Mouse Osteoblastic Cell Line Research Articles

Liver-type of tissue non-specific alkaline phosphatase is induced during mouse bone and tooth cell differentiation

Background and objective: Tissue non-specific alkaline phosphatase (TNSALP) contains two types—bone- and liver-type—which are produced from the same gene due to differences in splicing. These two differ in their promoter, but the amino acid sequences of the mature proteins are identical. In this study, we examined the relationship between the two types of TNSALP expression and osteoblast differentiation.Design: Gene expression of the two types of TNSALP was observed by reverse transcription-polymerase chain reaction. MC3T3-NM4 was sub-cloned from an established mouse osteoblastic cell line in which osteoblast characters do not appear without dexamethasone. The C2C12 mouse myoblastic cell line, which can be induced to osteoblasts with bone morphogenic protein 2, and organ-cultured tooth germs were also used in this work.Results: The gene expression of liver-type TNSALP was observed in only MC3T3-NM4 activated by dexamethasone. For C2C12, the gene expression of bone-type TNSALP was observed even in non-induced conditions where myotubes were formed, whereas the liver-type TNSALP mRNA was only expressed when C2C12 differentiated into osteoblasts by bone morphogenic protein 2. Furthermore, in the organ-cultured tooth germs, the liver-type TNSALP mRNA was expressed according to differentiation of tooth germs.Conclusion: These results suggest that the liver-type TNSALP mRNA is induced according to differentiation of bone and tooth.

Strontium doped hydroxyapatite from Mercenaria clam shells: Synthesis, mechanical and bioactivity study

Synthesis of strontium-doped hydroxyapatite from Mercenaria clam shells has been carried out by hydrothermal method. The doping of bioceramic, processed from biogenic resources is mostly unexplored. The objective is to understand the effect of strontium (Sr) incorporation on phase stability, sintering behaviour, mechanical properties and cytotoxicity of hydroxyapatite (HAp) derived from clam shells. The different molar concentrations of Sr, varies from 10, 30, 50, 70% of Ca, were substituted into the HAp. The synthesized powders were sintered at 1200 °C in air. The as synthesized powders and sintered specimens were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and high resolution transmission electron microscopy. The crystallite size and cell parameters of sintered specimens were analyzed from XRD. The XRD of hydrothermally synthesized powders mostly matched with HAp with slight shifting due to Sr doping. However, some distinct Sr based compounds were also observed where Sr substitution is more that 50% of Ca. The XRD of sintered specimen showed increasing β-tricalcium phosphate (β-TCP) phase with Sr substitution. The sintered density of solid samples gradually increased from 3.04 g/cc to 3.50 g/cc and surface energy decreased with increasing Sr substitution. Similarly, microhardness, fracture toughness and nanohardness of solid samples found to be enhanced with Sr substitution. The elastic modulus gradually increased from 130 to 137 GPa for HAp and Sr substituted HAp (70% of Ca). The in vitro cytotoxicity of sintered specimen against mouse osteoblast cell line showed that all the samples were nontoxic. However cell proliferation found low for the solid samples containing more than 50% Sr substitution.

Fluorine substituted nano hydroxyapatite: Synthesis, bio-activity and antibacterial response study

In the present investigation, nano hydroxyapatite (HAp) and fluorine substituted hydroxyapatite (FHAp) with varying fluorine concentrations have been successfully prepared by hydrothermal method. The as synthesized powders were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), high resolution transmission electron microscope (HRTEM) and ion chromatography (IC) for fluorine content analysis. XRD results showed shifting of HAp peaks with increasing fluorine substitution and detected calcium fluoride (CaF2) phase in the synthesized powders where theoretically 100% or more OH ions substituted by F ions. The crystallinity of HAp powder gradually increased and cell volume decreased with fluorine substitution. HRTEM of the synthesized powders showed the nano rod like morphology. The IC was carried out to understand the degree fluoridation in the synthesized powder. The IC result of in vitro fluorine ion leaching showed that fluorine concentration in aqueous medium depends on the degree of fluoridation in the synthesized powder. The as synthesized powders were checked for antimicrobial activity by using both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) strains. The powder having ~33% fluorine substitution showed bactericidal effect. The in vitro fluorine ion release study showed significantly high fluorine concentration in aqueous medium for the HAp powders having more than 50% fluorine substituted. The in vitro cell viability assay using mouse osteoblast cell line (MC3T3-E1) confirmed that the powders were non-cytotoxic.

