摘要
第一部分:低剂量X线照射(Low Dose Irradiation, LDI)骨折模型制作中骨折专用造模支架的研制及应用
研究背景和目的:研究大鼠股骨骨折愈合的模型通常有两种:开放截骨模型和闭合折骨模型。开放截骨模型具有截面光整,骨折角度和位置可控等优点,在国内应用较多。但该方法中易于损伤软组织和骨膜、电动工具对骨皮质有热损伤、冲洗带走较多骨髓以及破坏骨折处血运等不利因素,大大增加了骨折愈合的差异,不能再现骨折愈合(特别是重要的骨痂形成期)的真实过程。目前国外广泛采用的是闭合折骨模型,具有手术简便,骨折周围组织破坏小等优点,但需要专用造模支架。为此,我们参考相关文献,改良并制作了专用造模支架,用于不同月龄的大鼠,探索标准股骨骨折闭合模型的制作方法。方法:简化设计并制作骨折造模支架。对80个雄性Sprague-Dawley大鼠(2月龄和3月龄各40只)手术行逆行性克氏针固定股骨,然后使用造模支架制造股骨中段闭合骨折,通过X线摄片、组织学检查了解骨折类型、移位程度以及骨折的愈合情况。结果:除2例术中不明原因死亡外,共78个右侧股骨。骨刀撞击大腿后无表皮裂伤例。摄片证实横断或短斜形无明显移位骨折67例,骨折粉碎6例,长斜形骨折1例,部位偏离中段4例。造模成功率为85.9%。骨折后第1周组织学切片见骨折为横形,除骨折处外其余骨膜基本完整,软组织破坏不明显。第2周骨折两端的骨膜下已出现膜内化骨,骨折端较多纤维性骨痂和软骨性骨痂并存;第3周骨折端软骨性骨痂变少,纤维性骨痂少见,骨折处有大量编织骨骨痂出现;第4周软骨性骨痂已少见,小梁骨骨痂减少,皮质骨出现较多吸收腔隙,并见板层骨;第8周时髓腔已再通,皮质骨以板层骨为主;第12周则与正常骨无异。所有骨折无内固定脱落及骨不连等现象。结论:改良自制造模支架制作大鼠股骨骨折模型操作简便、成功率高、软组织损伤小、能最大限度减少外在因素对骨折的影响。制作的模型呈现典型的骨折二期愈合过程,符合标准闭合骨折模型的要求。
第二部分:LDI促进大鼠骨折骨痂矿化的研究
研究背景和目的:大量研究显示中高剂量X线照射抑制骨折愈合,但是对于低剂量照射后骨折修复的情况尚不清楚。最近有研究表明LDI可以促进缺血肢体的恢复、加速创伤组织愈合。原因是LDI可以促进有利于血管新生的基因的表达而加速例如血管内皮生长因子等的分泌,进而刺激血管新生,加速组织修复。骨折愈合过程中骨痂处血管的新生和重建是骨折愈合的前提和关键,骨痂新生血管的增多能明显促进骨折愈合。因此,本实验应用常规摄片、微焦点CT(μCT)、肢体定量CT(pQCT)、激光共聚焦显微镜(CLSM)、组织学切片以及生物力学测试等对大鼠股骨中段闭合照射骨折模型在不同愈合时期分别进行评测,以研究LDI对骨折愈合是否有益。方法:大鼠随机分为两组低剂量照射组(实验组)和假照射组(对照组)。实验组采用医用直线加速器对大鼠进行总量为1Gy的低剂量X线照射。对照组不予照射。然后马上按照第一部分内容制造单侧标准的股骨中段骨折模型。分别于术后2、3、4、8、12周各处死动物。股骨标本使用常规摄片和μCT观察骨痂桥接情况,pQCT定量分析骨矿含量及骨痂面积,CLSM测定骨痂矿化率,四点弯曲检测骨痂的力学性能并对部分标本作组织学分析。结果:骨折后第2周,低剂量照射组的骨痂面积、骨矿化率、组织学评分及最大负荷均较对照组小(p<0.05);在第3周,则实验组的骨痂桥接评分值、骨矿含量、骨矿化率、组织学评分及三项力学测试参数均大于对照组(p<0.05)。4周后两组的各种检测结果无统计学差别。结论:结果表明尽管LDI在早期(2周内)对骨痂形成有害,但是在随后的硬骨痂形成期则对骨痂形成和矿化具有促进作用。
第三部分:LDI促进骨痂矿化的初步机理
研究背景和目的:第二部份研究显示,LDI对硬骨痂形成期的骨痂形成和矿化有明显的促进作用,表现在骨痂桥接、骨矿含量、骨痂矿化率以及骨痂力学性能的增加。但机理尚不明了。由于骨折愈合好坏主要与血管新生和成骨前体细胞有关。本部份通过对照射骨折模型的周围血细胞的检测间接了解LDI对前体细胞的增殖能力的影响,以及肢体血管固化造影三维重建了解骨痂微血管新生。方法:按第一部分所述造模,造模后的即刻(0.1天)以及7、14、21、28、56、84天分别采血送检血常规。然后对大鼠行腹主动脉以远固化造影,使用μCT扫描骨痂,计算机重建三维血管网以及计算总血管体积、体积分数和平均直径等各项数据。结果:实验组(照射+手术)术后即刻所测白细胞及血小板数均明显下降达67%和41%,对照组(手术但无照射)虽也下降约47%和23%,但降幅比实验组小(P<0.05)。两组随后均缓慢恢复,实验组白细胞在术后第14、21天增幅超过对照组;两组血小板恢复较快,第14天时已正常。血管固化造影见两组随着时间的增长,血管总量及直径均明显上升,骨痂体积增大,骨折线部位界限逐渐模糊。造模后第1周,实验组较对照组血管体积总量和体积分数值低,但无统计学差异。造模后第2周,实验组血管体积总量及血管体积分数明显高于对照组。造模后第3周两组各参数无明显差异。结论:结果显示低剂量全身照射后大鼠骨折模型早期血细胞减少、后期则代偿性增生;照射后骨痂部位血管体积总量、体积分数增加。证实了LDI可动员前体细胞的增殖并促进骨痂微血管新生。
