Additive manufacturing of photosensitive hydrogels for tissue engineering applications

Design considerations

To design appropriate hydrogel precursors for potential AMT applications in tissue engineering, several considerations should be addressed.

First, from a chemical point of view, hydrogel precursors should possess sufficient water solubility. To achieve this goal, polyethylene glycol (PEG) based synthetic polymers as well as certain naturally-derived polymers (e.g., gelatin, hyaluronan or HA) have drawn much attention due to their superior hydrophilicity. On the other hand, the photo reactivity of hydrogel precursors plays an important role in the feasibility to AMT. To fabricate an object with size scales that are clinically relevant (mm to cm scale), the photoreactivity of monomers/macromers directly determines both the writing speed and time costs of the manufacturing process.

Second, from a biological point of view, hydrogel precursors must meet further requirements.

• Cytocompatibility: For in situ cell encapsulation, cells are directly exposed to a series of materials at varying stages: initially hydrogel precursors, then radical-mediated polymerization process, subsequent crosslinked networks, photoinduced byproducts and finally degradation products. Therefore, all of these involved chemicals and related polymer chemistry must be cytocompatible or at least of very low cytotoxicity.

• Bioactivity: Apart from cytocompatibility, hydrogel scaffolds produced by AMTs should provide cells with a physiologically-relevant environment and thus support all cell functions that are occurring in vivo (adhesion, migration, proliferation, differentiation). In a natural environment of cells, cell attachment is regulated by the crosstalk between integrin receptors on cell membranes and integrin-binding motifs (e.g., RGD motif peptides) distributed on ECM components. To this end, researchers have utilized either synthetic materials in combination with RGD motifs or naturally-derived proteins containing RGD motifs (e.g., gelatin, fibrin, fibrinogen) to create biomimetic hydrogels for cell culture [8].

• Biodegradability: Since the ultimate goal of implantable hydrogels is to replace or repair damaged tissues, the degradation kinetics of printed hydrogels should be tunable to match the growth rate of new tissues.

In the next sections, we discuss photopolymerizable hydrogel precursors that have been used in tissue engineering, proceeding from the chemistry of hydrogel formation, to synthetic hydrogels and naturally-derived hydrogels while discussing structure-property relationships and their applications in AMT.

Chemistry of hydrogel formation

The formation of hydrogel networks is based on the presence of multifunctional crosslinker during network formation. Thus, synthetic approaches to light induced network formation are not only limited to classical free-radical chain polymerization, but also alternative approaches based on click chemistry are currently investigated.

Chain polymerization: Free-radical chain polymerization is a facile approach to create hydrogels where a chain reaction propagates through vinyl-group containing monomers. The reactivity of vinyl monomers towards chain polymerization is dependent on the electronic environment of vinyl groups. Generally, it is accepted that the reactivity of vinyl monomers towards free-radical chain polymerization follows this sequence (Scheme 1): acrylate > vinyl ester ∼ vinyl carbonate > methacrylate > fumarate [33].

Scheme 1

Schematic showing the formation of hydrogel networks via free-radical chain-growth polymerization (FG: functional group).

To date, acrylated monomers are the state-of-the-art materials for AMT applications due to their high reactivity. A general method to synthesize acrylated macromers is based on esterification reaction between acryloyl chloride and hydroxyl groups of several synthetic building blocks [PEG, polyvinyl alcohol (PVA), etc.]. Although acrylates have superior reactivity, irritancy and potential cytotoxicity of unreacted acrylate groups is posing a challenge for their potential clinical-relevant applications. In contrast, methacrylates are much less cytotoxic as evidenced by their wide use as dental filling materials [34]. But the moderate photoreactivity of methacrylates raises many practical problems in most photolithography-based AMT. To address this challenge, we recently developed alternative photopolymers (vinyl esters) that represent a good trade-off between high reactivity and low toxicity [35]. Interestingly, there are very few monofunctional vinyl ester derivatives and only one hydrophobic difunctional vinyl esters (divinyl adipate) commercially available and a very limited number of photopolymerization studies related to vinyl esters have been reported so far [36]. Furthermore, hydrogels formed by vinyl ester chemistry remain largely unexplored. We therefore synthesized PEG divinyl esters (PEGDVE) through a transesterification reaction between PEG dicarboxylic acids and vinyl acetate under the catalysis of palladium (II) acetate [37].