Inhibition of osteoblast differentiation by ritonavir.

Osteoporosis is one of the chronic complications seen in human immunodeficiency virus (HIV)-infected patients, and affects patients at high prevalence. The causes of osteoporosis in HIV-infected patients are multiple, and include chronic HIV infection, living habits such as smoking and alcohol consumption, and antiretroviral drug use. Among antiretroviral drugs, protease inhibitors have been reported to be associated with osteoporosis. However, it remains to be determined how anti-HIV drugs affect osteoblast differentiation. In the current study, MC3T3-E1 cells, a mouse osteoblastic cell line, were cultured in osteoblast differentiation medium with or without different protease inhibitors (ritonavir, lopinavir, darunavir or atazanavir), and alkaline phosphatase (ALP) activity and the expression of Runt-related transcription factor 2 (Runx2) were analyzed. The ALP activity in MC3T3-E1 cells cultured with ritonavir was significantly reduced compared with that in cells in only osteoblast inducer reagent, indicating that ritonavir inhibited osteoblast differentiation. Meanwhile, ALP activity was not reduced in cells cultured with any of the other inhibitors. In addition, ritonavir inhibited the expression of Runx2, a key regulator of osteoblast differentiation, in the early period of osteoblast differentiation. To the best of our knowledge, this is the first study to demonstrate that ritonavir inhibits osteoblast differentiation in vitro. The present findings may explain the mechanism of osteopenia induced by combination antiretroviral therapy involving protease inhibitors.

Abstract 2136: Transcriptome analysis of osteoblasts fused with cancer-derived exosomes

Abstract Breast cancer tumors have a high rate of metastasis and certain aggressive subtypes favor the bone environment. Clinically, it is estimated that metastatic breast cancer has a prevalence of up to 70% of bone tumors. Exosomes are important in cell-cell communication, and are increasingly being recognized as key contributors for priming targeted tissues for metastasis. Despite numerous advances in exosome detection and cargo characterization, transcriptional effects of cancer exosomes on targeted cells remain difficult to characterize at physiologically relevant levels. This is largely due to a combination of limited exosome secretion/uptake and an inability to segregate affected cells from the population. Conventional approaches to circumvent these limitations include spiking cell environments with exosome quantities far in excess of physiological concentrations. We sought to analyze the transcriptional effect of cancer exosomes on cells that have uptaken cancer-derived exosomes in a dynamic co-culture environment. In order to accomplish this, we first engineered a human breast cancer cell line (MDA-MB-231) to express GFP labeled exosomes. This novel cell line expresses three GFP fusion proteins for tetraspanin exosome markers (CD9, CD63, and CD81) known to be found on exosomes derived from MDA-MB-231 cells. Stable, intrinsic, fluorescent labeling of three exosome markers allows for an improved, more sensitive method for the analysis of exosome transfer. We can now examine transfer representing a more diverse pool of exosomes requiring no manipulation post exosome isolation prior to visualization. To investigate breast cancer exosome-mediated alterations on gene expression in bone cells to mimic breast cancer bone metastasis, this GFP exosome expressing line (MDA-MB-231 exo-GFP) was co-cultured with a mouse osteoblast cell line (MC3T3) then analyzed via flow cytometry to quantify the uptake of cancer exosomes in target cell population over time. MC3T3 cells that become GFP positive, indicative of exosome mediated transfer, increased from 0.91% of the population on day 3 post co-culture to 17.3% on day 14. Subsequent cell sorting and RNA-seq of GFP positive MC3T3 populations yielded novel insights into the progressive transcriptional effect of cancer exosomes on target cells. Future research will expand upon this approach to examine the effects on bone cells non-metastatic and metastatic cancer cell lines have on the metastatic niche through exosome-mediated signaling. This approach introduces a novel method for prolonged transcriptional analysis of cancer and target cell co-culture allowing the segregation of exosome affected and unaffected subpopulations of target cells. This study was funded by DOD grant BC151687. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas H. Hum, Kelly A. Martin, Gabriela G. Loots. Transcriptome analysis of osteoblasts fused with cancer-derived exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2136.

Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis

Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell differentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.

