Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario.

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.

hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.

Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.

We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Bone tissue is known to possess an intrinsic regeneration potential. However, in cases of major injury, trauma, and disease, bone loss is present, and the regeneration potential of the tissue is often impaired. The process of bone regeneration relies on a complex interaction of molecules. MicroRNAs (miRNA) are small, non-coding RNAs that inhibit messenger RNAs (mRNA). One miRNA can inhibit several mRNAs and one mRNA can be inhibited by several miRNAs. Functionally, miRNAs regulate the entire proteome via the local inhibition of translation. In fact, miRNA modulation has been shown to be involved in several musculoskeletal diseases¹. In those pathologies, they modulate the transcriptional activity of mRNAs important for differentiation, tissue-specific activity, extracellular matrix production, etc. Because of their function in inhibiting translation, miRNAs are being researched in many diseases and are already being used for interventional treatment². Bone tissue and its related conditions have been widely investigated up to this day^1,3. This talk will focus on the relevancy of miRNAs to bone tissue, its homeostasis, and disease. After, examples will be given of how miRNAs can be used in bone regeneration and diseases such as osteoporosis and osteosarcoma. The use of miRNAs in both, detection and therapy will be discussed.