Insulin effects on fracture healing and cytokines in the osteotylus in experimental diabetic rats

doi:10.3969/j.issn.2095-4344.2016.29.002

Abstract

Abstract:

BACKGROUND: Fracture healing in diabetic patients is usually unsatisfactory because of hormones and metabolic disorder, and an eventual multiple organ dysfunction resulting from high blood glucose.
OBJECTIVE: To dynamically observe the changes of cytokines during the fracture healing process in diabetic rats before and after insulin treatment.
METHODS: A total of 120 Sprague-Dawley rats were included in this study. Of them, 90 rats intravenously injected with 5% tetraoxypyrimidine to induce rat models of diabetes were randomized into insulin treatment and diabetes groups, respectively. The remaining 30 rats were intravenously injected with equal volume of saline and selected as control group. The next day, blood glucose was determined. Healing at 1, 4, and 8 weeks after fracture were observed by the X-ray film. Biomechanical strength of the injured right tibia was measured at 4, 6, and 8 weeks after modeling. Cytokines in the osteotylus were determined by immunohistochemical staining and in situ hybridization technique.
RESULTS AND CONCLUSION: The X-ray films showed that the speed of fracture healing in the diabetes group was slower than insulin treatment and control groups. Biomechanical strength of the osteotylus in the diabetes group was significantly decreased compared with the insulin treatment and control groups. However, there were no significant differences in above-mentioned parameters between the control and insulin treatment groups. Bone morphogenetic protein 2, basic fibroblast growth factor, transforming growth factor-beta, and vascular endothelial growth factor were widely expressed in the osteotylus and their expressions in diabetes group were significantly lower and slower than those in the control and insulin treatment groups. There was no statistical difference between control and insulin treatment groups. These results indicate that osteotylus formation speed, biomechanical strength, and growth factor expressions at the fracture site in diabetes rats were decreased compared with normal rats. Insulin treatment can enhance cytokine levels at the fracture site, thereby promoting the osteoblast proliferation and fracture healing.

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

Key words: Diabetes Mellitus, Insulin, Fracture Healing, Cytokines, Tissue Engineering

CLC Number:

R318

Zhou Qiang, Lu Hua, Wang Zhan-chao, Zhang Hao-jie, Jiang Lei-sheng. Insulin effects on fracture healing and cytokines in the osteotylus in experimental diabetic rats[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(29): 4269-4276.

Figures/Tables 3

静脉注射5%四氧嘧啶，选取连续3 d血糖> 16.7 mmol/L的糖尿病造模成功大鼠进行实验。成功造模74只，建模成功率82.2%。将建模成功的大鼠分为2组，糖尿病组37只，胰岛素治疗组37只。 2.2 不同时期的血糖水平变化分别于建模前和建模后的48 h以及第1，4，6，8周测量空腹血糖值，见表1。造模前，对照组、糖尿病组和胰岛素治疗组之间的基础血糖没有差异。大鼠注射四氧嘧啶48 h后，血糖浓度与对照组比较显著升高(P < 0.01)，达到(28.71±2.20) mmol/L。骨折造模1周后，胰岛素治疗组血糖值明显低于糖尿病组，且糖尿病组相较于胰岛素治疗组和对照组，血糖值差异有显著性意义(P < 0.01)。结果表明，实验成功建立了糖尿病和胰岛素治疗组的大鼠骨折模型。 2.3 各组大鼠X射线片观察骨折愈合情况见图1。骨折后第1周(图1A)骨折端骨折线清楚，骨膜增厚，对照组和胰岛素治疗组可见形成少量骨痂，然而糖尿病组中并未观察到明显的骨痂形成现象。骨折后第4周(图1B)对照组和胰岛素治疗组可以看到，骨折区域的X射线片透亮度显著下降，骨量较多，并能观察到骨小梁发生连接，而在糖尿病组观察到骨折区域的X射线片透亮度较高，骨量较少，仍能观察到清晰的骨折线。骨折后第8周(图1C)对照组和胰岛素治疗组可以看到，骨折区域的骨量进一步增加，观察到的骨折线模糊不清，而在糖尿病组的骨折区域可见大量的骨痂包裹，但相比对照组和胰岛素治疗组骨折线仍较清晰。 2.4 生物力学检测结果将对照组、糖尿病组、胰岛素治疗组造模后的第4，6，8周大鼠取出完整胫骨干置于力学测试机的夹具上固定测定。破坏性三点弯曲力学测试结果见图2A，B，扭转强度测试结果见图2C，D。由计算机软件自动计算弯曲强度、扭转强度和每对标本的参数比率。结果显示，糖尿病组生物力学特征的数值相比对照组差异有显著性意义(P < 0.01)，而胰岛素治疗后检测数值明显上升，差异有显著性意义(P < 0.01)。 2.5 免疫荧光检测相关细胞因子的表达为了检测糖尿病和胰岛素干预治疗对糖尿病大鼠骨折愈合的影响，实验通过免疫荧光技术检测骨痂内多种常见细胞因子的表达情况。在第4周，分别选取对照组、糖尿病组、胰岛素治疗组中骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子的免疫荧光结果(图3)。骨形态发生蛋白2、碱性成纤维细胞生长因子、血管内皮生长因子呈绿色荧光的阳性表达，可以明显观察到对照组和胰岛素治疗组的荧光强度较高，糖尿病组中则较弱；转化生长因子呈红色荧光的阳性表达，同样可以观察到对照组和胰岛素治疗组的荧光强度较高，糖尿病组中则较弱。结果表明，糖尿病组中各细胞因子骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子相比于对照组免疫染色结果均下降，而糖尿病组进行胰岛素治疗后，骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子的免疫染色显著增强，且与对照组染色结果区别不大。 2.6 原位杂交检测相关细胞因子的表达为了进一步观察3组大鼠骨折愈合过程中细胞因子的动态变化，实验运用原位杂交技术检测骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子在造模后1，2，3，4，6，8周的表达情况(图4A)，并计算平均灰度值，代表各细胞因子的含量。对照组和胰岛素治疗组的骨形态发生蛋白2在第1周和第2周的阳性表达逐步升高，第2周达顶峰，第3，4，6，8周逐渐呈下降趋势，而糖尿病组骨形态发生蛋白2阳性表达第3周达顶峰，且阳性表达明显低于对照组(P < 0.01)(图4B)。对照组和胰岛素治疗组的碱性成纤维细胞生长因子表达自第1周至第3周逐步提高，呈现高阳性的持续表达，第3周达表达顶峰，在第4，6，8周表达逐渐降低，而糖尿病组碱性成纤维细胞生长因子阳性表达在第4周达顶峰，且阳性表达明显低于对照组(P < 0.01)(图4C)。对照组和胰岛素治疗组的转化生长因子阳性表达自第1周至第3周逐步升高，第3周达顶峰，第4，6，8周逐渐下降，而糖尿病组转化生长因子阳性表达在第4周达顶峰，且阳性表达明显低于对照组(P < 0.01)(图4D)。对照组和胰岛素治疗组的血管内皮生长因子表达第1至3周逐步升高，呈持续性的高阳性表达，在第3周时达顶峰，第4，6，8周逐渐下降，而糖尿病组血管内皮生长因子阳性表达在第4周达顶峰，且阳性表达明显低于对照组(P < 0.01)(图4E)。"

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