Insights into the antiosteoporotic mechanism of the soy-derived isoflavone genistein: Modulation of the Wnt/beta-catenin signaling.

Bone remodeling is a process that involves osteoblasts, osteoclasts, and osteocytes, and different intracellular signaling, such as the canonical Wnt/ß-catenin pathway. Dysregulations of this pathway may also occur during secondary osteoporosis, as in the case of glucocorticoid-induced osteoporosis (GIO), which accelerates osteoblast and osteocyte apoptosis by reducing bone formation, osteoblast differentiation and function, accelerates in turn osteoblast, and osteocyte apoptosis. Genistein is a soy-derived nutrient belonging to the class of isoflavones that reduces bone loss in osteopenic menopausal women, inhibiting bone resorption; however, genistein may also favor bone formation. The aim of this study was to investigate whether estrogen receptor stimulation by genistein might promote osteoblast and osteocyte function during glucocorticoid challenge. Primary osteoblasts, collected from C57BL6/J mice, and MLO-A5 osteocyte cell line were used to reproduce an in vitro model of GIO by adding dexamethasone (1 µM) for 24 h. Cells were then treated with genistein for 24 h and quantitative Polymerase Chain Reaction (qPCR) and western blot were performed to study whether genistein activated the Wnt/ß-catenin pathway. Dexamethasone challenge reduced bone formation in primary osteoblasts and bone mineralization in osteocytes; moreover, canonical Wnt/ß-catenin pathway was reduced following incubation with dexamethasone in both osteoblasts and osteocytes. Genistein reverted these changes and this effect was mediated by both estrogen receptors α and ß. These data suggest that genistein could induce bone remodeling through Wnt/ß-catenin pathway activation.

Inhibitory Effect of Ulmus davidiana and Cornus officinalis Extracts on Osteoporotic Bone Loss In Vitro and In Vivo.

Background and Objectives: Traditional herbal medicines are becoming more popular as a complementary medication as they have the advantages of being mostly harmless and safe, causing fewer side-effects than conventional medications. Here, we demonstrate the inhibitory effects of the combination of Ulmus davidiana (UD) and Cornus officinalis (CO) extracts on osteoporotic bone loss. Materials and Methods: This study presented osteogenic effects in primary cultured osteoblasts, pre-osteoblastic MC3T3-E1 cell lines, and osteoclastogenic effects in osteoclasts derived from bone marrow monocytes, and finally, protective effects on bone loss in an ovariectomy (OVX)-induced osteoporotic animal model. Results: A significant increase in alkaline phosphatase (ALP) activity was observed following treatment with UD and CO mixtures (8:2, 7:3, and 5:5 ratios) and individual UD and CO extracts, with the highest ALP activity being detected for the treatment with UD and CO extracts at a 5:5 ratio. An optimal ratio of UD and CO (UC) extract promoted osteoblast differentiation in both pre-osteoblastic cells and primary osteoblasts by increasing osteoblastic markers such as Alpl, Runx2, and Bglap. However, treatment with the UC extract inhibited osteoclast differentiation with a decreased expression of osteoclastogenesis-related genes, including Ctsk, Acp5, Mmp9, and Nfatc1. In addition, UC treatment prevented osteoporotic bone loss in OVX mice and improved impaired skeletal structure parameters. Conclusions: This study suggests that combined UD and CO extracts may be a beneficial traditional medicine for the prevention of postmenopausal osteoporosis.

Interleukin-17 induces pyroptosis in osteoblasts through the NLRP3 inflammasome pathway in vitro.

