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Cell aggregates are widely used to study heterotypic cellular interactions during the development of vascularization in vitro. In this study, we examined heterotypic cellular spheroids made of adipose-derived stem cells and CD34+/CD31− endothelial progenitor cells induced by the transfection of miR-148b mimic for de novo induction of osteogenic differentiation and miR-210 mimic for de novo induction of endotheliogenesis, respectively. The effect of the microRNA (miRs) mimic treatment group and induction time on codifferentiation was assessed in spheroids formed of transfected cells over the course of a 4-week culture. Based on gene and protein markers of osteogenic and endotheliogenic differentiation, as well as mineralization assays, our results showed that miRs directed cell differentiation and that progenitor maturity influenced the development of heterotypic cellular regions in aggregates. Overall, the success of coculture to create a prevascularized bone model is dependent on a number of factors, particularly the induction time of differentiation before combining the multiple cell types in aggregates. The approach that has been proposed could be valuable in creating vascularized bone tissue by employing spheroids as the building blocks of more complex issues through the use of cutting-edge methods such as 3D bioprinting. 

Abstract The engineering of osteochondral interfaces remains a challenge. MicroRNAs (miRs) have emerged as significant tools to regulate the differentiation and proliferation of osteogenic and chondrogenic formation in the human musculoskeletal system. Here, we describe a novel approach to osteochondral reconstruction based on the three-dimensional (3D) bioprinting of miR-transfected adipose-derived stem cell (ADSC) spheroids to produce a heterotypic interface that addresses the intrinsic limitations of the traditional approach to inducing zonal differentiation via the use of diffusible cytokines. We evaluated the delivery of miR-148b for osteogenic differentiation and the codelivery of miR-140 and miR-21 for the chondrogenic differentiation of ADSC spheroids. Our results demonstrated that miR-transfected ADSC spheroids exhibited upregulated expression of osteogenic and chondrogenic differentiation related gene and protein markers, and enhanced mineralization and cell proliferation compared to spheroids differentiated using a commercially-available differentiation medium. Upon confirmation of the osteogenic and chondrogenic potential of miR-transfected ADSC spheroids, using aspiration-assisted bioprinting, these spheroids were 3D bioprinted into a dual-layer heterotypic osteochondral interface with a stratified arrangement of distinct osteogenic and chondrogenic zones. The proposed approach holds great promise for the biofabrication of stratified tissues, not only for the osteochondral interfaces presented in this work, but also for other composite tissues and tissue interfaces, such as, but not limited to, the bone-tendon-muscle interface and craniofacial tissues. 

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore–quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.