A. Subramanian (Huntsville, US)
University of Alabama-Huntsville Chemical EngineeringPresenter Of 2 Presentations
12.1.9 - Continuous Low Intensity Ultrasound Enhances Cartilage Repair Outcomes Upon Microfracture
Abstract
Purpose
The lack of cues to guide the chondrogenic differentiation of egressed mesenchymal stromal cells (MSCs) can be attributed to the poor outcomes upon microfracture. This study evaluates the ability of cLIUS when applied at the tissue resonant frequency of 3.8 MHz to yield superior repair outcomes upon microfracture in a rabbit model. The ability of cLIUS to maintain chondroinductive and chondroprotective ability in a pro-inflammatory environment was studied.
Methods and Materials
Bilateral microfracture defects were created on the femoral medial condyle of female New Zealand white rabbits (n=12) and the left joint received cLIUS treatment (3.8 MHz, 3.8 Vpp, 8 minutes/application/day) and the contralateral right joint served as the control (Fig.1A). Rabbits were euthanized at 8-weeks and outcomes were assessed histologically. The ability of cLIUS to maintain MSC chondrogenesis in a proinflammatory milieu was evaluated by assaying for NF-kB pathway markers.
Results
Defect areas in the right joints exhibited irregular cartilage surface and loss of glycosaminoglycan (GAG). The defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte-morphology, and columnar organization (Fig.1B/D). Significantly higher O’Driscoll scores were obtained for the left knee joints (Fig. 1E). No adverse impact on bone or change in the joint space was noted (Fig. 1C). Synovial fluid collected from cLIUS treated left knee joints demonstrated lower levels of cytokines when compared to the synovial fluid untreated right knee joints (Fig. 1F). The presence of TNFα reduced the levels of SOX9 in the nucleus when compared to control and cLIUS samples (Fig. 2). cLIUS stimulation in the presence of TNFα inverted the localization of these markers, where the levels of nuclear pNFκB were decreased and SOX9 was upregulated.
Conclusion
Our results demonstrate that healing of chondral defects treated with microfracture can be accelerated by employing cLIUS regimen that possess chondroinductive and chondroprotective properties.
16.1.1 - Continuous Low-Intensity Ultrasound Preserves Chondrogenesis of MSCs and Mitochondrial Potential While Inhibiting NFκB Activation
Abstract
Purpose
Dysregulation of the anabolic processes in a proinflammatory joint environment coupled with impeded chondrogenic differentiation of mesenchymal stromal cells (MSCs) led to inferior cartilage repair outcomes. The preponderance of proinflammatory cytokines activated nuclear factor kappa B (NFκB) impedes the chondrogenesis of MSCs. Strategies that minimize the deleterious effects of activated NFκB while promoting MSC chondrogenesis are of interest. The present study establishes the ability of continuous low-intensity ultrasound (cLIUS) to preserve MSC chondrogenesis impacted by a proinflammatory environment.
Methods and Materials
MSCs were seeded in alginate: collagen hydrogels and cultured for 21-days in ultrasound (US)-assisted bioreactor 14 kPa (4-applications/day) in the presence of IL1β and evaluated by qRT-PCR and immunofluorescence (IF). The differential expression of markers associated with the NFκB pathway under cLIUS was evaluated upon a single exposure of cLIUS and assayed by qRT-PCR, IF, 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 (Fig.1B).
Results
Chondroinductive potential of cLIUS was preserved as noted by the increased expression of SOX9 and deposition of collagen II (Fig. 2A-I & III). cLIUS extended its chondroprotective effects by stabilizing the NFκB complex in the cytoplasm via engaging the IκBα feedback mechanism, thus preventing its nuclear translocation (Fig. 2A-II). cLIUS acted as a mitochondrial protective agent by restoring the mitochondrial potential and the mitochondrial mRNA expression in a proinflammatory environment (Fig. 2C-VI). Altogether, our results demonstrated the potential of cLIUS for cartilage repair and regeneration under proinflammatory conditions (Fig.1A).
Conclusion
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.