Advances in Gelatin Bioinks to Optimize Bioprinted Cell Functions

Abstract

Gelatin is a widely utilized bioprinting biomaterial due to its cell-adhesive and enzymatically cleavable properties, which improve cell adhesion and growth. Gelatin is often covalently cross-linked to stabilize bioprinted structures, yet the covalently cross-linked matrix is unable to recapitulate the dynamic microenvironment of the natural extracellular matrix (ECM), thereby limiting the functions of bioprinted cells. To some extent, a double network bioink can provide a more ECM-mimetic, bioprinted niche for cell growth. More recently, gelatin matrices are being designed using reversible cross-linking methods that can emulate the dynamic mechanical properties of the ECM. This review analyzes the progress in developing gelatin bioink formulations for 3D cell culture, and critically analyzes the bioprinting and cross-linking techniques, with a focus on strategies to optimize the functions of bioprinted cells. This review discusses new cross-linking chemistries that recapitulate the viscoelastic, stress-relaxing microenvironment of the ECM, and enable advanced cell functions, yet are less explored in engineering the gelatin bioink. Finally, this work presents the perspective on the areas of future research and argues that the next generation of gelatin bioinks should be designed by considering cell-matrix interactions, and bioprinted constructs should be validated against currently established 3D cell culture standards to achieve improved therapeutic outcomes.

Keywords: 3D bioprinting; covalent cross-linking; extracellular matrix; gelatin bioinks; stress relaxation; viscoelasticity.

Figure 1.

a) Schematic description of the collagen structure and hydrolysis to gelatin, b) The application of gelatin in bioprinting in terms of the number of publications as per Scopus database [search string: ABS ( gelatin/hyalur/chitosan/alginate/silk* AND bioprint* OR biofabr* )]. c) The amino acids present in gelatin. Adapted with permission [41]. Copyright 2022, Wiley-VCH Verlag GmBH & Co. d) The amount of carboxylic acid and amine functional groups in gelatin type A and type B [42, 43, 45, 59]

Figure 2.

a) Schematic showing the 3D bioprinting of organoid laden and perfusable GelMA construct using volumetric bioprinting. b) Liver organoids maintained self-organization and expressed key liver markers. c) Metabolic activity of liver organoids compared to extrusion bioprinting, casting with and without iodixanol and in Matrigel. Adapted with permission [144]. Copyright 2022, Wiley-VCH Verlag GmBH & Co, d) Optical images and confocal images of various patterned cellular structures using acoustic bioprinting (i-vi) (all scale bars: 500 μm), Viability of acoustic bioprinted cells vs. control group (vii). Adapted with permission [146], Copyright 2021, Royal Society of Chemistry.

Figure 3:

a) Schematic diagram for thiol-ene crosslinking of gelatin – norbornene. Adapted with permission [178], Copyright 2021, Wiley-VCH GmbH. b) Comparison of cell viability of HUVECs in GelMA vs GelNB/SH. Adapted with permission [174], Copyright 2021, American Chemical Society. c) Low irradiation time required for the curing of GelNB with GelS crosslinker compared to DTT crosslinker. d) The effect of the degradation products of GelMA, GelNB/DTT and GelNB/GelS on the viability of hepatocarcinoma (HepG2) cells. Adapted with permission [177], Copyright 2021, Wiley-VCH GmbH, Weinheim.

Figure 4.

a) Schematic representing preparation of GelMA construct using alginate as a sacrificial support material. b) Reduction in the intensity of alginate within GelMA when alginate is gradually dissolved. Adapted with permission [133], Copyright 2018 WILEY-VCH Verlag GmbH & Co, c) Frequency sweep of hybrid hydrogels, i) increasing frequency responsiveness in hybrid hydrogels with increasing βCD % (from lowest in AAG1 to highest in AAG5, AAG7 represents GelMA only), ii) increased storage modulus and decreased frequency responsiveness as a result of increased GelMA amounts (from lowest in AAG4 to highest in AAG6), iii) Reduced polymer density increases frequency responsiveness but reduces storage modulus (AAG-5–75%, AAG-5–50%, and AAG-5–50% represents hybrid hydrogel prepared from 75%, 50%, and 25% substances of AAG-5, respectively. Solid symbols (G′) & hollow symbols (G′′)), Adapted with permission [201], Copyright 2022, Elsevier.

Figure 5.

a) Static covalent networks confine cells, restricting their cellular functions. b) Stress relaxation rates of different natural tissues. Adapted with permission [273], Copyrights 2020, Springer Nature. c) schematics representing the reversible covalent and non-covalent bonds that can be introduced in gelatin-bioinks to improve viscoelasticity. d) Stress relaxation rates of different dynamic bonds (Increasing from left to right).

Figure 6.

Guest-host crosslinking: a) A schematic representation of host-guest interactions between β-cyclodextrin (host moiety) and adamantane (guest molecule). Adapted with permission [59], Copyright 2020, Wiley Periodicals. b) Mechanical characterization of GelMA, HG DN and MEHG DN hydrogels. i) Compressive stress – strain curve, ii) change in viscosity as function of temperature, iii) tensile stress-strain curve, iv) shear thinning behavior of hydrogels (viscosity vs shear rate), Adapted with permission [284], Copyrights 2022, Wiley-VCH GmbH.

Figure 7.

(a i – iii) Step strain-sweeps of composite hydrogels (phenyl boronic acid (PBA) modified gelatin and polyvinyl alcohol) with different amounts of PBA (PBA amounts in Gel 3 > Gel 2 > Gel 1). b) Self-healing behavior of composite hydrogel system, c) In-vitro degradation rate of dynamic gels with respect of increasing PBA amount, Adapted with permission [271], Copyright 2022, Elsevier. d) A schematic representation of reversible boronate-ester-diol bonding between GelNB-BA and PEG4SH. Adapted with permission [270], Copyright 2021, American Chemical Society. e) Live/Dead and F-actin staining of human MSCs encapsulated in GelNB-BA / PEG4SH hydrogels in the absence or presence of PVA, which improved viscoelasticity due to boronate-ester bonding of PVA and gelatin. Adapted with permission [306], Copyright 2021, American Chemical Society.