Mierisch Cay M, Wilson Heather A, Turner Maria A, Milbrandt Todd A, Berthoux Lionel, Hammarskjöld Marie-Louise, Rekosh David, Balian Gary, Diduch David R
Department of Orthopaedic Surgery, Orthopaedic Research Laboratory, University of Virginia, Charlottesville, 22908, USA.
J Bone Joint Surg Am. 2003 Sep;85(9):1757-67. doi: 10.2106/00004623-200309000-00015.
The fate of transplanted chondrocytes used to elicit the repair of osteochondral defects is unknown. The objective of this study was to examine the fate and the expression of cartilage-specific genes in chondrocytes when the chondrocyte phenotype was maintained preoperatively by alginate suspension culture, the cells were labeled with enhanced green fluorescent protein, and the chondrocytes in alginate were then implanted into full-thickness osteochondral defects in rabbits.
To determine the effect of alginate on rabbit chondrocytes in vitro, cells were grown in monolayer or in alginate suspension culture, and gene expression for aggrecan, type-I collagen, and type-II collagen was analyzed by reverse transcription-polymerase chain reaction. Cells were genetically labeled with the gene for enhanced green fluorescent protein, and the effect of transfer of the gene for enhanced green fluorescent protein on chondrocyte phenotype was assessed in vitro. Chondrocytes labeled with enhanced green fluorescent protein that were embedded in alginate were implanted into osteochondral defects in rabbit knees, either immediately after creation of the defects or after the cells had been preconditioned in alginate suspension culture for two weeks. The repair tissue within the osteochondral defects was assessed at one to four weeks. Cells labeled with enhanced green fluorescent protein were quantified by confocal microscopy, and the repair tissue was examined histologically with safranin O.
Gene expression by chondrocytes demonstrated a selective upregulation of cartilage-specific genes in alginate suspension culture. This effect was less pronounced in cells that were transduced with enhanced green fluorescent protein. Chondrocytes transplanted in vivo were detected in the repair tissue for the entire period of observation with diminishing cell density over time. At one week, the cell density of the transplanted chondrocytes was 100% of the initial density; at two and three weeks, the cell density was 70%; and, after four weeks, the cell density had decreased to 15%. Safranin-O staining of histological sections indicated cartilage-specific matrix production in vitro and in vivo. Integration of transplanted cells into the host repair tissue was not observed. The two-week period of preconditioning in alginate suspension culture had no apparent influence on the temporal fate of the cells or the histological appearance of the repair tissue.
Alginate promotes expression of cartilage-specific genes and allows delivery of chondrocytes into osteochondral defects. Transgenic chondrocytes labeled with enhanced green fluorescent protein are detectable in the defect, but they do not appear to form repair tissue and they decrease in number with time. In view of the clinical application of cell-based cartilage repair, understanding the fate of transplanted cells becomes increasingly relevant. Transgenic chondrocytes are an effective tool to study the role of transplanted chondrocytes in articular cartilage repair.
用于诱导修复骨软骨缺损的移植软骨细胞的命运尚不清楚。本研究的目的是在术前通过海藻酸钠悬浮培养维持软骨细胞表型、用增强型绿色荧光蛋白标记细胞，然后将海藻酸钠中的软骨细胞植入兔的全层骨软骨缺损后，研究软骨细胞的命运及软骨特异性基因的表达。
为了确定海藻酸钠对兔软骨细胞的体外作用，将细胞在单层或海藻酸钠悬浮培养中生长，通过逆转录 - 聚合酶链反应分析聚集蛋白聚糖、I型胶原和II型胶原的基因表达。用增强型绿色荧光蛋白基因对细胞进行基因标记，并在体外评估增强型绿色荧光蛋白基因转移对软骨细胞表型的影响。将嵌入海藻酸钠中的增强型绿色荧光蛋白标记的软骨细胞植入兔膝关节的骨软骨缺损处，要么在缺损形成后立即植入，要么在细胞在海藻酸钠悬浮培养中预处理两周后植入。在1至4周时评估骨软骨缺损内的修复组织。通过共聚焦显微镜对增强型绿色荧光蛋白标记的细胞进行定量，并使用番红O对修复组织进行组织学检查。
软骨细胞的基因表达显示在海藻酸钠悬浮培养中软骨特异性基因有选择性上调。这种作用在用增强型绿色荧光蛋白转导的细胞中不太明显。在整个观察期内，在修复组织中均检测到体内移植的软骨细胞，但其细胞密度随时间逐渐降低。在1周时，移植软骨细胞的细胞密度为初始密度的100%；在2周和3周时，细胞密度为70%；4周后，细胞密度降至15%。组织学切片的番红O染色表明在体外和体内均有软骨特异性基质产生。未观察到移植细胞与宿主修复组织的整合。在海藻酸钠悬浮培养中进行两周的预处理对细胞的时间命运或修复组织的组织学外观没有明显影响。
海藻酸钠可促进软骨特异性基因的表达，并允许将软骨细胞递送至骨软骨缺损处。用增强型绿色荧光蛋白标记的转基因软骨细胞在缺损处可被检测到，但它们似乎未形成修复组织，且数量随时间减少。鉴于基于细胞的软骨修复的临床应用，了解移植细胞的命运变得越来越重要。转基因软骨细胞是研究移植软骨细胞在关节软骨修复中作用的有效工具。
