Homicz Mark R, Schumacher Barbara L, Sah Robert L, Watson Deborah
Division of Head and Neck Surgery, University of California, San Diego and San Diego Veterans Affairs Healthcare System, California 92161, USA.
Otolaryngol Head Neck Surg. 2002 Nov;127(5):398-408. doi: 10.1067/mhn.2002.129730.
Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage.
Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 [P(0)]) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P(1)) and passage 2 (P(2)). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay).
Histologic sections of constructs seeded with P(0) cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P(1) and P(2) cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P(0) constructs was higher than in either P(1) (P < 0.05) or P(2) (P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages.
Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation.
The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs.
用于重建手术的软骨移植物未来某天或许可由采集的自体软骨细胞制成，这些细胞在体外扩增后接种到可生物降解支架上。本研究旨在量化由人鼻中隔软骨细胞构建的新软骨的生化组成，并研究单层培养中细胞增殖对新软骨最终组成的影响。
来自10名供体的人鼻中隔软骨细胞在收获后立即（第0代[P(0)]）接种到聚乙醇酸（PGA）支架上，或在单层培养中增殖，然后在第1代（P(1)）和第2代（P(2)）接种到支架上。细胞/支架构建体在体外培养7、14和28天。对新软骨构建体进行组织学分析，检测基质硫酸化糖胺聚糖（S-GAG）和II型胶原蛋白，并对细胞数量（Hoescht 33258检测法）、S-GAG含量（二甲基亚甲基蓝检测法）和胶原蛋白含量（羟脯氨酸检测法）进行定量评估。
接种P(0)细胞的构建体的组织学切片对S-GAG和II型胶原蛋白染色强烈，而在源自P(1)和P(2)细胞的构建体中，两种基质成分的染色均减少。构建体的细胞数量、S-GAG含量和总胶原蛋白含量从第7天到第28天显著增加。P(0)构建体中S-GAG的积累高于P(1)（P < 0.05）或P(2)（P < 0.01）构建体，而细胞数量和总胶原蛋白含量在各代之间无差异。
由在单层培养中经过连续传代的软骨细胞构建的新软骨表现出基质S-GAG和II型胶原蛋白减少，表明细胞去分化。
去分化软骨细胞产生的基质组成改变可能会限制新软骨构建体的机械稳定性。