OBJECTIVE: Interleukin-17 (lL-17), a pro-inflammatory cytokine produced by Th17 cells, is also considered to play an important role in bone metabolism, but the exact mechanism of bone destruction remains unclear. In this study, we explored whether IL-17 could induce osteoblasts pyroptosis in vitro. METHODS: The murine primary osteoblasts were isolated from the calvarial bones of mice. The proliferation of osteoblasts was evaluated by cell counting kit-8 (CCK-8) assay. The mRNA levels of NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis associated speck like protein containing a card (ASC), caspase-1, gasdermin-D (GSDMD), IL-1ß and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured by real-time quantitative PCR. Pyroptosis after IL-17 treatment was evaluated by lactate dehydrogenase (LDH) Release Assay Kit and the morphological characteristics of osteoblasts were observed via Scanning Electron Microscopy (SEM). Pyroptosis associated proteins, cleaved IL-1ß and RANKL were evaluated through western blot. The release of IL-1ß and RANKL was measured by ELISA. In addition, calcium nodule was tested by alizarin red staining. RESULTS: High concentration IL-17 (100 ng/mL) could affect the proliferation of osteoblasts, promote the gene expression of NLRP3, caspase-1, GSDMD, IL-1ß and RANKL. In contrast to control group, osteoblasts treated with IL-17 had the appearance of numerous pores, swelling and rupture. Also, the release of LDH, IL-1ß and RANKL increased in the presence of IL-17. However, inhibition of NLRP3 prevented activation of the NLRP3 inflammasome, thereby restoring osteoblasts morphology and function. CONCLUSION: IL-17 induced osteoblasts pyroptosis, and the pyroptosis of osteoblasts may prompt the release of IL-1ß and RANKL,which may further contribute to disruption of bone metabolism. Besides, the NLRP3 inflammasome pathway was involved in the pyroptosis of osteoblasts.

RANKL expression of primary osteoblasts is enhanced by an IL-17-mediated JAK2/STAT3 pathway through autophagy suppression.

Objective: Interleukin-17 (IL-17), produced by T helper (Th)-17 cells, is a potent regulator of bone homeostasis. Osteoblasts are key cells that orchestrate inflammatory bone destruction and bone remodeling. This study examines the effect of different concentrations of IL-17 on osteogenesis and receptor activator of nuclear factor-kappa B ligand (RANKL) expression of primary osteoblasts.Methods: First, the growth of primary osteoblasts was evaluated. Second, we assessed the effects of IL-17 on the level of autophagy and the related Janus activated kinase 2 (JAK2) and downstream signal transducer and activator of transcription 3 (STAT3) signaling pathway. Next, osteogenic activity in different concentrations of IL-17 was tested. Finally, the specific JAK2/STAT3 signaling pathway inhibitor AG490 and autophagy inhibitor 3-MA were used to investigate the involvement of this pathway and autophagy in IL-17-induced regulation of RANKL expression.Results: Initially, we found that IL-17 treatment promoted growth of osteoblasts in a time- and dose-dependent manner. Next, we showed that low levels of IL-17 promoted autophagy activity, whereas the opposite was observed at high levels of IL-17. Moreover, high levels of IL-17 activated the JAK2/STAT3 signaling pathway, although this effect was reversed by upregulation of autophagy. Furthermore, our findings indicated that high concentrations of IL-17 promoted the differentiation, calcification, and RANKL expression of murine osteoblasts via activation of the JAK2/STAT3 pathway. Importantly, downregulation of autophagy at high IL-17 concentrations further enhanced RANKL expression via suppressing the JAK2/STAT3 cascade.Conclusion: Overall, our findings demonstrate, for the first time, that IL-17 modulates RANKL expression of osteoblasts through an autophagy-JAK2-STAT3 signaling pathway, thus affecting bone metabolism.

In vitro osteogenic performance of two novel strontium and zinc-containing glass polyalkenoate cements.

Glass polyalkenoate cements (GPCs) are under investigation as potential bone adhesives, as they may provide an alternative to polymethylmethacrylate-based cements. GPCs containing strontium (Sr) and zinc (Zn) in place of aluminium (Al) are of particular interest because these ions are known stimulators of osteoprogenitor differentiation. GPCs have been manufactured from a novel bioactive glass (SiO2 :0.48, ZnO:0.36, CaO:0.12, SrO:0.04) in the past, but, while such materials have been assessed for their influence on viability, their influence on osteogenic function has not been investigated until now. For this study, two GPCs were formulated from the same glass precursor evaluated in previous studies. These GPCs were named GPC A and GPC B, and they differed in glass particle size, polyacrylic acid molecular weight, and their powder: liquid ratios. The effect of these two GPCs on osteogenic differentiation of primary rat osteoblasts were evaluated using three culture systems: culture with dissolution extracts, indirect contact with transwell-inserts and direct contact. Additionally, the degradation characteristics of GPCs were assessed, including their interfacial pH and surrounding pH. The experimental outcomes revealed that collagen deposition, alkaline phosphatase expression, and mineralization were largely dependent on GPC composition as well as the mode of interaction with cells. These markers were found to be significantly elevated in response to GPC A's dissolution products. However, osteogenic differentiation was inhibited when osteoblasts were cultured indirectly and directly with GPCs, with, overall, GPC B significantly outperforming GPC A. These results suggest that GPC degradation products effect osteogenic differentiation in a dose-dependent manner.

