Systematic Analysis Of Cartilage Hypertrophic Layer Differentiation By Single Cell RNA-seq And Evaluation Of Tissue Engineering Repair Performance

Tissue engineering is a highly interdisciplinary field,and it requires a systematic understanding of the development and regeneration of tissues,as well as the engineering of biomaterial scaffolds and seed cells.This thesis focuses on cartilage tissue engineering,and it encompasses a systematic investigation of the molecular cascade guiding the hypertrophic development process of the growth plate,using single-cell genomics and bioinformatics.We also studied and evaluated the application potential of a novel DNA hydrogel scaffold and controllable engineered seed cells in cartilage tissue engineering.The hypertrophic development of the growth plate is critical for individual development and tissue regeneration in mammals,including humans.The growth plate generally consists of continuous cell layers sequentially undergoing hypertrophic differentiation.Here,we sequenced the transcriptomes of 217 single cells isolated from mouse growth plates,and reconstructed the hypertrophic development process using bioinformatic in silico approaches.The analysis revealed significant gene expression tendencies and the activation of critical signaling pathways during the process,both temporally and spatially,and identified hitherto unknown surface markers able to distinguish cells from different growth plate stages.We further developed an algorithm to predict the transcription factors contributing to the hypertrophic development process,which were in turn validated via in vitro screening.In the domain of tissue engineering,the thesis focuses on a comprehensive strategy encompassing a novel biomaterial scaffold and seed cell enhancement.DNA hydrogels,which show excellent biocompatibility and high permeability,are promising materials for tissue engineering applications.We comprehensively evaluated the performance of a DNA hydrogel scaffold in cartilage repair,while also integrating modules for sustained release and gene engineering to controllably enhance the cartilage seed cell performance and repair outcomes in an animal model.The results clearly demonstrate the application potential of this DNA hydrogel scaffold and engineered seed cells in cartilage tissue engineering,implying the tantalizing possibility of future clinical applications.In conclusion,this thesis deals with multiple aspects of cartilage tissue engineering,from the systematic understanding of normal cartilage development,via tissue engineering and analysis of repaired tissues using the latest single-cell genomics methods,to seed-cell optimization incorporating rational gene circuit design for the tissue engineering repair process.Taken as a whole,this work reveals how these techniques could be applied more widely,providing a roadmap for other tissue engineering scenarios.