Barou O, Mekraldi S, Vico L, Boivin G, Alexandre C, Lafage-Proust M H
Laboratoire de Biologie et de Biochimie du Tissu Osseux, Equipe Mixte INSERM 9901 Faculté de Médecine, Saint-Etienne, France.
Bone. 2002 Apr;30(4):604-12. doi: 10.1016/s8756-3282(02)00677-4.
Beside its well-known role in bone development, vascularization plays a major role in bone cell migration for bone remodeling and metastatic tumor invasion. However, the various techniques used to identify vessels in bone have never been tested for trabecular bone vessel quantification, whereas bone remodeling quantitative parameters are commonly assessed. In this context, we developed and compared various histological techniques used to visualize blood vessels in rat bone in order to quantify them. First, several products were tested by intracardiac infusion to opacify the bone vascular network. The best results were obtained using either an India ink-1% agarose solution or an India ink-saturated barium sulfate solution followed by X-ray microradiography. Second, to identify the types of vessels, we also performed histoenzymology and immunohistochemistry stainings. Neither alkaline phosphatase (for endothelial cells) nor adenosine triphosphatase (ATPase) stainings (for smooth muscle cells) provided a low enough background to allow for vessel identification and quantification. For immunohistochemistry, various specific vessel constituents were analyzed: laminin, smooth muscle cell alpha-actin, factor VIII, and lectin Griffonia simplifolia. Anti-laminin and anti-smooth muscle cell alpha-actin antibodies gave the best results for quantification. Third, after optimization of these techniques, we performed quantitative bone and vessel histomorphometry on two groups of 12 rats each, for which bone remodeling and vessel number and area parameters were measured. No statistical differences were observed between the two groups, confirming the reproducibility of our measurements. A significant relationship was found between vessel number and histodynamic parameters; that is, bone formation rate correlated positively with India ink-positive vessel area (p < 0.009, r2 = 0.54) and alpha-actin-positive vessel number (p < 0.05, r2 = 0.66). Furthermore, we report reproducible techniques for visualization and quantification of vessels in bone that also allowed for simultaneous conventional bone histomorphometry. This methodology should help researchers to better understand the functional and anatomical relationship between trabecular bone and its vascularization during normal or pathological processes.
除了在骨骼发育中广为人知的作用外，血管生成在骨细胞迁移以进行骨重塑和转移性肿瘤侵袭方面也起着重要作用。然而，用于识别骨中血管的各种技术从未针对小梁骨血管定量进行过测试，而骨重塑定量参数通常是可以评估的。在此背景下，我们开发并比较了用于可视化大鼠骨中血管以对其进行定量的各种组织学技术。首先，通过心内灌注测试了几种产品以使骨血管网络不透明。使用印度墨水 - 1%琼脂糖溶液或印度墨水饱和硫酸钡溶液然后进行X射线微放射摄影获得了最佳结果。其次，为了识别血管类型，我们还进行了组织酶学和免疫组织化学染色。碱性磷酸酶（用于内皮细胞）染色和三磷酸腺苷酶（ATP酶）染色（用于平滑肌细胞）都没有提供足够低的背景以允许血管识别和定量。对于免疫组织化学，分析了各种特定的血管成分：层粘连蛋白、平滑肌细胞α - 肌动蛋白、因子VIII和简叶豆凝集素。抗层粘连蛋白和抗平滑肌细胞α - 肌动蛋白抗体在定量方面给出了最佳结果。第三，在优化这些技术后，我们对两组各12只大鼠进行了定量骨和血管组织形态计量学分析，测量了骨重塑以及血管数量和面积参数。两组之间未观察到统计学差异，证实了我们测量的可重复性。发现血管数量与组织动力学参数之间存在显著关系；也就是说，骨形成率与印度墨水阳性血管面积呈正相关（p < 0.009，r2 = 0.54）以及与α - 肌动蛋白阳性血管数量呈正相关（p < 0.05，r2 = 0.66）。此外，我们报告了用于可视化和定量骨中血管的可重复技术，这些技术还允许同时进行传统的骨组织形态计量学分析。这种方法应有助于研究人员更好地理解正常或病理过程中小梁骨与其血管生成之间在功能和解剖学上的关系。
