Abstract: BACKGROUND Osteoarthritis (OA) is marked by progressive cartilage degeneration and limited intrinsic repair. Mesenchymal stem cells (MSCs) show promise for cartilage regeneration, yet unstable chondrogenesis and a tendency towards hypertrophy constrain their efficacy. Dysregulated osteoarthritic microRNAs (miRNAs), including miR-16-5p, miR-30b-5p, miR-146a-5p, and miR-193b-3p, negatively influence chondrogenic programmes and matrix homeostasis. We hypothesized that ex vivo priming of MSCs with locked nucleic acid-antisense oligonucleotides (LNA-ASOs) targeting these anti-chondrogenic miRNAs would stabilize early chondrogenesis and enhance matrix formation.
AIM To determine whether transient ex vivo LNA-ASO silencing of OA-related miRNAs could improve the chondrogenesis of MSCs.
METHODS Rat bone-marrow MSCs were transfected with LNA-ASOs against miR-16-5p, miR-30b-5p, miR-146a-5p, and miR-193b-3p (single or combined) and induced into high-density micromass cultures. On day 11, quantitative real-time polymerase chain reaction assessed Sox9, Acan, Runx2, Mef2C. On day 21, histology (Safranin O, Alcian Blue/Fast Red, Masson?s trichrome, Picrosirius Red under polarised light) and DMMB (sulphated glycosaminoglycans normalised to dsDNA) quantified cartilage-like matrix. Transfection efficiency was measured by flow cytometry/confocal microscopy. Statistics compared each condition with a power inhibitor control.
RESULTS All LNA-ASOs achieved transfection efficiencies greater than 85%. The combination miR-30b-5p + miR-193b-3p produced the most favourable molecular profile at day 11, with higher Sox9 and Acan expression and lower Runx2 compared with the power inhibitor control, outperforming single-inhibitor conditions. miR-30b-5p alone and miR-30b-5p + miR-146a-5p also improved chondrogenic markers, whereas the miR-16-5p/miR-30b-5p combination showed a pattern prone to hypertrophy. At day 21, inhibition of miR-30b-5p/miR-193b-3p yielded a stronger Safranin O and Alcian Blue staining and more abundant, better-organized collagen on Masson?s trichrome and Picrosirius Red. DMMB analysis (sulphated glycosaminoglycans normalised to dsDNA) was highest with miR-30b-5p + miR-193b-3p, consistent with the histology results.
CONCLUSION Brief ex vivo priming of MSCs with LNA-ASOs targeting anti-chondrogenic miRNAs promoted a hyaline-like programme and strengthened matrix deposition. Notably, the miR-30b-5p/miR-193b-3p pairing showed the most favourable profile, indicating promise for MSC-based strategies in cartilage regeneration.
