Bone Marrow Stromal ST2 Cells Research Articles

Lipids of Platelet-Rich Fibrin Reduce the Inflammatory Response in Mesenchymal Cells and Macrophages.

Platelet-rich fibrin (PRF) has a potent anti-inflammatory activity but the components mediating this effect remain unknown. Blood lipids have anti-inflammatory properties. The question arises whether this is also true for the lipid fraction of PRF. To answer this question, lipid fractions of solid and liquid PRF were tested for their potential to lower the inflammatory response of ST2 bone marrow stromal cells and primary bone marrow macrophages exposed to IL1β and TNFα, and LPS, respectively. Cytokine production and the underlying signalling pathway were analysed by RT-PCR, immunoassays, and Western blotting. We report here that lipids from solid and liquid PRF substantially lowered cytokine-induced expression of IL6, CCL2 and CCL5 in ST2 cells. Moreover, the inflammatory response induced by Pam3CSK4, the agonist of Toll-like receptor (TLR) TLR2, was partially reduced by the lipid extracts in ST2 cells. The PRF lipids further reduced the LPS-induced expression of IL1β, IL6 and CCL5 in macrophages at the transcriptional level. This was confirmed by showing the ability of PRF lipids to diminish IL6 at the protein level in ST2 cells and macrophages. Likewise, PRF lipid extracts reduced the phosphorylation of p38 and JNK and moderately decreased the phosphorylation of NFκB-p65 in ST2 cells. These findings suggest that the lipid fraction is at least partially responsible for the anti-inflammatory activity of PRF in vitro.

TGF-β Signalling Mediates the Anti-Inflammatory Activity of Enamel Matrix Derivative In Vitro.

Enamel matrix derivative (EMD) prepared from extracted porcine fetal tooth material can support the regrow of periodontal tissues. Previous findings suggest that EMD has anti-inflammatory properties and TGF-β activity in vitro. However, the anti-inflammatory activity of EMD is mediated via TGF-β has not been considered. To this aim, we first established a bioassay to confirm the anti-inflammatory activity of EMD. The bioassay was based on the RAW 264.7 macrophage cell line and proven with primary macrophages where EMD significantly reduced the forced expression of IL-6. We then confirmed the presence of TGF-β1 in EMD by immunoassay and by provoking the Smad2/3 nuclear translocation in RAW 264.7 macrophages. Next, we took advantage of the TGF-β receptor type I kinase-inhibitor SB431542 to block the respective signalling pathway. SB431542 reversed the anti-inflammatory activity of EMD and TGF-β in a bioassay when IL-6 and CXCL2 expression was driven by the LPS stimulation of RAW 264.7 macrophages. This central observation was supported by showing that SB431542 reversed the anti-inflammatory activity of EMD using IL-1β and TNF-α-stimulated ST2 bone marrow stromal cells. Together, these findings implicate that the TGF-β activity mediates at least part of the anti-inflammatory activity of EMD in vitro.

Genetic response to low‑intensity ultrasound onmouse ST2 bone marrow stromal cells.

Although low-intensity ultrasound (LIUS) is a clinically established procedure, the early cellular effect of LIUS on a genetic level has not yet been studied. The current study investigated the early response genes elicited by LIUS in bone marrow stromal cells (BMSCs) using global-scale microarrays and computational gene expression analysis tools. Mouse ST2 BMSCs were treated with LIUS [ISATA, 25 mW/cm2 for 20 min with a frequency of 1.11 MHz in a pulsed-wave mode (0.2-s burst sine waves repeated at 1 kHz)], then cultured for 0.5, 1 and 3 h at 37°C. The time course of changes in gene expression was evaluated using GeneChip® high-density oligonucleotide microarrays and Ingenuity® Pathway Analysis tools. The results were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A single exposure of LIUS did not affect cell morphology, cell growth or alkaline phosphatase activity. However, 61 upregulated and 103 downregulated genes were identified from 0.5 to 3 h after LIUS treatment. Two significant gene networks, labeled E and H, were identified from the upregulated genes, while a third network, labeled T, was identified from the downregulated genes. Gene network E or H containing the immediate-early genes FBJ osteosarcoma oncogene and early growth response 1 or the heat shock proteins heat shock protein 1a/b was associated mainly with the biological functions of bone physiology and protein folding or apoptosis, respectively. Gene network T containing transcription factors fos-like antigen 1 and serum response factor was also associated with the biological functions of the gene expression. RT-qPCR indicated that the expression of several genes in the gene networks E and H were elevated in LIUS-treated cells. LIUS was demonstrated to induce gene expression after short application in mouse ST2 BMSCs. The results of the present study provide a basis for the elucidation of the detailed molecular mechanisms underlying the cellular effects of LIUS.

