【作者】 向明；

【导师】 邓友章；

【作者基本信息】 成都中医药大学 ， 中医骨伤科学， 2013， 博士

【摘要】 目的：本研究利用自行研制的微型外固定架,制备标准兔股骨骨折端微动模型,通过动态观察各时间点的X线表现,了解骨痂的大小以及生长情况,检测骨痂骨密度(BMD)和骨痂力学刚度、分析组织学和免疫组化结果,探讨不同时间开始可控性微动对骨折愈合的影响及机制。方法：本课题制备标准兔股骨骨折模型48只,随机平均分成四组(每组12只),第一组：持续固定组：兔股骨骨折模型建立后,用单边双杆外固定架固定,骨折断端间持续固定不诱导微动；第二组：即刻微动组：兔股骨骨折模型建立后,用单边双杆外固定架固定,骨折断端间即刻通过两连接杆之间的滑动而诱导0.5mm的微动。第三组：1周微动组：兔股骨骨折模型建立后,用单边双杆外固定架固定,骨折断端间于固定1周后通过两连接杆之间的滑动而诱导0.5mm的微动。第四组：2周微动组：兔股骨骨折模型建立后,用单边双杆外固定架固定,骨折断端间于固定2周后通过两连接杆之间的滑动而诱导0.5mm的微动。术后1、2、3、5周摄取股骨正侧位片,观察骨痂生长情况,并用外骨痂及骨折线模糊程度进行评分；术后5周分批处死动物,每组各3只,取出完整股骨干后,清除其周围软组织,纱布包裹,生理盐水浸透后,用双层塑料袋封存于-20。C冰箱内。检测时标本解冻后,通过8874型Instron生物力学试验机测量骨折断端处骨痂的最大载荷、挠度和刚度；在进行生物力学之前,测量骨痂的大小,并用GE Prodigy双能X线骨密度测定仪测定骨折处的骨密度。术后1、2、3周处死动物,在每时间点每组处死3只动物,以骨折断端为中心,切取1cm长标本,单面刀片将骨标本分割成0.5cm×0.2cm×0.2cm小块,生理盐水冲洗干净后,立即置入4%多聚甲醛中固定72小时,然后脱钙、包埋、制成石蜡切片,HE染色及其它染色观察骨痂的相关组织学形态并用免疫组织化学方法在蛋白质水平上检测骨痂中骨钙素和Ⅱ型胶原(osteocalcin (oc) and collagen type II(col2))的表达和分布。结果：一、X线观察：2周后即刻微动组骨折断端间的外骨痂较持续固定组明显增多,但骨折线模糊程度和桥接骨外骨痂形成却晚于持续固定组,而1周和2周微动组骨折断端间的外骨痂较持续固定组明显增多,骨折线模糊程度和桥接骨痂形成加快。按外骨痂和骨折线模糊程度半定量标准进行评分后统计学分析证实：2周时即刻微动组、1周微动组骨折处外骨痂及骨折线模糊程度评分较持续固定组和2周微动组明显增高(P<0.05),3周和5周时即刻微动组外骨痂及骨折线模糊程度评分明显低于持续固定组(P<0.05),1周和2周微动组外骨痂及骨折线模糊程度评分明显高于持续固定组(P<0.05)微动的时间效应对实验组动物骨折愈合有不同的影响,即刻微动组虽骨折处外骨痂多于其它各组,但骨折线模糊程度却明显慢于其它组；1周和2周骨折处外骨痂多于持续固定组而少于即刻微动组,但骨折线模糊程度却快于持续固定组和即刻微动组。二、骨痂大小的测量：5周时即刻微动组外骨痂前后径和内外径较持续固定组明显增加(P<0.05),而1周和2周微动组外骨痂前后径和内外径虽小于持续固定组却无统计学意义(P>0.05)。三、骨密度测量结果：5周时即刻微动组骨折处骨密度值及比率与持续固定组相比无显著差异(P>0.05),而1周和2周微动组骨折处骨密度值及比率高于正常水平,较持续固定组显著增高(P<0.05)。四、生物力学测量结果：术后5周骨痂的最大载荷1周微动组和2周微动组均显著高于持续固定组、即刻微动组(P<0.05)；而即刻微动组其最大载荷则明显低于持续固定组(P<0.05)。术后5周骨痂的挠度各组间均无明显差异(P>0.05)。术后5周骨痂的刚度1周微动组显著高于其它各组(P<0.05),2周微动组其刚度则显著高于即刻微动组和持续固定组(P<0.05),而即刻微动组和持续固定组之间其刚度无明显差异(P>0.05)。五、组织学观察：术后一周：四组均见骨折断端间血肿机化,大量细胞聚集在骨折断端间及周围而形成纤维骨痂,可见间充质细胞、成纤维细胞、成骨细胞以及粒细胞、单核—巨噬细胞等炎性细胞和新生的毛细血管,骨折断端的骨膜外间充质细胞增殖、分化为软骨细胞与成骨细胞而形成新生骨小梁,在骨膜下成骨区域的边缘,成骨细胞增殖活跃,出现1-2层的成骨细胞,开始出现软骨细胞,分泌软骨基质,并且可见即刻微动组新生的软骨细胞明显多于其它组。各组均可见出现少量膜内骨性骨痂。术后两周：骨折断端间已有部分软骨骨痂连接。可见四组均在骨膜下形成的骨小梁与软骨细胞交界处出现钙化前缘,软骨内成骨开始,一周微动组可见较多的破骨细胞,成骨细胞更加活跃,出现3-4层的成骨细胞；而即刻微动组新生的骨小梁较少,软骨细胞成熟较慢,软骨基质较少,仅见少量的软骨内成骨。一周微动组已可见少许编织骨存在。术后三周：持续固定组和2周微动组相似,可见破骨细胞出现,纤维性骨痂内的软骨岛不断扩展,血管周围多为体积较小的圆形幼稚软骨细胞,软骨痂周边部的软骨细胞肥大,可见变性坏死的软骨细胞被原始骨小梁所取代。一周微动组仍有较多的破骨细胞,成骨细胞活跃,软骨内成骨活跃,可见较多的编织骨膜性骨痂延伸到骨折端,其内血管增生明显,部分骨痂已越过骨折间隙。而即刻微动组软骨细胞成熟较慢,软骨内骨化延迟,新生的骨小梁较稀疏,尚无骨痂越过骨折断端。六、免疫组织化学观察各组外骨痂中的骨钙素和Ⅱ型胶原染色强度随时间延长而均有不同程度的增加；术后一周时,即刻微动组骨钙素染色强度明显弱于1周微动组(P<0.05),而Ⅱ型胶原染色强度强于其余各组(P<0.05)；术后二周时,即刻微动组骨钙素染色强度明显弱于持续固定组、1周微动组和2周微动组(P<0.05),但持续固定组Ⅱ型胶原染色强度明显弱于即刻微动组和1周微动组(P<0.05)：术后三周时,即刻微动组骨钙素染色强度明显弱于持续固定组、1周微动组和2周微动组(P<0.05),而1周微动组骨钙素染色强度明显强于持续固定组(P<0.05),持续固定组Ⅱ型胶原染色强度明显弱于即刻微动组和1、2周微动组(P<0.05)。结论：1.微动的时机是影响骨折愈合的一项重要因素,不同时间点开始可控性微动有不同的时间效应,过早和过晚的微动均不利于骨折的愈合。2.即刻微动虽然有最多、最大的外骨痂,但其愈合时间、骨折处的骨密度和愈合组织的刚度却逊于持续固定组。3.1周和2周开始微动能明显增加骨折处的外骨痂量、骨折线模糊程度,加快桥接骨痂的形成。4.1周和2周开始微动能明显增加骨折处外骨痂的骨密度和愈合组织的刚度。5.1周和2周微动组可见成骨细胞活跃,甚至出现3-4层的成骨细胞,软骨细胞成熟、肥大以及矿化加快,使软骨内成骨过程加快。即刻微动组虽然早期刺激软骨细胞的形成,但软骨细胞的成熟、肥大和矿化则反而减慢。6.各组外骨痂中的骨钙素和Ⅱ型胶原染色强度随时间延长而有不同程度的增加；即刻微动组骨钙素染色强度明显减弱；术后三周时,1周微动组骨钙素染色强度明显强于持续固定组；持续固定组Ⅱ型胶原染色强度明显弱于微动各组。7.1周和2周开始微动除了使成骨细胞活跃外,还能增加破骨细胞数量和密度,明显加快软骨细胞的成熟和矿化；成骨细胞和破骨细胞耦合作用的加强促进了软骨痂向硬骨痂转化以及硬骨痂的重塑。更多还原

