OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model
OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model
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DOI：
10.14670/hh-26.1265
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发表时间：
2011-10-01
影响因子：
2
通讯作者：
Elisseeff, Jennifer
中科院分区：
文献类型：
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作者：
Musumeci, Giuseppe;Loreto, Carla;Elisseeff, Jennifer
骨关节炎是以软骨磨损、软骨下骨改建、骨赘形成和滑膜炎症为特征的疾病。炎症、氧化应激、线粒体功能障碍和促凋亡/抗凋亡失调所引起的动态平衡紊乱被认为损害关节微环境中软骨细胞的存活，并参与了骨关节炎的发病。然而，软骨细胞程序性死亡(凋亡)的分子机制尚不清楚。本研究旨在探讨骨性关节炎患者膝关节软骨细胞的凋亡情况。本研究的目的是观察和比较caspase-3在组织外植体、单层培养的细胞和聚乙二醇二丙烯酸酯(PEGDA)支架包裹的细胞中的表达对细胞凋亡的影响。具体地说，关节软骨标本通过组织学(苏木素和曙红)和组织化学(藏红素-O和阿尔新蓝)进行评估。采用免疫组织化学、免疫细胞化学和免疫荧光等方法对细胞凋亡效应因子caspase-3进行研究。用原位末端标记法检测新鲜分离的人骨关节炎软骨细胞的DNA链断裂，并用Hoechst 33258染色检测凋亡细胞的核形态变化。结果显示，培养5周后，caspase-3在组织块、融合前细胞和培养4代后表达增加，而在水凝胶包裹的软骨细胞(PEGDA)中caspase-3表达下降，新鲜分离的软骨细胞呈TUNEL阳性。软骨细胞在水凝胶中培养5周后形成新的透明软骨，细胞生长、细胞聚集和细胞外基质(ECM)生成增加。这与在患者的治疗方法中使用骨关节炎细胞和组织工程学特别相关。
Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold.Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture.The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients.