Modular multifunctional poly(ethylene glycol) hydrogels for stem cell differentiation

Abstract

Synthetic polymers are employed to create highly defined microenvironments with controlled biochemical and biophysical properties for cell culture and tissue engineering. Chemical modification is required to input biological or chemical ligands, which often changes the fundamental structural properties of the material. Here, we report on a simple modular biomaterial design strategy that employs functional cyclodextrin nanobeads threaded onto poly(ethylene glycol) polymer necklaces to form multifunctional hydrogels. Nanobeads with desired chemical or biological functionalities can be simply threaded onto the PEG chains to form hydrogels, creating an accessible platform for users. We describe the design and synthesis of these multifunctional hydrogels, elucidate structure-property relationships, and demonstrate applications ranging from stem cell culture and differentiation to tissue engineering.

Keywords: Functional biomaterials; Hydrogels; Poly(ethylene glycol); Tissue Engineering; α-cyclodextrin.

Figure 1.. Design of a molecular-necklace system to create tunable, multifunctional hydrogels with independent control of mechanics, cell adhesion properties, and chemical functionality.

a, Alpha-cyclodextrin (α-CD), with its nanobead-like structure, forms an inclusion complex with poly(ethylene glycol)-diacrylate (PEGDA) (R = hydroxyl or other functional groups). After threading α-CD onto polymer chains, PEGDA is crosslinked to form a hydrogel. b, The mechanical properties of α-CD-PEG hydrogel can be varied independent of α-CD by manipulating the cross-linking density of PEG, which is directly related to the stiffness of the hydrogel. c, The α-CD can be substituted with cell adhesion peptides before threading and hydrogel formation. The concentration of cell integrin-binding peptide conjugated to α-CD can be varied independent of the cross-linking density. d, The chemical functionality of α-CD can be varied (i.e., hydrophobic, hydrophilic or charged groups) to create specific microenvironments.

Figure 2.. Synthesis and characterization of cell-interactive molecular necklace, α-CD-YRGDS-PEG hydrogels.

a, MALDI-TOF spectrum to confirm peptide conjugation, and synthesis of α-CD-YRGDS (mol wt ~1595 Da) (see in Supplementary Fig. S1a). b, XPS spectrum for α-CD-YRGDS to determine the presence of nitrogen with peptide conjugation to α-CD. c, FTIR spectra for α-CD-YRGDS threading onto PEGDA chains. The spectrum for PEGDA provides a baseline before threading, followed by the α-CD-YRGDS alone, mixed with PEGDA, and α-CD-YRGDS-PEGDA after threading. A peak at 1750 cm⁻¹ arises for the amide stretching of the peptide conjugated to α-CD, and a broader peak at ~3550 cm⁻¹ is for the hydroxyl groups of α-CD and YRGDS. Threading efficiency was ~20% as determined by the ninhydrin assay on rigorously washed and dried hydrogels (see in Supplementary Fig. S1b-d). d, PEGDA (10% w/v) was threaded with α-CD-YRGDS nanobeads and cross-linked to form hydrogels, which were then seeded with MSCs. e, MSCs cultured on the surface of the α-CD-YRGDS-PEGDA hydrogels decorated with varying numbers of α-CD-YRGDS nanobeads (0.1%, 0.5%, and 2.0%, w/v) had significantly greater cell areas and complex actin structures compared to the respective α-CD and PEGDA controls after 4 days of culture (TCP = tissue culture plate; F-actin staining with Texas Red^®-X phalloidin).

Figure 3.. α-CDYRGDS-PEG hydrogels with independently tunable mechanical and cell adhesion properties.

An array of hydrogels was synthesized with independently varied cross-linking densities (% of PEGDA, necklace) and cell adhesions (α-CD-YRGDS, nanobeads). a, The shear storage moduli of the hydrogels varied only with the crosslinking density and did not change with the incorporation of α-CD nanobeads. The PEGDA 15% (w/v) hydrogels with shear storage modulus ~30 kPa are defined as stiff substrates, while PEGDA 10% (w/v) with shear storage modulus ~7–10 kPa as moderately stiff, and PEGDA 5% (w/v) with shear storage modulus ~0.5 kPa as soft substrates. b, The amount of water absorbed into the hydrogels (swelling ratio) also varied with respect to the crosslinking density, and it did not change with α-CD nanobeads. c, MSCs cultured on the surfaces of stiff and soft hydrogels with varying concentrations of cell adhesive nanobeads have different morphological and organization characteristics (F-actin staining with Texas Red^®-X phalloidin at 16 h). Cells were able to spread on the soft surfaces and aggregated with increasing adhesion compared to cells seeded on the stiffer substrates that had longer cell extensions in isolation (little aggregation). Data collected throughout the study of compression modulus and swelling ratio are presented as a mean ± standard deviation of three or more data samples (*p ≤ 0.05).

Figure 4.. Stem cell response to materials with independently controlled stiffness and adhesion.

The morphological changes of cells cultured on surfaces with independently controlled stiffness and adhesion were quantified over time in terms of inverse shape ratio (perimeter²/4π*Area) and projected perimeter. Inverse shape ratio characterizes cell spreading and deviation from non-adherent circular shape. Cells were characterized on: a, low adhesion (α-CD-YRGDS) and b, high adhesion (α-CD-YRGDS) content surfaces with variable cross-linking density or mechanical properties. Larger differences in both cell shape parameters developed when the hydrogels were decorated with higher concentrations of α-CD-YRGDS nanobeads. Cell differentiation was related to cell shape, and gene expression was evaluated after 21 days of culture for markers related to c, adipogenesis, d, myogenesis, e, chondrogenesis, and f, osteogenesis. Data collected throughout the study of cell morphology is presented as a mean ± standard deviation of multiple data samples (n = 15–20), except for PEGDA samples.

Figure 5.

Chemical functionality of molecular necklace directs differentiation of stem cells in 3D hydrogels. Stem cells encapsulated in hydrogels with α-CD nanobeads functionalized with polar, hydrophobic, or charged chemical groups were cultured for 3 weeks to evaluate differentiation. a, α-CD (1%, w/v) with hydroxyl groups stimulated chondrogenesis of MSCs compared to control PEG hydrogels with increased early expression of Sox9 transcription factor and (matrix molecules or ECM proteins) aggrecan and type II collagen. Increased cartilage matrix production is visible by safranin-O histological staining. b, The chemical structure of hydrophobic α-CD-CH₃ was confirmed by MALDI-TOF spectrometry. Human adipose-derived stem cells (hADSCs) encapsulated in the α-CD-CH₃/PEG hydrogels increased expression of adipose-related genes several-fold. Genes include fatty acid-binding protein (FABP), lipoprotein lipase (LPL) and CEBPA (also see Supplementary Figure S2b) and produced more lipid droplets visualized by Oil red O staining, compared to control α-CD hydrogels. c, Cells cultured in hydrogels with α-CD-PO₄⁻ functionalized nanobeads (the chemical structure confirmed by MALDI-TOF) produced more mRNA for osteogenic genes COL X, OCN, and OPN compared to control hydrogels (also see Supplementary Figure S2e). Morphologically, mineralization characterized by alizarin red staining also increased in α-CD-PO₄⁻/PEG hydrogels compared to controls.