Chinese Journal of Tissue Engineering Research ›› 2017, Vol. 21 ›› Issue (14): 2227-2233.doi: 10.3969/j.issn.2095-4344.2017.14.016
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Lu Jing1, Wang Yu-jia1, Ling Wei-shi1, Chen Min-xiao1, Li Chang-hong1, Guan Shuang1, Deng Xu-ming2
Received:
2017-01-23
Online:
2017-05-18
Published:
2017-06-10
Contact:
Deng Xu-ming, Professor, Doctoral supervisor, College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin Province, China
About author:
Lu Jing, College of Food Science and Engineering, Jilin University, Changchun 130062, Jilin Province, China
Supported by:
the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period, No. 2013BAD16B09
CLC Number:
Lu Jing, Wang Yu-jia, Ling Wei-shi, Chen Min-xiao, Li Chang-hong, Guan Shuang, Deng Xu-ming. Preparation and properties of collagen/chitosan composite sponge from forest frog skin[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(14): 2227-2233.
2.1 胶原海绵的吸水倍数 海绵的吸水性能够侧面反映海绵的预期止血效果,较高的吸水能力为海绵的止血奠定了初步基础,是海绵止血性能优劣的一个重要指标;此外吸水能力可以在一定程度上反映胶原海绵的品质,比如海绵孔径大小和杂质如盐分含量的多少[19]。 由图1可以看出,胶原蛋白的量和海绵的交联程度直接影响胶原海绵的吸水倍数。胶原蛋白含量越多,海绵融水越快,吸水倍数越低。戊二醛的浓度越高,交联越好,吸水倍数越高。当壳聚糖与胶原蛋白的质量比为1∶1、戊二醛浓度为1.5%和2%时,海绵的吸水倍数最高。与此次研究结果相似,有研究发现壳聚糖与胶原蛋白的比例为1∶1时胶原海绵吸水性好,并且戊二醛的浓度(0.1-0.5%)越高,胶原海绵吸水性越好[20]。也有研究发现聚糖与胶原蛋白的质量比为3∶7胶原海绵吸水性好,且在一定范围内,随着戊二醛浓度的增加,吸水倍数呈先增加后下降的趋势,在戊二醛浓度为 0.015%时吸水倍数最大[14]。因此,壳聚糖与胶原蛋白比例及戊二醛的浓度均与吸水倍数密切相关,不同研究之所以获得差异结果可能与胶原蛋白种类有关。 "
2.2 胶原海绵的机械性能 材料的力学性能与材料的内部结构密切相关,是胶原海绵材料的重要性能指标。胶原海绵是疏松多孔的结构,并且交联程度不同的海绵其内部的空隙不同,因此,海绵的机械性能与海绵的交联度有很大的关系。研究以断裂伸长率为指标考察海绵的机械性能。研究认为戊二醛交联能显著提升胶原材料的拉伸强度和断裂伸长率[21],海绵的断裂伸长率随戊二酸浓度的增加先增加后降低,并在戊二醛浓度为1.5%时达到最大值[20],与此次研究结果相似。此外,胶原蛋白的含量和种类也影响胶原海绵的机械性能[22],断裂伸长率随胶原蛋白比例的增加及壳聚糖比例的减少而降低[20]。由图2可以看出当壳聚糖与胶原蛋白的质量比为1∶1、戊二醛的浓度为1%时,林蛙胶原海绵的机械性能最好。"
2.4 胶原海绵制备工艺优化 由表1可以看出,壳聚糖/胶原蛋白质量比对海绵吸水倍数的影响大于戊二醛的浓度,当壳聚糖/胶原蛋白质量比为1∶1、戊二醛的浓度为2%时,海绵的吸水倍数最大。戊二醛的浓度对海绵机械性能的影响大于壳聚糖/胶原蛋白质量比,当壳聚糖/胶原蛋白质量比为1∶1、戊二醛的浓度为1%时,海绵的机械性能最好。戊二醛的浓度对海绵热变性温度的影响大于壳聚糖/胶原蛋白质量比,当壳聚糖/胶原蛋白质量比为1∶2、戊二醛的浓度为1%时,海绵的热变性温度最佳。综合考虑以上各种影响因素以及对复合海绵的指标要求,采用壳聚糖/胶原蛋白为1∶1,戊二醛添加量为1%,可获得吸水性,机械性,热变性较理想的胶原海绵。"
2.5 胶原海绵的细胞相容性 体外细胞相容性实验由于实验条件易于控制、能分析单一因素对细胞的影响、具有标准化、定量和可重复性等优点而成为研究生物材料相容性的主要方法[27]。MTT法检测细胞毒性是体外细胞相容性检测的首要方法[28],因此采用MTT法检测胶原海绵的体外细胞相容性。 由图4可以看出,所有的林蛙皮胶原海绵浸提液对人包皮成纤维细胞均没有抑制作用,说明胶原海绵具有良好的体外细胞相容性,并且除了壳聚糖与胶原蛋白质量比为 1∶4、戊二醛浓度为1%和5%的胶原海绵浸提液外,其他胶原海绵对对人包皮成纤维细胞的细胞活性均有促进作用,有利于细胞的活性和生长,具有良好的生物相容性,符合生物材料应用要求。"
2.6 胶原海绵形貌观察 胶原的微观形貌对于有效的聚集、吸附血小板完成止血作用机制具有重要的作用,是影响材料止血性能的关键因素之一。海绵的孔径过大,则机械强度差;海绵孔径过小,影响创面初次渗血的吸收,因此,海绵孔径大小是衡量海绵质量的重要指标[29]。溶液酸度、预冻温度、溶液浓度、预冻时间、胶原浓度、无二醛交联等制备过程中的参数影响胶原海绵的微观形貌[30]。随着胶原质量的上升,林蛙皮胶原/壳聚糖复合海绵微结构中的丝状结构逐渐减少,出现了较大片状结构,同时海绵的孔径变大;随着戊二醛浓度的上升,海绵的孔结构减少。由图5可以看出优化后的海绵孔径大小为200-400 µm,网孔大小基本一致,排列有序。"
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