骨碎补总黄酮促进股骨缺损牵张成骨新骨形成的实验研究
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摘要
目的：
牵张成骨技术已经在临床应用几十年,多用于治疗各种原因引起的双下肢肢体不等长畸形,如创伤性骨缺损、骨骺损伤、骨肿瘤和骨感染等导致的骨缺损、脊髓灰质炎后遗肢短畸形,以及先天性肢短畸性和肢体肥大症等。牵张成骨术以其独有的成骨方式和其他技术无可比拟的临床效果,一直备受医学界的青睐。近年来,随着牵张器不断在改进,手术设计的不断完善,该技术得到了突飞猛进的发展,在临床应用的范围也越来越广。但是临床上主要应用于胫骨,文献已有较多报道。这主要是因为胫骨延长技术相对简单,但在某些特定的情况下对患者只能实施股骨延长术,文献已有报道股骨延长病例,治疗效果令人满意,但是治疗过程也同样存在不足,如病程长,骨形成不良等。
本研究通过自行设计用于家兔股骨的牵张支架,建立家兔股骨缺损牵张成骨模型,在牵张成骨过程中应用中药骨碎补总黄酮,可以促进股骨骨缺损牵张成骨(DO)过程中新骨的形成、矿化及塑型改建,加速牵张成骨新骨形成的速度以及成骨质量,从而可以缩短牵张成骨的治疗周期,从而为临床上运用股骨延长术寻找一种快速有效的促进成骨的治疗方法。
方法：
一、自行设计牵张支架系统,建立家兔股骨缺损牵张成骨模型。取兔股骨标本,肉眼观察牵张间隙内新生骨痂生长情况及进行X线观察。
二、将42只新西兰大白兔随机分模型组、治疗组(骨碎补总黄酮)、对照组(rhBMP-2)三组。所有动物行双侧股骨手术截骨,截骨长度为1.4cm,每组14只兔(N=14),28个股骨标本(n=28)。分别在固定第14天、28天取材,标本通过：1、一般情况及大体观察；2、X线观察；3、MICRO-CT定量骨扫描；4、生物力学测试；5、RT-QPCR检测目的基因BMP-2mRNA水平；6、组织学光镜观察；7、扫描电镜观察等指标进行评价新骨形成速度及质量。
结果：
一、牵张成骨模型大体标本观察
随着时间推移,肉眼可分辨牵张间隙内骨痂生长由少到多,逐渐钙化；
固定第7天：牵张间隙处新生骨组织颜色鲜红,与正常骨两端界限非常清晰,两端骨痂生长较多,中间尚未生长；
固定第14天：牵张间隙内新生骨组织颜色变浅,较前致密、匀称,呈圆柱状贯通骨折两端,与正常骨两端界限较前模糊；
固定第28天：牵张间隙内可见新生骨组织表面与正常骨颜色、纹理相同,肉眼较难以分清正常骨组织和牵张间隙新生骨区的界限,骨密度明显增高。
二、牵张成骨模型X线观察
术后当天即刻行X光检查,牵张支架固定稳定,骨折两端对位对线可；
牵张结束行X光：牵张间隙远端可见有云雾状阴影；固定第7天：牵张间隙远端云雾状阴影较前加深,范围扩大；固定第14天：牵张间隙内可见连接正常骨两端的云雾状阴影,密度进一步增高；固定第21天：牵张间隙内已有明显的新生骨组织连接正常骨两端；固定第28天：新生骨组织钙化完全,骨皮质完全形成,髓腔畅通。
三、治疗后大体标本观察
固定第14天：肉眼观察下三组新生骨组织从质量或者硬度上由高到低排列为：对照组>治疗组>模型组。
固定第28天：肉眼观察下可初步判断,成骨质量由高到低排列为：对照组>治疗组>模型组。
四、治疗后X片观察结果
各组手术当天X光侧位片显示牵张支架固定稳定,骨折端对位对线可。
牵张结束当天：模型组牵张间隙无明显阴影；治疗组及对照组有新骨及类骨质形成。
固定第7天：模型组牵张间隙远端可见较明显的云雾状阴影；治疗组牵张间隙内云雾状阴影较前加深,贯穿股骨两骨折端；对照组牵张间隙内圆柱状阴影较前加深,近端部分皮质密度增高,部分钙化。
固定第14天：对照组牵张间隙内阴影明显加深,并出现骨皮质阴影；但治疗组及模型组新生骨骨密度较对照组低。
固定第21天：新生骨密度由低到高为模型组、治疗组、对照组。
固定第28天：对照组及治疗组新生骨骨密度明显高于模型组。
五、治疗后Micro-CT测试结果
模型组、治疗组、对照组分别在固定第14天和28时取材进行MICRO-CT图像比较和定量骨扫描。
MICRO-CT的扫描图像上看,固定第14天：对照组的成骨与骨塑型均明显强于模型组与治疗组,治疗组的成骨与骨塑型稍好于模型组；固定第28天：对照组与治疗组的成骨与骨塑型均强于模型组,对照组的成骨与骨塑性稍好于治疗组。
BMC、TMC、BMD、TMD、Tb.N、Tb.Pf等指标,固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组同模型组及对照组无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
BV/TV在固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。固定第28天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。
Tb.Sp、Tb.Th、SMI其中固定第14天：各组间无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
六、治疗后力学测试结果
截面惯性矩、最大载荷、最大挠度、弹性模量、弹性应力、最大应变等指标,固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组同模型组及对照组比较无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
