Matsusaki Michiya, Kadowaki Koji, Tateishi Kosuke, Higuchi Chikahisa, Ando Wataru, Hart David A, Tanaka Yoshinari, Take Yasuhiro, Akashi Mitsuru, Yoshikawa Hideki, Nakamura Norimasa
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
Tissue Eng Part A. 2009 Jan;15(1):55-63. doi: 10.1089/ten.tea.2007.0424.
Mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC)-hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl(2)/Tris-HCl and Na(2)HPO(4)/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of approximately 1 mum were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. approximately 500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.
通过交替浸泡法制备了基于间充质干细胞（MSC）的组织工程构建体（TEC）-羟基磷灰石（HAp）复合材料。将源自培养的滑膜间充质干细胞的TEC交替浸入不同浓度的CaCl₂/Tris-HCl和Na₂HPO₄/Tris-HCl缓冲液中，并通过傅里叶变换红外光谱（FT-IR）、广角X射线衍射和扫描电子显微镜（SEM）分析HAp的形成。这些分析清楚地证明了TEC中HAp的形成。具体而言，SEM评估表明，约1μm的球形HAp晶体直接在细胞表面和细胞外基质（ECM）纤维上形成。通过活/死染色和总DNA测定评估，暴露于浓度>100 mM的钙或磷酸盐缓冲液会产生细胞毒性，但暴露于浓度<50 mM时未检测到这种细胞毒性。在10 mM钙或磷酸盐缓冲液中经过20个循环后形成了HAp纳米晶体（约500 nm），并证实了复合材料中的细胞存活。此外，将源自兔滑膜间充质干细胞的TEC-HAp复合材料初步植入兔骨软骨缺损处显示出加速的骨诱导作用。这些复合材料可能是由ECM、HAp纳米晶体和活间充质干细胞组成的混合材料的首个实例，并且TEC-HAp复合材料可能是用于骨组织工程的独特且有用的材料。
