Developmentally inspired tissue engineering strategies are increasingly being employed to generate biomimetic articular cartilage (AC) grafts. One such approach leverages the capacity of stem or progenitor cells to self-organize and generate microtissues or organoids, which can then be used as biological building blocks to fabricate larger grafts of clinically relevant size. While human mesenchymal stem/stromal cells (hMSCs) can be used to generate cartilage-like microtissues, they are often fibrocartilaginous in nature and/or have an inherent tendency to become hypertrophic and progress along an endochondral pathway. In this study, a gene silencing approach was explored to engineer hyaline cartilage microtissues by delivering the prochondrogenic factor, antimicro ribonucleic acid 221 (anti-miR-221), using a polymeric nonviral vector. Effective silencing of micro ribonucleic acid 221 (miR-221) was observed for a range of doses, while selected anti-miR-221 concentrations supported type II collagen deposition while simultaneously suppressing the production of type X collagen within the cartilage microtissues. In addition, large numbers of such “silenced” chondrogenic microtissues could be fused into larger grafts, with the resulting constructs again showing no signs of early hypertrophy. To conclude, miR-221-silenced hMSCs support the development of hyaline cartilage microtissues rich in type II collagen, which could be used as in vitro models of AC or as biological building blocks in the engineering of scaled-up regenerative grafts.
Impact Statement
Ensuring stable chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) remains a major challenge in the field of articular cartilage tissue engineering. Here we investigated the delivery of the prochondrogenic factor, anti-miR-221, to hMSCs to support the generation of hyaline cartilage microtissues. The treatment improved the deposition of type II collagen while suppressing the deposition of type X collagen. This gene inhibition approach supports the development of microtissues as in vitro models of AC and their use in the engineering of scaled-up regenerative grafts.
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