Monocytes Do Not Transdifferentiate into Proper Osteoblasts

Recent publications suggested that monocytes might be an attractive cell type to transdifferentiate into various cellular phenotypes. Aim was, therefore, to evaluate the potential of blood monocytes to transdifferentiate into osteoblasts. Monocytes isolated from peripheral blood were subjected to two previously published treatments to obtain unique, multipotent cell fractions, named programmable cells of monocytic origin (PCMOs) and monocyte-derived mesenchymal progenitor cells (MOMPs). Subsequently, MOMPs and PCMOs were treated with osteogenic differentiation medium (including either vitamin D or dexamethasone) for 14 days. Regarding a variety of surface markers, no differences between MOMPs, PCMOs, and primary monocytes could be detected. The treatment with osteogenic medium neither resulted in loss of hematopoietic markers nor in adoption of mesenchymal phenotype in all cell types. No significant effect was observed regarding the expression of osteogenic transcription factors, bone-related genes, or production of mineralized matrix. Osteogenic medium resulted in activation of monocytes and appearance of osteoclasts. In conclusion, none of the investigated monocyte cell types showed any transdifferentiation characteristics under the tested circumstances. Based on our data, we rather see an activation and maturation of monocytes towards macrophages and osteoclasts

Quercetin Protects Primary Human Osteoblasts Exposed to Cigarette Smoke through Activation of the Antioxidative Enzymes HO-1 and SOD-1

Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS). The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM) rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO-) 1 and superoxide-dismutase- (SOD-) 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers

5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation

The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors

Signaling pathway STAT1 is strongly activated by IFN-β in the pathogenesis of osteoporosis

BACKGROUND: Despite extensive research, the underlying pathological mechanisms of osteoporosis are not completely understood. Recent studies have indicated a distinct role for the IFN-β/STAT1 pathway in bone metabolism. An inhibitory effect of IFN-β on osteoclastogenesis has been detected and STAT1/2 has been shown to influence osteoblastic bone metabolism. So far, no data concerning the IFN-β/STAT1 pathways in osteoblasts and osteoclasts from osteoporotic and non-osteoporotic patients are available. The aim of the study was to analyze these pathways in both cell types. METHODS: Osteoblasts were isolated from the femoral heads of 12 osteoporotic and 11 non-osteoporotic patients and monocytes were differentiated into osteoclasts. After the differentiation period, cells were stimulated once with 20 and 100 ng/mL IFN-β for 4 days. Viability, activity, bone metabolism-related genes, and the proteins Fra1, SOCS1, STAT1, p-STAT1, and TRAF6 were analyzed. RESULTS: Viability, activity, and gene expressions were not affected by stimulating the osteoblasts. However, in osteoporotic osteoclasts, which display a significantly higher basal osteoclastic activity, the stimulation with IFN-β lead to significant inhibition. Further, an increased STAT1 activation was detected in both cell types with no significant differences between the groups. Regarding the phosphorylation of STAT1, no significant influence was detected in osteoblasts but the IFN-β stimulation led to a significant increase of p-STAT1 in osteoclasts of both groups. CONCLUSIONS: IFN-β is a principal mediator in the pathogenesis of osteoporosis by inhibiting osteoclasts and inducing and activating STAT1. Our results also confirm this in cells from osteoporotic and non-osteoporotic patients. Strong inhibitory effects on the osteoclastogenesis of osteoporotic osteoclasts were detectable. Nevertheless, osteoblast activity was not negatively affected by IFN-β stimulation. These results may contribute to a better understanding of the underlying pathological signaling pathways of osteoporosis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40001-014-0074-4) contains supplementary material, which is available to authorized users

Green tea protects human osteoblasts from cigarette smoke-induced injury: possible clinical implication

Artículo de publicación ISIPurpose Recent reports discuss the altered bone homeostasis in cigarette smokers, being a risk factor for osteoporosis and negatively influencing fracture healing. Cigarette smoke is known to induce oxidative stress in the body via an increased production of reactive oxygen species (ROS). These increases in ROS are thought to damage the boneforming osteoblasts. Naturally occurring polyphenols contained in green tea extract (GTE), e.g., catechins, are known to have anti-oxidative properties. Therefore, the aim of this study was to investigate whether GTE and especially catechins protect primary human osteoblasts from cigarette smoke-induced damage and to identify the underlying mechanisms. Methods Primary human osteoblasts were isolated from patients’ femur heads. Cigarette smoke medium (CSM) was obtained using a gas-washing bottle and standardized by its optical density (OD320) at λ0320 nm. ROS formation was measured using 2′7′dichlorofluorescein diacetate, and osteoblasts’ viability was detected by resazurin conversion. Results Co-, pre-, and post-incubation with GTE and catechins significantly reduced ROS formation and thus improved the viability of CSM-treated osteoblasts. Besides GTE’s direct radical scavenging properties, pre-incubation with both GTE and catechins protected osteoblasts from CSM-induced damage. Inhibition of the anti-oxidative enzyme HO-1 significantly reduced the protective effect of GTE and catechins emphasizing the key role of this enzyme in GTE anti-oxidative effect. Conclusions Our data suggest possible beneficial effects on bone homeostasis, fracture healing, and bone mineral density following a GTE-rich diet or supplementation