【Abstract】 Objective:In this study, we prepared the experimental micromovement model of rabbit femur fracture using self-designed mini-external-fixator. and realize the size and formation of callus through dynamic observation of X-ray’s performance on different time point, and detected the BMD and callus mechanical characteristics, and analyzed the histology and immunohistochemistry results in order to tried to explore the influence and mechanism of time effect of the controlled micromovement on fracture healing.Methods:we prepared48experimental micromovement model of rabbit femur fracture, which were divided randomly into four groups(with12rabbits for each group), all were fixed with unilateral external fixator connected with two bars. Group one:continuing immobilization group, There exists no micromovement between the fragment after the fracture model was made and fixed with unilateral two bars external fixator. Group two:instant micromovement group, the0.5mm micromovement was induced immediately by the sliding between the two connection bars. Group three:micromovement began after1week, the0.5mm micromovement was induced after1week’s immobilization. Group four:micromovement began after2week, the0.5mm micromovement was induced after2week’s immobilization. The AP and lateral view of the femur were obtained at1、2、3and5weeks postoperatively, and observe the callus formation according to the score of the external callus formation and the vagueness level of fracture line. After5weeks postoperatively, all animals were executed in batch, with3animals out of each group, the integrated femurs were obtained and draped with sterile bandage after all soft tissues removed, saturated with saline, sealed with double layer plastic envelope, contained in refrigerator under-20℃circumstances. The specimens were thawed and contained in saline for2hours, then the maximum load and rigidity of the callus were measured using8874-Instron biomechanical testing machine. The size of the callus was measured and the bone mineral density surrounding the fracture site were measured using GE Prodigy double-energy X-ray bone densitometry machine before the biomechanical test. Three animals were executed on every observation time point in each group after1,2and3weeks postoperatively,1cm long specimen was abtained, centering on the fracture site, then divided into pieces about0.5cm×0.2cm×O.2cm using single plane razor blade, washed up with saline and put in4%paraformaldehyde fixed for72hours. Paraffin sections were made after decalcification and embedding. Histology of callus was observed after HE and others staining and expression and distribution of osteocalcin(oc) and collagen type Ⅱ(col2) were analyzed with immunohistochemical methods.Results:1. X-ray observations:After2weeks, the external callus increased obviously in Group two comparing with Group one, while the vagueness level of fracture line and bridging callus formation occurred later than the latter. The external callus increased obviously in Group three and Group four than that in the Group one, while the vagueness level of fracture line and bridging callus formation accelerated in the former groups.Statistically analysis was carried out after semiquantitative score was done according to the external callus formation and the vagueness level of fracture line. Two weeks after experiment was done, the score of the external callus formation and the vagueness level of fracture line were increased obviously in Group two and Group three than that in Group one and Group four(P<0.05). While three and five weeks after experiment, the score of the external callus formation and the vagueness level of fracture line were obviously lower in Group two than that in Group one(P<0.05), on the other hand, the scores were much higher in Group three and Group four than that in Group one (P<0.05)The time effect of controlled micromovement had various influence on the