Full length articleDual alginate crosslinking for local patterning of biophysical and biochemical properties
Graphical abstract
Introduction
Hydrogels have found widespread application in the field of tissue engineering due to their similarity to the native extracellular matrix (ECM) and their suitability for physical and chemical modifications. One main trend in the discipline has been to transition away from passive to bioinstructive matrices with the motivation of stimulating desired cell responses in a spatially-controlled manner [1,2]. One way of achieving this goal is through the use of photopatterning by controlling the site of light-induced reactions using photomasks, thereby changing the biophysical or biochemical characteristics locally.
Research in this field has involved different light-based chemistries, including azide-alkyne cycloadditions and Michael additions, but thiol-ene (TE) coupling has proven especially suitable due to its fast, specific and cytocompatible reaction scheme [3], [4], [5]. Photopatterning permits the fabrication of cell-instructive matrices presenting mechanical and chemical cues in select, well-defined microenvironments. In the context of stiffness modulation, light exposure can be used to form stiffness patterns of various geometries or gradients, and this has been accomplished in different material systems, affecting such diverse cell behaviors as differentiation potential, gene expression, migration, morphology and proliferation [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Similarly, topographical cues have been exploited to influence differentiation, fibronectin fibrillogenesis and migration, among other cell behaviors [16], [17], [18], [19], [20].
In the realm of biochemical modification, reactions can broadly be divided into those that rely on photochemistry to attach a molecule of interest, and those that take advantage of light exposure to de-protect a caged moiety [21], [22], [23], [24], [25]. Though covalent immobilization of the cell-adhesive molecule Arginine-Glycine-Aspartate (RGD) to control cell attachment is most common, T-cell receptor ligands, antibodies, and different growth factors such as sonic hedgehog, bone morphogenetic protein-2 and vascular endothelial growth factor, have also been coupled to different types of surfaces [26], [27], [28], [29], [30]. In addition to affecting the initial adhesion of cells, subsequent migration in 2D and 3D, chondrogenic differentiation potential and neuronal growth have been controlled on a local level using different spatial patterns of biomolecules [31], [32], [33], [34].
Patterns in hydrogel degradation have mostly been achieved by spatially restricting laser light exposure of photo-degradable materials to locally trigger this process. The locally-induced material changes have been shown to control cell attachment, induce cell spreading, guide neuronal outgrowth and network formation, steer vasculogenesis and stimulate directed migration [35], [36], [37], [38], [39], [40]. Another approach applies two orthogonal crosslinking chemistries combined in a hyaluronic acid-based network, in which regions shielded from ultraviolet (UV) light feature primary addition crosslinks remaining “permissive”, and those regions subsequently exposed to UV obtain secondary radical crosslinks becoming “inhibitory”, thereby allowing control over cell spreading and differentiation, as well as in vitro blood vessel formation [41,42].
Recent efforts have focused on merging biophysical and biochemical cues or applying reversible chemistries to create sophisticated matrices with spatiotemporal control over these properties that more accurately mimic the native microenvironment [43], [44], [45]. These presented investigations, though certainly promising, have mostly been limited to in vitro settings, albeit select in vivo proof-of-concept studies have also been performed. For example, cord-like endothelial structures cultured on micropatterned substrates have been shown to provide a template to define neo-vascularization in vivo with cord dimensions dictating vessel density, location and growth dynamics [46,47]. In the context of bone regeneration, aligned microscale mechanical cues on 3D-printed poly(ε-caprolactone) scaffolds have led to coordinated tissue growth, while aligned nanotopographies have enhanced host osteoblast recruitment, migration and differentiation, ultimately accelerating new bone formation in both a mouse calvarial and tibia defect [48,49].
Our biomaterial system features the naturally-occurring polysaccharide alginate that has previously been covalently crosslinked using either Diels-Alder (DA) or TE chemistry. The former relies on modification of the polymer backbone with norbornene or tetrazine functional groups and covalent crosslinking occurs spontaneously when these two constituents are combined [50]. The latter involves the same norbornene-modified alginate combined with a dithiol peptide linker and covalent crosslinking occurs in the presence of photoinitiator upon UV light exposure [51]. As this second reaction is UV-mediated, its precise spatial location can be controlled using photomasks. This same TE reaction can also be exploited for biomolecule conjugation to residual norbornene groups not involved in crosslinking using any monothiol biomolecule of interest. Furthermore, patterns in degradation can be achieved by choosing an enzymatically-degradable peptide sequence as the dithiol linker.
The aim of this investigation is to engineer a hydrogel system that combines DA and TE crosslinking orthogonally, thereby permitting the formation of three different types of patterns: stiffness, biomolecule presentation or degradation. We hypothesized that these patterns in biophysical and biochemical properties in one system could be employed to stimulate different cell behaviors on a local level, laying the groundwork for a material platform offering multiple degrees of freedom for in vitro studies and, more importantly, to guide endogenous regeneration processes in vivo.
Section snippets
Synthesis of 3-(p-benzylamino)-1,2,4,5 tetrazine
3-(p-benzylamino)-1,2,4,5-tetrazine was synthesized, as previously described, by mixing 4-(aminomethyl) benzonitrile hydrochloride and formamidine acetate before adding anhydrous hydrazine [50]. Following gas evolution, the reaction was covered and stirred at 200 rpm for 40 min at 90°C until the color changed to deep red. The mixture was subsequently cooled on ice. Then, sodium nitrite solution was added before 10% hydrochloric acid solution was introduced drop-wise to acidify the reaction.
Chemical reaction scheme of patterned alginate hydrogels
Norbornene- and tetrazine-modified alginates were synthesized via carbodiimide chemistry allowing for spontaneous DA crosslinking when combined. The success of these reactions was determined and the extent of the modification was quantified via NMR measurement (Supplementary Figure S1-4; Supplementary Table S1-2). The same norbornene-modified alginate was simultaneously crosslinked with non-degradable dithiol molecules in the presence of photoinitiator and UV light by TE chemistry. Performing
Discussion
The presented biomaterial system featuring norbornene- and tetrazine-modified alginate, and a dithiol crosslinker or monothiol peptide, invites the fabrication of hydrogels with different kinds of patterns. First, when a dithiol crosslinker is incorporated into the pre-polymer solution and the mixture is immediately exposed to UV light, patterns in stiffness are formed. Second, when, instead of a dithiol crosslinker, a molecule of interest with a pendant thiol is introduced,
Conclusion
We have developed an alginate-based material platform implementing two different complementary crosslinking schemes enabling the fabrication of hydrogels with patterns in biophysical and biochemical cues. These materials or subsets thereof were characterized for their topographical features, their mechanical properties and then subjected to different cellular assays and in vivo testing. Stiffness patterns were confirmed by microindentation and investigation of fibroblast seeding on these
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft (DFG) grant CI 203/1-2 and the Berlin-Brandenburg School for Regenerative Therapies (BSRT) Extension grant. The authors would like to thank Alexander Stafford, Olaf Niemeyer, Hubert Taieb, Dag Wulsten and Mario Thiele for technical assistance with tetrazine synthesis, NMR, Amira software, microindentation and image analysis, respectively. Additionally, the authors would like to acknowledge Claudia Fleck and Peter Fratzl for scientific
Author contribution
A.C., D.J.M. and G.N.D. conceived the idea. A.L. and A.C. wrote the manuscript. A.L. and D.S.G. carried out the experiments and evaluated the data. A.E. performed the surgeries. All authors contributed to the interpretation of the results and commented on the manuscript.
Data availability
The raw/processed data required to reproduce these findings are available from the authors upon request.
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