Mineralization Of SaOS-2 Cells Research Articles

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Evaluation of the cargo contents and potential role of extracellular vesicles in osteoporosis.

Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). Extracellular vesicles (EVs) remain unexplored in the context of osteoporosis. Towards this, EVs were isolated from plasma of a discovery cohort with 8 non-osteoporotic and 8 osteoporotic individuals, and nanoparticle tracking analysis (NTA) revealed a significantly higher EV concentration in osteoporotic individuals (P = 0.003). Moreover, EVs concentration showed a linear correlation with bone mineral density (BMD) values (linear correlation coefficient r = 0.9542, deviation from zero, p < 0.001). Results using a mouse model of osteoporosis confirmed that the number of EVs in mice from hindlimb unloading group was significantly higher than that from the age-matched control group (p = 0.015). TaqMan Real-Time PCR demonstrated that miR-335-5p, -320a, -483-5p, and miR-21-5p, were significantly higher expressed in osteoporotic patients compared with non-osteoporotic individuals. Quantitative real-time PCR shown that Wnt1, Wnt5a, Wnt7a, and Wnt9a mRNAs were lower expressed in osteoporosis derived EVs. In vitro functional assay indicated that osteoporosis derived EVs resulted in reduced mineralization in SaOS-2 cells. In conclusion, these results suggest that osteoporosis increased the secretion of EVs which carry higher expression of miRNAs and decreased expression of Wnt signals, further decreased the mineralization capacity in human osteoblasts.

Production of hydroxyapatite–bacterial cellulose composite scaffolds with enhanced pore diameters for bone tissue engineering applications

Bone tissue engineering scaffolds used for the treatment of bone defects are required to be osteoconductive, osteoinductive, osteogenic, biocompatible, and have enough porosity to allow osteointegration, as well as vascularization. It is known that addition of the hydroxyapatite (HAp) to bone tissue scaffolds promotes bone formation by increasing osteoconductivity. Bacterial cellulose (BC) is a highly biocompatible material, and its mechanical properties and fibrous structure allow that it can be used as a bone tissue scaffold; yet, the nano-porous structure of BC (50–200 nm) prevents or limits cell migration and vascularization. In this study, it is intended to take advantage of the porous structure and mechanical strength of BC and osteoconductive properties of HAp for the production of tissue engineering scaffolds. Pore sizes of BC were enhanced to 275 μm by a novel shredded agar technique, and SaOs-2 cells were shown to migrate between the fibers of the modified BC. It was observed that mineralization of SaOs-2 cells was enhanced on in situ produced HAp-BC nano-composites compared to BC scaffolds.

Fabrication of amorphous strontium polyphosphate microparticles that induce mineralization of bone cells in vitro and in vivo.

In this manuscript, we fabricated amorphous strontium-polyphosphate microparticles ("Sr-a-polyP-MP") and studied their effects on bone mineral formation in vitro as well as in vivo. In vitro, those particles substantially increased the expression of the genes encoding for alkaline phosphatase, the bone morphogenetic protein 2 and the mineralization. In vivo, the "Sr-a-polyP-MP" packed into PLGA microspheres and implanted into critical-size calvarial defects in rats resulted in a speeded up of the healing/mineralization of the bone defect. Those properties qualify Sr-a-polyP as a suitable biomaterial for bone regenerative implants.

Supraphysiological Levels of Quercetin Glycosides are Required to Alter Mineralization in Saos2 Cells.

Flavonoid intake is positively correlated to bone mineral density (BMD) in women. Flavonoids such as quercetin exhibit strong anti-oxidant and anti-inflammatory activity that may be beneficial for bone health. Quercetin, previously shown to positively influence osteoblasts, is metabolized into glycosides including rutin and hyperoside. We compared the effects of these glycosides on mineralization in human osteoblast (Saos2) cells. Administration of rutin (≥25 µM) and hyperoside (≥5 µM) resulted in higher mineral content, determined using the alizarin red assay. This was accompanied by higher alkaline phosphatase activity with no cell toxicity. The expression of osteopontin, sclerostin, TNFα and IL6, known stimuli for decreasing osteoblast activity, were reduced with the addition of rutin or hyperoside. In summary, rutin and hyperoside require supraphysiological levels, when administered individually, to positively influence osteoblast activity. This information may be useful in developing nutraceuticals to support bone health.

