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.
"While the effects of the OM supplements have been largely studied with BMSCs [15,19-21], there are only limited studies with ASCs [16,22,39]. One of the major shortcomings in all of these in vitro studies has been the lack of comparison between different serum conditions. "
[Show abstract][Hide abstract] ABSTRACT: Introduction
Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening, two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control.
To compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-based medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP).
In HS-based medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-based medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant upregulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-based medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1 to 3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1 to 3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3.
Serum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-based medium, although osteogenic markers were upregulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro.
[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 · 0.71 Impact Factor
"ASC are readily accessible, easily expandable and have low donor site morbidity; therefore, applying ASC as an alternative cell source for cartilage repair is an area of intense interest   . Most of our current research is focusing on studying the chondrogenic and osteogenic capacities of these cells      . Because of the low proliferation capability of explanted chondrocytes our group and others are investigating several in vitro chondrogenesis differentiation systems using ASC. "
[Show abstract][Hide abstract] ABSTRACT: Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.
Biochemical and Biophysical Research Communications 08/2007; 359(2):311-6. DOI:10.1016/j.bbrc.2007.05.098 · 2.30 Impact Factor
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