Abrams G A, Goodman S L, Nealey P F, Franco M, Murphy C J
Department of Surgical Sciences, School of Veterinary Medicine, UW-Madison, WI 53706, USA.
Cell Tissue Res. 2000 Jan;299(1):39-46. doi: 10.1007/s004419900074.
This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149 +/- 60 nm, 191 +/- 72 nm, and 147 +/- 73 nm, respectively. Mean fiber diameter as measured by SEM was 77 +/- 44 nm and pore diameter was 72 +/- 40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.
本文定量定义了猕猴角膜前上皮下基底膜的纳米级形貌。将从猕猴身上切除的角膜纽扣置于2.5 mM乙二胺四乙酸（EDTA）中2.5小时，之后小心去除上皮，以暴露其下的基底膜。使用荧光显微镜结合针对层粘连蛋白和IV型胶原的抗体染色以及透射电子显微镜来验证剩余基底膜的完整性。使用透射电子显微镜、高分辨率低电压扫描电子显微镜和原子力显微镜对基底膜表面进行表征。通过这三种成像技术获得定量数据并进行比较。基底膜具有复杂的形貌，由紧密交联的纤维与孔隙交织而成。通过透射电子显微镜、扫描电子显微镜和原子力显微镜测量的特征平均高度分别为149±60 nm、191±72 nm和147±73 nm。通过扫描电子显微镜测量的平均纤维直径为77±44 nm，平均孔径为72±40 nm，孔隙约占总表面积的15%。对于基质胶（一种市售的基底膜样复合物）也发现了类似的特征类型和尺寸，这支持角膜制备程序去除上层上皮时引入的人为因素最少。形貌特征使细胞与基质相互作用发生的表面积估计扩大了400%。基底膜的三维结构呈现出由尺寸范围为30至400 nm的孔隙和纤维组成的丰富复杂的个体特征形貌。这些纳米级基质特征可能调节诸如黏附、迁移、增殖和分化等基本细胞行为。
