The classic bone tissue engineering model for bone regeneration combines three elements: scaffolds, biomaterials, and mesenchymal stem cells (MSCs). Incorporation of MSCs and growth factors into a scaffold implanted into the area of bone injury is a proven strategy to achieve successful bone regeneration as demonstrated in the literature. However, a major limitation of using bone grafts or scaffolds is oxygen (O₂) deprivation in the inner sections of the construct, due to lack of adequate vascularization. To address this limitation, we proposed two treatment strategies for MSC-seeded constructs or adipose tissue scaffolds before implantation: (1) O₂ enrichment and (2) acclimation to hypoxia. Based on previous studies, the significance of the different O₂ concentrations on MSC biological characteristics remains controversial. Therefore, the optimal O₂ condition for engineered bone tissues should be determined. Thus, we designed an innovative multichamber gas system aimed to simultaneously assess the effects of different O₂ levels on cell culture. This system was assembled using three isolated chambers integrated into a single incubator. To explore the efficacy of our method, we investigated the effect of hyperoxia, normoxia, and hypoxia, (50–60%, 21%, and 5–7.5% O₂, respectively) on the biological characteristics of human adipose-derived MSCs: immunophenotyping, adhesion, proliferation, and osteogenic, and angiogenic differentiation. Our findings demonstrated that hypoxic adipose-derived mesenchymal stem cells (ASCs) conditions exhibited significantly lower levels of CD34 (p = 0.014), with significantly higher osteogenic and angiogenic differentiation capacities (p = 0.023 and p = 0.0042, respectively) than normoxia. Conversely, hyperoxia-cultured ASCs demonstrated significantly higher levels of CD73 and CD90 expression than both normoxic ASCs (p = 0.006 and p = 0.025, respectively) and hypoxic ASCs (p = 0.003 and p = 0.003, respectively). In addition, hyperoxic ASCs showed significantly reduced proliferation capacity by day 11 (p = 0.032) and significantly enhanced migration rates after 48 h (p = 0.044). The newly developed controllable multichamber gas system was cost-effective and easy to use. Different assays can be performed concurrently while preserving all other conditions identical, and the use of other ranges of O₂ concentrations is feasible and also necessary to determine the ideal O₂ concentration. Furthermore, the multichamber gas system has the potential for wide application, including other cell cultures, grafts, or scaffolds for in vitro and in vivo experimentation. This study was approved by the Galilee Medical Center Helsinki Committee (No. 0009-19-NHR).

中文翻译：

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用于检查多种氧气条件对细胞培养的影响的多室气体系统

用于骨再生的经典骨组织工程模型结合了三个要素：支架、生物材料和间充质干细胞 (MSC)。如文献所示，将 MSCs 和生长因子纳入植入骨损伤区域的支架是实现成功骨再生的一种行之有效的策略。然而，由于缺乏足够的血管化，使用骨移植物或支架的主要限制是结构内部的氧气 (O ₂ ) 剥夺。为了解决这一限制，我们在植入前为 MSC 种子构建体或脂肪组织支架提出了两种治疗策略：（1）O ₂富集和（2）适应缺氧。根据以往的研究，不同 O ₂MSC 生物学特性的浓度仍然存在争议。因此，应确定工程骨组织的最佳 O ₂条件。因此，我们设计了一种创新的多室气体系统，旨在同时评估不同 O ₂水平对细胞培养的影响。该系统使用集成到单个培养箱中的三个隔离室组装而成。为了探索我们方法的有效性，我们研究了高氧、常氧和缺氧（50-60%、21% 和 5-7.5% O ₂，分别）关于人脂肪来源的 MSC 的生物学特性：免疫表型、粘附、增殖、成骨和血管生成分化。我们的研究结果表明 ，与 常氧相比，低氧脂肪源性间充质干细胞 (ASC) 条件下的 CD34 水平显着降低 ( p = 0.014)，具有显着更高的成骨和血管生成分化能力（分别为p  = 0.023 和p = 0.0042）。相反，高氧培养的 ASC 的 CD73 和 CD90 表达水平显着高于常氧 ASC（分别为p  = 0.006 和p  = 0.025）和缺氧 ASC（p  = 0.003 和p = 0.003，分别）。此外，高氧 ASC 在第 11 天显示出显着降低的增殖能力 ( p  = 0.032)，并在 48 小时后显着提高迁移率 ( p  = 0.044)。新开发的可控多室气体系统具有成本效益且易于使用。不同的检测可以同时进行，同时保持所有其他条件相同，使用其他范围的 O ₂浓度是可行的，也是确定理想的 O ₂浓度所必需的。此外，多室气体系统具有广泛应用的潜力，包括用于体外和体内的其他细胞培养物、移植物或支架实验。这项研究得到了赫尔辛基加利利医学中心委员会 (No. 0009-19-NHR) 的批准。