Elsevier

Acta Biomaterialia

Volume 115, 1 October 2020, Pages 185-196
Acta Biomaterialia

Full length article
Dual alginate crosslinking for local patterning of biophysical and biochemical properties

https://doi.org/10.1016/j.actbio.2020.07.047Get rights and content

Abstract

Hydrogels with patterned biophysical and biochemical properties have found increasing attention in the biomaterials community. In this work, we explore alginate-based materials with two orthogonal crosslinking mechanisms: the spontaneous Diels-Alder reaction and the ultraviolet light-initiated thiol-ene reaction. Combining these mechanisms in one material and spatially restricting the location of the latter using photomasks, enables the formation of dual-crosslinked hydrogels with patterns in stiffness, biomolecule presentation and degradation, granting local control over cell behavior. Patterns in stiffness are characterized morphologically by confocal microscopy and mechanically by uniaxial compression and microindentation measurement. Mouse embryonic fibroblasts seeded on stiffness-patterned substrates attach preferably and attain a spread morphology on stiff compared to soft regions. Human mesenchymal stem cells demonstrate preferential adipogenic differentiation on soft surfaces and osteogenic differentiation on stiff surfaces. Patterns in biomolecule presentation reveal favored attachment of mouse pre-osteoblasts on stripe regions, where thiolated cell-adhesive biomolecules have been coupled. Patterns in degradation are visualized by microindentation measurement following collagenase exposure. Patterned tissue infiltration into degradable regions on the surface is discernible in n=5/12 samples, when these materials are implanted subcutaneously into the backs of mice. Taken together, these results demonstrate that our hydrogel system with patterns in biophysical and biochemical properties enables the study of how environmental cues affect multiple cell behaviors in vitro and could be applied to guide endogenous tissue growth in diverse healing scenarios in vivo.

Statement of Significance

Hydrogels with patterns in biophysical and biochemical properties have been explored in the biomaterials community in order to spatially control or guide cell behavior. In our alginate-based system, we demonstrate the effect of local substrate stiffness and biomolecule presentation on the in vitro cell attachment, morphology, migration and differentiation behavior of two different mouse cell lines and human primary cells. Additionally, the effect of degradation patterns on the in vivo tissue infiltration is analyzed following subcutaneous implantation into a mouse model. The achievement of patterned tissue infiltration following the hydrogel template represents an important step towards guiding endogenous healing responses, thus inviting application in various tissue engineering contexts.

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.

References (70)

  • S.H. Lee et al.

    Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration

    Biomaterials

    (2008)
  • S. Khetan et al.

    Patterning network structure to spatially control cellular remodeling and stem cell fate within 3-dimensional hydrogels

    Biomaterials

    (2010)
  • D. Hanjaya-Putra et al.

    Spatial control of cell-mediated degradation to regulate vasculogenesis and angiogenesis in hyaluronan hydrogels

    Biomaterials

    (2012)
  • A. Lueckgen et al.

    Hydrolytically-degradable click-crosslinked alginate hydrogels

    Biomaterials

    (2018)
  • A. Lueckgen et al.

    Enzymatically-degradable alginate hydrogels promote cell spreading and in vivo tissue infiltration

    Biomaterials

    (2019)
  • W.H. Guo et al.

    Substrate rigidity regulates the formation and maintenance of tissues

    Biophys. J.

    (2006)
  • A.J. Engler et al.

    Matrix elasticity directs stem cell lineage specification

    Cell

    (2006)
  • A. Cipitria et al.

    In-situ tissue regeneration through SDF-1alpha driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect

    Acta Biomater.

    (2017)
  • J. Vaithilingam et al.

    Multifunctional Bioinstructive 3D Architectures to Modulate Cellular Behavior

    Adv. Function. Mater.

    (2019)
  • C.E. Hoyle et al.

    Thiol-ene click chemistry

    Angewandte Chemie (International ed in English)

    (2010)
  • M.A. Azagarsamy et al.

    Bioorthogonal Click Chemistry: An Indispensable Tool to Create Multifaceted Cell Culture Scaffolds

    ACS Macro Lett.

    (2013)
  • J. Van Hoorick et al.

    Highly Reactive Thiol-Norbornene Photo-Click Hydrogels: Toward Improved Processability

    Macromol. Rapid Commun.

    (2018)
  • R.A. Marklein et al.

    Spatially controlled hydrogel mechanics to modulate stem cell interactions

    Soft Matter

    (2010)
  • S. Nemir et al.

    PEGDA hydrogels with patterned elasticity: Novel tools for the study of cell response to substrate rigidity

    Biotech. Bioeng.

    (2010)
  • J.R. Tse et al.

    Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate

    PloS One

    (2011)
  • O. Jeon et al.

    Regulation of Stem Cell Fate in a Three-Dimensional Micropatterned Dual-Crosslinked Hydrogel System

    Adv. Function. Mater.

    (2013)
  • K.A. Mosiewicz et al.

    Microscale patterning of hydrogel stiffness through light-triggered uncaging of thiols

    Biomater. Sci.

    (2014)
  • C. Yang et al.

    Spatially patterned matrix elasticity directs stem cell fate

    Proc. Natl. Acad. Sci. U. S. A.

    (2016)
  • Y. Zheng et al.

    Optoregulated Biointerfaces to Trigger Cellular Responses

    Langmuir

    (2018)
  • J. Ballester-Beltran et al.

    Effect of topological cues on material-driven fibronectin fibrillogenesis and cell differentiation

    J. Mater. Sci. Mater. Med.

    (2012)
  • W.S. Dillmore et al.

    A Photochemical Method for Patterning the Immobilization of Ligands and Cells to Self-Assembled Monolayers

    Langmuir

    (2004)
  • Y. Kikuchi et al.

    Arraying heterotypic single cells on photoactivatable cell-culturing substrates

    Langmuir

    (2008)
  • C.A. DeForest et al.

    Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments

    Nat. Mater.

    (2009)
  • M. Wirkner et al.

    Photoactivatable caged cyclic RGD peptide for triggering integrin binding and cell adhesion to surfaces

    Chembiochem Eur. J. Chem. Biol.

    (2011)
  • J. Doh et al.

    Immunological synapse arrays: patterned protein surfaces that modulate immunological synapse structure formation in T cells

    Proc. Natl. Acad. Sci. U. S. A.

    (2006)
  • Cited by (13)

    • Manufacturing and post-engineering strategies of hydrogel actuators and sensors: From materials to interfaces

      2022, Advances in Colloid and Interface Science
      Citation Excerpt :

      In addition, mechanically and electrically anisotropic hydrogels can be obtained by adjusting the orientation of the internal structure of hydrogels [33,34]. Traditional synthesis and processing techniques such as molding [13,35,36] and photomask [37–39] have been widely used in manufacturing various hydrogel materials. However, most of these techniques can neither adjust the internal structure of the hydrogel, nor improve the mechanical or electrical properties of hydrogels directly, herein these traditional manufacturing techniques are not discussed in this review.

    • Composite alginate-gelatin hydrogels incorporating PRGF enhance human dental pulp cell adhesion, chemotaxis and proliferation

      2022, International Journal of Pharmaceutics
      Citation Excerpt :

      Gelatin has the potential to release several growth factors and cytokines. On the other hand, alginate has been used in medical devices for healing of wounds, scars, injuries of bones, and as scaffold for cell growth and in drug delivery (Lueckgen, et al., 2020). Furthermore, to improve the functionality of engineered tissues, scientists are applying 3D bioprinting techniques with the aim of providing higher precision in cell and matrix deposition, and rapidly fabricating complex structures (Ostrovidov, et al., 2019).

    • Restoring Carboxylates on Highly Modified Alginates Improves Gelation, Tissue Retention and Systemic Capture

      2022, Acta Biomaterialia
      Citation Excerpt :

      Chemical functionalization with small molecules regulates immune and foreign body response [39–41], functionalization with peptides mediates cellular and tissue responses [42–44], and modification with reactive chemical groups enables new modes of drug delivery [24,45–49]. Alginate polymers conjugated to bioorthogonal “click” chemical motifs enhance cross-linking [15,50–54] and expedite polymer modification [55–57]. More recently, alginates modified with click motifs have been used as targetable drug depots [18,58–60], capable of repeatedly capturing and releasing drugs.

    View all citing articles on Scopus
    View full text