osteoblasts

 Photobiomodulation with low-level laser treatment can enhance bone formation by stimulating the cell division of osteoblasts and increasing the amount of protein deposition, thus encouraging the formation of new bone. This study aimed to evaluate the effects of photobiomodulation with a low-level laser on proliferation and gene expression related to calcium signaling in human osteoblasts.

 Osteoblastic cell lines of the hFOB1.19 lineage, human osteoblasts, were grown and assigned into two groups, control (C; n=78 cultured wells) and photobiomodulation (L; n=78 cultured wells) with n=6 per day of the experimental period. Cells were cultured (immature at 34 ºC), and after maturation at 37 ºC, group L cells were exposed to laser irradiation with a low-level laser device (gallium and aluminum arsenide), at a wavelength of 808 nm, a power output of 200 mW, and a power density of 200 mW/cm2. The energy delivered to the cells was 37 J/cm2, with a beam area of 0.02 mm2 and an exposure time of 5 seconds. This treatment was applied daily for 13 days. Following this, the number of cells was counted, and RNA was isolated, measured, and then converted into cDNA for further quantification using a comparative Ct method with real-time polymerase chain reaction. The results were then subjected to statistical analysis through a Mann-Whitney test, with a significance level of P<0.05.

 The cell count in the L group (37.25x10±4±22.02) was statistically higher compared to the control group (22.75x10±4±7.660) with a P value of 0.0259. The values of 2- ΔΔCt for S100A6, plasma membrane calcium ATPase (PMCA), and calmodulin genes indicated hyperexpression on the thirteenth day, while the osteocalcin gene showed hypo-expression.

 The study suggests that the photobiomodulation mechanism with a low-level laser may regulate gene expression in human osteoblasts in a dose-dependent and cumulative manner.

We previously reported that cadmium (Cd) inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In addition, gallic acid (GA) improves BMSC differentiation. Here, we aim to study the ability of GA to prevent osteogenic inhibition induced by Cd.

In this experimental study, BMSCs were extracted and purified from Wistar rats and their viability was determined in the presence of Cd and GA. The results indicated that 1.5 μM Cd and 0.25 μM of GA were appropriate for further investigation. After 20 days in osteogenic medium, matrix production was analysed by alizarin red, calcium content, and alkaline phosphatase (ALP) activity. Osteogenic-related genes and collagen 1A1 (COL1A1) protein expressions were investigated. The preventive effect of GA on oxidative stress and metabolic change induced by Cd was estimated.

GA counteracted the inhibitory effect of Cd on matrix production and significantly (P=0.0001) improved the osteogenic differentiation ability of BMSCs. Also, GA prevented the toxic effect of Cd on osteogenic-related gene expressions and nullified the reducing effect of Cd on COL1Al and ALP activity. A significant reduction (P=0.0001) in malondialdehyde and lactic acid concentration showed that GA counteracted both oxidative stress and metabolic changes caused by Cd.

GA prevented the toxic effect of Cd, an environmental pollutant and a factor in osteoporosis.

A variety of laser treatments have been applied in numerous medical fields. In dentistry, laser treatments are used for caries, root canals, and periodontal disease, as well as surgical resection. Numerous reports have recently been published on the use of lasers for bone regeneration. If laser irradiation is found to promote the activation of bone metabolism, it might also be effective for periodontal treatment, peri-implantitis, and bone regeneration. Therefore, the present in vitro study aimed to elucidate the mechanisms underlying the effects of erbium-doped yttrium aluminum garnet (Er: YAG) laser irradiation on the bone using osteoblast-like cells.

Osteoblast-like Saos 2 cells (5.0 × 104 cells) were seeded in 24-well plates. 24 hours after being seeded, the cells were subjected to 0.3 W, 0.6 W, and 2.0 W Er: YAG laser irradiation and then allowed to recover for 48 hours. The expression levels of bone metabolism-related factorsalkaline phosphatase (ALP), bone sialoprotein (BSP), and osteoprotegerin (OPG)were then evaluated using reverse transcription–quantitative polymerase chain reaction and western blot analyses.

Saos 2 cells subjected to Er: YAG laser irradiation at 0.3 W, 0.6 W, and 2.0 W showed normal growth. When the Er: YAG laser irradiation and control groups were compared after 48 hours, increases were observed in ALP, BSP, and OPG gene and protein expression in the 2.0 W group. Similar results were obtained in the western blot analysis.

