Osteoblast Matrix Mineralization Research Articles

Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1β and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

ASPN Synergizes with HAPLN1 to Inhibit the Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells and Extracellular Matrix Mineralization of Osteoblasts.

Bone marrow mesenchymal stromal cells (BMSCs) are major sources of osteogenic precursor cells in bone remodeling, which directly participate in osteoporosis (OP) progression. However, the involved specific mechanisms of BMSCs in OP warrant mass investigations. Initially, our bioinformatics analysis uncovered the prominent up-regulation of Asporin (ASPN) and proteoglycan link protein 1 (HAPLN1) in osteoblasts (OBs) of OP patients and their possible protein interaction. Hence, this study aimed to explore the effects of ASPN and HAPLN1 on osteogenic differentiation of BMSCs, extracellular matrix (ECM) mineralization of OBs, and osteoclastogenesis, hoping to offer research basis for OP treatment. GSE156508 dataset was used for analysis and screening to acquire the differentially expressed genes in OBs of OP patients, followed by the predicative analysis via STRING. OP mouse models were induced by ovariectomy (OVX), and ASPN and HAPLN1 expression was determined. BMSCs and bone marrow macrophages (BMMs) were isolated from OVX mice and induced for osteogenic differentiation and osteoclastogenesis, respectively. After knockdown experiments, we assessed adipogenic differentiation and osteogenic differentiation in BMSCs. Osteogenic (OPN, OCN, and COL1A1) and osteoclast (Nfatc1 and c-Fos) marker protein expression was determined. The binding of ASPN to HAPLN1 was analyzed. High expression of ASPN and HAPLN1 and their protein interaction were observed in OBs of OP patients via bioinformatics and in bone tissues of OVX mice. ASPN interacted with HAPLN1 in BMSCs of OVX mice. ASPN/HAPLN1 knockdown increased ALP, OPN, OCN, and COL1A1 protein expression and ECM mineralization in BMSCs while decreasing Nfatc1 and c-Fos expression in BMMs. These effects were aggravated by the simultaneous knockdown of ASPN and HAPLN1. Our results indicate that ASPN synergises with HAPLN1 to suppress the osteogenic differentiation of BMSCs and ECM mineralization of OBs and promote the osteoclastogenesis in OP.

In vitro efficacy of whole-grain finger millet (Eleusine coracana) extract on primary rat osteoblasts

To improve the primary osteoblast cells of neonatal rats for mineralization, Finger millet grain (Eleusine coracana) extract was employed. In vitro digested extract (IVD) and citric acid buffered extract (CAB) were evaluated for cell proliferation, regeneration and matrix mineralization. The cytotoxic effect and the osteogenic differentiation were examined using MTT (3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, Giemsa stain and Von-Kossa respectively. Cell progression and osteogenic differentiation markers like Osteocalcin (OCN), Osteopontin (OPN), collagen type I (Col-I), collagen type II (Col-II) and collagen type III (Col-III), RUNX family transcription factor 2 (Runx2), Matrix metallo peptidase 9 (MMP-9) and peroxisome proliferator-activated receptor (PPARG) expression were made using reverse transcriptase-polymerase chain reaction. Exposure of different concentrations of the extract (100-800μg/ml of CAB and 25-400 μg/ml of IVD) on cells showed the characteristic morphology change in osteoblastic cells including the increase in cell number. In a dose-dependent way (p<0.005), osteoblast matrix mineralization revealed that CAB stimulates osteoblasts. In light of these findings, finger millet (CAB extract) could be used for osteoporosis prevention and bone rebuilding.

Dikkopf-1 promotes matrix mineralization of osteoblasts by regulating Ca+-CAMK2A- CREB1 pathway

Other SectionsABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGEMENTSCONFLICTS OF INTERESTFIGURESREFERENCES

Molecular Mechanisms Involved in Hypoxia-Induced Alterations in Bone Remodeling.

Bone is crucial for the support of muscles and the protection of vital organs, and as a reservoir of calcium and phosphorus. Bone is one of the most metabolically active tissues and is continuously renewed to adapt to the changes required for healthy functioning. To maintain normal cellular and physiological bone functions sufficient oxygen is required, as evidence has shown that hypoxia may influence bone health. In this scenario, this review aimed to analyze the molecular mechanisms involved in hypoxia-induced bone remodeling alterations and their possible clinical consequences. Hypoxia has been associated with reduced bone formation and reduced osteoblast matrix mineralization due to the hypoxia environment inhibiting osteoblast differentiation. A hypoxic environment is involved with increased osteoclastogenesis and increased bone resorptive capacity of the osteoclasts. Clinical studies, although with contradictory results, have shown that hypoxia can modify bone remodeling.

