Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells
Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells
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DOI：
10.1002/jgm.826
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发表时间：
2006-01-01
影响因子：
3.5
通讯作者：
Schneider, H
中科院分区：
文献类型：
--
作者：
Park, J;Gelse, K;Schneider, H
背景：不同来源的成体间充质细胞可用于关节软骨修复，且供体部位发病率低。本研究旨在比较它们的成软骨潜能。方法采用逆转录聚合酶链反应（RT-PCR）方法，从成年大鼠的软骨膜/骨膜、骨髓或脂肪中分离出干细胞相关抗原Sca-1、c-Kit、CD10、CD13和CD90阳性的间充质细胞。采用重组骨形态发生蛋白2 （BMP-2）或携带BMP-2 cDNA的腺病毒载体诱导成软骨分化，然后进行微块培养。通过RT-PCR、细胞增殖和凋亡检测对刺激后的细胞进行表征。采用RT-PCR、免疫荧光和免疫组织化学等方法分析聚合蛋白、ⅰ型、ⅱ型、LX和X型胶原蛋白及碱性磷酸酶基因的表达。将腺病毒刺激的细胞移植到大鼠股骨髌骨沟机械生成的部分厚度软骨病变中。采用组织化学和免疫组织化学方法评估修复组织的质量和完整性。结果BMP-2或AdBMP-2刺激导致所有三种细胞群中软骨特异性基因表达上调，其中在软骨膜/骨膜细胞中表达最为迅速和显著，II型胶原mRNA增加3200倍，刺激后II型和IX型胶原转录物达到最高的绝对水平。骨髓基质细胞（BMSC）也获得了类似的结果，而脂肪基质细胞中相应的转录物水平在最初超过30倍的升高后下降。在体内移植后，adbmp -2感染的软骨膜/骨膜细胞产生了富含蛋白聚糖的II型胶原阳性基质，只有微弱的I型胶原染色。来自adbmp -2感染的骨髓间充质干细胞的修复组织显示出较弱的II型胶原染色，但具有相对丰富的蛋白聚糖基质，I型胶原呈弱阳性。转基因激活的脂肪基质细胞形成了以I型胶原为主的纤维组织。三个不同群体的未受刺激的细胞只产生纤维组织。结论软骨膜/骨膜来源细胞和骨髓间充质干细胞在形成透明软骨组织方面优于脂肪分离细胞。软骨形成的刺激，如BMP-2 cDNA的转移，似乎是启动和支持软骨分化所必需的。版权所有(c) 2005 John Wiley & Sons, Ltd。
Background Adult primary mesenchymal cells of different origin which can be obtained with minor donor site morbidity are considered for articular cartilage repair. This study aims at a comparison of their chondrogenic potential.Methods Mesenchymal cells were isolated from perichondrium/periosteum, bone marrow or fat of adult rats and found to be positive for the stem-cell-related antigens Sca-1, c-Kit, CD10, CD13 and CD90 by reverse transcription polymerase chain reaction (RT-PCR). Chondrogenic differentiation was induced by applying recombinant bone morphogenetic protein-2 (BMP-2) or adenoviral vectors carrying BMP-2 cDNA, followed by micromass culture. The stimulated cells were characterized by RT-PCR, cell proliferation and apoptosis assays. Expression of aggrecan, collagen type I, II, LX and X and alkaline phosphatase genes was analyzed by RT-PCR, immunofluorescence and immunohistochemistry in comparison with unstimulated control cells. Adenovirally stimulated cells were transplanted into mechanically generated partial-thickness cartilage lesions in the patellar groove of the rat femur. Quality and integration of the repair tissues were assessed by histochemical and immunohistochemical methods.Results Stimulation with BMP-2 or AdBMP-2 led to an up-regulation of cartilage-specific gene expression in all three cell populations studied, most rapidly and prominently in the perichondrial/periosteal cells, which showed a 3200-fold increase of type II collagen mRNA and reached the highest absolute levels of type II and IX collagen transcripts after stimulation. Similar results were obtained for the bone marrow stromal cells (BMSC), while the respective transcript levels in fat stromal cells declined after an initial more than 30-fold elevation. Following transplantation in vivo, AdBMP-2-infected perichondrial/periosteal cells produced a proteoglycan-rich, type II collagen-positive matrix with only faint staining for type I collagen. The repair tissue originating from AdBMP-2-infected BMSC showed less intense type II collagen staining, but a relatively proteoglycan-rich matrix, weakly positive for type I collagen. Transgene-activated fat stromal cells formed rather fibrous tissue mainly composed of type I Collagen. Unstimulated cells of the three different populations gave only rise to fibrous tissue.Conclusions Perichondrium/periosteum-derived cells and BMSC seem superior to cells isolated from fat with respect to forming hyaline cartilaginous tissue. A chondrogenic stimulus, e.g. by transfer of BMP-2 cDNA, appears to be required for initiation and support of chondrogenic differentiation. Copyright (c) 2005 John Wiley & Sons, Ltd.