Page 432 - IJB-9-3

International Journal of Bioprinting Gelatin-PVA crosslinked genipin bioinks for skin tissue engineering

bioprinting. Besides having excellent capability to (GE) powder from (Nitta-Gelatin Ltd., Osaka, Japan)
improve mechanical strength, high swelling ratio, as was weighed and added to the dissolved PVA mixture
well as the attributes of being biocompatible and non- after the temperature cooled down at 40°C and stirred
toxic, PVA also has the benefit of post-polymerization for 30 min until fully dissolved. Next, 0.1% of genipin
modification because of its secondary hydroxyl groups. (GNP) (FUJIFILM Wako Pure Chemical Corporation,
On the other hand, PVA has limitations in cell- Japan) was prepared by dissolving 0.01 g (w/v) of GNP
biomaterial interactions and should be supplemented powder in 70% of ethanol (EtOH; MERCK, Darmstadt,
with other tissue-inducing materials to speed up the Germany). After the mixture became homogenous, the
healing process [23] . Other research has shown that PVA GNP was added into the GPVA mixture to obtain a
has low protein affinities, which restrict cell binding final formulation of GE_GNP (0.1% GNP), GPVA3_
or attachment and potentially lead to a rounded GNP (3% PVA _0.1% GNP), and GPVA5_GNP (5%
morphology. Therefore, this study incorporated PVA PVA_0.1% GNP) while the non-crosslinked hydrogels
with gelatin to provide a conducive environment for were represented as GE_NC, GPVA3_NC (3% PVA), and
cells to survive due to an arginine-glycine-aspartic acid GPVA5_NC (5% PVA).
integrin-binding sequence in the gelatin that involves
both the A-chain and the B-chain [24] . 2.2. Synthesis of gelatin-PVA composite bioinks
The crosslinking process is one of the sustainable and Sterilized bioinks were prepared under the biosafety
well-known approaches to ensuring excellent stability cabinet to maintain sterility using sterilized GE and PVA
of fabricated bioscaffolds before implantation. It can powder. Next, autoclaved distilled water (dH O) was
2
be divided into irradiation-, physical- and chemical- used to dissolve the PVA powders at 60°C followed by
based methods. Genipin derived from the Gardenia dissolving the GE powder at 40°C. Sterilized GNP powder
jasminoides plant was used in fabricating gelatin- was dissolved in 70% ethanol before adding into the
PVA hydrogels as a crosslinking agent. It was utilized GPVA solution to obtain different hydrogels formulation
as a crosslinking agent due to its potential to increase GE_GNP, GPVA3_GNP, and GPVA5_GNP solution.
mechanical strength, stability and non-toxicity [25] . Dissolved GPVA solutions were thoroughly mixed with
Briefly, the genipin combines with primary amine human dermal fibroblasts (HDFs) with 1.5 × 10 of cell
6
groups to fix biological tissues, and it is substantially density per mL of gels for the subsequent 3D bioprinting
less harmful than other chemical crosslinkers, such process.
as glutaraldehyde, diisocyanates, and epoxides [26] .
This study aims to develop a simple, accessible, and 2.3. 3D bioprinting of gelatin-PVA hydrogel
reproducible bioinks formulation for wound healing A model was built by Autodesk fusion 360 software (stl.
applications, and to evaluate the influence of genipin file format). The pre-defined structures were input into
as a chemical crosslinker against gelatin-PVA bioinks. Simplify3D software (version 4.1). A 3D bioprinter,
Besides, the physicochemical and rheological properties Biogens XI (3D Gens, Malaysia), was used to print GPVA
of the bioinks were evaluated after fabricated via 3D bioinks. Sterilized GPVA bioinks were loaded into a
bioprinting approach. Finally, by varying the amount of sterilized printing syringe (inner diameter: 0.3 mm) at
PVA, this study aims to identify a composite bioscaffold the tip, and the printing process was conducted using an
with acceptable performance and biocompatibility for extrusion-based printhead. The hydrogel was deposited
skin tissue engineering through in vitro testing. via an extrusion-based bioprinting approach, and the
material flow for the printhead was controlled by pressure
2. Materials and methods regulators. The printability of different GPVA hydrogels
The study design was approved by the Universiti was evaluated using a combination of different printing
Kebangsaan Malaysia Research Ethics Committee temperatures (27 – 19°C) and speed rates (4000 mm/s)
(Code no. FF-2021-376 and JEP-2021-605). The research using a constant nozzle diameter of 0.3 mm. The adjacent
was performed in certified facilities under ISO 9001:2015 filaments were printed with 2.5 cm in length at 0.3 mm
quality management system. of retraction. Multi-layer layered hydrogel construct
with grid-like patterns was fabricated by printing each
2.1. Preparation of gelatin-PVA hydrogels layer of grid-like patterns directly over the previous layer
0.3 g and 0.5 g of PVA powder (MERCK KGaA, Germany; using an optimal combination of speed rates and printing
partially hydrolyzed [≥85%], MW 70,000 g/mol) were temperature. Figure 1 demonstrates the functional block
dissolved in 10 mL of distilled water (dH O) for 1 h at 60°C diagram for 3D bioprinting process from the beginning
2
to obtain 3% and 5% (w/v) concentration. 6 g of gelatin until 3D model is constructed.

Volume 9 Issue 3 (2023) 424 https://doi.org/10.18063/ijb.677