Shafaei Hajar, Esfandiari Ebrahim, Esmaeili Abolghasem, Razavi Shahnaz, Hashemibeni Batool, Nasr Esfahani Mohammad Hossien, Shiran Mohammad Bagher, Isfahani Sayed Hamid Zarkesh, Mardani Mohammad
Department of Anatomical Sciences, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Adv Biomed Res. 2013 Oct 30;2:79. doi: 10.4103/2277-9175.120867. eCollection 2013.
Hyaline cartilage tissue of joints is susceptible to injuries due to avascularity. Mesenchymal stem cells (MSCs) are used for cartilage tissue engineering. Among MSCs, adipose stem cells (ASCs) are attractive because of accessibility, their large number, and rapid growth. Common in vitro protocols successfully induce chondrogenic differentiation by expression of multiple cartilage-specific molecules. However, transforming growth factor β (TGFβ) promotes chondrogenesis to terminal stages. Despite much attention being given to the influences of biochemical factors on chondrogenesis of MSCs, few studies have examined the chondrogenic effect of mechanical factors such as ultrasound as a feasible tool.
In this study, we focused on inducing chondrogenesis in the early stages of differentiation by using low-intensity ultrasound (LIUS). Four groups of ASC pellets (control, ultrasound, TGFβ, and ultrasound/TGF) were cultured under chondrogenic (10 ng/ml of TGFβ3) and ultrasound conditions (200 mW/cm(2), 10 min/day). After 2 weeks, differentiation was evaluated by histology, quantitative gene expression analysis, and immunohistochemistry.
Our data demonstrated that ultrasound differentiated pellets showed increased expression of early chondrogenesis marker, Col2A, than those in TGFβ groups (P < 0.001), and Col2B and Col10 expression were more prominent in TGFβ groups. Immunostaining of sections showed Col2 fibrils around lacuna in LIUS and TGFβ treated groups.
Using LIUS resulted in early chondrogenesis in comparison with terminally differentiated chondrocytes by TGFβ. Therefore, LIUS might provide an applicable, safe, efficient, and cheap tool for chondrogenic differentiation of ASCs in cartilage tissue engineering.
关节透明软骨组织因无血管而易受损伤。间充质干细胞（MSCs）被用于软骨组织工程。在MSCs中，脂肪干细胞（ASCs）因其易于获取、数量众多和生长迅速而备受关注。常见的体外方案通过多种软骨特异性分子的表达成功诱导软骨形成分化。然而，转化生长因子β（TGFβ）促进软骨形成至终末阶段。尽管生化因素对MSCs软骨形成的影响受到了广泛关注，但很少有研究将超声等机械因素作为一种可行工具来研究其软骨形成作用。
在本研究中，我们聚焦于使用低强度超声（LIUS）诱导分化早期的软骨形成。四组ASC微球（对照组、超声组、TGFβ组和超声/TGF组）在软骨形成（10 ng/ml的TGFβ3）和超声条件（200 mW/cm²，每天10分钟）下培养。2周后，通过组织学、定量基因表达分析和免疫组织化学评估分化情况。
我们的数据表明，超声分化的微球显示早期软骨形成标志物Col2A的表达高于TGFβ组（P < 0.001），而Col2B和Col10的表达在TGFβ组中更显著。切片的免疫染色显示，LIUS组和TGFβ处理组的腔隙周围有Col2纤维。
与TGFβ诱导的终末分化软骨细胞相比，使用LIUS可导致早期软骨形成。因此，LIUS可能为软骨组织工程中ASCs的软骨形成分化提供一种适用、安全、高效且廉价的工具。
