A photo-crosslinkable cartilage-derived extracellular matrix bioink for auricular cartilage tissue engineering

Abstract

Three-dimensional (3D) bioprinting of patient-specific auricular cartilage constructs could aid in the reconstruction process of traumatically injured or congenitally deformed ear cartilage. To achieve this, a hydrogel-based bioink is required that recapitulates the complex cartilage microenvironment. Tissue-derived decellularized extracellular matrix (dECM)-based hydrogels have been used as bioinks for cell-based 3D bioprinting because they contain tissue-specific ECM components that play a vital role in cell adhesion, growth, and differentiation. In this study, porcine auricular cartilage tissues were isolated and decellularized, and the decellularized cartilage tissues were characterized by histology, biochemical assay, and proteomics. This cartilage-derived dECM (cdECM) was subsequently processed into a photo-crosslinkable hydrogel using methacrylation (cdECMMA) and mixed with chondrocytes to create a printable bioink. The rheological properties, printability, and in vitro biological properties of the cdECMMA bioink were examined. The results showed cdECM was obtained with complete removal of cellular components while preserving major ECM proteins. After methacrylation, the cdECMMA bioinks were printed in anatomical ear shape and exhibited adequate mechanical properties and structural integrity. Specifically, auricular chondrocytes in the printed cdECMMA hydrogel constructs maintained their viability and proliferation capacity and eventually produced cartilage ECM components, including collagen and glycosaminoglycans (GAGs). The potential of cell-based bioprinting using this cartilage-specific dECMMA bioink is demonstrated as an alternative option for auricular cartilage reconstruction.

Keywords: Bioink; Bioprinting; Cartilage tissue engineering; Decellularization; Extracellular matrix; Methacrylation.

Figure 1.

Schematic diagram of the auricular cartilage-derived ECM (cdECM) bioink development. (A) Methacrylation of cdECM obtained by decellularization and solubilization process. (B) Illustration of cdECMMA bioink formulation containing cells. (C) 3D bioprinting process using cell-laden cdECMMA bioink. (D) 3D bioprinting workflow from 3D CAD/CAM model to bioprinted ear construct for personalized auricular reconstruction.

Figure 2.

Characterizations of cdECM. (A) DNA contents before and after decellularization of cartilage tissue (n=3). Data are represented mean ± SD. The p-value by Student t-test is indicated. (B) Second-harmonic generation and two-photon autofluorescence (SHG/2-PEF) images of collagenous fiber bundles of native and decellularized auricular cartilage tissues. Scale = 100 μm. (C) Histological analyses of native and decellularized auricular cartilage tissues by H&E, MTE, and Alcian Blue staining. (D) Collagen contents (n=5) and (E) GAG contents (n=5) of native and decellularized auricular cartilage tissues. Data are represented mean ± SD. The p-value by Student t-test is indicated.

Figure 3.

Proteomic analysis of native and decellularized auricular cartilage tissues. (A) Venn diagram showing numbers of unique proteins found in either native (683) or decellularized (21), and numbers of proteins found in both tissues (412) (in at least 2 out of 3 samples). (B) Data are represented mean ± SD of numbers of proteins identified using mass spectrometry (MS/MS) of native cartilage tissue and decellularized cartilage tissue (n=3). (C) Heat map and supervised cluster analysis using protein expression data from native and decellularized cartilage tissue. Notice biological variation in both native and decellularized tissue samples (orange). Only proteins found in 2 out of 3 samples were processed for further analysis. (D) Protein-protein network of significantly upregulated proteins in decellularized cartilage (p<0.05, fold change ≥1.5, detected in at least 2 out of 3 samples). The networks were generated using default settings in String and visualized using Cytoscape. (E) Percentage (%) of gene hits against total number of genes associated with a certain cellular location in native cartilage tissue and (F) decellularized cartilage tissue (Panther GO - Slim cellular component, detected in at least 2 out of 3 samples).

Figure 4.

Synthesis and characterization of photo-crosslinkable cdECM bioinks. (A) Schematic illustration of methacrylation and UV crosslinking of cdECM and (B) degree of methacrylation (%) of GelMA and cdECM-MA (n=9). (C) Gross appearance of cdECM-MA bioink constructs with different concentrations (scale bar: 5 mm). (D) Swelling ratios and (E) gel stiffness of cdECMMA hydrogels with different concentrations. GelMA hydrogel served as a control. All data are represented mean ± SD. The p-values by Student t-test are indicated. (F) Lattice structure (top, scale bar: 5 mm) and ear-shape of 3D printed cdECM-MA bioink constructs (30 mg/ml) (bottom, scale bar: 10 mm).

Figure 5.

Cell viability and proliferation in the bioprinted cdECMMA constructs at 1, 3, and 7 days in culture. (A) Live/Dead™ stained images and (B) quantification (n=3, *p<0.05). (C) AlamarBlue™ assay for cell proliferation (n=6). GelMA construct served as a control. All data are represented mean ± SD. The p-values by one-way ANOVA followed by Tukey’s test are indicated.

Figure 6.

In vitro cartilage tissue formation of 3D bioprinted cdECMMA constructs containing rabbit auricular chondrocytes after 28 days in culture. (A) Histological evaluations by H&E, Safranin O/Fast Green, and Alcian Blue/Sirius Red staining. Scale bar: 100 μm. Biochemical assay for (B) collagen (n=3) and (C) GAG (n=3) production in the 3D printed constructs. GelMA construct served as a control. All data are represented mean ± SD. The p-values by one-way ANOVA followed by Tukey’s test are indicated.