These findings suggest that the Er: YAG laser irradiation of osteoblast-like cells is effective for activating bone metabolism factors.

Osteoporosis is regarded as a silent disorder affecting bone slowly, leading to an increased risk of fractures. Lately, selenium has been found to be associated with the acquisition and maintenance of bone health by affecting the bone remodeling process. However, the mechanism of action of selenium on bone is poorly understood. Here, the protective effects of sodium selenite on the differentiation process of osteoblasts as well as under oxidative stress-induced conditions were evaluated. MC3T3-E1 cells, were treated with a various doses (0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 ug/ml) of sodium selenite. Cell viability and cytotoxicity were observed by MTT and LDH assay. The osteogenic activity and expression level of osteogenic markers were confirmed through ALP activity, real-time RT-PCR and sirius red staining. Role of sodium selenite and involvement of WNT signaling was assessed by Axin-2 reporter assay and western blotting. It was observed that the sodium selenite could promote the ALP activity and collagen synthesis in pre-osteoblasts. Moreover, sodium selenite increased the mRNA expression levels of osteogenic transcriptional factors, such as runt-related transcription factor 2 (Runx2) and osterix (OSX). In addition, the terminal differentiation markers, such as osteocalcin (OCN), and collagen 1α (Col1α), were also increased. Treatment of sodium selenite recused the H2O2-induced inhibition of osteoblastic differentiation of pre-osteoblasts cells. Furthermore, sodium selenite restored the H2O2 repressed β-catenin stability and axin-2 reporter activity in MC3T3-E1 cells implicating involvement of WNT signaling pathway. It may be concluded that selenite can stimulate bone formation and rescue the oxidative repression of osteogenesis by activating WNT signaling pathway and may act as a potential therapeutic intervention for osteoporosis.

According to reports, icariin (ICA) is a bone anabolic agent able to prevent osteoporosis in both ovariectomized rats and postmenopausal women. However, its effect on osteoblast proliferation remains to be determined, and the underlying mechanism remains to be elucidated.

Icariin-bovine serum albumin (BSA) conjugates were purified by Sephadex G-25 gel chromatography technology. Primary osteoblasts from neonatal rats were used to evaluate the effects of ICA, ICA-BSA, ICA-BSA + ICI182780, and ICA-BSA + PD98059. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI)-staining assays were used to detect the proliferation of osteoblasts after drug exposure. The intracellular calcium ions were detected using a confocal microscope with Fluo-3/AM as the fluorescent indicator. Western blot was capitalized on to measure the relative content of phospho-extracellular signal-regulated kinase (p-ERK).

Primary osteoblasts in culture were detected by histochemical staining of alkaline phosphatase, and calcified nodules were obtained by sequential digestion. Icariin and bovine serum albumin could form conjugate, which could be purified by Sephadex G-25 gel chromatography technology. MTT and flow cytometry results show that ICA-BSA conjugate significantly facilitated the proliferation of osteoblasts (P < 0.05). The intracellular calcium ions also ascended vastly in the cells treated with ICA-BSA conjugate (P < 0.01). Icariin-bovine serum albumin exposure rapidly activated the extracellular signal-regulated kinase (ERK) signaling. Furthermore, ICA- and ICA-BSA-mediated actions on osteoblasts were signally alleviated after dealing with ERK inhibitor PD98059 or estrogen receptor (ER) antagonist ICI182780, which might have a relation to the repression of ERK phosphorylation.

Icariin could serve as estrogen in osteoblast cells by the rapid nongenomic ER signaling pathway independent of ligand and estrogen response element (ERE) and mediated by mitogen-activated protein kinase (MAPK).

Photomodulation is a promising strategy for optimizing tissue healing, butits photomodulatory effects on the synergistic cellular metabolism of gingival and bony tissues remain largely unknown. The aim of the present study was to evaluate the photomodulatory effects of a diode laser (810 nm) on osteoblasts, HGFs and their co-cultures in vitro.

Primary cultures of HGFs, cultures of immature osteoblastic cells (MG63) and their co-cultures were irradiated with a diode laser (810 nm), 15 J/cm2. Cell cultures were examined for cellular proliferation (MTT assay), viability (FDA/PI staining) after 24, 48 and 72 hours and cell differentiation (qPCR of collagen type 1a – COL1a and alkaline phosphatase expressions - ALP) after 7 days.

Photomodulation with an 810-nm diode laser increased cell proliferation at all time points. COL1a gene expression increased both in HGF and co-cultures. ALP expression was up-regulated in osteoblastic cultures, but co-cultures with fibroblasts negated this response.

