Limits...
Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis.

Guha Thakurta S, Budhiraja G, Subramanian A - J Tissue Eng (2015)

Bottom Line: Expression of miR-145 was used as a metric to qualitatively assess the efficacy of human mesenchymal stem cell conversion.The combination of growth factor and ultrasound stimulation (group 3) resulted in enhanced COL2A1, SOX-9, and ACAN protein expression when compared to growth factor alone (group 2).No COL10A1 protein expression was noted.

View Article: PubMed Central - PubMed

Affiliation: Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA.

ABSTRACT
Ultrasound at 5.0 MHz was noted to be chondro-inductive, with improved SOX-9 gene and COL2A1 protein expression in constructs that allowed for cell-to-cell contact. To achieve tissue-engineered cartilage using macroporous scaffolds, it is hypothesized that a combination of ultrasound at 5.0 MHz and transforming growth factor-β3 induces human mesenchymal stem cell differentiation to chondrocytes. Expression of miR-145 was used as a metric to qualitatively assess the efficacy of human mesenchymal stem cell conversion. Our results suggest that in group 1 (no transforming growth factor-β3, no ultrasound), as anticipated, human mesenchymal stem cells were not efficiently differentiated into chondrocytes, judging by the lack of decrease in the level of miR-145 expression. Human mesenchymal stem cells differentiated into chondrocytes in group 2 (transforming growth factor-β3, no ultrasound) and group 3 (transforming growth factor-β3, ultrasound) with group 3 having a 2-fold lower miR-145 when compared to group 2 at day 7, indicating a higher conversion to chondrocytes. Transforming growth factor-β3-induced chondrogenesis with and without ultrasound stimulation for 14 days in the ultrasound-assisted bioreactor was compared and followed by additional culture in the absence of growth factors. The combination of growth factor and ultrasound stimulation (group 3) resulted in enhanced COL2A1, SOX-9, and ACAN protein expression when compared to growth factor alone (group 2). No COL10A1 protein expression was noted. Enhanced cell proliferation and glycosaminoglycan deposition was noted with the combination of growth factor and ultrasound stimulation. These results suggest that ultrasound at 5.0 MHz could be used to induce chondrogenic differentiation of mesenchymal stem cells for cartilage tissue engineering.

No MeSH data available.


Related in: MedlinePlus

miR-145 gene expression was quantified after 7, 14, and 21 days of culture according to culture conditions listed in Table 2. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene snU6. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05, **p < 0.01).qRT-PCR: quantitative real-time polymerase chain reaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
getmorefigures.php?uid=PMC4300305&req=5

fig6-2041731414566529: miR-145 gene expression was quantified after 7, 14, and 21 days of culture according to culture conditions listed in Table 2. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene snU6. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05, **p < 0.01).qRT-PCR: quantitative real-time polymerase chain reaction.

Mentions: miRNA gene regulation is often not a decisive on and off switch but a subtle function that fine-tunes cellular phenotypes.46 As miR-145 is significantly downregulated during MSC chondrogenesis and is silenced in differentiated MSCs,46 we have used miR-145 expression to track the differentiation process under US and TGFβ3. Figure 6 shows the differential expression of miRNA 145 during chondrogenic differentiation of hMSCs under US. Our results suggest that in group 1 (No TGFβ3, No US), as anticipated, hMSCs were not efficiently differentiated into chondrocytes, judging by the lack of decrease in the level of miR-145 expression. hMSCs differentiated into chondrocytes in group 2 (TGFβ3, No US) and group 3 (TGFβ3, US), with group 3 having a 2-fold lower miR-145 when compared to group 2 at day 7, indicating a higher conversion to chondrocytes. Overall, a 20% higher rate of conversion may be inferred based on a linear fit to the data.


Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis.

Guha Thakurta S, Budhiraja G, Subramanian A - J Tissue Eng (2015)

miR-145 gene expression was quantified after 7, 14, and 21 days of culture according to culture conditions listed in Table 2. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene snU6. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05, **p < 0.01).qRT-PCR: quantitative real-time polymerase chain reaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4300305&req=5

fig6-2041731414566529: miR-145 gene expression was quantified after 7, 14, and 21 days of culture according to culture conditions listed in Table 2. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene snU6. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05, **p < 0.01).qRT-PCR: quantitative real-time polymerase chain reaction.
Mentions: miRNA gene regulation is often not a decisive on and off switch but a subtle function that fine-tunes cellular phenotypes.46 As miR-145 is significantly downregulated during MSC chondrogenesis and is silenced in differentiated MSCs,46 we have used miR-145 expression to track the differentiation process under US and TGFβ3. Figure 6 shows the differential expression of miRNA 145 during chondrogenic differentiation of hMSCs under US. Our results suggest that in group 1 (No TGFβ3, No US), as anticipated, hMSCs were not efficiently differentiated into chondrocytes, judging by the lack of decrease in the level of miR-145 expression. hMSCs differentiated into chondrocytes in group 2 (TGFβ3, No US) and group 3 (TGFβ3, US), with group 3 having a 2-fold lower miR-145 when compared to group 2 at day 7, indicating a higher conversion to chondrocytes. Overall, a 20% higher rate of conversion may be inferred based on a linear fit to the data.

Bottom Line: Expression of miR-145 was used as a metric to qualitatively assess the efficacy of human mesenchymal stem cell conversion.The combination of growth factor and ultrasound stimulation (group 3) resulted in enhanced COL2A1, SOX-9, and ACAN protein expression when compared to growth factor alone (group 2).No COL10A1 protein expression was noted.

View Article: PubMed Central - PubMed

Affiliation: Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA.

ABSTRACT
Ultrasound at 5.0 MHz was noted to be chondro-inductive, with improved SOX-9 gene and COL2A1 protein expression in constructs that allowed for cell-to-cell contact. To achieve tissue-engineered cartilage using macroporous scaffolds, it is hypothesized that a combination of ultrasound at 5.0 MHz and transforming growth factor-β3 induces human mesenchymal stem cell differentiation to chondrocytes. Expression of miR-145 was used as a metric to qualitatively assess the efficacy of human mesenchymal stem cell conversion. Our results suggest that in group 1 (no transforming growth factor-β3, no ultrasound), as anticipated, human mesenchymal stem cells were not efficiently differentiated into chondrocytes, judging by the lack of decrease in the level of miR-145 expression. Human mesenchymal stem cells differentiated into chondrocytes in group 2 (transforming growth factor-β3, no ultrasound) and group 3 (transforming growth factor-β3, ultrasound) with group 3 having a 2-fold lower miR-145 when compared to group 2 at day 7, indicating a higher conversion to chondrocytes. Transforming growth factor-β3-induced chondrogenesis with and without ultrasound stimulation for 14 days in the ultrasound-assisted bioreactor was compared and followed by additional culture in the absence of growth factors. The combination of growth factor and ultrasound stimulation (group 3) resulted in enhanced COL2A1, SOX-9, and ACAN protein expression when compared to growth factor alone (group 2). No COL10A1 protein expression was noted. Enhanced cell proliferation and glycosaminoglycan deposition was noted with the combination of growth factor and ultrasound stimulation. These results suggest that ultrasound at 5.0 MHz could be used to induce chondrogenic differentiation of mesenchymal stem cells for cartilage tissue engineering.

No MeSH data available.


Related in: MedlinePlus