van Osch G J, van der Veen S W, Verwoerd-Verhoef H L
Department of Otorhinolaryngology, Erasmus University Medical Center, Rotterdam, The Netherlands.
Plast Reconstr Surg. 2001 Feb;107(2):433-40. doi: 10.1097/00006534-200102000-00020.
To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.
为了利用组织工程构建自体软骨移植物，细胞必须在体外增殖；然后它们会失去软骨特异性表型。本研究的目的是评估增殖后的耳软骨细胞在不同培养条件下重新表达其软骨表型的能力。从三只幼年和三只成年兔的耳部软骨中分离细胞，在单层培养中增殖后，将它们接种到藻酸盐中，并在无血清培养基中与胰岛素样生长因子1（IGF-1）和转化生长因子-β2（TGF-β2）以三种不同剂量组合培养3周。作为对照，细胞在10%胎牛血清中培养，先前的实验表明该血清无法诱导再分化。当血清被胰岛素样生长因子-1和转化生长因子-β2替代时，幼年兔而非成年兔耳部的软骨细胞合成的糖胺聚糖显著增多。II型胶原阳性细胞的数量在幼年细胞中从10%增加到97%，在成年细胞中增加到33%。使用12例患者（年龄7至60岁）的人耳细胞，用胰岛素样生长因子和转化生长因子-β替代血清也能刺激糖胺聚糖的合成。尽管在这些条件下II型胶原阳性细胞的数量可以增加，但从未超过10%。来自五例患者的数据表明，通过添加ITS+和皮质醇进一步优化培养条件可显著增加（加倍或三倍）糖胺聚糖的合成和II型胶原的表达。总之，本研究证明了一种使增殖后的耳软骨细胞恢复软骨表型的方法。这提高了生成用于外耳重建或鼻中隔缺损修复的人软骨移植物的可能性。
