Review article(Photo-)crosslinkable gelatin derivatives for biofabrication applications
Graphical abstract
Introduction
During the last two decades, biofabrication has gained increasing attention within the field of tissue engineering and regenerative medicine. This is a consequence of the potential to fabricate complex, patient-specific constructs that closely resemble the complexity and heterogeneity of native tissues and organs in an automated way according to a computer-aided design (CAD) [1], [2], [3]. Within the field of biofabrication, two main strategies are currently explored, which are either based on the use of a (bio)material support resembling the extracellular matrix (ECM) or solely on cells along with cell secreted materials. When a biomaterial is applied, it can either be used as a biomaterial ink or a bioink depending on the composition. The term biomaterial ink refers to material processing via additive manufacturing and subsequent cell seeding, while the term bioink corresponds to the use of a mixture which already contains cells prior to processing via additive manufacturing [1], [3].
Gelatin as a bioink or a biomaterial ink – throughout the present manuscript, the term bio(material)ink is used when referring to both – has attracted considerable attention over the years as it is derived from collagen, which is the main constituent of the natural ECM of mammals [4], [5]. It is a denatured protein constituting 18 different amino acids characterized by a repetitive unit of glycine – X – Y in which X and Y can be several different amino acids [6], [7]. However, X and Y predominantly consist of proline and hydroxyproline which enables the formation of triple helices or physical crosslinks via interchain hydrogen bonds [6], [7]. As a consequence, the material is characterized by a dissociation temperature around 30–35 °C [6], [8], [9]. This implies that it dissolves at elevated temperatures, while forming a swollen hydrogel below this phase change temperature [6], [9], [10], [11]. Additionally, the presence of the tripeptide arginine-glycine-aspartic acid (RGD) in the protein backbone results in cell-interactive properties [12], [13]. Furthermore, it is enzymatically degradable by metalloproteases such as collagenase, which cleaves sequences such as Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln between Gly and Ile allowing cells to remodel it [10], [11], [14], [15], [16]. Due to the harsh acidic or basic denaturation process during the conversion of collagen to gelatin, concerns regarding immunogenicity and pathogen transmittance associated with the use of collagen are circumvented [4], [17]. In addition, it is considered safe by the Food and Drug administration (FDA) with a wide track record in the food and pharmaceutical industry [6], [7], [18]. Furthermore, gelatin is a by-product from the meat industry making it very attractive from an economical point of view [19].
However, due to the solubility at body temperature, the material was originally only applied as a temporary cell carrier to enable more straightforward cell manipulation [19]. To overcome this limitation, strategies were developed to stabilise the material at physiological conditions via the formation of chemical crosslinks. A common approach in this respect, consists of coupling the primary amines present in (hydroxy)lysine and ornithine with the carboxylic acids from aspartic and glutamic acid using carbodiimide chemistry thereby resulting in a zero length crosslinked hydrogel network [17], [20]. Alternatively, the nucleophilic functionalities of gelatin can be crosslinked using glutaraldehyde [21]. However, these stabilisation techniques offer limited control over the design of the obtained construct, as the material manipulation window is limited in time with little control over the crosslinking process.
A realm shift occurred in 2000 when Van den Bulcke et al. developed and patented the first photo-crosslinkable gelatin derivative (i.e. gelatin-methacrylamide (gel-MA)) [21], [22]. Photopolymerization exhibits attractive capabilities in terms of material processing including highly controllable gelation kinetics and predictable degradation capabilities, enabling convenient and straightforward material processing for biofabrication purposes [9], [22], [23]. The functionalization occurs by reacting the primary amines in the side chains of (hydroxy)lysine and ornithine with methacrylic anhydride, resulting in the formation of methacrylamide moieties [9]. Ever since, gel-MA has been applied for a plethora of biofabrication and tissue engineering strategies either as a standalone material or co-crosslinked with other (synthetic) materials (e.g. PEG) to form biohybrid hydrogels. As a result, it became one of the gold standards in the field [2], [8], [24], [25], [26], [27], [28], [29], [30], [31], [32]. Following this success, it has even started to bridge the gap between academia and industry as it is offered commercially by several companies as a bio(material)ink for research purposes [33], [34]. Besides gel-MA, several other (photo-)crosslinkable gelatin derivatives have emerged (Fig. 1). These derivatives can be subdivided into different classes based on the applied crosslinking mechanism including chain-growth (Fig. 1 blue) and step-growth polymerization. Within the step-growth classification, several other subclasses can be distinguished based on the applied crosslinking chemistry: thiol-ene (photo-)click chemistry (Fig. 3 red): (thiols: Fig. 1 purple; enes: Fig. 1 red), disulphide linkages (Fig. 1, Fig. 3 purple), Diels-Alder click (Fig. 1, Fig. 3: light green), Schiffs-base formation (Fig. 3 grey), π-π cycloaddition (Fig. 1, Fig. 3: yellow), photooxidation (Fig. 1, Fig. 3: green) and enzymatic based crosslinking (Fig. 1, Fig. 3: white).
