The extracellular matrix (ECM) is a complex, hierarchical material containing structural and bioactive components. This complexity makes decoupling the effects of biomechanical properties and cell-matrix interactions difficult, especially when studying cellular processes in a 3D environment. Matrix mechanics and cell adhesion are both known regulators of specific cellular processes such as stem cell proliferation and differentiation. However, more information is required about how such variables impact various neural lineages that could, upon transplantation, therapeutically improve neural function after a central nervous system injury or disease. Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are one biomaterial approach to meet these goals, consisting of a family of peptide sequences that assemble into physical hydrogels in physiological media. In this study, we studied our previously reported supramolecularly-assembling RAPID hydrogels functionalized with the ECM-derived cell-adhesive peptide ligands RGD, IKVAV, and YIGSR. Using molecular dynamics simulations and experimental rheology, we demonstrated that these integrin-binding ligands at physiological concentrations (3–12 m
- Award ID(s):
- 1655740
- NSF-PAR ID:
- 10228947
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 6
- Issue:
- 28
- ISSN:
- 2375-2548
- Page Range / eLocation ID:
- eaaz5894
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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m ) did not impact the assembly of the KYFIL peptide system. In simulations, molecular measures of assembly such as hydrogen bonding and pi-pi interactions appeared unaffected by cell-adhesion sequence or concentration. Visualizations of clustering and analysis of solvent-accessible surface area indicated that the integrin-binding domains remained exposed. KYFIL or AYFIL hydrogels containing 3 mm of integrin-binding domains resulted in mechanical properties consistent with their non-functionalized equivalents. This strategy of doping RAPID gels with cell-adhesion sequences allows for the precise tuning of peptide ligand concentration, independent of the rheological properties. The controllability of the RAPID hydrogel system provides an opportunity to investigate the effect of integrin-binding interactions on encapsulated neural cells to discern how hydrogel microenvironment impacts growth, maturation, or differentiation. -
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Abstract Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell‐applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin‐like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine‐modified ELP (ELP‐HYD) and aldehyde/benzaldehyde‐modified polyethylene glycol (PEG‐ALD/PEG‐BZA). The reversible DCC crosslinks in ELP‐PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast‐relaxing or slow‐relaxing hydrogels with a range of stiffness (500–3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two‐dimensional substrates, on which ECs exhibited greater cell spreading on fast‐relaxing hydrogels up through 3 days, compared with slow‐relaxing hydrogels at the same stiffness. In three‐dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast‐relaxing, low‐stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast‐relaxing, low‐stiffness hydrogel produced significantly more vascularization compared with the slow‐relaxing, low‐stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast‐relaxing, low‐stiffness hydrogels supported the highest capillary density in vivo.
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Synthetic matrices that are cytocompatible, cell adhesive and cell responsive are needed for the engineering of implantable, secretory salivary gland constructs to treat radiation induced xerostomia or dry mouth. Here, taking advantage of the bioorthogonality of the Michael-type addition reaction, hydrogels with comparable stiffness but varying degrees of degradability (100% degradable: 100DEG; 50% degradable: 50DEG; and non-degradable: 0DEG) by cell-secreted matrix metalloproteases (MMPs) were synthesized using thiolated HA (HA-SH), maleimide (MI)-conjugated integrin-binding peptide (RGD-MI) and MI-functionalized peptide crosslinkers that are protease degradable (GIW-bisMI) or non-degradable (GIQ-bisMI). Organized multicellular structures developed readily in all hydrogels from dispersed primary human salivary gland stem/progenitor cells (hS/PCs). As the matrix became progressively degradable, cells proliferated more readily and the multicellular structures became larger, less spherical, and more lobular. Immunocytochemical analysis showed positive staining for stem/progenitor cell markers CD44 and keratin 5 (K5) in all three types of cultures, and positive staining for the acinar marker α-amylase under 50DEG and 100DEG conditions. Quantitatively at the mRNA level, the expression levels of key stem/progenitor markers KIT, KRT5, and ETV4/5 were significantly increased in the degradable gels as compared to the non-degradable counterparts. Western blot analyses revealed that imparting matrix degradation led to >3.8-fold increase in KIT expression by day 15. The MMP-degradable hydrogels also promoted the development of a secretary phenotype, as evidenced by the upregulation of acinar markers α-amylase (AMY), aquaporin-5 (AQP5), and sodium-potassium-chloride cotransporter 1 (SLC12A2). Collectively, we show that cell-mediated matrix remodeling is necessary for the development of regenerative pro-acinar progenitor cells from hS/PCs.more » « less
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Abstract Vasculogenesis is the
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Abstract Xeno‐free, chemically defined poly(ethylene glycol) (PEG)‐based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene‐click chemistries are integrated to form synthetic, dynamically tunable PEG–peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol–norbornene hydrogels crosslinked by multiarm PEG–norbornene (PEG–NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine–norbornene (Tz–NB) click reaction is then employed to dynamically stiffen the cell‐laden hydrogels. Fast reactive Tz and its stable derivative methyltetrazine (mTz) are tethered to multiarm PEG, yielding mono‐functionalized PEG‐Tz, PEG‐mTz, and dualfunctionalized PEG‐Tz/mTz that react with PEG–NB to form additional crosslinks in the cell‐laden hydrogels. The versatility of Tz‐NB stiffening is demonstrated with different Tz‐modified macromers or by intermittent incubation of PEG‐Tz for temporal stiffening. Finally, the Tz–NB‐mediated dynamic stiffening is explored for 4D culture and definitive endoderm differentiation of hiPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno‐free and dynamic stem cell niche.
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/sciadv.aaz5894
