Anti-Scar Effects of Micropatterned Hydrogel after Glaucoma Drainage Device Implantation

Abstract

Excessive fibrosis is the primary factor for the failure of glaucoma drainage device (GDD) implantation. Thus, strategies to suppress scar formation in GDD implantation are crucial. Although it is known that in implanted medical devices, microscale modification of the implant surface can modulate cell behavior and reduce the incidence of fibrosis, in the field of ophthalmic implants, especially the modification and effects of hydrogel micropatterns have rarely been reported. Here, we designed the patterned gelatin/acrylamide double network hydrogel and developed an innovative GDD with micropattern to suppress inflammatory and fibroblast activation after GDD implantation. Pattern topography suppressed F-actin expression and mitigated actin-dependent nuclear migration of myocardin-related transcription factor A (MRTF-A) during the proliferative phase after GDD implantation. Ultimately, the expression of α-smooth muscle actin (α-SMA), a key fibrosis-related gene product, was suppressed. Moreover, the modified GDD effectively controlled intraocular pressure (IOP), mitigated fibrous formation, and remodeled extracellular matrix (ECM) collagen distribution in vivo. Therefore, the novel GDD with surface patterning interventions provides a promising strategy to inhibit scar formation after GDD implantation and raise the efficacy of GDD implantation.

Fig. 1.

The synthesis process of patterned PG hydrogel. (A) Synthesis mechanism of PG hydrogel. (B) Schematic diagram of patterned hydrogel synthesis. (C) Multifunction of patterned hydrogel including anti-inflammatory, promoting proliferation, and preventing scarring.

Fig. 2.

(A) Stress–strain curves of PG hydrogels containing different amounts of gelatin. (B) Stress–strain curves of PG hydrogels with varying levels of acrylamide. (C) Water contact angle data of PG hydrogel. (D) SEM of the patterned PG hydrogels with different stripe-to-spacing ratios (5/20, 10/20, 15/20, and 20/20). (E) Water contact angle diagram of PG hydrogels. (F) Images of produced patterned hydrogel and its magnified surface (10 μm). (G) Optical images and variation of pattern interval of hydrogels after immersion in PBS for 30 d. Scale bars, 50 μm.

Fig. 3.

The biocompatibility and cytotoxicity in vitro. (A) Live/dead staining of RTFs and RAWs co-incubated with AGV silicon and PG hydrogels. Scale bar, 250 μm. (B) CCK-8 assay detected cell viability at 1, 3, and 5 d. Scale bar, 250 μm.

Fig. 4.

Pattern topography regulates macrophage morphology and phenotype in vitro. (A) Images and elongation factor of RAWs on PG hydrogels with different micropatterns. (B) Fluorescence images of RAWs on 10/20, 15/20, and flat gel immunostained for iNOS (M1 macrophage marker, green), Arg-1 (M2 macrophage marker, red), and nuclei [4′,6-diamidino-2-phenylindole (DAPI), blue]. Scale bars, 100 μm. (C) Expression of IL-6, iNOS, IL-10, and Arg-1 in RAWs cultured on hydrogel. Data are shown as fold change over glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2^−ΔΔCt). Scale bars, 100 μm.

Fig. 5.

Pattern topography regulates fibroblast morphology and activation in vitro. (A) Images of RTFs on PG hydrogels after culturing for 72 h. (B) Immunofluorescence staining of F-actin. The illustration below shows the magnified cell morphology. (C) Statistical analysis of cell density and cell spreading area. (D) Optical density (O.D) values of RTFs on silicon, flat, 5-μm, 10-μm, 15-μm, and 20-μm pattern gels after 1, 3, and 5 d of culture. (E) Images of RTFs adhering to the pattern topography after culturing for 72 h with and (F) without profibrotic TGF-β. RTFs were stained for α-SMA (green) and nuclei (blue). (G) Statistical results of α-SMA relative expression. (H) Western blotting analysis of α-SMA expression of RTFs on flat hydrogel and 10/20 hydrogel before and after TGF-β stimulation. Scale bars, 100 μm.

Fig. 6.

RTFs were stained for MRTF-A (red) and nuclei (blue) to observe the regulation of MRTF-A localization by patterned surfaces, both with and without TGF-β stimulation. Scale bar, 50 μm.

Fig. 7.

In vivo pattern PG implantation. (A) Image of the process of GDD implantation. (B) AS-OCT scan position (yellow line). (C) IOP measurements in the days 0, 1, 7, 14, 30, 60, and 90. (D) AS-OCT images of the filter bleb. (E) Maximum and minimum capsule thickness of filter bleb.

Fig. 8.

Histological and IHC graphics of the filter bleb. (A) Representative images stained with hematoxylin and eosin (H&E) and Masson staining. (B) Typical IHC images stained with α-SMA and PCNA. (C) Fibrous capsule thickness surrounding implants on their roof and baseline side. (D) Quantitative analysis of α-SMA and PCNA relative expression. (E) Typical IHC images stained with MRTF-A and analyzed MRTF-A nuclear positive expression at 30 and 90 d after operation. Scale bars, 100 μm.

Fig. 9.

The distributions of different collagen types. (A) Typical immunofluorescence images stained with Col-I (green) and Col-III (red). # represents the location of the implant. (B) Relative intensity of Col-I/Col-III. Scale bar, 150 μm.

Fig. 10.

Proposed mechanism of PG hydrogel modified with surface patterning suppressed capsular fibrosis after GDD implantation.