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Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

ABSTRACT: Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way. STATEMENT OF SIGNIFICANCE: Fibrin hydrogels are used as a naturally derived model to study interactions between cells and their surrounding extracellular matrix (ECM). ECM stiffness influences cell state. Cells in 3D culture considerably modify the stiffness of their pericellular space, which can be quite heterogeneous at the micron-scale. Here we present and evaluate a method to pattern stiffness within fibrin hydrogels using a laser scanning confocal microscope and selective photo crosslinking. We believe that this technique can aid future studies of cell-ECM interactions by enabling researchers to modify the pericellular distribution of stiffness.

SUBMITTER: Keating M

PROVIDER: S-EPMC6684034 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

Keating Mark M Lim Micah M Hu Qingda Q Botvinick Elliot E

Acta biomaterialia 20190117

Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a ...[more]

PMID: 30660778

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Project description:Hydrogels with dynamically tunable crosslinking are invaluable for directing stem cell fate and mimicking a stiffening matrix during fibrosis or tumor development. The increases in matrix stiffness during tissue development are often accompanied by the accumulation of extracellular matrices (e.g., collagen, hyaluronic acid (HA)), a phenomenon that has received little attention in the development of dynamic hydrogels. In this contribution, we present a gelatin-based cell-laden hydrogel system capable of being dynamically stiffened while accumulating HA, a key glycosaminoglycans (GAG) increasingly deposited by stromal cells during tumor progression. Central to this strategy is the synthesis of a dually-modified gelatin macromer - gelatin-norbornene-carbohydrazide (GelNB-CH), which is susceptible to both thiol-norbornene photopolymerization and hydrazone click chemistry. We demonstrate that the crosslinking density of cell-laden thiol-norbornene hydrogels can be dynamically tuned via simple incubation with aldehyde-bearing macromers (e.g., oxidized dextran (oDex) or oHA). The GelNB-CH hydrogel system is highly cytocompatible, as demonstrated by in situ encapsulation of pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF). This unique dynamic stiffening scheme provides a platform to study tandem accumulation of HA and elevation in matrix stiffness in the pancreatic tumor microenvironment. STATEMENT OF SIGNIFICANCE: Hydrogels permitting on-demand and secondary crosslinking are ideal for mimicking a stiffening tumor microenvironment (TME). However, none of the current dynamic hydrogels account for both stiffening and accumulation of hyaluronic acid (HA), a major extracellular matrix component increasingly deposited in tumor stromal tissues, including pancreatic ductal adenocarcinoma (PDAC). The current work addresses this gap by developing a dynamic hydrogel system capable of simultaneously increasing stiffness and HA accumulation. This is achieved by a new gelatin macromer permitting sequential thiol-norbornene (for primary network crosslinking) and hydrazone click chemistry (for bioinert or biomimetic stiffening with oxidized dextran (oDex) or oHA, respectively). The results of this study provide new insights into how dynamically changing physicochemical matrix properties guide cancer cell fate processes.

Project description:The complex network of biochemical and biophysical cues in the pancreatic desmoplasia not only presents challenges to the fundamental understanding of tumor progression, but also hinders the development of therapeutic strategies against pancreatic cancer. Residing in the desmoplasia, pancreatic stellate cells (PSCs) are the major stromal cells affecting the growth and metastasis of pancreatic cancer cells by means of paracrine effects and extracellular matrix protein deposition. PSCs remain in a quiescent/dormant state until they are 'activated' by various environmental cues. While the mechanisms of PSC activation are increasingly being described in literature, the influence of matrix stiffness on PSC activation is largely unexplored. To test the hypothesis that matrix stiffness affects myofibroblastic activation of PSCs, we have prepared cell-laden hydrogels capable of being dynamically stiffened through an enzymatic reaction. The stiffening of the microenvironment was created by using a peptide linker with additional tyrosine residues, which were susceptible to tyrosinase-mediated crosslinking. Tyrosinase catalyzes the oxidation of tyrosine into dihydroxyphenylalanine (DOPA), DOPA quinone, and finally into DOPA dimer. The formation of DOPA dimer led to additional crosslinks and thus stiffening the cell-laden hydrogel. In addition to systematically studying the various parameters relevant to the enzymatic reaction and hydrogel stiffening, we also designed experiments to probe the influence of dynamic matrix stiffening on cell fate. Protease-sensitive peptides were used to crosslink hydrogels, whereas integrin-binding ligands (e.g., RGD motif) were immobilized in the network to afford cell-matrix interaction. PSC-laden hydrogels were placed in media containing tyrosinase for 6h to achieve in situ gel stiffening. We found that PSCs encapsulated and cultured in a stiffened matrix expressed higher levels of αSMA and hypoxia-inducible factor 1α (HIF-1α), suggestive of a myofibroblastic phenotype. This hydrogel platform offers a facile means of in situ stiffening of cell-laden matrices and should be valuable for probing cell fate process dictated by dynamic matrix stiffness.Statement of significanceHydrogels with spatial-temporal controls over crosslinking kinetics (i.e., dynamic hydrogel) are increasingly being developed for studying mechanobiology in 3D. The general principle of designing dynamic hydrogel is to perform cell encapsulation within a hydrogel network that allows for postgelation modification in gel crosslinking density. The enzyme-mediated in situ gel stiffening is innovative because of the specificity and efficiency of enzymatic reaction. Although tyrosinase has been used for hydrogel crosslinking and in situ cell encapsulation, to the best of our knowledge tyrosinase-mediated DOPA formation has not been explored for in situ stiffening of cell-laden hydrogels. Furthermore, the current work provides a gradual matrix stiffening strategy that may more closely mimic the process of tumor development.

Dataset Information

Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

Publications

Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

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