Orthogonally crosslinked gelatin-norbornene hydrogels for biomedical applications

Abstract

The thiol-norbornene photo-click reaction has exceptionally fast crosslinking efficiency compared with chain-growth polymerization at equivalent macromer contents. The orthogonal reactivity between norbornene and thiol/tetrazine permits crosslinking of synthetic and naturally derived macromolecules with modularity, including PEG-norbornene (PEGNB), gelatin-norbornene (GelNB), among others. For example, collagen-derived gelatin contains both cell adhesive motifs (e.g., RGD) and protease-labile sequences, making it an ideal macromer for forming cell-laden hydrogels. First reported in 2014, GelNB has increasingly been used in orthogonal crosslinking of biomimetic matrices in various applications. GelNB can be crosslinked into hydrogels using multi-functional thiol linkers (e.g., dithiothreitol (DTT) or PEG-tetra-thiol (PEG4SH) via visible light or longwave UV light step-growth thiol-norbornene reaction or through an enzyme-mediated crosslinking (i.e., horseradish peroxidase, HRP). GelNB-based hydrogels can also be modularly crosslinked with tetrazine-bearing macromers via inverse electron-demand Diels-Alder (iEDDA) click reaction. In this review, we survey the various methods for preparing GelNB macromers, the crosslinking mechanisms of GelNB-based hydrogels, and their applications in cell and tissue engineering, including crosslinking of dynamic matrices, disease modeling and tissue regeneration, delivery of therapeutics, as well as bioprinting and biofabrication.

Graphical Abstract

This review surveys the various methods for preparing gelatin-norbornene (GelNB), the crosslinking mechanisms of GelNB-based hydrogels, and their applications in cell and tissue engineering, including crosslinking of dynamic matrices, disease modeling and tissue regeneration, delivery of therapeutics, as well as bioprinting and biofabrication.

1. Introduction

Hydrogels crosslinked by naturally derived biomolecules have been used in various biomedical applications, including as vehicles for the loading and release of disease-modifying therapeutics, as carriers for delivering cells to promote tissue healing and regeneration, and as three-dimensional (3D) scaffolds for studying cell-material interactions. Gelatin, a polypeptide derived from denatured collagen, is one of the most used bioactive macromolecules in cell and tissue engineering applications. Like collagen, gelatin contains many bioactive sequences that bind to cell surface receptors (e.g., Arg-Gly-Asp or RGD for integrins). Different from collagen, gelatin lacks triple helical structures and has an upper critical solution temperature (UCST) between 30°C to 35°C, leading it to undergo gel-sol transition when the surrounding temperature is above the UCST. As such, gelatin itself does not have the structural stability to support 3D mammalian cell culture, which is typically conducted at 37°C. While gelatin can still be used as a coating material to improve cell adhesion onto an otherwise cell-repelling surface, chemical modification on the gelatin backbone is required if one wishes to use gelatin in 3D cell culture. As mentioned previously, gelatin is a polypeptide composed of many amino acids with reactive side groups (e.g., primary amine group on lysine, carboxylic acid on glutamic acid, aspartic acid, etc.), it is highly amenable to chemical modification, a necessary step for crosslinking gelatin into 3D hydrogels with tunable mechanical properties. Early examples of forming gelatin hydrogels include using glutaraldehyde^[1,2] or transglutaminase^[3–7] to chemically or enzymatically crosslink unmodified gelatin. Unfortunately, these crosslinking reagents/enzymes were cytotoxic even at low concentrations, precluding their use in live cell encapsulation. Other more cytocompatible reagents (e.g., genipin)^[1,8,9] have also been used to crosslink unmodified gelatin into stable 3D hydrogels.

In the engineered biomaterials space, one early example of gelatin modification was carried out by Van Den Bulcke et al. where they synthesized methacrylamide-modified gelatin via reacting gelatin with methacrylic anhydride.^[10] The synthesis protocol starts with dissolving type B gelatin in phosphate buffer (pH7.5) at 50°C, followed by slowly adding methacrylic anhydride and vigorous stirring for 1 hour. The reaction was stopped by dilution with additional buffer and the mixture was dialyzed for 24h against distilled water at 40°C to prevent physical gelation. The reaction product was freeze-dried to a white solid and the degree of substitution (DS) was determined by quantifying the percentage of converted amino groups. Gelatin-methacrylamide (or gelatin-methacryloyl, GelMA) with a range of DS was prepared by adding different amounts of methacrylic anhydride. The derivation of gelatin-methacrylamide with different DS permitted the crosslinking of gelatin into hydrogels with different mechanical properties (e.g., storage modulus) suitable for 3D cell culture.^[11–15] For example, Anseth and colleagues first introduced GelMA as a photocrosslinkable macromer for in situ cell encapsulation, where they used these tunable hydrogels to study the myofibroblastic activation of valvular interstitial cells (VICs).^[16] Khademhosseini et al. later expanded the use of chemically crosslinked GelMA hydrogels into various 3D cell culture and microfabrication applications.^[17–31] A common feature of the above examples is that GelMA crosslinks into hydrogels via chain-growth photopolymerization, which is initiated by a photoinitiator (e.g., Irgacure-2959 (I-2959), lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP), eosin-Y) and an appropriate light source (365 nm for I-2959; 365–405 nm for LAP, 440–575 nm for eosin-Y). While this is a simple crosslinking mechanism, evidence has shown that chain-growth polymerization produces high concentrations of propagating radical species that could induce cytotoxicity for sensitive cell types.^[32] Chain-growth polymerization is sensitive to dissolved oxygen and yields hydrophobic crosslinks with heterogeneous molecular weights, the former delays the on-set of gelation whereas the latter promotes undesired protein-polymer conjugates. Alternatively, GelMA hydrogels can be crosslinked by light-independent Michael-type addition with multi-functional thiol linkers. However, the methacrylate-thiol reaction does not allow spatial-temporal tunability in crosslinking kinetics.

