The relationship between the change of Osx and Dlx5 expressed in the callus and fracture healing in type 2 diabetes rats

doi:10.3969/j.issn.2095-4344.2015.11.012

Abstract

Abstract:

BACKGROUND: Experiments have shown that the expression levels of Osx and Dlx5 may have some relationships with fracture healing disorder.

OBJECTIVE: To probe into the relationship between the change of expression of Osx and Dlx5 in the callus of type 2 diabetes rats and fracture healing disorder.

METHODS: We selected 32 Sprague-Dawley rats with proper weight in 8 weeks age. They were randomly assigned into two groups: the experimental group (n=18) was fed with high fat and sugar diet for 8 weeks, and then received intraperitoneal injection of streptozotocin to induce diabetic models; the control group was fed with normal diet and received intraperitoneal injection of the same dose of citric acid. Two weeks later, all the rats were used to establish left tibia traction fracture models with orthopedic external fixator. We extended the external fixator at a proper length per day. The animals were sacrificed at 15 days after establishment of traction fracture models. The blood specimens were evaluated for biochemical detection. X-ray was used to observe the growth of callus. The callus tissues from the left tibia were taken for histological observation and the expression of Dlx5 and Osx in the callus was detected.

RESULTS AND CONCLUSION: X-ray results showed that the amount of callus tissues in the experimental group was significantly reduced. The hematoxylin-eosin staining of the callus tissue revealed that the microcolumn formation in the experimental group was significantly reduced compared with the control group; the area of primary matrixfront was lightly colored. The callus histological quantitative analysis showed that in the experimental group the area of new callus decreased and more adipose cells presented in the proximal fracture end compared with the control group (P < 0.01). QPCR displayed that the expression of Osx and Dlx5 was lower in the experimental group than the control group (P < 0.05). These findings indicate that the poorer fracture healing in type 2 diabetes than the normal rats may be associated with the decrease of Osx and Dlx5 expression.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: Diabetes Mellitus, Type 2, Bony Callus, Fracture Healing

CLC Number:

R318

Liu Zhen-dong, Cai Xu, Wang Kui-xiang, Zhang Yuan-jun, Liu Zhi-tao. The relationship between the change of Osx and Dlx5 expressed in the callus and fracture healing in type 2 diabetes rats[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(11): 1699-1706.

Figures/Tables 6

2.1 实验动物数量分析实验选用大鼠32只，分为2组，实验过程中无脱失，全部进入结果分析。 2.2 实验组与对照组大鼠血液生化指标 2.2.1 给药前血液生化指标分组饲养8周后，实验组大鼠与对照组大鼠相比较总胆固醇明显升高(P < 0.01)，三酰甘油和空腹胰岛素差异有显著性意义(P < 0.05)，空腹血糖差异无显著性意义(P > 0.05)，见表1。 2.2.2 腹腔注射药物2周后血液生化指标与对照组大鼠比，实验组大鼠血清总胆固醇以及空腹血糖明显升高，差异有非常显著性意义(P < 0.01)；三酰甘油水平升高，差异有显著性意义(P < 0.05)；空腹胰岛素两组比较差异无显著性意义(P > 0.05)，见表2。 2.2.3 牵引模型持续牵引2周后的血液生化指标牵引2周后，实验组大鼠与对照组大鼠相比，总胆固醇、三酰甘油、空腹血糖的检测结果则显示差异均有显著性意义(P < 0.05)，而两组大鼠空腹胰岛素比较差异无显著性意义(P > 0.05)，见表3。通过对比8周时，链脲佐菌素注射2周后，牵引2周后3个不同时期血液生化指标(总胆固醇、三酰甘油、空腹胰岛素、空腹血糖)，可知大鼠骨折牵引模型以及骨折愈合是在典型2型糖尿病病理条件下进行。 2.3 X射线片牵引骨痂生长情况对比骨折牵引模型采取持续牵引，均匀的间断延长14 d，牵引后第15天采集标本，行X射线检查，见图2。X射线照射条件：40 kV，4 mA•s，将实验组、对照组大鼠随机分为4组统计，对于照射后X射线照片，采用imagej2.1软件分析，将X射线图像转化为8位图像，通过灰度值，对骨折间隙内骨痂面积以及骨痂密度进行量化统计，发现实验组大鼠及对照组大鼠均有骨痂生成，而实验组大鼠与对照组正常大鼠相比骨痂面积明显减少(P < 0.05)，实验组平均灰度值较对照组下降(P < 0.05)，见表4。 2.4 苏木精-伊红染色牵引骨痂生长情况对比随机选取实验组、对照组大鼠各5只，并行编号1-5号，行苏木精-伊红染色。检测其苏木精-伊红染色显示：实验组大鼠与对照组大鼠相比，实验组大鼠微骨柱排列紊乱，部分甚至未见明显微骨柱，初始基质前沿大部分明显浅染，证明实验组大鼠骨痂形成较正常大鼠明显减慢，部分出现骨痂形成障碍，而骨痂生长，分化能力明显下降，见图3。组织学定量对骨痂面积分析显示：实验组大鼠骨痂面积相对减少(P < 0.05)，见表4。靠近骨折端骨髓内脂肪细胞数量在单位面积内明显增多，见图4。2.5 Western blot结果实验组和对照组相比，实验组大鼠骨折牵引间隙内骨痂组织中Dlx5、Osx蛋白的灰度值较对照组明显降低，表达下调，经统计学分析差异有显著性意义(P < 0.05)，见图5，表5。 "

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