弹性载荷、刚性系数、弹性应变等指标,固定第14天：治疗组、对照组与模型组,治疗组与对照组有统计学意义(P<0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
弹性挠度,固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。固定第28天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。
最大应力,固定第14天：各组间无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
七、治疗后RT-QPCR检测目的基因BMP-2mRNA水平
固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组同模型组及对照组无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组之间有统计学意义(P<0.05),治疗组与对照组之间无统计学意义(P>0.05)。
八、治疗后组织学光镜观察结果
固定第14天：牵张间隙内有大量成骨细胞分布,对照组延长区见少量纤维组织,新生骨小梁延牵张方向生长。治疗组及模型组延长区可见大量纤维组织,新生骨小梁较少。固定第28天：在牵张间隙内可见有大量新骨形成,治疗组及对照组牵张间隙内基本被延牵张方向排列的新生骨小梁充填,排列密集,散在分布纤维区带；模型组牵张间隙内新生骨小梁排列相对稀疏,纤维组织较多。
九、治疗后扫描电镜观察结果
固定第14天：对照组可见大量沿牵张方向排列的胶原纤维束,其间可见很多新生类骨质开始形成；模型组及治疗组见到大量沿牵张方向排列的胶原纤维束,新生类骨质则较少。固定第28天：对照组及治疗组可见骨表面光滑平整,骨小梁较为致密,钙化较完全,基本接近正常骨表面；模型组骨小梁较纤细,没有前两组骨小梁成熟,胶原纤维束含量较多,骨表面尚不平整钙化不完全。
结论：
1、通过进行动物实验证明自行设计的单臂式牵张支架其结构稳定、使用方便、价格便宜、手术操作简单,成功率高等特点。
2、将牵张支架应用于兔股骨牵张成骨实验,通过设计合理的手术步骤以及规范的术后牵张成骨操作,能够顺利建立兔股骨牵张成骨模型。
3、应用骨碎补总黄酮在牵张成骨中能够起到加快新生骨组织的塑形与重建的作用,且用药方便,为临床缩短骨牵张疗程提供了一种方便、经济的方法。
Objective:
Distraction osteogenesis (DO) adopted in clinical applications for several decades has been used to treat leg length discrepancy caused by various disease such as traumatic bone defect, epiphyseal injury, and bone defect caused by bone tumors and bone infection, short limb deformity caused by polio heritage, and Congenital short limb deformities and limb hypertrophy and so on.Distraction osteogenesis gains great popularity in clinical applications for its unique way of osteogenesis and incomparable clinical efficacy Compared with other techniques. In recent years, with the constantly improving of distractor and the continuous improvement of the operation design, the technology has been developing by leaps and bounds, the scope also more and more widely in clinical application.But clinical application is more mainly in tibia, many of literatures have been reported. Mainly because the tibic lengthening is relatively simple, but in some specific cases the femoral lengthening must be used to patient, femoral lengthening cases has been reported in literature, the treatment effect is satisfactory, but the treatment process also exists shortage, such as long duration and poor bone formation, etc.
In our study, the rabbit model of bone defect was treated by distraction osteogenesis with a novel distractor invented by us. Total flavonoids from rhizoma drynariae was used during the process of DO, in order to achieve accelerated the new bone formation, mineralization and model reconstruction of femur bone defects during the process of DO, accelerate the speed of new bone formation and osteogenesis quality, which can shorten the entire treatment cycle, so as to provide femoral distraction osteogenesis an efficient and effective method by promoting the process of osteogenesis.
Methods:
一、The distractor was designed and made, then, it was applied on the rabbit model of femoral bone defect. The femoral samples of rabbits were taken and assessed by the callus of distracted zone observed by perusal and X ray.
二、42new zealand white rabbits was randomly divided into three experimental groups:model group, treatment group (intervened by Total flavonoids from rhizoma drynariae and control group(intervened by rhBMP-2). The osteotomy length was1.4cm in all the experimental animals.14rabbits in each group (N=14),28femoral specimens (N-28). The speed and quality of new formatting bone was observed and assessed by:1. general observation;2. X ray;3.scanning of Micro CT;4. Bone biomechanical testing;5.detecting the expression level of BMP-2mRNA gene by RT-QPCR.5. observing histologic features by light microscopy.7. observation by scanning electron microscope.
Results:
一. Gross specimen observation of the model of DO
Over time, the naked eye can distinguish callus growth from less to more in the area of distraction, and gradually calcification;
At7days of consolidation, the new bone have a bright red color in the area of distraction, the normal boundaries of both ends of the bone is very clear, both ends of the callus growth more, the middle has not been grown;
at14days of consolidation, the color of the new bone is becoming lighter、 compact, well-proportioned in the area of distraction compared with the previous. Fracture callus was cylindrical through both ends of the fracture, the normal boundaries of both ends of the bone is fuzzy over the previous;
At28days of consolidations, the surface of New bone have normal bone color and same texture in the area of distraction, the naked eye is more difficult to distinguish the boundaries between normal bone and new bone, bone mineral density was significantly higher.
二、X-ray observation of the model of DO
X-ray inspection was instantly taken after operation, the distractor was fixed stably, counterpoint is good to the line.
X-ray inspection was taken at the end day of the distraction, cloudy shadows was visible at the Remote of distraction gap; at7days of consolidation, cloudy shadows was deepen and extended;At14days of consolidation, the cloudy shadows connected the two ends of the normal bone, density is further increased; fixed the third week, a marked new bone tissue connected both ends of the normal bone;At28days of consolidation, new bone tissue calcified completely, cortical formed fully, medullary cavity was unimpeded
三. General observation of samples after treatment
At14days of consolidation:three groups of new bone from high to low quality or hardness:the control group>the treatment group>the model group.
At28days of consolidationks:The osteogenesis quality high to low:the control group>the treatment group>the model group.
四. X-ray observation after treatment
X-ray inspection was instantly taken after operation, the distractor was fixed stably, counterpoint is good to the line.
At the end day of distraction, the model group had no significant shadow, the treatment group and the control group had the formation of new bone and osteoid.
At7days of consolidation, cloudy shadows was markedly visible at the Remote of distraction gap, cloudy shadows was deepen in the treatment group and throughout both the ends of femur fracture, the cylindrical cloudy shadows was deepen in the control group compared with the previous.The proximal portion of the cortical density increased, partially calcified.
At14days of consolidation, cloudy shadow was deepened in the control group, and the emergence of the shadow of the cortical bone, new bone mineral density of the treatment group compared with the control group and the treatment group is lower.
At21days of consolidation, the bone mineral density of the new born from low to high as the model group, treatment group and control group.
At28days of consolidations, the new bone mineral density of the control group and the treatment group was significantly higher than that in the model group.
五. Micro-CT test results after treatment
Model group, treatment group and control group were drawn at14and21days of consolidation and did MICRO-CT quantitative bone scan and image comparison.
From Micro-CT scanning images, osteogenesis and bone modeling of the control group was significantly stronger than the model group and the treatment group at14days of consolidation, osteogenesis and bone modeling of the treatment group was slightly better than the control group;At28days of consolidations, osteogenesis and bone modeling of the control group and the treatment group were stronger than the model group, osteogenesis and bone modeling of the control group was slightly better than the treatment group.