Direct-writing of 3D periodic TiO2 bio-ceramic scaffolds with a sol-gel ink for in vitro cell growth

In this work, three-dimensional (3D) periodic TiO2 bio-ceramic scaffolds were created by continuous filament writing with TiO2 sol-gel ink followed by sintering. The TiO2 sol-gel ink has the advantage of creating 3D bio-ceramic scaffolds with finer feature size than commonly used ceramic powder based ink systems. The structure, pore sizes, and interconnectivity could be controlled through 3D structure design and printing parameter adjustment. To evaluate the potential of these 3D TiO2 bio-ceramic scaffolds for bone tissue engineering, the mouse osteoblastic cell line MC3T3-E1 was cultured on them. The observed good viability demonstrated that 3D TiO2 bio-ceramic scaffolds created by direct-writing technique provide a biocompatible environment that favors cell growth and attachment.

The potential use of thermosensitive chitosan/silk sericin hydrogels loaded with longan seed extract for bone tissue engineering.

In this study, hydrogels that were thermosensitive at body temperature were developed using chitosan (CS)/silk sericin (SS)/β-glycerophosphate (β-GP) loaded with longan seed extract (LE) for use in bone tissue engineering. These hydrogels were transformed into a gel at 37 °C within 10 min via interactions between CS and β-GP. The incorporation of SS resulted in a shorter gelation time of 5–7 min. The morphological structure of the thermosensitive CS/β-GP hydrogels exhibited an irregular pore structure, whereas the morphological structure of the thermosensitive CS/SS/β-GP hydrogels became more slender and porous. The incorporation of SS affected the network structure of the CS hydrogels, which degraded more rapidly. Moreover, the cumulative amounts of both gallic acid (GA) and ellagic acid (EA) released from the hydrogels loaded with LE increased with an increase in the SS content. Finally, these thermosensitive hydrogels were non-toxic to both a mouse fibroblast cell line (NCTC clone 929) and a mouse osteoblast cell line (MC3T3-E1) and promoted the attachment of MC3T3-E1 cells to the surface of the hydrogels. Therefore, these thermosensitive hydrogels might be a promising candidate for bone tissue engineering.

Magnesium phosphate ceramics incorporating a novel indene compound promote osteoblast differentiation in vitro and bone regeneration in vivo

Incorporating bioactive molecules into synthetic ceramic scaffolds is challenging. In this study, to enhance bone regeneration, a magnesium phosphate (MgP) ceramic scaffold was incorporated with a novel indene compound, KR-34893. KR-34893 induced the deposition of minerals and expression of osteoblast marker genes in primary human bone marrow mesenchymal stem cells (BMSCs) and a mouse osteoblastic MC3T3-E1 cell line. Analysis of the mode of action showed that KR-34893 induced the phosphorylation of MAPK/extracellular signal-regulated kinase and extracellular signal-regulated kinase, and subsequently the expression of bone morphogenetic protein 7, accompanied by SMAD1/5/8 phosphorylation. Accordingly, KR-34893 was incorporated into an MgP scaffold prepared by 3D printing at room temperature, followed by cement reaction. KR-34893-incorporated MgP (KR-MgP) induced the expression of osteoblast differentiation marker genes in vitro. In a rat calvaria defect model, KR-MgP scaffolds enhanced bone regeneration and increased bone volume compared with MgP scaffolds, as assessed by micro-computed tomography and histological analyses. In conclusion, we developed a method for producing osteoinductive MgP scaffolds incorporating a bioactive organic compound, without high temperature sintering. The KR-MgP scaffolds enhanced osteoblast activation in vitro and bone regeneration in vivo.

IL-1β suppresses nephronectin expression in osteoblasts via ERK1/2 and JNK

Nephronectin (Npnt), an extracellular matrix protein, is considered to play critical roles in development of various tissues and their functions. In basic science experiments, we found that interleukin-1β (IL-1β), well known to have an important role in inflammatory response, inhibited Npnt gene expression in MC3T3-E1 cells, a mouse osteoblastic cell line. The purpose of this study was to investigate mechanisms that govern the regulation of Npnt gene expression by IL-1β in osteoblasts.

Effect of trace elements on the sintering effect of fish scale derived hydroxyapatite and its bioactivity

