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The Effect of Low Intensity Pulsed Ultrasound on the Anterior Cruciate Ligament Injury

低强度脉冲超声治疗前交叉韧带损伤的效果

基本信息

批准号：
14370463
负责人：
KUROSAKA Masahiro
金额：
$ 5.44万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370463/
关键词：
Anterior Cruciate Ligament Low Intensity Pulsed Ultrasound Tenomodulin GDF-5 bFGF GDF-5

项目摘要

Muscle-derived cells (MDC) have been reported to differentiate into osteogenic and adipogenic cells. We investigated that low intensity pulsed ultrasound (LIPUS) acting as mechanical stress make MDCs differentiate into ligament cell lineage. Muscle-derived cells were isolated from the muscle of beagle dogs. Minced muscle was digested with collagenase and trypsinized. The cells were cultivated on T25 flask. Then, the attached cells were passaged for cell expansion. MDCs were plated in 6-well culture plates at a density of 2x10^5 and after incubation for 24 hrs, the medium was replaced. Following this the MDCs were exposed to LIPUS, consisting of 200 microseconds burst sine waves of 1.5 MHz repeating at 1.0 kHz, for 20 minutes. The incident intensity was 30 mW/cm^2 At 0 (immediately after 20 minutes LIPUS exposure) and one hour after LIPUS exposure, total RNA was extracted for the following RT-PCR. We semi-quantified mRNA of growth factors (TGFb1, bFGF, PDGF, GDF-5). We also investigated mRNA of type I and III collagen, which is major components of ligament fiber, and tenomodulin, as a differentiation marker for ligament. The up-regulations of bFGF was observed after the 20 minutes of LIPUS exposure. Type I and III collagen mRNA was expressed constitutively in both the MDCs exposed to LIPUS and the unexposed cells but that tenomodulin mRNA was induced in the MDCs exposed to LIPUS. An up-regulation of tenomodulin mRNA was observed in the MDCs exposed to LIPUS at immediately after the 20 minutes of LIPUS exposure. MDCs differentiated into another tissue lineage like tendon or ligament as a result of LIPUS exposure. This study can contribute to a strategy to successfully culture replacement human ligament tissues in vitro.

肌源性细胞（MDC）可分化为成骨细胞和成脂细胞。我们研究了低强度脉冲超声（LIPUS）作为机械应力作用下，MDCs向韧带细胞系分化。从比格犬的肌肉中分离肌源性细胞。用胶原酶消化和胰蛋白酶消化切碎的肌肉。将细胞在T25烧瓶上培养。然后，将附着的细胞传代用于细胞扩增。将MDCs以2x10^5的密度接种在6孔培养板中，孵育24小时后，更换培养基。此后，将MDC暴露于LIPUS，LIPUS由以1.0 kHz重复的1.5 MHz的200微秒脉冲正弦波组成，持续20分钟。入射强度为30 mW/cm ^2，在0（LIPUS暴露20分钟后立即）和LIPUS暴露后1小时，提取总RNA用于以下RT-PCR。我们半定量的生长因子（TGF β 1，bFGF，PDGF，GDF-5）的mRNA。我们还研究了I型和III型胶原蛋白的mRNA，这是韧带纤维的主要成分，以及肌腱调节蛋白，作为韧带的分化标志物。LIPUS作用20 min后bFGF表达明显上调。I型和III型胶原mRNA在暴露于LIPUS的MDCs和未暴露的MDCs中均组成性表达，但在暴露于LIPUS的MDCs中，Tenomodulin mRNA被诱导。在LIPUS暴露20分钟后立即观察到MDCs中的Tenomodulin mRNA的上调。由于LIPUS暴露，MDCs分化成另一种组织谱系，如肌腱或韧带。本研究为成功地在体外培养人韧带替代组织提供了一种策略。