Table 2 presents a comparative analysis on the biocompatibility of acrylates and vinyl esters. Importantly, upon hydrolytic degradation, the major degradation products out of poly(vinyl esters) are FDA-approved poly(vinyl alcohol) (PVA) while polyacrylates result in high molecular weight poly (acrylic acids) which have proven very difficult to be excreted from the human body [35]. On the other hand, since it is unlikely to reach complete conversion of vinyl groups in radical photopolymerization, careful attention should be paid to the remaining unpolymerized vinyl groups (at least 10–20%). It is noteworthy that the remaining acrylate groups would form acrylic acids as degradation products that are highly cytotoxic and irritant while the residual vinyl ester groups can be easily hydrolyzed into acetaldehyde that can be easily metabolized into acetic acid in vivo under the catalysis of acetaldehyde dehydrogenase. Although vinyl esters are generally not as reactive as their acrylate analogs, our recent work demonstrated that the thiol-ene photo-click chemistry could greatly improve the reactivity of vinyl esters even to the level of acrylates [37]. Moreover, it is important to mention that the poor storage stability related to thiol-ene systems have recently been solved by using a combination of radical and acidic stabilizing system [38]. All these aspects suggest that water-soluble vinyl esters are promising hydrogel precursors for photolithography-based AMTs and tissue engineering applications.

Table 2

Comparative analysis of biocompatibility: polyacrylates vs. polyvinyl esters.

Monomers		Major degradation products	Hydrolyzed product of unpolymerized group
Acrylates
Vinyl esters

Click-chemistry based polymerization: Besides chain polymerization, click chemistry is an emerging methodology to synthesize chemically-crosslinked hydrogels (Scheme 2). According to the initial concept proposed by Sharpless, click chemistry refers to unique chemical reactions between orthogonal groups that react with high selectivity under mild conditions [39]. For instance, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is highly specific and occurs in the presence of competing functional groups (Scheme 2). Researchers have successfully used CuAAC chemistry in combination with a series of hydrophilic macromers (PEG, PVA, HA) to prepare hydrogels [40–42]. However, spatiotemporal control is not readily achieved in these hydrogel systems. Interestingly, a collaborative study from Anseth and Bowman groups explored photoinitiated CuAAC reaction to create hydrogels with spatiotemporal control [43]. In this work, the CuAAC reaction was catalyzed by the photoreduction of Cu(II) to Cu(I) whereby the reduction was initiated by free radicals from activated photoinitiators. This method demonstrated comprehensive spatiotemporal control over the process of hydrogel formation, thus showing great promise for photopatterning or fabrication of hydrogels. But utilization of this method in AMT remains unexplored. Although hydrogels could also be formed via other click approaches like tetrazine-norbornene Diels-Alder reaction and oxime ligation [44, 45], these approaches generally lack of spatiotemporal control and thus are not suitable for direct use in AMT applications.

Scheme 2

Schematic showing the formation of hydrogel networks via step-growth click-polymerization.

Radical-mediated thiol-ene photopolymerizations are alternative attractive click-approaches to create chemically-crosslinked hydrogels with spatiotemporal control. Such reactions are chemo-selective, robust and applicable to a wide range of macromers containing multiple ene or thiol groups. For instance, the pioneering work of Anseth and co-workers demonstrated that thiol-norbornene step-growth photopolymerizations are extremely robust systems for preparing hydrogels [46]. In their work, 4-arm PEG norbornenes could efficiently ‘click’ with a dithiol (i.e., a peptide with two cysteines), forming ideal hydrogel networks in <1 min [46].

The underlying mechanism of step-growth thiol-ene photopolymerizations is proposed below (Scheme 3A). Upon irradiation, free radicals are generated from photoinitiators followed by the H-abstraction reaction from thiols. The resulting thiyl radicals propagate across the ene functionalities, forming the carbon-centered radicals. Ideally, chain-transfer reactions are much more competitive than further chain-propagation reactions. Thus, the thiyl radicals are generated again upon highly efficient chain-transfer reactions.

Scheme 3

(A) Proposed mechanism of radical-mediated thiol-ene photopolymerization and (B) reactivity sequence of variable “ene” groups towards thiol-ene conjugation.