Fructus Ligustri Lucidi preserves bone quality through induction of canonical Wnt/ß-catenin signaling pathway in ovariectomized rats.

Fructus Ligustri Lucidi (FLL) has been preclinically and clinically used to treat musculoskeletal diseases. However, whether and how FLL affect the canonical Wnt/ß-catenin signaling in the management of osteoporosis remains largely unknown. To this end, ovariectomized (OVX) rats and primary osteoblasts were administrated with FLL aqueous extract and medicated serum, respectively. Supplement of FLL to OVX rats maintains bone quality by attenuating the reduction in bone mineral density, strength and microstructure. The maintenance may be associated with upregulating the expression of insulin-like growth factor-1, osteoprotegerin, phospho (p)-low-density lipoprotein receptor-related protein 6, p-glycogen synthase kinase 3 beta (GSK3ß), ß-catenin, Runx2 and c-Myc, and downregulating the expressions of sclerostin (SOST), dickkopf-related protein 1 (DKK1), GSK3ß and p-ß-catenin in rat femurs and tibias. In addition, the medicated serum promotes osteoblastic bone formation through activation of Wnt/ß-catenin signaling via inhibition of DKK1 and SOST overexpression. Salidroside may be one of the active ingredients in FLL that are beneficial for bone homeostasis. In summary, our results suggest that FLL may preserve bone quality through induction of canonical Wnt/ß-catenin signaling via inhibition of DKK1 and SOST overexpression. And FLL may offer a new source of the DKK1 or SOST inhibitors in protection against osteoporosis.

A Review of the Effect of a Nanostructured Thin Film Formed by Titanium Carbide and Titanium Oxides Clustered around Carbon in Graphitic Form on Osseointegration.

Improving the biocompatibility of implants is an extremely important step towards improving their quality. In this review, we recount the technological and biological process for coating implants with thin films enriched in titanium carbide (TiC), which provide improved cell growth and osseointegration. At first, we discuss the use of a Pulsed Laser Ablation Deposition, which produced films with a good biocompatibility, cellular stimulation and osseointegration. We then describe how Ion Plating Plasma Assisted technology could be used to produce a nanostructured layer composed by graphitic carbon, whose biocompatibility is enhanced by titanium oxides and titanium carbide. In both cases, the nanostructured coating was compact and strongly bound to the bulk titanium, thus particularly useful to protect implants from the harsh oxidizing environment of biological tissues. The morphology and chemistry of the nanostructured coating were particularly desirable for osteoblasts, resulting in improved proliferation and differentiation. The cellular adhesion to the TiC-coated substrates was much stronger than to uncoated surfaces, and the number of philopodia and lamellipodia developed by the cells grown on the TiC-coated samples was higher. Finally, tests performed on rabbits confirmed in vivo that the osseointegration process of the TiC-coated implants is more efficient than that of uncoated titanium implants.

TNF­α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR­335­5p.

