AO Research Institute Davos, Switzerland.
Osteoarthritis Cartilage. 2012 Apr;20(4):288-95. doi: 10.1016/j.joca.2011.12.010. Epub 2012 Jan 10.
Functional cartilage tissue engineering aims to generate grafts with a functional surface, similar to that of authentic cartilage. Bioreactors that stimulate cell-scaffold constructs by simulating natural joint movements hold great potential to generate cartilage with adequate surface properties. In this study two methods based on atomic force microscopy (AFM) were applied to obtain information about the quality of engineered graft surfaces. For better understanding of the molecule-function relationships, AFM was complemented with immunohistochemistry.
Bovine chondrocytes were seeded into polyurethane scaffolds and subjected to dynamic compression, applied by a ceramic ball, for 1h daily [loading group 1 (LG1)]. In loading group 2 (LG2), the ball additionally oscillated over the scaffold, generating sliding surface motion. After 3 weeks, the surfaces of the engineered constructs were analyzed by friction force and indentation-type AFM (IT-AFM). Results were complemented and compared to immunohistochemical analyses.
The loading type significantly influenced the mechanical and histological outcomes. Constructs of LG2 exhibited lowest friction coefficient and highest micro- and nanostiffness. Collagen type II and aggrecan staining were readily observed in all constructs and appeared to reach deeper areas in loaded (LG1, LG2) compared to unloaded scaffolds. Lubricin was specifically detected at the top surface of LG2.
This study proposes a quantitative AFM-based functional analysis at the micrometer- and nanometer scale to evaluate the quality of cartilage surfaces. Mechanical testing (load-bearing) combined with friction analysis (gliding) can provide important information. Notably, sliding-type biomechanical stimuli may favor (re-)generation and maintenance of functional articular surfaces and support the development of mechanically competent engineered cartilage.
功能性软骨组织工程旨在生成具有类似于天然软骨功能表面的移植物。通过模拟自然关节运动来刺激细胞-支架构建体的生物反应器具有生成具有足够表面性能的软骨的巨大潜力。在这项研究中，应用了两种基于原子力显微镜（AFM）的方法来获取有关工程化移植物表面质量的信息。为了更好地理解分子-功能关系，AFM 辅以免疫组织化学。
将牛软骨细胞接种到聚氨酯支架中，并每天进行 1 小时的动态压缩，由陶瓷球施加[加载组 1（LG1）]。在加载组 2（LG2）中，球还在支架上振荡，产生滑动表面运动。3 周后，通过摩擦力和压痕型原子力显微镜（IT-AFM）分析工程化构建体的表面。结果进行了补充并与免疫组织化学分析进行了比较。
加载类型显著影响机械和组织学结果。LG2 组的构建体表现出最低的摩擦系数和最高的微纳米硬度。所有构建体中均容易观察到 II 型胶原和聚集蛋白的染色，并且与未加载的支架相比，在加载（LG1、LG2）的支架中似乎到达了更深的区域。LG2 的顶部表面特异性检测到润滑素。
本研究提出了一种基于 AFM 的定量功能分析，可在微米和纳米尺度上评估软骨表面的质量。机械测试（承载）与摩擦分析（滑动）相结合可以提供重要信息。值得注意的是，滑动型生物力学刺激可能有利于（重新）生成和维持功能性关节表面，并支持具有机械能力的工程化软骨的发展。
