(10e) Integrating Fibrous Structure within Hydrogel Biomaterials to Support Stem Cell Migration for Collagenous Tissue Regeneration | AIChE

(10e) Integrating Fibrous Structure within Hydrogel Biomaterials to Support Stem Cell Migration for Collagenous Tissue Regeneration


Ford, E. M. - Presenter, University of Delaware
Kloxin, A., University of Delaware
Extracellular matrix (ECM) properties (e.g., mechanics, multiscale structure, and biochemical content including ligand presentation and soluble factors) are essential for proper connective tissue function and repair.1,2 The aforementioned physical and chemical properties instruct cell behavior including migration, proliferation, and cytoskeletal organization, initiating tissue regeneration after injury.3,4,5 Tissue engineering approaches that present extracellular cues in a defined manner offer an opportunity to study and direct connective tissue regeneration both in vitro and in vivo. Methodologies that have been investigated to mimic native tissue structural properties have included wrinkled topologies,6 electrospun fibers,7 and self-assembling collagen mimetic peptides within hydrogels.8 Toward imparting biochemical content into hydrogel systems, controlled release of soluble proteins from hydrogels provides the opportunity to use soluble factors to direct cell behavior.9 Though much progress has been made toward better recapitulating native ECMs, questions still remain regarding the interplay of structure, mechanics, and biochemical content.

To address these questions, we have engineered synthetic hydrogel matrices that mimic aspects of the collagen-rich ECM found in early stages of tissue healing and aim to encourage stem cell migration with the ultimate goal of improved wound healing. These materials promote high human mesenchymal stem cell (hMSC) viability (over 85%) during long-term (10 days) 3D cell culture, and the incorporation of fibrous structure leads to an increase in cell elongation and cell-cell interactions.8 Recently, we collaboratively have demonstrated that these hydrogel matrices can be formed in vivo and ex vivo within tendon tissues and are retained for weeks, indicating that these materials have the potential to act as a stable scaffold and promote tissue regeneration (in preparation for submission). Here, we build on these previous works to investigate the synergistic roles of structure, mechanics, and biochemical content for promoting migration of hMSCs into these scaffolds for both fundamental insights into the cell-matrix interactions associated with migration and identification of materials design for translation that have the potential to enhance connective tissue regeneration. Further, we refine synthetic protocols to better support fabrication of the larger quantities of self-assembling peptides materials needed for these applications.

Poly(ethylene glycol) (PEG) hydrogels containing cell-degradable moieties and integrin-binding peptides were crosslinked via photoinitiated step growth polymerization. To impart fibrillar structure, a multifunctional collagen mimetic peptide (mfCMP) was preassembled and covalently incorporated into the hydrogel network via an allyloxycarbonyl reactive handle. Self-assembly of the mfCMP is driven by hydrogen bonding (via the Proline-Hydroxyproline-Glycine repeat unit) to form triple helices and ionic interactions between charged amino acid residues promote larger fibril formation.8 Using updated synthesis and purification techniques, we have established a protocol to synthesize this same mfCMP sequence in a fraction of the time. Toward characterization of the assembled mfCMP structure, circular dichroism (CD) and transmission electron microscopy (TEM) were implemented. Through CD, the melting temperature (Tm, the point where approximately fifty percent of the triple helices have disassembled into individual peptide strands) of the mfCMP triple helices was determined to be physiologically relevant at 37.2 ± 0.06 °C.

Hydrogels were formed with and without mfCMP. The storage moduli of the resulting hydrogels were characterized using parallel plate rheometry, where no statistical difference in modulus was observed between hydrogels with mfCMP (2.5 mM, 3650 ± 230 Pa) when compared to hydrogels without mfCMP (0 mM, 3480 ± 90.2 Pa). Toward studying cell migration in vitro, a Boyden chamber assay was modified to monitor 3D cell migration into hydrogels in response to an induced protein gradient. hMSCs were seeded on top of hydrogels either with (2.5 mM) or without (0 mM) mfCMP and migration in response to platelet-derived growth factor-BB (PDGF-BB, a chemotactic growth factor important in connective tissue healing) was monitored using confocal microscopy. hMSCs demonstrated affinity for both material compositions even in the absence of the PDGF-BB protein gradient and showed increased directional migration in response to a PDGF-BB gradient within 48 hours. Importantly, with increasing concentrations of mfCMP, enhanced cell motility was observed.

In these studies, we demonstrate that beyond providing a hospitable environment, mfCMP-laden hydrogels support cell-matrix interactions that give rise to heightened cell motility and promote directional cell invasion. This work supports the relevance of this collagen mimetic material for both fundamental studies to examine multidimensional cell response in fibrous wound-healing environments and translational research toward implementing these hydrogels as a biomimetic matrix for enhanced tissue regeneration.


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