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Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds

View Article: PubMed Central - PubMed

ABSTRACT

Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters—excitation intensity, frequency and duty cycle—to find 30 mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering.

No MeSH data available.


Effects of 3-min LIPUS (30 mW/cm2;1.5 MHz; 20% duty cycle; 200 μs pulse length unless otherwise specified for each parameter variation) stimulation at varying (a) intensity, (b) duty cycle, (c) frequency and (d) excitation duration in the presence of 0.5% (v/v) MB suspension on day 1 (Data are mean ± StdEM, n = 9). Values significantly different from control group are indicated by *for p < 0.05 and **for p < 0.01.
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f6: Effects of 3-min LIPUS (30 mW/cm2;1.5 MHz; 20% duty cycle; 200 μs pulse length unless otherwise specified for each parameter variation) stimulation at varying (a) intensity, (b) duty cycle, (c) frequency and (d) excitation duration in the presence of 0.5% (v/v) MB suspension on day 1 (Data are mean ± StdEM, n = 9). Values significantly different from control group are indicated by *for p < 0.05 and **for p < 0.01.

Mentions: The optimal LIPUS excitation was determined by evaluating hMSC proliferation under various intensities (10, 30, 70, 100, 150 and 300 mW/cm2). Other acoustic parameters (1.5 MHz, 20% duty cycle and 200 μs pulse length) were kept the same. After 24-hour culture, hMSC proliferation was evaluated in response to 3 minutes of exposure to LIPUS in the presence of 0.5% MB suspension. As demonstrated in Fig. 6 (a), cell proliferation increased approximately by 20% at 30 mW/cm2 on day 1. At higher intensities, the proliferation diminished. The intensity was kept constant at 30 mW/cm2 for the rest of the studies. It is noteworthy to mention that our previous study showed that 100 mW/cm2 intensity resulted in the maximum hMSC proliferation rate without the presence of MBs (data not shown here). As expected, addition of MBs has lowered the energy threshold required for enhancing cell growth.


Lipid Coated Microbubbles and Low Intensity Pulsed Ultrasound Enhance Chondrogenesis of Human Mesenchymal Stem Cells in 3D Printed Scaffolds
Effects of 3-min LIPUS (30 mW/cm2;1.5 MHz; 20% duty cycle; 200 μs pulse length unless otherwise specified for each parameter variation) stimulation at varying (a) intensity, (b) duty cycle, (c) frequency and (d) excitation duration in the presence of 0.5% (v/v) MB suspension on day 1 (Data are mean ± StdEM, n = 9). Values significantly different from control group are indicated by *for p < 0.05 and **for p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121887&req=5

f6: Effects of 3-min LIPUS (30 mW/cm2;1.5 MHz; 20% duty cycle; 200 μs pulse length unless otherwise specified for each parameter variation) stimulation at varying (a) intensity, (b) duty cycle, (c) frequency and (d) excitation duration in the presence of 0.5% (v/v) MB suspension on day 1 (Data are mean ± StdEM, n = 9). Values significantly different from control group are indicated by *for p < 0.05 and **for p < 0.01.
Mentions: The optimal LIPUS excitation was determined by evaluating hMSC proliferation under various intensities (10, 30, 70, 100, 150 and 300 mW/cm2). Other acoustic parameters (1.5 MHz, 20% duty cycle and 200 μs pulse length) were kept the same. After 24-hour culture, hMSC proliferation was evaluated in response to 3 minutes of exposure to LIPUS in the presence of 0.5% MB suspension. As demonstrated in Fig. 6 (a), cell proliferation increased approximately by 20% at 30 mW/cm2 on day 1. At higher intensities, the proliferation diminished. The intensity was kept constant at 30 mW/cm2 for the rest of the studies. It is noteworthy to mention that our previous study showed that 100 mW/cm2 intensity resulted in the maximum hMSC proliferation rate without the presence of MBs (data not shown here). As expected, addition of MBs has lowered the energy threshold required for enhancing cell growth.

View Article: PubMed Central - PubMed

ABSTRACT

Lipid-coated microbubbles are used to enhance ultrasound imaging and drug delivery. Here we apply these microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5% (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters&mdash;excitation intensity, frequency and duty cycle&mdash;to find 30&thinsp;mW/cm2, 1.5&thinsp;MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering.

No MeSH data available.