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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

(a) Enhancement of hMSC proliferation using US. MSCs were grown on glass coverslips for 4 days in M2 medium with and without US (14 kPa; 3 min; 1, 2, or 4 times/day). Cells were fixed and stained for Ki67 (mitosis marker) and Hoechst (nuclear marker). Five pictures were randomly taken on three coverslips per condition (n = 15), and Ki67 and Hoechst positive cells were counted using ImageJ™. Data were expressed as percent Ki67 positive. (b) hMSC proliferation is increased with US application. hMSC-seeded scaffolds were cultured in CDM for 14 days and in M3 media for another 7 days in the US-assisted bioreactor according to the culture conditions outlined in Table 2. dsDNA was assayed by Picogreen assay. Data were presented as average ± standard deviation (n = 5–8 constructs). Statistically significant data were accounted with respect to respective control and shown as *p < 0.05, **p < 0.01, and ***p < 0.001.
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fig3-2041731414566529: (a) Enhancement of hMSC proliferation using US. MSCs were grown on glass coverslips for 4 days in M2 medium with and without US (14 kPa; 3 min; 1, 2, or 4 times/day). Cells were fixed and stained for Ki67 (mitosis marker) and Hoechst (nuclear marker). Five pictures were randomly taken on three coverslips per condition (n = 15), and Ki67 and Hoechst positive cells were counted using ImageJ™. Data were expressed as percent Ki67 positive. (b) hMSC proliferation is increased with US application. hMSC-seeded scaffolds were cultured in CDM for 14 days and in M3 media for another 7 days in the US-assisted bioreactor according to the culture conditions outlined in Table 2. dsDNA was assayed by Picogreen assay. Data were presented as average ± standard deviation (n = 5–8 constructs). Statistically significant data were accounted with respect to respective control and shown as *p < 0.05, **p < 0.01, and ***p < 0.001.

Mentions: In an attempt to isolate the impact of US-induced MSC chondrogenesis from growth factors and to ascertain the chondro-inductive ability of US at 5.0 MHz, MSC media (M2, no exogenous growth factors) was used to culture hMSC pellets, MSCs encapsulated in Glycosan, and MSCs seeded on macroporous BM scaffolds in these initial sets of experiments. The SOX-9 gene expression was assessed and shown in Figure 2. In comparison to controls (day-0), MSC pellets had a 7-fold higher expression of SOX-9 and MSC pellets exposed to US stimulation had an 11-fold higher expression of SOX-9. MSC-Glycosan exposed to US stimulation had 2.4-fold higher gene expression of SOX-9 when compared to unstimulated MSCs encapsulated in Glycosan, where MSC-Glycosan had a 13-fold higher expression compared to day-0 control. SOX-9 gene expression levels were similar between day-0 controls and unstimulated cell-seeded BM constructs; however, an 11-fold higher gene expression of SOX-9 was noted on US-stimulated cell-seeded BM constructs. Inserts show the protein expression of COL2A1 by western blotting in MSCs encapsulated in Glycosan and MSCs seeded in BM constructs. The successful differentiation of MSCs to chondrocytes in pellet cultures in the presence of TGFβ3 (10 ng/mL) is also demonstrated. Irrespective of the cellular microenvironment, Figure 2 shows that SOX-9 (a type-II collagen transcription factor) expression was enhanced by US, in the absence of exogenously added growth factors. The ability of US to impact hMSC proliferation in the absence of TGFβ3 was also evaluated and depicted in Figure 3(a). Our results indicate that US impacts hMSC proliferation in the absence of exogenously added growth factors. Although the standard method of pellet culture for in vitro MSC chondrogenesis is effective and mimics the MSC condensation phase in the limb bud (i.e. high density and cell-to-cell contact), the requirement for pellet culture makes scale-up problematic in addition to the limitations in pellet size, diffusional limitations, and hypertrophy in the pellet core. As an alternative to pellet cultures, hydrogels have been developed as they can provide higher cell density upon encapsulation and mimic the initial phase of mesenchymal condensation. Indeed, our results with MSC-Glycosan show that the gene expression of SOX-9 was comparable to pellet cultures, with additional increase in SOX-9 gene expression upon US stimulation.


