显微CT多阈值分析评价骨折愈合效果

Evaluation of bone fracture healing by micro-CT analysis with multiple thresholds

目的研究显微CT多阈值分析对骨折愈合过程中不同类型骨组织表达的可靠性,为采用显微CT影像学方法有效评价骨折愈合奠定实验基础。方法制备大鼠股骨中段闭合性骨折模型,5 w后采用显微CT对骨折部位进行扫描,分别于225～700(代表全骨组织),225～330(代表钙化软骨和骨痂)和331～700(代表成熟骨组织)3个阈值范围内,测量和分析骨折标本的骨体积(bonevolume,BV)、组织体积(tissue volume,TV)和骨体积分数(BV/TV)。然后,对标本进行四点弯曲生物力学测试,分别测量和计算标本的刚度(stiffness,S)和弹性模量(elastic modulus,E)。最后,制作上述标本的不脱钙组织切片,分别行苏木精-伊红(H&E)和Von Kossa(VK)染色,并采用Osteomea-sure软件对切片的骨体积分数(bone volume/tissue volume,BV/TV)进行测量和分析。上述实验均以正常大鼠股骨作为对照。结果显微CT重建:实验组骨折已完全连接,髓腔再通。显微CT测量结果示:在225～700阈值内,实验组BV值与正常对照组无显著差异(P>0.05),其BV/TV也略低于正常对照组(P=0.054);在225～330阈值内,实验组BV和BV/TV均明显高于正常对照组(P<0.05);在331～700阈值内,实验组BV和BV/TV明显低于正常对照组(P<0.05)。生物力学测量示:实验组S和E均明显低于正常对照组(P<0.05)。H&E染色示:实验组骨折已完全被新生组织桥接修复,但修复组织多为软骨样或编织骨组织。VK染色示:实验组骨折部位填充组织钙盐含量较低,部分组织VK染色阴性。采用Osteomeasure软件对VK染色阳性区域进行测量和统计学分析示:实验组BV/TV明显低于正常对照组(P<0.05)。结论①采用显微CT单阈值分析会将部分不成熟的骨组织成分当做正常骨组织进行评价,造成对骨修复效果和骨强度误判。②通过显微CT多阈值分析可以明确表示不同的骨质成分,相关结果与组织学和生物力学检测存在较好的依从性。③采用显微CT多阈值分析技术能够有效评价骨折愈合效果。

Objective To investigate the feasibility of micro-CT analysis with multiple thresholds for the expression of different types of bone tissues during bone fracture healing and toprovide an experimental ba- sis for effective evaluation of bone fracture healing by micro-CT method. Method A rat model of closed transverse mid-diaphyseal fracture in the right femur was established using a three-point guillotine-like bending device and Micro-CT scan was performed for each fractured region 5 weeks later. Different thresh- olds,225 ~ 700, 225 ~ 330 and 331 ~ 700 were programmed into the micro-CT software to include all min- eralized tissue （whole bone）, callus and calcified cartilage, very dense cortical bone, respectively. Bone volume （BV）, tissue volume （TV） and bone volume fraction （BV/TV） were measured and analysed with the above three thresholds. Then a four-point bending was used to assess the mechanical properties including stiffness （S） and elastic modulus （E）. Finally, undecalcified samples of the fracture healing areas were sectioned and stained with hematoxylin-eosin （H＆E） and Von Kossa （VK） methods. Bone volume fraction （BV/TV） was measured using the Osteomeasure software. Result Fractures in the ex- perimental group were completely united and the bone marrow cavities were recanalization revealed by mi- cro-CT scan. with the threshold of 225 -700, there was no significant difference of BV between the ex- perimental and control groups （P 〉 O. 05 ） and the BV/TV of the former was a little lower than that of the later （P = O. 054）. With the threshold of 225 ~ 330, both the BV and BV/TV of the experimental group were significantly higher than those of the control group （ P 〈 0.05 ）. In contrast, with the threshold of 331 ~700, the BV and BV/TV of the experimental group were significantly lower than those of the control group （P 〈 0. 05 ）. The S and E of the experimental group were obviously lower than those of the control group （P 〈 0. 05）. The fractures of the experimental group were completely repaired as evidenced by H＆E staining. By the VK staining, the newly formed tissues in by new formed tissues the fracture area were proved to contain less calcium. Based on the Osteomeasure analysis with VK staining, the BV/TV in the ex- perimental group was significantly lower than that of the control group （P 〈 0. 05）. Conclusions （1） The difference between immature and mature bone tissues cannot be distinguished by micro-CT analysis with a single threshold, which will cause the misjudgment of bone repair and bone strength. （2） Different types of bone tissues can be revealed with multiple thresholds which show better compliance with histological and biomechanical testing. （3） Micro-CT analysis with multiple thresholds can be used for effective evaluation of fracture healing.