Low-intensity pulsed ultrasound promotes the expression of immediate-early genes in mouse ST2 bone marrow stromal cells.

The effects of low-intensity pulsed ultrasound (LIPUS) on the expression of immediate-early genes (IEGs) in bone marrow stromal cells (BMSCs) were evaluated to elucidate the early cellular response to LIPUS. Mouse ST2 BMSCs were treated with LIPUS (ISATA, 12-34 mW/cm2 for 20min), then cultured at 37°C. The expression levels of four IEGs (Fos, Egr1, Jun, and Ptgs2) and ERK1/2, a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), were assessed using real-time quantitative PCR and Western blot analyses, respectively. A single exposure of LIPUS at an intensity of 25 mW/cm2 significantly and transiently increased the expression levels of all four IEGs, and the peak expression was detected at 30-60min after LIPUS stimulation. LIPUS exposure also significantly increased the phosphorylation level of ERK1/2. U0126, an inhibitor of MAPK/ERK, significantly prevented LIPUS-induced expression of Fos and Egr1, but not that of Jun and Ptgs2. On the other hand, treatment of the cells with LIPUS did not affect cell growth or alkaline phosphatase activity, a marker of osteoblast differentiation. These results suggest that LIPUS exposure significantly induces expression of IEGs such as Fos and Egr1 via the MAPK/ERK pathway in ST2 BMSCs.

Effect of Functionally-Graded Calcium Titanate Film, Prepared by Metal-Organic Chemical Vapor Deposition, on Titanium Implant

Calcium Titanate (CaTiO3) has been introduced as an attractive biomaterial for the enhancement of calcium phosphate deposition in vivo and in vitro. We hypothesized that CaTiO3 directly coated on titanium (Ti) by metal-organic chemical vapor deposition (MOCVD) could be a suitable candidate for biocompatible coatings for medical devices, particularly dental implants. To prove this hypothesis, surface characterization, cell culture, and animal study were completed in this study. The result of this study showed that CTO 800, a CaTiO3 film prepared by heating at 800 °C, had a high hydrophilic surface. Mouse bone marrow stromal ST-2 cells cultured on substrates and CTO 800 exhibited cell differentiation, represented by alkaline phosphatase activity, compared with cells cultured on non-coated Ti and CTO 700 (a CaTiO3 film prepared by heating at 700 °C). The push-in test value of CTO 800, a parameter that indicates the degree of osseointegration, was significantly higher than that of Ti. Calcium titanate coated on Ti by MOCVD has the potential to accelerate the process of osseointegration; thus, our results support the use of CaTiO3 coating for biocompatible biomaterial for medical applications, particularly dental implants.

MicroRNA-200a regulates adipocyte differentiation in the domestic yak Bos grunniens

The domestic yak (Bos grunniens) is a culturally important animal that lives at high altitude and is farmed by Tibetan herders for its meat, milk, and other animal by-products. Within the animal, adipose tissue is an important store and source of energy and is used to maintain adequate body temperature during the extended cold seasons. Exploring the biomolecular role of microRNAs (miRNAs) in the regulation of growth, development, and metabolism of yak adipocytes may provide valuable insights into the physiology of adipogenesis in the yak. This study investigated whether and how miR-200a (a miRNA recently reported to promote adipogenesis in ST2 bone marrow stromal cells) regulates adipocyte differentiation in the yak. Expression levels of miR-200a gradually increased during day 0 to day 8 of adipocyte differentiation, and transfection of adipocytes with miR-200a enhanced lipid accumulation and triglyceride content compared to control (un-transfected) adipocytes. We additionally verified (using qRT-PCR analysis) that miR-200a increased the expression of adipocyte-specific genes involved in lipogenic transcription (PPARγ, ELVOL, and C/EBPα), fatty acid synthesis (ACC, ACS, SCD, and FAS), and fatty acid transport (DGAT, LPL, and FABP4). We also found that transfection of adipocytes with miR-200a resulted in suppression of the levels of noncanonical Wnt signaling transcription factors (Wnt5a, TAK1, and NLK). These results indicate that miRNA-200a plays an important role in promoting yak adipocyte differentiation that may operate via the suppression of noncanonical Wnt signaling.

