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. 2023 Apr 13;15(8):1868.
doi: 10.3390/polym15081868.

Chondrogenic Differentiation of Adipose-Derived Stromal Cells Induced by Decellularized Cartilage Matrix/Silk Fibroin Secondary Crosslinking Hydrogel Scaffolds with a Three-Dimensional Microstructure

Jing Zhou  1 , Nier Wu  2 , Jinshi Zeng  1 , Ziyu Liang  3 , Zuoliang Qi  1 , Haiyue Jiang  1 , Haifeng Chen  3 , Xia Liu  1   4
Affiliations

Chondrogenic Differentiation of Adipose-Derived Stromal Cells Induced by Decellularized Cartilage Matrix/Silk Fibroin Secondary Crosslinking Hydrogel Scaffolds with a Three-Dimensional Microstructure

Jing Zhou et al. Polymers (Basel). .

Abstract

Finding an ideal scaffold is always an important issue in the field of cartilage tissue engineering. Both decellularized extracellular matrix and silk fibroin have been used as natural biomaterials for tissue regeneration. In this study, a secondary crosslinking method of γ irradiation and ethanol induction was used to prepare decellularized cartilage extracellular matrix and silk fibroin (dECM-SF) hydrogels with biological activity. Furthermore, the dECM-SF hydrogels were cast in custom-designed molds to produce a three-dimensional multi-channeled structure to improve internal connectivity. The adipose-derived stromal cells (ADSC) were seeded on the scaffolds, cultured in vitro for 2 weeks, and implanted in vivo for another 4 and 12 weeks. The double crosslinked dECM-SF hydrogels exhibited an excellent pore structure after lyophilization. The multi-channeled hydrogel scaffold presents higher water absorption ability, surface wettability, and no cytotoxicity. The addition of dECM and a channeled structure could promote chondrogenic differentiation of ADSC and engineered cartilage formation, confirmed by H&E, safranin O staining, type II collagen immunostaining, and qPCR assay. In conclusion, the hydrogel scaffold fabricated by the secondary crosslinking method has good plasticity and can be used as a scaffold for cartilage tissue engineering. The multi-channeled dECM-SF hydrogel scaffolds possess a chondrogenic induction activity that promotes engineered cartilage regeneration of ADSC in vivo.

Keywords: cartilage tissue engineering; decellularized cartilage extracellular matrix; multi-channeled; secondary crosslinking; silk fibroin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Graphical representation of the hydrogel scaffold preparation procedure.
Figure 2
Figure 2
Mechanism of the γ-ray and ethanol crosslinking of SF-ECM hydrogel. (A). Mechanism of the γ-ray crosslinking. Active groups such as hydroxyl radicals were first generated from water by irradiation (1), after which polypeptide chains were attacked and turned to radicals (2). These peptide radicals could finally combine to form a crosslinking network (3). (B). Mechanism of ethanol crosslinking.
Figure 3
Figure 3
Structure and characterization of the scaffolds. (A). Gross view and ultrastructure of scaffolds, bar = 200 μm. (B). Pore size. (C). Porosity. (D). Surface wettability. (E). Swelling index of hydrogel scaffolds. (F). Water uptake. (G). DAPI and light microscopy images, bar = 100 μm. (H). FTIR spectra. (I). Cytotoxicity assay. “*” indicates a significant difference between the two groups with p < 0.05. Original magnification: ×250.
Figure 4
Figure 4
The cytocompatibility of the hydrogel scaffolds in vitro. SEM, bar = 30 μm. Fluorescence microscopy, bar = 100 μm.
Figure 5
Figure 5
Gross view and histological examinations of engineered cartilage at 4 weeks in vivo. Bar = 20 μm.
Figure 6
Figure 6
Gross view and histological examinations of the regenerated cartilage in nude mice for 12 weeks. Bar = 20 μm.
Figure 7
Figure 7
mRNA expression of chondrogenic-related genes examined with qPCR in solid hydrogel and MC hydrogel. “**” indicates significant difference between the two groups with p < 0.01.
Figure 8
Figure 8
Comparison of the chondrogenic induction effects of hydrogels with or without dECM by qPCR. “**” indicates a significant difference between the two groups with p < 0.01.
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