Within the scope of ene groups, the conjugation rate with thiol groups differ greatly from one to another according to the electron density of the ‘ene’ groups [47]. For a specific thiol, electron-rich enes polymerize much faster than electron-poor enes. It is accepted that reactivity of ene groups towards radical-mediated thiol-ene polymerization follows this sequence (Scheme 3B): norbornene > vinyl ether > vinyl ester > allyl ether > vinyl carbonate > acrylate > N-substituted maleimide > methacrylate. It is important to note that some of these groups (e.g., norbornene, maleimide) undergo ideal step-growth polymerization while other functional groups (e.g., vinyl ester, vinyl carbonate, acrylate, methacrylate) undergo a mixed-mode (step-growth and chain-growth) polymerization mechanism. More information related to thiol-ene photo-click chemistry could be found in comprehensive reviews of Hoyle and Bowman [47, 48].

Synthetic hydrogel precursors

Synthetic hydrogels are increasingly explored for tissue engineering applications due to numerous advantages, such as easy access, cost efficacy, biologically blank state, and so forth. It is feasible to molecularly tailor the mechanical properties and degradation profiles of synthetic hydrogels and to match requirements for a specific application. Precursors of synthetic hydrogels include PEG, PVA, and poly(2-hydroxyethyl methacrylate) (PHEMA) (Figure 3). Considering that very few examples of PHEMA could be found in AMT applications, this section focuses on precursors based on PEG and PVA only.

Figure 3

Examples of common synthetic hydrogels.

Poly(ethylene glycol): PEG and its derivatives have proved extremely versatile for tissue engineering applications, such as non-fouling coatings that are resistant to protein adsorption [49]. In recent years researchers have witnessed the wide use of PEG in the field of hydrogels due to its superior hydrophilicity, biocompatibility, and negligible immunogenicity [50]. PEG-based hydrogel precursors could be synthesized by introducing multifunctional vinyl or clickable groups to the pendant hydroxyl groups (Figure 4). For example, high M_w (3–20 kDa) (meth)acrylated PEG (PEGDA/PEGDMA) can be synthesized in one step by reacting (meth)acryloyl chloride with pendant hydroxyl groups [51] while lower M_w ones (0.3–1 kDa) are commercially available.

Figure 4

Chemical structures of PEG-based hydrogel precursors

Researchers have also developed bioerodible PEG hydrogels using photopolymerizable macromers that contain a PEG central core but terminate with oligomers of alpha-hydroxy acids such as PLA [13, 52–54]. Specifically, the PLA-b-PEG-b-PLA triblock copolymers were firstly synthesized via classical ring-opening polymerization. An excess of (meth)acryloyl chloride was then reacted with PLA-b-PEG-b-PLA. The degradation rate of formed hydrogels can be tailored from <1 day to up to 4 months by controlling the crosslinking density of the gel or by selecting the type and amount of α-hydroxy esters. It is noteworthy to mention the reactivity differences between acrylated PEG and methacrylated PEG. For instance, 2 min exposure time was required for the former while 10 min was needed for the latter [52, 53].

As mentioned before, although PEGDA are the most widely used hydrogel precursor in the biomaterials community, their irritancy and potential cytotoxicity of unreacted acrylate groups might preclude them from further clinical-relevant applications [35]. To address this challenge, we recently developed PEG-based vinyl ester (PEGDVE) [37]. It was demonstrated that cytotoxicity of PEGDVE was more than one magnitude lower than its PEGDA/PEGDMA analogues. Furthermore, it is important to note that degradation products of PEGDVE hydrogels should be based on non-cytotoxic PVA and PEG.

Although the photoreactivity of PEGDVE are not comparable to PEGDA due to the presence of abstractable hydrogens, we recently circumvented this problem by using the robust thiol-ene chemistry [37]. Based on systematic photo-differential-scanning-calorimetry (Photo-DSC) studies, we suppose that vinyl esters are less prone to chain-growth homopolymerization when compared to acrylates, presumably because the formed radicals lack of resonance stabilization. In contrary, the low resonance stabilization effect might explain why these radicals show high reactivity towards chain transfer reactions with thiols. High reactivity and high conversion could be achieved in thiol-vinyl ester photopolymerizations.