Elucidation of the underlying mechanisms governing osteogenic differentiation is of significant importance to the improvement of therapeutics for bone­related inflammatory diseases. Tumor necrosis factor­α (TNF­α) is regarded as one of the major agents during osteogenic differentiation in an inflammatory environment. miR­335­5p post­transcriptionally downregulates the Dickkopf WNT signaling pathway inhibitor 1 (DKK1) protein level by specifically binding to the DKK1 3'UTR and activating Wnt signaling. The role of miR­335­5p in TNF­α­induced post­transcriptional regulation of DKK1 remains to be elucidated. In the present study, the mRNA and protein levels of DKK1 and the level of miR­335­5p were determined in MC3T3­E1 cells and the primary calvarial osteoblasts treated with or without TNF­α. The role of NF­κB signaling in TNF­α­induced post­transcriptional regulation of DKK1 was also evaluated. The present study determined that although TNF­α treatment exhibited cell­specific effects on DKK1 mRNA expression, the stimulation of TNF­α time­ and concentration­dependently upregulated the protein levels of DKK1. In primary calvarial osteoblasts, the decreased miR­335­5p level induced by TNF­α­activated NF­κB signaling served an important role in mediating the post­transcriptional regulation of DKK1 by TNF­α treatment. In MC3T3­E1 cells, the post­transcriptional regulation of DKK1 by TNF­α treatment was more complicated and involved other molecular signaling pathways in addition to the NF­κB signaling. In conclusion, TNF­α treatment served an important role in the post­transcriptional regulation of DKK1 expression, which requires further investigation. The results of the present study not only provided new insights into the regulatory effects of miR­335­5p on osteogenic differentiation in an inflammatory microenvironment, but may also promote the development of potential therapeutic strategies for the treatment of bone­related inflammatory diseases.

Primary osteoblasts, osteoblast precursor cells or osteoblast-like cell lines: Which human cell types are (most) suitable for characterizing 45S5-bioactive glass?

The question how bioactive glasses (BGs) influence the viability and osteogenic differentiation of human osteogenic cells has already been addressed by several studies. However, a literature review revealed great differences in the type of cells used for these experiments. Primary human osteoblasts (hOBs) represent the desired standard, but possess the limitation of patient variability and time-consuming isolation protocols. Therefore, several alternative cell types have been used including primary mesenchymal stromal cells (BMSCs) and the "osteoblast-like" cell lines MG-63, Saos-2, HOS, and U2OS. The aim of our study was the identification of the cell type most suitable for tissue engineering projects involving BGs by comparative analysis of cell viability and osteogenic differentiation in response to crystallized 45S5-BG. We observed that hOBs, BMSCs, and MG-63 cells were resistant to 45S5-BG induced cytotoxicity, while the viability of Saos-2, HOS, and U2OS cells was significantly reduced. In addition, we detected alkaline phosphatase activity, except in U2OS cells, that increased upon 45S5-BG cocultivation, demonstrating the induction of osteogenic differentiation. Our data and the fact that the donor-dependent variations can be avoided when using MG-63 cells suggest that these are a promising alternative to primary cells and remain an important cell line for future BG related studies.

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway.

Microtubule actin cross-linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3-E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast-specific Osterix (Osx) promoter-driven Macf1 conditional knockout mice (Macf1f/f Osx-Cre). The Macf1f/f Osx-Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1f/f Osx-Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1f/f Osx-Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1f/f Osx-Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.

Flaxseed oil ameliorated high-fat-diet-induced bone loss in rats by promoting osteoblastic function in rat primary osteoblasts.

