Functions of vitamin D, retinoic acid, and dexamethasone in mouse adipose-derived mesenchymal cells.
ABSTRACT Adipose-derived mesenchymal cells (AMCs) offer great promise for tissue engineering of bone. Previously, 1,25-dihydroxyvitamin D3, retinoic acid (RA), and dexamethasone had been shown to promote osteogenesis in bone marrow-derived mesenchymal cells (BMSCs). To study the osteogenic characteristics of mouse AMCs, we applied these 3 hormones alone and in combination to the AMCs and examined markers of osteogenic differentiation. Interestingly, vitamin D and RA demonstrated a consistent, dose-dependent enhancement of osteogenesis and upregulated osteoblast specific markers including osteopontin and osteocalcin. However, in AMCs, dexamethasone clearly inhibited osteogenic differentiation in a dose dependent fashion and greatly increased the adipogenic marker peroxisome proliferator activated receptor gamma (PPAgamma). In summary, we show in vitro that vitamin D and RA are potential candidates to serve as enhancers of osteogenesis of AMCs and may be incorporated into future cell-based strategies for bone tissue engineering.
SourceAvailable from: Aušra Unguryte[Show abstract] [Hide abstract]
ABSTRACT: More effective techniques should be employed for isolation of human mesenchymal stromal cells derived from adipose tissue (ADSC), seeking to make adipose tissue biopsies smaller in volume and thus less invasive. In this study, we compared properties of ADSC isolated by several different methods from the same samples of adipose tissue in order to enhance yields of potential ADSC. The mature adipocyte fraction was investigated using the ceiling culture method, including both ceiling and bottom cell fractions, and the control culture method with standard amount of medium. The results were also compared using the stromal vascular fraction from the same samples. The most efficient was the bottom cell population isolated from the mature adipocyte fraction by ceiling culture method. These cells readily differentiated into osteogenic, adipogenic and chondrogenic lineages and, similar to stromal vascular fraction cells, displayed high proliferation potential. Cultures of mature adipocyte fractions with standard amount of medium were considerably less effective. Mature adipocyte fractions yields large quantities of adipose-derived stem cells that have properties comparable with stromal vascular fraction cells suitable for tissue regeneration, especially when only small biopsies can be taken.Central European Journal of Biology 02/2010; 5(1):47-58. DOI:10.2478/s11535-009-0073-6 · 0.63 Impact Factor
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ABSTRACT: An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.PLoS ONE 12/2013; 8(12):e82388. DOI:10.1371/journal.pone.0082388 · 3.53 Impact Factor
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ABSTRACT: The aim of this study was to develop and test a controlled delivery system of two adipogenic factors, (insulin and dexamethasone), to generate stable adipose tissue when mixed with disaggregated human fat. Both drugs were encapsulated in poly(lactic-co-glycolic acid), (PLGA) microspheres and mixed with human lipoaspirate to induce adipogenesis in vivo. It was hypothesized that the slow release of insulin and dexamethasone would enhance both adipogenesis and angiogenesis, thus retaining the fat graft volume in a nude mouse model. Insulin/dexamethasone-loaded PLGA microspheres (Insulin/Dex MS) were prepared using both single and double emulsion/solvent extraction techniques. The bioactivity of the drugs was assessed by mixing the microspheres with human lipoaspirate and injecting subcutaneously into the dorsal aspect of an athymic mouse. Five doses of the drugs were examined and samples were analyzed grossly and histologically after 5 weeks in vivo. Mass and volume of the grafts were measured, with the microsphere-containing samples demonstrating increased mass and volume with increasing drug dose. Histological analysis including H&E and CD31 indicated increased vascularization within the insulin/dex MS-containing samples compared to the lipoaspirate only samples. This study demonstrates that the controlled delivery of adipogenic factors such as insulin and dexamethasone via polymer microspheres can significantly enhance tissue formation and vascularization, therefore presenting a potentially clinically relevant model of adipose retention.Tissue Engineering Part A 03/2014; 20(17-18). DOI:10.1089/ten.TEA.2012.0701 · 4.64 Impact Factor