Alternatively, PEG norbornenes are ideal hydrogel precursors in terms of high reactivity for thiol-ene step-growth photopolymerization. Fairbanks and Anseth demonstrated that thiol-norbornene photopolymerizations are suitable models to assemble hydrogels with CRGDS peptide in cell photo-encapsulation studies [46]. The robustness of thiol-norbornene click reactions even minimized the dose of photoinitiators required for cell-encapsulation and thus optimized the precursor’s cytocompatibility. A recent study by McCall et al. proved that thiol-ene photopolymerizations are promising methods to form hydrogels for protein encapsulation while maintaining most bioactivities of proteins [55].

Poly(vinyl alcohol): PVA are hydrophilic polymers originated from partial hydrolysis of poly (vinyl acetate). PVA-based hydrogels have been widely used in space filling and drug delivery systems because of their superior biocompatibility (FDA-approved). In addition, PVA hydrogels are uniquely stronger than most other synthetic hydrogels [16]. Although aqueous PVA solutions can be physically crosslinked by crystallites formed by repeated freeze-thawing [56], the resultant hydrogels lack of stability at high temperature because of the melting crystallites. To get better stability, chemically crosslinked PVA hydrogels have been synthesized by using glutaraldehyde [57] or succinyl chloride [58]. However, these crosslinking agents are highly toxic and exclude the feasibility for in situ cell encapsulation applications.

In the last decade, researchers have extensively explored PVA-based hydrogels formed by photopolymerization in a minimally invasive fashion. Anseth and co-workers have developed photopolymerizable PVA macromers with varying degree of degradable ester linkages and reactive acrylate groups [59]. In this work, an acrylate terminated ester-anhydride was prepared and subsequently reacted with pendant hydroxyl groups of PVA to form the desired acrylated ester-PVA (Figure 5). Synthetic hydrogels with tunable degradation profiles are important for most biodegradable hydrogel scaffolds in tissue engineering. To this end, Martens et al. have copolymerized methacrylated PEG and acrylated PVA macromers that are hydrolytically degradable to produce hydrogels [60]. In this work, it was found that degradation rates of copolymerized PEG-PVA gel were faster than the rates of PEG gel, but slower than the rates of PVA gel.

Figure 5

Synthetic scheme of acrylated ester-PVA.

Although PVA hydrogels are not cell-adhesive like most other neutral polymers, they can be rendered cell-adhesive by covalently conjugating biological molecules (e.g., RGD motif, fibronectin) to the pendant hydroxyl groups of PVA (Figure 6). For instance, Schmedlen et al. have prepared bioactive PVA hydrogels that were not only non-cytotoxic but also supported the attachment and spreading of fibroblasts [61].

Figure 6

Synthesis of methacrylated-RGDS-PVA.

In this study, PVA was firstly modified with methacrylamide (for photocrosslinking) and amino groups (for RGD conjugation) via reaction of methacrylamidoacetaldehyde dimethyl acetal and aminobutyraldehyde diethyl acetal. Acetylated RGDS peptide was then immobilized on the modified PVA through an aminolysis reaction. It was found that the extent to which these hydrogels can support cell attachment and spreading was dose-dependent on the immobilized RGDS. In another work, Nuttelman et al. have covalently attached fibronectin on the surface of preformed PVA hydrogels through carbonyl diimidazol (CDI) chemistry [57]. The biofunctionalized PVA hydrogels drastically increased the rate of NIH3T3-fibroblast attachment and proliferation, and promoted 2D cell migration.

Naturally-derived hydrogel precursors

When it comes to biocompatibility issues, naturally-derived hydrogels are generally thought to be advantageous over synthetic hydrogels since natural gels may offer better biochemical and biological cues to surrounding cells. Most naturally-derived hydrogels are either components of natural ECM or provide similar properties that mimic the natural situations. Researchers have used a variety of naturally-derived materials, including collagen, gelatin, hyaluronic acid (HA), chitosan, alginate, and so forth [62–65]. Importantly, collagen is the major component of the proteins in the ECM of human tissues and takes up approx. 25% of the total protein mass. Furthermore, HA is another important ECM component and widely distributed throughout connective, epithelial, and neural tissues. Chemically, HA is a non-sulfated, anionic glycosaminoglycan consisting of repeating disaccharide units. Similar to HA, both chitosan and alginate are linear, hydrophilic polysaccharides with superior biocompatibility (Figure 7). They have been shown to present favorable in vivo characteristics and thus they have been widely accepted as excellent hydrogel precursors for various tissue engineering applications [66–68].

