Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering.

Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-κB-dependent SEAP reporter gene.

BACKGROUND: NF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening. METHOD: We recombined the reporter gene vector with inserting the "neo" transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela. RESULT: In this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes. CONCLUSION: In conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.

Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.

2,4,5-Trimethoxyldalbergiquinol promotes osteoblastic differentiation and mineralization via the BMP and Wnt/ß-catenin pathway.

Dalbergia odorifera has been traditionally used as a medicine to treat many diseases. However, the role of 2,4,5-trimethoxyldalbergiquinol (TMDQ) isolated and extracted from D. odorifera in osteoblast function and the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate the effects and possible underlying mechanisms of TMDQ on osteoblastic differentiation of primary cultures of mouse osteoblasts as an in vitro assay system. TMDQ stimulated osteoblastic differentiation, as assessed by the alkaline phosphatase (ALP) activity, ALP staining, mineralized nodule formation, and the levels of mRNAs encoding the bone differentiation markers, including ALP, bone sialoprotein (BSP), osteopontin, and osteocalcin. TMDQ upregulated the expression of Bmp2 and Bmp4 genes, and increased the protein level of phospho-Smad1/5/8. Furthermore, TMDQ treatment showed the increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and the expression of ß-catenin protein. The TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, Dickkopf-1 (DKK1), and bone morphogenetic protein (BMP) antagonist, noggin. TMDQ-induced runt-related transcription factor 2 (Runx2) expression was attenuatted by noggin and DKK1. These data suggest that TMDQ acts through the activation of BMP, Wnt/ß-catenin, and Runx2 signaling to promote osteoblast differentiation, and we demonstrate that TMDQ could be a potential agent for the treatment of bone loss-associated diseases such as osteoporosis.

Acclimation of Emiliania huxleyi (1516) to nutrient limitation involves precise modification of the proteome to scavenge alternative sources of N and P.

Limitation of marine primary production by the availability of nitrogen or phosphorus is common. Emiliania huxleyi, a ubiquitous phytoplankter that plays key roles in primary production, calcium carbonate precipitation and production of dimethyl sulfide, often blooms in mid-latitude at the beginning of summer when inorganic nutrient concentrations are low. To understand physiological mechanisms that allow such blooms, we examined how the proteome of E. huxleyi (strain 1516) responds to N and P limitation. We observed modest changes in much of the proteome despite large physiological changes (e.g. cellular biomass, C, N and P) associated with nutrient limitation of growth rate. Acclimation to nutrient limitation did however involve significant increases in the abundance of transporters for ammonium and nitrate under N limitation and for phosphate under P limitation. More notable were large increases in proteins involved in the acquisition of organic forms of N and P, including urea and amino acid/polyamine transporters and numerous C-N hydrolases under N limitation and a large upregulation of alkaline phosphatase under P limitation. This highly targeted reorganization of the proteome towards scavenging organic forms of macronutrients gives unique insight into the molecular mechanisms that underpin how E. huxleyi has found its niche to bloom in surface waters depleted of inorganic nutrients.

A host-microbiome interaction mediates the opposing effects of omega-6 and omega-3 fatty acids on metabolic endotoxemia.

Metabolic endotoxemia, commonly derived from gut dysbiosis, is a primary cause of chronic low grade inflammation that underlies many chronic diseases. Here we show that mice fed a diet high in omega-6 fatty acids exhibit higher levels of metabolic endotoxemia and systemic low-grade inflammation, while transgenic conversion of tissue omega-6 to omega-3 fatty acids dramatically reduces endotoxemic and inflammatory status. These opposing effects of tissue omega-6 and omega-3 fatty acids can be eliminated by antibiotic treatment and animal co-housing, suggesting the involvement of the gut microbiota. Analysis of gut microbiota and fecal transfer revealed that elevated tissue omega-3 fatty acids enhance intestinal production and secretion of intestinal alkaline phosphatase (IAP), which induces changes in the gut bacteria composition resulting in decreased lipopolysaccharide production and gut permeability, and ultimately, reduced metabolic endotoxemia and inflammation. Our findings uncover an interaction between host tissue fatty acid composition and gut microbiota as a novel mechanism for the anti-inflammatory effect of omega-3 fatty acids. Given the excess of omega-6 and deficiency of omega-3 in the modern Western diet, the differential effects of tissue omega-6 and omega-3 fatty acids on gut microbiota and metabolic endotoxemia provide insight into the etiology and management of today's health epidemics.

NF-κB, CRE and YY1 elements are key functional regulators of CMV promoter-driven transient gene expression in CHO cells.

