EDTA-Na_2脱钙模拟椎体骨质疏松体外模型的建立及其临床意义
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摘要
第一部分：
EDTA-Na2脱钙模拟椎体骨质疏松体外模型的选择
研究背景：随着人口寿命的延长及老龄化社会的到来,老年人骨质疏松发病率较高,骨质疏松严重影响老年人的生活质量,其最严重的并发症就是骨质疏松性骨折,一旦患者经历了第一次骨质疏松性骨折,继发性骨折的危险明显加大。该病的防治已经成为一个迫切需要解决的问题,那么对骨质疏松的研究也在逐步深入,骨质疏松的模型也广泛应用。正确选择和制备一个理想的骨质疏松实验动物的模型,是开展骨质疏松研究工作的基础。目前建立骨质疏松模型常用的方法分绝经后骨质疏松模型和局部骨质丢失模型,具体包括去势、药物(激素,维甲酸,羟基脲,同型半胱氨酸)、废用、失重、甲状旁腺切除术、增龄、低钙、转基因、基因敲除、酒精、放射、细胞等模型,但是常规建立模型的方法耗时较多,无法提供在初期大量使用动物模型进行设计修改和测试的要求,同时在动物的购买、饲养、药物、手术费用投入较大,而且动物的数量有限,差异性,重复性差。快速有效的建立模型将为骨质疏松症的深入研究提供帮助。EDTA脱钙方法是一种比较合适的方法,能够制备出满足骨质疏松实验的大量动物模型。相较于活体动物模型其优点：(1)快速、简便、费用低；(2)可靠性好；(3)通过调整脱钙的时间,还可以调控模型的骨质疏松程度。而试验动物的选择也是关键,目前常用的有灵长类、啮齿目动物、兔形目、食肉目、偶蹄目等,绵羊在形态计量学方面,做为脊柱动物模型与人类相比均具有良好的相似性和可比性。短期内制备脱钙骨质疏松体外模型国内张智海等曾有描述,其用椎弓根钻孔EDTA-Na2脱钙的方法达到骨质疏松的目的,但没有阐明骨密度随时间的变化及椎弓根钻孔对椎体骨密度的影响,本试验正是研究经椎弓根钻孔并EDTA-Na2脱钙的绵羊椎骨体外骨质疏松模型、脱钙时间与骨密度的变化及椎弓根钻孔对骨密度变化的影响。此种快速制备骨质疏松模型的方法可以为骨质疏松症的科研及防治节省宝贵时间。
目的：观察利用EDTA-Na2脱钙后椎体骨密度的变化,以及打孔对椎体骨密度的影响,探讨一种制作骨质疏松模型的方法。
方法：取70个新鲜绵羊腰1椎体,随机分成10小组,每组7个标本,1-5组全部经椎弓根打孔,记A大组,6-10组不行椎弓根打孔,记为B大组,然后把标本侵入0.4916mmol/L的EDTA-Na2脱钙液体中,分别在0(未浸)、3、6、9、12天分别取出A大组与B大组各一小组行骨密度测试及每组取一个标本大体剖开。观察脱钙时间与A.B两组骨密度变化及两组间的差异。
结果：A.B两组随着脱钙时间的延长,大体观察可见各组标本骨小梁逐渐变细、间隙增宽,骨密度检测结果显示逐渐减低的趋势,并且各组脱钙前后骨密度变化均有明显的差异性。A与B大组间在骨密度的变化无明显的差异(P>0.05)。
结论：采用0.4916mmol/L EDTA-Na2脱钙12天可模拟轻、中、重度骨质疏松,经椎弓根打孔对骨密度变化没有影响,但钻孔可能对制造易压缩的骨质疏松骨折模型有实际意义。
第二部分：
EDTA-Na2脱钙模拟椎体骨质疏松体外模型的建立
研究背景：越来越多的人口患有骨质疏松疾病,由骨质疏松导致的脊柱压缩性骨折已严重危害老人的健康和严重影响老人的生活质量,加重了社会和家庭的负担。随着对骨质疏松研究的不断深入,骨质疏松模型的应用越来越广泛,并且骨质疏松性骨折手术器械的试制及药品的研制,都需要在大量、可重复的动物模型上进行试验。而建立骨质疏松的动物模型受到诸多限制,并且骨质疏松的活体动物模型,常用来研究骨质疏松的病理生理机制及治疗,所以目前迫切需要一种可短期内制作的体外骨质疏松模型的方法。我们正是建立这种经EDTA-Na2脱钙的绵羊椎骨体外骨质疏松模型,并且是可以改变骨密度和骨强度的大量、可重复的骨质疏松椎骨体外模型,而且标本在脱钙程度与脱钙时间上都能得到较好的控制。
骨质疏松症是以骨强度降低导致骨折危险性增加的一种骨骼疾病,骨密度和骨质量的好坏反应了骨强度的高低。临床用来检测骨质疏松的方法有骨代谢生化标志物检测、骨矿密度(BMD)测量、定量超声测定、骨强度分析、常规X线检查、磁共振成像检查、放射性核素骨显像等。目前,骨质疏松的诊断主要是依赖骨形态学测量和骨矿物密度(BMD)值的测定。而根据现有的条件,多种方法联合应用是必不可少的。本实验对体外骨质疏松模型同时进行了骨灰化测定。
目的：利用乙二胺四乙酸二钠盐(EDTA-Na2)体外脱钙后的椎体模拟椎体骨质疏松,对椎体骨质疏松模型进行DR、DEXA、Micro-CT、骨灰化等检测,观察短期制造骨质疏松模型的结果,探讨一种短期制造骨质疏松性椎体压缩骨折模型的方法。
方法：取84个新鲜绵羊胸腰椎3联体,随机分成7组,记为A. B.C. D.E.F.G每组12个标本,中间椎体钻孔后,在室温下将椎体浸入0.4916mmol/L的EDTA-Na2脱钙液体中,分别在0(未浸)、3、6、9、12天每组各取一个标本测骨密度,其中每组在脱钙0、9天行X拍片(n=7)后大体剖开观察,中间椎体Micro-CT检查(n=7)及骨灰化测定(n=7)。
结果：随着脱钙时间的延长,大体观察可见各组标本骨小梁逐渐变细、间隙增宽,骨密度检测结果显示逐渐减低的趋势,并且各组脱钙前后骨密度变化均有明显的差异性。Micro-CT检测脱钙9天椎体的骨小梁表观密度、骨体积分数、骨小梁厚度、骨小梁数量、骨小梁分离度、结构模型指数、各向异性的程度、欧拉数、骨小梁连结密度均有明显的变化(p<0.05),骨灰化测定脱钙0、9天骨矿物质含量有明显的变化(p<0.05)。
结论；采用0.4916mmol/L EDTA-Na2脱钙9-12天可模拟中、重度骨质疏松性椎体压缩骨折,是一种快速、可行、重复性高的方法。
第三部分：
骨质疏松体外模型的生物力学试验及临床意义
研究背景：1885年,Pommer最早提出“骨质疏松”这一名词,意思是骨质减少,可见布满空隙的骨骼,1990年在丹麦举行的第三届骨质疏松大会上明确提出其定义,之后在1993年的第四届大会上得到完善,并为世界各国所公认,2001年,美国国家卫生学院(NIH)提出,骨质疏松症是以骨强度降低导致骨折危险性增加的一种骨骼疾病,骨密度和骨质量的好坏反应了骨强度的高低。
1987年,法国学者Galibert和Deramond根据外科手术填塞骨水泥的经验采用经皮穿刺椎体后注射骨水泥治疗血管瘤,此可谓将经皮椎体成形术运用于临床的最初报道。1990年,Galibert和Deramond进一步提出该技术可扩大应用于骨质疏松性椎体骨折,这标志着椎体成形术的突破性进展。此后该技术在脊柱外科领域得到了广泛的应用和发展。
目的：探讨骨质疏松及椎体成形术后绵羊胸腰段三联椎体的生物力学特性变化,为临床试验提供依据。
方法：取0.4916mmol/L的EDTA-Na2脱钙液体中的A. B. C. D. E. F. G组绵羊胸腰椎三联体每组6个,共42个,分别在第0(未浸)、3、6、12天每组各取一个标本,而在第9天每组取两个标本,以供试验使用。测量每个椎体的高度,计算压缩50%所需形变量。以最大压力为压缩强度,将各标本上下椎体卡在压缩模具上,然后置于生物力学实验机上,以5mm/min的加载速率进行垂直加载,直到标本屈服为止,测出最大抗压力。记录载荷和形变(高度)的变化,得到载荷-形变曲线,以最大载荷为压缩强度,以载荷-形变曲线的斜率为压缩刚度。在压缩过程中不断用注射器向标本上注射0.9%氯化钠溶液,保持椎体湿润。
取第9天压缩标本行椎体成形术,分为PMMA组和活性骨水泥组,每组各7个标本,在C形臂X线机监视下,模拟临床球囊扩张椎体后凸成形术操作。用3.5mm钻头作椎弓根钻孔,直至椎体前中1/3交界处,置入导针后沿导针放入一根套管针,沿套管放入可扩张球形气囊,然后向气囊内充气扩张。球囊扩张及复位满意后将其复原后撤出。调配骨水泥,PMMA组使用zimmer骨水泥,活性骨水泥组使用CPC/PMMA复合组成的生物活性骨水泥。将调配好的骨水泥装入注射器中,在团状期将骨水泥经工作套管推入椎体内空腔。根据球囊扩张体积确定骨水泥注射量。各椎体拍摄正侧位X线片,测量各椎体高度、压缩刚度及压缩强度(方法同前)。
观察随脱钙时间延长BMD与椎体压缩强度和刚度的变化,以及三者之间的关系,椎体成形前后椎体压缩强度和刚度的变化。
结果：随着脱钙时间的延长,各组的BMD、椎体压缩强度和刚度逐渐降低,存在显著性差异(P<0.005)；椎体成形术后各组的椎体压缩强度明显高于术前(P<0.05)；PMMA组的椎体压缩刚度与术前无统计学意义(P>0.05),活性骨水泥组的刚度高于术前组和PMMA组(P<0.01)。
