Page 201 - IJB-10-2

International Journal of Bioprinting Optimizing inkjet bioprinting

an increase in cell concentration results in reduced impact Another study has suggested the utilization of porous
velocity of the ejected droplets, ultimately enhancing the hydrogels created from aqueous two-phase systems (ATPS),
viability of the printed cells and eliminating the occurrence which consist of photocrosslinkable gelatin methacryloyl
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of droplet splashing. (GelMA) and dextran. The phase behavior, either
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monophasic or biphasic, can be adjusted by controlling the
6. Applications pH and dextran concentration. The manipulation of these
two parameters leads to the development of hydrogels
6.1. Incorporating biophysical and biological cues with three distinct microstructures: homogeneous
within 3D hydrogel matrices nonporous (NOP), regular disconnected pores (RDP),
The utilization of inkjet bioprinting offers a valuable and bicontinuous interconnected pores (ICP). Notably, the
method for precise DOD deposition of cell-laden droplets sizes of the pores in the latter two types of hydrogels can
and manipulation of desired cell density by controlling be finely tuned within the range of 4–100 µm by adjusting
the number of cell-laden droplets at specific locations. A the pH and dextran concentration, ensuring a high level of
printable bio-ink has stringent requirements for its physical reproducibility in the microstructure. Various types of cells
properties in terms of viscosity, surface tension, and including human bone marrow-derived mesenchymal
density; the printed cells are usually encapsulated within stem cells (hMSC), human periodontal ligament fibroblasts
low-viscosity bio-inks with viscosities ranging from 3 to 10 (hPDLF), and human neuroblastoma (hNB) cells were used
mPa·s. Various hydrogel-based bio-inks are employed for to investigate how the microenvironment influences cellular
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diverse bioprinting applications; the hydrogel-based bio- behavior. The ICP hydrogels, with the largest pore sizes
ink comprises a 3D polymeric network with a significant ranging from 40 to 60 µm, facilitated a more homogeneous
water content and provides a 3D microenvironment that distribution of cells and the formation of a dense network
closely mimics the natural extracellular matrix (ECM). of cell-to-cell connections for both hMSC and hPDLF.
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The biomimetic 3D microenvironment plays a critical In contrast, hNB cells maintained their characteristic
role in providing the necessary signals and guiding rosette-like organization with minimal or no spreading,
cellular responses to facilitate proper tissue development even in the presence of interconnected pores within the
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and function; 88-90 some important considerations for hydrogels. Another work delved into the impact of 3D
fabricating such biomimetic 3D microenvironment matrix stiffness on potential alterations in the morphology
include the incorporation of biophysical (porosity/pore of stem cells and their subsequent differentiation
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size and mechanical) and biological (growth factors) cues capabilities. By combining thermo-responsive agarose
within the 3D hydrogel matrices. hydrogels with collagen type I, it was possible to enhance
the mechanical stiffness and precision of the contours in
In the context of 3D hydrogel matrices used in in vitro printed constructs compared to pure collagen hydrogels,
tissue engineering, a significant concern revolves around which are commonly used as the standard materials for
the limited porosity of most hydrogels. This limited porosity differentiating hMSC into osteogenic lineages. The hMSC
can constrain the mobility and spreading of encapsulated exhibited the highest degree of spreading and branching
cells. To address these limitations, PVP macromolecule- when encapsulated within the softest 3D hydrogels,
based bio-ink was used to modulate the collagen whereas those encapsulated within the rigid 3D matrix
fibrillogenesis process and fabricate 3D hierarchical maintained a spherical morphology. Although further
porous collagen-based hydrogel constructs. Increasing analysis revealed that the stiffness of the 3D hydrogel
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the number of printed PVP droplets led to the formation has no significant effect on calcium accumulation as
of thicker collagen fibers and larger pore sizes within the indicated by Alizarin Red staining or alkaline phosphatase
3D collagen matrices. This effect occurred because an (ALP) activity, there was an increase in the expression of
increased PVP concentration has a more pronounced osteogenic genes (COL1 and RUNX2) in the softer 3D
excluded volume effect (EVE) on the surrounding collagen matrix. As a result, the study concluded that the preferable
fibrils during the fibrillogenesis process (Figure 5). conditions for hMSC osteogenic differentiation were found
Additionally, the study noted a slower growth rate for the in less stiff agarose-collagen hydrogels that did not hinder
human skin fibroblasts within 3D collagen matrices with cell spreading and branching.
larger pore sizes, aligning with findings from previous To date, there are limited studies that investigated
studies that indicated that larger pores generally lead to a the influence of growth factor patterning within 3D
reduction in cell proliferation rates. 92,93 Further analysis matrix. This is a key research area that is valuable for
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also revealed that 3D collagen matrices with larger pore translating 3D growth factor patterning into potential in
sizes exhibited higher fibronectin deposition. vivo applications. A study utilized inkjet bioprinting to

Volume 10 Issue 2 (2024) 193 doi: 10.36922/ijb.2135