fracture healing of experimental animals. Although the external callus in Group two was more than that in the other Groups, but the vagueness of fracture line was obviously lower than other Groups. While in Group three and Group four, the external callus was larger than that in Group one but less than that in Group two. but the vagueness of fracture line was faster in Group three and Group four than that in Group one and Group two.2. Measurement of callus sizeFive weeks after experiment was done, the anteroposterior diameter and the exterior and interior diameter of the callus were obviously larger in Group two than that in Group one(P<0.05),but the diameters in Group three and Group four were smaller than that in Group one, although there was not statistically significant.3. Measurement result of bone mineral density.There was no significant difference of bone mineral density value and proportion rate in tne fracture site between Group two and Group one(P>0.05).But the value and proportion rate in tne fracture site were notably higher in Group there and Group four comparing with that in Group one (P<0.05)4. Biomechanical measurement results.The maximum load of the callus five weeks postoperatively was significantly higher in Group three and Group four than that in Group one and Group two(P<0.05), while the maximum load of the callus was notably lower in Group two than that in Group one (P<0.05). The deflection five weeks postoperatively was no difference between groups (P>0.05).The callus rigidity five weeks postoperatively was significantly higher in Group three than that in the other groups (P<0.05), while the callus rigidity was higher in Group four than that in Group one and Group two(P<0.05), but there was no difference between group one and two (P>0.05)5. Histology observation.One week postoperatively:organization hematoma between the fracture sides could be seen in all four experimental groups. Mass of cell aggregated together around the fracture ends to form fibrous callus, lots of inflammatory cells like mesenchyme cell、fibroblast、osteoblast、granulocyte、mononuclear macrophage and neonatal capillaries could be seen. Mesenchyme cells beyond the periosteum proliferated, differentiated to chondrocytes and osteoblasts, then to form neonatal bone trabecula. Osteoblasts proliferated actively in the margin of ossification zone under the periosteum, appearing1-2layers of osteoblasts. Chondrocytes began to appear, secreting cartilage matrix. And the neonatal chondrocytes in Group two were more than that in the other groups obviously. A small quantity of intramembrane osseous callus could be seen in all four experimental groups.Two weeks postoperatively:Partial cartilaginous callus connected between the fracture ends. Calcification front appeared in the juncture of chondrocytes and bone trabeculas formed under periosteum in all four groups. Endochondral ossification began to start, lots of osteoclasts occurred in Group three, osteoblasts activated all the more, appearing3-4layers of osteoblasts. Less neonatal bone trabeculas appeared in Group two, chondrocytes matured slower with fewer endochondral ossification. A few of woven bone presented.Three weeks postoperatively:similar to Group Four, osteoclasts presented in Group One, cartilage islands in the fibrous callus expanded gradually, majority of the chondrocytes surrounding blood vessel were spherical naive one with small volume. Chondrocytes arounding cartilaginous callus got to hypertrophy, and