Injectable, porous, and cell-responsive gelatin cryogels

Abstract

The performance of biomaterials-based therapies can be hindered by complications associated with surgical implant, motivating the development of materials systems that allow minimally invasive introduction into the host. In this study, we created cell-adhesive and degradable gelatin scaffolds that could be injected through a conventional needle while maintaining a predefined geometry and architecture. These scaffolds supported attachment, proliferation, and survival of cells in vitro and could be degraded by recombinant matrix metalloproteinase-2 and -9. Prefabricated gelatin cryogels rapidly resumed their original shape when injected subcutaneously into mice and elicited only a minor host response following injection. Controlled release of granulocyte-macrophage colony-stimulating factor from gelatin cryogels resulted in complete infiltration of the scaffold by immune cells and promoted matrix metalloproteinase production leading to cell-mediated degradation of the cryogel matrix. These findings suggest that gelatin cryogels could serve as a cell-responsive platform for biomaterial-based therapy.

Keywords: Cryogel; Degradable; Gelatin; Injectable; Matrix metalloproteinase.

Figure 1

Fabrication of gelatin cryogels with highly interconnected pores. (A) Schematic of GelMA synthesis and crosslinking. Pendant methacrylate groups are added primarily to the free amines of gelatin by reaction with methacrylic anhydride. Free radical polymerization results in crosslink formation between methacrylate groups. (B) Cryopolymerization of methacrylated gelatin. Freezing of methacrylated gelatin in the presence of radical initiators (APS and TEMED) allows polymerization to occur in the partially frozen state (cryopolymerization). Ice crystals formed during the freezing process and thawing after cryopolymerization results in the formation of a hydrogel with micron-scale pores. (C) Volume of interconnected pores in gelatin cryogels (normalized to total gel volume). Values represent mean and standard deviation (n=10). Data were compared using ANOVA with Bonferroni’s post-hoc test (**p<0.01, ***p<0.001).

Figure 2

Bulk mechanical behavior and injectability of gelatin cryogels. (A) Images demonstrating ability of an individual rhodamine-gelatin cryogel to be compressed between forceps (dashed white line) to large strain, followed by release and resumption of its original shape. (B) Rhodamine-gelatin cryogel following collapse due to wicking of free water (0 ms), and rapid rehydration following exposure to excess DPBS. (C) Rhodamine-gelatin cryogel exiting the bore of a 16 G needle (0 ms, outer needle wall outlined with dashed line), and its expansion to original size and shape after injection. All scale bars = 2 mm.

Figure 3

Microarchitecture of gelatin cryogels. (A) Surface and cross-sectional SEM micrographs of highly porous 1.0% (w/v) gelatin cryogels (scale bar = 50 μm). (B) 2-photon imaging at a depth of 150 μm below the surface of a rhodamine-gelatin cryogel (scale bar = 1 mm). The inset shows a magnified view at the center of the scaffold diameter (scale bar = 100 μm). Images are representative of at least 5 gels imaged using each modality.

Figure 4

Cell attachment, survival, and proliferation on gelatin cryogels in vitro. (A) Representative 2-photon microscopy image of CMFDA-labeled cells at 150 μm depth below the surface of a rhodamine-gelatin cryogel 2 h after seeding (n=5, scale bar = 200 μm). (B) SEM micrograph of 3T3 cells spread on cryogel surface at 1 d post-seeding (n=5, scale bar = 10 μm). Cells are false-colored for emphasis. (C) Staining of F-actin on histological sections showing cell spreading within scaffolds after one day of culture (n=3, scale bar = 25 μm). (D) Analysis of the number of live cells, overall metabolic activity as measured by alamarBlue reduction, and viability of cells recovered from gelatin cryogels over time in culture. Values represent the mean and standard deviation (n=3). (E) Staining for new DNA synthesis within the scaffold using EdU incorporation (n=3, scale bar = 100 μm).

Figure 5

Enzymatic degradation of gelatin cryogels in vitro. (A) In vitro degradation of rhodamine-gelatin cryogels (n=3) in the presence of 25 U/ml collagenase type II. (B) Zymography with mouse and human pro-MMP-2 and pro-MMP-9 using GelMA-polyacrylamide gels. (C) Quantification of degradation of rhodamine-gelatin cryogels (n=4) in the presence of 10 μg/ml recombinant mouse and human MMP-2 and -9 for 18 hours. One-way ANOVA with Dunnett’s test was performed to compare all MMP-treated conditions with the control gels incubated in buffer alone (n=4, ****p<0.0001). Values represent the mean and standard deviation in all plots.

Figure 6

In vivo injection of gelatin cryogels. (A) In vivo fluorescence image of rhodamine-gelatin cryogel in a C57Bl/6J mouse immediately following subcutaneous injection. (B) In situ image of rhodamine-gelatin cryogel under the skin at 1 w and 2 m following implant. (C) H&E at 1 w and (D) 2 m following implant at the cryogel-tissue interface (left) and the cryogel interior (right) (n=3, scale bar = 50 μm). Arrows indicate the cryogel-host border.

Figure 7

In vivo cell recruitment to gelatin cryogels by sustained release of GM-CSF. (A) Schematic of cell recruitment to GM-CSF-releasing gelatin cryogels. Sustained release of GM-CSF from the cryogel implant creates a chemoattractant gradient to attract host immune cells. (B) In vitro cumulative GM-CSF release and (C) average release rate from gelatin cryogels. (D) Recruited cell numbers in blank and GM-CSF-releasing gelatin cryogels at 14 d post-implant (Student’s t-test, n=3 mice, **p<0.01). (E) Representative H&E staining from blank and GM-CSF-releasing cryogels 14 d after implantation in C57/Bl6J mice (n=3, scale bar = 500 μm). Inset shows a magnified view of the scaffold interior (scale bar = 20 μm). Arrows indicate the cryogel-tissue borders. Values represent the mean and standard deviation in all plots.

Figure 8

Gelatin cryogel degradation by recruited cells in vivo. (A) Images and (B) quantification of fluorescence signal from longitudinal in vivo imaging of the degradation of blank and GM-CSF-releasing rhodamine-gelatin cryogels in C57Bl/6J-Tyr^c-2J mice (n=5). (C) Gelatin zymography of cellular lysates from 1 w implanted blank and GM-CSF-releasing rhodamine-gelatin cryogels. Representative in vivo (D) fluorescence imaging, and (E) quantitation (paired t-test, n=3, **p<0.01), of MMPSense 750 FAST activation in mice 7 d following injection in opposite flanks with blank and GM-CSF-releasing rhodamine-gelatin cryogels. Scaffold borders are outlined in white in (D–E). Values represent the mean and standard deviation in all plots.