| Cartilage is highly prone to injury,which can lead to knee joint dysfunction,significant pain,and even disability.However,mature cartilage tissue has very limited self-repairing ability due to the lack of vascular and nervous tissue.The current clinical treatment including medication,physiotherapy and surgical intervention still cannot achieve the satisfied regeneration,as the biochemical and mechanical properties of the neo-tissue are inferior to normal cartilage tissue.Thus,more effective therapeutic techniques for cartilage repair are urgently requiredIn recent years,tissue engineering technique with three key elements including stem cells,biological agents and biomaterials emerges a promising strategy for cartilage regeneration.However,there remains three key issues need to be addressed when applying tissue engineered techniques for cartilage repair:(1)improving the efficacy of stem cells therapy;(2)achieving safe and precise application of biological agents;(3)fabrication biocompatible biomaterials scaffold for cell delivery and promotion of tissue growth.Our study focused on the fabrication of engineered adipose stem cells using chemically modified m RNA(mod RNA),the fabrication of silk fibroin microsphere for cell delivery and the biocompatibility evaluation of silk firoin,aiming at providing a reliable and safe strategy for the cartilage regenerationBased on our study,we successfully constructed engineered stem cell with the ability to secrete functional insulin-like growth factor-1(IGF-1)by transfected adipose derived stem cells(ADSCs)with mod RNA in vitro and then evaluated its therapeutic effect on cartilage repair.Then we fabricated silk fibroin microsphere and assessed the biocompatibility of silk fibroin by biochemical analysis,histological analysis,whole body multi-tissue high-throughput m RNA sequencing and single-cell mass spectrometry.Finally,the therapeutic effect of the combination of silk fibroin microsphere and ADSCs transfected with IGF-1 mod RNA(IGF-1-ADSCs)was tested on a mouse cartilage defect model.We discovered that(1)mod RNA can transfect murine ADSCs with high efficiency.IGF-1-ADSCs can secrete functional growth factor IGF-1 to promote matrix synthesis of chondrocytes;The injection of IGF-1-ADSCs can reduce the loss of cartilage matrix and ameliorate the development of knee osteoarthritis(OA);(2)The silk firoin microsphere prepared by electrospray has porous structure which was suitable for cell adhesion and proliferation.The implantation of silk firoin would not cause acute renal and liver dysfunction.Compared to clinically approved polypropylene scaffold,silk fibroin scaffold showed better biocompatibility.Silk firoin induced less immune cell activation with lower proportions of macrophages,monocytes,and neutrophils in the innate system and lower proportions of CD4+ T cells and CD8+ T cells in the adaptive system.Genes(CD115 and CD27)involved in the immune cell activation and migration were also downregulated in the silk fibroin group.Histological analysis confirmed that silk fibroin scaffold implantation led to decreased neutrophils and macrophages recruitment and less fibrosis formation;(3)The combination of silk fibroin microsphere enhanced the therapeutic effect of IGF-1-ADSCs for cartilage injury.Overall,this study combined stem cell therapy technique and gene therapy technique to fabricate engineered cells.To the best of our knowledge,it was the first time for the application of mod RNA technology in cartilage repair.In addition,this study proposed a novel strategy for biocompatibility analysis of biomaterials by high-throughput methods including multi-tissue m RNA sequencing and single-cell mass cytometry.What's more,the combination of IGF-1-ADSCs and silk fibroin microsphere could be a promising strategy for cartilage repair. |
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