Exosomes exhibit great therapeutic potential in bone tissue engineering. The study aimed to investigate whether the exosomes derived from human adipose-derived stem cells (hADSCs-Exos) during different time-span of osteogenic differentiation could promote osteogenesis. The appropriate concentrations of hADSCs-Exos to enhance the proliferation, migration and osteogenesis of hADSCs-Exos were also examined. PKH67 labelled hADSCs-Exos was used to detect the internalization ability of hADSCs. The osteogenic differentiation abilities of hADSCs after treatment with hADSCs-Exos was evaluated by Alizarin red staining (ARS). The proliferation and migration of hADSCs was examined by cell counting kit-8 and wound healing assay, respectively. The expression of exosomal surface markers and osteoblast-related protein of hADSCs was assessed by Western blot. PKH67-labelled exosomes were internalized by hADSCs after 4 h incubation. ARS showed that the amount of mineralized nodules in Exo^1−14d group was significantly higher than that in Exo^15−28d group. hADSCs-Exos could promote the proliferation and migration capacity of hADSCs. Western blot analysis showed that after hADSCs-Exos treatment, ALP and RUNX2 were significantly enhanced. Specially, the Exo^1−14d group of 15 μg/mL significantly upregulated the expression of RUNX2 than the other exosomes treated groups. Our findings suggest that exosomes secreted by hADSCs during osteogenic induction for 1–14 days could be efficiently internalized by hADSCs and could induce osteogenic differentiation of hADSCs. Moreover, administration of Exo^1−14d at 15 μg/mL promoted the proliferation and migration of hADSCs. In conclusion, our research confirmed that comprised of hADSCs-Exos and hADSCs may provide a new therapeutic paradigm for bone tissue engineering.

外泌体在骨组织工程中表现出巨大的治疗潜力。该研究旨在研究在不同的成骨分化时间跨度期间源自人类脂肪干细胞（hADSCs-Exos）的外泌体是否可以促进成骨。还检查了适当浓度的 hADSCs-Exos，以增强 hADSCs-Exos 的增殖、迁移和成骨。PKH67标记的hADSCs-Exos用于检测hADSCs的内化能力。通过茜素红染色 (ARS) 评估 hADSCs-Exos 处理后 hADSCs 的成骨分化能力。hADSCs 的增殖和迁移分别通过细胞计数试剂盒 8 和伤口愈合试验进行检测。通过蛋白质印迹评估外泌体表面标志物和 hADSCs 的成骨细胞相关蛋白的表达。PKH67 标记的外泌体在孵育 4 小时后被 hADSC 内化。ARS 显示 Exo 中矿化结核的数量^1-14d组显着高于Exo ^15-28d组。hADSCs-Exos 可以促进 hADSCs 的增殖和迁移能力。Western印迹分析显示，hADSCs-Exos处理后，ALP和RUNX2显着增强。特别是，与其他外泌体处理组相比，15 μg/mL的 Exo ^1-14d组显着上调了 RUNX2 的表达。我们的研究结果表明，在 1-14 天的成骨诱导过程中，hADSCs 分泌的外泌体可以被 hADSCs 有效内化，并可以诱导 hADSCs 的成骨分化。此外，Exo ^{1−14d 的管理}15 μg/mL 促进 hADSCs 的增殖和迁移。总之，我们的研究证实，由 hADSCs-Exos 和 hADSCs 组成可能为骨组织工程提供新的治疗范式。