BMC, TMC, BMD, TMD, Tb. N, Tb. Pf:at14days of consolidation, the control group was statistically significant (P<0.05)with the model group, treatment group was not statistically significant (P>0.05) with model group and the control group. At28days of consolidations, the treatment group and the control group was statistically significant (P<0.05) with model group, the treatment group was not statistically significant (P>0.05)with the control group.
BV/TV:the control group was statistically significant (P<0.05)with the model group at14days of consolidation, the treatment group was not statistically significant (P>0.05) with the model group and control group. At28days of consolidations, the control group was statistically significant (P<0.05)with the model group, the treatment group was not statistically significant (P>0.05) with the model group and control group
Tb. Sp, Tb. Th, SMI, At14days of consolidation there was not statistically significant (P>0.05)between the groups. At28days of consolidations, the treatment group and the control group was statistically significant (P <0.05)with the model group, the treatment group was not statistically significant (P>0.05)with the control group
六. Mechanical testing after treatment
Sectional moment of inertia, maximum load, maximum deflection, modulus of elasticity, elastic stress, strain and other indicators, At14days of consolidation the control group was statistically significant (P<0.05) with the model group, the treatment group was not statistically significant (P>0.05) with model group and the control group.At28days of consolidations, the treatment group and the control group was statistically significant (P<0.05) with model group, the treatment group was not statistically significant (P>0.05)with the control group
Elastic load and the stiffness elastic strain indicators,at14days of consolidation, the treatment group and the control group was statistically significant (P<0.05)with model group, the treatment group was statistically significant (P<0.05)with the control group. At28days of consolidation, the treatment group and the control group was statistically significant (P<0.05)with the model group, the treatment group was not statistically significant (P>0.05) with the control group
Elastic deflection, fixed two weeks, the treatment group was statistically significant (P<0.05)with the model group,the treatment group was not statistically significant (P>0.05)with the model group and control group. At28days of consolidation, the control group was statistically significant (P<0.05)with the model group, the treatment group was not statistically significant (P>0.05)with the model group and control group.
Maximum stress, at14days of consolidation there was not statistically significant (P>0.05)between the groups. At28days of consolidation, the treatment group and the control group was statistically significant (P<0.05) with the model group, the treatment group was not statistically significant (P>0.05)with the control group
七. RT-QPCR detect purpose genes BMP-2mRNA level after treatment
At14days of consolidation, the treatment group was not statistically significant (P>0.05) with model group and the control group. At28days of consolidations, the treatment group and the control group was statistically significant (P<0.05) with model group, the treatment group was not statistically significant (P>0.05)with the control group
八.Histology by light microscopy observations after treatment
At14days of consolidation,a large number of osteoblasts in the area of distraction, in the control group there was a small amount of fibrous tissue, new bone trabecular grown extended stretch direction. In the treatment group and model group there was a large number of fibrous tissue, new bone trabecular was less.
At28days of consolidation, there was a large number of new bone formation, treatment group and control groups was filled with new trabecular bone filling stretch aligned, densely arranged, scattered distribution of fiber zone; in model group, new bone trabeculae relatively was sparse, and filled with more fibrous tissue.
九. Scanning electron microscopy results after treatment
At14days of consolidation, the control group arrangement of the collagen fiber bundles was visible along the stretch direction, during which can be seen a lot of new osteoid began to form; model group and treatment group saw a large number of collagen fibers aligned along the stretch, newborn bone the quality is less.
At28days of consolidation, control group and treatment group had smooth on the surface of the bone, trabecular bone is more dense, more completely calcified, close to the normal bone surface; thin trabecular bone in the model group, the first two groups there was not more mature trabecular bone compared with other group, the collagen fibers was more, bone surface is not smooth calcification incomplete.
Conclusion:
1. Through animal experiments show that self-designed single-arm distractor have stable structure, convenient to use, cheap, simple operation, high success rate.
2. The distractor applied to the rabbit femur distraction osteogenesis, through the rational design of surgical procedures, it is possible to successfully establish rabbit femoral distraction osteogenesis model.
3. Application total flavonoids from rhizoma drynariae in distraction osteogenesis can accelerate new bone remodeling and reconstruction, and the medication is convenient, provides a convenient and economical method to shorten the clinical distraction treatment.
