Crosbie Michelina, Vanderboom Kailey, Souccar-Young Jamie, Pearson Wendy
Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada.
Methods Protoc. 2025 Aug 6;8(4):91. doi: 10.3390/mps8040091.
In vitro cartilage explant culture has been used to assess nutraceuticals on cartilage responses to inflammatory stimuli. However, applying extracts of nutraceuticals directly to cartilage explants does not account for effects of digestion and hepatic biotransformation, or selective exclusion of product metabolites from joint fluid by the synovial membrane. The current study produced a simulated biological extract of a common nutraceutical (glucosamine; G) by exposing it to a simulated upper gastrointestinal tract digestion, hepatic biotransformation by liver microsomes, and purification to a molecular weight cut-off of 50 kDa. This extract was then used to condition cartilage explants cultured for 120 h in the presence or absence of an inflammatory stimulus (lipopolysaccharide). Media samples were analyzed for prostaglandin E (PGE), glycosaminoglycan (GAG), and nitric oxide (NO). Tissue was digested and analyzed for GAG content and stained for viability. Conditioning of explants with G significantly reduced media GAG in stimulated and unstimulated explants and reduced nitric oxide production in unstimulated explants. These data provide evidence for the value of glucosamine in protecting cartilage from deterioration following an inflammatory challenge, and the model improves applicability of these in vitro data to the in vivo setting.
体外软骨外植体培养已被用于评估营养保健品对软骨炎症刺激反应的影响。然而，将营养保健品提取物直接应用于软骨外植体并不能说明消化和肝脏生物转化的影响，也不能说明滑膜对关节液中产品代谢物的选择性排除。本研究通过将一种常见的营养保健品（葡萄糖胺；G）暴露于模拟的上消化道消化、肝微粒体的肝脏生物转化以及纯化至截留分子量为50 kDa，制备了一种模拟生物提取物。然后，将该提取物用于在有或无炎症刺激（脂多糖）的情况下培养120小时的软骨外植体的预处理。分析培养基样本中的前列腺素E（PGE）、糖胺聚糖（GAG）和一氧化氮（NO）。消化组织并分析GAG含量，并进行活力染色。用G预处理外植体显著降低了刺激和未刺激外植体培养基中的GAG，并降低了未刺激外植体中的一氧化氮产生。这些数据为葡萄糖胺在保护软骨免受炎症挑战后恶化方面的价值提供了证据，并且该模型提高了这些体外数据在体内环境中的适用性。
