低强度脉冲超声波影响兔骨髓基质干细胞与β-磷酸三钙复合修复骨缺损的实验研究

1、低强度脉冲超声波影响兔骨髓基质干细胞与-磷酸三钙复合修复骨缺损的实验研究         10-05-04 15:59:00     编辑：studa20             作者：梁伟，苟三怀，欧阳跃平，刘岩，严力生，陈洪勋【摘要】  目的 探讨低强度脉冲超声波(LIPUS)影响-磷酸三钙(-TCP)与兔骨髓基质干细胞(BMSCs)复合体修复

2、兔桡骨骨缺损的效果。方法 从兔股骨粗隆部抽取骨髓，体外培养扩增，取第3代BMSCs接种于-TCP培养1周，分为两组，一组予以LIPUS作用，另一组作为对照。以20只成年新西兰兔为研究对象，制作双侧桡骨远端骨缺损模型。一侧桡骨骨缺损处植入LIPUS作用过的细胞支架复合体做为实验组，另一侧植入LIPUS未作用过的细胞支架复合体做为对照组。分别于术后4周、8周处死新西兰兔，运用计算机图像分析X线片上骨痂的灰度密度，行组织学切片检查骨痂生长情况。结果X线片骨痂灰度密度测量提示实验组骨痂较对照组生长明显增多。组织学切片显示实验组血肿机化、吸收、骨小梁和骨基质形成早于对照组。结论在骨缺损修复早期，LIPU

3、S对BMSCs与-TCP复合体修复兔骨缺损有明显的促进作用；而在骨缺损修复晚期，LIPUS的作用相应减弱。 【关键词】  骨组织工程； 细胞支架复合体； 骨缺损； 低强度脉冲超声波    Abstract: ObjectiveTo explore the effect of low intensity pulsed ultrasound(LIPUS) on the ability of repairing rabbit radius defect by cell-scaffold complex composed with bone marrow str

4、omal cells (BMCs) and -tricalcium phosphate (-TCP). MethodBone marrow of a rabbit was drawn and cultured by differentiation culture medium to harvest BMCs and the third generation BMCs were cultivated on -TCP for one week in vitro.Bilateral distal radial defects were made using a 1-mm saw in 20 matu

5、re New Zealand white rabbits.The cell-scaffold complexs stimulated by LIPUS were implanted in rabbit radial defects (cell-scaffold complexs without stimulation as control).All the rabbits were killed at 4 or 8 weeks after operation.The defect healing state was observed with X-ray and histological te

6、chnic.The gray density of CA was analyzed by computer image analysis system.ResultIt was found that the callus in the experimental group grew more quickly and apparently than that in the control group.At 4 weeks after operation,there was significantly statistical difference (P<0.05) in CA hetween

7、 the two groups with image analysis system.But at 6 weeks after operation,the bone density difference was not statistically significant (P>0.05).Histological slices in the experimental group showed that the absorption,organization of hematoma,the formation of trabeculae and matrix preceded those

8、in the control group.There were more fibroblast,osteoblasts and callus in the experimental group.ConclusionCell-scaffold complex composed with BMCs and -TCP stimulated by LIPUS can repair bone defect more effectively than that without LIPUS stimulation at the early stage of healing,but the effect

9、60; tend to decrease at the late stage.    Key  words：bone tissue engineering;  cell-scaffold complex;  bone defect;  low intensity pulsed ultrasound    骨缺损的治疗是骨科临床医师面临的难题之一，创伤、肿瘤和重建常常导致骨缺损。尽管骨组织有强大的自我修复能力，但是骨缺损的面积较大时，常常导致骨不连和机体功能的丧失。自体骨移植是公认的金标准，它具有

10、骨诱导、骨传导和提供成骨细胞的优点，但移植骨的大小和形状受到限制，而且往往带来供区并发症，限制其应用。    组织工程骨的出现为克服上述不足提供了新方法：其基本思路是将活细胞体外培养增殖后，与支架材料相复合修复骨缺损。LIPUS作为一种超过人类听阈范围的高频声波，可对骨和周围组织产生微型压力作用，导致细胞生化性能发生改变，从而促进骨折愈合1、2。临床上，LIPUS已经被成功用于促进骨折愈合和治疗骨不连。    既然LIPUS被广泛应用于临床刺激骨增生，而组织工程骨在修复骨缺损方面的应用又备受关注，将这两种治疗方法结合起来治疗骨缺损有可

11、能产生更好的效果。本实验采用经LIPUS刺激的兔BMSCs和-TCP组成的细胞支架复合体修复兔桡骨骨缺损的方法，为评估LIPUS应用到骨组织工程上的可行性提供实验依据。    1  材料与方法    1.1  实验材料    3个月龄成年雄性新西兰大耳白兔，体重(2.33.2 kg)，由上海市伤骨科研究所动物实验中心提供。    DMEM培养液(GIBCO)，10%胎牛血清(杭州四季青公司)。条件培养基：在10%胎牛血清(FBS)低糖DMEM培养基中加入以

12、下试剂(Sigma,USA)：1×10-8 mmolL地塞米松：（10 mmolL -甘油磷酸钠；50 mgL维生素C）。    立刻生TM低强度脉冲超声波治疗仪，由杭州核新生物医疗技术有限公司提供。超声波强度为40 mWcm2，超声波频率为1.5 MHz，脉冲频率为1 kHz，脉冲宽度为200 ms，间隙时间为800 ms。    1.2  实验方法    1.3  观测指标    1.4  统计学分析    应用SPSS 10.0软件对实验组和对照组骨痂灰度密度的差异进行t检验，P<0.05认为差异有统计学意义。    2  结果