Another light-mediated crosslinking mechanism that could address many challenges mentioned above is the thiol-norbornene photo-click reaction.^[33] The thiol-norbornene photo-click reaction is not oxygen-inhibited and has exceptionally fast crosslinking efficiency compared with chain-growth polymerization at equivalent macromer contents.^[34] The orthogonal reactivity between thiol and norbornene permits crosslinking of synthetic and naturally derived macromolecules with modularity.^[35] The first thiol-norbornene hydrogels were crosslinked by PEG-norbornene (PEGNB) and peptide crosslinkers bearing cysteine termini.^[33,34,36] The PEG-based thiol-norbornene hydrogels have been highly useful for in situ encapsulation of therapeutically relevant cells, such as human mesenchymal stem cells (hMSCs),^[36,37] VICs,^[38,39] pancreatic β-cells,^[34] and cancer cells.^[40–42] A typical measure in crosslinking PEG-based thiol-norbornene hydrogels includes the use of cysteine-containing integrin-binding peptides (e.g., CRGDS) to afford critical cell-matrix interaction. On the other hand, the bis-cysteine-bearing peptide crosslinkers typically contain protease-sensitive sequences (e.g., CGPQG↓IWGQC, arrow: matrix metalloproteinase or MMP cleavage site).^[36] The presence of these two bioactive components (e.g., integrin-binding and protease-labile sites) in the otherwise inert PEG-based network is required to create a promoting microenvironment for cellular processes, such as adhesion, migration, proliferation, and differentiation.^[35] As gelatin contains both RGD motif and MMP-labile sequences, it is an ideal macromer for forming cell-laden hydrogels. In this regard, we described the synthesis of the first gelatin-norbornene (GelNB) for orthogonal crosslinking of biomimetic cell-laden hydrogels.^[43] The original reaction scheme to acquire GelNB emulated that of GelMA synthesis, except that carbic anhydride was used in place of methacrylic anhydride.^[43] Briefly, norbornene moiety was conjugated to gelatin via reaction with carbic anhydride in aqueous buffer solutions at 40°C. During the reaction, the solution pH was maintained at 8 to facilitate the dissolution of carbic anhydride, which improved the conjugation efficiency. We demonstrated orthogonal crosslinking of GelNB into hydrogels using multi-functional thiol linkers (e.g., dithiothreitol (DTT) or PEG-tetra-thiol (PEG4SH) via visible light or longwave (λ ~ 365nm) UV light step-growth thiol-norbornene reactions^[43–45] or through an enzyme-mediated crosslinking (i.e., horseradish peroxidase, HRP).^[46] Since then, GelNB has been increasingly used by us and others in the space of orthogonal and dynamic crosslinking of gelatin-based hydrogels.^[47] In this review, we survey the various methods for preparing GelNB macromer, the crosslinking mechanisms of GelNB-based hydrogels, and their applications in cell and tissue engineering.

2. Synthesis of GelNB

2.1. Nucleophilic acyl substitution with carbic anhydride

The first norbornene-modified gelatin (i.e., GelNB) was synthesized by reacting gelatin with carbic anhydride (i.e., cis-5-norbornene-endo-2,3-dicarboxylic anhydride) following a nucleophilic acyl substitution reaction.^[43] In this reaction scheme, a nucleophilic attack by a primary amine (from gelatin) to a carbonyl group (from the dicarboxylic anhydride on carbic anhydride) affords an amide linkage and a norbornene group with a carboxylic acid tether (Figure 1). It is important to note that this reaction should be performed at a higher temperature (40°C) to facilitate the dissolution of gelation. Furthermore, to enhance the functionalization, GelNB synthesis is catalyzed by sodium hydroxide (NaOH) or trimethylamine (TEA), which enhances the deprotonation of primary amine groups. If NaOH is used, the solution pH is adjusted to 7.5 to 8 as carbic anhydride demonstrates complete solubility and optimal reactivity at basicity. However, pH adjustment is not necessary when a TEA catalyst is employed. The degree of norbornene substitution can be altered by adjusting the amount of carbic anhydride added to the reaction. Since this reaction is carried out in an aqueous solution, a high degree of variation can happen, especially when NaOH is used to adjust the solution’s pH. In contrast, a more stringent control of norbornene functionalization can be achieved when TEA is used to catalyze the reaction.^[48,49] Kim et al. reported comprehensive methods for this synthesis scheme.^[49]

Figure 1.

Nucleophilic acyl substitution of carbic anhydride with an amino group (R-NH₂) for introducing norbornene on gelatin.

