Manickavasagam A, Hirvonen L M, Melita L N, Chong E Z, Cook R J, Bozec L, Festy F
Biomaterial, Biomimetics & Biophotonics Division, King's College London Dental Institute, London, UK.
Analyst. 2014 Dec 7;139(23):6135-43. doi: 10.1039/c4an01523a. Epub 2014 Oct 15.
Collagen is a structural component of the human body, as a connective tissue it can become altered as a result of pathophysiological conditions. Although the collagen degradation mechanism is not fully understood, it plays an important role in ageing, disease progression and applications in therapeutic laser treatments. To fully understand the mechanism of collagen alteration, in our study photo-disruptive effects were induced in collagen I matrix by point-irradiation with a femtosecond Ti-sapphire laser under controlled laser ablation settings. This was followed by multi-modal imaging of the irradiated and surrounding areas to analyse the degradation mechanism. Our multi-modal methodology was based on second harmonic generation (SHG), scanning electron microscope (SEM), autofluorescence (AF) average intensities and the average fluorescence lifetime. This allowed us to quantitatively characterise the degraded area into four distinct zones: (1) depolymerised zone in the laser focal spot as indicated by the loss of SHG signal, (2) enhanced crosslinking zone in the inner boundary of the laser induced cavity as represented by the high fluorescence ring, (3) reduced crosslinking zone formed the outer boundary of the cavity as marked by the increased SHG signal and (4) native collagen. These identified distinct zones were in good agreement with the expected photochemical changes shown using Raman spectroscopy. In addition, imaging using polarisation-resolved SHG (p-SHG) revealed both a high degree of fibre re-orientation and a SHG change in tensor ratios around the irradiation spot. Our multi-modal optical imaging approach can provide a new methodology for defining distinct zones that can be used in a clinical setting to determine suitable thresholds for applying safe laser treatments without affecting the surrounding tissues. Furthermore this technique can be extended to address challenges observed in collagen based tissue engineering and used as a minimally invasive diagnostic tool to characterise diseased and non-diseased collagen rich tissues.
胶原蛋白是人体的一种结构成分，作为一种结缔组织，它可能会因病理生理状况而发生改变。尽管胶原蛋白的降解机制尚未完全明确，但它在衰老、疾病进展以及治疗性激光治疗的应用中发挥着重要作用。为了全面了解胶原蛋白改变的机制，在我们的研究中，在受控的激光烧蚀设置下，用飞秒钛宝石激光对I型胶原蛋白基质进行点照射，诱导光破坏效应。随后对照射区域及周围区域进行多模态成像，以分析降解机制。我们的多模态方法基于二次谐波产生（SHG）、扫描电子显微镜（SEM）、自发荧光（AF）平均强度和平均荧光寿命。这使我们能够将降解区域定量地分为四个不同区域：（1）激光焦点处的解聚区域，表现为SHG信号消失；（2）激光诱导腔体内边界处的交联增强区域，由高荧光环表示；（3）腔体外边界形成的交联减少区域，以SHG信号增加为标志；（4）天然胶原蛋白区域。这些确定的不同区域与使用拉曼光谱显示的预期光化学变化高度一致。此外，使用偏振分辨SHG（p-SHG）成像显示，照射点周围既有高度的纤维重新定向，又有张量比的SHG变化。我们的多模态光学成像方法可以提供一种新的方法来定义不同区域，可用于临床环境中确定应用安全激光治疗的合适阈值，而不影响周围组织。此外，该技术可以扩展到解决基于胶原蛋白的组织工程中观察到的挑战，并用作微创诊断工具来表征患病和未患病的富含胶原蛋白的组织。
