(25f) Continuous Low-Intensity Ultrasound Rescues Chondrogenesis of Mesenchymal Stromal Cells By Inhibiting NF?B Activation and Preserving Mitochondrial Potential

Background: Cartilage repair strategies that rely on mesenchymal stromal cells (MSCs) hold therapeutic promise. A cytokine-rich proinflammatory environment present in a diseased joint or in a surgically operated joint impedes chondrogenic differentiation of MSCs, leading to inferior cartilage repair outcomes (1). It is established that cytokines activate the inflammatory regulator, nuclear factor kappa B (NFκB), which inhibits the cartilage repair process by negatively impacting MSC chondrogenesis (2). Thus, clinically translatable strategies that minimize the deleterious effects of activated NFκB while promoting MSC chondrogenesis are of interest. Continuous low-intensity ultrasound (cLIUS), at the resonant frequency of 5 MHz and constant pressure amplitude of 14 kPa, has been established to induce the chondrogenic differentiation of MSCs, albeit in the absence of cytokine insults (3). There is an impetus to devise strategies to rescue MSC chondrogenesis (4,5). The focus of this work is to demonstrate the ability of cLIUS to rescue MSC chondrogenesis using an in vitro model of MSC chondrogenesis and show the ability of cLIUS to specifically deactivate the NFkB pathway.

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 IL1β and evaluated by qRT-PCR, immunofluorescence, western blotting (WB), and immunohistochemistry. The differential expression of markers associated with the NFκB 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 IL1β and cLIUS treatment. Study groups included appropriate non-cytokine treated and non-cLIUS treated controls.

Results: The gene expression of catabolic markers MMP13 and NFκB were significantly upregulated (>3-fold) in samples exposed to IL1β. 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 IL1β and treated with cLIUS (Fig. 1A and B). In contrast, samples treated with IL1β alone showed low levels of collagen II expression. Enhanced nuclear localization of NFκB (>50-fold) was observed in samples treated with IL1β, suggesting the activation of the NFκB pathway. The inclusion of cLIUS reduced the levels of nuclear NFκB to control levels, indicating the ability of cLIUS to deactivate the NFκB pathway (Fig. 1C). A western blot analysis of the key markers associated with the NFκB pathway indicated that cLIUS muted the expression of NFκB by overexpressing its inhibitor, total IκBa ( Fig. 1D). Furthermore, cLIUS restored the mitochondrial potential that was dampened by IL1β treatment (Fig. 1E). Thus, our work demonstrates that cLIUS is able to direct MSC chondrogenesis in the presence of cytokines by inactivating the NFκB 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.

References:

1. Kondo M, Yamaoka K, Tanaka Y. Acquiring chondrocyte phenotype from human mesenchymal stem cells under inflammatory conditions. Int J Mol Sci. 2014;15(11):21270-21285. doi:10.3390/ijms151121270.
2. Wojdasiewicz P, Poniatowski ŁA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators Inflamm. 2014;2014. doi:10.1155/2014/561459.
3. Sahu N, Budhiraja G, Subramanian A. Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways. Stem Cell Res Ther. 2020;11(1):1-15. doi:10.1186/s13287-019-1532-2.
4. Gao B, Gao W, Wu Z, et al. Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells. Stem Cell Res Ther. 2018;9(1):1-12. doi:10.1186/s13287-018-0892-3.
5. Armbruster N, Krieg J, Weißenberger M, Scheller C, Steinert AF. Rescued chondrogenesis of mesenchymal stem cells under interleukin 1 challenge by foamyviral interleukin 1 receptor antagonist gene transfer. Front Pharmacol. 2017;8(MAY):1-15. doi:10.3389/fphar.2017.00255.