Osteogenic activity of Na2Ti3O7/SrTiO3 hybrid coatings on titanium

Titanium (Ti) and its alloys have been widely adopted as implant materials due to their satisfactory biocompatibility and favorable mechanical property. However, they usually show poor osteogenic ability, hindering its clinical application. Herein, Na2Ti3O7/SrTiO3 hybrid coatings on Ti were fabricated via one-step alkali etching in a mixed solution of NaOH and Sr(OH)2 to enhance its osteogenic activity. Scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and transmission electron microscopy were used to characterize the samples. Biological experiments were conducted to evaluate their osteogenic activity. The results showed Na2Ti3O7 nanowire coatings could be grown on Ti surface and Sr could be incorporated into the coatings. Incorporation of Sr clustered the nanowires and high Sr content lead to the formation of spherical SrTiO3 with diameter of about 1 μm. Sr2+ could continuously release from the samples and the release amount increased with Sr content in the coatings. Although adhesion, spreading, viability and proliferation of osteoblasts on all the samples had no significant difference when compared with Sr-free one, they could significantly promote osteogenic differentiation as verified by enhanced alkaline phosphatase (ALP) activity, collagen secretion, and extracellular matrix mineralization of osteoblasts. Further studies depicted that the expression levels of osteogenic-related genes including ALP, type I collagen and transforming growth factor-β were significantly upregulated in Sr-containing samples. The desirable osteogenic capacity may be ascribed to the synergistic effect of continuous Sr2+ release and nanostructured morphology. The samples containing Sr of 5.65–11.07 at.% showed the best osteogenic capacity therefore possess great potential in hard-tissue implant field.

LncRNA H19 promotes matrix mineralization through up-regulating IGF1 by sponging miR-185-5p in osteoblasts

BackgroundMatrix mineralization is a key stage in bone formation involving in many bone-specific genes and signaling pathways. Emerging evidence indicate that long non-coding RNA (lncRNA) and microRNAs (miRNAs) play crucial roles in regulating the mineralization process of osteoblasts. This study aims to characterize the function and mechanism of lncRNA H19/miR-185-5p/IGF1 axis in modulating matrix mineralization of osteoblasts.ResultsH19 and IGF1 were highly expressed while miR-185-5p was lowly expressed in mineralized cells. Knocking down H19 inhibited matrix mineralization of osteoblasts, yet miR-185-5p had opposite effects. Moreover, H19 directly targeted miR-185-5p, whereas miR-185-5p repressed IGF1 expression. Meanwhile, miR-185-5p inhibition compensated the suppression of the matrix mineralization in osteoblasts by H19 knockdown.ConclusionsThe findings of this study showed that lncRNA H19 was upregulated in mineralized osteoblasts and promoted matrix mineralization through miR-185-5p/IGF1 axis in osteoblasts for the first time. This study may provide a new perspective for the diagnosis and treatment of diseases related to bone metabolism.

Glutamine Metabolism Is Essential for Stemness of Bone Marrow Mesenchymal Stem Cells and Bone Homeostasis.

Skeleton has emerged as an endocrine organ which is both capable of regulating energy metabolism and being a target for it. Glutamine is the most bountiful and flexible amino acid in the body which provides adenosine 5′-triphosphate (ATP) demands for cells. Emerging evidences support that glutamine which acts as the second metabolic regulator after glucose exerts crucial roles in bone homeostasis at cellular level, including the lineage allocation and proliferation of bone mesenchymal stem cells (BMSCs), the matrix mineralization of osteoblasts, and the biosynthesis in chondrocytes. The integrated mechanism consisting of WNT, mammalian target of rapamycin (mTOR), and reactive oxygen species (ROS) signaling pathway in a glutamine-dependent pattern is responsible to regulate the complex intrinsic biological process, despite more extensive molecules are deserved to be elucidated in glutamine metabolism further. Indeed, dysfunctional glutamine metabolism enhances the development of degenerative bone diseases, such as osteoporosis and osteoarthritis, and glutamine or glutamine progenitor supplementation can partially restore bone defects which may promote treatment of bone diseases, although the mechanisms are not quite clear. In this review, we will summarize and update the latest research findings and clinical trials on the crucial regulatory roles of glutamine metabolism in BMSCs and BMSC-derived bone cells, also followed with the osteoclasts which are important in bone resorption.

