Department of Trauma Surgery, Regensburg University Medical Center, Franz-Josef-Strauss-Allee 11, 93042, Regensburg, Germany.
Int Orthop. 2013 May;37(5):945-51. doi: 10.1007/s00264-013-1800-1. Epub 2013 Jan 31.
Mesenchymal stem cells (MSCs) express markers of hypertrophic chondrocytes during chondrogenic differentiation. We tested the suitability of parathyroid hormone-related protein (PTHrP), a regulator of chondrocyte hypertrophy in embryonic cartilage development, for the suppression of hypertrophy in an in vitro hypertrophy model of chondrifying MSCs.
Chondrogenesis was induced in human MSCs in pellet culture for two weeks and for an additional two weeks cultures were either maintained in standard chondrogenic medium or transferred to a hypertrophy-enhancing medium. PTHrP(1-40) was added to the medium throughout the culture period at concentrations from 1 to 1,000 pM. Pellets were harvested on days one, 14 and 28 for biochemical and histological analysis.
Hypertrophic medium clearly enhanced the hypertrophic phenotype, with increased cell size, and strong alkaline phosphatase (ALP) and type X collagen staining. In chondrogenic medium, 1-100 pM PTHrP(1-40) did not inhibit chondrogenic differentiation, whereas 1,000 pM PTHrP(1-40) significantly reduced chondrogenesis. ALP activity was dose-dependently reduced by PTHrP(1-40) at 10-1,000 pM in chondrogenic conditions. Under hypertrophy-enhancing conditions, PTHrP(1-40) did not inhibit the induction of the hypertrophy. At the highest concentration (1,000 pM) in the hypertrophic group, aggregates were partially dedifferentiated and differentiated areas of these aggregates maintained their hypertrophic appearance.
PTHrP(1-40) treatment dose-dependently reduced ALP expression in MSC pellets cultured under standard chondrogenic conditions and is thus beneficial for the maintenance of the chondrogenic phenotype in this medium condition. When cultured under hypertrophy-enhancing conditions, PTHrP(1-40) could not diminish the induced enhancement of hypertrophy in the MSC pellets.
间充质干细胞(MSCs)在软骨分化过程中表达肥大软骨细胞的标志物。我们测试了甲状旁腺激素相关蛋白(PTHrP)的适用性,它是胚胎软骨发育中软骨细胞肥大的调节剂,用于抑制软骨形成的 MSC 体外肥大模型中的肥大。
在微球培养中诱导人 MSC 软骨分化两周,然后再培养两周,培养基分别为标准软骨形成培养基或增强肥大培养基。在整个培养过程中,将 PTHrP(1-40)添加到培养基中,浓度为 1 至 1000 pM。在第 1、14 和 28 天收获微球进行生化和组织学分析。
肥大培养基明显增强了肥大表型,细胞体积增大,碱性磷酸酶(ALP)和 X 型胶原染色强烈。在软骨形成培养基中,1-100 pM PTHrP(1-40) 不会抑制软骨形成,而 1000 pM PTHrP(1-40) 则显著降低了软骨形成。在软骨形成条件下,10-1000 pM PTHrP(1-40) 剂量依赖性地降低了 ALP 活性。在增强肥大的条件下,PTHrP(1-40) 没有抑制肥大的诱导。在肥大组的最高浓度(1000 pM)下,聚集体部分去分化,这些聚集体的分化区域保持其肥大外观。
PTHrP(1-40)处理剂量依赖性地降低了在标准软骨形成条件下培养的 MSC 微球中的 ALP 表达,因此有利于维持该培养基条件下的软骨形成表型。在增强肥大的条件下,PTHrP(1-40) 不能减少 MSC 微球中诱导的肥大增强。