2.2. Norbornene substitution with carbodiimide chemistry

In addition to nucleophilic acyl substitution, GelNB can be synthesized by reacting amino groups on gelatin with 5-norbornene-2-carboxylic acid (Figure 2A), 5-norbornene-2-succinimidyl ester (Figure 2B), or 5-norbornene-2-acetic acid succinimidyl ester (Figure 2C) via standard carbodiimide chemistry. Truong et al.^[50] described the synthesis of GelNB using 5-norbornene-2-carboxylic acid (norbornene-acid) by first converting norbornene-acid into norbornene-N-hydroxysuccinimide (norbornene-NHS) in CH₂Cl₂, followed by reacting the norbornene-NHS with amine groups on gelatin in a water/dimethylformamide (DMF) mixture and with N,N-diisopropylethylamine as a base catalyst. After 12 hours of reaction at ambient temperature, the product was retrieved via dialysis and lyophilization. Later, Van Hoorick et al. used a similar approach by activating norbornene-acid with 1-ethyl-3-[3-dimethylaminopropyl)carbodiimide (EDC) and NHS, yielding a reactive 5-norbornene-2-succinimidyl ester.^[51,52] This activation step was carried out for at least 25 hours to eliminate any unreacted EDC that could result in gelatin self-crosslinking. The actual norbornene conjugation step was performed in dry DMSO for 5 to 20 hours under an inert atmosphere and reflux conditions. Anseth and colleagues took a more straightforward method of GelNB synthesis by reacting gelatin with commercially available 5-norbornene-2-acetic acid succinimidyl ester in pH 8.5 sodium bicarbonate buffer.^[53] The reaction was carried out at 37°C for 1 hour and purified by dialysis against pH 8.5 sodium bicarbonate for 4 hours at room temperature.

Figure 2.

Chemical structure of (A) 5-norbornene-2-carboxylic acid, (B) 5-norbornene-2-succinimidyl ester, (C) 5-norbornene-2-acetic acid succinimidyl ester, and (D) 5-norbornene-2-methylamine.

In addition to using norbornene-acid-based reagents, GelNB can also be synthesized by using 5-norbornene-2-methylamine (i.e., norbornene-amine, Figure 2D), which reacts with carboxylic acid groups on gelatin (e.g., aspartic acid, glutamic acid). For example, the Mooney group synthesized GelNB by reacting carboxylic acid groups on type A gelatin with norbornene-amine in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH6, 37°C) via the EDC/NHS activation route.^[54] More recently, Luo et al. pre-reacted gelatin with succinic anhydride to increase the available carboxylic acid groups for facilitating the subsequent functionalization of norbornene-amine and to prevent cross-reaction of amine-carboxylic acid moieties within gelatin itself.^[55]

The determination of norbornene functionalization on gelatin is typically carried out using NMR spectroscopy and colorimetric/fluorometric quantification methods, such as ninhydrin and fluoraldehyde reagents. NMR analysis does not require the use of standards with known amine concentrations to determine the degree of functionalization. The successful modification of norbornene on gelatin can be readily observed through the appearance of unsaturated caron-carbon double bonds typically appearing at ppm 6 on the NMR spectrum, which is not present in unmodified gelatin. However, due to the presence of various amino acid residues on gelatin, it is difficult to quantitatively determine the degree of norbornene functionalization using proton NMR. In this regard, colorimetric/fluorometric assays should be used in tandem to determine the quantitative degree of norbornene functionalization. In the ninhydrin assay, primary amines react with ninhydrin, resulting in the formation of Ruhemann’s Purple, which can be quantified by measuring its absorbance at a wavelength of 570nm. On the other hand, the fluoraldehyde assay involves the reaction of primary amines with o-phthalaldehyde (OPA) reagent, leading to the formation of a fluorescent product that can be quantitatively determined (excitation/emission = 340 nm/455 nm).

3. Crosslinking of GelNB hydrogels

GelNB-based hydrogels can be crosslinked by several mechanisms, including light-initiated (UV or visible light) photopolymerization, enzyme-induced crosslinking, and iEDDA click reaction. Table 1 summarizes the main characteristics and potential challenges for these crosslinking mechanisms.

Table 1.

Comparison of GelNB hydrogel crosslinking mechanisms.

Crosslinking mechanism	Gelation speed	Catalyst or Initiator	Spatial-temporal control	Potential Challenges
Longwave UV light-Initiated thiol-norbornene polymerizations	Fast	Photo-initiator	High	• Cytotoxicity of radical species • UV light exposure & light attenuation
Visible light-initiated thiol-norbornene polymerizations	Fast to moderate	Photo-initiator	High	• Cytotoxicity of radical and initiator species
Enzyme-catalyzed thiol-norbornene click reactions	Moderate	Enzyme & cofactor	Low	• Require hydrogen peroxide as a cofactor
Tetrazine-norbornene iEDDA click reactions	Moderate	None	Low	• Produce nitrogen gas

3.1. UV light-Initiated thiol-norbornene photopolymerization

Norbornene is a highly strained bridged cyclic hydrocarbon that can react with various functional groups under defined conditions. In the space of hydrogel crosslinking, norbornene is routinely used in longwave ultraviolet (UV) light-initiated thiol-norbornene photocrosslinking (Figure 3A).^[33] In this regard, gelatin-derived GelNB was developed for its multiple inherent bioactivities (i.e., cell adhesion and protease-labile) and crosslinkability afforded by the additional norbornene moiety.^[43] Unlike GelMA which could chain-polymerize and self-crosslink into covalent hydrogels in the presence of a photoinitiator, the crosslinking of GelNB into hydrogels also requires the use of a multifunctional thiol linker (e.g., DTT or PEG4SH).^[43] This gives modularity control of the physicochemical properties of the resulting hydrogels. For example, Shih et al. utilized thiol-norbornene photocrosslinking to modularly crosslink GelNB and thiolated hyaluronic acid (THA) into hydrogels with adaptable properties for mimicking the pancreatic tumor microenvironment.^[45] The thiol-norbornene photo-click reaction is initiated by a photoinitiator, such as lithium aryl phosphinate (LAP). Upon 365 nm light irradiation, LAP was photolyzed into two radical species, each can abstract hydrogen from nearby sulfhydryl group (-SH), creating a thiyl radical that can propagate through the strained cyclic hydrocarbon on norbornene ^[56,57]. The carbon radical terminates by abstracting another hydrogen on the available sulfhydryl group while continuing the propagation step.^[33] The step-growth thiol-norbornene photopolymerization proceeds with high efficiency, with a gel point, which is defined as the crossover time when the storage modulus (G’) supersedes the loss modulus (G’), occurs as rapidly as a few seconds upon light exposure.^[33,34] The typical storage modulus of gelatin-based thiol-ene hydrogels can range between sub-100’s of Pa to a few tens of kPa.^[58,59] The step-growth thiol-norbornene photo-click hydrogels, including those crosslinked by GelNB, are generally more cytocompatible for in situ cell encapsulation than their chain-growth polymerized counterparts.^[34,43] Unlike chain-growth GelMA hydrogels, step-growth GelNB hydrogels do not contain crosslinks with heterogeneous molecular weights, which may hinder the accessibility of cell-secreted proteases (e.g., MMPs) to the cleavage sites on the gelatin backbone.^[43] For example, Meeremans et al. synthesized GelNB with 55% and 85% norbornene substitution and crosslinked with either DTT or thiolated gelatin (GelSH) of 75% thiol substitution to form hydrogels for tenocyte culture.^[60] It was found that the GelNB hydrogels were more ideal for tenocyte culture than the “gold standard” GelMA hydrogels. Nonetheless, longwave (λ ~ 365nm) UV-light initiated GelNB hydrogel crosslinking, while having fast gelation kinetics, may experience the same challenges as crosslinking of other hydrogels, including the potential cytotoxicity of radical species and light attenuation in dark samples (Table 1).