Indirect rapid prototyping of sol-gel hybrid glass scaffolds for bone regeneration – Effects of organic crosslinker valence, content and molecular weight on mechanical properties

We present a series of organic/inorganic hybrid sol-gel derived glasses, made from a tetraethoxysilane-derived silica sol (100% SiO2) and oligovalent organic crosslinkers functionalized with 3-isocyanatopropyltriethoxysilane. The material was susceptible to heat sterilization. The hybrids were processed into pore-interconnected scaffolds by an indirect rapid prototyping method, described here for the first time for sol-gel glass materials. A large panel of polyethylene oxide-derived 2- to 4-armed crosslinkers of molecular weights ranging between 170 and 8000Da were incorporated and their effect on scaffold mechanical properties was investigated. By multiple linear regression, ‘organic content’ and the ‘content of ethylene oxide units in the hybrid’ were identified as the main factors that determined compressive strength and modulus, respectively. In general, 3- and 4-armed crosslinkers performed better than linear molecules. Compression tests and cell culture experiments with osteoblast-like SaOS-2 cells showed that macroporous scaffolds can be produced with compressive strengths of up to 33±2MPa and with a pore structure that allows cells to grow deep into the scaffolds and form mineral deposits. Compressive moduli between 27±7MPa and 568±98MPa were obtained depending on the hybrid composition and problems associated with the inherent brittleness of sol-gel glass materials could be overcome. SaOS-2 cells showed cytocompatibility on hybrid glass scaffolds and mineral accumulation started as early as day 7. On day 14, we also found mineral accumulation on control hybrid glass scaffolds without cells, indicating a positive effect of the hybrid glass on mineral accumulation. Statement of SignificanceWe produced a hybrid sol-gel glass material with significantly improved mechanical properties towards an application in bone regeneration and processed the material into macroporous scaffolds of controlled architecture by indirect rapid prototyping. We were able to produce macroporous materials of relevant porosity and pore size with compressive moduli, covering the range reported for cancellous bone while an even higher compressive strength was maintained. By multiple linear regression, we identified crosslinker parameters, namely organic content and the content of ethylene oxide units in the hybrids that predominantly determined the mechanics of the hybrid materials. The scaffolds proved to be cytocompatible and induced mineralization in SaOS-2 cells. This provides new insight on the critical parameters for the design of the organic components of covalent hybrid sol-gel glasses.

Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption.

AimThe cysteine protease cathepsin K (CatK), abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs) to test their effects on osteoblasts and osteoclasts.Research Design and MethodsCell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices.ResultsTreatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10–100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed down by the addition of CKI-8 and CKI-13.Conclusion CKI-8 and CKI-13 screened here show promise as antiresorptive osteoporosis therapeutics but some off target effects on osteoblasts were found with CKI-13.

AP2 suppresses osteoblast differentiation and mineralization through down-regulation of Frizzled-1.

Transcription factor activating protein 2 (AP2) plays an important role in cellular differentiation. Although profound craniofacial and long bone developmental abnormalities have been observed in AP2-knockout mice, the molecular effects of AP2 on osteoblasts are poorly defined. We demonstrated that AP2 regulates the expression of human Frizzled 1 (FZD1), a co-receptor for the Wnt signalling pathway, in human osteoblast cell lines and primary bone marrow stromal cells (BMSCs). We also identified a putative AP2-binding site in the FZD1 proximal promoter in silico and characterized this binding element further in Saos2 invitro by ChIP, electrophoretic mobility shift and promoter reporter assays. The transcriptional repression of the FZD1 promoter by AP2 was confirmed in normal human fetal osteoblasts (hFOB). Furthermore, overexpression of AP2 resulted in a significant reduction in both differentiation and mineralization of Saos2 cells. Knockdown of FZD1 expression before AP2 up-regulation diminished the AP2-dependent inhibition of Saos2 cell differentiation and mineralization. Similarly, overexpressing FZD1 before AP2 treatment in both Saos2 and BMSCs diminished the inhibitory effect of AP2 on osteoblast differentiation and mineralization. Taken together, these results demonstrate that AP2 is a negative regulator of osteoblast differentiation and mineralization, and its inhibitory effect may be mediated in part through down-regulation of FZD1 expression.

