Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV were observed. After 56 days in culture, the bioprinted constructs were still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results showed a promising procedure to obtain 3D cartilage-like constructs that could be potential use as stable chondral tissue implants for future therapies.
Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates. The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture. Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes. RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment. RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes.
