Examination of dynamic changes of different-type collagens in bone fracture healing with a polarized light microscopy / 中国组织工程研究

ABSTRACT

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.