Pharmacological inhibition of S6K1 impairs self-renewal and osteogenic differentiation of bone marrow stromal cells.

mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt-induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self-renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony-Forming Unit-Fibroblast (CFU-F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a-induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self-renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a-induced osteoblast differentiation and protein anabolism.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.

Structural characteristics and surface properties of fluoride-containing calcium phosphates derived from octacalcium phosphate

Event Abstract Back to Event Structural characteristics and surface properties of fluoride-containing calcium phosphates derived from octacalcium phosphate Yukari Shiwaku1, 2, Takahisa Anada1 and Osamu Suzuki1 1 Tohoku University Graduate School of Dentistry, Division of Craniofacial Function Engineering, Japan 2 Tohoku University Graduate School of Dentistry, Liaison Center for Innovative Dentistry, Japan Introduction: Fluoride ion (F-) is well known to be incorporated in hydroxyapatite (HA) and form fluoridated hydroxyapatite (FHA). Fluoride ion stimulates osteoblastic cells[1],[2] and modifies protein adsorption on the calcium phosphate. We previously reported that synthetic octacalcium phosphate (OCP), a precursor of HA, promotes osteoblastic cell differentiation and facilitates bone regeneration[3],[4]. Because of the structure similarity to HA, OCP prepared with F- possibly improves osteogenic capability of original OCP. The aim of this study is to investigate the structural characteristics, protein adsorption, and cellular response of fluoride-containing calcium phosphate (F-CaP) derived from OCP. F-CaPs were prepared two different methods, such as hydrolysis of OCP in F- solution and co-precipitation of OCP with F- . Materials and Methods: F-CaP prepared by hydrolysis (HF-CaP) was synthesized by incubating OCP in 150 mM Tris buffer (pH = 7.1~9.9) containing 50 ppm F- at 37oC. F-CaP prepared by co-precipitation (CF-CaP) was synthesized by adding calcium solution into phosphate solution including 12 and 96 ppm F- at 70oC[5]. The chemical composition and crystal structure of the F-CaPs were examined using inductively coupled plasma (ICP), fluoride electrode and x-ray diffraction (XRD), and field emission scanning electron microscope (FE-SEM). The solubility of the products was evaluated by Ca2+ concentration released after immersing them at pH = 7.4 and 4.0. Protein adsorption on F-CaPs was analyzed using bovine serum albumin (BSA) and cytochrome c. For cell attachment and proliferation test, mouse bone marrow stromal ST-2 cells were seeded onto coatings of F-CaPs and OCP. The number of cells on the coatings or polystyrene culture plate (no coating) was determined using the WST-8 assay. Results: Chemical composition and XRD analysis showed that HF-CaP tended to convert to FHA depending on the F- content of the solids. On the other hand, CF-CaP possessed not only the reflection of OCP, but also that of fluoridated hydroxyapatite (FHA). The solubility of HF-CaP was decreased depending on the fluoride content, whereas that of CF-CaP was comparable to OCP regardless of fluoride contens. HF-CaP adsorbed more cytochrome c than CF-CaP and OCP, whereas BSA adsorption was inhibited on HF-CaP. The difference of protein adsorption behaviors between two types of F-CaPs affected cell attachment and proliferation of ST-2 cells. CF-CaP with high F- content promoted cell attachment and proliferation more than OCP. In contrast, the cell numbers on the HF-CaP with middle F- content were lowest among all the groups. Discussion and Conclusion: The present study confirmed that one of the CF-CaP with high F- content enhances the attachment and proliferation of mouse bone marrow stromal cells. The synthesis processes of incorporating F- to OCP had crucial effects on the crystal structure and morphology as well as the protein adsorption on F-CaP. CF-CaP preserved a small amount of OCP in FHA and maintained and Ca2+ concentration in the medium at a level similar to the original OCP. It seems likely that CF-CaP with high F- content may work as a better substrate for coating an implant surface or bone substitute material.