Tea flavonoids stimulate mineralization in osteoblast‐like cells (259.7)

Epidemiological studies suggest that habitual tea consumption is associated with higher bone mineral density. Rooibos tea, in particular, is a rich source of flavonoids, including rutin, orientin, hyperoside, and luteolin, that may favorably modulate cellular processes such as hydroxyapatite production. The study objective was to determine if individual flavonoids (0.001µM‐100µM) promoted mineralization in Saos2 cells. Mineralization was quantified by alizarin red staining at day 21, while alkaline phosphatase activity, a marker of osteoblast cell activity, was determined at days 3, 7 and 10. Rutin (25µM), orientin (0.01µM), hyperoside (5µM) and luteolin (5µM) resulted in higher (p&lt;0.05) mineral production than control. Higher mineral production was associated with higher alkaline phosphatase activity (p&lt;0.05) for rutin (25µM), orientin (0.1µM), hyperoside (1µM) and luteolin (1µM) as early as 3 days into the mineralization process. Maximum enzyme activity was dependent on the flavonoid. Mitochondrial activity, a marker of cellular proliferation, reflected the mineralization data. In conclusion, flavonoids can positively influence mineral production in osteoblasts and this is, in part, due to higher osteoblast activity. Thus, tea flavonoids are a promising area for future research investigating dietary approaches for bone health.Grant Funding Source: Supported by a NSERC Discovery Grant for W. Ward

Water Soluble Components of 'Osteocare' Promote Cell Proliferation, Differentiation, and Matrix Mineralization in Human Osteoblast-Like SaOS-2 Cells.

Osteocare, a herbal formulation, has been found to be very effective in bone mineralization and support of the microstructure of bone tissue. The water-soluble components of Osteocare (WSCO) induced osteogenic activity in human osteoblast-like SaOS-2 cells. The addition of WSCO (100 μg/ml) to SaOS-2 cells was effective in increasing the cell proliferation by 41.49% and DNA content by 1.9-fold. WSCO increased matrix mineralization in SaOS-2 cells by increased alkaline phosphatase levels and calcium-rich deposits as observed by Alizarin red staining. WSCO markedly increased mRNA expression for osteopontin (OPN), osteocalcin (OCN), type I collagen (Col I) in SaOS-2 cells, and it down-regulated IL-6 mRNA levels in SaOS-2 cells. The present study showed that WSCO plays an important role in osteoblastic bone formation through enhanced activities of ALP, Col I, bone matrix proteins such as OPN and OCN, down-regulation of cytokines like IL-6, as well as promoting mineralization in SaOS-2 cells.

High xylosyltransferase activity in children and during mineralization of osteoblast-like SAOS-2 cells

Skeletal growth and tissue remodelling processes are characterized by an elevated collagen and proteoglycan biosynthesis. The xylosyltransferases I and II are the rate-limiting step enzymes in proteoglycan biosynthesis and serum xylosyltransferase (XT) activity has been shown to be a biomarker for the actual proteoglycan biosynthesis rate. Here, XT, alkaline phosphatase (ALP), bone ALP (BALP) activities were measured in 133 juvenile Caucasians. Serum XT activities in juveniles were elevated and significantly correlated with ALP and BALP. In an osteoblast-like cell model using SAOS-2 cells mineralization and bone nodule formation were induced and XT-I, XT-II and ALP were monitored. Induction of mineralization in SAOS-2 cells resulted in a long-term increase of XT-I mRNA and enzyme activity, which could be paralleled with elevated ALP activity. In addition, HGH and IGF-I treatment of SAOS-2 cells led to an increased expression of XT-I and ALP. These results point to skeletal growth and tissue remodeling as a cause of the high XT activity in children.

Puerariae radix promotes differentiation and mineralization in human osteoblast-like SaOS-2 cells

Puerariae radix (PR) is a traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify whether Puerariae radix extract induces osteogenic activity in human osteoblast-like SaOS-2 cells. Puerariae radix had no effect on the viability of osteoblastic cells, and dose-dependently increased alkaline phosphatase (ALP) activity. Puerariae radix markedly increased mRNA expression for vascular endothelial growth factor (VEGF), osteocalcin (OCN), osteopontin (OPN), and type I collagen (Col I) in SaOS-2 cells. Extracellular accumulation of proteins such as VEGF and Col I was increased in a dose-dependent manner. Also, Puerariae radix significantly induced mineralization in the culture of SaOS-2 cells. In conclusion, this study showed that Puerariae radix had no effect on viability, but enhanced ALP activity, VEGF, bone matrix proteins such as OCN, OPN, and Col I, and mineralization in SaOS-2 cells. These results propose that Puerariae radix can play an important role in osteoblastic bone formation, and may possibly lead to the development of bone-forming drugs.