Differentially circulating miRNAs after recent osteoporotic fractures can influence osteogenic differentiation

Abstract

Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level and are known to take part in the control of bone formation and bone resorption. In addition, it is known that miRNAs are secreted by many cell types and can transfer "messages" to recipient cells. Thus, circulating miRNAs might not only be useful as surrogate biomarkers for the diagnosis or prognosis of pathological conditions, but could be actively modulating tissue physiology. Therefore, the aim of this study was to test whether circulating miRNAs that exhibit changes in recent osteoporotic fracture patients could be causally related to bone metabolism. In the first step we performed an explorative analysis of 175 miRNAs in serum samples obtained from 7 female patients with recent osteoporotic fractures at the femoral neck, and 7 age-matched female controls. Unsupervised cluster analysis revealed a high discriminatory power of the top 10 circulating miRNAs for patients with recent osteoporotic fractures. In total 6 miRNAs, miR-10a-5p, miR-10b-5p, miR-133b, miR-22-3p, miR-328-3p, and let-7g-5p exhibited significantly different serum levels in response to fracture (adjusted p-value<0.05). These miRNAs were subsequently analyzed in a validation cohort of 23 patients (11 control, 12 fracture), which confirmed significant regulation for miR-22-3p, miR-328-3p, and let-7g-5p. A set of these and of other miRNAs known to change in the context of osteoporotic fractures were subsequently tested for their effects on osteogenic differentiation of human mesenchymal stem cells (MSCs) in vitro. The results show that 5 out of 7 tested miRNAs can modulate osteogenic differentiation of MSCs in vitro. Overall, these data suggest that levels of specific circulating miRNAs change in the context of recent osteoporotic fractures and that such perturbations of "normal" levels might affect bone metabolism or bone healing processes.

Serum miRNA Signatures Are Indicative of Skeletal Fractures in Postmenopausal Women With and Without Type 2 Diabetes and Influence Osteogenic and Adipogenic Differentiation of Adipose Tissue–Derived Mesenchymal Stem Cells In Vitro

“…We favored collection and usage of hASCs over bone marrowderived mesenchymal stem cells (BMSCs) because hASCs are readily accessible in our laboratory under well-established and well-standardized harvesting conditions, and because we had been able to demonstrate in an earlier study by our group that, at least with respect to the osteogenic effect of miR-637, our hASC model and a BMSC model showed comparable results. (21) Successful overexpression compared to nontargeting miRNA transfections was controlled by qPCR 48 hours after transfection (Fig. 6A).…”

“…(21,39,40) All patients had given prior written consent. For isolation of hASCs, the harvested lipoaspirate was first washed with PBS (PAA, Dartmouth, MA, USA) to remove cell debris, then digested with collagenase NB4 (Serva, Heidelberg, Germany) for 1 hour to further disintegrate adipose tissue agglomerates.…”

“…Standard protocols and eight replicate wells each were utilized as described. (21) After alcohol fixation, cells were stained for 20 min with 40 mM Alizarin red solution (Sigma-Aldrich, Munich, Germany) and washed with PBS to remove all traces of unbound dye. Alizarin was then extracted for 30 min using 200 mL 0.1 M HCL/0.5% SDS solution and the amount of mineralized surface per well was determined by measuring the OD of the Alizarin red-stained matrix via spectrophotometer at 425 nm against a standard curve.…”

“…(17) Recent studies suggest a crucial role of miRNAs in bone development and homeostasis (18) and have found close association between (serum) miRNA dysregulation and bone diseases, including osteoporosis. (19)(20)(21) There is also first, limited evidence that miRNAs may be involved in the pathophysiology of T2D. (22) However, to date, no data are available on the role of miRNAs in diabetic bone disease and fragility.…”

Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis

“…For osteogenic differentiation, cells were reseeded at 10 5 cells/cm 2 and induced in osteogenic induction medium (OIM) [DMEM supplemented with 10% FBS, 0.1 μM dexamethasone (Dex, Sigma, USA), 50 μM ascorbate acid (Sigma, USA), and 10 mM β-glycerophosphate sodium (Sigma, USA)] which contained a low dose of Dex to promote osteogenic differentiation (high concentrations of Dex inhibit osteogenic differentiation) [15,16] under 20% O 2 or 2% O 2 conditions. After induction in OIM, cells were fixed and then evaluated by Alizarin red-S staining and ALP staining to show osteogenic differentiation.…”