椎体BMD与椎体压缩强度存在着明显的正相关关系(γ=0.976,P<0.01)。BMD的大小直接影响椎体承载能力的大小。椎体刚度反映椎体在轴向载荷下,抵抗变形能力的大小,与BMD亦成正相关关系(γ=0.922,P<0.01)
结论：1.BMD可以反映椎体骨质疏松的程度及椎体的生物力学性能,为临床上预防骨质疏松性骨折提供帮助。
2.活性骨水泥在椎体成形术后可以恢复椎体的生物力学强度,可达到正常椎体的生理需求。
3. EDTA快速脱钙制备骨质疏松模型法可以为生物力学及临床研究提供基础。
PartⅠ:
The selection of a model to simulate vertebral osteoporosis in vitro EDTA-Na2 by demineralization
Background As the aging society comes with life prolongation of the population, the osteoporosis attacked in elderly group becomes higher,which seriously harms the life quality of old people, and its most serious complications is osteoporotic fractures. once the patients get the fracture, the possibility of its recidivation is obviously high. It is urgent that the prevention of the disease has to be solved, thus the research of osteoporosis is carrying out, and there is a wide range of application of the model of osteoporosis. The correct choice and preparation an ideal osteoporosis model of experimental animals in research work is the basis of osteoporosis. Establish the osteoporotic model at present commonly used methods points postmenopausal osteoporosis model and local bone loss model,and specific include gonadectomy, drugs which include hormone, retinoic acid, hydrea and HCY, disuse, agravity, Parathyroid resection, increasing age,, low calcium, transgenic, gene knock-out,alcohol,radiation, cell model and etc. But those mentioned methods above cost a lot of time, can not satisfy the demand of design, adjustment and test in animal model in the initial stage, and at the same time, the cost taken in the pruchasing, feeding of animals, also the input of the drugs and operation is large, and the number of animals is limited, which has low otherness and repeatability. To build model for osteoporosis quickly and effectively offers a help in lucubrating the disease. EDTA decalcification method is an approach that satisfies the demand of a large number of animal models for osteoporosis experiment. Compare to the living body model, the model manufactured by EDTA method is:(1) Rapid, simple, low in the cost; (2) High reliability; (3) Via adjusting the demineralization time, can control the extent of osteoporosis of the model. Meanwhile it's also a key for the choice of experimental animals,and now the primates, the rodent mesh animals, lagomorpha, carnivorous mesh, cloven hooves, etc are commonly used. Sheep As the spine animal models are good comparability and comparability compared with humans in morphometric aspects. There was a description of the manufacture of the osteoporosis model in vitro by decalcification in short terms from Zhang Zhihai's group, that with its method to get osteoporosis by punching hole on pedicle of vertebral arch with EDTA-Na2 decalcification, but it is not clarified the BMD changing along time and the influence of punching hole on pedicle of vertebral arch to centrum's BMD. in this research, the aim is to study the osteoporosis model of sheep vertebrae by punching hole on pedicle of vertebral arch and EDTA-Na2 demineralization in vitro,and to investigate the time of decalcification versus the change of BMD, and the changes of BMD via punching hole on pedicle of vertebral arch. Thanks to this method that it can rapidly manufacture the model of osteoporosis, which saves valuable time for the development of prevention and treatment of osteoporosis.
Objective Develope a osteoporosis model by observing the change of the bone mineral density in vertebrae with the demineralization that occurred via the Ethylene Diamine Tetraacetic Acid disodium salt (EDTA-Na2) process and the the influence of the bone mineral density in vertebrae in which two hole made.
