掺钴磷酸钙骨水泥的制备及其诱导骨血管生成的研究

发布时间：2018-06-23 21:29

  本文选题：磷酸钙 + 碳酸钴　； 参考：《南方医科大学》2017年硕士论文

【摘要】：实验目的:低氧诱导因子(HIF-1α)是调控组织和细胞适应低氧环境的核心转录因子,二价钴离子作为低氧诱导剂可以明显促进其表达。本实验将微量元素钴负载于磷酸钙骨水泥材料中,以持久模拟细胞缺氧状态,诱导细胞自主产生促血管因子,如:VEGF-A等,可望研制出具有超长诱导血管生成活性的可降解骨修复材料,促进材料周围组织的血管大量形成,进而有利于新骨的生成及改建,加快骨缺损修复的进程。实验方法:1、制备并表征该材料的可注射性、凝固时间、降解性能及钴离子缓释效果。2、评价材料的细胞毒性与粘附性及其对细胞形态及骨架的影响3、实时荧光定量PCR检测MC3T3-E1细胞中HIF-1α的表达情况,ELISA检测VEGF-A的表达情况,观察HUVEC成管效果。4、通过组织病理学和免疫组化方法评估体内成血管成骨能力。实验结果:1、骨水泥早期可注射性未受到明显的影响,推注时间超过3min时,随着碳酸钴含量的增加,可注射性逐渐变差且水泥初凝时间逐渐缩短,终凝时间无明显变化;骨水泥降解率比较:0%1%5%10%;掺钴的骨水泥均能够缓释钴离子,且缓释长达56天。2、0%、1%、5%及10%组细胞毒性依次为:100.4%、123.2%、96.7%、83.1%;所有细胞均能粘附,生长,不同浓度钴离子促进细胞增殖的增殖速率比较:1%0%5%10%;0%、1%、5%的细胞形态无明显改变,10%组的细胞质萎缩,突触明显减少,细胞形态变化较大。3、掺钴磷酸钙能够模拟细胞缺氧状态;随着碳酸钴含量的提高,细胞表达VEGE-A的量逐渐增加;4h时的各组血管标志物表达情况:空白对照组≈0%1%5%,6h后各组成管效果差距更加明显,空白对照组0%1%5%。4、皮下免疫组化结果:1%组出现大量毛细血管,5%组血管密度最高,但管径略小于1%组,其余各组血管很少;骨组织免疫组化结果:术后2月,空白组和0%组未见明显毛细血管,1%组和5%组在新生骨质内可见明显血管状结构,5%组的毛细血管状结构密度最高。术后3个月,血管状结构退化,1%和5%组新生骨生长充分,此外,5%组可见相当成熟的哈弗氏管;骨组织HE染色:成骨效果比较可见,空白对照组0%1%5%组;骨组织Masson染色:空白对照和0%组骨胶原稀疏,1%和5%组骨胶原大量充满缺损处,其中5%组新生的骨胶原更加密实、成熟,已可见明显的哈弗氏管。结论:1、采用微量金属元素钴元素提高磷酸钙材料的生物学功能具有一定的可行性。2、少量的碳酸钴(≤1%)有利于细胞的增殖粘附,明显提高材料的生物相容性;但是,大量的碳酸钴(≥10%)反而增加该水泥的细胞毒性,抑制细胞增殖粘附。3、掺钴的磷酸钙骨水泥均能够明显促进细胞分泌VEGF-A并直接作用于血管内皮细胞,促进其增殖、迁移、分化及芽生毛细血管。4、在动物体内,掺钴的磷酸钙骨水泥能够明显促进局部毛细血管再生及骨胶原的合成,加快骨痂的生成、改建和重塑。
[Abstract]:Objective: hypoxia inducible factor (HIF-1 伪) is a core transcription factor regulating the adaptation of tissues and cells to hypoxia. In this experiment, the trace element cobalt was loaded into calcium phosphate cement material to simulate the anoxic state of cells and induce the spontaneous production of vascular stimulating factors, such as: VEGF-A, et al. It is expected that a biodegradable bone repair material with super-long angiogenic activity will be developed, which can promote the formation of blood vessels in the surrounding tissues of the material, thus facilitate the formation and remodeling of new bone and accelerate the process of bone defect repair. Experimental method: 1, preparation and characterization of the injectability, solidification time of the material, Biodegradability and sustained release effect of cobalt ion. The cytotoxicity and adhesion of the materials and their effects on cell morphology and cytoskeleton were evaluated. The expression of HIF-1 伪 in MC3T3-E1 cells was detected by real-time fluorescence quantitative PCR and the expression of VEGF-A in MC3T3-E1 cells was detected by Elisa. To observe the effect of HUVEC tube formation. 4. To evaluate the ability of vascular formation by histopathology and immunohistochemistry. The results showed that the early injectability of bone cement was not affected obviously. When the injection time was longer than 3min, the injectability became worse with the increase of cobalt carbonate content, and the initial setting time of cement decreased gradually, but the final setting time did not change obviously. The degradation rate of bone cement was 1: 015. The bone cement doped with cobalt was able to release cobalt ion slowly, and the cytotoxicity of 5% and 10% groups was 100.4% and 123.2% respectively. All the cells were able to adhere and grow. Comparison of proliferation rates of different concentrations of cobalt ions on cell proliferation; the cell morphology of 5% of the cells in the 10% group did not change significantly, the synapses decreased significantly, and the morphology of the cells changed significantly. 3. Calcium cobalt phosphate could mimic the anoxic state of the cells. With the increase of cobalt carbonate content, the expression of VEGE-A in the cells increased gradually at 4 h. The difference in the effect of each component tube was more obvious in the blank control group 鈮，

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