The Analysis Of Gene Expression Profile And Identification Of Potential Markers For Chondrocyte Dedifferentiation

Objective Dedifferentiation of chondrocyte greatly restricts its function and application,however,it is poorly understood except a small number of canonical markers.The objective of this study is to explore the global alteration of gene expression during chondrocyte' 2D culture,compare the effects of different culture conditions on gene expression of chondrocytes,and then identify potential markers of chondrocyte dedifferentiation by combining with 3D culture of chondrocyte in hydrogel using high-throughput RNA sequencing(RNA-seq).Methods Morphology detection is first carried out on articular chondrocytes at primary(P0)and passage 1(P1)stages to explore the effect of monolayer culture and passage on chondrocytes.The change of chondrocyte morphology inevitably will accompany the significant change of gene expression,and therefore,RNA-seq was performed on articular chondrocytes at P0 and P1 stages to explore the global alteration of gene expression,and then the results of RNA-seq were verified by q PCR detection.The non-cell-adhesive property endows polysaccharide hydrogel with the ability to maintain chondrocyte phenotype,which is used as a platform to identify new molecular markers and potential therapeutic targets of chondrocyte dedifferentiation.P0 chondrocytes were cultured by 2D monolayer and in 3D constructs(i.e.chondrocyte-laden alginate hydrogel and HA-MA hydrogel)respectively to demonstrate the potential markers or therapeutic targets of chondrocyte dedifferentiation by q PCR detection and immunofluorescence staining.Results After monolayer passage,the morphology,the composition of extracellular matrix(ECM)and the gene expression as well as protein expression are changed dramatically in chondrocytes.Dedifferentiated chondrocyte was similar with osteoarthritis(OA)chondrocyte at the transcriptome level,including pathways such as NF-?B signaling pathway,Wnt signaling pathway and HIF-1 signaling pathway,as well as expression of genes such as KDM6 B and PFKFB3.In the early stage of chondrocyte dedifferentiation,Type II collagen(COL2)was significantly decreased,and Type I collagen(COL1)was significantly increased.Interestingly,monolayer culture without passage results in high expression of both COL1 and COL2.PFKFB3,KDM6 b and MEF2 C were significantlydown-regulated during monolayer culture and passage,WNT5 B was significantly upregulated during monolayer culture and passage.However,PFKFB3,KDM6 B,MEF2C and WNT5 B were effectively maintained when chondrocyte is cultured in hydrogel.Both alginate hydrogel and HA-MA hydrogel can effectively maintain the phenotype of chondrocytes,and alginate hydrogel is superior than that of HA-MA hydrogel.Both hydrogels had similar swelling and permeability.However,alginate hydrogel was more effectively enhance the secretion of ECM compared with HA-MA hydrogel.Conclusion This study explored the global alteration of gene expression along with chondrocyte dedifferentiation and developed the use of hydrogel as a platform to investigate chondrocyte dedifferentiation.In addition,several potential marker genes,such as PFKFB3,KDM6 B,had been identified via comparatively analyzing their expression in P0 and P1 chondrocytes as well as in 3D constructs at both m RNA and protein level.The results provided new molecular markers and potential therapeutic targets of chondrocyte dedifferentiation.