Dell'Accio F, De Bari C, Luyten F P
University Hospitals Katholieke Universiteit Leuven, Belgium.
Arthritis Rheum. 2001 Jul;44(7):1608-19. doi: 10.1002/1529-0131(200107)44:7<1608::AID-ART284>3.0.CO;2-T.
To establish a model and associated molecular markers for monitoring the capacity of in vitro-expanded chondrocytes to generate stable cartilage in vivo.
Adult human articular chondrocytes (AHAC) were prepared by collagenase digestion of samples obtained postmortem and were expanded in monolayer. Upon passaging, aliquots of chondrocyte suspensions were either injected intramuscularly into nude mice, cultured in agarose, or used for gene expression analysis. Cartilage formation in vivo was documented by histology, histochemistry, immunofluorescence for type II collagen, and proteoglycan analysis by 35S-sulfate incorporation and molecular sieve chromatography of the radiolabeled macromolecules. In situ hybridization for species-specific genomic repeats was used to discriminate human-derived from mouse-derived cells. Gene expression dynamics were analyzed by semiquantitative reverse transcription-polymerase chain reaction.
Intramuscular injection of freshly isolated AHAC into nude mice resulted in stable cartilage implants that were resistant to mineralization, vascular invasion, and replacement by bone. In vitro expansion of AHAC resulted in the loss of in vivo cartilage formation. This capacity was positively associated with the expression of fibroblast growth factor receptor 3, bone morphogenetic protein 2, and alpha1(II) collagen (COL2A1), and its loss was marked by the up-regulation of activin receptor-like kinase 1 messenger RNA. Anchorage-independent growth and the reexpression of COL2A1 in agarose culture were insufficient to predict cartilage formation in vivo.
AHAC have a finite capacity to form stable cartilage in vivo; this capacity is lost throughout passaging and can be monitored using a nude mouse model and associated molecular markers. This cartilage-forming ability in vivo may be pivotal for successful cell-based joint surface defect repair protocols.
建立一种模型及相关分子标志物，用于监测体外扩增的软骨细胞在体内生成稳定软骨的能力。
通过对死后获取的样本进行胶原酶消化制备成人关节软骨细胞（AHAC），并在单层中进行扩增。传代时，将软骨细胞悬液的等分试样要么肌肉注射到裸鼠体内，在琼脂糖中培养，要么用于基因表达分析。通过组织学、组织化学、II型胶原免疫荧光以及通过35S-硫酸盐掺入和放射性标记大分子的分子筛色谱法进行蛋白聚糖分析来记录体内软骨形成情况。使用物种特异性基因组重复序列的原位杂交来区分人源细胞和鼠源细胞。通过半定量逆转录-聚合酶链反应分析基因表达动态。
将新鲜分离的AHAC肌肉注射到裸鼠体内可形成稳定的软骨植入物，该植入物对矿化、血管侵入和骨替代具有抗性。AHAC的体外扩增导致体内软骨形成能力丧失。这种能力与成纤维细胞生长因子受体3、骨形态发生蛋白2和α1（II）胶原（COL2A1）的表达呈正相关，其丧失以激活素受体样激酶1信使核糖核酸的上调为标志。在琼脂糖培养中不依赖贴壁生长和COL2A1的重新表达不足以预测体内软骨形成。
AHAC在体内形成稳定软骨的能力有限；这种能力在传代过程中丧失，可使用裸鼠模型和相关分子标志物进行监测。这种体内软骨形成能力对于基于细胞的关节表面缺损修复方案的成功可能至关重要。