Osteonectin regulates the extracellular matrix mineralization of osteoblasts through P38 signaling pathway.

Osteonectin binds strongly to type I collagen and hydroxyapatite and plays a crucial role in extracellular matrix mineralization. Previous studies have also shown that p38 signaling pathway is an important regulator for osteoblast mineralization. This study focused on the role of osteonectin in regulating extracellular matrix mineralization via the p38 signaling pathway. Osteoblasts were isolated and cultured from parietal bones of neonatal Sprague-Dawley rats. The gene and protein expressions of noncollagen proteins (BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin), p38 mitogen-activated protein kinase, and SIBLINGs (Small Integrin-Binding LIgand N-linked Glycoproteins) members (DMP1, dentine matrix protein 1, DSPP, dentin sialophosphoprotein, and MEPE, matrix extracellular phosphoglycoprotein) were detected by reverse-transcription quantitative polymerase chain reaction and western blot analysis. Alizarin red staining, intracellular calcium assay, and transmission electron microscopy were used to detect mineralization. Initially, by adding osteonectin at different concentrations in osteoblasts and detecting the above mineralization indexes, 1 µg/ml was determined to be the optima osteonectin concentration, which significantly increased gene expressions of BSP, OPN, OCN, DMP1, MEPE, DSPP, and p38 in osteoblasts, p38 and p-p38 protein expressions were also significantly increased, mineralized nodules were significantly enhanced; when added with SB203580 (a specific inhibitor for p38) these effects were inhibited. Furthermore, osteoblasts transfected with Ad-p38 also significantly upregulated the protein and gene expressions of noncollagens and SIBLINGs members, whereas transfection of p38-rhRNA showed the opposite effect. Our data suggest that osteonectin regulates the extracellular matrix mineralization of osteoblasts through the P38 signaling pathway.

Tissue-Nonspecific Alkaline Phosphatase Is Required for MC3T3 Osteoblast-Mediated Protection of Acute Myeloid Leukemia Cells from Apoptosis.

The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.

Inhibitory Effects of Human Primary Intervertebral Disc Cells on Human Primary Osteoblasts in a Co-Culture System.

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.

Icariin attenuates titanium particle-induced inhibition of osteogenic differentiation and matrix mineralization via miR-21-5p.

Inhibition of bone regeneration by wear debris is the main cause of peri-prosthetic osteolysis. Here, we investigated the effect of icariin on cell proliferation, apoptosis, osteogenic differentiation and matrix mineralization of osteoblasts in an in vitro model of titanium (Ti) particle-induced osteolysis. In the present study, MC3T3-E1 cells were pretreated with 10-8 M icariin for 4 h and then incubated with Ti particles (0.1 mg/mL). The results showed that Ti particles inhibited cell proliferation and promoted cell apoptosis of MC3T3-E1 cells, whereas icariin pretreatment blocked the effect of Ti particles. In addition, we found that icariin stimulation alone increased ALP activity, accelerated matrix mineralization and upregulated the levels of bone morphogenetic protein 2 (BMP2), Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and miR-21-5p; whereas, Ti particles alone exerted the opposite effects. Icariin partly reversed the effect of Ti particles on cell differentiation and mineralization. Twenty hours after transfection with antagomiR-21-5p or antagomiR-NC, the cells were pretreated with icariin for 4 h and then incubated with Ti particles. Further studies showed that partial knockdown of miR-21-5p abolished the promotion effect of icariin on osteoblast differentiation and matrix mineralization in Ti particle-stimulated MC3T3-E1 cells. In conclusion, miR-21-5p may be a potential pro-osteogenesis regulator and icariin may protect against Ti particle-induced inhibition of osteogenic differentiation and mineralization through upregulation of miR-21-5p.

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways.

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3-E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3-E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF-ERK1/2 and BMP-Smad pathways, and suppresses the induction of markers of osteoblast differentiation.