Method 70 fresh first lumbar vertebrae of sheep were randomly divided into 10 groups, each group 7 specimens, each vertebrae was made two holes in Vertebral pedicle by a dill with 3mm in Diameter in 1-5 group marked A big group,and 6-10 group without pedicle hole marked B big group,then all the specimens were dropped into 0.4916mmol/L of EDTA-Na2 demineralized liquid, a group was removed respectively in the day 0,3,6,9,12 in both A and B.after bone mineral density were tested a sample was cut open in each group. Observation relationship between the change of bone mineral density and the decalcified time in A.B group and that bwteen A and B in bone mineral density.
Results A.B the gross observation showed trabecular bone of specimens of each group thinnering, the gap widening, bone mineral density test showed the trend of gradual reduction, and each group had significant difference in bone mineral density before and after decalcification. the change in bone mineral density in A and B group were no significant differente (P> 0.05).
Conclusion Vertebraes decalcified for 12 days by the EDTA-Na2 of 0.4916mmol/L EDTA-Na2 was to simulate the light, moderate and severe osteoporosis in degree and that the holes drilled in Vertebral pedicle did not affect the change of bone mineral density,but drilling holes may be have practical sense of creating the compressed fracture model of osteoporosis.
Part II:
Development of the model to simulate vertebral osteoporosis in vitro by EDTA-Na2 demineralization
Background More and more people suffer from osteoporosis disease,and the vertebral compressibility fracture already serious harm the old man's health and seriously affect the old man's life quality,meanwhile increase social and family burden. With the deepening of the research for osteoporosis, the osteoporotic models are used more and more widely,and the research of surgical instrument test and drug development for osteoporotic fractures both need a large number and repeatable animal model to experiment. While the establishment of osteoporotic animal model is affected by many restrictions, and the living osteoporotic animal model are used to study the pathophysiology of osteoporosis and treatment. There is an urgent need to a method of short-term producted osteoporotic model in vitro.It is our aim to found this osteoporosis model of sheep vertebrae by EDTA-Na2 demineralization in vitro,which can change BMD and bone strength and can be a large number and repeatable animal model.And for the specimens in demineralization extent and the time on the demineralization we can get better control.
Osteoporosis is a bone disease that increase fracture risk caused by bone strength reduce. And the stand or fall of BMD and bone quality level determine the bone strength. Clinical used to detect osteoporosis methods include bone metabolism biochemical markers detection,BMD,quantitative ultrasound, bone strength analysis, conventional X-ray, magnetic resonance imaging, radionuclide bone scintigraphy,etc. At present, the diagnosis of Osteoporosis mainly rely on morphometric aspects and BMD. On the basis of the conditions now having got, it is necessary to unify the multiple methodes. In this experiment, it also processes the bone ash determination.
Objective To mimic vertebral osteoporosis in vitro by demineralization EDTA-Na2, process image test like DR,DEXA and Micro-CT,and bone ashes detection on the vertebral osteoporosis model, observe the result of the model that manufactured in short term, and then discuss a kind of method that can manufacture the osteoporotic vertebral compression fracture model in short period.
Method Take 84 fresh sheep thoracic and lumbar vertebraes 3 concatemers and randomly divide to 7 groups, and marked as A.B.C.D.E.F.G,12 samples in each group, drill a hole in the vertebraes body, at room temperature immerse the samples into 0.4916mmol/L EDTA-Na2 demineralized liquid, separately withdraw samples from 0 (not immersed),3,6,9 and 12 perday for BMD. For the demineralised groups after 0,9days, take X rays (n=7), and then divide for general observation (n= 7),and the intermediate vertebras make Micro-CT examination (n=7) and ashes of measurement (n=7).
Results With the decalcification, general observation shows that bone trabecula of each group became thinner and the gap between bone trabecula became wider gradually,the bone mineral density test show a trend that reduced, and the difference of BMD measurement bwteen pre-and post-demineraling process were statistically significant. After demineraling for 9 days Micro-CT detection showed that vBMD, BVF, Tb.Th, Tb.N, Tb.Sp, SMI, DA, Eu.N, Conn.Dn were significantly different compared to pre-demineraling (p<0.05). After Ashes of determination demineralization, at 0,9 day (days), the BMC apparently changed (p<0.05).
Conclusion The vertebraes decalcified for 9-12 days to simulate osteoporotic vertebral compression fractures is a fast, feasible and reproducible method.
PartⅢ:
The biomechanical experiment of osteoporosis model in vitro and its clinical significancee
Background In 1885, Pommer suggested that the word of osteoporosis, which describes the reducing of sclerotin, voided bones, and it is defined by the 3rd of osteoporosis meeting held in denmark in 1990, and then it is completed in the 4th meeting in 1993, and confirmed by every country in the world, in 2001, NIH suggested that the osteoporosis is a kind of bone diesease that results in fracture by bone strength reducing, the BMD and bone mass reflect the bone strength.
In 1987, Galibert and Deramond from Franch adopted the treatment for hemangioma by inject bone cement after skin puncture the centrum according to the experience of filling bone cement in surgical operation, this is the first case of application in clinical. In 1990, they further suggested that this technique can be applied in osteoporosis centrum fracture (OCF), which is a milestone of Vertebroplasty. And this technique is widely applied and developed in the field.
Objective Probe into sheep thoracic and lumbar vertebraes biomechanical characteristics change after Vertebroplasty and osteoporosis, provide evidence for clinical experiment.
Method Take 6 of each group (A.B.C.D.E.F.G) of sheep thoracic and lumbar vertebraes in 0.4916mmol/L EDTA-Na2 decalcification liquid in total of 42, a sample is with drawed from day 0 (not immersed),3,6,12,and two samples from day 9 for experimental use. Measure the height and weight of the centrum of each group in the front, rear, left, right direction, calculate the average out by the measuring tool of vernier caliper, accurated to 0.02mm.The compressive strength is the maximum pressure, that compresses the upper and down centrums of samples on the mould, and then put into the biomechanic apparatus, process vertical load in the rate of 5mm/min, when the sample is submited, measure the max-compressive resistance.Record the strength and shape-change (height) 10times/min, draw the curve of strength vs shape-change, find out the initial compressive strength and stiffness. Set the maximum pressure as the compressive strength, take the gradient of the curve of Strength vs Shape-change as the compressive stiffness.During the compression inject the 0.9% sodium chloride solution continuously on the sample, keep the sample moist.
Take vertebroplasty for ninth day compressive sample, divided into PMMA group and bioactive bone cement group,7 specimens in each group; imitate the clinical operation of PKP under c-arm X-ray machine monitoring.Drill hole on pedicle of vertebral arch by 3.5mm drill, into 1/3 of the centrum, after placing the guidepin, insert a trocar along the guidepin, along the trocar, place in an expandable spherical gasbag, and then aerate the gasbag. After the balloon expansion and reset satisfaction after recovering the withdrawal. Mix the bone cement, PMMA Group using the zimmer bone cement, and bioactive bone cement group with CPC/PMMA composite consisting of bioactive bone cement. Put bone cement into the injector, taking injection to the cavity of the centrum during the bone cement is in round state according to the volume of the gasbag. Shooting a side X-ray image of each centrum, measure the height, compressive stiffness and compressive strength (same method as having said).
Analysis the BMD, compressive strength and stiffness with correlation analysis by statistical software SPSS 17.0, Vertebral molding before and after of the vertebral compression strength and stiffness, comparison with single factor analysis of variance (ANOVA), the level of significance in P<0.05.
Results The centrums are in total of 42 in this experiment, as the decalcification time the BMD, vertebral compression strength and stiffness of each group reduce gradually, differ from each other obviously(P<0.005), that the groups after the vertebroplasty are higher than before in the compressive strength(P<0.05); The PMMA group were not significantly different compared to vertebral molding before in vertebral compression stiffness(P>0.05);The active bone cement group are higher than before and PMMA group in vertebral compression stiffness, that (P<0.01).
The BMD of the centrum and the compressive strength of the centrum are obvious in relationship of positive correlation (y= 0.976, P<0.01).The magnitude of BMD direct influences the Vertebral bearing capacity. The stiffness of the centrum means the deformability resistance of the centrum under the axial load, is in relationship of positive correlation (y=0.922, P<0.01).
Conclusion The first, BMD can reflect the level of osteoporosis, predict the fracture, and in an extent that could show the sclerotin situation of the centrum, offering help in clinical treatment and early diagnosis.The second, Bioactive bone cement in vertebroplasty surgery can restore the biomechanical strength of vertebral body, that can be achieved by the normal physiological needs of vertebral.The end, Rapid Preparation of EDTA decalcification method for osteoporosis model provide the basis for Biomechanical and clinical studies.