BACKGROUND: α-Linolenic acid (ALA) is a plant-derived omega-3 unsaturated fatty acid that is rich in flaxseed oil (FO). The effect of FO on bone health is controversial. This study aims to evaluate the effect of FO on bone damage induced by a high-fat diet (HFD) and to explore the possible mechanism. METHODS: Male Sprague-Dawley rats were fed a normal control diet (NC, 10% fat), FO diet (NY, 10% fat), HFD (60% fat), or HFD containing 10% FO (HY, 60% fat) for 22 weeks. Micro CT and three-point bending tests were conducted to evaluate bone microstructure and biomechanics. Serum was collected for the detection of ALP, P1NP, and CTX-1. Rat primary osteoblasts (OBs) were treated with different concentrations of ALA with or without palmitic acid (PA) treatment. The ALP activity, osteogenic-related gene and protein expression were measured. RESULTS: Rats in the HFD group displayed decreased biomechanical properties, such as maximum load, maximum fracture load, ultimate tensile strength, stiffness, energy absorption, and elastic modulus, compared with the NC group ( p  < 0.05). However, HY attenuated the HFD-induced decreases in bone biomechanical properties, including maximum load, maximum fracture load, and ultimate tensile strength (p < 0.05). Trabecular bone markers such as trabecular volume bone mineral density (Tb. vBMD), trabecular bone volume/total volume (Tb. BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) were decreased, trabecular separation (Tb. Sp) and the structure model index (SMI) were increased in the HFD group compared with the NC group, and all parameters were remarkably improved in the HY group compared to the HFD group (p < 0.05). However, cortical bone markers such as cortical volume bone mineral density (Ct. vBMD), cortical bone volume/total volume (Ct. BV/TV) and cortical bone thickness (Ct. Th) were not significantly different among all groups. Moreover, the serum bone formation markers ALP and P1NP were higher and the bone resorption marker CTX-1 was lower in the HY group compared with levels in the HFD group. Compared with the NC group, the NY group had no difference in the above indicators. In rat primary OBs, PA treatment significantly decreased ALP activity and osteogenic gene and protein (ß-catenin, RUNX2, and osterix) expression, and ALA dose-dependently restored the inhibition induced by PA. CONCLUSIONS: FO might be a potential therapeutic agent for HFD-induced bone loss, most likely by promoting osteogenesis.

MiR-152 influences osteoporosis through regulation of osteoblast differentiation by targeting RICTOR.

Context: Evidence suggests that microRNA (miRNA) regulate gene expression and bone tissue homoeostasis of osteoporosis. MiR-152 has found to be abnormally expressed in osteoporosis, but its role in osteoblast differentiation has not been elucidated. Objective: To understand the potential mechanism of miR-152 in osteoblast differentiation via regulation of RICTOR. Materials and methods: The expression of miR-152 and RICTOR were tested in ovariectomized rat models of osteoporosis. Primary osteoblasts and MC3T -E1 cells were assigned into four groups, namely Control, miR-152 inhibitor, miR-control and miR-152 inhibitor + siRICTOR groups. qRT PCR and Western blot were performed to detect the expressions of miR-152 and RICTOR, respectively. MTT assay was used to evaluate cell viability, and ALP activity determination and mineralization analyses were also conducted. Results: In ovariectomy-induced osteoporotic rats, miR-152 (3.06 ± 0.35) in femoral tissues increased significantly, while RICTOR (0.31 ± 0.04) decreased. Compared with Control group, miR-152 inhibitor group presented appreciable reduction of miR-152 in primary osteoblasts and MC3T3-E1 cells, as well as remarkable increases in RICTOR, p-Akt(s473)/Akt ratio, and osteogenesis-related genes, with enhanced cell viability, ALP activity and mineralization. In comparison with cells in the miR-152 inhibitor group, those in the miR-152 inhibitor + siRICTOR group had no observable difference in miR-152, but were dramatically up-regulated in RICTOR, as well as the corresponding opposite tendencies of other factors. Conclusion: Inhibiting miR-152 promoted osteoblasts differentiation and alleviated osteoporosis by up-regulating RICTOR. Therefore, miR-152 may be an essential mediator of osteoblast differentiation and a new therapeutic strategy for osteoporosis.

Aberrant miR-145-5p/ß-catenin signal impairs osteocyte function in adolescent idiopathic scoliosis.