Figure 3.

(A) Schematic of thiol-norbornene photoclick reaction. P.I. = photoinitiator; hv = light source. (B) Schematic of tetrazine-norbornene iEDDA click reaction.

3.2. Visible light-initiated thiol-norbornene photopolymerization

In addition to longwave UV light, thiol-norbornene photopolymerization can also be initiated by photoinitiators sensitive to visible light. For example, type I (or cleavage-type) photoinitiator LAP exhibits slight absorbance between 400 and 405 nm (molar absorbability ε ≈ 30 m⁻¹ cm⁻¹ at 405 nm), allowing it to be used as a visible light initiator for hydrogel crosslinking.^[61] Eosin-Y, a type II photoinitiator that absorbs light between ~430 nm to ~550nm and with a peak absorbance at ~515 to 525 nm (ε ≈ 100,000 m⁻¹ cm⁻¹ at 525 nm), can also be used to initiate hydrogel crosslinking. The ability of eosin-Y to initiate a chain-growth polymerization is, in most cases, contingent upon the presence of a co-initiator, such as triethanolamine (TEOA).^[62] The use of TEOA in traditional eosin-Y mediated visible light-based gelation is particularly problematic as it is a strong base that could induce cytotoxicity at higher concentrations. However, the Lin research group discovered that thiol-norbornene photopolymerization can be initiated by white light (400 – 700 nm) using eosin-Y as the only initiator.^[45,63–67] Mechanistically, visible light exposure excites eosin-Y to a higher energy state, allowing it to abstract hydrogen from thiol-containing crosslinkers and form a thiyl radical needed for the thiol-norbornene click reaction (Figure 3A). The use of eosin-Y as the sole initiator in thiol-norbornene photocrosslinking simplifies the formulation of hydrogel compositions. With this crosslinking mechanism, Shih et al. exploited the visible light-based modular crosslinking of GelNB hydrogels,^[45] with thiolated poly(vinyl alcohol) (TPVA) or THA to enable modular cross-linking of bioinert (i.e., purely synthetic), bioactive (i.e., using gelatin), and biomimetic (i.e., using gelatin and hyaluronic acid) hydrogels. The benefits of this crosslinking scheme lie in the ability to tune hydrogel stiffness without affecting the concentrations of the bioactive components.

Another emerging type II photoinitator system consists of ruthenium (Ru) and sodium persulfate (SPS). Ru absorbs light at 400–500 nm (ε ≈ 14,600 m⁻¹ cm⁻¹ at 450 nm) and oxidizes into Ru³⁺ by donating electrons to SPS, which then dissociates into sulfate anions and sulfate radicals. The latter are responsible for initiating the hydrogel crosslinking. Ru/SPS system is increasingly used in phtotocrosslinking of GelMA^[68] and GelNB (i.e., Gel-NOR) hydrogels.^[69,70] At equivalent gelatin macromer concentration, Ru/SPS-crosslinked GelNB hydrogels were demonstrated to have higher crosslinking efficiency than the GelMA counterparts, as demonstrated by the lower sol fraction and higher compressive modulus of the GelNB hydrogels.^[70] In general, while visible light wavelengths (λ ~400nm to 700nm) are arguably safer than UV light wavelengths, visible light-initiated crosslinking is not as fast as longwave UV light and it may require or produce some cytotoxic species.

3.3. Enzyme-catalyzed thiol-norbornene click reaction

While UV or visible light-initiated reactions are often adopted for thiol-norbornene hydrogel crosslinking, it is possible to use enzymes to catalyze this reaction. One shortage of light-based hydrogel crosslinking is the limitation of sample thickness due to light attenuation. In this regard, the use of enzyme to catalyze hydrogel crosslinking affords precise control of gelation kinetics without limitation of sample thickness. For example, Nguyen et al. discovered that thiol-norbornene photo-click reaction can be initiated by horseradish peroxide (HRP) and low concentration of hydrogen peroxide.^[46] Mechanistically, HRP generates the thiyl radicals from thiol-containing linkers (e.g., bis-cysteine-containing peptide or PEG4SH) to initiate crosslinking of NB-containing macromers (e.g., PEG8NB or GelNB) into injectable hydrogels. It was also found that addition of tyrosine residue in the peptide crosslinker facilitated HRP-initiated PEG-peptide hydrogel crosslinking. Furthermore, the additional tyrosine residues did not form permanent dityrosine cross-links following HRP-induced gelation and remained available for a secondary enzymatic reaction (i.e., tyrosinase) for dynamically increased hydrogel stiffness. The use of enzyme to initiate GelNB hydrogel crosslinking eliminates the light attenuation issue, but the spatiotemporal gelation control provided by photopolymerization is lost in enzymatic reaction. Additionally, the use of hydrogen peroxide in HRP-initiated thiol-norbornene crosslinking may be a source of concern for in situ cell encapsulation.

