A Transcriptome Sequencing Study On Genome-wide Gene Expression Differences Of 3d Cultured Chondrocytes In Hydrogel Scaffolds With Different Gel Density

ObjectiveTo explore the changes of chondrocyte transcriptome level in chondrocyte-alginate constructsMethodsChondrocytes were encapsulated in hydrogels made of 0.8% and 4% sodium alginate solutions,respectively,to make cell-gel constructs and cultured for a period of time.The physical properties,such as cross-linking density,swelling rate and modulus,of the two gel materials were characterized,and then the difference of chondrocyte transcriptome was detected and analyzed by RNA sequencing technology under two culture conditions.Finally,the sequencing results were verified by q PCR and immunohistochemistry.ResultsWe found that 0.8% constructs exhibited lower cross-linking density,higher swelling rate as well as higher nutrient exchange permeability compared to 4% constructs.The differences in chondrocyte transcriptome were detected from these two culture conditions by RNA sequencing,and it was found that the differentially expressed genes(DEGs)are reflected in extracellular matrix(ECM)secretion,cell cycle,cell proliferation,cartilage development,etc.It is worth noting that the expression of DEGs related to cartilage ECM and cell proliferation was up-regulated in 0.8% constructs,while the expression of DEGs involved in cell cycle and matrix degradation was up-regulated in 4% constructs;Moreover,the expressions of chondrocyte hypertrophy markers are upregulated in the 0.8% constructs;while 4% constructs seemingly favor the long-term maintenance of chondrocyte phenotype.Besides,the chondrocyte transcriptome is also remarkably affected by the length of time in tissue culture.These results have also been verified by q PCR and immunohistochemistry.ConclusionIn cartilage tissue engineering,gels of different density lead to different physical properties,which will create different microenvironment for chondrocytes,thereby alter the transcriptional level of chondrocytes.These alterations are reflected in cell proliferation,matrix secretion,cell phenotype maintenance and so on.