Enhancement of osteoblastic differentiation in alginate gel beads with bioactive octacalcium phosphate particles

The present study investigated whether alginate (Alg) hydrogel microbeads have a role in maintaining mouse bone marrow stromal ST-2 cells and release the cells after being stimulated by synthetic octacalcium phosphate (OCP), which is a mineral crystal capable of stimulating osteoblastic differentiation during a conversion process to hydroxyapatite (HA). The ST-2 cell suspension in the alginate solution, which contained various concentrations of OCP granules with diameters less than 53 μm, was extruded drop-wise into a stirred gelation solution containing BaCl2 using an encapsulator with nitrogen gas stream. The Alg-microbeads (Alg/OCP · ST-2 microbeads) that were generated, which had a diameter of approximately 400 μm, were incubated for up to 14 d and then assessed for osteoblastic differentiation. Alg-microbeads with cells were also incubated to identify the possible conversion from OCP to HA. Osteoblast differentiation markers in ST-2 cells, alkaline phosphatase (ALP) and collagen type I, were up-regulated in the presence of higher amounts of OCP. X-ray diffraction analysis and Fourier transform infrared spectroscopy confirmed that the OCP tended to convert to HA over time, suggesting that the OCP in Alg-microbeads interacts three-dimensionally with ST-2 cells and stimulates its osteoblastic differentiation. The release of ST-2 cells from the microbeads was also estimated. ST-2 cells were identified outside of the microbeads, although the cell number tended to decrease with increasing OCP. These results suggest that Alg/OCP microbeads could be used as a vehicle to activate osteoblastic cells and deliver them to sites where bone regeneration is needed.

Characterization of partially hydrolyzed OCP crystals deposited in a gelatin matrix as a scaffold for bone tissue engineering

The present study was designed to investigate how hydrolysis of octacalcium phosphate (OCP) into hydroxyapatite is affected by the presence of gelatin (Gel) molecules and how osteoblastic cells respond to the resultant OCP hydrolyzate/Gel composites as the hydrolysis advances. OCP was prepared from a solution containing calcium and phosphate ions and Gel molecules, having a composition to produce a 40 wt% OCP as a final co-precipitate as the OCP/Gel. The precipitate was further incubated up to 40 h to advance the hydrolysis of OCP. These precipitates were processed to mold OCP/Gel sponges through lyophilization and dehydrothermal treatment. Chemical analysis, X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and selected area electron diffraction revealed that the hydrolysis of OCP/Gel composite in hot water advanced in a time-dependent manner and faster than hydrolysis of OCP alone. The effect of Gel on the OCP hydrolysis was further examined in the presence of distinct concentrations of Gel molecules in hot water, showing that the Gel enhanced the hydrolysis as the concentration increased. Proliferation and differentiation of mouse bone marrow stromal ST-2 cells on the hydrolyzed OCP/Gel composites were compatible with Gel sponge alone after 21 days of culture, suggesting that these composites could be a candidate as a scaffold in bone tissue engineering.

Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways.

Amphirionin-4 with potent proliferation-promoting activity on bone marrow stromal cells from a marine dinoflagellate amphidinium species.

A linear polyketide, amphirionin-4 (1), has been isolated from cultivated algal cells of the marine dinoflagellate Amphidinium species. The structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data, and the absolute configurations of C-4 and C-8 were determined using the modified Mosher's method. Amphirionin-4 (1) exhibited extremely potent proliferation-promoting activity on murine bone marrow stromal ST-2 cells (950% promotion) at a concentration of 0.1 ng/mL.

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase

To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells. Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP). HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells. These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.