Transient gene expression (TGE) in CHO cells is utilized to produce material for use in early stage drug development. These systems typically utilize the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. In this study, we have mechanistically dissected CMV-mediated TGE in CHO cells in order to identify the key regulators of this process. An in silico analysis of the promoter composition of transcription factor regulatory elements (TFREs) and the CHO cell repertoire of transcription factors identified eight TFREs as likely effectors of CMV activity. We determined the regulatory function of these elements by preventing their cognate transcription factors from binding at the CMV promoter. This was achieved by both scrambling promoter binding site sequences and using decoy molecules to sequester intracellular transcription factors. We determined that the vast majority of CMV activity is mediated by just two discrete TFREs, showing that simultaneous inhibition of NF-κB and CRE-mediated transactivation reduced CMV-driven transient secreted alkaline phosphatase (SEAP) production by over 75%. Further, we identified a mechanism by which CMV-mediated TGE is negatively regulated in CHO cells, showing that inhibition of YY1-mediated transrepression increased SEAP production 1.5-fold. This work enables optimization and control of CMV-mediated TGE in CHO cells, in order to improve transient protein production yields.

Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study.

The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 µM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.

5-aza-2'-Deoxycytidine, a DNA methyltransferase inhibitor, facilitates the inorganic phosphorus-induced mineralization of vascular smooth muscle cells.

AIM: Vascular calcification, an independent risk factor for cardiovascular disease in patients with chronic kidney disease(CKD), refers to the mineralization of vascular smooth muscle cells(VSMCs) caused by phenotypic changes toward osteoblast-like cells. DNA methylation, mediated by DNA methyltransferases(DNMTs), plays an important role in the differentiation of osteoblasts. We herein assessed the effects of a DNMT inhibitor on phenotypic changes in VSMCs and the development of vascular calcification. METHODS: The effects of 5-aza-2'-deoxycytidine(5-aza-dC), a DNMT inhibitor, on human aortic smooth muscle cells(HASMCs) were evaluated. The expression and DNA methylation status of osteogenic genes were determined using RT-qPCR and bisulfite sequencing, respectively. Mineralization of HASMCs was induced by high concentrations of inorganic phosphate(Pi), as confirmed by quantitation of the calcium levels and von Kossa staining. Moreover, we examined the effects of the suppression of DNMT1 and/or alkaline phosphatase(ALP) on the mineralization of HASMCs. RESULTS: 5-aza-dC increased the expression and activity of ALP and reduced the DNA methylation levels of the ALP promoter region in the HASMCs. In addition, both treatment with 5-aza-dC and downregulation of the DNMT1 expression promoted the Pi-induced mineralization of HASMCs. Moreover, both treatment with phosphonoformic acid(PFA), a sodium-dependent phosphate transporter inhibitor, and suppression of the ALP expression inhibited the 5-aza-dC-promoted mineralization of HASMCs. CONCLUSIONS: The present study showed that DNMT inhibitors facilitate the Pi-induced development of vascular calcification via the upregulation of the ALP expression along with a reduction in the DNA methylation level of the ALP promoter region.

In vitro comparative analysis of monocrotophos degrading potential of Aspergillus flavus, Fusarium pallidoroseum and Macrophomina sp.

Fungal degradation is emerging as a new powerful tool for the removal of potent neurotoxin pesticide, monocrotophos. Therefore, the present study is aimed at comparative characterization of monocrotophos degrading ability of three different fungal strains. Fungal strains were isolated from local agricultural soil by enrichment culture method, screened by gradient culture and identified as Aspergillus flavus, Fusarium pallidoroseum and Macrophomina sp. Growth kinetics revealed a direct positive influence of monocrotophos on the viability of fungal isolates. Fungal degradation was studied in phosphorus free liquid culture medium supplemented with 150 mg L(-1) concentration of monocrotophos for a period of 15 days under optimized culture conditions. Degradation of MCP followed first order kinetics with kdeg of 0.007, 0.002 and 0.005 day(-1) and half life (t1/2) of 4.21, 12.64 and 6.32 days for A. flavus, F. pallidoroseum and Macrophomina sp. respectively. To the best of our knowledge, it is the first report signifying the potential of monocrotophos degradation by Fusarium and Macrophomina sp. The results were further confirmed by HPTLC and FTIR which indicates disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. Degradation of monocrotophos by fungal isolates was accompanied by the release of extracellular alkaline phosphatases, inorganic phosphates and ammonia. The overall comparative analysis followed the order of A. flavus > Macrophomina sp. > F. pallidoroseum. Therefore, it could be concluded from the study that these three different fungal strains could be effectively used as a potential candidate for the removal of monocrotophos from contaminated sites.