Role of Ox-PAPCs in the Differentiation of Mesenchymal Stem Cells (MSCs) and Runx2 and PPARγ2 Expression in MSCs-Like of Osteoporotic Patients

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. METHODOLOGY: We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a "sentinel" that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). PRINCIPAL FINDINGS: Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. CONCLUSIONS: Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs

A Subset of Osteoblasts Expressing High Endogenous Levels of PPARγ Switches Fate to Adipocytes in the Rat Calvaria Cell Culture Model

Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)gamma.Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically) when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2), PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment.We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively mature developmental stages implies an unexpected plasticity with important implications in normal and pathological bone development

Bone Marrow Stromal Cell Assays: In Vitro and In Vivo

Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") contain a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). The biological properties of BMSC cultures are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provides a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in diseas

Osteocompatibility of Biofilm Inhibitors

The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics