Continuous Low-Intensity Ultrasound Improves Cartilage Repair in Rabbit Model of Subchondral Injury

Subchondral drilling (SD), a bone marrow stimulation technique, is used to repair cartilage lesions that lack regenerative potential. Cartilage repair outcomes upon SD are typically fibrocartilaginous in nature with inferior functionality. The lack of cues to foster the chondrogenic differentiation of egressed mesenchymal stromal cells upon SD can be attributed for the poor outcomes. Continuous low-intensity ultrasound (cLIUS) at 3.8 MHz is proposed as a treatment modality for improving cartilage repair outcomes upon marrow stimulation. Bilateral defects were created by SD on the femoral medial condyle of female New Zealand white rabbits (n = 12), and the left joint received cLIUS treatment (3.8 MHz, 3.5 Vpp, 8 min/application/day) and the contralateral right joint served as the control. On day 7 postsurgery, synovial fluid was aspirated, and the cytokine levels were assessed by Quantibody™ assay. Rabbits were euthanized at 8 weeks and outcomes were assessed macroscopically and histologically. Defect areas in the right joints exhibited boundaries, incomplete fill, irregular cartilage surfaces, loss of glycosaminoglycan (GAG), and absence of chondrocytes. In contrast, the repaired defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte morphology, COL2A1 staining, and columnar organization. Synovial fluid collected from cLIUS-treated left knee joints had lower levels of IL1, TNFα, and IFNγ when compared to untreated right knee joints, alluding to the potential of cLIUS to mitigate early inflammation. Further at 8 weeks, left knee joints (n = 12) consistently scored higher on the O'Driscoll scale, with a higher percent hyaline cartilage score. No adverse impact on bone or change in the joint space was noted. Upon a single exposure of cLIUS to TNFα-treated cells, nuclear localization of pNFκB and SOX9 was visualized by double immunofluorescence and the expression of markers associated with the NFκB pathway was assayed by quantitative real-time polymerase chain reaction. cLIUS extends its chondroprotective effects by titrating pNFκB levels, preventing its nuclear translocation, while maintaining the expression of SOX9, the collagen II transcription factor. Our combined results demonstrate that healing of chondral defects treated with marrow stimulation by SD can be accelerated by employing cLIUS regimen that possesses chondroinductive and chondroprotective properties. Impact statement Repair of cartilage represents an unsolved biomedical burden. In vitro, continuous low-intensity ultrasound (cLIUS) has been demonstrated to possess chondroinductive and chondroprotective potential. To our best knowledge, the use of cLIUS to improve cartilage repair outcomes upon marrow stimulation, in vivo, has not been reported and our work reported here fills that gap. Our results demonstrated enhanced cartilage repair outcomes under cLIUS (3.8 MHz) in a rabbit model of subchondral injury by subchondral drilling. Enhanced repair stemmed from mesenchymal stem cell differentiation in vivo and the subsequent synthesis of articular cartilage-specific matrix.