Klinder Annett, Kussauer Sophie, Hiemer Bettina, Wree Andreas, Bader Rainer, Jonitz-Heincke Anika
Department of Orthopedics, Biomechanics and Implant Technology Research Laboratory, Rostock University Medical Center, 18057 Rostock, Germany.
Department of Anatomy, Rostock University Medical Center, 18057 Rostock, Germany.
J Clin Med. 2020 Aug 30;9(9):2798. doi: 10.3390/jcm9092798.
A major challenge of cell-based therapy for cartilage lesions is the preservation of the chondrogenic phenotype during ex vivo cell cultivation. In this in vitro study, the chondro-inductive capacity of two different hyaline cartilage-conditioned cell culture media on human chondrocytes in 3D spheroids was determined. Media were conditioned by incubation of 200 mg/mL vital or devitalized cartilage matrix in growth media over 35 days. The media were analyzed for the content of soluble procollagen type (Col) II and glycosaminoglycans (GAGs) as well as released TGF-β1, IGF-1 and IGFBP3. Unconditioned medium served as a negative control while the positive medium control was supplemented with TGF-β1 and IGF-1. Spheroid cultures prepared from human chondrocytes were cultivated at 37 °C, 5% CO and 21% O in the respective media and controls. After 14 and 35 days, the deposition of ECM components was evaluated by histological analysis. Vital cartilage-conditioned medium contained significantly higher levels of Col II and active TGF-β1 compared to medium conditioned with the devitalized cartilage matrix. Despite these differences, the incubation with vital as well as devitalized cartilage conditioned medium led to similar results in terms of deposition of proteoglycans and collagen type II, which was used as an indicator of re-differentiation of human chondrocytes in spheroid cultures. However, high density 3D cell cultivation showed a positive influence on re-differentiation.
基于细胞的软骨损伤治疗面临的一个主要挑战是在体外细胞培养过程中保持软骨生成表型。在这项体外研究中，测定了两种不同的透明软骨条件细胞培养基对三维球体中人类软骨细胞的软骨诱导能力。通过在生长培养基中孵育200mg/mL的活软骨或失活软骨基质35天来制备条件培养基。分析培养基中可溶性II型前胶原（Col）和糖胺聚糖（GAGs）的含量以及释放的转化生长因子-β1（TGF-β1）、胰岛素样生长因子-1（IGF-1）和胰岛素样生长因子结合蛋白3（IGFBP3）。未处理的培养基作为阴性对照而阳性培养基对照添加了TGF-β1和IGF-1。从人类软骨细胞制备的球体培养物在各自的培养基和对照中于37°C、5%二氧化碳和21%氧气条件下培养。14天和35天后，通过组织学分析评估细胞外基质成分的沉积。与用失活软骨基质制备的条件培养基相比，活软骨条件培养基中Col II和活性TGF-β1的水平显著更高。尽管存在这些差异，但用活软骨和失活软骨条件培养基孵育在蛋白聚糖和II型胶原沉积方面导致了相似的结果，蛋白聚糖和II型胶原沉积用作球体培养中人类软骨细胞再分化的指标。然而，高密度三维细胞培养对再分化显示出积极影响。
