Suppression of osteoblast-related genes during osteogenic differentiation of adipose tissue derived stromal cells.

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p < 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p < 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p < 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications.

Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering.

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors.

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, ß-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.