3.4. Tetrazine-norbornene inverse-electron demand Diels-Alder (iEDDA) click reactions

In addition to participating in light or enzymatic thiol-norbornene reactions as discussed above, norbornene can also click with tetrazine (Tz) or methyltetrazine (mTz) via iEDDA click reaction. iEDDA click reaction is a cycloaddition between an electron-rich dienophile (e.g., norbornene or trans-cyclooctene) and an electron-poor diene (e.g., Tz or mTz).^[71] iEDDA click reactions between TCO (or norbornene) and tetrazines are widely used in bioconjugation and live cell labeling as neither moiety exists within the body and crosslink efficiently with each other without the risk of disrupting cellular processes.^[72–76] iEDDA click reaction, by itself or together with other crosslinking mechanism, has also been increasingly exploited in dynamic hydrogel crosslinking.^[77–91] For example, Mooney and colleagues first described the iEDDA click crosslinking of GelNB and GelTz (i.e., tetrazine-conjugated gelatin) into covalent hydrogels.^[54] While complete gelation of iEDDA-crosslinked GelNB-GelTz hydrogels is substantially slower (10 – 70 minutes) than light-initiated thiol-norbornene click crosslinking (seconds to a few minutes),^[92] the iEDDA click reaction generates no radical species and produces nitrogen gas as the only byproduct.^[93,94] In general, GelNB-GelTz iEDDA click hydrogels can be formulated to have different stiffness and can be injectable through needles for minimally invasive delivery in vivo.^[92]

While tetrazine–norbornene iEDDA ligation has been highly useful in bioorthognal hydrogel crosslinking, free thiol in a biological environment may cause reduction of tetrazine, and hence lowering the crosslinking efficiency. To circumvent this potential shortcoming, Carthew et al. synthesized 4-arm PEG-dihydrogentetrazine (PEG-dHTz) and used HRP to oxidize dHTz into a Tz group that can then be reacted with norbornene via standard iEDDA click reaction.^[95] They found that HRP-mediated dHTz oxidation is highly efficient at a relatively low HRP concentration (23 nM) without the need of using hydrogen peroxide. Rapid gelation (5 to 10 minutes) of PEG-Tz and GelNB was achieved when HRP was added to the polymer precursor solution. The gel fractions were not as high as light-crosslinked hydrogels (~73% to 82%) but the storage modulus could be readily tuned in a physiologically relevant range (1.2 and 3.8 kPa) by adjusting the concentration of the crosslinker. Regardless of traditional tetrazine-norbornene or HRP-catalyzed iEDDA click chemistry, these reactions produce nitrogen gas as the by-product. The N₂ gas is typically entrapped in hydrogels, forming uncontrolled gas bubbles that may be a source of concern.

4. Biological applications of GelNB hydrogels

4.1. Dynamic hydrogel crosslinking

GelNB hydrogels are mostly used as a platform for 3D cell culture. Typical thiol-norbornene photocrosslinking, including crosslinking of GelNB hydrogels, leads to hydrogels with covalent and static crosslinks that may not recapitulate the dynamics of cellular microenvironment. To circumvent the non-dynamic nature of thiol-norbornene crosslinks, our group has formulated dually modified gelatin macromers for dynamic hydrogel crosslinking, including GelNB-hydroxyphenyl acetic acid (GelNB-HPA),^[96,97] GelNB-carbohydrazide (GelNB-CH),^[98] and GelNB-boronic acid (GelNB-BA).^[99] Kim et al. summarized the detailed synthetic schemes of these dually modified gelatin macromer in a recent protocol paper (Figure 4).^[49] The conjugation of HPA to GelNB produces a macromer bearing abundant tyrosyl groups that can be further conjugated by secondary enzymatic reaction (e.g., mushroom tyrosinase). To achieve dynamic crosslinking or patterning, crosslinked hydrogels were equilibrated with tyronsinase, followed by enzymatic reaction. As such, hydrogels crosslinked by GelNB-HPA can be dynamically stiffened or spatiotemporally patterned with bioactive ligands.^[96,97] Similar to GelNB-HPA, we prepared GelNB-CH so that the hydrogels can be dynamically stiffened via simple diffusion of aldehyde-bearing macromers, such as sodium periodate-oxidized dextran (oDex) or hyaluronic acid (oHA). oHA-induced dynamic stiffening was used to mimic concurrent tumor stromal stiffening and accumulation of HA.^[98] Finally, hydrogels crosslinked by GelNB-BA were found to exhibit tunable stress-relaxation, a physical property increasingly studied in disease progression. For GelNB-BA hydrogels to possess tunable stress-relaxation, diol-containing polymers (e.g., poly(vinyl alcohol), PVA) was incorporated in the primary hydrogel network. The reversible boronate-ester diol bonds formed between BA and diol moieties give rise to the high loss modulus and stress-relaxation properties of the hydrogels.^[99]

Figure 4.

