Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue-derived mesenchymal stromal cells (MSCs) and composite 3D collagen-nano-hydroxyapatite (nanoHA) scaffolds. Xeno- and DMSO-free cryopreserved MSCs were perfusion-seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post-thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen-nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell-free collagen-nanoHA scaffolds. The established combination of xeno-free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.

中文翻译：

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使用冷冻保存的间充质基质细胞和大孔胶原-纳米羟基磷灰石冷冻凝胶生成骨移植物

骨组织工程策略涉及 3D 支架和促进受损区域替换的适当细胞类型。在这项工作中，我们旨在使用冷冻保存的脂肪组织衍生的间充质基质细胞 (MSCs) 和复合 3D 胶原-纳米-羟基磷灰石 (nanoHA) 支架，开发一种快速可靠的临床相关方案，用于工程可行的骨移植物。将不含异种物质和 DMSO 的冷冻保存的 MSC 灌注接种到通过冷冻凝胶制备的仿生胶原蛋白/纳米 HA 支架中，并在体外和体内评估其骨再生潜力。在解冻后培养的 7 天内，冷冻保存的 MSC 保留了均匀地重新填充整个支架体积的能力。此外，该支架为诱导细胞成骨分化提供了合适的微环境，碱性磷酸酶活性和矿化作用证实。与无细胞胶原-纳米HA支架相比，在免疫抑制大鼠中，植入冷冻保存的MSCs的胶原-纳米HA冷冻凝胶加速了编织骨组织的形成、骨小梁的成熟和股骨缺损的血管化。已建立的无异种细胞培养和冷冻保存技术与适当的支架设计和细胞再增殖方法相结合，加速了可行骨移植物的产生。