Alginate Conjugation Increases Toughness in Auricular Chondrocyte Seeded Collagen Hydrogels

Abstract

Current auricular cartilage replacements for pediatric microtia fail to address the need for long-term integration and neocartilage formation. While collagen hydrogels have been successful in fostering neocartilage formation, the toughness and extensibility of these materials do not match that of native tissue. This study used the N-terminal functionalization of collagen with alginate oligomers to improve toughness and extensibility through metal-ion complexation. Alginate conjugation was confirmed via FTIR spectroscopy. The retention of native collagen fibrillar structure, thermal gelation, and helical conformation in functionalized gels was confirmed via scanning electron microscopy, oscillatory shear rheology, and circular dichroism spectroscopy, respectively. Alginate-calcium complexation enabled a more than two-fold increase in modulus and work density in functionalized collagen with the addition of 50 mM CaCl₂, whereas unmodified collagen decreased in both modulus and work density with increasing calcium concentration. Additionally, the extensibility of alginate-functionalized collagen was increased at 25 and 50 mM CaCl₂. Following 2-week culture with auricular chondrocytes, alginate-functionalization had no effect on the cytocompatibility of collagen gels, with no effects on cell density, and increased glycosaminoglycan deposition. Custom MATLAB video analysis was then used to quantify fracture toughness, which was more than 5-fold higher following culture in functionalized collagen and almost three-fold higher in unmodified collagen.

Keywords: collagen; complexation; extensibility; functionalization; toughness.

Figure 1

Reaction scheme (A) for alginate functionalization of collagen n-termini (i) to enable calcium complex formation (ii). FTIR spectra of alginate-functionalized and unmodified collagen, alginate stock (B), and sugar/amide II peak area calibration to determine extent of functionalization (C). n = 6 for all spectra and samples.

Figure 2

SEM micrographs of collagen (A) and ColAlg (B). Storage modulus (C), gelation time (D), and circular dichroism spectra (E) of Col and ColAlg. n = 3 for all samples. Shared letters denote no significant difference.

Figure 3

Representative stress–strain curves for Col (A) and ColAlg (B) gels, with calculated moduli (C), strain at ultimate tensile stress (D), and work density (E). n = 2–8. Shared letters denote no significant difference.

Figure 4

Alcian blue histology with fast red counterstaining of auricular chondrocyte-seeded Col (A) and ColAlg (B) constructs. Quantification of construct contraction (C) by area ratio, DNA (D), hydroxyproline (E), and glycosaminoglycans (F) according to sample wet weight. n = 3–6. Shared letters denote no significant difference.

Figure 5

Calculated modulus (A) and strain at ultimate tensile stress (B) of acellular and cell-seeded constructs. Representative crack edge images (C) with automated crack tracking outlined in red. Calculated fracture toughness of acellular and cell-seeded constructs (D). n = 2–8. Shared letters denote no significant difference.