Antibacterial, osteogenic, and angiogenic activities of SrTiO3 nanotubes embedded with Ag2O nanoparticles

Biomedical titanium (Ti) implants with good anti-infective, osteogenic, and angiogenic properties are in great demand. SrTiO3 nanotubes (NTs) are embedded with silver oxide (Ag2O) nanoparticles (NPs) (denoted as NT-Sr-Ag) by a hydrothermal treatment of TiO2 NTs containing Ag2O NPs (denoted as NT-Ag) in a Sr(OH)2 solution. The morphology, composition, microstructure, ion release phenomenon, as well as antibacterial, osteogenic, and angiogenic activities are investigated in details. During the hydrothermal treatment, the amorphous TiO2 in the NTs morphs into cubic SrTiO3 gradually and the ordered nanotubular architecture is preserved. Some Ag2O NPs are incorporated into the structure although some of them dissolve in the solution. Long-term bacterial resistance against Staphylococcus aureus is observed as a result of the prolonged and controllable Ag+ release. NT-Sr-Ag can also release Sr2+ similarly to stimulate osteoblasts to secrete the vascular endothelial growth factor (VEGF). Both the released Sr2+ and secreted VEGF upregulate the alkaline phosphatase (ALP) activity and extracellular matrix mineralization of osteoblasts. Furthermore, better angiogenic activity is observed when endothelial cells are cultured in NT-Sr-Ag conditioned media when compared with that in NT-Ag conditioned media, which is believed to be ascribed to the positive regulation of VEGF secretion of Sr2+. NT-2Sr-Ag and NT-3Sr-Ag (Hydrothermal treatment for 2 and 3h, respectively) exhibit excellent antibacterial, osteogenic, and angiogenic activities and are promising in biomedical implants.

The influence of MicroRNA-150 in Osteoblast Matrix Mineralization.

This study investigated the influence of miR-150 expression on osteoblast matrix mineralization and its mechanisms. The mouse osteoblast cell line MC3T3-E1 was used as an in vitro model of bone formation. On the fifth day of mineralization, transfection experiments using agomiR-150, agomiR-NC, antagomiR-150 antagomiR-NC, and mock groups were set up to test the effects of miR-150 in MC3T3-E1 model. The mRNA and protein levels of OC, ALP, type I collagen, and OPN were measured by qRT-PCR and ELISA. Matrix mineralization was detected by alizarin red S (ARS) staining and flow cytometry was employed to quantify apoptosis in each group. RT-PCR and Western blot were applied to detect the expression of target gene MMP14. Our results demonstrated that the endogenous expression levels of miR-150, OC, ALP, type I collagen, and OPN in MC3T3-E1 cells increased steadily. Exogenous expressions of agomiR-150 and antagomiR-150 can significantly up-/down-regulate, respectively, the expression level of miR-150 in MC3T3-E1 cells. Compared with the mock group, higher expression levels of OC, ALP, type I collagen, and OPN mRNA were observed in the agomiR-150 group, while lower mRNA expression levels of OC, ALP, type I collagen, and OPN were found in the antagomiR-150 group. Based on these results, potential miR-150 targeted genes are discussed. Our results showed that miR-150 supports the osteoblastic phenotype related to osteoblast function and bone mineralization. Thus, miR-150 may have potential therapeutic applications in promoting bone formation in certain disease settings, such as in osteoporosis and in elderly patients.

Cissus quadrangularis Linn exerts dose-dependent biphasic effects: osteogenic and anti-proliferative, through modulating ROS, cell cycle and Runx2 gene expression in primary rat osteoblasts.

This report highlights phytoconstituents present in Cissus quadrangularis (CQ) extract and examines biphasic (proliferative and anti-proliferative) effects of its extract on bone cell proliferation, differentiation, mineralization, ROS generation, cell cycle progression and Runx2 gene expression in primary rat osteoblasts. Phytoconstituents were identified using gas chromatography-mass spectroscopy (GC-MS). Osteoblasts were exposed to different concentrations (10-100 μg/ml) of CQ extract and cell proliferation and cell differentiation were investigated at different periods of time. Subsequently, intracellular ROS intensity, apoptosis and matrix mineralization of osteoblasts were evaluated. We performed flow cytometry for DNA content and real-time PCR for Runx2 gene expression analysis. CQ extract's approximately 40 bioactive compounds of fatty acids, hydrocarbons, vitamins and steroidal derivatives were identified. Osteoblasts exposed to varying concentrations of extract exhibited biphasic variation in cell proliferation and differentiation as a function of dose and time. Moreover, lower concentrations (10-50 μg/ml) of extract slightly reduced ROS intensity, although they enhanced matrix mineralization, DNA content in S phase of the cell cycle, and levels of Runx2 expression. However, higher concentrations (75-100 μg/ml) considerably induced the ROS intensity and nuclear condensation in osteoblasts, while it reduced mineralization level, proportion of cells in S phase and Runx2 level of the osteogenic gene. These findings suggest that CQ extract revealed concentration-dependent biphasic effects, which would contribute notably to future assessment of pre-clinical efficacy and safety studies.

Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts.

BackgroundOsteoporosis is one of the prevalent diseases in ageing populations. Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder. Punica granatum (PG) represent a potent fruit-bearing medicinal herb which exerted valuable anti-osteoporotic activities. The present study was carried out to validate the in vitro osteogenic effects of the PG seed extract in primary calvarial osteoblast cultures harvested from neonatal rats.MethodsThe ethanolic extract of PG was subjected to evaluate cell proliferation, regeneration, mineralization and formation of collagen matrix using MTT, alkaline phosphatase, Alizarin Red-S staining and Sirius Red dye, respectively. Cell cycle progression and osteogenic gene Runx2 expression were carried out by flow cytometry and real time PCR, respectively.ResultsExposure of different concentrations (10–100 μg/ml) of the extract on osteoblastic cells showed characteristic morphological changes and increment in cell number. A significant growth in cell proliferation, ALP activity, collagen contents and matrix mineralization of osteoblasts in a dose dependent manner (p < 0.05), suggested that PG has a stimulatory effect on osteoblastic bone formation or potential activity against osteoporosis. In addition, PG extract also enhanced DNA content in S phase of cell cycle and Runx2 gene expression level in osteoblasts.ConclusionThe data clearly indicated that PG promoting bone cell proliferation and differentiation in primary osteoblasts might be due to elevating the osteogenic gene Runx2 expression. The present study provides an evidence for PG could be a promising herbal medicinal candidate that able to develop drugs for osteoporosis.

Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.

Identification of novel regulators of osteoblast matrix mineralization by time series transcriptional profiling

Bone mineralization is a carefully orchestrated process, regulated by a number of promoters and inhibitors that function to ensure effective hydroxyapatite formation. Here we sought to identify new regulators of this process through a time series microarray analysis of mineralising primary osteoblast cultures over a 27 day culture period. To our knowledge this is the first microarray study investigating murine calvarial osteoblasts cultured under conditions that permit both physiological extracellular matrix mineralization through the formation of discrete nodules and the terminal differentiation of osteoblasts into osteocytes. RT-qPCR was used to validate and expand the microarray findings. We demonstrate the significant up-regulation of >6,000 genes during the osteoblast mineralization process, the highest-ranked differentially expressed genes of which were those dominated by members of the PPAR-γ signalling pathway, namely Adipoq, Cd36 and Fabp4. Furthermore, we show that the inhibition of this signalling pathway promotes matrix mineralisation in these primary osteoblast cultures. We also identify Cilp, Phex, Trb3, Sox11, and Psat1 as novel regulators of matrix mineralization. Further studies examining the precise function of the identified genes and their interactions will advance our understanding of the mechanisms underpinning biomineralization.

Comparison of bone tissue properties in mouse models with collagenous and non-collagenous genetic mutations using FTIRI

Understanding how the material properties of bone tissue from the various forms of osteogenesis imperfecta (OI) differ will allow us to tailor treatment regimens for affected patients. To this end, we characterized the bone structure and material properties of two mouse models of OI, the osteogenesis imperfecta mouse (oim/oim) and fragilitas ossium (fro/fro), in which bone fragility is due to a genetic defect in collagen type I and a defect in osteoblast matrix mineralization, respectively. Bones from 3 to 6month old animals were examined using Fourier transform infrared spectroscopic imaging (FTIRI), microcomputed tomography (micro-CT), histology, and biochemical analysis. The attributes of oim/oim bone tissue were relatively constant over time when compared to wild type animals. The mineral density in oim/oim cortices and trabecular bone was higher than wild type while the bones had thinner cortices and fewer trabeculae that were thinner and more widely spaced. The fro/fro animals exhibited osteopenic attributes at 3months. However, by 6months, their spectroscopic and geometric properties were similar to wild type animals. Despite the lack of a specific collagen defect in fro/fro mice, both fro/fro and oim/oim genotypes exhibited abnormal collagen crosslinking as determined by FTIRI at both time points. These results demonstrate that abnormal extracellular matrix assembly plays a role in the bone fragility in both of these models.