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Tissue Engineering. Part A Jun 2017Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address...
Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).
Topics: Bioreactors; Cell Culture Techniques; Cells, Cultured; Humans; Osteoblasts; Rotation; Tissue Scaffolds
PubMed: 28125920
DOI: 10.1089/ten.TEA.2016.0357 -
Brazilian Journal of Medical and... Sep 2014The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of...
The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, and 5-HT2C) were found to exist in rat osteoblasts. Of these, 5-HT2A and 5-HT1B receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.
Topics: Animals; Calcification, Physiologic; Cell Differentiation; Cell Proliferation; DNA Primers; Gene Expression; Osteoblasts; Primary Cell Culture; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Receptors, Serotonin; Serotonin
PubMed: 25098615
DOI: 10.1590/1414-431x20143565 -
Journal of Cellular and Molecular... Nov 2021Steroid-induced osteoblast apoptosis is a crucial pathological process in steroid-induced osteonecrosis of the femoral head (SONFH). Autophagy can resist apoptosis and...
Steroid-induced osteoblast apoptosis is a crucial pathological process in steroid-induced osteonecrosis of the femoral head (SONFH). Autophagy can resist apoptosis and AMPK plays an important role in autophagy regulation. Aucubin from the small tree Eucommia ulmoides Oliv., which has a long history of use in orthopaedics and traumatology in Asian medicine, can promote bone formation, but whether it can slow or prevent steroid-osteoblast apoptosis is unclear. Therefore, we investigated the pathogenesis of SONFH and how the osteoblast responds to aucubin under the dexamethasone stimulation. In human femoral head osteonecrosis specimens, we found that the autophage and apoptosis level were increased, and the AMPK signalling was crucial to autophagy. We observed that aucubin could prevent dexamethasone-induced apoptosis in osteoblasts by enhancing the level of autophagy. Further, we confirmed that the regulatory effect of aucubin on autophagy and apoptosis was achieved by activating AMPK signalling. We have demonstrated a mechanism of disease progression and shown that aucubin could enhance autophagy through AMPK signalling to prevent osteoblast apoptosis. These findings provide a basis for the further investigation of the potential therapeutic role of aucubin in the SONFH.
Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Cell Line; Dose-Response Relationship, Drug; Humans; Immunophenotyping; Iridoid Glucosides; Mice; Osteoblasts; Phosphorylation; Protective Agents; Steroids
PubMed: 34612603
DOI: 10.1111/jcmm.16954 -
Journal of Bone and Mineral Research :... Jan 2019Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone...
Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorβ) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorβ expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorβ in mice results in preservation of bone mass during aging. These data establish that Rorβ inhibits osteogenesis and that strict control of Rorβ expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorβ expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorβ and miRNA expression. As Rorβ levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorβ expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorβ expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorβ expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorβ mRNA in osteoblasts, defining Rorβ as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorβ in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.
Topics: Aging; Animals; Cell Differentiation; Gene Expression Regulation; Humans; Mice; MicroRNAs; Nuclear Receptor Subfamily 1, Group F, Member 2; Osteoblasts; Osteoporosis
PubMed: 30321475
DOI: 10.1002/jbmr.3586 -
PloS One 2018Low bone mineral density (BMD) is a risk factor of osteoporotic fracture (OF). Peripheral blood monocytes (PBM) can differentiate into osteoclasts to resorb bone. It was...
Low bone mineral density (BMD) is a risk factor of osteoporotic fracture (OF). Peripheral blood monocytes (PBM) can differentiate into osteoclasts to resorb bone. It was known that PBM-expressed Anxa2 protein is associated with BMD, and extracellular Anxa2 protein promotes osteoclastogenesis. This study aimed to test 1) whether Anxa2 protein level in PBM differs significantly between subjects with OF and without fracture history (NF); 2) whether Anxa2 level in plasma is associated with BMD; 3) how Anxa2 protein at various concentrations would affect osteoblastic activity in vitro. All the study subjects were Chinese Han elderly. Firstly, Anxa2 protein in PBM was identified and quantitated by LC-MS/MS and compared between 45 OF cases and 42 healthy controls. Secondly, plasma Anxa2 protein level was quantitated by ELISA and compared between unrelated subjects with extremely low vs. high hip BMD (0.63±0.10 vs. 1.05±0.10 g/cm2, n = 75). Furthermore, in vitro functional assay was utilized to test the effects of extracellular Anxa2 protein on osteoblastic growth. We found that Anxa2 protein expression in PBM was significantly up-regulated in OF vs. NF subjects (fold change [FC)] = 1.16, P<0.05). Plasma Anxa2 protein concentration (range: 31.69-227.35ng/ml) was significantly elevated in low vs. high BMD subjects (84.85 vs. 66.15ng/ml, FC = 1.28, P<0.05). Cellular dynamical monitoring demonstrated that the general shape of dose-response relationship is the inverse U-shaped curve. Specifically, lower dose of Anxa2 protein may promote osteoblast growth and the optimal concentration for osteoblastic growth was around 50ng/ml, but even higher concentration could attenuate hFOB1.19 osteoprogenitor cell growth. We concluded that Anxa2 protein could attenuate osteoblast growth and be associated with hip BMD and OF in Chinese elderly.
