Early microRNA and metabolite changes after anterior cruciate ligament reconstruction surgery

Objectives: Anterior cruciate ligament (ACL) reconstruction after injury does not prevent post-traumatic osteoarthritis (PTOA). Circulating microRNA (miRNA) and metabolite changes emerging shortly after ACL injury and reconstruction remain insufficiently defined, potentially harbouring early cues contributing to PTOA evolution. Moreover, their differential expression between females and males also may influence PTOA's natural trajectory. This study aims to determine alterations in plasma miRNA and metabolite levels in the early stages following ACL reconstruction and between females and males.

Methods: A cohort of 43 ACL reconstruction patients was examined. Plasma was obtained at baseline, 2 weeks, and 6 weeks post-surgery (129 biospecimens in total). High-throughput miRNA sequencing and metabolomics were conducted. Differentially expressed miRNAs and metabolites were identified using negative binomial and linear regression models, respectively. Associations between miRNAs and metabolites were explored using time and sex as co-variants, (pre-surgery versus 2 and 6 weeks post-surgery). Using computational biology, miRNA-metabolite-gene interaction and pathway analyses were performed.

Results: Levels of 46 miRNAs were increased at 2 weeks post-surgery compared to pre-surgery (baseline) using miRNA sequencing. Levels of 13 metabolites were significantly increased while levels of 6 metabolites were significantly decreased at 2 weeks compared to baseline using metabolomics. Hsa-miR-145-5p levels were increased in female subjects at both 2 weeks (log₂-fold-change 0.71, 95%CI 0.22,1.20) and 6 weeks (log₂-fold-change 0.75, 95%CI 0.07,1.43) post-surgery compared to males. In addition, hsa-miR-497-5p showed increased levels in females at 2 weeks (log₂-fold-change 0.77, 95%CI 0.06,1.48) and hsa-miR-143-5p at 6 weeks (log₂-fold-change 0.83, 95%CI 0.07,1.59). Five metabolites were decreased at 2 weeks post-surgery in females compared to males: L-leucine (-1.44, 95%CI -1.75,-1.13), g-guanidinobutyrate (-1.27, 95%CI 1.54,-0.99), creatinine (-1.17, 95%CI -1.44,-0.90), 2-methylbutyrylcarnitine (-1.76, 95%CI -2.17,-1.35), and leu-pro (-1.13, 95%CI -1.44,-0.83). MiRNA-metabolite-gene interaction analysis revealed key signalling pathways based on post-surgical time-point and in females versus males.

Conclusion: MiRNA and metabolite profiles were modified by time and by sex early after ACL reconstruction surgery, which could influence surgical response and ultimately risk of developing PTOA.