Recently, noncoding RNAs have been thought to play important roles in the sporadic occurrence of spinal deformity of adolescent idiopathic scoliosis (AIS). As a prognostic factor for curve progression, low bone mass has been hypothesized to crosstalk with AIS pathogenesis. Abnormal osteoblasts activities are reported in AIS without a clear mechanism. In this study, bone biopsies from patients with AIS and control subjects and the primary osteoblasts derived from those samples were used to identify the potential microRNA (miRNA) candidates that interfere with osteoblasts and osteocytes function. Microarray analysis identified miRNA-145-5p (miR-145) as a potential upstream regulator. miR-145 and ß-catenin mRNA ( CTNNB1) were overexpressed in AIS bone tissues and primary osteoblasts, and their expression correlated positively in AIS. Knockdown of miR-145 restored impaired osteocyte activity through the down-regulation of active ß-catenin expression and its transcriptional activity. Significant negative correlations between circulating miR-145 and serum sclerostin, osteopontin, and osteoprotegerin were noted in patients with AIS, which was in line with our cellular findings. This is the first study to demonstrate the effect of aberrant miRNA expression and its effect on osteocyte function in AIS, which may contribute to the low bone mass. Our findings also provide insight into the development of circulating microRNAs as a bone quality biomarker or even a prognostic biomarker for AIS.-Zhang, J., Chen, H., Leung, R. K. K., Choy, K. W., Lam, T. P., Ng, B. K. W., Qiu,Y., Feng, J. Q., Cheng, J. C. Y., Lee, W. Y. W. Aberrant miR-145-5p/ß-catenin signal impairs osteocyte function in adolescent idiopathic scoliosis.

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.

The positive effects of secreting cytokines IL-17 and IFN-γ on the early-stage differentiation and negative effects on the calcification of primary osteoblasts in vitro.

OBJECTIVE: Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) are all pro-inflammatory cytokines produced by specific subsets of T-cells and are also considered crucial regulators in bone remodeling, but their effects on osteogenesis have not been carefully studied. So, this study aimed to investigate the effects of secreting cytokines IL-17 and IFN-γ on the osteogenesis of primary osteoblasts and to clarify the potential roles of the related Janus activated kinase 2 (JAK2) and downstream signal transducer and activator of transcription 3 (STAT3) signaling pathway in bone remodeling. METHODS: The proliferation of osteoblasts was evaluated by MTT assay. Osteogenic activity was tested by alkaline phosphatase (ALP) activity assay and alizarin red staining. The mRNA levels of ALP, osteocalcin, osteoprotegerin (OPG), Runt-related transcription factor 2 (Runx2) and receptor activator of nuclear factor-kappa B ligand (RANKL) were also measured by real-time quantitative PCR. The JAK2-STAT3 pathway was evaluated by Western blot. RESULTS: Osteoblasts showed no obvious proliferation when treated with IL-17 and/or IFN-γ, but higher ALP activities were observed in primary osteoblasts treated with IL-17 or IL-17 + IFN-γ in induction medium. We also found that IL-17 could promote the gene expression of Alp, Runx2, Osteocalcin, Opg, and Rankl, while IFN-γ might attenuate this effect. Nevertheless, IL-17 and IFN-γ exhibited an inhibitory effect on the calcification of primary osteoblasts. We also found that IL-17 could directly facilitate RANKL expressions by JAK2-STAT3 pathway. CONCLUSION: The positive effects of IL-17 and IFN-γ on the early-stage differentiation and the negative effects on the calcification of murine calvarial osteoblasts contribute to our understanding of the role and interaction of inflammatory factors in the bone remodeling and as fundamental mechanisms involved in the destruction of alveolar bone.

Multiple calcium patterns of rat osteoblasts under fluidic shear stress.

The intracellular calcium ([Ca2+ ]i ) response induced by external forces can be diverse and complex. Using primary osteoblasts from Wistar rats, we found multiple patterns of [Ca2+ ]i responses induced by fluidic shear stress (Fss), including homogeneous non-oscillation and heterogeneous oscillations. These multiple-patterned [Ca2+ ]i responses could be influenced by Fss intensity, cell density, and cell differentiation. Our real-time measurements with free calcium, ATP, ATP without calcium, suramin, apyrase, and thapsigargin confirmed homogeneous [Ca2+ ]i patterns and/or heterogeneous [Ca2+ ]i oscillations with respect to the combined degree of external Ca2+ influx, and intracellular Ca2+ release. Our theoretical model supported diverse Fss-induced calcium activities as well. We concluded that a singular factor of Ca2+ influx or release dominated to produce smooth homogeneous patterns, but combined factors produced oscillatory heterogeneous patterns. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2039-2051, 2018.

Culture of Human Primary Bone Cells and Phenotype Assessment.

