Department of Orthopaedic Surgery, CAPHRI School for Public Health and Primary Care, Maastricht University Medical Center, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
Osteoarthritis Cartilage. 2012 Oct;20(10):1170-8. doi: 10.1016/j.joca.2012.06.016. Epub 2012 Jul 10.
Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation.
Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation.
3D cultures specifically expressed chondrogenic mRNAs [collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures.
For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.
三维（3D）培养广泛用于软骨细胞的再分化。然而，选择 3D 而非二维（2D）培养的基本原理尚未得到系统研究，主要基于 mRNA 表达和糖胺聚糖（GAG）含量。本研究的目的是通过研究 mRNA 表达、蛋白表达、GAG 含量和细胞增殖，确定人关节软骨细胞（HAC）在单层、藻酸盐珠和微球培养中的差异再分化特征。
来自六个人的去分化 HAC 在相同的培养基条件下，分别在单层、藻酸盐珠或微球培养中进行 7 天的再分化。检测参数包括软骨形成和肥大 mRNA 和蛋白的表达、GAG 含量和细胞增殖。
3D 培养物特异性表达软骨形成 mRNAs [胶原类型 II（COL2A1）、性别决定区 Y 盒 9（SOX9）、聚集蛋白聚糖（ACAN）]，而 2D 培养物则不表达。肥大 mRNAs（胶原类型 X（COL10A1）、 runt 相关转录因子 2（RUNX2）、基质金属蛋白酶 13（MMP13）、血管内皮生长因子 A（VEGFA）、骨桥蛋白（OPN）、碱性磷酸酶（ALP））在 2D 培养物中高度增加，而在 3D 培养物中则较低。COL1A1 mRNA 的表达在 3D 培养物中最高。蛋白表达支持大部分 mRNA 数据，但在单层培养物中 COL2A1 和 SOX9 的 mRNA 和蛋白表达之间存在重要差异，这强调了蛋白表达分析的重要性。GAG 含量在 3D 培养物中最高，而软骨细胞增殖几乎是 2D 培养物的特异性。
对于去分化 HAC 的再分化，3D 培养物表现出最强的软骨形成潜能，而 2D 培养物则最能获得肥大表型。这是第一项系统评估常用 2D 和 3D 软骨细胞培养技术之间增殖、GAG 含量、蛋白表达和 mRNA 表达差异的人体研究。
