Carbon Dots Crosslinked Egg White Hydrogel for Tissue Engineering

Abstract

Egg white (EW)-derived hydrogels hold promise as biomaterials for in vitro cell culture due to their ability to mimic the extracellular matrix. However, their highly cross-linked structures restrict their potential for in vivo applications, as they are unable to integrate dynamically with tissues before degradation. In this study, this limitation is addressed by introducing carbon dots (CDs) as cross-linking agents for EW in a dilute aqueous solution. The resulting CDs-crosslinked EW hydrogel (CEWH) exhibits tensile strength comparable to that of skin tissue and features a large pore structure that promotes cell infiltration. Subcutaneous implantation of CEWH demonstrated excellent integration with surrounding tissue and a degradation rate aligned with the hair follicles (HFs) regeneration cycle. This allows the long-term regeneration and establishment of an M2 macrophage-dominated immune microenvironment, which in turn promotes the re-entry of HFs into the anagen phase from the telogen phase. Additionally, CEWH demonstrated potential as a wound dressing material. Overall, this study paves the way for utilizing EW as a versatile biomaterial for tissue engineering.

Keywords: carbon dots; egg white; hair follicle regeneration; protein hydrogels; wound healing.

Figure 1

Schematic illustration of CEWH formation process. a) The actual synthetic steps of CEWH. The CDs were combined with a diluted pure‐EW solution to create an EW + CD mixture, which was subsequently heated to produce a transparent hydrogel‐CEWH. b) Depiction of the possible molecular‐level interactions during the formation of EW‐based hydrogels with or without CDs addition.

Figure 2

The mechanistic investigation of CDs cross‐linked EW proteins. a) Absorption and fluorescence spectra (excitation at 589 nm) of a pure‐EW solution, EW + CDs solution, and CEWH. b) Fluorescence images of a pure‐EW solution, EW + CDs solution, and CEWH (excited at 488, 546, and 647 nm). c) Circular dichroism spectra of a pure‐EW solution before and after heating, EW + CDs solution, and CEWH. d–g) SEM images of xerogels: 1CDs@EW, 4CDs@EW, CEWH (8CDs@EW), and 12CDs@EW. Scale bars: 20 µm. h) SEM images of EW hydrogel. Scale bar: 20 µm. The inset in (h) provided a magnified view of the pore structure of the EW hydrogel. i) Tensile stress–strain curves for 4CDs@EW, 8CDs@EW (CEWH), and 12CDs@EW. The initial dimensions of the specimen were 16.0 mm × 4.0 mm × 1.0 mm. Tensile tests were conducted at a constant rate of 20 mm min⁻¹ at room temperature. j) Circular dichroism spectra of exudates from CEWH and EW hydrogel, obtained by immersing 7 g of each in 6 mL of water for 48 h. k) Photographs of large‐scale CEWH (20 cm × 15 cm) applied to a human elbow joint undergoing elbow flexion. l) A proposed mechanism for the formation of a stretchable and transparent network structure using CDs and EW peptide chains.

Figure 3

Characterization of CEWH biocompatibility and its interaction with cells. a) Optical images of MDA‐MB‐231 cells incubated on the stripped surface of CEWH, captured on days 1 and 3. b) 3D fluorescence image (TPF) of Hela‐GFP cells (green) cultured on CEWH (red), with fluorescence generated by a 960 nm femtosecond laser. c) SEM image of CEWH xerogel after a 3‐day incubation with MDA‐MB‐231 cells. d) In vivo fluorescence images of a mouse with a subcutaneous CEWH implant, excited with a 589 nm laser. Images were collected using a 610 nm long‐pass optical filter at various time points post‐implantation. Excised mouse skin with e) an EW hydrogel implant and f) a CEWH implant, respectively, captured using a stereomicroscope (Leica MZ10 F) on day 21 post‐implantation. All implants were 5 mm × 5 mm × 2 mm. g) SEM images of the CEWH implant xerogel after 21 days in vivo post‐implantation. The yellow region corresponds to the CEWH implant, and the red regions represent red blood cells and blood vessels. Inset shows a high‐definition magnified SEM image. h–j) Hematoxylin and eosin staining of the subcutaneous (h) EW hydrogel implant on day 14 post‐implantation and (i, j) CEWH implant on days 14 and 21 post‐implantation (scale bars: 50 µm for all histological images). The pinkish‐purple regions represent the stained hydrogel implants.

Figure 4

Deep tissue imaging of 3 mm × 3 mm × 1 mm CEWH implants in LysM‐Cre‐mT/mG mouse ears using TPF microscopy. a) TPF images (scale bar: 1 mm) of a tissue section from the mouse ear containing a subcutaneous CEWH implant. The section was collected on day 12 post‐implantation. Two femtosecond lasers with distinct wavelengths were used for excitation: 960 nm for the green channel capturing macrophages and collagen fibers signals, and 1100 nm for the red channel capturing blood vessel signals. The inset shows a photograph of the experimentally operated mouse ear. b) TPF images (scale bars: 50 µm) of the HFs at the implant site, captured at different time points post‐CEWH implantation.

Figure 5

Analysis of skin tissues response to 5 mm × 5 mm × 2 mm CEWH implants. a) Masson's trichrome staining of mouse skin sections containing CEWH implants (scale bars: 1 mm; enlarged scale bars: 250 µm). Sections were collected on days 14, 21, and 28 post‐CEWH implantation. b) Volcano plots showing the gene set enrichment analysis (GSEA)‐enriched pathways in a mouse skin section with a CEWH implant, collected on day 14 post‐implantation. c–f) GSEA‐enriched signaling pathways associated with HFs development, macrophage activation, IL6/JAK/STAT3 signaling, and Wnt/β‐Catenin signaling in mouse skin sections with a CEWH implant (collected on day 14 post‐implantation). g) Venn diagrams illustrating the overlap of GSEA results from mouse skin sections with CEWH implants, collected at different time points post‐implantation. h) Expression levels of selected genes involved in the pathways.

Figure 6

CEWH promotes wound closure and HFs regeneration. a) Representative images (scale bars: 5 mm) showing wound closure in BALB/c mice at days 3, 6, 9, and 13 post‐wounding. Four groups were investigated: CEWH‐treated group, EW hydrogel‐treated group, UEH alginate gel‐treated group, and untreated control group. Medical tape was applied around the wounds to prevent abnormal wound contraction. b) Representative images (scale bars: 500 µm) of CEWH‐treated healing wound sections on day 13 post‐wounding, stained with H&E. Asterisks indicate epidermal cysts, and arrows point to newly formed hair follicles (HFs). c) The graph depicts the wound closure rates for the four groups. d) Representative images (scale bars: 500 µm) of wound sections for the four groups on days 6, 9, and 13 post‐wounding, stained with Masson's trichrome. e) Immunohistochemistry (IHC) staining for the four groups of CD68 (macrophage marker), CD86 (M1 macrophage marker), and CD206 (M2 macrophage marker) on day 6 post‐wounding. Scale bars: 100 µm.