Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Hybrid extracellular vesicles-liposome incorporated advanced bioink to deliver microRNA.

In additive manufacturing, bioink formulations govern strategies to engineer 3D living tissues that mimic the complex architectures and functions of native tissues for successful tissue regeneration. Conventional 3D-printed tissues are limited in their ability to alter the fate of laden cells. Specifically, the efficient delivery of gene expression regulators (i.e. microRNAs (miRNAs)) to cells in bioprinted tissues has remained largely elusive. In this study, we explored the inclusion of extracellular vesicles (EVs), naturally occurring nanovesicles (NVs), into bioinks to resolve this challenge. EVs show excellent biocompatibility, rapid endocytosis, and low immunogenicity, which lead to the efficient delivery of miRNAs without measurable cytotoxicity. EVs were fused with liposomes to prolong and control their release by altering their physical interaction with the bioink. Hybrid EVs-liposome (hEL) NVs were embedded in gelatin-based hydrogels to create bioinks that could efficiently encapsulate and deliver miRNAs at the target site in a controlled and sustained manner. The regulation of cells' gene expression in a 3D bioprinted matrix was achieved using the hELs-laden bioink as a precursor for excellent shape fidelity and high cell viability constructs. Novel regulatory factors-loaded bioinks will expedite the translation of new bioprinting applications in the tissue engineering field.

Understanding the impact of crosslinked PCL/PEG/GelMA electrospun nanofibers on bactericidal activity.

Herein, we report the design of electrospun ultrathin fibers based on the combination of three different polymers polycaprolactone (PCL), polyethylene glycol (PEG), and gelatin methacryloyl (GelMA), and their potential bactericidal activity against three different bacteria Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the morphology, chemical structure and wettability before and after UV photocrosslinking of the produced scaffolds. Results showed that the developed scaffolds presented hydrophilic properties after PEG and GelMA incorporation. Moreover, they were able to significantly reduce gram-positive, negative, and MRSA bacteria mainly after UV photocrosslinking (PCL:PEG:GelMa-UV). Furthermore, we performed a series of study for gaining a better mechanistic understanding of the scaffolds bactericidal activity through protein adsorption study and analysis of the reactive oxygen species (ROS) levels. Furthermore, the in vivo subcutaneous implantation performed in rats confirmed the biocompatibility of our designed scaffolds.