EDTA-Na2脱钙模拟椎体骨质疏松体外模型的选择
研究背景：随着人口寿命的延长及老龄化社会的到来,老年人骨质疏松发病率较高,骨质疏松严重影响老年人的生活质量,其最严重的并发症就是骨质疏松性骨折,一旦患者经历了第一次骨质疏松性骨折,继发性骨折的危险明显加大。该病的防治已经成为一个迫切需要解决的问题,那么对骨质疏松的研究也在逐步深入,骨质疏松的模型也广泛应用。正确选择和制备一个理想的骨质疏松实验动物的模型,是开展骨质疏松研究工作的基础。目前建立骨质疏松模型常用的方法分绝经后骨质疏松模型和局部骨质丢失模型,具体包括去势、药物(激素,维甲酸,羟基脲,同型半胱氨酸)、废用、失重、甲状旁腺切除术、增龄、低钙、转基因、基因敲除、酒精、放射、细胞等模型,但是常规建立模型的方法耗时较多,无法提供在初期大量使用动物模型进行设计修改和测试的要求,同时在动物的购买、饲养、药物、手术费用投入较大,而且动物的数量有限,差异性,重复性差。快速有效的建立模型将为骨质疏松症的深入研究提供帮助。EDTA脱钙方法是一种比较合适的方法,能够制备出满足骨质疏松实验的大量动物模型。相较于活体动物模型其优点：(1)快速、简便、费用低；(2)可靠性好；(3)通过调整脱钙的时间,还可以调控模型的骨质疏松程度。而试验动物的选择也是关键,目前常用的有灵长类、啮齿目动物、兔形目、食肉目、偶蹄目等,绵羊在形态计量学方面,做为脊柱动物模型与人类相比均具有良好的相似性和可比性。短期内制备脱钙骨质疏松体外模型国内张智海等曾有描述,其用椎弓根钻孔EDTA-Na2脱钙的方法达到骨质疏松的目的,但没有阐明骨密度随时间的变化及椎弓根钻孔对椎体骨密度的影响,本试验正是研究经椎弓根钻孔并EDTA-Na2脱钙的绵羊椎骨体外骨质疏松模型、脱钙时间与骨密度的变化及椎弓根钻孔对骨密度变化的影响。此种快速制备骨质疏松模型的方法可以为骨质疏松症的科研及防治节省宝贵时间。
目的：观察利用EDTA-Na2脱钙后椎体骨密度的变化,以及打孔对椎体骨密度的影响,探讨一种制作骨质疏松模型的方法。
方法：取70个新鲜绵羊腰1椎体,随机分成10小组,每组7个标本,1-5组全部经椎弓根打孔,记A大组,6-10组不行椎弓根打孔,记为B大组,然后把标本侵入0.4916mmol/L的EDTA-Na2脱钙液体中,分别在0(未浸)、3、6、9、12天分别取出A大组与B大组各一小组行骨密度测试及每组取一个标本大体剖开。观察脱钙时间与A.B两组骨密度变化及两组间的差异。
结果：A.B两组随着脱钙时间的延长,大体观察可见各组标本骨小梁逐渐变细、间隙增宽,骨密度检测结果显示逐渐减低的趋势,并且各组脱钙前后骨密度变化均有明显的差异性。A与B大组间在骨密度的变化无明显的差异(P>0.05)。
结论：采用0.4916mmol/L EDTA-Na2脱钙12天可模拟轻、中、重度骨质疏松,经椎弓根打孔对骨密度变化没有影响,但钻孔可能对制造易压缩的骨质疏松骨折模型有实际意义。
第二部分：
EDTA-Na2脱钙模拟椎体骨质疏松体外模型的建立
研究背景：越来越多的人口患有骨质疏松疾病,由骨质疏松导致的脊柱压缩性骨折已严重危害老人的健康和严重影响老人的生活质量,加重了社会和家庭的负担。随着对骨质疏松研究的不断深入,骨质疏松模型的应用越来越广泛,并且骨质疏松性骨折手术器械的试制及药品的研制,都需要在大量、可重复的动物模型上进行试验。而建立骨质疏松的动物模型受到诸多限制,并且骨质疏松的活体动物模型,常用来研究骨质疏松的病理生理机制及治疗,所以目前迫切需要一种可短期内制作的体外骨质疏松模型的方法。我们正是建立这种经EDTA-Na2脱钙的绵羊椎骨体外骨质疏松模型,并且是可以改变骨密度和骨强度的大量、可重复的骨质疏松椎骨体外模型,而且标本在脱钙程度与脱钙时间上都能得到较好的控制。
骨质疏松症是以骨强度降低导致骨折危险性增加的一种骨骼疾病,骨密度和骨质量的好坏反应了骨强度的高低。临床用来检测骨质疏松的方法有骨代谢生化标志物检测、骨矿密度(BMD)测量、定量超声测定、骨强度分析、常规X线检查、磁共振成像检查、放射性核素骨显像等。目前,骨质疏松的诊断主要是依赖骨形态学测量和骨矿物密度(BMD)值的测定。而根据现有的条件,多种方法联合应用是必不可少的。本实验对体外骨质疏松模型同时进行了骨灰化测定。
目的：利用乙二胺四乙酸二钠盐(EDTA-Na2)体外脱钙后的椎体模拟椎体骨质疏松,对椎体骨质疏松模型进行DR、DEXA、Micro-CT、骨灰化等检测,观察短期制造骨质疏松模型的结果,探讨一种短期制造骨质疏松性椎体压缩骨折模型的方法。
方法：取84个新鲜绵羊胸腰椎3联体,随机分成7组,记为A. B.C. D.E.F.G每组12个标本,中间椎体钻孔后,在室温下将椎体浸入0.4916mmol/L的EDTA-Na2脱钙液体中,分别在0(未浸)、3、6、9、12天每组各取一个标本测骨密度,其中每组在脱钙0、9天行X拍片(n=7)后大体剖开观察,中间椎体Micro-CT检查(n=7)及骨灰化测定(n=7)。
结果：随着脱钙时间的延长,大体观察可见各组标本骨小梁逐渐变细、间隙增宽,骨密度检测结果显示逐渐减低的趋势,并且各组脱钙前后骨密度变化均有明显的差异性。Micro-CT检测脱钙9天椎体的骨小梁表观密度、骨体积分数、骨小梁厚度、骨小梁数量、骨小梁分离度、结构模型指数、各向异性的程度、欧拉数、骨小梁连结密度均有明显的变化(p<0.05),骨灰化测定脱钙0、9天骨矿物质含量有明显的变化(p<0.05)。
结论；采用0.4916mmol/L EDTA-Na2脱钙9-12天可模拟中、重度骨质疏松性椎体压缩骨折,是一种快速、可行、重复性高的方法。
第三部分：
骨质疏松体外模型的生物力学试验及临床意义
研究背景：1885年,Pommer最早提出“骨质疏松”这一名词,意思是骨质减少,可见布满空隙的骨骼,1990年在丹麦举行的第三届骨质疏松大会上明确提出其定义,之后在1993年的第四届大会上得到完善,并为世界各国所公认,2001年,美国国家卫生学院(NIH)提出,骨质疏松症是以骨强度降低导致骨折危险性增加的一种骨骼疾病,骨密度和骨质量的好坏反应了骨强度的高低。
1987年,法国学者Galibert和Deramond根据外科手术填塞骨水泥的经验采用经皮穿刺椎体后注射骨水泥治疗血管瘤,此可谓将经皮椎体成形术运用于临床的最初报道。1990年,Galibert和Deramond进一步提出该技术可扩大应用于骨质疏松性椎体骨折,这标志着椎体成形术的突破性进展。此后该技术在脊柱外科领域得到了广泛的应用和发展。
目的：探讨骨质疏松及椎体成形术后绵羊胸腰段三联椎体的生物力学特性变化,为临床试验提供依据。
方法：取0.4916mmol/L的EDTA-Na2脱钙液体中的A. B. C. D. E. F. G组绵羊胸腰椎三联体每组6个,共42个,分别在第0(未浸)、3、6、12天每组各取一个标本,而在第9天每组取两个标本,以供试验使用。测量每个椎体的高度,计算压缩50%所需形变量。以最大压力为压缩强度,将各标本上下椎体卡在压缩模具上,然后置于生物力学实验机上,以5mm/min的加载速率进行垂直加载,直到标本屈服为止,测出最大抗压力。记录载荷和形变(高度)的变化,得到载荷-形变曲线,以最大载荷为压缩强度,以载荷-形变曲线的斜率为压缩刚度。在压缩过程中不断用注射器向标本上注射0.9%氯化钠溶液,保持椎体湿润。
取第9天压缩标本行椎体成形术,分为PMMA组和活性骨水泥组,每组各7个标本,在C形臂X线机监视下,模拟临床球囊扩张椎体后凸成形术操作。用3.5mm钻头作椎弓根钻孔,直至椎体前中1/3交界处,置入导针后沿导针放入一根套管针,沿套管放入可扩张球形气囊,然后向气囊内充气扩张。球囊扩张及复位满意后将其复原后撤出。调配骨水泥,PMMA组使用zimmer骨水泥,活性骨水泥组使用CPC/PMMA复合组成的生物活性骨水泥。将调配好的骨水泥装入注射器中,在团状期将骨水泥经工作套管推入椎体内空腔。根据球囊扩张体积确定骨水泥注射量。各椎体拍摄正侧位X线片,测量各椎体高度、压缩刚度及压缩强度(方法同前)。
观察随脱钙时间延长BMD与椎体压缩强度和刚度的变化,以及三者之间的关系,椎体成形前后椎体压缩强度和刚度的变化。
结果：随着脱钙时间的延长,各组的BMD、椎体压缩强度和刚度逐渐降低,存在显著性差异(P<0.005)；椎体成形术后各组的椎体压缩强度明显高于术前(P<0.05)；PMMA组的椎体压缩刚度与术前无统计学意义(P>0.05),活性骨水泥组的刚度高于术前组和PMMA组(P<0.01)。