PartⅠEstablishment of a standard closed fracture model on rat femur
Introduction: Two distinct animal models, open osteotomy and closed fracture are used to study fracture healing. The results from studies using osteotomy models are often extrapolated to explain fracture repair, even though the healing process is affected by many variables including the extent of soft and hard tissue damage, vascular supply, and hematoma formation. A smooth cut surface with controlled angularity can be consistently reproduced with osteotomy, but it is accompanied by laceration of the peri-osseous soft tissues, including the periosteum, and by thermal damage to the cortical bone. While the closed fractures are easy to be established on small animals as rats with a special apparatus. Objective: To develop a technique for the production of a standard closed experimental fracture on rat femurs with a custom-built fracture apparatus. Methods: A reformed simplified fracture apparatus was designed and tested on 78 male Sprague-Dawley rats. First, the femur was treated with an intramedullary Kirschner’s wire retrogradely through a medial parapatellar approach. The femoral diaphysis was then fractured by means of a blunt guillotine driven by a dropped weight. Results were assessed by plain radiographs and histographs. Results: A highly reproducible transverse fracture with a success rate of 85.9% was produced. Minimal comminution and angulation of the intramedullary wires were found radiographically. And after 12 weeks follow up, a typical secondary fracture healing process was observed histologically. Conclusions: The reformed apparatus is simple to build and easy to use. Through its application, a highly reproducible closed fracture model with minimal soft tissue damage is established.
