Department of Biomedical Engineering, University of California, Irvine, Irvine, California, USA.
Invest Ophthalmol Vis Sci. 2011 Nov 11;52(12):8818-27. doi: 10.1167/iovs.11-8070.
To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these findings with mechanical data obtained by indentation testing of corneal flaps.
Twelve corneas from 10 donors were studied. Vibratome sections, 200 μm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 μm lateral, 2 μm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer.
The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowman's layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma.
The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.
利用非线性光学高分辨率宏观技术（NLO-HRMac）对人眼角膜的三维胶原纤维（板层）结构进行特征描述和量化，并将这些发现与角膜瓣压痕测试获得的力学数据相关联。
研究了 10 位供体的 12 只眼角膜。从 5 位供体的 5 只眼睛中，使用振动切片机沿垂直子午线从角膜缘到角膜缘（弧长 12mm）切取 200μm 厚的切片。从这些切片中收集背散射二次谐波产生（SHG）的 NLO 信号，作为一系列重叠的 3D 图像，这些图像被拼接在一起形成单个 3D 镶嵌图（像素分辨率：0.44μm 横向，2μm 轴向）。通过确定基质深度的分支点密度来量化胶原纤维的缠绕程度。对来自另外 7 只眼睛的角膜瓣进行力学测试。使用飞秒手术激光系统将角膜切成三层（前部、中间部和后部），并进行压痕测试以确定各层的弹性模量。
3D 重建显示前部角膜的胶原纤维分支模式复杂，纤维从前部限制层（ALL，Bowman 层）延伸，与深部纤维交织，并重新插入到 ALL，形成弓状弹簧样结构。在前部角膜的前三分之一处测量到的分支点密度是后三分之一处的四倍，并且随着与 ALL 的距离增加而呈对数下降。压痕测试显示前部基质的弹性模量增加了 8 倍。
板层交织的轴向梯度似乎与角膜有效弹性模量的轴向梯度相关，这表明胶原纤维交织和弓状弹簧样结构的形成提供了类似于桥梁和大型结构中的横梁的结构支撑。未来的研究需要确定径向和轴向结构力学异质性在控制角膜形状和圆锥角膜、散光和其他屈光不正发展中的作用。