Figure 7

Structures of common substrates for naturally-derived hydrogels: hyaluronic acid (HA) and chitosan.

Collagen: Collagen is one type of fibrous proteins and the most abundant protein in mammals. Collagen has a triple helical structure formed by polypeptide helices made of Gly-X-Y repeating units [69]. As a major structural protein of most ECM, Type-I collagen plays important passive roles in stress-bearing by providing skin, bone, tendon, and ligament with their characteristic tensile strengths. Since Type-I collagen covers a wide range of domains that could crosstalk with integrin receptors on the cell surfaces and other matrix proteins, collagen-based hydrogels are cell adhesive and favourable for cell migration [70]. Hydrogels based on collagen can be formed by either physically crosslinking treatments (i.e., irradiation, freeze-drying) [71, 72], or chemically crosslinking agents (i.e., glutaraldehyde, carbodiimide) [72, 73]. Yet, there are few examples of polymerizable collagen derivatives, due to the difficulty of collagen dissolution.

Gelatin: Compared to native collagen, gelatin is more cost-effective and has easier access. Gelatin products with different isoelectronic points can be prepared via acidic or alkaline denaturation [74]. Gelatin is derived from collagen Type I via heat denaturation and has gained increasing popularity in the biomaterials community. For this point gelatin hydrogels have been widely used as drug carriers in controlled delivery systems [75].

A general problem related to gelatin-based hydrogels is their poor mechanical properties which can be improved by physical or chemical crosslinking methods. Among them, photopolymerizable gelatin derivatives have received the most attention due to their great advantage for in situ cell photo-encapsulation. To render gelatin polymerizable, target functional groups could be introduced by modifying lysine units or glutamic/aspartic acid units (Figure 8). For instance, Van den Bulcke et al. have reported a derivative of gelatin modified with methacrylamide groups (Gel-MA) [76]. In this work Gel-MA was prepared through an amidation reaction between ε-amino groups of lysine units of gelatin and methacrylic anhydride. Gel-MA hydrogels have enabled in situ cell encapsulation by providing favorable cell viability and cell adhesion [77]. Klein, Malda, and co-workers exploited Gel-MA in combination with other photocrosslinkable derivatives of HA, alginate, chondroinite sulfate for cartilage tissue engineering applications [78, 79]. Notably, Ovsianikov et al. explored the two-photon polymerization of Gel-MA by using I2959 as photoinitiator [80]. Although proof-of-concept structuring succeeded, it is important to note that the photoreactivity of methacrylated macromers are generally too low for most photolithography-based AMT applications.

Figure 8

Structures of photopolymerizable gelatin derivatives: gelatin methacrylamide (Gel-MA), gelatin acrylamide (Gel-AC) and gelatin vinyl ester (GH-VE) for naturally-derived hydrogels.

To tackle the reactivity issue, acrylamide derivative of gelatin (Gel-AC) was developed in the Liska group (unpublished results). Although photo-rheometry studies proved that Gel-AC indeed possessed high photoreactivity, cell-encapsulation attempts revealed that Gel-AC induced a drastic decrease of MG63 cell viability. It is presumably due to the Michael-addition side reactions between acrylamide moieties and amine groups of cell-surface proteins. To achieve a good trade-off between high reactivity and low toxicity, Qin et al. recently reported the first hydrogel precursor with vinyl ester groups, i.e., gelatin vinyl esters (GH-VE), which was proved to be low cytotoxic and enzymatically degradable [81]. Specifically, low MW gelatin (GH) was functionalized with VE groups through an aminolysis reaction between ε-amino groups of lysine units and an excessive amount of divinyl adipate (DVA). MTT assay revealed that GH-VE macromer presented negligible cytotoxicity on MG63 cells, which suggests that the modification did not negatively influence the cytocompatibility of the substrate. Although ¹H-NMR and MALDI-TOF analysis showed that there are more than two VE groups per macromer, the photoreactivity of GH-VE towards homopolymerization was quite limited. To overcome this limitation, the more efficient thiol-ene click chemistry was adopted in this study [81].

