Institute of Clinical Medicine, University of Tromsø, Norway.
Department of Physics, Norwegian University of Science and Technology, Norway.
Osteoarthritis Cartilage. 2017 Oct;25(10):1729-1737. doi: 10.1016/j.joca.2017.06.008. Epub 2017 Jun 29.
Current systems to evaluate outcomes from tissue-engineered cartilage (TEC) are sub-optimal. The main purpose of our study was to demonstrate the use of second harmonic generation (SHG) microscopy as a novel quantitative approach to assess collagen deposition in laboratory made cartilage constructs.
Scaffold-free cartilage constructs were obtained by condensation of in vitro expanded Hoffa's fat pad derived stromal cells (HFPSCs), incubated in the presence or absence of chondrogenic growth factors (GF) during a period of 21 d. Cartilage-like features in constructs were assessed by Alcian blue staining, transmission electron microscopy (TEM), SHG and two-photon excited fluorescence microscopy. A new scoring system, using second harmonic generation microscopy (SHGM) index for collagen density and distribution, was adapted to the existing "Bern score" in order to evaluate in vitro TEC.
Spheroids with GF gave a relative high Bern score value due to appropriate cell morphology, cell density, tissue-like features and proteoglycan content, whereas spheroids without GF did not. However, both TEM and SHGM revealed striking differences between the collagen framework in the spheroids and native cartilage. Spheroids required a four-fold increase in laser power to visualize the collagen matrix by SHGM compared to native cartilage. Additionally, collagen distribution, determined as the area of tissue generating SHG signal, was higher in spheroids with GF than without GF, but lower than in native cartilage.
SHG represents a reliable quantitative approach to assess collagen deposition in laboratory engineered cartilage, and may be applied to improve currently established scoring systems.
目前评估组织工程软骨（TEC）结果的系统并不理想。本研究的主要目的是展示二次谐波产生（SHG）显微镜作为一种评估实验室制造的软骨构建体中胶原沉积的新定量方法的用途。
通过体外扩增的Hoffa 脂肪垫基质细胞（HFPSC）的凝聚获得无支架软骨构建体，在存在或不存在软骨生成生长因子（GF）的情况下孵育 21 天。通过阿利新蓝染色、透射电子显微镜（TEM）、SHG 和双光子激发荧光显微镜评估构建体中的软骨样特征。采用新的评分系统，使用二次谐波产生显微镜（SHGM）指数评估胶原密度和分布，以适应现有的“Bern 评分”，从而评估体外 TEC。
具有 GF 的球体由于适当的细胞形态、细胞密度、组织样特征和糖胺聚糖含量而具有相对较高的 Bern 评分值，而没有 GF 的球体则没有。然而，TEM 和 SHGM 均揭示了球体中的胶原框架与天然软骨之间的明显差异。与天然软骨相比，SHGM 观察球体中的胶原基质需要将激光功率增加四倍。此外，具有 GF 的球体中的胶原分布（确定为产生 SHG 信号的组织面积）高于没有 GF 的球体，但低于天然软骨。
SHG 是一种可靠的定量方法，可用于评估实验室工程软骨中的胶原沉积，并可能应用于改进现有的评分系统。
