How Cross-Linking Mechanisms of Methacrylated Gellan Gum Hydrogels Alter Macrophage Phenotype

Abstract

In tissue engineering scaffolds, macrophages play a critical role in determining the host response to implanted biomaterials. Macrophage phenotype is dynamic throughout the host response, and a balance of phenotypes is essential for timely progression from injury to proper wound healing. Therefore, it is important to predict how materials will modulate the response of macrophages. In this study, we investigated the effect of methacrylated gellan gum hydrogels on macrophage phenotype and proliferation with the ultimate goal of improving rational design of biomedical implants. Naïve, along with classically and alternatively activated RAW 264.7 macrophages were seeded on methacrylated gellan gum hydrogels that were fabricated with different thiol-ene ratios and crosslinking mechanisms. Live/dead assays showed that all hydrogels supported cell attachment and proliferation. Stiffer substrates enhanced anti-inflammatory production of nitrites from both naïve and classically activated macrophages compared to the softer substrates. Moreover, arginine and CD206 expression-markers for alternatively activated macrophages-were inhibited by higher thiol content. Introducing ionic crosslinks using calcium did not influence the proliferation or polarization for any of the three macrophage phenotypes. Our results suggest that the macrophage phenotype shift from M1 to M2 is controlled by the different crosslinking mechanisms, physical properties, and the chemistry of methacrylated gellan gum hydrogels. Disciplines Biochemical and Biomolecular Engineering | Biology and Biomimetic Materials | Biomaterials Comments

Hydrolytically Degradable Microgels with Tunable Mechanical Properties Modulate the Host Immune Response

“…Although changes in mechanical cues have demonstrated an effect on M1‐like macrophage activation, [ 32,33 ] changes in surface chemistry have been shown to have a higher influence on M2‐like polarization. [ 34,35 ] In this study, neither the presence of EGBMA crosslinking units nor changes in mechanical properties had an impact on M2‐associated CD206 marker expression in vitro. We postulate that most of the unreacted crosslinking molecules were removed from the microgel suspension through the centrifugation/washing steps.…”

Macrophage Phenotypic Changes on FN-Coated Physical Gradient Hydrogels

“…Previously, we studied how macrophage phenotype was altered by methacrylated gellan gum crosslinking mechanisms. The stiffer substrates modulated classically activated macrophages toward an M2-like phenotype . Classically activated primary human macrophages were polarized toward an anti-inflammatory phenotype on the more rigid hyaluronic acid hydrogel .…”

“…We use 25 ng/mL IL-4 (denoted M2 cells, eBioscience Inc., San Diego, CA) or 500 ng/mL LPS (M1, Sigma-Aldrich) to stimulate macrophages to desired phenotypes. Then, the plate was incubated for 48 h. Live and dead assay result was obtained using 7.5 μM 7-aminoactinomycin d (Tonbo Biosciences, San Diego, CA) and 2 μM calcein AM (AnaSpec, Fremont, CA) based on previously published study …”

“…RAW 264.7 cells were fluorescently labeled using a previously described protocol . Briefly, activated macrophages were seeded at 1 × 10 6 cells/cm 2 on different gel samples, which adhesive to poly- l -lysine-coated glass coverslips.…”

“…Previously, we tuned the mechanical properties, including compressive modulus, swelling rate, and degradation rate, of gellan gum hydrogels by adjusting the crosslinking conditions using chain, step, and mixed-mode crosslinking mechanisms . We have shown that macrophage phenotype changes on gels fabricated using different crosslinking mechanisms and were directly altered by both substrate stiffness and surface chemistry . To better elucidate the influence of gellan gum substrate stiffness on macrophage response, the step-growth mechanism was used to crosslink the hydrogel with a stiffness gradient.…”