Therefore, the present review aims to provide a helicopter view on all aspects related to the use of gelatin for biofabrication applications starting with raw materials and ending with final applications. Throughout the review, attention is paid to the physical and chemical properties, different chemical modification strategies and their implications on material and processing properties. Furthermore, an overview of different applied additive manufacturing technologies is provided including some final biomedical applications. Finally, a non-exhaustive overview of all presented gelatin derivatives and their processability potential towards specific additive manufacturing technologies is presented.
Section snippets
Crosslinking via chain-growth polymerization
The most commonly used crosslinkable gelatin derivatives take advantage of a chain growth polymerization crosslinking approach. Here, crosslinking occurs by polymerizing reactive functionalities (typically (meth)acrylates/(meth)acrylamides) immobilized onto gelatin resulting in the formation of short oligomer/polymer kinetic chains in between the gelatin chains [9], [10], [25], [37], [61], [63] (Fig. 2. A & C). Consequently, a polymer network is generated containing both gelatin polypeptide
Overview of applied photoinitiators
When using light-based crosslinking chemistries, typically a photo-initiator is required to initiate the crosslinking reaction. Since gelatin is a hydrogel material, suitable photo-initiators need to be water- soluble, thereby rendering the options limited. A non-exhaustive overview of photo-initiators applied for gelatin crosslinking is presented below, which can be classified according to their activation behaviour.
Important considerations during gelatin modification strategies
Since gelatin is a biopolymer consisting of around 18 different amino acids with various functionalities in different ratios, the material is characterised by a specific behaviour towards solvents, reaction conditions, temperature, pH, etc based on the relative amino acid composition.
Therefore, it is important to take a closer look at some of the aspects which are required to be taken into consideration when modifying or processing gelatin (-based) materials.
Controlling the mechanical properties of crosslinked gelatin hydrogels
The mechanical properties of photo-crosslinkable gelatin hydrogels can be tuned in various ways either during gelatin modification or during material processing.
Gelatin processing via additive manufacturing
Gelatin-based hydrogels have frequently been processed via additive manufacturing (AM) techniques. These methods are mostly based on the layer-by-layer fabrication of 3D constructs according to a CAD model. Printing of the materials can either occur by following a direct approach during which the material is directly processed in the AM step [26] or by an indirect approach in which a template is fabricated using AM to control the shape of the secondary material [8], [159].
Indirect approaches
Processability of gelatin derivatives using additive manufacturing technologies.
Table 1 provides a non-exhaustive overview of the processability of all reported gelatin derivatives using additive manufacturing technologies. Furthermore, if a specific derivative hasn’t been reported for a certain processing technology to date, a reasonable estimation of processability using that particular technology is presented based on the properties of the derivative. The symbol ‘✓’ is applied if no difficulties towards processing are anticipated based on previous experiments with
Conclusions
Throughout the past two decades, a plethora of photo-crosslinkable gelatins suitable for tissue engineering purposes have emerged. Although a large number of crosslinkable chemistries, each characterized by their specific benefits and drawbacks, have been reported, the majority of the reported gelatin derivatives apply a chain growth crosslinking system (e.g. gel-MA). However, a second important chemistry which is gaining increasing attention in the field is thiol-ene (photo-)click chemistry,
Acknowledgements
Jasper Van Hoorick and Liesbeth Tytgat were granted an FWO-SB PhD grant provided by the Research Foundation Flanders (FWO, Belgium). The FWO-FWF grant (a bilateral Research foundation Flanders – Austrian Science fund project) is acknowledged for financial support. S. Van Vlierberghe would like to thank the FWO for financial support under the form of research grants (G005616N, G0F0516N, FWOKN273, G044516N) as well as Ghent University for funding a starting grant through the Special Research Fund.
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- 1
Both authors contributed equally.
- 2
Austrian Cluster for Tissue Regeneration, (http://www.tissue-regeneration.at).