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International Journal of Bioprinting Internally-crosslinked ADA/Alg/Gel bioinks

1. Introduction biopolymer thickens and forms gel bodies that remain in
the heart muscle as permanent internal support implants
3D bioprinting has radically transformed the manufacturing for the injured heart, preventing disease progression. 12,14
process of scaffolds for tissue engineering (TE) applications The device has been tested in three clinical trials: two were
by converting computer-aided design models into layered completed (ClinicalTrials.gov identifier: NCT00847964;
structures, bypassing traditional molding methods. Highly NCT01311791 [AUGMENT-HF]) and one has been
1
reproducible and customizable 3D-printed scaffolds published but is still not recruiting (ClinicalTrials.gov
can be employed as a potential approach to meet the identifier: NCT03082508 [AUGMENT-HF II]). However,
increasing global demand for organ replacement and tissue Algysil-LVR is defined as a permanent device that only
regeneration. In this scenario, the heart ranks as the third offers mechanical support to the left ventricle without
2,3
most transplanted organ worldwide, primarily because it providing full restoration of cardiac functionality. 12,13
represents the only therapeutic option in case of end-stage
heart failure due to its poor intrinsic regenerative capacity. One of the most interesting properties of Alg is the
In the last three decades, cardiac TE has emerged as a ability to complex with divalent cations, particularly
potential new therapeutic and in vitro modeling approach, calcium ions, to form hydrogels through physical
based on the use of biomaterials, eventually combined crosslinking under physiological conditions, forming an
15
with bioactive factors, with/without cells for regeneration. “egg box” model. Depending on the source of calcium
4
3D-bioprinted constructs can be exploited as therapeutic ions, two different modes of ionic crosslinking have been
patches to favor heart regeneration or as in vitro models investigated: internal and external methods. 16-18 External
5,6
of human cardiac tissue for preclinical validation of drugs gelation is characterized by the use of highly soluble
and therapies, in agreement with the 3Rs principles. 7 calcium salts (i.e., CaCl ) and consists of the diffusion
2
of multivalent cations from the outside into the Alg
Among 3D bioprinting techniques, microextrusion solution phase, forming a crosslinked Alg matrix. Most
bioprinting is based on the layer-by-layer deposition of studies on microextrusion bioprinting of Alg bioinks have
cell-laden bioinks to create intricate 3D structures. This exploited such external mechanism. 19-21 The use of external
technique has emerged as a promising tool in TE, owing to crosslinking leads to the formation of a non-homogeneous
its scalability, versatility, and the capability to incorporate filament, characterized by a highly crosslinked surface,
various cell types, growth factors, and biomolecules within as the rapid gelation is confined at the interface between
the bioink matrix. Microextrusion printing relies on the calcium solution and Alg phase. 22,23 Such highly
8
hydrogels as ideal bioink materials for 3D printing cell- crosslinked surface might affect the behavior of embedded
laden constructs, allowing the production of hydrated cells, limiting the diffusion of nutrients and oxygen. 24,25
constructs mimicking the natural extracellular matrix Moreover, the application of the external crosslinking
(ECM) of tissues. Despite considerable efforts, one key technique is usually associated with the use of coagulation
9
challenge in micro-extrusion bioprinting is the availability baths, in which the Alg phase is extruded. Such baths
of suitable bioinks able to fulfill all the following (e.g., freeform reversible embedding of suspended
requirements: (i) biocompatibility; (ii) biomimetic hydrogels [FRESH] printing ) physically support the
19
properties comparable to the ECM of the target tissue, in extruded bioink filaments and allow crosslinking of
order to favor cell adhesion, spreading, and differentiation; its polymeric chains to obtain constructs with high
(iii) controlled degradability, ideally matching the cell- shape fidelity and resolution. However, the use of such a
mediated ECM deposition rate; and (iv) proper rheological support bath, which involves a microparticle slurry, adds
characteristics for printing. 10-12 complexity to the bioprinting process, as particle size and
Among the materials investigated for microextrusion shape can significantly affect bioprinting resolution and
26
bioprinting applications, alginate (Alg) has been the morphology of the printed filaments. Lastly, the use
widely employed due to its tunable properties, cost- of external gelation can be a drawback for drug delivery
effectiveness, 10,11 non-immunogenicity, and non- applications, as the cargo embedded within the bioink
toxicity. Hence, Alg represents an excellent candidate for might be partially released during the coagulation step.
regenerative medicine and has been recently tested in Recently, internal gelation of Alg solutions has been
preclinical research and clinical trials for cardiovascular studied to develop 3D self-standing constructs without
diseases. 12,13 Algysil-LVR (LoneStar Heart Inc., United the need for a support bath. Internal gelation involves the
States of America [USA]) is an Alg-based injectable dispersion of water-insoluble calcium salt, such as calcium
hydrogel designed for the treatment of heart failure carbonate (CaCO ), within the Alg solution, followed by
3
by reshaping the left ventricle geometry, preventing or the gradual release of calcium ions through a decrease
reversing ventricular enlargement. 12,14 Once injected, the in pH that is induced by an acidic compound, usually

Volume 10 Issue 6 (2024) 545 doi: 10.36922/ijb.4014