Synthesis of nano-crystalline hydroxyapatite (HAp) from fish scale (Catla catla) was carried out by calcining of raw fish scale between 200 °C and 1200 °C. The synthesized powders were characterized using thermogravimetry – differential thermal analysis (TGA-DTA), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, inductively coupled plasma atomic emission spectroscopy (ICP-AES), scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM). The crystallinity, crystallite size, strain and cell parameters of fish scale derived calcined powders and chemically synthesized HAp (S-HAp) powders were estimated from XRD data. The average crystallite size of fish scale derived HAp powder increased with increasing calcination temperature. The fish scale derived HAp (FSH800) and S-HAp, calcined at 800 °C, showed crystallite size of 30 nm and 60 nm, respectively. ICP-AES analysis revealed that the fish scale derived HAp powder calcined at 1200 °C (FSH1200) contain significantly high amount of magnesium and strontium ions compared to S-HAp. After sintering at 1200 °C for 1 h, the relative density of FSH1200 was 20% higher than that of S-HAp due to better sinterability as a result of its small crystallite size and the presence of trace elements. In vitro cytotoxicity studies performed using human osteoblast-like cells (MG63) and mouse osteoblast cell line (MC3T3-E1) demonstrated that fish scale derived HAp (FSH1200) is non-toxic.

MicroRNA-433-3p promotes osteoblast differentiation through targeting DKK1 expression.

Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3’UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/β-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.

Triptolide inhibits proliferation, differentiation and induces apoptosis of osteoblastic MC3T3‑E1 cells.

Ankylosing spondylitis (AS) is characterized by the formation of bony spurs. Treatment of the resulting ankylosis, excessive bone formation and associated functional impairment, remain the primary therapeutic aims in research regarding this condition. Triptolide is the primary active component of the perennial vine Tripterygium wilfordii Hook. f., and has previously been demonstrated to exert anti-tumor activities including inhibition of cell growth and the induction of apoptosis, however, the effect of triptolide on osteoblasts remains to be elucidated. In the present study, the MC3T3-E1 mouse osteoblast cell line was treated with differing concentrations of triptolide for various intervals. Cell proliferation was detected using the bromodeoxyuridine assay, cell cycle and apoptosis were measured by flow cytometry, nuclear apoptosis was observed by Hoechst staining and associated proteins were determined via western blot analysis. The cells were then further incubated with osteogenic induction medium supplemented with triptolide for 7 or 12 days and the differentiation to osteoblasts was examined by picrosirius staining, observation of alkaline phosphatase activity and a calcium deposition assay. It was demonstrated that treatment with triptolide significantly inhibited osteoblast proliferation and induced cell cycle arrest and apoptosis of the osteoblasts. Furthermore, treatment with triptolide reduced collagen formation, alkaline phosphatase activity and calcium deposition. The present study demonstrated an inhibitory effect of triptolide on osteoblast proliferation and differentiation, and therefore suggests a potential therapeutic agent for the treatment of AS in the future.

Degradation behavior and compatibility of micro, nanoHA/chitosan scaffolds with interconnected spherical macropores

Hydroxyapatite/Chitosan (HA/CS) composite have significant application in biomedical especially for bone replacement. Inorganic particle shape and size of composite affect the scaffold mechanical property, biological property, and degradation. The aim of this study was to fabricate HA/CS scaffold with good pore connectivity and analyze their biological, degradation properties. Microhydroxyapatite/chitosan (mHA/CS) and nanohydroxyapatite/chitosan (nHA/CS) composite scaffolds with interconnected spherical pore architectures were fabricated. Composite scaffolds structure parameters were analyzed using micro CT. Cell proliferation and morphology were tested and compared between two scaffolds using mouse osteoblastic cell line MC3T3-E1. To research the composite degradation in lysozyme PBS solution, degradation rate and reducing sugar content were tested, and scaffolds morphology were observed by SEM. The results showed that microHA and nanoHA were fabricated by being calcined and synthesis methods, and their infrared spectra are very similar. EDAX composition analysis demonstrated that both of microHA and nanoHA were calcium deficiency HA. Micro-CT results demonstrated the scaffolds had interconnected spherical pores, and the structure parameters were similar. Cell viabilities were significant increased with cultured time, but there were no significant difference between microHA/CS and nanoHA/CS scaffolds. Scaffold structure was gradually destroyed and inorganic composition HA particles are more prominent with degradation time.Significance: (1) Inorganic particle shape and size of composite affect the scaffold mechanical property, biological property, and degradation. NanoHA/CS and microHA/CS scaffolds with good pore connectivity were fabricated and their biological, degradation properties were studied in this manuscript. (2) The scaffold with interconnected porosity construct provides the necessary support for cells to proliferate and maintain their differentiated function, and its architecture related to the structure and morphology of new bone. Polymer scaffolds were fabricated by the technique of compression molding and particulate leaching method, and paraffin microspheres were used as the porogen. (3) MicroHA/CS and nanoHA/CS composite scaffolds are potential materials for use in bone tissue engineering.