Since water-soluble multifunctional macrothiols are not commercially available, the authors selected reduced BSA (BSA-SH) as a model macrothiol to donate multiple free cysteine units for subsequent photo-click reactions with vinyl ester groups of GH-VE (Figure 9). The extent of cysteine units was controlled by changing the stoichiometry between disulfide bridges and reducing agent such as tris(2-carboxyethyl)phosphine (TCEP). It was found that both photoreactivity and crosslinking density of these hydrogels were dependent on the thiol/ene ratio, as proved by photo-rheometry and swelling studies [81]. Importantly, it was shown that the extent of cell attachment on these protein-based hydrogels could be regulated by the relative ratio between GH-VE (adhesive) and BSA-SH (repellent). By using a highly efficient initiator (i.e., WSPI) and two-photon thiol-ene lithography, it was feasible to produce complex hydrogel constructs in 3D at a writing speed as high as 50 mm/s.

Figure 9

Schematic showing the hydrogel formation via thiol-ene photopolymerization using GH-VE and BSA-SH as precursors.

Hyaluronic acid: HA or hyaluronan is a linear polysaccharide consisting of D-glucuronic acid and D-N-acetylglucosamine as repeating units. As the major component of ECM, HA plays an important role in wound healing, cell signaling and tissue morphogenesis. Due to its unique biological functions, HA has been widely used in biomedical applications such as for treatment of osteoarthritis and regeneration of vocal cord [82, 83]. Nevertheless, the native HA suffers from poor mechanical properties and short half-lives in vivo which makes it inappropriate for certain clinical applications such as articular cartilage regeneration. As such, many researchers have explored a variety of methods to chemically modify HA with polymerizable groups in order to obtain covalently crosslinked hydrogel networks [84–86]. These methods could effectively improve the mechanical strength and prolong the half-lives of HA hydrogels in vivo.

The principle targets for chemical modification in HA are primary and secondary hydroxyl groups and the carboxyl groups (Figure 10). For instance, photopolymerizable methacrylated HA (HA-MA) were synthesized by reacting either methacrylic anhydride (or glycidyl methacrylate) with the primary hydroxyl groups in HA [87, 88]. Khademhosseini et al. have explored the micromolding-based photopatterning of HA-MA hydrogels for cell encapsulation applications [89]. In this work, the irradiation conditions were harsh (300 mW/cm² UV light, 3 min), presumably as a result of the limited reactivity of HA-MA. Recently, Khetan and Burdick developed acrylated HA (HA-AC) [90]. Specifically, 2-hydroxy ethyl acrylate was firstly conjugated with succinate followed by a further esterification reaction with HA. However, the use of HA-AC in AMT has not been reported yet.

Figure 10

Chemical structures of hyaluronic acid (HA) derivatives: HA-MA, HA-AC and HA-VE.

While (meth)acrylated HA has been developed as photocrosslinkable hydrogel precursors, several optimizations could be addressed. Like most methacylate-based monomers, the methacrylated HA lack of photoreactivity which poses a challenge for potential uses in AMT applications. Additionally, it is well known that the efficient thiol-ene click chemistry could not improve the reactivity of methacrylates [47, 48, 91]. Although acrylated HA exhibits higher photoreactivity, the irritancy and potential cytotoxicity of acrylates are very likely problematic for FDA-approval. Very recently, Qin et al. reported a HA alternative with pendant vinyl ester groups (HA vinyl esters, HA-VE) to tackle these limitations [92]. A series of HA-VE with tunable degree of substitution were synthesized via lipase-catalyzed transesterification. The development of HA-VE was based on previous work: 1) vinyl esters are much less cytotoxic than their acrylates references [35], and 2) the moderate photoreactivity of vinyl esters could be greatly improved by using the robust thiol-ene chemistry [37]. Owing to the unique molecular design, degradation products of HA-VE hydrogels through hydrolysis are PVA and adipic acid (both FDA-approved). Importantly, the cytotoxicity of HA-VE on L929 fibroblasts was found to be significantly lower than that of HA-AC (p<0.01) and HA-MA (p<0.05) [92]. Although the reactivity of HA-VE towards homopolymerization was insufficient for 2PP, it was proved that thiol-ene chemistry could substantially improve its reactivity. This optimization enabled 2PP microfabrication of a complex hydrogel construct of HA-VE with μm-scale accuracy and high writing speed (50 mm/s).