椎体BMD与椎体压缩强度存在着明显的正相关关系(γ=0.976,P<0.01)。BMD的大小直接影响椎体承载能力的大小。椎体刚度反映椎体在轴向载荷下,抵抗变形能力的大小,与BMD亦成正相关关系(γ=0.922,P<0.01)
结论：1.BMD可以反映椎体骨质疏松的程度及椎体的生物力学性能,为临床上预防骨质疏松性骨折提供帮助。
2.活性骨水泥在椎体成形术后可以恢复椎体的生物力学强度,可达到正常椎体的生理需求。
3. EDTA快速脱钙制备骨质疏松模型法可以为生物力学及临床研究提供基础。
PartⅠ:
The selection of a model to simulate vertebral osteoporosis in vitro EDTA-Na2 by demineralization
Background As the aging society comes with life prolongation of the population, the osteoporosis attacked in elderly group becomes higher,which seriously harms the life quality of old people, and its most serious complications is osteoporotic fractures. once the patients get the fracture, the possibility of its recidivation is obviously high. It is urgent that the prevention of the disease has to be solved, thus the research of osteoporosis is carrying out, and there is a wide range of application of the model of osteoporosis. The correct choice and preparation an ideal osteoporosis model of experimental animals in research work is the basis of osteoporosis. Establish the osteoporotic model at present commonly used methods points postmenopausal osteoporosis model and local bone loss model,and specific include gonadectomy, drugs which include hormone, retinoic acid, hydrea and HCY, disuse, agravity, Parathyroid resection, increasing age,, low calcium, transgenic, gene knock-out,alcohol,radiation, cell model and etc. But those mentioned methods above cost a lot of time, can not satisfy the demand of design, adjustment and test in animal model in the initial stage, and at the same time, the cost taken in the pruchasing, feeding of animals, also the input of the drugs and operation is large, and the number of animals is limited, which has low otherness and repeatability. To build model for osteoporosis quickly and effectively offers a help in lucubrating the disease. EDTA decalcification method is an approach that satisfies the demand of a large number of animal models for osteoporosis experiment. Compare to the living body model, the model manufactured by EDTA method is:(1) Rapid, simple, low in the cost; (2) High reliability; (3) Via adjusting the demineralization time, can control the extent of osteoporosis of the model. Meanwhile it's also a key for the choice of experimental animals,and now the primates, the rodent mesh animals, lagomorpha, carnivorous mesh, cloven hooves, etc are commonly used. Sheep As the spine animal models are good comparability and comparability compared with humans in morphometric aspects. There was a description of the manufacture of the osteoporosis model in vitro by decalcification in short terms from Zhang Zhihai's group, that with its method to get osteoporosis by punching hole on pedicle of vertebral arch with EDTA-Na2 decalcification, but it is not clarified the BMD changing along time and the influence of punching hole on pedicle of vertebral arch to centrum's BMD. in this research, the aim is to study the osteoporosis model of sheep vertebrae by punching hole on pedicle of vertebral arch and EDTA-Na2 demineralization in vitro,and to investigate the time of decalcification versus the change of BMD, and the changes of BMD via punching hole on pedicle of vertebral arch. Thanks to this method that it can rapidly manufacture the model of osteoporosis, which saves valuable time for the development of prevention and treatment of osteoporosis.
Objective Develope a osteoporosis model by observing the change of the bone mineral density in vertebrae with the demineralization that occurred via the Ethylene Diamine Tetraacetic Acid disodium salt (EDTA-Na2) process and the the influence of the bone mineral density in vertebrae in which two hole made.
