Selective and Improved Photoannealing of Microporous Annealed Particle (MAP) Scaffolds

Abstract

Microporous annealed particle (MAP) scaffolds consist of a slurry of hydrogel microspheres that undergo annealing to form a solid scaffold. MAP scaffolds have contained functional groups with dual abilities to participate in Michael-type addition (gelation) and radical polymerization (photoannealing). Functional groups with efficient Michael-type additions react with thiols and amines under physiological conditions, limiting usage for therapeutic delivery. We present a heterofunctional maleimide/methacrylamide 4-arm PEG macromer (MethMal) engineered for selective photopolymerization compatible with multiple polymer backbones. Rheology using two classes of photoinitiators demonstrates advantageous photopolymerization capabilities. Functional assays show benefits for therapeutic delivery and 3D printing without impacting cell viability.

Keywords: 3D printing; MAP; photopolymerization; porous hydrogel.

Figure 1.

Synthesis and characterization of three microgel types. A) The three gel formulations were composed of a PEG-Vinyl Sulfone backbone (VS), a PEG-Maleimide backbone (Mal), and a PEG-Maleimide with 1mM MethMal (MethMal). All gel formulations were crosslinked with a 4-arm PEG Thiol and had RGD cell adhesive peptide. B) The three gel formulations were mechanically matched to have a Young’s modulus of ~46kPa determined by Instron testing of macroscale gels. Note: All microgel compositions were formulated to stoichiometrically provide a theoretical 1mM excess photoannealing functional group for all conditions. C) Microgels were produced using a high throughput microfluidics device. D) Biotinylated microgels were fluorescently visualized with streptavidin-488. Scale bar represents 100μm. E) Microgels were size matched to approximately ~75μm in diameter. All graphs show mean +/− standard deviation. One-way ANOVA was used to determine significance in mechanical moduli.

Figure 2.

Quantification of annealing across photoinitiators via rheological analysis. A) Change in storage moduli compared to light energy introduced to the system. Horizontal lines indicate maximum ΔG’. B) Early annealing kinetics determined by maximum rates of change following toe regions of curve. C) Light energy required to reach one-half of the maximum increases in storage moduli. All graphs show mean +/− standard deviation. One-way ANOVAs followed by post-hoc multiple comparisons tests (Tukey HSD) were used to determine significance. **** p-value < 0.0001, *** p < 0.001, ** p < 0.01.

Figure 3.

Primary cell viability in response to annealing chemistries and photoinitiators. A) Representative maximum intensity projection (MIP) images from each condition showing both live (green) and dead (red) HDFs. Scale bar represents 300μm. B) HDF viability at 24 hours shown as a fold-change from 2D tissue culture plastic controls. C) HDF viability at 24 hours following culture in MethMal gel and annealed with 20μM Eosin-Y using a range of exposure times. All graphs show mean +/− standard deviation. One-way ANOVAs followed by post-hoc multiple comparison tests (Tukey HSD) were used to determine significance. *** p-value < 0.001, * p < 0.05.

Figure 4.

Functional assays to compare annealing chemistries. A) BSA release was characterized by loading MAP particles with fluorescently tagged BSA and monitoring release in infinite sink conditions for 72 hours. B) Release profiles over 72 hours. C) 3D printing experiments consisted of printing single-layer lines and 5-layer squares and submerging them in PBS to test the quality of crosslinking. D) Line thickness was compared between groups to evaluate the kinetics of annealing. Scale bars represent 500μm. All graphs show mean +/− standard deviation. One-way ANOVAs followed by post-hoc multiple comparisons tests (Tukey HSD) were used to determine significance. *** p < 0.001, ** p < 0.01, * p < 0.05.