Bone engineering scaffolds and biomimetic substitutes are currently the leading techniques to repair and regenerate bone defects. Ideally, bone grafts should imitate the structure and properties of bone extracellular matrix, house osteoprogenitor cells, and provide all the necessary environmental cues to orchestrate the functions of osteoblast and osteoclast cells. Consequently, there is an increasing demand for preclinical models based on in vitro bone-derived cell cultures for screening of novel biomaterials. In this chapter, we provide the protocols for culture of primary human bone cells from explants of cortical mandible bone and for characterization of cell behavior on biomimetic surfaces in terms of strength of adhesion, proliferation, differentiation, and matrix mineralization.

[Effect of Aralia echinocaulis containing serum on Wnt/ß-catenin signaling pathway of primary osteoblast].

This paper was aimed to investigate the effect of Aralia echinocaulis containing serum on expression of ß-catenin, Wnt-1, Frizzed-2, TCF and Axin in Wnt/ß-catenin signaling pathway of primary osteoblasts. SD healthy female rats (n=80) were used to make A. echinocaulis containing serum by gastric perfusion for seven days with distilled water, A. echinocaulis decoction high dosage, middle dosage, and low dosage. In vitro, primary osteoblasts were cultured and identified. The third generation primary osteoblasts were taken and cultured for 48 h, then cells were treated with the different drug serums for 10 days and calcified nodules were counted by alizarin red staining. The cells were collected after treatment for 48 h and the expression levels of ß-catenin, Wnt-1, Frizzled-2, TCF and Axin were detected by Real-time PCR and Western blot. The results suggested that the in vitro cells were primary osteoblasts; and after treatment, various doses groups could promote the mineralization ability of primary osteoblasts, up-regulate the mRNA and protein expression levels of ß-catenin, Wnt-1, Frizzled-2, and TCF, and down-regulate the mRNA and protein expression levels of Axin. These findings indicated that A. echinocaulis containing serum can enhance the differentiation and proliferation of osteoblasts by regulating the expression levels of ß-catenin, Wnt-1, Frizzled-2, TCF and Axin in Wnt/ß-catenin signaling pathway of primary osteoblasts.

Ex vivo construction of human primary 3D-networked osteocytes.

A human bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via the biomimetic assembly of primary human osteoblastic cells with 20-25µm microbeads and subsequent microfluidic perfusion culture. The biomimetic assembly: (1) enabled 3D-constructed cells to form cellular network via processes with an average cell-to-cell distance of 20-25µm, and (2) inhibited cell proliferation within the interstitial confine between the microbeads while the confined cells produced extracellular matrix (ECM) to form a mechanically integrated structure. The mature osteocytic expressions of SOST and FGF23 genes became significantly higher, especially for SOST by 250 folds during 3D culture. The results validate that the bone tissue model: (1) consists of 3D cellular network of primary human osteocytes, (2) mitigates the osteoblastic differentiation and proliferation of primary osteoblast-like cells encountered in 2D culture, and (3) therefore reproduces ex vivo the phenotype of human 3D-networked osteocytes. The 3D tissue construction approach is expected to provide a clinically relevant and high-throughput means for evaluating drugs and treatments that target bone diseases with in vitro convenience.

An O-acetyl-glucomannan from the rhizomes of Curculigo orchioides: Structural characterization and anti-osteoporosis activity in vitro.

Osteoporosis is characterized by a reduction in bone mass and bone mineral density, which weakens the bone. Due to the side effects associated with drugs that are currently used to treat this disease, an increasing number of studies have focused on the research of effective ingredients derived from natural products. In particular, polysaccharides extracted from Chinese herbal medicines have received increasing attention. In this study, we isolated a homogeneous polysaccharide (COP90-1) from the dried rhizomes of Curculigo orchioides, a famous traditional Chinese medicine that is widely used in China, and determined its structure using the combined methods of chemical and spectral analyses. In addition, its effects on the proliferation and differentiation of primary mouse osteoblasts were assessed. The results showed that COP90-1 can effectively promote the proliferation and differentiation of primary osteoblasts in vitro. Additional studies are warranted to study the effects of this compound in vivo.