Two-photon-induced biofunctionalization

In a natural environment, cells are embedded in the ECM, which is a complex 3D hydrogel network containing a variety of biochemical and biophysical signals. Within the ECM context, spatial and temporal access of integrin ligands, morphogens and growth factors regulate a number of biological processes [4, 112–114]. For instance, formation of new blood vessels (neovascularization) is dictated by the highly delicate biochemical and biophysical environments of different cell types, and in particular the spatial and temporal presentation of a number of growth factors [115–117]. An excellent overview of channeled hydrogels for neovascularization goes to a very recent review by Redl and co-workers [118]. Apart from creating channeled hydrogel structures, spatiotemporal patterning of bioactive molecules in preformed 3D hydrogel constructs is an extremely useful tool to precisely control cell behavior. West and co-workers have shown that the two-photon patterning of RGD motifs in PEG-based hydrogels could effectively confine cell adhesion and cell migration in the area functionalized with RGD [119, 120]. In these studies hydrogel pellets were firstly formed by SP photopolymerization and then soaked in a solution of photoinitiator and acrylated RGD peptides.

Besides, researchers have also explored the two-photon uncaging concept for biofunctionalization of 3D hydrogels. In a recent study of Shoichet and co-workers, multiple growth factors were sequentially immobilized within agarose hydrogels (Figure 13) [121]. Specifically, agarose hydrogel matrices were firstly activated with carbonyldiimidazole (CDI) and then modified with 2-nitrobenzyl-protected cysteines [122]. Upon two-photon irradiation, cysteine pendant groups were liberated for thiol-ene conjugation with maleimide-containing barnase/streptavidin. Finally, growth factors terminated with orthogonal counterparts (barstar/biotin) were immobilized via affinity-binding within the 3D pattern.

Figure 13

Two-photon biofunctionalization of alginate gels with various growth factors: (A) schematics of the protocol and (B) 3D projection of a confocal micrograph illustrating the patterned volumes and user-defined localization of two growth factors. Scale bar, 100 μm. (Reprinted with permission, Copyright Nature Publishing Group [121].)

A recent study by Ovsianikov et al. has established that bioorthogonal groups such as azides can be precisely immobilized within PEG matrices via 3D photografting [123]. Specifically, the grafting process was based on the decomposition of a commercially available aromatic diazide under three-photon excitation (3PA) and subsequent nitrene insertion, finally leaving pendant azide groups for further functionalization. However, it is noteworthy that three-photon absorption (3PA) is often less efficient and thus requires much higher laser intensity than TPA due to the lower probability of 3PA [124]. Li et al. recently developed a novel fluoroaryl azide compound (FAF-3) for efficient TPA grafting (Figure 14A). The rational molecular design provides FAF-3 with a large TPA cross section (178 GM at 800 nm) while the alkyne tail facilitates further bioorthogonal conjugation. Specifically, FAF-3 was precisely grafted within PEG matrices via TPA-induced photolysis and subsequent nitrene insertion. Since the grafting reactions are highly localized in a confined volume within the laser focal spot, arbitrary Yin-Yang pattern of alkyne groups (Figure 14B) was created in the PEG matrices with high spatial resolution (∼3.6 μm) [125].

Figure 14

3D site-specific functionalization of PEG matrices via photografting: (A) grafting mechanism and subsequent CuAAC reaction; and (B) schematics of the grafting procedure. (Scale bar: 50 μm [125].)

In addition, ultrafast scanning (∼550 mm s^–1) was achieved due to the highly efficient grafting reactions. To realize 3D site-specific functionalization, a model compound (i.e., red fluorophore terminated with azide group) was further conjugated on the “clickable” Yin-Yang pattern through CuAAC reactions. After that the Yin-Yang pattern changed from green to red, indicating the success of site-specific functionalization. This work shows great potential for further biologically-relevant studies where matrix biofunctionalization is necessary. In theory, any bioactive molecule of interest (e.g., VEGF-2) with alkyne groups could be readily incorporated through CuAAC-based click reactions.

