羟基磷灰石/壳聚糖复合二甲双胍用于大鼠骨缺损的生物学特点

Biological characteristics of hydroxyapatite/chitosan combined with metformin for bone defect in rats

背景:二甲双胍能够刺激机体干细胞的成骨分化,促进骨组织再生修复。目的:观察羟基磷灰石/壳聚糖复合二甲双胍在大鼠颅骨骨缺损中的修复再生作用。方法:将壳聚糖溶于二甲双胍去离子水溶液中,随后加入一定质量比的羟基磷灰石,制备羟基磷灰石/壳聚糖/二甲双胍复合材料,同理制备羟基磷灰石/壳聚糖复合材料。将羟基磷灰石/壳聚糖/二甲双胍复合材料浸泡于细胞培养液中,体外检测复合材料中的二甲双胍释放速率。取30只8周龄雄性SD大鼠,制作直径5 mm颅骨缺损,分3组干预:空白组不植入材料,对照组、实验组分别植入羟基磷灰石/壳聚糖复合材料与羟基磷灰石/壳聚糖/二甲双胍复合材料,术后3个月,进行颅骨缺损部位X射线片、Micro-CT与组织学检测。动物实验通过湖北医药学院伦理委员会通过(批准号:20170539)。结果与结论:①羟基磷灰石/壳聚糖/二甲双胍复合材料在细胞培养液中可持续释放二甲双胍,至21 d时二甲双胍累计释放量为(95.37±4.31)%,可见二甲双胍持续释放贯穿于整个成骨细胞诱导分化过程中;②X射线片、Micro-CT检测显示,3组骨缺损部位均有不同程度的修复,骨折线模糊,实验组骨缺损新生骨面积与体积大于对照组、空白组,且实验组新生骨面积与体积大于对照组;③苏木精-伊红染色显示,实验组与对照组材料不同程度的降解,新生骨主要填充于支架材料间隙,周围伴有少量纤维细胞,实验组、对照组新生骨面积百分数及新生血管密度多于空白组(P<0.05),实验组新生骨面积百分数及新生血管密度多于对照组(P<0.05);④结果表明,羟基磷灰石/壳聚糖复合二甲双胍复合生物材料具有良好的促成骨作用。

BACKGROUND:Metformin can stimulate the osteogenic differentiation of stem cells and promote the regeneration and repair of bone tissue.OBJECTIVE:To observe the restorative effect of hydroxyapatite/chitosan combined with metformin on skull defects in rats.METHODS:The hydroxyapatite/chitosan/metformin composite was prepared by dissolving chitosan into metformin deionized water solution and adding hydroxyapatite with certain mass ratio.Hydroxyapatite/chitosan composite was prepared by the same method.The hydroxyapatite/chitosan/metformin composite was immersed in cell culture medium,and the release rate of metformin in the composite was detected in vitro.Thirty 8-week-old male Sprague-Dawley rats were used to make skull defects at a diameter of 5 mm.Samples were randomly divided into three groups.No material was implanted in the blank group.The control group and experimental group were implanted with hydroxyapatite/chitosan composite and hydroxyapatite/chitosan/metformin composite respectively.At 3 months after operation,X-ray,micro CT and histological examination were performed.The animal experiment was approved by the Ethics Committee of Hubei Medical University(approval No.20170539).RESULTS AND CONCLUSION:(1)Hydroxyapatite/chitosan/metformin composite sustained release of metformin in the cell culture medium,and the cumulative release of metformin was(95.37±4.31)%at 21 days,which showed that metformin continued to release throughout the induction and differentiation of osteoblasts.(2)X-ray and micro-CT results showed that the skull bone defects of the three groups were repaired to different degrees,and the fracture line was fuzzy.The area and volume of new bone in the experimental group were larger than those in the control group and blank group,and those in the experimental group were significantly larger compared with the control group.(3)Hematoxylin-eosin staining results showed that in the experimental group and the control group,the materials were degraded in different degrees.The new bone was mainly filled in the gap between the scaffold materials,with a small number of fiber cells around.The percentage and density of new bone area in the experimental group and the control group were significantly higher than those in the blank group(P<0.05),while the percentage and density of new bone area in the experimental group were significantly higher than those in the control group(P<0.05).(4)The results indicate that hydroxyapatite/chitosan/metformin composite biomaterial has a good osteogenic promoting effect.