Elsevier

Acta Biomaterialia

Volume 97, 1 October 2019, Pages 46-73
Acta Biomaterialia

Review article
(Photo-)crosslinkable gelatin derivatives for biofabrication applications

https://doi.org/10.1016/j.actbio.2019.07.035Get rights and content

Abstract

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for biofabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs.

Statement of Significance

Gelatin and more specifically gelatin-methacryloyl has emerged to become one of the gold standard materials as an extracellular matrix mimic in the field of biofabrication. However, also other modification strategies have been elaborated to take advantage of a plethora of crosslinking chemistries. Therefore, a review paper focusing on the different modification strategies and processing of gelatin is presented. Particular attention is paid to the underlying chemistry along with the benefits and drawbacks of each type of crosslinking chemistry. The different strategies were classified based on their basic crosslinking mechanism including chain- or step-growth polymerization. Within the step-growth classification, a further distinction is made between click chemistries as well as other strategies. The influence of these modifications on the physical gelation and processing conditions including mechanical properties is presented. Additionally, substantial attention is put to the applied photoinitiators and the different biofabrication technologies including inkjet, deposition or light-based technologies.

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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