The 810-nm diode laser positively affected cell proliferation and viability in all experimental groups. The statistically significant increased COL1a gene expression at 7 days after irradiation both in the irradiated HGF and co-cultures suggests that low-level laser therapy (LLLT) stimulated extracellular matrix (ECM) formation signaling in both cell types

Radiation induced bystander effects (RIBEs) occurs in unirradiated cells exhibiting indirect biological effect as a consequence of signals from other irradiated cells in the population.

In this study, bystander effects in MCF-7 breast cancer cells and hFOB 1.19 normal osteoblast cells irradiated with gamma emitting HDR Brachytherapy Ir-192 source were investigated.

In this in-vitro study, bystander effect stimulation was conducted using medium transfer technique of irradiated cells to the non-irradiated bystander cells. Cell viability, reactive oxygen species (ROS) generation and colony forming assay was employed to evaluate the effect.

Results indicate that the exposure to the medium irradiated MCF-7 induced significant bystander killing and decreased the survival fraction of bystander MCF-7 and hFOB from 1.19 to 81.70 % and 65.44 %, respectively. A significant decrease in survival fraction was observed for hFOB 1.19 bystander cells (p < 0.05). We found that the rate of hFOB 1.19 cell growth significantly decreases to 85.5% when added with media from irradiated cells. The ROS levels of bystander cells for both cell lines were observed to have an increase even after 4 h of treatment. Our results suggest the presence of bystander effects in unirradiated cells exposed to the irradiated medium.

These data provide evidence that irradiated MCF-7 breast cancer cells can induce bystander death in unirradiated MCF-7 and hFOB 1.19 bystander cells. Increase in cell death could also be mediated by the ROS generation during the irradiation with HDR brachytherapy.

Low levels of estrogen can cause osteoporosis and usually occur during a woman’s menopausal phase. Osteoporosis can lead to bone resorption, the absence of osseointegration, and implant failure. The aim of this study is to determine the expression of transforming growth factor‑beta 1 (TGF‑β1), runt‑related transcription factor (RUNX2), and osteoblasts in mandibular rats with low levels of estrogen. This study is an in vivo experimental research. Female Wistar rats (n = 18) were divided into two groups: (1) Postsham surgery and (2) ovariectomy group. After 12 weeks, the rats were sacrificed to identify the level of estrogen, while histological analysis was conducted to determine the level of osteoblast and the expression of TGF‑β1 and RUNX2. The data were analyzed using t‑test (P < 0.05). There were significant lower levels of estrogen and osteoblast among the ovariectomy group compared to the postsham group (P < 0.05). RUNX2 levels were found to be significantly higher in the ovariectomy group than that in the postsham group (P < 0.05). However, there were no significant differences between TGF‑β1 levels within the ovariectomy and postsham groups (P > 0.05). Ovariectomy can lead to decreased osteoblastogenesis in mandibular bone by the reduced level of osteoblast and the increased expression of TGF‑β1 and RUNX2.

The prognosis of osteoporosis is very poor, and it is very important to identify a biomarker for prevention of osteoporosis. In this study, we aimed to identify candidate markers in osteoporosis and to investigate the role of candidate markers in osteogenic differentiation. Using Weighted Gene Co-Expression Network analysis, we identified three hub genes might associate with osteoporosis. The mRNA expression of hub genes in osteoblasts from osteoporosis patients or healthy donor was detected by qRT-PCR. Using siRNA and overexpression, we investigated the role of hub gene BRCC3 in osteogenic differentiation by alkaline phosphatase staining and Alizarin red staining. Moreover, the role of β-catenin signaling in the osteogenic differentiation was detected by using β-catenin signaling inhibitor XAV939. We identified three hub genes that might associate with osteoporosis including BRCC3, UBE2N, and UBE2K. UBE2N mRNA and UBE2K mRNA were not changed in osteoblasts isolated from osteoporosis patients, compared with healthy donors, whereas BRCC3 mRNA was significantly increased. Depletion of BRCC3 promoted the activation of alkaline phosphatase and formation of calcified nodules in osteoblasts isolated from osteoporosis patients and up-regulated β-catenin expression. XAV939 reversed the BRCC3 siRNA-induced osteogenic differentiation. Additionally, inhibited osteogenic differentiation was also observed after BACC3 overexpression, and this was accompanied by decreased β-catenin expression. BRCC3 is an important regulator for osteogenic differentiation of osteoblasts through β-catenin signaling, and it might be a promising target for osteoporosis treatment.

