Vielreicher Martin, Bozec Aline, Schett Georg, Friedrich Oliver
Institute of Medical Biotechnology, Department of Chemical and Biological Engineering, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.
Department of Internal Medicine 3 - Rheumatology and Immunology, University Clinic, Erlangen, Germany.
Front Bioeng Biotechnol. 2021 Jun 11;9:608383. doi: 10.3389/fbioe.2021.608383. eCollection 2021.
Chronic inflammatory disease of bones and joints (e.g., rheumatoid arthritis, gout, etc.), but also acute bone injury and healing, or degenerative resorptive processes inducing osteoporosis, are associated with structural remodeling that ultimately have impact on function. For instance, bone stability is predominantly orchestrated by the structural arrangement of extracellular matrix fibrillar networks, i.e., collagen-I, -IV, elastin, and other proteins. These components may undergo distinct network density and orientation alterations that may be causative for decreased toughness, resilience and load bearing capacity or even increased brittleness. Diagnostic approaches are usually confined to coarse imaging modalities of X-ray or computer tomography that only provide limited optical resolution and lack specificity to visualize the fibrillary collagen network. However, studying collagen structure at the microscopic scale is of considerable interest to understand the mechanisms of tissue pathologies. Multiphoton Second Harmonic Generation (SHG) microscopy, is able to visualize the sterical topology of the collagen-I fibrillar network in 3D, in a minimally invasive and label-free manner. Penetration depths exceed those of conventional visible light imaging and can be further optimized through employing decalcification or optical clearing processing . The goal of this proof-of-concept study was to use SHG and two-photon excited fluorescence (2-PEF) imaging to mainly characterize the fibrillary collagen organization within decalcified normal mouse metatarsus bone and joint. The results show that the technique resolved the fibrillar collagen network of complete bones and joints with almost no artifacts and enabled to study the complex collagen-I networks with various fiber types (straight, crimped) and network arrangements of mature and woven bone with high degree of detail. Our imaging approach enabled to identify cavities within both cortical and trabecular bone architecture as well as interfaces with sharply changing fiber morphology and network structure both within bone, in tendon and ligament and within joint areas. These possibilities are highly advantageous since the technology can easily be applied to animal models, e.g., of rheumatoid arthritis to study structural effects of chronic joint inflammation, and to many others and to compare to the structure of human bone.
骨骼和关节的慢性炎症性疾病（如类风湿性关节炎、痛风等），以及急性骨损伤与愈合，或导致骨质疏松的退行性吸收过程，都与最终影响功能的结构重塑有关。例如，骨稳定性主要由细胞外基质纤维网络（即I型、IV型胶原蛋白、弹性蛋白和其他蛋白质）的结构排列来协调。这些成分可能会经历不同的网络密度和方向改变，这可能导致韧性、弹性和承重能力下降，甚至脆性增加。诊断方法通常局限于X射线或计算机断层扫描等粗略成像方式，这些方式仅提供有限的光学分辨率，且缺乏可视化纤维状胶原网络的特异性。然而，在微观尺度上研究胶原蛋白结构对于理解组织病理学机制具有重要意义。多光子二次谐波产生（SHG）显微镜能够以微创和无标记的方式三维可视化I型胶原纤维网络的空间拓扑结构。其穿透深度超过传统可见光成像，并且可以通过采用脱钙或光学清除处理进一步优化。本概念验证研究的目的是使用SHG和双光子激发荧光（2-PEF）成像来主要表征脱钙正常小鼠跖骨和关节内的纤维状胶原组织。结果表明，该技术能够解析完整骨骼和关节的纤维状胶原网络，几乎没有伪影，并能够高度详细地研究具有各种纤维类型（直的、卷曲的）以及成熟骨和编织骨的复杂I型胶原网络及其网络排列。我们的成像方法能够识别皮质骨和小梁骨结构内的空洞，以及骨骼、肌腱和韧带以及关节区域内纤维形态和网络结构急剧变化的界面。这些可能性非常有利，因为该技术可以轻松应用于动物模型，例如类风湿性关节炎模型，以研究慢性关节炎症的结构影响，也可应用于许多其他模型，并与人类骨骼结构进行比较。