Synthesis of dually modified gelatin derivatives from GelNB. Reproduced with permission from American Chemical Society.^[49]

An alternative strategy to introduce dynamic crosslinks in GelNB hydrogels is through integrating supramolecular interaction with thiol-norbornene photocrosslinking. Myung and colleagues formulated a supramolecular crosslinker composed of a ternary complex between one cucurbit[8]uril (CB[8]) and two FGGC peptides.^[100] The supramolecular crosslinker presents two free thiol groups (from the cysteine residues on FGGC peptides) for thiol-norbornene photocrosslinking with GelNB, producing a bulk hydrogels with both covalent thiol-ene crosslinks and dynamic CB[8]·FGGC complexes. As the complexation between CB[8] and FGGC is reversible, the resulting CB[8]·FGGC-GelNB hydrogels exhibited shear-thinning and injectable properties with potential in bioprinting applications. One notable difference between this example and the conventional thiol-norbornene crosslinking of GelNB hydrogels is that hydrogels in the presence of CB[8]·FGGC required 10 minutes of light exposure, as compared to the quicker 2-minute UV exposure needed for thiol-ene click reactions. The reduction of crosslinking kinetics is likely caused by the presence of bulky CB[8] group that hinders the accessibility of the free thiol groups on cysteine residues.

4.2. Stem cells encapsulation and differentiation

A unique feature of orthogonally crosslinked GelNB hydrogels is that the crosslinking density could be tuned, via using crosslinkers with different functionality, independently from the content of biological motifs (i.e., gelatin concentration. Fig. 5A). To this end, GelNB hydrogels have been used to study the influence of matrix properties on stem cell fate. For example, the Lin research group first demonstrated high cytocompatibility of GelNB hydrogels for 3D culture of human mesenchymal stem cells (hMSCs).^[43] Compared with chain-polymerized GelMA hydrogels with similar crosslinking density, GelNB hydrogels supported higher degree of cell spreading (Fig. 5B). GelNB hydrogels crosslinked by PEGdHT via HRP-induced iEDDA click reaction was supported spreading of hMSCs.^[95] Forsythe and colleagues utilized microfluidic devices and visible light-cured GelNB-PEGdiSH microgels to encapsulate human bone marrow derived stem cells (hBMSCs) and showed high level of chondrogenesis, as evidenced by significant upregulation of type II collagen compared with bulk hydrogels and the “gold standard” pellet culture.^[101] These GelNB-based microgels were also found to support in vivo maturation of human articular chondrocytes (hACs) and human fetal chondroprogenitor cells (hCCs).^[102] Van Damme et al. showed that thiol-norbornene GelNB hydrogels supported higher degree of adipogenesis as compared to GelMA hydrogels.^[59] Arkenberg et al. expanded the use of heparinized GelNB (GelNB-Hep) hydrogels for neuroectoderm, mesoderm, and endoderm differentiation of human induced pluripotent stem cells (hiPSCs).^[72,103] GelNB or GelNB-Hep (synthesized by reacting tetrazine-modified heparin with GelNB via the iEDDA click reaction) was modularly cross-linked with either inert PEG4SH or bioactive thiolated hyaluronic acid (THA) to afford biomimetic matrices with similar stiffness but varying bioactive components. Both GelNB and GelNB-Hep hydrogels were found to support hiPSC growth and neuroectodermal, mesodermal, and endodermal differentiation (Fig. 5C).^[72] In a follow-up work, GelNB hydrogels were further used for pancreatic progenitor (PP) differentiation of hiPSC.^[103] It was found that differentiation of hiPSCs in GelNB hydrogels supported prominent branching ductal network formation and generated diverse endoderm populations. Through single-cell RNA-sequencing (scRNA-seq), it was revealed that 3D differentiation in GelNB hydrogels resulted in enrichments of pan-endodermal cells, ductal cells, and a group of extraembryonic cells, which were absent in 2D differentiation and were likely a result of overly activated Wnt and BMP pathways. Through inhibiting Wnt signaling pathway at the beginning of the posterior foregut stage, the expressions of PP signature genes PDX1 and NKX6.1 were restored.^[103]

Figure 5. Modular control of GelNB hydrogels for stem cell encapsulation and differentiation.

(A) By changing the functionality of the thiol-containing crosslinker (i.e., DTT or PEG4SH), the stiffness and crosslinking density of GelNB hydrogels could be controlled independently of its biological components (i.e., gelatin content). Reproduced with permission from the Royal Society of Chemistry.^[43] (B) Compared with GelMA-based hydrogels, GelNB-DTT hydrogels supported more extensive spreading and intercellular connectivity of hMSCs. Reproduced with permission from the Royal Society of Chemistry.^[24] (C) GelNB-derived hydrogels (with or without heparin conjugation) could be used to support trilineage (neuroectoderm, mesoderm, and endoderm) differentiation of hiPSCs. Reproduced with permission from American Chemical Society.^[72]

