Spatial Confinement Modulates Macrophage Response in Microporous Annealed Particle (MAP) Scaffolds

Abstract

Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes such as foreign body response and wound healing. Mounting evidence suggests that physical factors also modulate macrophage activation. 2D in vitro systems demonstrate that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, how dimensionality and pore size affect macrophage phenotype is less explored. In this work, the change in macrophage M1/M2 polarization when confined in microporous annealed particle (MAP) scaffolds is studied. Particles sizes (40, 70, and 130 µm) are selected using outputs from software LOVAMAP that analyzes the characteristics of 3D pores in MAP gels. As the size of building block particle correlates with pore size inside the scaffolds, the three types of scaffold allow us to study how the degree of spatial confinement modulates the behavior of embedded macrophages. Spatially confining macrophages in scaffolds with pore size on the scale of cells leads to a reduced level of the inflammatory response, which is correlated with a change in cell morphology and motility.

Exploring the Role of Spatial Confinement in Immune Cell Recruitment and Regeneration of Skin Wounds

“…Microfluidic devices and microgels were made as previously described. [ 34 ] Briefly, 8 arm PEG‐VS microgels were formulated at a final concentration of 5 wt% w/v PEG‐VS (JenKem technology) in 0.3 m triethanolamine (Sigma) with 500 µ m RGD (Ac‐RGDSPGERCG‐NH2, GenScript), 500 µ m K‐peptide (Ac‐FKGGERCG‐NH2, GenScript), and 500 µ m Q‐peptide (Ac‐NQEQVSPLGGERCG‐NH2, GenScript) and crosslinked at a 0.6 VS to thiol ratio with di‐thiol matrix metalloproteinase sensitive peptide (Ac‐GCRDGPQGIWGQDRCG‐NH2, GenScript) plus 10 µ m AlexaFluor‐647 (ThermoFisher). For 40 and 70 µm microgel generation, a four‐inlet microfluidic device was used.…”

Accelerating Patterned Vascularization Using Granular Hydrogel Scaffolds and Surgical Micropuncture

“…[78,79] Previous research has shown that GHS comprising varying microgel sizes influence cell infiltration and macrophage polarization. [80,81] GHS made up of ≈48 or ≈146 μm microgels resulted in higher M1 polarization or M2 polarization in macrophages, respectively. [79] The inflammatory response of macrophages has also been shown to decrease when the GHS pore size is within the same range of cell size.…”

“…[80] GHS with a similar pore size to our GHS-M (average microgel diameter ≈ 70-80 μm) had a significantly higher infiltrated cell area after 21 days of subcutaneous implantation compared with GHS made up of smaller (≈40 μm) or larger (≈130 μm) microgels. [81] Furthermore, the cytotoxicity of scaffolds was assessed clinically via weight, and there was no associated weight loss in any animal cohort. Histology was used to demonstrate end organ normalcy, as shown in Figure 5C.…”

Engineering the microstructure and spatial bioactivity of MAP scaffolds in vitro instructs neovascularization in vivo

“…The fabrication of the PEG μgels used in this work has been described previously by Liu, et al [32] The precursor solution for these μgels included 8-arm PEG Vinylsulfone (PEG-VS), K-peptide (Ac-FKGGERCG-NH2, GenScript), Q-peptide (Ac-NQEQVSPLGGERCG-NH2, GenScript), and RGD (Ac-RGDSPGERCG-NH2, GenScript) in 0.3 m triethylamine (Sigma) buffer. The crosslinker solution was prepared by dissolving the MMP-cleavable peptide (Ac-GCRDGPQGIWGQDRCG-NH2, GenScript) in distilled water at 12 mm and 10 μm Alexa-Fluor 647-maleimide (Invitrogen).…”