Since the low concentration and short-time treatment with sodium nitroprusside (SNP), a nitric oxide (NO)–donor, cause no harm to rat bone marrow mesenchymal stem cells (MSCs), we studied the impact of SNP on MSCs differentiation. MSCs were treated with 100 and 1000 µM of SNP for 1 hour in every 48 hours and after 5, 10, 15, and 21 days in osteogenic media. The viability and the level of mineralization were determined using MTT assay and alizarin red staining, respectively. Morphology of the cells was studied using fluorescent dye. Concentration of calcium and the activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were evaluated by commercial kits. SNP with the concentration of 1000 µM significantly reduced viability from day 5 to day 20, but 100 µM did not affect the viability until the day 15. The low concentration of SNP increased matrix deposition from day 10 and reached almost its maximum (4.40 ± 2.4) at the day 15. Also, increasing the activity of ALP (419 ± 2.2), due to low concentration of SNP, started at day 10 and continued till the day 20, while LDH (2026 ± 11) and AST (25.6 ± 0.4) elevations were observed from day 5 onwards. In case of ALT, we observed a significant decrease (36%) from day 5 till day 20. Based on our findings, low concentrations of SNP might be useful in the promotion of bone repair.

Mesenchymal stromal cells (MSCs) have high differentiation potential into different cell lines like osteoblasts. Osteogenic differentiation can be regulated through various and complex mechanisms, such as changing the microRNAs expression level. Although platelet-rich plasma (PRP) has been used in MSCs’ osteogenic differentiation process, the molecular mechanisms underlying the effect of PRP induction of osteogenesis by microRNAs is not well understood.

We evaluated the effect of PRP on the expression of miR-29a, miR-29b, and miR-155 in the PRP-based osteogenic differentiation of human MSCs.

This experimental study was conducted on healthy individuals referred to Taleghani Hospital in Ahvaz, Iran, for abdominoplasty from September 2017 to April 2018. Stromal cells were isolated from human adipose tissue and differentiated into osteoblasts. Effect of 10% PRP on osteoblasts differentiation was monitored by the measurement of alkaline phosphatase activity and calcium deposition. We also evaluated gene expression of the Runx2 and the OPN along with the expression of miRNAs. All tests were performed in triplicate.

Treatment of MSCs with 10% PRP resulted in induction of osteogenic differentiation by a significant upregulation of the miR-29a/b (miR-29a: 5.27 (0.77), P < 0.01 (day 3) and 3.76 (0.124), P < 0.01 (day 14); miR-29b: 3.11 (0.35), P < 0.001 (day 3) and 4.25 (0.304), P < 0.01 (day 14)) and a significant downregulation in the miR-155 expression (0.62 (0.006), P < 0.001 (day 3) and 0.55 (0.114), P < 0.05 (day 14)).

The remarkable rise in the expression of important osteoblast genes, alkaline phosphatase activity, and calcium deposition verified accelerated differentiation. The present study showed that microRNAs such as miR-29a/b and miR-155 play an active role in the process of bone differentiation during PRP treatment, which in turn, affects mesenchymal cells signaling pathways

Bone surgery as a current bone treatment method is not always successful to fulfil bone repair in bone degenerative diseases or extensive injuries. Due to the limited capacity of bone remodeling, the demand for alternative approaches remains to be met. Thus, efforts in ex vivo generation of bone forming cells, osteoblasts, and their further application in cell therapy as a promising approach are of vital prominence from a scientific perspective. Though several studies have focused on microRNA roles in osteoblast differentiation in various cell recourses, yet none has reported miR-210 enhancing role in human mesynchymal stem cells (MSCs) so far.

Hence, we wished to examine the nature of the relationship between osteoblast differentiation and miR-210 in unique human mesynchymal stem cells, unrestricted somatic stem cells (USSCs). Osteoblast markers at gene level namely, Runx2, col I in addition to osteocalcin were assessed using qRT-PCR, and Alizarin Red S staining was also carried out to observe histochemical changes 7 days following miR-210 transduction.

The conclusion that follows from our findings represents a marked increase in osteoblast differentiation markers. Interestingly, for the first time, human USSCs differentiation into osteoblasts was performed in our research.

our study may provide helpful insights into surmounting bone related issues by combination of both gene and cell therapy.