Method 70 fresh first lumbar vertebrae of sheep were randomly divided into 10 groups, each group 7 specimens, each vertebrae was made two holes in Vertebral pedicle by a dill with 3mm in Diameter in 1-5 group marked A big group,and 6-10 group without pedicle hole marked B big group,then all the specimens were dropped into 0.4916mmol/L of EDTA-Na2 demineralized liquid, a group was removed respectively in the day 0,3,6,9,12 in both A and B.after bone mineral density were tested a sample was cut open in each group. Observation relationship between the change of bone mineral density and the decalcified time in A.B group and that bwteen A and B in bone mineral density.
Results A.B the gross observation showed trabecular bone of specimens of each group thinnering, the gap widening, bone mineral density test showed the trend of gradual reduction, and each group had significant difference in bone mineral density before and after decalcification. the change in bone mineral density in A and B group were no significant differente (P> 0.05).
Conclusion Vertebraes decalcified for 12 days by the EDTA-Na2 of 0.4916mmol/L EDTA-Na2 was to simulate the light, moderate and severe osteoporosis in degree and that the holes drilled in Vertebral pedicle did not affect the change of bone mineral density,but drilling holes may be have practical sense of creating the compressed fracture model of osteoporosis.
Part II:
Development of the model to simulate vertebral osteoporosis in vitro by EDTA-Na2 demineralization
Background More and more people suffer from osteoporosis disease,and the vertebral compressibility fracture already serious harm the old man's health and seriously affect the old man's life quality,meanwhile increase social and family burden. With the deepening of the research for osteoporosis, the osteoporotic models are used more and more widely,and the research of surgical instrument test and drug development for osteoporotic fractures both need a large number and repeatable animal model to experiment. While the establishment of osteoporotic animal model is affected by many restrictions, and the living osteoporotic animal model are used to study the pathophysiology of osteoporosis and treatment. There is an urgent need to a method of short-term producted osteoporotic model in vitro.It is our aim to found this osteoporosis model of sheep vertebrae by EDTA-Na2 demineralization in vitro,which can change BMD and bone strength and can be a large number and repeatable animal model.And for the specimens in demineralization extent and the time on the demineralization we can get better control.
Osteoporosis is a bone disease that increase fracture risk caused by bone strength reduce. And the stand or fall of BMD and bone quality level determine the bone strength. Clinical used to detect osteoporosis methods include bone metabolism biochemical markers detection,BMD,quantitative ultrasound, bone strength analysis, conventional X-ray, magnetic resonance imaging, radionuclide bone scintigraphy,etc. At present, the diagnosis of Osteoporosis mainly rely on morphometric aspects and BMD. On the basis of the conditions now having got, it is necessary to unify the multiple methodes. In this experiment, it also processes the bone ash determination.
Objective To mimic vertebral osteoporosis in vitro by demineralization EDTA-Na2, process image test like DR,DEXA and Micro-CT,and bone ashes detection on the vertebral osteoporosis model, observe the result of the model that manufactured in short term, and then discuss a kind of method that can manufacture the osteoporotic vertebral compression fracture model in short period.