Starch-derived absorbable polysaccharide hemostat enhances bone healing via BMP-2 protein

Surgical hemostasis is critical in reducing the likelihood of excessive bleeding and blood transfusion. In treating some cases, commonly used hemostatic agent showed limited efficacy and prolonged degradation and clearance, causing an inhibition of bone healing. Starch absorbable polysaccharide (SAPH) is a novel hemostatic agent made from a plant starch, which can be completely absorbed and achieve better hemostatic effects than many commonly used hemostatic agents. However, whether SAPH can induce a promotion of bone healing remains unknown. In this study, we used a model of rabbit parietal bone defect and a mouse osteoblast cell line MC3T3-E1 to evaluate the effects of SAPH on bone healing. We found that SAPH significantly decreased bone healing scores, reduced defective area of parietal bone, and increased the areas of bone trabeculae and cavitas medullaris. In addition, SAPH enhanced MC3T3-E1 osteoblasts proliferation, up-regulated the expressions of alkaline phosphatase (ALP) and osteocalcin and increased the level of bone morphogenetic protein 2 (BMP-2) in MC3T3-E1 osteoblasts. These SAPH-induced benefits in MC3T3-E1 osteoblasts were significantly abolished by the application of BMP-2-siRNA. These findings suggested that SAPH enhances bone healing, promotes the proliferation, differentiation and maturation of osteoblast by up-regulating BMP-2 expression in osteoblastic cells.

Expression of nephronectin is enhanced by 1α,25-dihydroxyvitamin D3.

The extracellular matrix protein nephronectin (Npnt), also called POEM, is considered to play critical roles as an adhesion molecule in development and functions of various tissues, such as the kidneys, liver, and bone. In the present study, we examined the molecular mechanism of Npnt gene expression and found that vitamin D3 (1α,25‐dihydroxyvitamin D3,VD 3) strongly enhanced Npnt mRNA expression in MC3T3‐E1 cells from a mouse osteoblastic cell line. The VD 3‐induced increase in Npnt expression is both time‐ and dose‐dependent and is mediated by the vitamin D receptor (VDR).

Linarin promotes osteogenic differentiation by activating the BMP-2/RUNX2 pathway via protein kinase A signaling.

Linarin (LIN), a flavonoid which exerts both anti-inflammatory and antioxidative effects, has been found to promote osteogenic differentiation. However, the molecular mechanism of its effect on osteoblast differentiation was unclear. In the present study, LIN from Flos Chrysanthemi Indici (FCI) was isolated in order to investigate the underlying mechanisms of LIN on MC3T3-E1 cells (a mouse osteoblastic cell line) and the osteoprotective effect of LIN in mice which had undergone an ovariectomy (OVX). The results revealed that LIN enhanced osteoblast proliferation and differentiation in MC3T3-E1 cells dose-dependently, with enhanced alkaline phosphatase (ALP) activity and mineralization of extracellular matrix. LIN upregulated osteogenesis-related gene expression, including that of ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), bone sialoprotein (BSP), and type I collagen (COL-I). Pretreatment with noggin, a bone morphogenetic protein-2 (BMP-2) antagonist, meant that LIN-induced gene expression levels of COL-1, ALP, OCN, BSP and RUNX2 were significantly reduced, as shown by RT-qPCR. Western blot analysis showed that LIN dose-dependently increased the protein levels of BMP-2 and RUNX2 and enhanced the phosphorylation of SMAD1/5. In addition, LIN dose-dependently upregulated protein kinase A (PKA) expression. H-89 (a PKA inhibitor) partially blocked the LIN-induced protein increase in BMP-2, p-SMAD1/5 and RUNX2. We noted that LIN preserved the trabecular bone microarchitecture of ovariectomized mice in vivo. Moreover, pretreatment with LIN significantly lowered serum levels of ALP and OCN in ovariectomized mice. Our data indicated that LIN induced the osteogenic differentiation and mineralization of MC3T3-E1 osteoblastic cells by activating the BMP-2/RUNX2 pathway through PKA signaling in vitro and protected against OVX-induced bone loss in vivo. The results strongly suggest that LIN is a useful natural alternative for the management of postmenopausal osteoporosis.

Biocompatibility of artificial bone based on vancomycin loaded mesoporous silica nanoparticles and calcium sulfate composites.

