Tailoring the Degradation Time of Polycationic PEG-Based Hydrogels toward Dynamic Cell Culture Matrices

Abstract

Poly(ethylene glycol)-based (PEG) hydrogels provide an ideal platform to obtain well-defined and tailor-made cell culture matrices to enhance in vitro cell culture conditions, although cell adhesion is often challenging when the cells are cultivated on the substrate surface. We herein demonstrate two approaches for the synthesis of polycationic PEG-based hydrogels which were modified to enhance cell-matrix interactions, to improve two-dimensional (2D) cell culture, and catalyze hydrolytic degradation. While the utilization of N,N-(bisacryloxyethyl) amine (BAA) as cross-linker for in situ gelation provides degradable scaffolds for dynamic cell culture, the incorporation of short segments of poly(N-(3-(dimethylamino)propyl)acrylamide) (PDMAPAam) provides high local cationic charge density leading to PEG-based hydrogels with high selectivity for fibroblastic cell lines. The adsorption of transforming growth factor (TGF-β) into the hydrogels induced stimulation of fibrosis and thus the formation of collagen as a natural ECM compound. With this, these dynamic hydrogels enhance in vitro cell culture by providing a well-defined, artificial, and degradable matrix that stimulates cells to produce their own natural scaffold within a defined time frame.

Keywords: 2D cell culture; degradable hydrogels; poly(ethylene glycol); polycationic hydrogel; transforming growth factor β.

Figure 1

Synthetic route toward the degradable cross-linker BAA.

Figure 2

Synthetic route toward the asymmetric star-shaped block copolymer [PEG₂₆-SH]₃[PEG₂₆-b-PDMAPAam₆₂].

Figure 3

Normalized SEC traces (in DMAc + 0.21 wt % LiCl) of the starting material [PEG₂₆-SH]₄, the asymmetric block copolymer [PEG₂₆-SH]₃[PEG-b-PDMAPAam₆₂], and the respective intermediates.

Figure 4

Synthetic route toward polycationic PDMAPAam-hydrogels (left) and degradable and cationic BAA-hydrogels (right).

Figure 5

(A) Swelling degree of PDMAPAam-containing hydrogels in DI-water and PBS. (B) Time sweep showing storage modulus (G′) and loss modulus (G″) of pristine PEG hydrogels and hydrogels with a PDMAPAam content of 10 wt %.

Figure 6

(A) Normalized swelling of degradable BAA-hydrogels and nondegradable hydrogels cross-linked with PEG-DA in PBS. (B) Storage modulus (G′) of degradable BAA-hydrogels and stable hydrogels cross-linked with PEG-DA in PBS.

Figure 7

(A) Normalized swelling of degradable hydrogels with different BAA/TEG ratios in DMEM. (B) Storage modulus (G′) of degradable hydrogels with different BAA/TEG ratios in DMEM.

Figure 8

Fluorescence images of hydrogel-specific cell growth: (A) 3T3-J2 cell growth on PDMAPAam hydrogels and (B) LX-2 cell growth on BAA hydrogels; after 4 days of cultivation, the cells were stained with the viability dye Calcein AM and Hoechst to stain the nuclei.

Figure 9

Results of EIA of pro-collagen measured in the cell culture supernatant of 3T3-J2 cell cultivated on PDMAPAam hydrogels (A) and LX-2 cells cultivated on BAA hydrogels (B) after 4 days with and without TGF-β stimulation (each measured value represents cell culture supernatant from a separate hydrogel; significance was determined via t-test).

Figure 10

Fluorescence images of produced collagen I (red) of 3T3-J2 cell cultivated on PDMAPAam hydrogels with (A) and without (B) TGF-β and of LX-2 cells cultivated on BAA hydrogels with (D) and without (E) TGF-β after 4 days; (C) and (F) mean fluorescence intensity of the fluorescence images (for each condition, n = 3 images were analyzed; significance was determined via t-test).