Comparison of cation and anion-mediated resolution enhancement of bioprinted hydrogels for membranous tissue fabrication.

Fabrication of engineered thin membranous tissues (TMTs) presents a significant challenge to researchers, as these structures are small in scale, but present complex anatomies containing multiple stratified cell layers. While numerous methodologies exist to fabricate such tissues, many are limited by poor mechanical properties, need for post-fabrication, or lack of cytocompatibility. Extrusion bioprinting can address these issues, but lacks the resolution necessary to generate biomimetic, microscale TMT structures. Therefore, our goal was to develop a strategy that enhances bioprinting resolution below its traditional limit of 150 µm and delivers a viable cell population. We have generated a system to effectively shrink printed gels via electrostatic interactions between anionic and cationic polymers. Base hydrogels are composed of gelatin methacrylate type A (cationic), or B (anionic) treated with anionic alginate, and cationic poly-L-lysine, respectively. Through a complex coacervation-like mechanism, the charges attract, causing compaction of the base GelMA network, leading to reduced sample dimensions. In this work, we evaluate the role of both base hydrogel and shrinking polymer charge on effective print resolution and cell viability. The alginate anion-mediated system demonstrated the ability to reach bioprinting resolutions of 70 µm, while maintaining a viable cell population. To our knowledge, this is the first study that has produced such significant enhancement in extrusion bioprinting capabilities, while also remaining cytocompatible.

4D Bioprinting via Molecular Network Contraction for Membranous Tissue Fabrication.

Generation of thin membranous tissues (TMT), such as the cornea, epidermis, and periosteum, presents a difficult fabrication challenge in tissue engineering (TE). TMTs consist of several cell layers that are less than 100 µm in thickness per layer. While traditional methods provide the necessary resolution for TMT fabrication, they require significant handling and incorporation of several layers is limited. Extrusion bioprinting offers precise control over deposition of different biomaterials and cell populations within the same construct but lacks the resolution to generate biomimetic TMTs. For the first time, a 4D bioprinting strategy that allows for the generation of cell-laden TMTs is developed. Anionic gelatin methacrylate (GelMA) hydrogels are treated with cationic poly-l-lysine (PLL), which induces charge attraction, microscale network collapse, and macroscale hydrogel shrinking. The impact of shrinking on hydrogel properties, print resolution, and cell viability is presented. Additionally, this work suggests that a novel mechanism is occurring, where PLL exhibits a contractile force on GelMA and PLL molecular weight drives GelMA shrinking capabilities. Finally, it is shown that this phenomenon can occur while maintaining an encapsulated cell population. These findings address a critical barrier by generating macroscale tissue structures with their microscale TMT counterparts in the same print.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.