Osteoblasts (OBs) and osteoclasts (OCs) are 2 major groups of bone cells. Their cell-to-cell interactions are important to ensure the continuity of the bone-remodeling process. Therefore, the present study was carried out to optimize an OB/OC co-culture system utilizing the human OB cell line hFOB 1.19 and OCs extracted from peripheral blood mononuclear cells (PBMNCs). It was a 2-step procedure, involving the optimization of the OB culture and the co-culture of the OBs with PBMNCs at an optimum ratio. Firstly, pre-OBs were cultured to 90% confluency and the time required for differentiation was determined. OB differentiation was determined using the van Gieson staining to detect the presence of collagen and Alizarin Red for calcium. Secondly, OBs and OCs were co-cultured at the ratios of 1 OC: 1 OB, 1 OC: 4 OBs, 2 OCs: 1 OB, and 1 OC: 2 OBs. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the differentiation of the OCs. The results showed that collagen was present on day 1, whereas calcium was detected as early as day 3. Based on the result of TRAP staining, 1 OC: 2 OBs was taken as the most appropriate ratio. No macrophage colony-stimulating factor and receptor activator of the nuclear factor-κB ligand were added because they were provided by the OBs. In conclusion, these optimization processes are vital as they ensure the exact time point and ratio of the OB/OC co-culture in order to produce a reliable and reproducible co-culture system.

The mechanism of cadmium induced osteoporosis is not well understood, so in this study, we examined the toxicity of bone marrow mesenchymal stem cell (MSCs) following treatment of rats with CdCl2 in drinking water, to revile the effect of this chemical on differentiation potential of MSCs.
At the end of third passage, MSCs were grown in the osteogenic medium for 21 days. To study the differentiation property the viability, morphology, intracellular calcium, and matrix mineralization via quantitative alizarin red were evaluated. Besides, biochemical parameters including activity of alkaline phosphatase (ALP), aspartate amino transaminase (AST), alanine amino transaminase (ALT) as well as antioxidant enzyme such as superoxide dismutase, catalase, and peroxidase were determined too. In addition, level of lipid peroxidation based on determination of malondialdehyde (MDA) content was studied.
The results showed significant reduction in the viability of cells after differentiation compared to control (P Oral consumption of cadmium affects osteogenic differentiation potential of MSCs via membrane damage, reduction of calcium deposition and metabolic changes. Thus, it might be considered as a probable factor involve in cadmium related osteoporosis.

In this paper, preparation, bioactivity, and osteoblast cell behavior of cortical bone derived nanobiphasic calcium phosphate (nano-BCP) are presented. The calcined bovine bone samples with the addition of di-ammonium hydrogen phosphate were heated at 700°C for 100 min, and thus nano-BCP with the composition of 63/37 hydroxyapatite (HA)/β-tricalcium phosphate (β-TCP) was produced. Scanning electron microscopy (SEM) images, energy dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD) analysis of immersed samples in simulated body fluid (SBF) solution showed that a uniform layer was formed on the surface after 7 days with the chemical composition of HA. The results indicated that the nano-BCP sample developed excellent bioactivity after 28 days. The nano-BCP samples showed better cell proliferation compared to pure HA samples. After 7 days in cell culture, the prepared nano-BCP (HA/β-TCP) exhibited the maximum proliferation of the MG-63 osteoblast cells.

A large number of phospholipase C (PLC) enzymes, both mRNA transcripts and proteins, have been detected in osteoblasts, corroborating the importance of calcium regulation in bone tissue. MG-63 and SaOS-2 human osteosarcoma cell lines are actually considered osteoblast-like cells, and are therefore widely used as experimental models for osteoblasts.
Our aim was to verify whether MG-63 or SaOS-2 cells might also represent appropriate experimental osteoblast models for signal transduction studies, with special regard to the phosphoinositide (PI) pathway. We analyzed the expression and the subcellular distribution of enzymes related to calcium signal transduction (the PI-specific PLC family), which are known to possess high cell/tissue specificity.
The expression of PLC genes was analyzed by performing RT-PCR experiments. The presence of PLC enzymes and their subcellular distribution within the cells was analyzed with immunofluorescence experiments.
Osteoblasts, MG-63 cells, and SaOS-2 cells have expression panels similar to those of PLC enzymes. However, slight differences were found in the expression of enzymes belonging to the PLCη subfamily.
MG-63 and SaOS-2 osteosarcoma cell lines might not represent appropriate experimental models for studies that aim to analyze signal transduction in osteoblasts.