Hydrocolloids of Egg White and Gelatin as a Platform for Hydrogel-Based Tissue Engineering

Abstract

Innovative materials are needed to produce scaffolds for various tissue engineering and regenerative medicine (TERM) applications, including tissue models. Materials derived from natural sources that offer low production costs, easy availability, and high bioactivity are highly preferred. Chicken egg white (EW) is an overlooked protein-based material. Whilst its combination with the biopolymer gelatin has been investigated in the food technology industry, mixed hydrocolloids of EW and gelatin have not been reported in TERM. This paper investigates these hydrocolloids as a suitable platform for hydrogel-based tissue engineering, including 2D coating films, miniaturized 3D hydrogels in microfluidic devices, and 3D hydrogel scaffolds. Rheological assessment of the hydrocolloid solutions suggested that temperature and EW concentration can be used to fine-tune the viscosity of the ensuing gels. Fabricated thin 2D hydrocolloid films presented globular nano-topography and in vitro cell work showed that the mixed hydrocolloids had increased cell growth compared with EW films. Results showed that hydrocolloids of EW and gelatin can be used for creating a 3D hydrogel environment for cell studies inside microfluidic devices. Finally, 3D hydrogel scaffolds were fabricated by sequential temperature-dependent gelation followed by chemical cross-linking of the polymeric network of the hydrogel for added mechanical strength and stability. These 3D hydrogel scaffolds displayed pores, lamellae, globular nano-topography, tunable mechanical properties, high affinity for water, and cell proliferation and penetration properties. In conclusion, the large range of properties and characteristics of these materials provide a strong potential for a large variety of TERM applications, including cancer models, organoid growth, compatibility with bioprinting, or implantable devices.

Keywords: egg white; gelatin; hydrocolloids; hydrogels; microfluidics; tissue engineering.

Figure 9

(A) Representative rheological profiles of the 3D hydrogel scaffolds. Graphs show the storage G’ and loss modulus G” (red and blue respectively, measured in Pa), phase angle δ (black, measured in °), and specific viscosity η* (green, measured in Pa.s) of each sample recorded against increasing temperature (T, measured in °C). (B) G’, G”, η*, and δ values at 37 °C of the 3D hydrogel scaffolds (mean ± SD of at least N = 3). (C) Swelling ratio of the 3D hydrogel scaffolds in both H₂O and PBS (mean ± SD of an N = 2) after 15 min at 37 °C. * p < 0.05 and ** p < 0.001.

Figure 1

(A) Major structural components of chicken egg. (B) Composition of egg white showing the relative abundance of the different components. (C) Relative abundance of proteins present in egg white. (D) Biological activities of the main proteins found in egg white.

Figure 2

Visual summary of the experimental work carried out in this study. (A) Hydrocolloid solutions of EW and gelatin. (B) 2D hydrocolloid films. (C) Miniaturized 3D hydrogels of EW and gelatin inside microfluidic platforms. (D) 3D hydrogel scaffolds of EW and gelatin. AFM: atomic force microscopy; SEM: scanning electron microscopy; o/n: overnight; and GTA: glutaraldehyde. A detailed experimental design summary can be found in Section 4.1.

Figure 3

(A) Macroscopic appearance of lyophilized EW powder. (B) Macroscopic appearance of the different hydrocolloid solutions. (C) Color of the different hydrocolloid solutions. (D) Color evaluation using the RGB (red, green, blue) and L*a*b* systems.

Figure 4

Rheological analysis of the hydrocolloid solutions using a plate-plate sensor (graphs on the left) or a cone-plate one (graphs on the right). Graphs show the storage G’ and loss modulus G” (blue and green respectively, measured in Pa), phase angle δ (black, measured in °), and specific viscosity η* (red, measured in Pa.s) of each sample recorded against increasing temperature (T, measured in °C).

Figure 5

(A) Representative 2D topography AFM images of the different hydrocolloid films. Scan size is 50 µm × 50 µm. The inset panels represent 3D topography maps of 3 µm × 3 µm areas. (B) Box charts represent the mean values of the roughness average (Ra) parameter for the 3 tested surface areas (N = 3) for each analyzed sample. * p < 0.05.

Figure 6

(A) Scheme showing 2D culture of primary normal human dermal fibroblasts (pnHDFs) on cell culture well plates coated with hydrocolloid films. (B) Representative phase contrast light microscopy of pnHDF on hydrocolloid films at low confluency (left) and as a confluent monolayer (right). Scale bar = 100 µm. (C) Heat map of cell viability and proliferation (alamarBlue^® assay) compared with the control showing mean of N = 3 per hydrocolloid type. Control is uncoated tissue-culture treated polystyrene (PS) wells.

Figure 7

(A) Scheme showing miniaturized 3D culture of primary normal human dermal fibroblasts (pnHDFs) inside microfluidic devices loaded with EW and gelatin hydrocolloids. (B,C) Phase contrast light microscopy of pnHDF inside microfluidic devices. Scale bar is 175 µm. * Indicates large hydrating channels while white arrows point to small channels. (B) Representative images showing cell morphology over time. (C) Additional representative images. For the 1% EW + 1% Gel panel, the last 2 photos are the exact same view at different planes of observation, showing cells on both planes. Blue arrows point at examples of cells with a flat morphology whilst red arrows point at rounded cells. Green arrows show lamellipodia. (D) alamarBlue^® assay results showing median (central line), interquartile range (box), and minimum and maximum values (whiskers) of N = 5 per hydrocolloid type. No statistical differences were found.

Figure 8

(A) Macroscopic appearance of freshly fabricated 3D hydrogel scaffolds before lyophilization. (B) Representative SEM images of lyophilized 3D hydrogel scaffolds. Images at the bottom (scale bar = 5 µm) show nano-structures. EW: egg white; Gel: gelatin; and GTA: glutaraldehyde.

Figure 10

(A) Scheme of 3D culture of primary normal human dermal fibroblasts (pnHDFs) in 3D hydrogel scaffolds. (B) alamarBlue^® assay results showing median (central line), interquartile range (box), and minimum and maximum values (whiskers) of N = 3 per 3D hydrogel scaffold. * p < 0.05 and ** p < 0.001. (C) Representative H and E-stained sections of seeded 3D hydrogel scaffolds at day 10 of culture (dark pink = matrix, purple dots = cell nuclei). Yellow dots indicate pores between consecutive lamellae filled with cell layers. Scale bar = 50 µm.