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

肌源性细胞(MDC)已被报道分化为成骨和成脂细胞。我们研究了低强度脉冲超声(LIPUS)作为机械应力使MDCs分化为韧带细胞系。从Beagle犬的肌肉中分离出肌源性细胞。切碎的肌肉用胶原酶消化，胰酶消化。细胞在T25培养瓶中培养。贴壁细胞传代扩增。将MDCs以2×10~(-5)密度接种于6孔培养板中，孵育24小时后更换培养液。随后，将MDC暴露在LIPUS中，LIPUS由200微秒的1.5 MHz正弦波组成，以1.0 kHz重复，持续20分钟。照射强度为30 mW/cm2，照射后即刻(照射后20min)和照射后1h，提取总RNA进行RT-PCR。半定量检测生长因子(TGFb1、bFGF1、PDGF、GDF-5)的表达。我们还研究了韧带纤维的主要成分I型和III型胶原的mRNA，以及作为韧带分化标志的肌腱调节蛋白。LIPUS照射20min后，观察碱性成纤维细胞生长因子的表达上调。LIPUS处理的MDCs和未暴露的MDCs均有I型和III型胶原mRNA的组成性表达，但LIPUS处理的MDCs可诱导肌腱调节蛋白mRNA的表达。LIPUS作用20min后，MDCs的Tenomodrin基因表达上调。由于LIPUS暴露，MDCs分化为另一种组织谱系，如肌腱或韧带。本研究为成功体外培养替代人韧带组织提供了一种策略。

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.