Involvement of geranylgeranylation of Rho and Rac GTPases in adipogenic and RANKL expression, which was inhibited by simvastatin

Simvastatin suppresses myoblast differentiation via inhibition of Rac GTPase, which is involved in the mevalonic acid pathway that produces cholesterol. Statins also inhibit adipogenic differentiation and receptor activator of NFκB ligand (RANKL) expression, possibly through the mevalonic acid pathway, although the involvement of that pathway and effector proteins in these cellular events has not been fully clarified. In the present study, we aimed to elucidate the mechanism of the effects of simvastatin on adipogenic differentiation and calcitriol-induced RANKL expression in bone marrow stromal ST2 cells. Adipogenesis and mRNA up-regulation of peroxisome proliferator-activated receptor γ and adipocyte fatty acid-binding protein were induced by troglitazone, and those events were efficiently inhibited by simvastatin. In addition, RANKL expression induced by calcitriol was abrogated by simvastatin in ST2 cells. The inhibitory effects of simvastatin were adequately compensated by the addition of either mevalonic acid or an intermediate of the mevalonic acid pathway, geranylgeranyl pyrophosphate, but not by another intermediate, farnesyl pyrophosphate. These findings suggest that protein geranylgeranylation is related to cellular differentiation in those two directions. Furthermore, inhibitor analysis demonstrated that Rac GTPase is involved in adipogenic differentiation, whereas Rho GTPase was found to be involved in RANKL expression. Taken together, the present findings suggest that geranylgeranylation of Rho family GTPase is involved in both adipogenesis and RANKL expression of stromal cells, while Rac GTPase is involved in adipogenesis and Rho GTPase in RANKL expression.

Structural, morphological and surface characteristics of two types of octacalcium phosphate-derived fluoride-containing apatitic calcium phosphates

Octacalcium phosphate (OCP) has been reported to stimulate bone regeneration during hydrolysis into hydroxyapatite (HA). The present study was designed to characterize structural, morphological and surface properties of fluoride-containing apatitic calcium phosphates (CaP) obtained through OCP hydrolysis or direct precipitation of OCP in the presence of 12–230ppm of fluoride (F). The products were characterized by chemical analysis, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Fourier transform infrared spectroscopy (FTIR) as well as measurements of surface area, solubility, osteoblastic activities and bovine serum albumin (BSA) adsorption. XRD analysis re-confirmed that both preparations yielded more apatitic CaP with a higher concentration of F. However, the co-precipitated products (CF-CaP) maintained the properties of OCP, in particular the solubility, whereas the hydrolysis products (HF-CaP) had the characteristics of fluoridated apatite. The crystals of plate-like OCP were changed to the crystals of rod-like CF-CaP and small irregular HF-CaP with the advance of the hydrolysis. The SAED analysis detected both OCP and apatite crystals even in the most hydrolyzed CF-CaP. Mouse bone marrow stromal ST-2 cells grew better on CF-CaP compared with HF-CaP. BSA adsorption was inhibited on HF-CaP more than on CF-CaP. These results show that OCP produces physicochemically distinct apatitic fluoridated CaP during hydrolysis, regarding the structure, the crystal morphology and the protein adsorption, depending on the fluoride introduction route, which provides biologically interesting material.

Comparative study on in vitro biocompatibility of synthetic octacalcium phosphate and calcium phosphate ceramics used clinically

The present study was designed to investigate the extent to which calcium phosphate bone substitute materials, including osteoconductive octacalcium phosphate (OCP), display cytotoxic and inflammatory responses based on their dissolution in vitro. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics, which are clinically used, as well as dicalcium phosphate dihydrate (DCPD) and synthesized OCP were compared. The materials were well characterized by chemical analysis, x-ray diffraction and Fourier transform infrared spectroscopy. Calcium and phosphate ion concentrations and the pH of culture media after immersion of the materials were determined. The colony forming rate of Chinese hamster lung fibroblasts was estimated with extraction of the materials. Proliferation of bone marrow stromal ST-2 cells and inflammatory cytokine TNF-α production by THP-1 cells grown on the material-coated plates were examined. The materials had characteristics that corresponded to those reported. DCPD was shown to dissolve the most in the culture media, with a marked increase in phosphate ion concentration and a reduction in pH. ST-2 cells proliferated well on the materials, with the exception of DCPD, which markedly inhibited cellular growth. The colony forming capacity was the lowest on DCPD, while that of the other calcium phosphates was not altered. In contrast, TNF-α was not detected even in cells grown on DCPD, suggesting that calcium phosphate materials are essentially non-inflammatory, while the solubility of the materials can affect osteoblastic and fibroblastic cellular attachment. These results indicate that OCP is biocompatible, which is similar to the materials used clinically, such as HA. Therefore, OCP could be clinically used as a biocompatible bone substitute material.