Increased oxidative stress response in granulocytes from older patients with a hip fracture may account for slow regeneration

Proximal femur fracture, a typical fracture of the elderly, is often associated with morbidity, reduced quality of life, impaired physical function and increased mortality. There exists evidence that responses of the hematopoietic microenvironment to fractures change with age. Therefore, we investigated oxidative stress markers and oxidative stress-related MAPK activation in granulocytes from the young and the elderly with and without fractured long bones. Lipid peroxidation levels were increased in the elderly controls and patients. Aged granulocytes were more sensitive towards oxidative stress induced damage than young granulocytes. This might be due to the basally increased expression of SOD-1 in the elderly, which was not further induced by fractures, as observed in young patients. This might be caused by an altered MAPK activation. In aged granulocytes basal p38 and JNK activities were increased and basal ERK1/2 activity was decreased. Following fracture, JNK activity decreased, while ERK1/2 and p38 activities increased in both age groups. Control experiments with HL60 cells revealed that the observed p38 activation depends strongly on age. Summarizing, we observed age-dependent changes in the oxidative stress response system of granulocytes after fractures, for example, altered MAPK activation and SOD-1 expression. This makes aged granulocytes vulnerable to the stress stimuli of the fracture and following surgery

Distribution of 5-hydroxymethylcytosine (5 hmC) and 5-methylcytosine (5 mC) in Ad-MSCs.

<p>Basal mRNA levels of (A) <i>TET2</i> and (B) <i>TET3</i> were determined by qRT-PCR in Ad-MSCs from young and old donors (each group: N = 6, n = 3). (C) Expression changes of <i>TET2</i> and <i>TET3</i> were confirmed on the protein level by Western blot analysis (N = 3/group). For both techniques <i>GAPDH</i> was used for normalization. (D) Young and old Ad-MSCs (each group: N = 3, n = 5) were stained for 5-hydroxymethylcytosine (5 hmC/red) and 5-methylcytosine (5 mC/green). Cell nuclei were counterstained with Hoechst 33342 (blue). (E&F) For quantification 5 hmC or 5 mC positive nuclei were counted. *p<0.05, **p<0.01 (student’s t-test).</p

5-Azacytidine treatment increased <i>TET2</i> and <i>TET3</i> gene expression.

<p>Young and old Ad-MSCs were treated with 5 µM 5-Azacytidine for 48 h. mRNA levels of (A) <i>TET2</i>, (B) <i>TET3</i>, (E) Sox2, (F) <i>Lin28A</i> (2 donors from the young Ad-MSCs had to be excluded from the data set, due to artefacts in the melting curve), (G) <i>Oct4</i> and (H) <i>Nanog</i> were determined by qRT-PCR (N = 6, n = 3/age group). (I) Expression changes of <i>TET2, TET3, Lin28A</i>, <i>Oct4</i>, <i>Nanog</i> and <i>Sox2</i> were confirmed on the protein level by Western blot analysis (Representative figure for N = 2/group). <i>GAPDH</i> was used for normalization. Data are presented as relative expression changes (log₁₀) of 5-Azacytidine treated Ad-MSCs compared to the corresponding untreated Ad-MSCs. Distribution of 5-hydroxymethylcytosine (5 hmC) and 5-methylcytosine (5 mC) in the 5-Azacytidine treated and untreated Ad-MSCs was determined by immunofluorescent staining (N = 3, n = 5/age group). Cell nuclei were counterstained with Hoechst 33342. For quantification (D) 5 hmC and (E) 5 mC positive nuclei were counted. Data are represented as log₁₀ (fold of control). *p<0.05, **p<0.01, ***p<0.001 as compared to untreated cells (student’s t-test).</p

Sequence of primers used in qRT-PCR.

<p>*PCR conditions were optimized with varying template concentrations to be in the quantifiable/linear range of the PCR.</p