Three-Dimensional-Printed GelMA-KerMA Composite Patches as an Innovative Platform for Potential Tissue Engineering of Tympanic Membrane Perforations

Abstract

Tympanic membrane (TM) perforations, primarily induced by middle ear infections, the introduction of foreign objects into the ear, and acoustic trauma, lead to hearing abnormalities and ear infections. We describe the design and fabrication of a novel composite patch containing photocrosslinkable gelatin methacryloyl (GelMA) and keratin methacryloyl (KerMA) hydrogels. GelMA-KerMA patches containing conical microneedles in their design were developed using the digital light processing (DLP) 3D printing approach. Following this, the patches were biofunctionalized by applying a coaxial coating with PVA nanoparticles loaded with gentamicin (GEN) and fibroblast growth factor (FGF-2) with the Electrohydrodynamic Atomization (EHDA) method. The developed nanoparticle-coated 3D-printed patches were evaluated in terms of their chemical, morphological, mechanical, swelling, and degradation behavior. In addition, the GEN and FGF-2 release profiles, antimicrobial properties, and biocompatibility of the patches were examined in vitro. The morphological assessment verified the successful fabrication and nanoparticle coating of the 3D-printed GelMA-KerMA patches. The outcomes of antibacterial tests demonstrated that GEN@PVA/GelMA-KerMA patches exhibited substantial antibacterial efficacy against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, cell culture studies revealed that GelMA-KerMA patches were biocompatible with human adipose-derived mesenchymal stem cells (hADMSC) and supported cell attachment and proliferation without any cytotoxicity. These findings indicated that biofunctional 3D-printed GelMA-KerMA patches have the potential to be a promising therapeutic approach for addressing TM perforations.

Keywords: DLP 3D printing; FGF-2; gelatin methacryloyl; gentamicin; keratin methacrylolyl; nanoparticle; tympanic membrane perforation.

Figure 1

Schematic diagram of nanoparticle coated-GelMA-KerMA patches development process: (A) Preparation of GelMA-KerMA composite ink; (B) CAD file preparation and slicing for the 3D printing process; (C) 3D printing of GelMA-KerMA patches containing microneedles; (D) the process of coating GelMA-KerMA patches with coaxial nanoparticles by EHDA method; (E) final version of the coaxial nanoparticle coated 3D-printed GelMA-KerMA patch.

Figure 2

(a) ¹H-NMR spectra of gelatin and GelMA. Peaks observed at 5.7 ppm, 5.4 ppm, and 1.9 ppm indicate the presence of methacrylation in the protein. The peak at 3.0 ppm signifies a reduction in lysine groups. (b) ¹H-NMR spectra of keratin and KerMA. Peaks observed at 5.8 ppm, 5.5 ppm, and 1.9 ppm indicate the presence of methacrylation in the protein. The peak between 3.0 and 3.3 ppm may signify a reduction in lysine groups. (c) FTIR spectra of GelMA-KerMA patches.

Figure 3

SEM images of 3D-printed GelMA-KerMA patches: (a) blank GelMA-KerMA patch, (b) GEN@PVA/GelMA-KerMA patch, (c) FGF-2@PVA/GelMA-KerMA, (d) FGF-2@GEN@PVA/GelMA-KerMA patch.

Figure 4

Mechanical properties of blank and nanoparticle-coated 3D-printed GelMA-KerMA hydrogels: (a) compressive strength, (b) strain (%). Data are expressed as mean ± standard deviation (SD, n = 3).

Figure 5

(a) Swelling ability of GelMA-KerMA patches incubated in PBS for different time intervals in comparison with the constructed models. (b) Degradation profiles of GelMA-KerMA patches. Data are expressed as mean ± standard deviation (SD, n = 3).

Figure 6

(a) Linear calibration curve of GEN. (b) The in vitro release profile of GEN from GEN@PVA/GelMA-KerMA patches. The first 12 h GEN release is detailed on the graph. (c) Linear calibration curve of FGF-2. (d) The in vitro release profile of FGF-2 from FGF-2@PVA/GelMA-KerMA patches. The first 12 h FGF-2 release is detailed on the graph. All the measurements were repeated three times; the errors were less than 5%.

Figure 7

Antibacterial activities of GelMA-KerMA and GEN@PVA/GelMA-KerMA patches against (a) S. aureus, (b) P. aeruginosa and (c) E. coli. (1) Blank GelMA-KerMA, (2) GEN@PVA/GelMA-KerMA, and (3) GEN (positive control).

Figure 8

Cell viability results of 1-day, 3-day, and 7-day treated GelMA-KerMA patches. The TCPS was accepted as the control. (* p ≤ 0.05, ** p < 0.01, *** p < 0.001; data presented are mean ± SD, n = 3).

Figure 9

(A) Fluorescence images of the GelMA-KerMA patches on the 7th day of cell growth: (a) blank GelMA-KerMA patch, (b) GEN@PVA/GelMA-KerMA patch, (c) FGF-2@PVA/GelMA-KerMA, (d) FGF-2@GEN@PVA/GelMA-KerMA patch. (B) SEM images of the cultured GelMA-KerMA patches on the 7th day of cell growth: (a) blank GelMA-KerMA patch, (b) GEN@PVA/GelMA-KerMA patch, (c) FGF-2@PVA/GelMA-KerMA, (d) FGF-2@GEN@PVA/GelMA-KerMA patch. Blue circles indicate round-shaped cells in all groups.