牵张成骨技术已经在临床应用几十年,多用于治疗各种原因引起的双下肢肢体不等长畸形,如创伤性骨缺损、骨骺损伤、骨肿瘤和骨感染等导致的骨缺损、脊髓灰质炎后遗肢短畸形,以及先天性肢短畸性和肢体肥大症等。牵张成骨术以其独有的成骨方式和其他技术无可比拟的临床效果,一直备受医学界的青睐。近年来,随着牵张器不断在改进,手术设计的不断完善,该技术得到了突飞猛进的发展,在临床应用的范围也越来越广。但是临床上主要应用于胫骨,文献已有较多报道。这主要是因为胫骨延长技术相对简单,但在某些特定的情况下对患者只能实施股骨延长术,文献已有报道股骨延长病例,治疗效果令人满意,但是治疗过程也同样存在不足,如病程长,骨形成不良等。
本研究通过自行设计用于家兔股骨的牵张支架,建立家兔股骨缺损牵张成骨模型,在牵张成骨过程中应用中药骨碎补总黄酮,可以促进股骨骨缺损牵张成骨(DO)过程中新骨的形成、矿化及塑型改建,加速牵张成骨新骨形成的速度以及成骨质量,从而可以缩短牵张成骨的治疗周期,从而为临床上运用股骨延长术寻找一种快速有效的促进成骨的治疗方法。
方法：
一、自行设计牵张支架系统,建立家兔股骨缺损牵张成骨模型。取兔股骨标本,肉眼观察牵张间隙内新生骨痂生长情况及进行X线观察。
二、将42只新西兰大白兔随机分模型组、治疗组(骨碎补总黄酮)、对照组(rhBMP-2)三组。所有动物行双侧股骨手术截骨,截骨长度为1.4cm,每组14只兔(N=14),28个股骨标本(n=28)。分别在固定第14天、28天取材,标本通过：1、一般情况及大体观察；2、X线观察；3、MICRO-CT定量骨扫描；4、生物力学测试；5、RT-QPCR检测目的基因BMP-2mRNA水平；6、组织学光镜观察；7、扫描电镜观察等指标进行评价新骨形成速度及质量。
结果：
一、牵张成骨模型大体标本观察
随着时间推移,肉眼可分辨牵张间隙内骨痂生长由少到多,逐渐钙化；
固定第7天：牵张间隙处新生骨组织颜色鲜红,与正常骨两端界限非常清晰,两端骨痂生长较多,中间尚未生长；
固定第14天：牵张间隙内新生骨组织颜色变浅,较前致密、匀称,呈圆柱状贯通骨折两端,与正常骨两端界限较前模糊；
固定第28天：牵张间隙内可见新生骨组织表面与正常骨颜色、纹理相同,肉眼较难以分清正常骨组织和牵张间隙新生骨区的界限,骨密度明显增高。
二、牵张成骨模型X线观察
术后当天即刻行X光检查,牵张支架固定稳定,骨折两端对位对线可；
牵张结束行X光：牵张间隙远端可见有云雾状阴影；固定第7天：牵张间隙远端云雾状阴影较前加深,范围扩大；固定第14天：牵张间隙内可见连接正常骨两端的云雾状阴影,密度进一步增高；固定第21天：牵张间隙内已有明显的新生骨组织连接正常骨两端；固定第28天：新生骨组织钙化完全,骨皮质完全形成,髓腔畅通。
三、治疗后大体标本观察
固定第14天：肉眼观察下三组新生骨组织从质量或者硬度上由高到低排列为：对照组>治疗组>模型组。
固定第28天：肉眼观察下可初步判断,成骨质量由高到低排列为：对照组>治疗组>模型组。
四、治疗后X片观察结果
各组手术当天X光侧位片显示牵张支架固定稳定,骨折端对位对线可。
牵张结束当天：模型组牵张间隙无明显阴影；治疗组及对照组有新骨及类骨质形成。
固定第7天：模型组牵张间隙远端可见较明显的云雾状阴影；治疗组牵张间隙内云雾状阴影较前加深,贯穿股骨两骨折端；对照组牵张间隙内圆柱状阴影较前加深,近端部分皮质密度增高,部分钙化。
固定第14天：对照组牵张间隙内阴影明显加深,并出现骨皮质阴影；但治疗组及模型组新生骨骨密度较对照组低。
固定第21天：新生骨密度由低到高为模型组、治疗组、对照组。
固定第28天：对照组及治疗组新生骨骨密度明显高于模型组。
五、治疗后Micro-CT测试结果
模型组、治疗组、对照组分别在固定第14天和28时取材进行MICRO-CT图像比较和定量骨扫描。
MICRO-CT的扫描图像上看,固定第14天：对照组的成骨与骨塑型均明显强于模型组与治疗组,治疗组的成骨与骨塑型稍好于模型组；固定第28天：对照组与治疗组的成骨与骨塑型均强于模型组,对照组的成骨与骨塑性稍好于治疗组。
BMC、TMC、BMD、TMD、Tb.N、Tb.Pf等指标,固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组同模型组及对照组无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
BV/TV在固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。固定第28天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。
Tb.Sp、Tb.Th、SMI其中固定第14天：各组间无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
六、治疗后力学测试结果
截面惯性矩、最大载荷、最大挠度、弹性模量、弹性应力、最大应变等指标,固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组同模型组及对照组比较无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
