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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

Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ™ software and relative protein expression levels were computed with respect to β-actin.
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fig8-2041731414566529: Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ™ software and relative protein expression levels were computed with respect to β-actin.

Mentions: The protein expression of ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9), and COL10A1 were assayed by western blotting and shown in Figure 8. COL10A1 was undetectable. When compared to groups 1 and 2, group 3 showed higher levels of COL2A1 and ACAN. COL1A1 expression was similar between all groups. Protein expression of both SOX-9 and p-SOX-9 was slightly higher in group 3 compared to groups 2 and 1. Our combined gene and protein expression analyses at the end of day 14 indicate that US is able to mediate chondrogenesis of hMSCs seeded on BM scaffolds and this likely happens via SOX-9 mediated pathways.


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

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

Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ™ software and relative protein expression levels were computed with respect to β-actin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig8-2041731414566529: Analysis of protein expression. Cell-seeded scaffolds were randomly collected, trypsinized, lysed with IP lysis buffer—1X Halt protease and phosphatase inhibitor cocktail and cell lysates were subjected to SDS-PAGE followed by western blotting. (a) In separate experiments, the membranes were incubated with primary antibodies against ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9). β-Actin was used as the loading control. (b) All protein bands were quantified by ImageJ™ software and relative protein expression levels were computed with respect to β-actin.
Mentions: The protein expression of ACAN, COL1A1, COL2A2, COL9A1, SOX-9, p-SOX-9 (i.e. p-S181-SOX-9), and COL10A1 were assayed by western blotting and shown in Figure 8. COL10A1 was undetectable. When compared to groups 1 and 2, group 3 showed higher levels of COL2A1 and ACAN. COL1A1 expression was similar between all groups. Protein expression of both SOX-9 and p-SOX-9 was slightly higher in group 3 compared to groups 2 and 1. Our combined gene and protein expression analyses at the end of day 14 indicate that US is able to mediate chondrogenesis of hMSCs seeded on BM scaffolds and this likely happens via SOX-9 mediated pathways.

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