A silk fibroin/decellularized extract of Wharton's jelly hydrogel intended for cartilage tissue engineering

Abstract

A hybrid hydrogel was obtained from decellularized extract from Wharton's jelly (DEWJ) and silk fibroin (SF) and characterized for cartilage tissue engineering. Wharton's jelly was used due to its similarity with articular cartilage in extracellular matrix composition. Also, silk fibroin has good mechanical properties which make this construct appropriate for cartilage repair. Decellularization of Wharton's jelly was verified by DAPI staining, DNA quantification, and PCR analysis. Then, the biochemical composition of DEWJ was determined by ELISA kits for total proteins, collagens, sulfated glycosaminoglycans (sGAG), and transforming growth factor β1 (TGF-β1). After fabricating pure SF and SF/DEWJ hybrid hydrogels, their physical and mechanical properties were characterized by FESEM, Fourier-transform infrared spectroscopy (FTIR) and rheological assays (amplitude and frequency sweeps). Furthermore, cell viability and proliferation were assessed by MTT assay. The results have shown that DEWJ in hybrid hydrogels enhances mechanical properties of the construct relative to pure SF hydrogels. Also, this extract at its 40% concentration in culture media and 20% or 40% concentrations in SF/DEWJ hybrid hydrogels significantly increases population of the cells compared to control and pure SF hydrogel after 7 days. In conclusion, this study proposes the potential of SF/DEWJ hybrid hydrogels for cartilage tissue engineering applications.

Keywords: Cartilage tissue engineering; Decellularization; Hydrogel; Silk fibroin; Wharton’s jelly.

Fig. 1

Verification of WJ decellularization by a DAPI staining (scale bar = 100 µm), b DNA quantification assay (data are shown as mean ± standard deviation, n = 3), and c agarose gel of the PCR products for native WJ, extract before centrifugation, extracts after centrifugation at 5000 rpm/15 min, 5000 rpm/30 min, and 10,000 rpm/15 min. DEWJ dcellularized extract from Wharton’s jelly, WJ Wharton’s jelly

Fig. 2

Biochemical analysis of native WJ, immediate extract, and overnight extract in terms of a total protein, b sGAG, c TGF-β1, d total collagen, e soluble collagen, f insoluble collagen. Data are shown as mean ± standard deviation (n = 3). * and ^# symbols, respectively, indicate comparison with native WJ and immediate extract groups (*P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.05, and ^##P < 0.01). WJ Wharton’s jelly, sGAG sulfated glycoseaminoglycan, TGF-β1 transforming growth factor-beta 1

Fig. 3

MTT results for different concentrations of DEWJ using hEnSCs. Data are shown as mean ± standard deviation (n = 3). * symbol indicates comparison with control group (0%) at same time points (*P < 0.05 and ***P < 0.001). DEWJ dcellularized extract from Wharton’s jelly, hEnSCs human endometrial mesenchymal stem cells

Fig. 4

FTIR spectra of DEWJ, SF, SF/20% DEWJ, and SF/40% DEWJ lyophilized hydrogels. Dotted lines demonstrate the center of amide A, amide I, amide II, amide III and GAGs peaks

Fig. 5

Rheological characterization including a amplitude sweeps and b frequency sweeps demonstrates storage modulus (G′) and loss modulus (G′′) of SF, SF/20% DEWJ and SF/40% DEWJ hydrogels

Fig. 6

SEM images of SF, SF/20% DEWJ, SF/40% DEWJ lyophilized hydrogels (scale bar = 200 μm)

Fig. 7

MTT results for SF, SF/20% DEWJ, SF/40% DEWJ hydrogls using hEnSCs. Data are shown as mean ± standard deviation (n = 3). * and ^# symbols, respectively, indicate comparison with control group and SF hydrogel at same time points (**P < 0.01, ***P < 0.001, ^#P < 0.05, and ^##P < 0.01, n = 3). SF silk fibroin, DEWJ dcellularized extract from Wharton’s jelly, hEnSCs human endometrial mesenchymal stem cells