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

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.

Ultrasound Elastography for Estimation of Regional Strain of Multilayered Hydrogels and Tissue-Engineered Cartilage

“…10 Similar results have been reported for chick and human chondrocytes 4,34,40 and MSCs. 6,31 In general, in these studies, the intensity of the ultrasound was of similar or greater magnitude to that used here, while the duration of the ultrasound application was much longer than in our studies, e.g., 20–40 min/per day or longer vs. 1–2 min/day. 2,31,34,40 At a transducer center-frequency of nominally 20 MHz, we are also outside of the 5 MHz peak resonant frequency described for mammalian cells, thus minimizing ultrasound-mediated cellular stress.…”

“…2,31,34,40 At a transducer center-frequency of nominally 20 MHz, we are also outside of the 5 MHz peak resonant frequency described for mammalian cells, thus minimizing ultrasound-mediated cellular stress. 6,16 Therefore, we do not anticipate any major effects on tissue development through our intermittent US probing; if any, we would predict that they would be in line with the existing literature, i.e. , beneficial, and certainly do not expect a destructive effect.…”

Chondro-protective effects of low intensity pulsed ultrasound

“…LIPUS stimulation has also been shown to increase Col II expression in a rat OA model 23 and downregulate MMP-13, ERK1/2, and p38 in a rabbit OA model 24 . LIPUS also enhances chondrogenesis by increasing Col II, Sox9, and aggrecan expression in human mesenchymal stem cells 25 . Overall LIPUS shows both anabolic and anti-catabolic effects in settings of cartilage degradation.…”

Low‐Intensity Ultrasound Upregulates the Expression of Cyclin‐D1 and Promotes Cellular Proliferation in Human Mesenchymal Stem Cells

“…Differently from all approaches that employ low-intensity pulsed-ultrasound at 1.5-MHz, our published work has documented via theoretical modeling and experimental verification that US-induced bioeffects are maximized at the cell resonance frequency of 5.0-MHz and, proliferation as assessed by Ki67 was noted to be higher in US stimulated samples. [21] To establish the proliferative effects of ultrasound (US) at 5.0-MHz, in this study, first, the time-dependent proliferation of hMSCs under LIUS stimulation at 5.0-MHz was undertaken over a culture period of 14-days. The trilineage differential potential of hMSCs expanded under LIUS stimulation was also evaluated.…”