Cui Ji Hao, Park So Ra, Park Kwideok, Choi Byung Hyune, Min Byoung-Hyun
Department of Orthopaedic Surgery, Ajou University School of Medicine, Wonchon-dong, Youngtong-gu, Suwon, Gyeonggi, Korea.
Tissue Eng. 2007 Feb;13(2):351-60. doi: 10.1089/ten.2006.0080.
The purpose of this study was to evaluate the benefits of in vitro preconditioning of mesenchymal stem cells (MSCs) using low-intensity ultrasound (US) in the induction of chondrogenic differentiation of MSCs in vivo. After rabbit bone marrow-derived MSCs were seeded onto a polyglycolic acid (PGA) scaffold, the PGA-MSCs constructs were divided into 4 subgroups: untreated control, low-intensity US group, transforming growth factor-beta [TGF]-treated group and low-intensity US/TGF group. The chondrocyte-seeded PGA construct served as a positive control. For 1 week before implantation, the low-intensity US groups were subjected to ultrasound treatment for 20 min daily at an intensity of 200 mW/cm(2). The TGF groups were treated with 10 ng/mL TGF-beta1. The cells were then implanted into the nude mouse subcutaneously. Retrieved 1, 2, 4, and 6 weeks after implantation, each construct underwent gross examination, histology, biochemical assays, mechanical testing, and reverse transcriptase polymerase chain reaction (RT-PCR). Substantial size reduction and blood invasion were found much earlier in the groups that did not undergo low-intensity US than in those that did. Safranin O/Fast green staining revealed that the chondrogenic differentiation of MSCs was more widespread throughout the constructs in the low-intensity US groups. In the biochemical and mechanical analyses, the low-intensity US and low-intensity US/TGF groups were significantly better in forming hyaline cartilage-like tissue by 4 weeks than the non-low-intensity US groups. Presented by von Kossa staining, the development of osteogenic phenotypes was highly suppressed until 4 weeks in the low-intensity US groups, along with compressive strength comparable to the positive control. In the RT-PCR analysis before implantation, the messenger RNA levels of Sox-9, aggrecan, and tissue inhibitors of metalloproteinase-2 were higher in the low-intensity US groups, while those of type I and type X collagens and matrix metalloproteinase-13 were higher in the non-low-intensity US groups. Blood invasion into the constructs was also considerably hindered in the low-intensity US groups. These results strongly indicate that low-intensity US preconditioning in vitro could be an effective cue to upregulate chondrogenic differentiation of MSCs in vivo.
本研究的目的是评估使用低强度超声（US）对间充质干细胞（MSC）进行体外预处理在诱导其体内软骨分化方面的益处。将兔骨髓来源的MSC接种到聚乙醇酸（PGA）支架上后，将PGA-MSC构建体分为4个亚组：未处理的对照组、低强度超声组、转化生长因子-β [TGF]处理组和低强度超声/TGF组。接种软骨细胞的PGA构建体作为阳性对照。在植入前1周，低强度超声组每天接受强度为200 mW/cm²的超声处理20分钟。TGF组用10 ng/mL TGF-β1处理。然后将细胞皮下植入裸鼠体内。在植入后1、2、4和6周取出，每个构建体进行大体检查、组织学检查、生化分析、力学测试和逆转录聚合酶链反应（RT-PCR）。与接受低强度超声处理的组相比，未接受低强度超声处理的组更早出现明显的尺寸减小和血液侵入。番红O/固绿染色显示，低强度超声组中MSC的软骨分化在整个构建体中更为广泛。在生化和力学分析中，低强度超声组和低强度超声/TGF组在4周时形成透明软骨样组织的情况明显优于非低强度超声组。通过冯·科萨染色显示，低强度超声组中直到4周时成骨表型的发展受到高度抑制，同时抗压强度与阳性对照相当。在植入前的RT-PCR分析中，低强度超声组中Sox-9、聚集蛋白聚糖和金属蛋白酶组织抑制剂-2的信使RNA水平较高，而非低强度超声组中I型和X型胶原蛋白以及基质金属蛋白酶-13的信使RNA水平较高。低强度超声组中血液侵入构建体的情况也受到显著阻碍。这些结果有力地表明，体外低强度超声预处理可能是上调体内MSC软骨分化的有效因素。
