Silk hydrogel for cartilage tissue engineering

Abstract

Cartilage tissue engineering based on cultivation of immature chondrocytes in agarose hydrogel can yield tissue constructs with biomechanical properties comparable to native cartilage. However, agarose is immunogenic and nondegradable, and our capability to modify the structure, composition, and mechanical properties of this material is rather limited. In contrast, silk hydrogel is biocompatible and biodegradable, and it can be produced using a water-based method without organic solvents that enables precise control of structural and mechanical properties in a range of interest for cartilage tissue engineering. We observed that one particular preparation of silk hydrogel yielded cartilaginous constructs with biochemical content and mechanical properties matching constructs based on agarose. This finding and the possibility to vary the properties of silk hydrogel motivated this study of the factors underlying the suitability of hydrogels for cartilage tissue engineering. We present data resulting from a systematic variation of silk hydrogel properties, silk extraction method, gel concentration, and gel structure. Data suggest that silk hydrogel can be used as a tool for studies of the hydrogel-related factors and mechanisms involved in cartilage formation, as well as a tailorable and fully degradable scaffold for cartilage tissue engineering.

FIGURE 1

Silk hydrogel and porous scaffolds. (A) DNA, GAG content, and compressive modulus (*p < 0.05 vs. the previous time point, n = 4–5). DNA and GAG contents are expressed per unit wet weight. (B) Live-dead staining on day 42 (insert-magnified for cell morphology, bar = 50 mm). (C) Construct gross appearance after 42 days of culture. (D) Picrosirius red (top) and alcian blue (bottom) staining for collagen and GAG content, respectively. Bar = 1 mm. In (C) and (D), images at the left are for silk hydrogel constructs, and the images at the right are for porous silk scaffold constructs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 2

Immunostaining of type specific collagen in silk hydrogel and porous silk scaffolds on day 42. Bar = 0.5 mm.

FIGURE 3

Effects of silk fibroin extraction method (A) and concentration (B) on cartilage tissue development. (*p < 0.05 compared with the previous time point, ^§p < 0.05 compared with the other group at the same time point, n = 6 for all groups).

FIGURE 4

Agarose (2%) and silk (4%) hydrogels. (ND, not detectable; *p < 0.05 compared with the previous time point, ^§p < 0.05 compared with the agarose group at the same time point, n = 3–6).

FIGURE 5

Intrinsic biomechanical properties of the materials. Equilibrium (A) and dynamic (B) modulus (at 1 Hz) (*p < 0.05 compared with the agarose hydrogel group, ^§p < 0.05 compared with the silk hydrogel detergent.