弹性载荷、刚性系数、弹性应变等指标,固定第14天：治疗组、对照组与模型组,治疗组与对照组有统计学意义(P<0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
弹性挠度,固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。固定第28天：对照组与模型组有统计学意义(P<0.05),治疗组与模型组及对照组无统计学意义(P>0.05)。
最大应力,固定第14天：各组间无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组有统计学意义(P<0.05),治疗组与对照组无统计学意义(P>0.05)。
七、治疗后RT-QPCR检测目的基因BMP-2mRNA水平
固定第14天：对照组与模型组有统计学意义(P<0.05),治疗组同模型组及对照组无统计学意义(P>0.05)。固定第28天：治疗组、对照组与模型组之间有统计学意义(P<0.05),治疗组与对照组之间无统计学意义(P>0.05)。
八、治疗后组织学光镜观察结果
固定第14天：牵张间隙内有大量成骨细胞分布,对照组延长区见少量纤维组织,新生骨小梁延牵张方向生长。治疗组及模型组延长区可见大量纤维组织,新生骨小梁较少。固定第28天：在牵张间隙内可见有大量新骨形成,治疗组及对照组牵张间隙内基本被延牵张方向排列的新生骨小梁充填,排列密集,散在分布纤维区带；模型组牵张间隙内新生骨小梁排列相对稀疏,纤维组织较多。
九、治疗后扫描电镜观察结果
固定第14天：对照组可见大量沿牵张方向排列的胶原纤维束,其间可见很多新生类骨质开始形成；模型组及治疗组见到大量沿牵张方向排列的胶原纤维束,新生类骨质则较少。固定第28天：对照组及治疗组可见骨表面光滑平整,骨小梁较为致密,钙化较完全,基本接近正常骨表面；模型组骨小梁较纤细,没有前两组骨小梁成熟,胶原纤维束含量较多,骨表面尚不平整钙化不完全。
结论：
1、通过进行动物实验证明自行设计的单臂式牵张支架其结构稳定、使用方便、价格便宜、手术操作简单,成功率高等特点。
2、将牵张支架应用于兔股骨牵张成骨实验,通过设计合理的手术步骤以及规范的术后牵张成骨操作,能够顺利建立兔股骨牵张成骨模型。
3、应用骨碎补总黄酮在牵张成骨中能够起到加快新生骨组织的塑形与重建的作用,且用药方便,为临床缩短骨牵张疗程提供了一种方便、经济的方法。
Objective:
Distraction osteogenesis (DO) adopted in clinical applications for several decades has been used to treat leg length discrepancy caused by various disease such as traumatic bone defect, epiphyseal injury, and bone defect caused by bone tumors and bone infection, short limb deformity caused by polio heritage, and Congenital short limb deformities and limb hypertrophy and so on.Distraction osteogenesis gains great popularity in clinical applications for its unique way of osteogenesis and incomparable clinical efficacy Compared with other techniques. In recent years, with the constantly improving of distractor and the continuous improvement of the operation design, the technology has been developing by leaps and bounds, the scope also more and more widely in clinical application.But clinical application is more mainly in tibia, many of literatures have been reported. Mainly because the tibic lengthening is relatively simple, but in some specific cases the femoral lengthening must be used to patient, femoral lengthening cases has been reported in literature, the treatment effect is satisfactory, but the treatment process also exists shortage, such as long duration and poor bone formation, etc.
