(25f) Continuous Low-Intensity Ultrasound Rescues Chondrogenesis of Mesenchymal Stromal Cells By Inhibiting NF?B Activation and Preserving Mitochondrial Potential
AIChE Annual Meeting
Wednesday, November 10, 2021 - 3:48pm to 4:06pm
Methods: Human bone marrow-derived MSCs were seeded in alginate: collagen hydrogels and cultured for 21-days in an ultrasound-assisted bioreactor 14 kPa (5.0 MHz, 2.5 Vpp; 4-applications/day) for 21 days in the presence of IL1Î² and evaluated by qRT-PCR, immunofluorescence, western blotting (WB), and immunohistochemistry. The differential expression of markers associated with the NFÎºB pathway under cLIUS were evaluated upon a single exposure of cLIUS and assayed by qRT-PCR, immunofluorescence, WB, and tetramethylrhodamine methyl ester (TMRM) assay was used to assess the mitochondrial potential under IL1Î² and cLIUS treatment. Study groups included appropriate non-cytokine treated and non-cLIUS treated controls.
Results: The gene expression of catabolic markers MMP13 and NFÎºB were significantly upregulated (>3-fold) in samples exposed to IL1Î². The inclusion of cLIUS abrogated the expression of these selected markers (Fig. 1A). A 15-fold higher and a 70-fold higher expression of the master collagen II transcription factor SOX9 and collagen-II protein respectively was observed in samples exposed to IL1Î² and treated with cLIUS (Fig. 1A and B). In contrast, samples treated with IL1Î² alone showed low levels of collagen II expression. Enhanced nuclear localization of NFÎºB (>50-fold) was observed in samples treated with IL1Î², suggesting the activation of the NFÎºB pathway. The inclusion of cLIUS reduced the levels of nuclear NFÎºB to control levels, indicating the ability of cLIUS to deactivate the NFÎºB pathway (Fig. 1C). A western blot analysis of the key markers associated with the NFÎºB pathway indicated that cLIUS muted the expression of NFÎºB by overexpressing its inhibitor, total IÎºBa ( Fig. 1D). Furthermore, cLIUS restored the mitochondrial potential that was dampened by IL1Î² treatment (Fig. 1E). Thus, our work demonstrates that cLIUS is able to direct MSC chondrogenesis in the presence of cytokines by inactivating the NFÎºB pathway and protecting the mitochondrial potential that is needed for cellular function; schematically represented in Fig. 1F.
Conclusions: This study establishes the potential of cLIUS to improve and enhance outcomes of in vivo cartilage repair therapies. Translation of promising in vitro findings with cLIUS requires an understanding of the cLIUS propagation in the joint space along with optimal transducer settings. Current efforts are focused on establishing relevant mathematical models to allow for translation to small animal cartilage repair models to demonstrate the utility of cLIUS to improve cartilage repair outcomes.
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