Charge-Controlled Synthetic Hyaluronan-Based Cell Matrices

Abstract

The extracellular matrix (ECM) represents a highly charged and hydrated network in which different cells in vertebrate tissues are embedded. Hydrogels as minimal ECM mimetics with a controlled chemistry offer the opportunity to vary material properties by varying the negative network charge. In this paper, a synthetic biology model of the ECM based on natural and highly negatively charged polyelectrolyte hyaluronic acid (HA) is characterized with specific emphasis on its charge-related bioactivity. Therefore, the thiol-Michael addition click reaction is used to produce HA hydrogels with defined network structure and charge density. The presented hydrogels show enzymatic degradability and cell attachment. These properties depend on both covalent and electrostatic interactions within the hydrogel network. Furthermore, no unspecific or specific attachment of proteins to the presented hydrogels is observed. In addition, these fundamental insights into charge-related ECM behavior and the influence of electrostatic properties could also lead to innovations in existing biomedical products.

Keywords: cell attachment; enzymatic degradation; glycosaminoglycans; hyaluronan; polyelectrolyte hydrogel; synthetic ECM; tissue engineering.

Figure 1

Schematic representation of the progression of extracellular matrix (ECM) mimetics. (A) First approaches of coating cell culture plastic with ECM proteins; (B) three-dimensional (3D) hydrogels based on synthetic polymers, (C) 3D hydrogels made of natural ECM polymers with the possibility of remodeling by cells, (D) hydrogels from (C) with incorporated stem cells in order to release their proteins, growth factors, etc., and (E) ECM extracted from living donors (Dissertation Patricia Hegger, 2017).

Figure 2

Charge-dependent physicochemical properties of hyaluronan (HA) hydrogels (with degrees of thiolation in % = 18, 25, and 33 and uncharged and charged crosslinker). (a) Formed stable HA hydrogels with pyridin-bisacrylamide crosslinker (neutral; top) and pyridinium-bisacrylamide crosslinker (positively charged; bottom) at increasing degrees of thiolation from left to right. Swelling differences are clearly visible in the size of the equilibrium-swollen hydrogels (PBS). (b) Linear correlation of stiffness and negative network charge of the presented hydrogel system [17].

Figure 3

Charge-dependent degradation of HA hydrogels (with degrees of thiolation in % = 18, 25, and 33 and uncharged and charged crosslinkers) with hyaluronidase IV (a) follows the general trends of increasing half-life with increasing degree of thiolation and longer half-life with the charged crosslinker compared to the uncharged crosslinker at one degree of thiolation. (b) In relation to the negative network charge, the half-lives adopt a first-order exponential decay. Charge dependence can additionally be underlined by the fact that hydrogels with a similar negative network charge show similar half-lives for degradation.

Figure 4

Charge-dependent degradation of HA hydrogels (with degrees of thiolation in % = 18, 25, and 33 and uncharged and charged crosslinkers) with hyaluronate lyase (a) follows the general trends of increasing half-life with increasing degree of thiolation and longer half-life with the charged crosslinker compared to the uncharged crosslinker at one degree of thiolation. (b) In relation to the negative network charge, the half-lives adopt a first-order exponential decay. Charge dependence can additionally be underlined by the fact that hydrogels with a similar negative network charge show similar half-lives for degradation.

Figure 5

The number of cells adhering to different HA hydrogels with degrees of thiolation in % = 18, 25, and 33 and an uncharged crosslinker (orange) or a charged crosslinker (turquoise) increases with the increasing degree of thiolation and is higher on charged compared to uncharged crosslinker hydrogels. These two general trends are analogous to all other evaluated hydrogel properties and reproducible in all used cell types. (a) Human dermal lymphatic endothelial cells (HDLEC), (b) the breast cancer cell line MCF7, and (c) normal human dermal fibroblasts (NHDF), (d) Additionally, blocking CD44 on NHDF with antibody or sHA does not significantly influence the number of attached cells. Statistical analysis is carried out with a Kruskal-Wallis test in combination with Dunn’s multiple comparison for all cell types (p-values: **** < 0.0001; ** = 0.001–0.01; * = 0.01–0.1).

Figure 5

The number of cells adhering to different HA hydrogels with degrees of thiolation in % = 18, 25, and 33 and an uncharged crosslinker (orange) or a charged crosslinker (turquoise) increases with the increasing degree of thiolation and is higher on charged compared to uncharged crosslinker hydrogels. These two general trends are analogous to all other evaluated hydrogel properties and reproducible in all used cell types. (a) Human dermal lymphatic endothelial cells (HDLEC), (b) the breast cancer cell line MCF7, and (c) normal human dermal fibroblasts (NHDF), (d) Additionally, blocking CD44 on NHDF with antibody or sHA does not significantly influence the number of attached cells. Statistical analysis is carried out with a Kruskal-Wallis test in combination with Dunn’s multiple comparison for all cell types (p-values: **** < 0.0001; ** = 0.001–0.01; * = 0.01–0.1).