per pulse, but no significant differences were found for MTS proliferation nor mineral deposition compared to a negative control group.82 Optimization of the different variables including post-treatment incubation time, current delivery, and number of pulses per treatment may be necessary to improve osteogenic activity. The use of mesenchymal stem cells from murine bone marrow may also offer a physiologically relevant model for osteoblastic regeneration of periapical lesions. Objectives: The goal of this study was to investigate and optimize the effects of electromagnetic wave stimulation (EMS) on murine bone marrow mesenchymal stem cells (MSCs) by evaluating the proliferation and differentiation of the cells after exposure to different EMS treatment regimens. Materials and Methods: 5 x104 stromal osteoblasts (SOBs) were cultured in 24-well plates in α-MEM containing 10% fetal bovine serum. Cells were then subjected to pulsed EMS treatments of 1 mA, 10 mA, and 50 mA. EMS was generated using an electromagnetic apical treatment (EMAT) device created by J. Morita MFG Corp. Proliferation was assessed via MTS assay 1 days after treatment. For osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media, and SOBs were cultured for 14 days. Afterwards, alkaline phosphatase (ALP) activity and Alizarin-red S mineral deposition were quantified as measures of osteoblast activity. Cells grown in osteogenic media without EMS treatment served as the negative control. Results: Although MSC proliferation was unaffected by different EMS treatment regimens, 50 mA EMS resulted in a decrease in ALP activity and mineral deposition by osteoblasts. Conclusions: Our findings suggest bone healing by EMS may involve a different cellular mechanism, that is not reproduced in vitro in our studies. Utilizing different amperage and EMS regimens may improve osteogenic differentiation.

BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

No / Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.

Defects in bone are a constant and serious problem. They occur as a result of high energy trauma, congenital conditions or are created surgically to treat bone tumours or infection. Currently the treatment for these conditions is awkward for the patient, takes a long time and has a high complication rate. An elegant solution would be to mend the bone defect using the patient own cells; osteoblasts or mesenchymal stem cells seeded onto a supportive material scaffold. For successful regeneration of bone structures, a scaffold production technique has to be adopted that can precisely control porosity, internal pore architecture and fibre thickness, as well as maximising media diffusion and optimising scaffold mechanical properties so that the scaffold can withstand bone bearing pressures. It would also be beneficial if the scaffold uniformly distributed surface strain along the fibres throughout the entire scaffold as this would encourage more even cell proliferation/differentiation in the structure. This was addressed by performing a series of finite element analyses on the computer aided design model where the mechanical properties of the natural or synthetic polymer used have been incorporated to yield an accurate strain profile of the entire scaffold. The process used here to generate the scaffolds is a Rapid Prototyping method that creates a three-dimensional object through the repetitive deposition of fibres in layers via extrusion. Due to the high accuracy and versatility of the extruder, the diameter of the pores can be precisely controlled to an accuracy of 10μm, in the manufactured scaffolds the pore size ranges from 100 to 300μm as that is what is found in trabecular bone. Natural and synthetic polymers were plotted which altered the biodegradability properties of the scaffold and the degrees of cell adhesion, proliferation and differentiation in the structure. Scaffolds were manufactured that demonstrated compatibility with cell adhesion, proliferation and osteogenic differentiation. On completion of the scaffolds, the latter were seeded with osteoblasts or marrow stromal cells and put into a mechanically stimulating bioreactor machine to induce a small strain in the scaffold; this was performed to encourage cell proliferation/differentiation. The structure was left until the osteoblasts or marrow stromal cells modified the scaffold through bone deposition. In-vivo experiments were then undertaken. Preliminary data indicated an effect of mechanical stimulation of the cell/scaffold construct on the degree of mineralization of cell matrix generated by human osteogenic cells.