Primary Human articular chondrocyte isolation and miR-199a-5p or miR-199b-5p level manipulation
Objective  Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here we aimed to compare the cartilage transcriptome responses of a non-surgical, mechanically induced rupture of the anterior cruciate ligament (ACL) model and the surgical destabilisation of the medial meniscus (DMM) model.   Methods  Skeletally mature male C57Bl6 mice were subjected to either the non-surgical, mechanical ACL rupture or surgical DMM models and transcriptome profiling performed on micro-dissected cartilage at day 7 and 42 post-procedure, respectively; in general, naïve animals served as controls. MicroRNA profiling was also performed on the ACL rupture model. Expression levels of a miRNA of interest, miR-199-5p, were inhibited in primary human articular chondrocytes (HAC) with RNA-seq and 3'UTR assays used to identify and valid potential target genes.    Results  The number of differentially expressed genes between the two models were comparable and highly correlative (Spearman R =0.8, P<2.2E-16).  Gene ontology enrichment analysis identified similarly enriched pathways, containing anabolic terms including 'extracellular matrix organisation' enriched for the upregulated genes. Within the ACL rupture miRNA transcriptome, miR-199-5p family members were amongst the most abundantly, and differentially expressed, which was replicated in the DMM cartilage by qRT-PCR. Inhibition of miR-199-5p in HAC led to a comparable transcriptome response to that observed in both human OA damaged vs intact cartilage and murine DMM cartilage datasets. Several genes, including GIT1, NCEH1, SOS2 and ECE1 were all experimentally verified as targets.  Conclusion  For the first time, we have characterised both the mRNA and miRNA articular cartilage signature in the ACL rupture model and demonstrated highly correlative responses with the DMM PTOA model. These data support the use of the ACL rupture model as a non-invasive alternative to DMM. Overall design: Human articular chondrocyte (HAC) were isolated from knee cartilage by enzymatic digestion (briefly, macroscopically normal cartilage was removed from the subchondral bone and dissected into ~1mm pieces using scalpel and forceps. Enzymatic digestion was performed using hyaluronidase, trypsin and then collagenase overnight at 37oC).  Tissue was donated by four patients (CIMA55, CIMA56, CIMA64, CIMA67) with diagnosed osteoarthritis and undergoing joint replacement surgery (age 59-85; 3 female, 1 male) with informed consent and ethics committee approval (REC 19/LO/0389). Briefly, macroscopically normal cartilage was removed from the subchondral bone and dissected into ~1mm pieces using scalpel and forceps. Enzymatic digestion was performed using hyaluronidase, trypsin and then collagenase overnight at 37oC (28).  For modulation of miR-199 levels in HAC, Dharmacon miRIDIAN hairpin inhibitor against miR-199a-5p or miR-199b-5p or Dharmacon miRIDIAN miRNA hairpin inhibitor nontargeting Control #2 (IN-002005-01) were transfected into 40%–50% confluent HAC using Dharmafect 1 lipid reagent (Horizon Discovery) at 100 nM final concentration.  24 hours later RNA was isolated following the miRVana protocol, quality assessed, and cDNA libraries generated using the Illumina TruSeq Stranded mRNA protocol and sequenced on an Illumina NextSeq500 instrument. Kallisto, Tximport and DeSeq2 were used for analysis as described above