degenerated necrotic chondrocytes were substituted with primitive bone trabeculas. Lots of osteoclasts were still to remain in Group three, osteoblasts and endochondral ossification still activated, much woven periosteous callus extended to the fracture ends, with apparent blood vessel hyperplasia, partial callus crossed the fracture gap. While in Group two, chondrocytes matured slowly, endochondral ossification delayed, with sparse neonatal bone trabeculas and no callus crossing the fracture gap.6. Immunohistochemistry observation.Different degree increase of Osteocalcin and Collagen II were observed along with time prolonged in each group. One week postoperatively, The stain degree of Osteocalcin in group two was distinctly weaker than group three (P<0.05),while the Stain degree of Collagen II was distinctly stronger than other three groups (P<0.05); Two week postoperatively, The stain degree of Osteocalcin in group two was distinctly weaker than other three groups (P<0.05),while the stain degree of Collagen II in group one was distinctly weaker than group two and three (P<0.05):Three week postoperatively. The stain degree of Osteocalcin in group two was distinctly weaker than other three groups (P<0.05), and the stain degree of Osteocalcin in group three was distinctly stronger than group one (P<0.05),while the stain degree of Collagen Ⅱ in group one was distinctly weaker than other three groups (P<0.05)Conclusion:1. Occasion of micromovement is an important factor to influence the fracture healing, different time effect related to different onset of controlled micromovement. Micromovement early or late is unfavourable to fracture healing.2. Instant micromovement group gets inferior results to persistent fixation group, including healing time, bone mineral density in the fracture site and callus rigidity,although it has maximal and largest external callus.3. Micromovement begins on one and two weeks can significantly raise external callus formation and vagueness level of fracture line, accelerating bridging callus formation.4. Micromovement begins on one and two weeks can significantly raise bone mineral density and rigidity of callus.5. Micromovement begins on one and two weeks activated the osteoblasts even makes3-4layers of osteoblasts appeared,It also accelerates the maturity> hypertrophy and mineralization of chondrocyte,resulting in the stimulation of the fracture healing through endochondral ossification. Though the instant micromovement stimulate the formation of chondrocyte in the early stage,but slower the process of the maturity and hypertrophy and mineralization of chondrocyte6. Different degree increase of Osteocalcin and Collagen Ⅱ were observed along with time prolonged in each group.In instant micromovement group, The stain degree of Osteocalcin was distinctly weaker; Three week postoperatively, The stain degree of Osteocalcin in one week postoperative micromovement group was distinctly stronger than continuing immobilization group, while the stain degree of Collagen Ⅱ in continuing immobilization group was distinctly weaker than other three groups.7. Except that the micromovement begins on one and two weeks activated the osteoblasts, it seemingly can improve the amount and density of osteoclasts in callus to stimulate the maturity and mineralization of chondrocyte; The strengthening coupling of osteoblasts and osteoclasts can promote the transformation from soft callus to hard callus and the remolding of hard callus.更多还原