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

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

(a) Enhancement of hMSC proliferation using US. MSCs were grown on glass coverslips for 4 days in M2 medium with and without US (14 kPa; 3 min; 1, 2, or 4 times/day). Cells were fixed and stained for Ki67 (mitosis marker) and Hoechst (nuclear marker). Five pictures were randomly taken on three coverslips per condition (n = 15), and Ki67 and Hoechst positive cells were counted using ImageJ™. Data were expressed as percent Ki67 positive. (b) hMSC proliferation is increased with US application. hMSC-seeded scaffolds were cultured in CDM for 14 days and in M3 media for another 7 days in the US-assisted bioreactor according to the culture conditions outlined in Table 2. dsDNA was assayed by Picogreen assay. Data were presented as average ± standard deviation (n = 5–8 constructs). Statistically significant data were accounted with respect to respective control and shown as *p < 0.05, **p < 0.01, and ***p < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig3-2041731414566529: (a) Enhancement of hMSC proliferation using US. MSCs were grown on glass coverslips for 4 days in M2 medium with and without US (14 kPa; 3 min; 1, 2, or 4 times/day). Cells were fixed and stained for Ki67 (mitosis marker) and Hoechst (nuclear marker). Five pictures were randomly taken on three coverslips per condition (n = 15), and Ki67 and Hoechst positive cells were counted using ImageJ™. Data were expressed as percent Ki67 positive. (b) hMSC proliferation is increased with US application. hMSC-seeded scaffolds were cultured in CDM for 14 days and in M3 media for another 7 days in the US-assisted bioreactor according to the culture conditions outlined in Table 2. dsDNA was assayed by Picogreen assay. Data were presented as average ± standard deviation (n = 5–8 constructs). Statistically significant data were accounted with respect to respective control and shown as *p < 0.05, **p < 0.01, and ***p < 0.001.
Mentions: In an attempt to isolate the impact of US-induced MSC chondrogenesis from growth factors and to ascertain the chondro-inductive ability of US at 5.0 MHz, MSC media (M2, no exogenous growth factors) was used to culture hMSC pellets, MSCs encapsulated in Glycosan, and MSCs seeded on macroporous BM scaffolds in these initial sets of experiments. The SOX-9 gene expression was assessed and shown in Figure 2. In comparison to controls (day-0), MSC pellets had a 7-fold higher expression of SOX-9 and MSC pellets exposed to US stimulation had an 11-fold higher expression of SOX-9. MSC-Glycosan exposed to US stimulation had 2.4-fold higher gene expression of SOX-9 when compared to unstimulated MSCs encapsulated in Glycosan, where MSC-Glycosan had a 13-fold higher expression compared to day-0 control. SOX-9 gene expression levels were similar between day-0 controls and unstimulated cell-seeded BM constructs; however, an 11-fold higher gene expression of SOX-9 was noted on US-stimulated cell-seeded BM constructs. Inserts show the protein expression of COL2A1 by western blotting in MSCs encapsulated in Glycosan and MSCs seeded in BM constructs. The successful differentiation of MSCs to chondrocytes in pellet cultures in the presence of TGFβ3 (10 ng/mL) is also demonstrated. Irrespective of the cellular microenvironment, Figure 2 shows that SOX-9 (a type-II collagen transcription factor) expression was enhanced by US, in the absence of exogenously added growth factors. The ability of US to impact hMSC proliferation in the absence of TGFβ3 was also evaluated and depicted in Figure 3(a). Our results indicate that US impacts hMSC proliferation in the absence of exogenously added growth factors. Although the standard method of pellet culture for in vitro MSC chondrogenesis is effective and mimics the MSC condensation phase in the limb bud (i.e. high density and cell-to-cell contact), the requirement for pellet culture makes scale-up problematic in addition to the limitations in pellet size, diffusional limitations, and hypertrophy in the pellet core. As an alternative to pellet cultures, hydrogels have been developed as they can provide higher cell density upon encapsulation and mimic the initial phase of mesenchymal condensation. Indeed, our results with MSC-Glycosan show that the gene expression of SOX-9 was comparable to pellet cultures, with additional increase in SOX-9 gene expression upon US stimulation.

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