Topics: Aged; Annexin A2; Asian People; Biomarkers; Bone Density; Case-Control Studies; Cell Line; China; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Female; Humans; Monocytes; Osteoblasts; Osteoporotic Fractures; Peptide Fragments; Procollagen; Tandem Mass Spectrometry; Up-Regulation
PubMed: 29570731
DOI: 10.1371/journal.pone.0194781 -
International Journal of Molecular... Dec 2022The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are...
The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are several restrictions to this method, e.g., moderate sensitivity makes it difficult to uncover slight but significant effects of potentially clinically relevant substances. Therefore, an adaptation of the staining method is appropriate and might be obtained by increasing the mineralization ability of osteoblasts. In this study, cell culture experiments with human (SaOs-2) and murine (MC3T3-E1) osteoblasts were performed under the addition of increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM). After three or four weeks, the mineralization matrix was stained with alizarin red S and the concentration was quantified photometrically. Only calcium chloride was able to significantly increase mineralization, and therefore enhanced the sensitivity of the alizarin red S staining in a dose-dependent manner in both osteoblastic cell lines as well as independent of the cell culture well surface area. This cost- and time-efficient optimization enables a more sensitive analysis of potentially clinically relevant substances in future bone research.
Topics: Animals; Mice; Humans; Cell Differentiation; Calcification, Physiologic; Calcium Chloride; Osteoblasts
PubMed: 36614166
DOI: 10.3390/ijms24010723 -
Journal of Bone and Mineral Research :... Mar 2018Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs...
Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine-tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV-mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence-activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro-osteoclastic cytokine, receptor activator of nuclear factor κ-B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl osteoblasts lost this pro-osteoclastic function, indicating its Rankl-dependence. They integrated ex vivo into murine calvariae, and EV-shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl mice lack the osteoclast lineage and are negative for its specific marker tartrate-resistant acid phosphatase (TRAcP). Treatment of rankl mice with wild-type osteoblast EVs induced the appearance of TRAcP-positive cells in an EV density-dependent manner. Finally, osteoblast EVs internalized and shuttled anti-osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro-osteoclastic effect, and shuttle anti-osteoclast drugs, representing a potential means of targeted therapeutic delivery. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Topics: Animals; Autocrine Communication; Bone and Bones; Cell Survival; Culture Media, Conditioned; Drug Delivery Systems; Extracellular Vesicles; Female; Gene Expression Regulation; Mice, Inbred C57BL; Osteoblasts; Osteoclasts; RANK Ligand; RNA; Tissue Distribution
PubMed: 29091316
DOI: 10.1002/jbmr.3332 -
The Journal of Biological Chemistry Aug 2020Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and...
Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and bone homeostasis is still unclear. It has been reported that runt-related transcription factor 1 () is expressed in osteoblast progenitors, pre-osteoblasts, and mature osteoblasts; yet, surprisingly, the exact function of RUNX1 in osteoblast maturation and bone formation remains unknown. Here, we generated and characterized a pre-osteoblast and differentiating chondrocyte-specific conditional knockout mouse model to study RUNX1's function in bone formation. ablation in osteoblast precursors and differentiating chondrocytes via osterix-Cre (Osx-Cre) resulted in an osteoporotic phenotype and decreased bone density in the long bones and skulls of mice compared with and mice. RUNX1 deficiency reduced the expression of SRY-box transcription factor 9 (SOX9), Indian hedgehog signaling molecule (IHH), Patched (PTC), and cyclin D1 in the growth plate, and also reduced the expression of osteocalcin (OCN), OSX, activating transcription factor 4 (ATF4), and RUNX2 in osteoblasts. ChIP assays and promoter activity mapping revealed that RUNX1 directly associates with the gene promoter and up-regulates expression. Furthermore, the ChIP data also showed that RUNX1 associates with the promoter. In conclusion, RUNX1 up-regulates the expression of and multiple bone-specific genes, and plays an indispensable role in bone formation and homeostasis in both trabecular and cortical bone. We propose that stimulating activity may be useful in therapeutic approaches for managing some bone diseases such as osteoporosis.
