Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes.
Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes.
复制标题
扩增和再分化条件对培养软骨细胞的软骨形成能力的影响。
作者：
K. G. A. Yang;D. Saris;R. E. Geuze;Y. J. Helm;M. V. Rijen;A. Verbout;W. Dhert;L. Creemers
在各种体外模型中研究了基于分离和培养扩增的软骨细胞的软骨再生，但合成的组织的形态质量各不相同。本研究的目的是通过比较两种扩增和再分化方法，探讨扩增和再分化条件对最终组织形态的影响程度。来自 9 名人类供体的软骨细胞在不添加生长因子的培养基（基本扩增条件 [BEC]）或添加碱性成纤维细胞生长因子 (bFGF) 的培养基（添加生长因子的扩增条件 [GFSEC]）中扩增。扩增后，细胞在沉淀培养物中再分化或接种在 II 型胶原蛋白包被的滤膜上。通过半定量实时聚合酶链反应 (PCR) 测量的 I 型和 II 型胶原以及 Sox-5、-6 和 9 的扩增后 mRNA 水平表明，与 BEC 相比，GFSEC 中的扩增会导致去分化增加。然而，再分化培养 28 天后，与 BEC 相比，GFSEC 扩增的软骨细胞合成的组织形态在伯尔尼量表上的得分显着高于 BEC（颗粒培养中为 6.4 +/- 0.3 分 vs. 4.5 +/- 0.3 分，胶原蛋白包被过滤器上为 6.0 +/- 0.4 分 vs. 4.5 +/- 0.3 分；p < 0.05）。与 BEC 相比，GFSEC 中的扩增增加了第 9 天的蛋白多糖 (PG) 合成率（沉淀培养物中为 4.0 倍，胶原蛋白包被过滤器上为 1.9 倍；p < 0.01）、PG 释放（沉淀物培养物中为 6.7 倍，胶原蛋白包被过滤器上为 3.2 倍；p < 0.001），以及第 28 天时的最终 PG 含量（沉淀物培养物中为 1.6 倍，p < 0.001）。胶原涂层过滤器上的 1.5 倍；p < 0.05）。与沉淀培养相比，胶原包被滤膜上的再分化增加了第 9 天的 PG 合成率（BEC 扩增的软骨细胞为 5.2 倍，GFSEC 扩增的软骨细胞为 2.6 倍；p < 0.01），PG 释放（BEC 扩增的软骨细胞为 4.2 倍，GFSEC 扩增的软骨细胞为 3.1 倍；p < 0.01）和最终 PG 含量（BEC 扩增软骨细胞中的 1.3 倍，GFSEC 扩增软骨细胞中的 1.9 倍；p < 0.01）。此外，通过电子显微镜观察，在过滤器上培养的软骨细胞和细胞外基质的组织与透明软骨中发现的更相似。总之，GFSEC 中的软骨细胞扩增和胶原涂层过滤器上的再分化导致了最佳的软骨形成。
Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.
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影响因子：
2.9
作者：
KIM, YJ;SAH, RLY;GRODZINSKY, AJ
通讯作者：
GRODZINSKY, AJ
影响因子：
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作者：
S. Trippel
通讯作者：
S. Trippel
影响因子：
--
作者：
Mandl, EW;van der Veen, SW;van Osch, GJVM
通讯作者：
van Osch, GJVM
影响因子：
--
作者：
Mandl, EW;Van der Veen, SW;Van Osch, GJVM
通讯作者：
Van Osch, GJVM