4.3. Disease modeling and regenerative medicine

GelNB hydrogels crosslinked by thiol-norbornene photo-click reaction have emerged as diverse matrices in modeling disease progression and promoting tissue regeneration. For example, Lin and colleagues formulated visible-light initiated GelNB hydrogels for mimicking the biophysical properties and biochemical compositions of the pancreatic tumor microenvironment (TME).^[45] The Lin Research Group also designed several multi-functional GelNB-based hydrogels for mimicking the stiffening (Fig. 6A) and stress-relaxing pancreatic TME.^[49,96–99] In addition to TME mimicry, GelNB hydrogels have also been used to facilitate therapeutic screening in 3D. For example, Dobos et al. exploited GelNB-DTT thiol-norbornene hydrogels as matrices for 3D culture of osteosarcoma cells. The cell-laden GelNB hydrogels were used in screening two-photon activated photodynamic therapy sensitizers.^[104] GelNB has also been used as a membrane coating to culture corneal endothelial cells in hopes of creating an artificial hydrogel to overcome the limited availability for donor descemet membrane.^[52] Meeremans et al. utilized GelNB to crosslink thiol-norbornene hydrogels for improving the low viability of tenocytes encountered when using GelMA hydrogels.^[60] It was found that GelNB hydrogels preserved the characteristic elongated morphology of tenocytes important for tendon regenreation. In Luo’s work, GelNB hydrogels was created to mimic the soft nucleus pulposus in the intervertebral disc space.^[55] The hydrogels also contained TGF-β, whose delivery improved recovery of tissue and functionality of the IVD space over a 6-week recovery period (Fig. 6B).^[55] Ionescu et al. compared GelNB hydrogels, alginate mathecryate (AlgMA) hydrogels, and commercially available AquacelAg dressing in dermal wound healing.^[105] It was found that, while all three promoted wound healing, GelNB films produced faster healing rate, as evidenced by both microscopic and macroscopic evaluations.

Figure 6. GelNB hydrogels for disease modeling and regenerative medicine.

(A) Dynamic stiffening of GelNB-HPA hydrogels via on-demand addition of tyrosinase. Pancreatic cancer cells were encapsulated in GelNB-based hydrogels with either PEG or HA-based crosslinker and only the latter induced EMT-like phenotype. Reproduced with permission from Elsevier Ltd.^[96] (B) T2-MRI images of the regenerated spine tissues. Among treatment conditions, TGFβ-loaded GelNB (BIOGEL) hydrogels led to greater IVD hydration and the lowest CGRP (Pain) signals. Reproduced with permission from KeAi Communications Co., Ltd.^[55]

4.4. Delivery carriers

Hydrogels are great carriers for delivering therapeutically relevant agents, including synthetic drugs, peptides, proteins, and cells. In this regard, GelNB hydrogels, either crosslinked by thiol-norbornene photocrosslinking or iEDDA click reaction, have been used for the in situ delivery and transfection of micro RNA (miRNA) in stem cells,^[106] for activating or releasing transforming growth factor β (TGFβ),^[55,107] and for delivery of chimeric antigen receptor (CAR-) T cells (CAR-T cells).^[108] Carthew et al. performed in situ transfection of pro-osteogenic miRNAs in hMSCs encapsulated within a LAP and light-crosslinked GelNB-PEGdiSH hydrogel.^[106] Higher mineralization and osteogenic gene expression were observed in hMSCs with in situ transfections of miR-100–5p and miR-143–3p. Wang et al. developed GelNB-GelTz iEDDA click hydrogels to encapsulate alkaline microspheres for the purpose of activating endogenous TGFβ1.^[107] The hydrogels were injected into the tissue defect and the alkaline substances were able to diffuse from the microspheres into the hydrogel, which then activated endogenous latent TGFβ1 to promote MSCs recruitment and tissue regeneration. Luo et al. loaded TGFβ in GelNB-GelTz iEDDA click hydrogels for its delivery in vivo for promoting intervertebral disc (IVD) regeneration.^[55] Finally, Suraiya et al. exploited the use of GelNB-PEGdiSH photocrosslinked micogels for encapsulation and delivery of CAR-T cells.^[108] The GelNB microgels supported high viability of CAR-T cells, retained the T cell phenotype and functionality, and demonstrated potent cytotoxicity against human ovarian cancer in vitro. The successful use of GelNB-based hydrogels in these examples have demonstrated the potential of these bioactive hydrogels to serve as carrier systems for diverse therapeutically relevant applications.

4.5. Bioprinting and biofabrication

As a highly water soluble macromer, GelNB has lent itself as a bioink for bioprinting and biofabrication. For example, Tytgat et al. used GelNB/GelSH system as a bioink for extrusion bioprinting.^[109] A 27 gauge nozzle and a printing speed of 300 mm/min were used to create printing resolution of 200–1000 μm and the printed scaffolds were subsequently crosslinked by LAP and 365 nm light irradiation for 10 min. Adipose tissue-derived stem cells were seeded onto the printed scaffolds and differentiated toward adipocytes. Compared with GelMA, 3D printed GelNB/GelSH hydrogels supported higher degree of triglycerides positive cells, which were attributed to the higher swelling properties and lower mechanical strength of the GelNB-based hydrogels. This work did not explore in situ printing of cell-laden hydrogels. Later, Alge and co-workers comprehensively compared the GelNB and GelMA in extrusion bioprinting.^[110] They found that, compared with chain-growth GelMA bioink, step-growth GelNB bioink had improved photo-crosslinking kinetics, higher Z stability, and decreased filament spreading. On the other hand, GelMA bioinks had lower stress and were more easily extruded. Further studies by Göckler et al.,^[111] Zhao et al.,^[112] Yao et al.,^[113] and Burchak et al.^[114] also revealed the outstanding properties of GelNB-based bioink, including substantially reduced amounts of photoinitiator needed in crosslinking, fast curing (1–2 s), modular crosslinking of bioactive components, higher network homogeneity, minimal cross-reactivity with cellular components, and the ability in performing secondary thiol-norbornene click reaction post-printing. While GelNB itself can be used as the major bioink in extrusion bioprinting, it can also be used to improve the printing resolution of other extrudable bioinks. For example, our group has synthesized methylcellulose-norbornene (MCNB) for extrusion bioprinting.^[115] We found that the printing resolution was greatly improved when GelNB was supplemented in the MCNB bioink, which was attributed to the improved rheological property of the mixed bioink.