In our study, the rabbit model of bone defect was treated by distraction osteogenesis with a novel distractor invented by us. Total flavonoids from rhizoma drynariae was used during the process of DO, in order to achieve accelerated the new bone formation, mineralization and model reconstruction of femur bone defects during the process of DO, accelerate the speed of new bone formation and osteogenesis quality, which can shorten the entire treatment cycle, so as to provide femoral distraction osteogenesis an efficient and effective method by promoting the process of osteogenesis.
Methods:
一、The distractor was designed and made, then, it was applied on the rabbit model of femoral bone defect. The femoral samples of rabbits were taken and assessed by the callus of distracted zone observed by perusal and X ray.
二、42new zealand white rabbits was randomly divided into three experimental groups:model group, treatment group (intervened by Total flavonoids from rhizoma drynariae and control group(intervened by rhBMP-2). The osteotomy length was1.4cm in all the experimental animals.14rabbits in each group (N=14),28femoral specimens (N-28). The speed and quality of new formatting bone was observed and assessed by:1. general observation;2. X ray;3.scanning of Micro CT;4. Bone biomechanical testing;5.detecting the expression level of BMP-2mRNA gene by RT-QPCR.5. observing histologic features by light microscopy.7. observation by scanning electron microscope.
Results:
一. Gross specimen observation of the model of DO
Over time, the naked eye can distinguish callus growth from less to more in the area of distraction, and gradually calcification;
At7days of consolidation, the new bone have a bright red color in the area of distraction, the normal boundaries of both ends of the bone is very clear, both ends of the callus growth more, the middle has not been grown;
at14days of consolidation, the color of the new bone is becoming lighter、 compact, well-proportioned in the area of distraction compared with the previous. Fracture callus was cylindrical through both ends of the fracture, the normal boundaries of both ends of the bone is fuzzy over the previous;
At28days of consolidations, the surface of New bone have normal bone color and same texture in the area of distraction, the naked eye is more difficult to distinguish the boundaries between normal bone and new bone, bone mineral density was significantly higher.
二、X-ray observation of the model of DO
X-ray inspection was instantly taken after operation, the distractor was fixed stably, counterpoint is good to the line.
X-ray inspection was taken at the end day of the distraction, cloudy shadows was visible at the Remote of distraction gap; at7days of consolidation, cloudy shadows was deepen and extended;At14days of consolidation, the cloudy shadows connected the two ends of the normal bone, density is further increased; fixed the third week, a marked new bone tissue connected both ends of the normal bone;At28days of consolidation, new bone tissue calcified completely, cortical formed fully, medullary cavity was unimpeded
三. General observation of samples after treatment
At14days of consolidation:three groups of new bone from high to low quality or hardness:the control group>the treatment group>the model group.
At28days of consolidationks:The osteogenesis quality high to low:the control group>the treatment group>the model group.
四. X-ray observation after treatment
X-ray inspection was instantly taken after operation, the distractor was fixed stably, counterpoint is good to the line.
At the end day of distraction, the model group had no significant shadow, the treatment group and the control group had the formation of new bone and osteoid.
At7days of consolidation, cloudy shadows was markedly visible at the Remote of distraction gap, cloudy shadows was deepen in the treatment group and throughout both the ends of femur fracture, the cylindrical cloudy shadows was deepen in the control group compared with the previous.The proximal portion of the cortical density increased, partially calcified.
At14days of consolidation, cloudy shadow was deepened in the control group, and the emergence of the shadow of the cortical bone, new bone mineral density of the treatment group compared with the control group and the treatment group is lower.
At21days of consolidation, the bone mineral density of the new born from low to high as the model group, treatment group and control group.
At28days of consolidations, the new bone mineral density of the control group and the treatment group was significantly higher than that in the model group.
五. Micro-CT test results after treatment
Model group, treatment group and control group were drawn at14and21days of consolidation and did MICRO-CT quantitative bone scan and image comparison.
From Micro-CT scanning images, osteogenesis and bone modeling of the control group was significantly stronger than the model group and the treatment group at14days of consolidation, osteogenesis and bone modeling of the treatment group was slightly better than the control group;At28days of consolidations, osteogenesis and bone modeling of the control group and the treatment group were stronger than the model group, osteogenesis and bone modeling of the control group was slightly better than the treatment group.