Topics: Animals; Cell Differentiation; Core Binding Factor Alpha 2 Subunit; Female; Male; Mice; Mice, Knockout; Osteoblasts; Osteogenesis; Osteoporosis
PubMed: 32571873
DOI: 10.1074/jbc.RA119.007896 -
Biochemical and Biophysical Research... Dec 2019The peroxisome proliferator-activated receptor (PPAR)-α agonist fenofibrate is used as a lipid-lowering agent to reduce cholesterol and triglyceride in blood. In this...
The peroxisome proliferator-activated receptor (PPAR)-α agonist fenofibrate is used as a lipid-lowering agent to reduce cholesterol and triglyceride in blood. In this study, we investigated whether fenofibrate affects osteoblast differentiation of osteogenic precursor cells. Quantitative real-time PCR and alkaline phosphatase (ALP) staining assays revealed that fenofibrate can enhance the osteoblast differentiation of C3H10T1/2 and MC3T3-E1 cells. In contrast with fenofibrate, the PPARγ agonist rosiglitazone decreased or did not affect the expression of osteogenic genes in these cells. Fenofibrate dose- and time-dependently increased PPARα expression, and concomitantly increased the expression of bone morphogenetic protein 2 (BMP2). Knockdown of PPARα abolished fenofibrate-induced BMP2 expression, activity of the BMP2 promoter gene, and calcium deposition. The chromatin immunoprecipitation assay demonstrated that fenofibrate increased BMP2 expression by inducing direct binding of PPARα to the BMP2 promoter region. Taken together, we suggest that fenofibrate has a stimulatory effect on osteoblast differentiation via the elevation of PPARα levels and the PPARα-mediated BMP2 expression. Our findings provide fenofibrate as a useful agent for controlling hypercholesterolemic patients with osteoporosis.
Topics: Animals; Bone Morphogenetic Protein 2; Cell Differentiation; Cell Line; Fenofibrate; Gene Expression Regulation; Mice; Osteoblasts; PPAR alpha; Promoter Regions, Genetic; Transcription, Genetic
PubMed: 31607484
DOI: 10.1016/j.bbrc.2019.10.048 -
Cells Dec 2022Gentianae Scabrae Radix is used in traditional medicine and is known to possess bioactive compounds, including secoiridoid glycosides, flavonoids, lignans, and...
Gentianae Scabrae Radix is used in traditional medicine and is known to possess bioactive compounds, including secoiridoid glycosides, flavonoids, lignans, and triterpenes. Trifloroside (TriFs) is a secoiridoid glycoside known for its antioxidant activity; however, its other effects have not been studied. In the present study, we investigated the biological effects of TriFs isolated from the roots of Gentianae Scabrae Radix using pre-osteoblast MC3T3E-1 cells. No cellular toxicity was observed with 1 μM TriFs, whereas 5-100 μM TriFs showed a gradual increase in cell viability. Alkaline phosphatase staining and microscopic observations revealed that 1-10 μM TriFs stimulated osteogenic activity during early osteoblast differentiation. Trifloroside also increased mineral apposition during osteoblast maturation. Biochemical analyses revealed that TriFs promoted nuclear RUNX2 expression and localization by stimulating the major osteogenic BMP2-Smad1/5/8-RUNX2 pathway. Trifloroside also increased p-GSK3β, β-catenin, p-JNK, and p-p38, but not Wnt3a, p-AKT, and p-ERK. Moreover, TriFs increased the MMP13 levels and promoted cell migration and adhesion. In contrast, TriFs-induced osteoblast differentiation and maturation had negligible effects on autophagy and necrosis. Our findings suggest that TriFs induces osteogenic effects through differentiation, adhesion, migration, and mineral apposition. Therefore, TriFs is suggested as a potential drug target in osteoblast-mediated bone diseases.
Topics: Osteoblasts; Cell Differentiation; Osteogenesis; Cells, Cultured; Cell Line
PubMed: 36497145
DOI: 10.3390/cells11233887