Two-photon-induced polymerization

Although arbitrary biochemical patterns can be created via two-photon-induced biofunctionalization, these methods are intrinsically limited by the preformation of hydrogel pellets and thus not suitable for fabrication of complex structures. In contrast, 2PP strategy presents great promise to fabricate 3D hydrogels with complex geometry. By scanning focused pulsed laser in a hydrogel precursor solution, 3D hydrogels with user-dictated structures can be fabricated with micrometer-scale resolution. Again, high resolution is accessible because of 2PP processes are localized in the focused volume. Although radical-mediated 2PP is fast, it is important to note that the resolution of 2PP is also influenced by: 1) optical parameters of the 2PP setup; 2) how far the free radicals can diffuse from the reaction center; and 3) the extent to which oxygen could quench the reactions.

Direct fabrication of 3D hydrogels via 2PP primarily relies on the access to cytocompatible hydrogel precursors with high photoreactivity. Synthetic hydrogel precursors that are suitable for 2PP fabrication are mostly based on acrylated macromers like PEGDA. Torgersen et al. reported the first example of 2PP fabrication of hydrogels in the presence of living organisms [32]. In this work, viable Caenorhabditis elegans were captured in a 3D woodpile hydrogel structure of PEGDA. Besides, Ober et al. reported the use of PEGDA in combination with 2-hydroxyethyl methacrylate (HEMA) for 2PP microfabrication [126]. Käpylä et al. recently reported the 2PP of (meth)acrylated poly (α-amino acid)s in comparison with commercial PEGDA (M_n=575, 10000 Da) [127]. These new precursors were applicable for 2PP microfabrication in a wide processing window. Stable and well-defined hydrogel microstructures were fabricated with 80% water content by using I2959 as the PI. However, cytocompatibility of these hydrogel systems were not reported in this study.

Alternatively, naturally-derived hydrogel precursors have also been used for 2PP fabrication of 3D hydrogels. For instance, Ovsianikov et al. reported the 2PP microfabrication of 3D hydrogel constructs with high resolution (10 μm) and defined porosity by using photopolymerizable methacrylated gelatin (Gel-MA) [80, 128]. It was found that Gel-MA enabled favourable cell-matrix interactions with mesenchymal stem cells, including proliferation, adhesion and osteogenic lineage (Figure 15) [128]. Very recently, Ovsianikov et al. reported the first example of 2PP processing of cell-containing hydrogel constructs [129]. In this study, Gel-MA and highly efficient two-photon PIs were exploited as the photosensitive precursor materials. After the 2PP encapsulation of MG63 cells in Gel-MA, cell damage was observed within the laser-exposed region though a small percentage of cells were found to be viable in the vicinity to the exposed region. Interestingly, control experiments (i.e., no PIs) revealed that the same laser radiation conditions for 2PP seemed not to induce cell damage. The authors thus propose that the localized cell damage during 2PP could be attributed to cytotoxic species such as initiating radicals and reactive oxygen species.

Figure 15

Osteogenic differentiation of mesenchymal stems cells (at day 20) on gelatin scaffolds produced by two-photon polymerization (2PP) technique: (A) and (B) Micro-CT images showing crystal-like mineral deposition within the pores; (C) alizarin red staining confirming calcific deposition by cells of an osteogenic lineage. (Reprinted with permission from Woodhead Publishing [128], scale bar: 1 mm.)

Besides chain-growth 2PP, Liska and Stampfl groups have recently explored the robust thiol-ene click chemistry for 2PP microfabrication of 3D hydrogels (Figure 16) [81, 92]. As depicted in Figure 9, vinyl ester derivative of gelatin (GH-VE) and reduced albumin (BSA-SH) were developed as the “ene” and “thiol” macromers. By integrating thiol-ene photo-click chemistry and two-photon lithography, it was feasible to simultaneously pattern two proteins in 3D at high writing speed (50 mm/s) and produce a 3D hydrogel construct with complex geometry with micrometer-scale resolution.

Figure 16

Illustration for 2PP microfabrication of 3D hydrogel scaffolds with well-defined architectures using protein-based hydrogel precursors [81].