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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

hMSC-seeded Biomerix™ scaffolds were cultured according to culture conditions listed in Table 2. (a) COL10A1 gene expression was presented after 7, 14, and 21 days of culture. (b) Expression for ACAN, TGFβ1, and TGFβ3 genes was shown at day 21. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene GAPDH. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05; **p < 0.01).
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fig7-2041731414566529: hMSC-seeded Biomerix™ scaffolds were cultured according to culture conditions listed in Table 2. (a) COL10A1 gene expression was presented after 7, 14, and 21 days of culture. (b) Expression for ACAN, TGFβ1, and TGFβ3 genes was shown at day 21. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene GAPDH. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05; **p < 0.01).

Mentions: The impact of US stimulation on the expression of COL10A1 gene as a function of culture duration was examined by qRT-PCR and is shown in Figure 7. On day 7, the gene expression of hypertrophic marker, COL10A1, was higher in group 2 (TGFβ3, No US) when compared to group 3 (TGFβ3, US) and considerably greater when compared to group 1 (No TGFβ3, No US). The gene expression of COL10A1 in group 1 stayed relatively unchanged during the course of this experiment. On day 21, groups 2 and 3 had similar levels of COL10A1 mRNA levels and were significantly different from mRNA levels in group 1. The relative gene expression of ACAN gene, a member of aggrecan/versican proteoglycan (ACAN), was also carried out. At day 21, compared to group 1, higher levels of ACAN expression were observed on groups 2 and 3; with group 3 presenting the highest gene expression of ACAN. No significant difference was noted between the groups with respect to the gene expression of TGFβ1.


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

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

hMSC-seeded Biomerix™ scaffolds were cultured according to culture conditions listed in Table 2. (a) COL10A1 gene expression was presented after 7, 14, and 21 days of culture. (b) Expression for ACAN, TGFβ1, and TGFβ3 genes was shown at day 21. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene GAPDH. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05; **p < 0.01).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig7-2041731414566529: hMSC-seeded Biomerix™ scaffolds were cultured according to culture conditions listed in Table 2. (a) COL10A1 gene expression was presented after 7, 14, and 21 days of culture. (b) Expression for ACAN, TGFβ1, and TGFβ3 genes was shown at day 21. Gene expression was analyzed by qRT-PCR and normalized to the expression of the house keeping gene GAPDH. Each bar represents the mean ± standard deviation (n = 3; *p < 0.05; **p < 0.01).
Mentions: The impact of US stimulation on the expression of COL10A1 gene as a function of culture duration was examined by qRT-PCR and is shown in Figure 7. On day 7, the gene expression of hypertrophic marker, COL10A1, was higher in group 2 (TGFβ3, No US) when compared to group 3 (TGFβ3, US) and considerably greater when compared to group 1 (No TGFβ3, No US). The gene expression of COL10A1 in group 1 stayed relatively unchanged during the course of this experiment. On day 21, groups 2 and 3 had similar levels of COL10A1 mRNA levels and were significantly different from mRNA levels in group 1. The relative gene expression of ACAN gene, a member of aggrecan/versican proteoglycan (ACAN), was also carried out. At day 21, compared to group 1, higher levels of ACAN expression were observed on groups 2 and 3; with group 3 presenting the highest gene expression of ACAN. No significant difference was noted between the groups with respect to the gene expression of TGFβ1.

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