BMC, TMC, BMD, TMD, Tb. N, Tb. Pf:at14days of consolidation, the control group was statistically significant (P<0.05)with the model group, treatment group was not statistically significant (P>0.05) with model group and the control group. At28days of consolidations, the treatment group and the control group was statistically significant (P<0.05) with model group, the treatment group was not statistically significant (P>0.05)with the control group.
BV/TV:the control group was statistically significant (P<0.05)with the model group at14days of consolidation, the treatment group was not statistically significant (P>0.05) with the model group and control group. At28days of consolidations, the control group was statistically significant (P<0.05)with the model group, the treatment group was not statistically significant (P>0.05) with the model group and control group
Tb. Sp, Tb. Th, SMI, At14days of consolidation there was not statistically significant (P>0.05)between the groups. At28days of consolidations, the treatment group and the control group was statistically significant (P <0.05)with the model group, the treatment group was not statistically significant (P>0.05)with the control group
六. Mechanical testing after treatment
Sectional moment of inertia, maximum load, maximum deflection, modulus of elasticity, elastic stress, strain and other indicators, At14days of consolidation the control group was statistically significant (P<0.05) with the model group, the treatment group was not statistically significant (P>0.05) with model group and the control group.At28days of consolidations, the treatment group and the control group was statistically significant (P<0.05) with model group, the treatment group was not statistically significant (P>0.05)with the control group
Elastic load and the stiffness elastic strain indicators,at14days of consolidation, the treatment group and the control group was statistically significant (P<0.05)with model group, the treatment group was statistically significant (P<0.05)with the control group. At28days of consolidation, the treatment group and the control group was statistically significant (P<0.05)with the model group, the treatment group was not statistically significant (P>0.05) with the control group
Elastic deflection, fixed two weeks, the treatment group was statistically significant (P<0.05)with the model group,the treatment group was not statistically significant (P>0.05)with the model group and control group. At28days of consolidation, the control group was statistically significant (P<0.05)with the model group, the treatment group was not statistically significant (P>0.05)with the model group and control group.
Maximum stress, at14days of consolidation there was not statistically significant (P>0.05)between the groups. At28days of consolidation, the treatment group and the control group was statistically significant (P<0.05) with the model group, the treatment group was not statistically significant (P>0.05)with the control group
七. RT-QPCR detect purpose genes BMP-2mRNA level after treatment
At14days of consolidation, the treatment group was not statistically significant (P>0.05) with model group and the control group. At28days of consolidations, the treatment group and the control group was statistically significant (P<0.05) with model group, the treatment group was not statistically significant (P>0.05)with the control group
八.Histology by light microscopy observations after treatment
At14days of consolidation,a large number of osteoblasts in the area of distraction, in the control group there was a small amount of fibrous tissue, new bone trabecular grown extended stretch direction. In the treatment group and model group there was a large number of fibrous tissue, new bone trabecular was less.
At28days of consolidation, there was a large number of new bone formation, treatment group and control groups was filled with new trabecular bone filling stretch aligned, densely arranged, scattered distribution of fiber zone; in model group, new bone trabeculae relatively was sparse, and filled with more fibrous tissue.
九. Scanning electron microscopy results after treatment
At14days of consolidation, the control group arrangement of the collagen fiber bundles was visible along the stretch direction, during which can be seen a lot of new osteoid began to form; model group and treatment group saw a large number of collagen fibers aligned along the stretch, newborn bone the quality is less.
At28days of consolidation, control group and treatment group had smooth on the surface of the bone, trabecular bone is more dense, more completely calcified, close to the normal bone surface; thin trabecular bone in the model group, the first two groups there was not more mature trabecular bone compared with other group, the collagen fibers was more, bone surface is not smooth calcification incomplete.
Conclusion:
1. Through animal experiments show that self-designed single-arm distractor have stable structure, convenient to use, cheap, simple operation, high success rate.
2. The distractor applied to the rabbit femur distraction osteogenesis, through the rational design of surgical procedures, it is possible to successfully establish rabbit femoral distraction osteogenesis model.
3. Application total flavonoids from rhizoma drynariae in distraction osteogenesis can accelerate new bone remodeling and reconstruction, and the medication is convenient, provides a convenient and economical method to shorten the clinical distraction treatment.
引文
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