Key Laboratory of Animal Breeding, Reproduction and Molecular Design for Jiangsu Provience, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China; College of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt.
College of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt.
Int J Biochem Cell Biol. 2019 Jan;106:84-95. doi: 10.1016/j.biocel.2018.11.008. Epub 2018 Nov 16.
Large bone defects and bone loss after fractures remain significant challenges for orthopedic surgeons. Our study aims to find an available, applicable and biological treatment for bone regeneration overcoming the limitations in ESC/iPSC technology. We directly reprogrammed the mouse embryonic fibroblast (MEF) into osteoblast cells using different combinations of Yamanaka factors with human lim mineralization protein-3 (hLMP-3). LMP is an intracellular LIM-domain protein acting as an effective positive regulator of the osteoblast differentiation. After transduction, cells were cultured in osteogenic medium, and then examined for osteoblast formation. The expression of osteogenic markers (BMP2, Runx2 and Osterix) during reprogramming and in vitro mineralization assay revealed that the best reprogramming cocktail was (c-Myc - Oct4) with hLMP-3. In addition, both immunofluorescent staining and western blot analysis confirmed that osteocalcin (OCN) expression increased in the cells treated with the c-Myc/Oct4/hLMP3 cocktail than using hLMP-3 alone. Furthermore, this reprogramming cocktail showed efficient healing in an induced femoral bone defect in rat animal model one month after transplantation. In the present study, we reported for the first time the effect of combining Yamanaka factors with hLMP-3 to induce osteoblast cells from MEF both in vitro and in vivo.
骨折后大骨缺损和骨丢失仍然是骨科医生面临的重大挑战。我们的研究旨在寻找一种可用的、适用的和生物性的治疗方法，以促进骨再生，克服 ESC/iPSC 技术的局限性。我们使用不同组合的 Yamanaka 因子和人 LIM 矿化蛋白-3（hLMP-3）直接将小鼠胚胎成纤维细胞（MEF）重编程为成骨细胞。LMP 是一种细胞内 LIM 结构域蛋白，作为成骨细胞分化的有效正调节剂。转导后，将细胞在成骨培养基中培养，然后检查成骨细胞的形成。在重编程和体外矿化试验过程中，成骨标志物（BMP2、Runx2 和 Osterix）的表达表明最佳重编程混合物是（c-Myc-Oct4）与 hLMP-3 组合。此外，免疫荧光染色和 Western blot 分析均证实，与单独使用 hLMP-3 相比，c-Myc/Oct4/hLMP3 鸡尾酒处理的细胞中骨钙素（OCN）的表达增加。此外，该重编程混合物在移植后一个月的大鼠诱导性股骨骨缺损动物模型中显示出有效的愈合作用。在本研究中，我们首次报道了将 Yamanaka 因子与 hLMP-3 结合用于体外和体内诱导 MEF 成骨细胞的效果。