Method Take 84 fresh sheep thoracic and lumbar vertebraes 3 concatemers and randomly divide to 7 groups, and marked as A.B.C.D.E.F.G,12 samples in each group, drill a hole in the vertebraes body, at room temperature immerse the samples into 0.4916mmol/L EDTA-Na2 demineralized liquid, separately withdraw samples from 0 (not immersed),3,6,9 and 12 perday for BMD. For the demineralised groups after 0,9days, take X rays (n=7), and then divide for general observation (n= 7),and the intermediate vertebras make Micro-CT examination (n=7) and ashes of measurement (n=7).
Results With the decalcification, general observation shows that bone trabecula of each group became thinner and the gap between bone trabecula became wider gradually,the bone mineral density test show a trend that reduced, and the difference of BMD measurement bwteen pre-and post-demineraling process were statistically significant. After demineraling for 9 days Micro-CT detection showed that vBMD, BVF, Tb.Th, Tb.N, Tb.Sp, SMI, DA, Eu.N, Conn.Dn were significantly different compared to pre-demineraling (p<0.05). After Ashes of determination demineralization, at 0,9 day (days), the BMC apparently changed (p<0.05).
Conclusion The vertebraes decalcified for 9-12 days to simulate osteoporotic vertebral compression fractures is a fast, feasible and reproducible method.
PartⅢ:
The biomechanical experiment of osteoporosis model in vitro and its clinical significancee
Background In 1885, Pommer suggested that the word of osteoporosis, which describes the reducing of sclerotin, voided bones, and it is defined by the 3rd of osteoporosis meeting held in denmark in 1990, and then it is completed in the 4th meeting in 1993, and confirmed by every country in the world, in 2001, NIH suggested that the osteoporosis is a kind of bone diesease that results in fracture by bone strength reducing, the BMD and bone mass reflect the bone strength.
In 1987, Galibert and Deramond from Franch adopted the treatment for hemangioma by inject bone cement after skin puncture the centrum according to the experience of filling bone cement in surgical operation, this is the first case of application in clinical. In 1990, they further suggested that this technique can be applied in osteoporosis centrum fracture (OCF), which is a milestone of Vertebroplasty. And this technique is widely applied and developed in the field.
Objective Probe into sheep thoracic and lumbar vertebraes biomechanical characteristics change after Vertebroplasty and osteoporosis, provide evidence for clinical experiment.
Method Take 6 of each group (A.B.C.D.E.F.G) of sheep thoracic and lumbar vertebraes in 0.4916mmol/L EDTA-Na2 decalcification liquid in total of 42, a sample is with drawed from day 0 (not immersed),3,6,12,and two samples from day 9 for experimental use. Measure the height and weight of the centrum of each group in the front, rear, left, right direction, calculate the average out by the measuring tool of vernier caliper, accurated to 0.02mm.The compressive strength is the maximum pressure, that compresses the upper and down centrums of samples on the mould, and then put into the biomechanic apparatus, process vertical load in the rate of 5mm/min, when the sample is submited, measure the max-compressive resistance.Record the strength and shape-change (height) 10times/min, draw the curve of strength vs shape-change, find out the initial compressive strength and stiffness. Set the maximum pressure as the compressive strength, take the gradient of the curve of Strength vs Shape-change as the compressive stiffness.During the compression inject the 0.9% sodium chloride solution continuously on the sample, keep the sample moist.
Take vertebroplasty for ninth day compressive sample, divided into PMMA group and bioactive bone cement group,7 specimens in each group; imitate the clinical operation of PKP under c-arm X-ray machine monitoring.Drill hole on pedicle of vertebral arch by 3.5mm drill, into 1/3 of the centrum, after placing the guidepin, insert a trocar along the guidepin, along the trocar, place in an expandable spherical gasbag, and then aerate the gasbag. After the balloon expansion and reset satisfaction after recovering the withdrawal. Mix the bone cement, PMMA Group using the zimmer bone cement, and bioactive bone cement group with CPC/PMMA composite consisting of bioactive bone cement. Put bone cement into the injector, taking injection to the cavity of the centrum during the bone cement is in round state according to the volume of the gasbag. Shooting a side X-ray image of each centrum, measure the height, compressive stiffness and compressive strength (same method as having said).
Analysis the BMD, compressive strength and stiffness with correlation analysis by statistical software SPSS 17.0, Vertebral molding before and after of the vertebral compression strength and stiffness, comparison with single factor analysis of variance (ANOVA), the level of significance in P<0.05.
Results The centrums are in total of 42 in this experiment, as the decalcification time the BMD, vertebral compression strength and stiffness of each group reduce gradually, differ from each other obviously(P<0.005), that the groups after the vertebroplasty are higher than before in the compressive strength(P<0.05); The PMMA group were not significantly different compared to vertebral molding before in vertebral compression stiffness(P>0.05);The active bone cement group are higher than before and PMMA group in vertebral compression stiffness, that (P<0.01).
The BMD of the centrum and the compressive strength of the centrum are obvious in relationship of positive correlation (y= 0.976, P<0.01).The magnitude of BMD direct influences the Vertebral bearing capacity. The stiffness of the centrum means the deformability resistance of the centrum under the axial load, is in relationship of positive correlation (y=0.922, P<0.01).
Conclusion The first, BMD can reflect the level of osteoporosis, predict the fracture, and in an extent that could show the sclerotin situation of the centrum, offering help in clinical treatment and early diagnosis.The second, Bioactive bone cement in vertebroplasty surgery can restore the biomechanical strength of vertebral body, that can be achieved by the normal physiological needs of vertebral.The end, Rapid Preparation of EDTA decalcification method for osteoporosis model provide the basis for Biomechanical and clinical studies.
引文
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