Departments of Orthopaedic Surgery and Clinical Research, Center of Regenerative Medicine for Skeletal Tissues, University of Bern, Bern, Switzerland.
PLoS One. 2013;8(1):e53086. doi: 10.1371/journal.pone.0053086. Epub 2013 Jan 3.
Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated.
METHODOLOGY/PRINCIPAL FINDINGS: Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume.
CONCLUSIONS/SIGNIFICANCE: TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.
滑膜外植体为关节软骨修复提供了原位间充质干细胞群体。虽然骨形态发生蛋白 2（BMP-2）诱导牛滑膜外植体的软骨形成，但形成的软骨既不均匀分布，也不是纯透明软骨类型。此外，软骨细胞的下游分化进展到终末肥大阶段，这与不可避免的基质矿化密切相关。为了优化 BMP-2 诱导的软骨形成，研究了成纤维细胞生长因子 2（FGF-2）和转化生长因子 β1（TGF-ß1）的调节作用。
方法/主要发现：牛犊掌指关节滑膜外植体暴露于 BMP-2（200ng/ml）4 或 6 周。在孵育开始时引入 FGF-2（10ng/ml）或 TGF-ß1（10ng/ml），单独存在于培养的第一周或整个培养过程中。只有当 FGF-2 仅在培养的第一周存在时，才能增强 BMP-2 诱导的糖胺聚糖（GAG）染色的增加。TGF-ß1 不仅增强了 BMP-2 诱导的变色（比 FGF-2 更明显），而且还增强了 GAG 的生化测定积累，当它存在于整个培养过程中；此外，它还阻止了细胞的下游分化进入早期肥大阶段。这些发现得到了关键软骨标志物的基因和蛋白表达水平分析以及单个细胞体积估计的证实。
结论/意义：TGF-ß1 增强了牛滑膜外植体中 BMP-2 诱导的软骨形成，改善了新软骨的透明软骨样特性，并阻止了细胞的下游分化进入早期肥大阶段。在临床修复的背景下，工程成熟的真正关节类型软骨的前景下，我们的发现对于微调滑膜外植体最佳软骨分化的刺激方案将具有重要意义。
