Chondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in hypoxia-inducible factor 1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. Primary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4–16 mL/10 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and C-metabolic flux analysis (C-MFA). Increasing media volumes resulted in higher accumulation of cartilaginous ECM (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. C-MFA utilizing [U-C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the tricarboxylic acid (TCA) cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-C] glutamine tracing supported this notion. Elevated media availability upregulates ECM synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in ECM accumulation.

中文翻译：

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提高营养利用率可增强组织工程软骨中的软骨细胞代谢和生物合成

软骨细胞通常依赖于无氧代谢，当处于引发混合有氧-无氧代谢的条件时，其生物合成活性上调。之前，我们观察到增加培养基体积会导致从无氧代谢转变为混合有氧-无氧代谢。由于缺氧诱导因子 1α 信号传导和相关缺氧基因表达的变化，在此转变期间发生了最大细胞外基质 (ECM) 积累。本研究旨在探讨进一步增加介质可用性对 ECM 合成和软骨细胞代谢的影响。原代牛软骨细胞在不同水平的培养基可用性（4-16 mL/10 个细胞）下在 3D 高密度组织培养物中生长。通过生化测定和 C-代谢通量分析 (C-MFA) 确定 ECM 积累和代谢的变化。增加培养基体积导致软骨 ECM（胶原蛋白和蛋白聚糖）和细胞结构的积累增加。细胞外代谢物测量表明，培养基可用性的提高导致葡萄糖和谷氨酰胺代谢增加，同时无氧活动增加。利用 [UC] 葡萄糖的 C-MFA 表明，增加的培养基可用性显着影响中心碳代谢，上调所有葡萄糖相关代谢途径（糖酵解、乳酸发酵、三羧酸 (TCA) 循环、己糖胺生物合成途径和苹果酸-天冬氨酸穿梭） ）。此外，C-MFA 表明谷氨酰胺向 TCA 循环提供碳，涉及 [UC] 谷氨酰胺追踪的其他研究也支持了这一观点。培养基可用性的提高会上调 ECM 合成并导致代谢表型发生显着变化。谷氨酰胺在软骨细胞代谢中发挥重要作用，谷氨酰胺代谢的增加与 ECM 积累的增加相关。