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Probing fibroblast activation in response to extracellular cues with whole protein- or peptide-functionalized step-growth hydrogels.

ABSTRACT: Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating the role of the elasticity or 'stiffness' of the matrix in promoting fibroblast activation in wound healing and fibrosis. However, the two methods of promoting integrin binding are not often directly compared in the same system, partly owing to differences in material designs, despite the potential differences in the way cells interact with whole proteins and protein mimetic peptides. We hypothesized that such a comparison could provide insight into the ways integrin binding affects fibroblast activation within commonly utilized in vitro cell culture models, and more broadly, to inform the design of materials to modulate fibroblast activation in studies of wound healing and disease. To enable this comparison, we developed a method to conjugate whole proteins to step-growth poly(ethylene glycol) (PEG) hydrogels and investigated fibroblast response to protein-peptide pairs: fibronectin and PHSRN(G)10RGDS or collagen I and (POG)3POGFOGER(POG)4, which are important in matrix remodeling and relevant to fibroblast activation. With this approach, we observed that human pulmonary fibroblasts adopted a similar morphology on fibronectin and PHSRN(G)10RGDS, although with a slight increase in the percentage of alpha smooth muscle actin (?SMA) expressing cells on PHSRN(G)10RGDS. Interestingly, we observed that fibroblasts formed activated clusters on the collagen mimic (POG)3POGFOGER(POG)4 while exhibiting less activation on collagen I. This cell activation and clustering is reminiscent of fibroblast foci that are observed in lung fibrosis, suggesting the relevance of these well-defined polymer-peptide hydrogels for investigating fibrosis and decoupling biochemical and biophysical cues.

SUBMITTER: Smithmyer ME

PROVIDER: S-EPMC7269182 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Probing fibroblast activation in response to extracellular cues with whole protein- or peptide-functionalized step-growth hydrogels.

Smithmyer Megan E ME Spohn Joseph B JB Kloxin April M AM

ACS biomaterials science & engineering 20180727 9

Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating ...[more]

PMID: 32494587

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Project description:Purpose: Degeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by reduced cellularity and a shift towards a fibroblast-like state. We have recently demonstrated an ability to manipulate the phenotypic expression of adult degenerative NP cells by culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides. In the present study, we seek to understand the transcriptome changes elicited by NP cell interactions with the presented hydrogel system. Methods: mRNA profiles of NP from 3 human tissue samples were cultured for 4 days on either tissue culture polysyrene (TCPS) or the functionalized gel before RNA was isolated and sequenced, using Illumina NovaSeq. Unaligned reads were trimmed based on quality score (minimum quality level (Phred) = 20, minimum read length = 25) and aligned to the whole human genome (STAR 2.6.1d; hg19) using Partek Flow software. This software suite was also used to calculate the counts/normalized counts of genes and to perform gene specific analysis (GSA), principle component analysis (PCA), hierarchical clustering, and gene set enrichment. Differentially regulated genes were considered to be those with a fold change value (gel/TCPS) that was greater than or equal to 2 or less than or equal to negative 2 and a p-value of less than or equal to 0.05. Results: The data corroborate and expand on the previous findings by demonstrating that degenerative adult human NP cells cultured upon these gels have upregulations in some markers of NP and notochordal cells but also downregulations of some NP-specific markers and several fibroblastic markers. Furthermore, through gene set enrichment analysis we have shown that pathways related to cell differentiation and notochord morphogenesis were upregulated in the gel condition. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as up or downregulated following periods of culture upon the gel condition. Conclusions: The data from this RNA-sequencing demonstrate that culture on laminin-mimetic-peptide presenting gels can modulate cell phenotype and promotes upregulation of NP and notochordal markers.

Project description:Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate-PEG-(peptide-PEG)(m)-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate-PEG-CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.

Dataset Information

Probing fibroblast activation in response to extracellular cues with whole protein- or peptide-functionalized step-growth hydrogels.

Publications

Probing fibroblast activation in response to extracellular cues with whole protein- or peptide-functionalized step-growth hydrogels.

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