Chen Guo, Deng Shaohui, Zuo Mingxiang, Wang Jin, Cheng Du, Chen Bin
Key Laboratory for Polymeric Composite & Functional Materials of Ministry of Education, School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou, 510275, PR China.
Division of Orthopaedics and Traumatology, Department of Orthopaedics, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, PR China.
Mater Today Bio. 2022 Jul 11;16:100356. doi: 10.1016/j.mtbio.2022.100356. eCollection 2022 Dec.
Healing of large calvarial bone defects remains challenge but may be improved by stimulating bone regeneration of implanted cells. The aim of this study is to specially co-activate transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF-A) genes expressions in pre-osteoblast MC3T3-E1 cells through the non-viral CRISPR activation (CRISPRa) system to promote osteogenesis. A cationic copolymer carrying nucleus localizing peptides and proton sponge groups dimethyl-histidine was synthesized to deliver CRISPRa system into MC3T3-E1 cells with high cellular uptake, lysosomal escape, and nuclear translocation, which activated VEGF-A and TGF-β1 genes expressions and thereby additively or synergistically induced several osteogenic genes expressions. A tunable dual-crosslinked hydrogel was developed to implant the above engineered cells into mice calvaria bone defect site to promote bone healing . The combination of multi-genes activation through non-viral CRISPRa system and tunable dual-crosslinked hydrogel provides a versatile strategy for promoting bone healing with synergistic effect.
大型颅骨骨缺损的愈合仍然是一项挑战，但通过刺激植入细胞的骨再生可能会得到改善。本研究的目的是通过非病毒CRISPR激活（CRISPRa）系统在成骨前体细胞MC3T3-E1中特异性地共同激活转化生长因子β1（TGF-β1）和血管内皮生长因子（VEGF-A）基因的表达，以促进骨生成。合成了一种带有核定位肽和质子海绵基团二甲基组氨酸的阳离子共聚物，将CRISPRa系统递送至MC3T3-E1细胞，实现高细胞摄取、溶酶体逃逸和核转位，从而激活VEGF-A和TGF-β1基因的表达，并进而累加或协同诱导多种成骨基因的表达。研发了一种可调节的双交联水凝胶，将上述工程细胞植入小鼠颅骨骨缺损部位以促进骨愈合。通过非病毒CRISPRa系统激活多基因与可调节双交联水凝胶相结合，为促进骨愈合提供了一种具有协同效应的通用策略。