Nemo‐like kinase inhibits osteoblastogenesis by suppressing bone morphogenetic protein and WNT canonical signaling

The bone morphogenetic protein/Signaling mothers against decapentaplegic (BMP/Smad) and the WNT signaling pathways regulate the commitment of mesenchymal cells to the osteoblastic lineage. Nemo-like kinase (Nlk) is an evolutionary conserved kinase that suppresses Smad transactivation and WNT canonical signaling. However, it is not clear whether these effects of Nlk have any consequence on the differentiation of mammalian cells. To study the function of Nlk during the commitment of ST-2 bone marrow stromal cells to the osteoblastic fate, Nlk was downregulated by RNA interference (RNAi), following transfection of a specific small interfering (si)RNA. Nlk downregulation increased alkaline phosphatase and osteocalcin expression and sensitized ST-2 cells to the effects of BMP2 and WNT3 on alkaline phosphatase mRNA expression and activity. Accordingly, Nlk downregulation enhanced the effect of BMP2 on the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3, and on the levels of phosphorylated Smad1/5/8, whereas it did not affect the transactivation of the transforming growth factor-β/Smad reporter pSBE-Luc. Nlk downregulation sensitized ST-2 cells to the effects of WNT3 on the transactivation of the WNT/T-cell factor (Tcf) reporter construct 16xTCF-Luc, whereas it did not affect cytosolic β-catenin levels. To understand the function of Nlk in cells committed to the osteoblastic lineage, Nlk was suppressed by RNAi in primary calvarial osteoblasts. Downregulation of Nlk increased alkaline phosphatase and osteocalcin transcripts and sensitized osteoblasts to the effects of BMP2 on alkaline phosphatase activity and Smad1/5/8 transactivation and phosphorylation. In conclusion, Nlk suppresses osteoblastogenesis by opposing BMP/Smad and WNT canonical signaling.

The effect of an octacalcium phosphate co-precipitated gelatin composite on the repair of critical-sized rat calvarial defects

This study was designed to investigate the extent to which an octacalcium phosphate/gelatin (OCP/Gel) composite can repair rat calvarial critical-sized defects (CSD). OCP crystals were grown with various concentrations of gelatin molecules and the OCP/Gel composites were characterized by chemical analysis, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), selected area electron diffraction (SAED) and mercury intrusion porosimetry. The OCP/Gel composite disks received vacuum dehydrothermal treatment, were implanted in Wistar rat calvarial CSD for 4, 8 and 16weeks, and then subjected to radiologic, histologic, histomorphometric and histochemical assessment. The attachment of mouse bone marrow stromal ST-2 cells on the disks of the OCP/Gel composites was also examined after 1day of incubation. OCP/Gel composites containing 24wt.%, 31wt.% and 40wt.% of OCP and with approximate pore sizes of 10–500μm were obtained. Plate-like crystals were observed closely associated with the Gel matrices. TEM, XRD, FTIR and SAED confirmed that the plate-like crystals were identical to those of the OCP phase, but contained a small amount of sphere-like amorphous material adjacent to the OCP crystals. The OCP (40wt.%)/Gel composite repaired 71% of the CSD in conjunction with material degradation by osteoclastic cells, which reduced the percentage of the remaining implant to less than 3% within 16weeks. Of the seeded ST-2 cells, 60–70% were able to migrate and attach to the OCP/Gel composites after 1day of incubation, regardless of the OCP content. These results indicate that an OCP/Gel composite can repair rat calvarial CSD very efficiently and has favorable biodegradation characteristics. Therefore, it is hypothesized that host osteoblastic cells can easily migrate into an OCP/Gel composite.