PartⅡLow Dose X-irradiation Promotes Mineralization of Fracture Callus in A Rat Model
Introduction: X-irradiation at high dose impairs fracture healing. On the contrary, it has been reported that X-irradiation at low dose (LDI) promoted tissue revascularization, which critically precedes callus mineralization during fracture healing process. However, it still remains unclear whether LDI exerted beneficial effect on fracture callus mineralization, which provides local mechanical stability for early rehabilitation of fracture patients. Objective: To investigate the effect of LDI on fracture callus formation and mineralization evaluated using radiography, micro-CT, peripheral quantitative computed tomography (pQCT) and biomechanical test in a standardized rat femoral fracture model. Methods: 120 male Sprague-Dawley rats were subjected to standard closed fracture on right femur according to the way of the first part. Sixty rats of which were irradiated with low dose X-ray (1Gy as LDI group) for total body irradiation right before fracture induction. The remaining 60 rats without irradiation is regarded as SHAM-Group . The animals were euthanized at different time points: 2, 3, 4, 8 and 12 weeks post fracture. Fracture callus was assessed by using radiography and MicroCT for callus bridging, peripheral quantitative computed tomography (pQCT) for quantifying bone mineral content (BMC) and cross sectional area (CSA), confocal laser scanning microscopy for measuring area fraction of fluorescence labeling (AFFL), and four-point bending test for examining mechanical properties. Results: In both groups, the pQCT parameters of fracture callus continuously increased post operation and peaked at 3 weeks, then decreased at 4 weeks and kept stable at 8 and 12 weeks. With different pattern, the mechanical parameters continuously increased throughout experimental period.The CSA and AFFL were found 22% and 33% smaller in the LDI group compared to the SHAM group at 2 weeks (P<0.05 for both), whereas the BMC and AFFL were 15% and 34% higher in the LDI group at 3 weeks (P<0.05 for both). The changing patterns were consistent with the findings in 3-D MicroCT reconstructions. The mechanical parameters (Max-Load, Stiffness and Energy) were also 18%, 30% and 24% higher in the LDI group than in the SHAM group at 3 weeks (P<0.05 for all). At 4, 8 and 12 weeks, there was no difference found for all assessments between the two groups. The reduced CSA in the LDI-Group at 2 weeks could be explained by an acute inhibition effect of LDI on repairing progenitors, whereas the significantly enhanced mineralization in the LDI-Group at 3 weeks could be explained by delayed stimulation effect of LDI on mobilization of repairing progenitors and expression of growth factors required for angiogenesis hence osteogenesis reported by others. Conclusion: The results indicated LDI promoted mineralization at the stage of hard callus formation in a rat fracture model.
PartⅢThe Possible Mechanisms of Promoted Callus Mineralization Induced by LDI
Introduction and Objective: At the study of second part, the results indicated LDI promoted mineralization at the stage of hard callus formation in a rat fracture model. Nevertheless, the possible mechanisms kept unclear. Fracture repair is highly related with blood flow and the number of osteoblastic progenitors, the present study will focus on assessing callus angiogenesis at a macroscopic level by vascular perfusion and MicroCT evaluation, and at the same time, examine the routine blood cells to know about the osteoblastic progenitors with an indirect way。Methods: Low dose irradiated fracture models on rats were established according to the method of the second part. Peripheral blood was assessed for blood cell accountings at the following intervals: immediately after model establishment (0.1 day), 7, 14, 21, 28, 56 and 84 days. After that, blood vessel casting with a radiopaque silicone rubber compound containing lead chromate and MicroCT scanning were performed on randomly selected rats(n=4). 3-D vascular images were reconstructed and then calculated for vessel volume, average diameter and vessel volume fraction. Results: The WBC and platelet numbers decreased 67% and 41% respectively right after LDI and fracture (LDI group). While those of the control groups(fracture without LDI) decreased much less than those of LDI group (P<0.05). The blood cells recovered slowly in both groups. On day 14 and 21, WBC increased more in LDI group than in control group. The platelet recovered much faster in both groups and on day 14 post operation the plated numbers have reached normal. At 1 week after model establishment, MicroCT images visibly showed reduced early neovascularization in LDI group, whereas at 2 weeks, the quantitative analysis revealed increased vessel volume and volume fraction in LDI group. No significant difference was found at 3 weeks though. Conclusions: LDI can induce cell mobilization and neovascularization on a rat fracture model, which, were the possible explanations for the accelerated callus mineralization.
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