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Moldovan NI, et al.
Direct encapsulation of spheroids in polymeric and stability . This concept has been recently applied to
[79]
cages , or incorporation of fibrillary matrix fragments bioprinting, as a means to manipulate the porosity within
[71]
into spheroids [72] are other examples of hybrid multilayered collagen-based hydrogels, by altering the
biofabrication, with the potential to improve spheroid- collagen fibrillogenesis process through controlling the
based tissue engineering. For example, polycaprolactone number of macromolecule-based bioink droplets printed
was prepared as a fibrillary mesh by electrospinning, on each collagen layer .
[80]
then fragmented by limited hydrolysis. The resulting In summary, “hybrid biofabrication” is the cross-
material was added to mesenchymal stromal cells during pollination between approaches, methods, and materials
centrifugation-assisted spheroid preparation, becoming already common in this field as elsewhere in technology,
uniformly distributed within their volume. Consequently, which could be further expanded and exploited as
the resulting spheroids were less compact, and thus better a proven method to programmatically increase the
aerated and nurtured, as compared to their scaffold-free “vitality” (i.e., applicability and value generation) of the
counter-parts, which positively impacted on cell survival resulting products.
and osteogenic differentiation . Following the basic structure of tissues which contain
[72]
cells, extracellular matrix, and fluids, the composition of
4. Post-printing Considerations biofabricated tissue analogs may vary from biomaterials-
One of the big questions for all modes of biofabrication only, to cell suspensions, or cells-only constructs,
is what happens after the initial assembly of cells and with numerous intermediaries or “hybrid” situations
spheroids, namely how will the cells captured in scaffolds (e.g., spheroids in hydrogels or fibers in spheroids as
survive, differentiate and proliferate? And how will illustrated here). Correspondingly, these constructs can be
spheroids hold together: Will there be sufficient intrinsic used in a spectrum of applications ranging from inorganic
ECM produced to generate tissue-specific and tissue- biocompatible prostheses to live cell-based implants.
preserving connective tissue? In fact, the post-printing Macromolecular crowding agents can be also added to
ECM formation and remodeling is one of the biggest cell preparations to bring their environment closer to the
current challenges in biofabrication. ECM components natural density. The methods of bioprinting may also
are made by quite a variety of cells, but the immobilization come in these two versions, namely biomaterial-based or
and their firm deposition around the cells is a challenge in cell-based (“scaffold-free).”
standard aqueous culture conditions. One way to enhance Acknowledgments
ECM accumulation is the application of macromolecular
crowding (MMC). Of relevance to this discussion is the We would like to acknowledge support from Indiana
use of polydisperse additives, some akin to “particulate” Institute for Medical Research (NIM) and Richard L.
[73]
hydrogels, known MMC agents . Roudebush VA Medical Center (NIM, LM).
A special class of polymers with hydrogel-like properties,
consisting of smaller molecular weight compounds References
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