In addition to extrusion bioprinting, GelNB has also been used in other types of bioprinting, including electrowriting, two-photon polymerization (2PP), digital light processing (DLP) bioprinting, and volumetric bioprinting. For example, Castilho et al. developed a cell electrowriting process to decrease the diameter of the printed GelNB-based hydrogel filaments from hundreds of micrometers to 5 – 50 micrometers.^[116] GelNB, silk fibroin, and PEG8SH were used as the bioinks, which were crosslinked by the Ru/SPS initiation system.^[116] Compared with silk fibroin, the printed GelNB scaffolds were stiffer, had higher printing fidelity, but with reduced structural integrity in multi-layer printing.^[116] Additionally, Dobos and Van Hoorick et al. used two-photon polymerization (2PP) to crosslink and pattern cell-laden GelNB hydrogels with remarkably high scanning speeds (1000 mm s⁻¹).^[117,118] While the 2PP process was generally cytocompatible, the gelatin backbone would be cleaved when 2PP light was used at high laser intensities (i.e. ⩾150 mW).^[117] This could be potentially leveraged to create internal channels or softer regions in the crosslinked hydrogels. Levato et al. developed fish gelatin based GelNB for DLP bioprinting of high-resolution hydrogel constructs with embedded channels. Fish gelatin has higher thermal stability in solution at room temperature, which is ideal for DLP bioprinting of complex structures.^[69] Our group has also used DLP bioprinting to print GelNB hydrogels for the purpose of enhancing vascularization.^[119] In addition to demonstrating the cytocompatibility of DLP-printed GelNB hydrogels for in situ encapsulation and vascularization of human umbilical vein endothelial cells (HUVECs), we showed that the encapsulated HUVECs formed microvascular networks with lumen structures. Furthermore, the GelNB bioink permitted both in situ conjugation and secondary photo-tethering of pro-angiogenic and pro-vasculogenic QK peptide, a biomimetic peptide derived from vascular endothelial growth factor (VEGF).

The Zenobi-Wong group exploited GelNB as a bioink for volumetric bioprinting (VP),^[48] another light-mediated bioprinting technique for rapid printing of complex and low-defect 3D objects. It was suggested that the use of GelNB and VP can address the limitations facing GelMA-based bioinks for high-throughput printing of complex tissue structures. The use of GelNB also provides another unique property in creating dynamic VP printed scaffolds. For example, Falandt et al. aimed to create an angiogenic model through the VP of GelNB-based hydrogels.^[120] VEGF was grafted to the HUVEC-laden constructs. Using various ratios between GelNB and the chosen crosslinkers, full print volumes could form in the duration anywhere between 15 and 36 seconds. Within the hollow regions of the printed scaffolds, HUVECs were perfused along with VEGF and LAP to allow the cells and growth factor to adhere to the surface of the channel. After three days of culture, it was shown that VEGF-grafted scaffolds could support HUVEC sprouting with sprout lengths roughly twice as long as the VEGF-free regions.

4. Conclusion

Gelatin-based hydrogels present a highly adaptable biomaterial for biomedical applications, with GelMA being the most used hydrogels in tissue engineering, regenerative medicine, and biofabrication space. Albeit similar to GelMA in many aspects, GelNB offers unique characteristics that may provide unique benefits, including its fast reaction kinetics and diverse crosslinking mechanisms. Many comparison studies have concluded that GelNB outperforms GelMA in crosslinking speed, network homogeneity, modular adaptability in matrix physicochemical properties, and cytocompatibility. However, whether to use GelMA or GelNB for hydrogel crosslinking may depend on the specific applications. GelMA is routinely crosslinked as single-component hydrogels, whereas the crosslinking of GelNB requires a thiol- or tetrazine-containing crosslinker. Unlike GelMA, the utility of GelNB-based hydrogels as cell and therapeutics carriers has not been tested extensively, particularly in tissue regeneration in vivo. Some less explored areas also include the use of GelNB and its derivatives in crosslinking of viscoelastic hydrogels, macroporous scaffolds, and granularly assembled hydrogels. Nonetheless, GelNB has proven useful in tissue engineering and regenerative medicine and is expected to continue be exploited as a diverse macromer for other biomedical applications.

Figure 7. GelNB as a bioink for bioprinting and biofabrication.

(A) The addition of GelNB improves printing fidelity of another bioink MCNB. Reproduced with permission from IOP Publishing.^[115] (B) GelNB permits printing cell electrowritten of hexagon-shaped 3D scaffolds, which shows higher printing accuracy than extrusion printing. Reproduced with permission from American Chemical Society.^[116] (C) Improved 2PP fabrication of thiol-norbornene formulations (with different thiol-containing linkers) as opposed to the conventional gel-MOD (i.e., GelMA). Reproduced from IOP Publishing.^[117] (D) GelNB permits post-printing patterning of thiol-containing ligands (red) within the 3D printed structures (green). Reproduced with permission from Wiley-VCH GmbH.^[120]

Biography

Dr. Chien-Chi Lin is the Thomas J. Linnemeier Guidant Foundation Endowed Chair and Professor in Biomedical Engineering at Indiana University-Purdue University Indianapolis. Dr. Lin’s research focuses on designing multifunctional polymeric biomaterials for releasing therapeutically relevant agents and for delivering adult, stem, and progenitor cells for tissue engineering and regenerative medicine applications. His research group receives support from multiple NIH grants, the NSF CAREER award, private foundations, industry, and international research institutes. Dr. Lin’s current research projects focus on designing dynamic and spatially graded hydrogels for studying pancreatic cancer progression and for directing pancreatic differentiation of induced pluripotent stem cells.