偏振光显微观察不同胶原纤维在骨折愈合过程中的动态变化(英文)

Examination of dynamic changes of different-type collagens in bone fracture healing with a polarized light microscopy

背景:天狼星红是一种强酸性阴离子染料,染色后不褪色且具特异性,是目前胶原染色的最佳染料。胶原作为细胞外基质的主要成分之一,具有许多特殊的生理功能,机体通过胶原的合成和改建使骨折修复得以完善。目的:采用苦味酸天狼星红偏振光显微观察骨折愈合过程各型胶原的比例、分布的动态变化过程。设计:观察对比实验。单位:武汉大学人民医院骨外科,天津医院创伤骨科,北京大学医学部积水潭医院创伤骨科,解放军军事医学科学院基础医学研究所组织工程中心。材料:实验于2002-03/2003-09在解放军军事医学科学院基础医学研究所组织工程中心完成。选取健康成年中国绵羊3只,雄性,体质量25～35kg。方法:全部动物麻醉消毒后在跖骨干中段截取1cm长的骨缺损,骨折端用6孔加压钢板固定。术后1,3,6个月取骨折部位标本,乙二胺四乙酸脱钙制备切片,苦味酸天狼星红染色,偏振光显微镜观察胶原的类型和分布。主要观察指标:骨折愈合不同时期骨缺损区胶原的类型及其分布。结果:实验纳入3只绵羊,全部进入结果分析。①术后不同胶原纤维偏振光显微镜下的形态学表现:Ⅰ型胶原纤维紧密排列,显示很强的双折光性,呈黄色、橙色和红色的粗纤维;Ⅱ型胶原纤维显示弱的双折光,呈各种不同颜色的疏松网状;Ⅲ型胶原纤维疏网状,显示弱的双折光,呈绿色的细纤维。②术后不同胶原纤维偏振光显微镜下的数量观察:术后1个月,骨折处红色和黄色的Ⅰ型胶原极少见,绿色的Ⅲ型胶原纤维占大多数,胶原排列杂乱;术后3个月,骨折处红色和黄色的Ⅰ型胶原纤维明显增加,Ⅲ型胶原纤维的比例下降,胶原纤维的排列方向开始向有序的方向发展;术后6个月,粗大而鲜艳的红黄色Ⅰ型胶原成分占据了绝大部分,细小的绿色Ⅲ型胶原纤维的数量急剧减少,呈现明显的斜行螺旋性交叉的三维排列。结论:苦味酸天狼星红偏振光显微观察不仅能区分骨折局部Ⅰ、Ⅲ型胶原的类型,还可以清晰显示Ⅰ、Ⅲ型胶原的形态、分布和比例关系,具有操作简便、特异性强、灵敏度高的特点。

BACKGROUND：Sirius red is a strong acid anionic dye. Being not-easyto-fade and specific, sirius red becomes the best dye for collagen staining.Collagen is a major component of extracellular matrix and has some specific physiological functions. Through synthesis and reconstruction of collagen, bone fracture repair will be accomplished.OBJECTIVE： Picric acid-Sirius red stained slides were observed under a polarized light microscopy for evaluation the dynamic changes in the ratio of different collagen types and their distributions in bone fracture healing.DESIGN： It was a controlled observation.SETTING： It was conducted in the Department of Orthopedics, Renmin Hospital, Wuhan University; Department of Traumatic Orthopaedics, Tianjin Hospital; Department of Traumatic Orthopaedics, Jishuitan Hospital,Medical Department, Peking University; Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLAMATERIALS： It was conducted at Tissue Engineering Center of Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA from March 2002 to September 2003. Three healthy adult Chinese sheep, male and in weight from 25 to 35 g, were selected.METHODS： All the animals were anesthesized and sterilized; a transverse osteotomy of the trunk of metatarsus was performed; and the end of fracture was fixed with a six-hole Medoff sliding plate. At the post-operative month 1, 3 and 6, samples were taken from bone fractures. After decalcification with EDTA, they were stained with Picric acid-sirius red, and the types and distribution of collagens were observed under a polarized light microscopy.MAIN OUTCOME MEASURES： Types and distributions of collagens in bone lesion in different period of bone healing were investigated.RESULTS： Three sheep used in this study entered the statistical analysis.①Morphological features of various collagens under a polarized light microscopy postoperatively： Type Ⅰ collagen packed tightly, with a strong refraction and yellow, orange or red thick fibres. Type Ⅱ collagen formed a loose reticulation with fibres exhibiting different colour and a weak refraction. Thin fibres of type Ⅲ collagen with weak refraction and green colour formed a loose reticulation. ②Quantitative studies on various collagens under a polarized light microscopy postoperatively： At postoperative month 1,red or orange fibres （type Ⅰ collagen） were rarely seen in bone fracture,while green fibres （typical of type Ⅲ collagen） were dominant with a disorder pack. At postoperative month 3, red or orange fibres increased significantly and the ratio of type Ⅲ collagen reduced. The collagen fibres assembled regularly. At postoperative month 6, thick yellow-red collagen became dominant and thin green type Ⅲ collagen decreased dramatically and arranged in an obvious oblique, spiral and crossed orientations.CONCLUSION： Picric acid-sirius red stain combined with polarized light microscopy technique is not only capable of identifing type Ⅰ and type Ⅲ collagens in bone fraction, but also can reflect the morphological features,distribution and the ratio of these two type collagens. This approach has the virtues of easiness in operation, strong specificity and high sensitivity.