Enhancing Meniscus Regeneration through Mechanical Priming of Fibrochondrocyte-Derived Extracellular Vesicles

Introduction: Meniscus tears, which affect approximately 65 per 100,000 individuals, are a leading cause of knee pain [1]. Altered joint mechanics and synovial environment following injury contribute to the development of osteoarthritis (OA) in nearly two-thirds of meniscus tear patients [2]. Poor innate healing capacity of the tissue and inadequate clinical treatments necessitate the development of innovative therapies to enhance endogenous meniscus repair. Extracellular vesicles (EVs) are nanoscale membrane-bound bodies secreted by all cell types that serve as mediators of cell-cell communication, and show promise as novel therapeutic agents [4]. However, how mechanical cues influence EV biogenesis and secretion from meniscus cells and the effects of these EVs on neighboring cells remain unknown. In this study, we investigate a new method of “priming” EVs by subjecting meniscal fibrochondrocyte (MFC)-laden scaffolds to cyclic tensile loads. Additionally, we aim to elucidate whether these “mechanically primed” EVs stimulate recipient mesenchymal stem cells (MSCs, a promising cell source for meniscus engineering [3]) to promote endogenous tissue regeneration.

Materials and

Methods: Outer zone MFCs and bone marrow MSCs were isolated from juvenile (~3 mo) bovine knee joints [3]. Aligned nanofibrous polycaprolactone (PCL)-based scaffolds were prepared according to previously established protocols [3] and subsequently seeded with MFCs. MFC-laden scaffolds were pre-cultured for 2 days before undergoing dynamic loading in a custom bioreactor (Fig. 1A) [5]. Scaffolds were loaded at 1 Hz and 3% strain in media containing exosome-depleted FBS (Exo-D BM). To assess EV yield, scaffolds were either loaded for 3 hours (L) or remained free swelling (NL) in Exo-D BM. GW4869 (20µM) was added to cells for 24 hours prior to dynamic loading to inhibit EV biogenesis. Additional experiments included 1-hour loading either once (1dL) or repeated over 3 days (3dL). After completion of loading, scaffolds were processed for MFC mRNA isolation and EVs were isolated from cell culture media via differential ultracentrifugation [6]. Particle concentrations and size distributions were determined by nanoparticle tracking analysis (NTA). MSCs treated with donor-matched EVs (Fig. 2B) were assessed for cell viability using the CCK-8 assay. RT-qPCR was performed to determine expression of type I collagen (Col1), aggrecan (ACAN), and housekeeping gene GAPDH in both loaded MFCs and recipient MSCs. Fold change was calculated by the delta-delta-CT method. Statistical analyses were performed using Student’s t-test and one-way ANOVA.

Results, Conclusions, and Discussions: Dynamic mechanical loading for 3 hours significantly increased EV yield compared to free swelling controls (Fig. 1B). NTA results demonstrate consistent particle size distribution between mechanically primed and control EVs (Fig 1C), suggesting dynamic loading as a promising strategy to increase EV yield, a key challenge in EV therapeutics. Treatment with GW4869, an ESCRT-independent EV biogenesis inhibitor, decreased overall EV yield (data not shown), but this effect was overcome with 3 hours of dynamic loading (Fig 1D), indicating that loading may enhance EV yield via an ESCRT-dependent biogenesis pathway. MFCs subjected to 1 hour of dynamic loading for 1 (1dL) or 3 days (3dL) showed increased aggrecan expression (Fig. 2A). Interestingly, MSCs treated with EVs secreted by these cells also exhibited a significant increase in aggrecan expression (Fig. 2C), suggesting that mechanically primed EVs influence MSC differentiation. Notably, culturing MSCs with EVs did not affect MSC viability or proliferation (Fig 2D), indicating the safety and biocompatibility of EVs. Overall, these findings suggest that dynamic loading stimulates EV biogenesis, and that EVs from loaded cells induce similar gene expression profiles in recipient cells. Ongoing studies focus on further characterizing mechanically primed EVs through proteomic and small RNA-seq analyses and elucidating the mechanism by which dynamic loading enhances EV production.

Acknowledgements (Optional):

References: [1] Fox+ 2015, Clinical Anatomy; [2] Riera+ 2011, Arthritis Research & Therapy; [3] Baker+ 2007 Biomaterials; [4] Magdy+ 2020, Applied Sciences 10; [5] Heo+ 2016, eLife; [6] Théry+ 2006, Curr. Protoc. Cell Biol.

This work is supported by NIH P50 AR080581 and NSF CMMI 1548571.