Three-dimensional (3D) hydrogel serves as a platform to identify potential markers of chondrocyte dedifferentiation by combining RNA sequencing

Abstract

Dedifferentiation of chondrocyte greatly restricts its function and application, however, it is poorly understood except a small number of canonical markers. The non-cell-adhesive property endows polysaccharide hydrogel with the ability to maintain chondrocyte phenotype, which can serve as a platform to identify new molecular markers and therapeutic targets of chondrocyte dedifferentiation. In this study, the high-throughput RNA sequencing (RNA-seq) was first performed on articular chondrocytes at primary (P0) and passage 1 (P1) stages to explore the global alteration of gene expression along with chondrocyte dedifferentiation. Significantly, several potential marker genes, such as PFKFB3, KDM6B, had been identified via comparatively analyzing their expression in P0 and P1 chondrocytes as well as in 3D constructs (i.e. chondrocyte-laden alginate hydrogel and HA-MA hydrogel) at both mRNA and protein level. Besides, the changes in cellular morphology and enriched pathway of differentially expressed genes during chondrocyte dedifferentiation was studied in detail. This study developed the use of hydrogel as a platform to investigate chondrocyte dedifferentiation; the results provided new molecular markers and potential therapeutic targets of chondrocyte dedifferentiation.

Keywords: Chondrocyte dedifferentiation; Gene expression; Hydrogel; RNA sequencing.

Graphical abstract

Fig. 1

A) Schematic illustration of experimental procedures. B) Heatmap of typical DEGs associated with chondrocyte dedifferentiation. C) qPCR analysis for expression of representative genes in the chondrocytes at P0 and P1, in HA-MA gel and alginate gel constructs as well as by 5 days of monolayer culture, respectively; * indicated p < 0.05, ** indicated p < 0.005, *** indicated p < 0.0005, **** indicated p < 0.0001.

Fig. 2

A) P0 and P1 chondrocyte observation under bright-field microscopy or fluorescence microscopy after staining with CMFDA and Phalloidin, respectively. In Phalloidin staining, the nucleus was counterstained with DAPI (blue). B) Live/Dead staining of chondrocytes encapsulated in alginate and HA-MA constructs. C) Equilibrium swelling ratio of alginate gel and HA-MA gel, respectively. Magnification was 100x. Scale bars were 200 μm ** indicated p < 0.005.

Fig. 3

A) Number of differentially expressed genes (DEGs), including up-regulated (UP) and down-regulated (DOWN) gens in P1 cells compared to P0 cells. B) Correlation analysis between P0 and P1 samples. C) Volcano plot of gene expression profile in P1 cells versus P0 cells; up, nosig, and down indicates up-regulated genes, non-significantly differentially expressed genes, and down-regulated genes, respectively. D) Heatmap of DEGs in P0 and P1 cells.

Fig. 4

A) Gene Ontology (GO) enrichment analysis of the down-regulated genes in P1 cells. B) GO enrichment analysis of the up-regulated genes in P1 cells. C) KEGG pathway analysis of down-regulated genes in P1 cells. D) KEGG pathway analysis of up-regulated genes in P1 cells.

Fig. 5

A) Heatmap of chondrocyte-related DEGs between P0 cells and P1 cells. B) Heatmap of OA-related DEGs between P0 cells and P1 cells. C) KEGG pathway analysis of chondrocyte-related DEGs between P0 and P1.

Fig. 6

qPCR analysis for representative gene expressions in P0 and P1 chondrocytes; * indicated p < 0.05, ** indicated p < 0.005, *** indicated p < 0.0005, and **** indicated p < 0.0001.

Fig. 7

Immunofluorescence staining of cartilage tissue, P0 cells, P1 cells, alginate constructs, and HA-MA constructs for COL2, COL1, PFKFB3, and KDM6B, respectively. The nucleus was counterstained with DAPI (blue). Magnification was 100x. Scale bars were 200 μm.

Fig. 8

qPCR analysis for expression of representative genes in P0 and monolayer cultured chondrocytes as well as HA-MA gel and alginate gel constructs, respectively. * indicated p < 0.05, ** indicated p < 0.005, *** indicated p < 0.0005, and **** indicated p < 0.0001.