The aim of this study was to evaluate the in vitro and in vivo biocompatibility of artificial bone based on vancomycin loaded mesoporous silica nanoparticles and calcium sulfate composites. In vitro cytotoxicity tests by cholecystokinin octapeptide (CCK-8) assay showed that the 5 %Van-MSN-CaSO4 and Van-CaSO4 bone cements were cytocompatible for mouse osteoblastic cell line MC3T3-E1. The microscopic observation confirmed that MC3T3-E1cells incubated with Van-CaSO4 group and 5 %Van-MSN-CaSO4 group exhibited clear spindle-shaped changes, volume increase and maturation, showing that these cements supported adhesion of osteoblastic cells on their surfaces. In addition, the measurement of alkaline phosphatase activity revealed the osteoconductive property of these biomaterials. In order to assess in vivo biocompatibility, synthesized cements were implanted into the distal femur of twelve adult male and female New Zealand rabbits. After implantation in artificial defects of the distal femur, 5 %Van-MSN-CaSO4 and Van-CaSO4 bone cements did not damage the function of main organs of rabbits. In addition, the Van-MSN-CaSO4 composite allowed complete repair of bone defects with new bone formation 3 months after implantation. These results show potential application of Van-MSN-CaSO4 composites as bone graft materials for the treatment of open fracture in human due to its mechanical, osteoconductive and potential sustained drug release characteristics and the absence of adverse effects on the body.

Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells

Excessive systemic exposure to fluoride leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation. In this study, we examined the effects of fluoride exposure on c-Fos expression and its regulatory signaling pathways in MC3T3-E1 mouse osteoblast cell line. c-fos mRNA level, c-Fos protein level and c-Fos DNA-binding activity were markedly increased, with a peak at 2 or 4 h, in MC3T3-E1 cells exposed to sodium fluoride (NaF). Fra-1 protein, another member of Fos family, was also elevated, whereas FosB and Fra-2 proteins remained unchanged. NaF further induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), ERK5, c-Jun NH2-terminal kinase and p38. NaF-induced expression of c-Fos protein was markedly suppressed with U0126, the inhibitor of both activated and non-activated forms of MAPK/ERK kinase 1/2 (MEK1/2) and BIX02189, the MEK5 inhibitor, but partially with SP600125, the JNK inhibitor and SB203580, the p38 inhibitor. Therefore, ERK1/2 and ERK5 signal transduction pathways are important for accumulating c-Fos. siRNA targeting against the mouse c-fos gene further enhanced NaF-induced up-regulation of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, suggesting that c-Fos might negatively regulate OPG expression induced by fluoride in osteoblastic cells.

Enhanced attachment and growth of periodontal cells on glycine-arginine-glycine-aspartic modified chitosan membranes

Background: Chitosan, a polymeric carbohydrate derived from the exoskeleton of arthropod, has been suggested to be an excellent biomaterial for improving wound healing, especially for bones. To improve the periodontal cell attachment and growth, the cell adhesive peptide glycine-arginine-glycine-aspartic acid (Gly-Arg-Gly-Asp, GRGD) grafted chitosan membrane was introduced in this study. Materials and Methods: Two types of commercial chitosan, three types of primary cultured cells, and two established cell lines were used. Human gingival and periodontal fibroblasts (hGF and hPDL), human root derived cell (hRDC), and rat calvaria bone cell (rCalB) were cultured on the GRGD-fixed by ultraviolet light photochemical method on the chitosan membrane. With (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay and propidium iodine (PI) staining, the cell adhesion and growth on GRGD-grafted chitosan were examined. Basal mRNA expressions of the receptors for GRGD, integrin αv (ITG αv) and ITG β3, in the human gingival fibroblast cell line and mouse osteoblast cell line (MC3T3-E1) were examined with real-time polymerase chain reaction. Results: Because the cell adhesion/growth patterns on two chitosan membranes were similar, the GRGD modification was performed on one membrane (Primex) only. For periodontal cells (hGFs, hPDLs, and hRDCs), the number of attached cells were increased on the membrane with the high concentration of GRGD than those on the membrane unmodified or modified with low concentration GRGD. For rCalBs cells, a different pattern was noted: GRGD modification did not enhance the calvaria cells attachment or growth. Moreover, mRNA expressions of ITG αv and β3 in AG09319 cells were significantly higher than those in MC3T3-E1 cells. Conclusions: With the limitation of this study, we suggested that GRGD-modified chitosan, especially at high concentration, could enhance the growth of various periodontal fibroblasts, but did not change those of osteoblasts. Therefore, chitosan might be an excellent biomaterial for periodontal use.