Page 329 - IJB-10-4

International Journal of Bioprinting 3D bioprinting of composite hydrogels

Polyethyleneimine (PEI) is another polymer that blends have been used previously for drug delivery, 63–65 to
holds the potential for use in corneal replacement and our knowledge, this is the first report of a GG–PEI bioink
drug delivery. PEI is a weakly basic, cationic synthetic loaded with BSP. The GG–PEI bioink was optimized for
polymer that contains primary, secondary, and tertiary extrusion-based 3D bioprinting with and without the
amine groups. 51,52 PEI has been used in many biomedical introduction of porous structures. We also evaluated the
applications, e.g., as a synthetic polymer for tissue degree of gelation of GG to obtain the optimal condition
engineering 20,53 and for encapsulation of nanoparticles, for crosslinking and stability of a transparent GG–PEI
drugs, and gene constructs. 54,55 Owing to its biofunctionality, composite hydrogel. The biocompatibility of the resulting
water solubility, thermal stability, low toxicity, cell affinity, GG–PEI hydrogel was assessed using corneal fibroblasts.
flexibility of its chains, and high charge density, PEI also Furthermore, we demonstrated controlled and sustained
facilitates and promotes cell proliferation, differentiation, release of BSP from the 3D-bioprinted GG–PEI hydrogels.
and tissue formation. The presence of positively charged Finally, the mechanical properties of the GG–PEI hydrogels
15
amine groups on PEI permits electrostatic interaction with were sufficient to support their use for corneal implantation
negatively charged polymers such as GG. Vancomycin- and prolonged wear in the ocular environment.
loaded N,N-dodecyl,methyl-PEI nanoparticles were used
as a controlled drug delivery system to treat bacterial 2. Methods
endophthalmitis in the cornea, with a synergistic effect
on bactericidal activity along with preservation of retinal 2.1. Bioink preparation
functions. Furthermore, the therapeutic potential of PEI Gellan gum (GG; Merck, United States of America [USA])
56
66
has been well characterized in vitro and in vivo. For example, bioinks were fabricated by ionotropic gelation. Firstly, the
the antibacterial and anti-inflammatory properties of PEI cross-linkers, CaCl (Merck, USA) and CA (Merck, USA),
2
aided in complete wound closure in a model of ocular were dissolved in deionized water with magnetic stirring
epithelial damage. The potential for PEI to be used in at 40°C for 20 min. The GG powder was then added to the
57
corneal gene therapy has also been demonstrated in vitro solution and heated in a water bath at 90°C until complete
and in vivo. 58–60 dissolution of GG was achieved. The solutions were then
kept in the 90°C water bath without stirring until air
One of the critical limitations of GG-based bioinks is bubbles were no longer visible and a clear solution was
that the 3D-bioprinted structures do not remain stable obtained. PEI (408,727, molecular weight [MW]: ~25,000;
without crosslinking. This contrasts with many other Merck, USA) was then added dropwise and stirred at
biopolymers used for 3D bioprinting, such as alginate, 40°C for 2 h to facilitate the interaction between the two
which can be rapidly crosslinked to form a hydrogel in the opposite-charged polymers. Finally, the GG–PEI blend was
presence of divalent cations, such as calcium. However, kept at 4°C for 48 h to complete the crosslinking reaction.
alginate generally lacks sufficient mechanical properties for The composition and abbreviation for all samples are listed
in vivo applications. 61,62 Thus, to improve the stability of GG- in Table 1.
based bioinks after printing, we explored the use of citric
acid (CA) as an efficient cross-linker for GG. Additionally, 2.2. 3D bioprinting
we incorporated a biologically active interpenetrating Two types of patterns were printed: (i) a cube pattern
network of PEI to improve cell adhesion to the printed to evaluate shape fidelity and (ii) a corneal model.
structure. We also incorporated betamethasone sodium Stereolithography (STL) files of the two models were
phosphate (BSP) into the GG–PEI bioink. While GG–PEI imported into Simplify3D software to be converted to

Table 1. Composition of bioinks
Bioink GG (% [w/v]) Cross-linker, CA or CaCl (% [w/v]) PEI (% [w/v])
2
2.5GG 2.5 - -
3GG 3 - -
2.5GG–CA 2.5 CA (0.5) -
2.5GG–C 2.5 CaCl (0.5) -
2
GG–3PEI 2.5 CA (0.5) 3
GG–4PEI 2.5 CA (0.5) 4
GG–5PEI 2.5 CA (0.5) 5
Abbreviations: C, CaCl ; CA, citric acid; GG, gellan gum; PEI: polyethyleneimine.
2

Volume 10 Issue 4 (2024) 321 doi: 10.36922/ijb.3440