Boosting cartilage repair with silk fibroin-DNA hydrogel-based cartilage organoid precursor

Abstract

Osteoarthritis (OA), a common degenerative disease, is characterized by high disability and imposes substantial economic impacts on individuals and society. Current clinical treatments remain inadequate for effectively managing OA. Organoids, miniature 3D tissue structures from directed differentiation of stem or progenitor cells, mimic native organ structures and functions. They are useful for drug testing and serve as active grafts for organ repair. However, organoid construction requires extracellular matrix-like 3D scaffolds for cellular growth. Hydrogel microspheres, with tunable physical and chemical properties, show promise in cartilage tissue engineering by replicating the natural microenvironment. Building on prior work on SF-DNA dual-network hydrogels for cartilage regeneration, we developed a novel RGD-SF-DNA hydrogel microsphere (RSD-MS) via a microfluidic system by integrating photopolymerization with self-assembly techniques and then modified with Pep-RGDfKA. The RSD-MSs exhibited uniform size, porous surface, and optimal swelling and degradation properties. In vitro studies demonstrated that RSD-MSs enhanced bone marrow mesenchymal stem cells (BMSCs) proliferation, adhesion, and chondrogenic differentiation. Transcriptomic analysis showed RSD-MSs induced chondrogenesis mainly through integrin-mediated adhesion pathways and glycosaminoglycan biosynthesis. Moreover, in vivo studies showed that seeding BMSCs onto RSD-MSs to create cartilage organoid precursors (COPs) significantly enhanced cartilage regeneration. In conclusion, RSD-MS was an ideal candidate for the construction and long-term cultivation of cartilage organoids, offering an innovative strategy and material choice for cartilage regeneration and tissue engineering.

Keywords: Cartilage organoid; Cartilage repair; Chondrogenesis; Microsphere; Silk fibroin-DNA hydrogel.

Graphical abstract

Scheme 1

Schematic diagram of (A) the synthesis of RGD-SF-DNA microspheres and (B) their application in the preparation of cartilage organoid precursors (COPs) and cartilage regeneration.

Fig. 1

Synthesis and characterization of RSD-MS. (A) Microscopic images of SD-MS and RSD-MS. (B) Particle size distribution of SD-MSs and RSD-MSs. The lower sections display lower magnification images (scale bars: 500 μm) in the upper sections (scale bars 100 μm). (C) 1H NMR spectrogram of SD-MSs and RSD-MSs. (D) SEM images of SD-MS and RSD-MS. The lower sections display higher magnification sections (scale bars: 50 μm) of the areas highlighted by white boxes in the upper panels (scale bars 100 μm). (E) Pore size of SD-MSs and RSD-MSs. (F) The swelling ratio of SD-MSs and RSD-MSs in PBS at 37 °C. (G) In vitro degradation curves of SD-MSs and RSD-MSs in PBS and protease K solution at 37 °C.

Fig. 2

Biocompatibility and cell adhesion of microspheres. (A) Live/dead staining images of BMSCs cocultured with blank, SD-MSs and RSD-MSs for 5 days (scale bars 100 μm). (B) BMSCs proliferation cocultured with blank, SD-MS and RSD-MS for 1, 3 and 5 days. (C) Schematic representation of BMSCs loaded with microspheres. (D) Number of BMSCs cocultured with SD-MS and RSD-MS for 1, 3, and 7 days. (E) Phalloidin-stained and Dapi-stained BMSCs cocultured with RSD-MS for 1, 3, 7 days imaged via confocal laser scanning microscopy (scale bars 100 μm). And phalloidin-stained and Dapi-stained BMSCs cocultured with RSD-MS for 14 days imaged via fluorescence microscope (scale bars 500 μm).

Fig. 3

The effect of SD-MSs and RSD-MSs on BMSCs chondrogenic differentiation. (A) Alcian staining of the BMSCs cultured with SD-MSs and RSD-MSs (transwell) for 3, 7, and 14 days (scale bars 200 μm). (B) Quantification of Alcian blue staining. (C and D) Relative production of GAG and Col II for BMSCs cultured on SD-MSs and RSD-MSs at 3, 7, and 14 days. (E–I) In vitro expression of Col I, Col II, SOX9, ACAN, and Col X) in BMSCs cocultured with blank, SD-MSs and RSD-MSs for 7, and 14 days. (J) Immunofluorescence images of Col II, SOX9 and ACAN on BMSCs loaded on RSD-MSs after 14 days of induction (scale bars 100 μm). (K) Representative Western blot results of Col II, SOX9 and ACAN. (L) Quantitative analysis of Western blot.

Fig. 4

Transcriptomic analysis. (A) PCA of DEGs distributed in the blank group and RSD-MS group. (B) Comparative assessment of mRNA expression in the blank group and RSD-MS group. (C) Volcano plot representation of genes exhibiting significant expression changes exceeding a 2-fold difference in transcriptome sequencing results. (D) Heatmap of gene expression clustering for DEGs. (E) GO analysis for upregulated genes in BMSCs. (F) KEGG analysis for upregulated genes in BMSCs. (G–I) GSEA enrichment analysis for integrin-mediated cell adhesion pathway, focal adhesion pathway and glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate in BMSCs.

Fig. 5

(A) Scheme of cartilage defect modeling and treatment protocol. (B) Gross observation of femur cartilage after treatment at 5 and 10 weeks (scale bars 2 mm). (C) Heat map of ICRS macroscopic assessment of cartilage at 5 and 10 weeks. (D) Roughness parameter of the cartilage surface regeneration zone at10 weeks after treatment. (E) The AFM images of the cartilage surface regeneration zone at10 weeks after treatment (10 × 10 mm).

Fig. 6

(A) Representative H&E staining of femur cartilage at 5 and 10 weeks after treatment. Scale bars, 500 μm and 200 μm. (B)Representative Safranin O/Fast Green staining of femur cartilage at 5 and 10 weeks after treatment. Scale bars, 500 μm and 200 μm.

Fig. 7

(A)Immunohistochemical staining of Col II in rat cartilage post-treatment defects. Scale bars, 500 μm and 100 μm. (B)Immunohistochemical staining of ACAN in rat cartilage post-treatment defects. Scale bars, 500 μm and 100 μm. (C) Quantitative analysis of the Col II content. (D) Quantitative analysis of the ACAN content.

Fig. 8

(A) Blood biochemistry markers on 10 weeks. (B) The toxicity of in situ microspheres at 10 weeks (scale bars 100 μm). (C) The scheme of IVIS evaluation protocol. (D) In vivo degradation behavior analysis of RSD-MS.