CN107903336B - 一种磷酸肌酸改性壳聚糖材料及其制备方法与应用 - Google Patents
一种磷酸肌酸改性壳聚糖材料及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种磷酸肌酸改性壳聚糖材料及其制备方法与应用。该方法首先将壳聚糖溶于MES缓冲液,再通过零位接枝技术将磷酸肌酸接枝在壳聚糖上,透析后冷冻‑冻干得到磷酸肌酸改性壳聚糖材料。该材料具有多种应用前景:未引入双键时,将材料溶解、冻干成型后加入交联剂‑京尼平交联得到多孔支架,可用于非承重骨缺损部位的修复;与甲基丙烯酸酐反应引入双键时,可在紫外光下交联聚合得到可注射型水凝胶,可用于复杂骨损伤创面的微创原位注射修复。本发明有效解决了单纯使用壳聚糖用于骨修复时效果差,形式单一的缺陷,改性壳聚糖材料不仅可以制备多孔支架,而且可以制备可注射型水凝胶,对于拓展壳聚糖材料在骨组织工程中的应用有重大的意义。
Description
技术领域
本发明属于生物医用骨修复材料制备领域,尤其涉及一种磷酸肌酸改性壳聚糖材料及其制备方法与应用于制备磷酸肌酸改性壳聚糖多孔支架和可注射型水凝胶。
背景技术
骨形成是体内生物矿化的结果,钙磷矿物粒子与胶原结合形成复杂有序的分层结构,获得极好的机械性能。无机材料如磷酸钙,羟基磷灰石和生物玻璃,和正常骨的矿物相具有相似的化学成分,展现出良好的骨传导性、成骨诱导性和机械强度,是骨修复和再生的极佳选择并被用于骨组织工程数十年。但是使用无机材料无法控制和调节材料的性能,尤其是降解性能;很难改变其化学结构以结合生物化学信号促进骨形成。复合具有成骨诱导性的磷酸钙和具有生物降解性的生物高分子成为骨组织工程的一个选择。无机材料-生物高分子复合材料机械强度虽然获得提高,但是在生物高分子中涂覆或复合无机材料也存在诸多缺点,包括两种材料间附着力差;材料表面和矿物层之间缺乏结构联系;无机填料在聚合物基体中分散不均匀,影响整体力学性能;材料降解不同步,聚合物降解后力学性能下降;没有充足的动物实验数据支持;当无机粒子浓度较高时,复合材料表现出易碎的特性等问题。为解决这些问题,希望可以通过生物高分子材料本身的化学改性,模拟天然骨的形成过程,即改性生物高分子材料在生物体内自发诱导矿化成核,形成纳米羟基磷灰石-生物高分子仿生复合材料,在促进骨细胞的粘附和增殖的同时,诱导干细胞成骨分化,最终实现理想的骨修复效果。
磷酸化生物高分子材料,可以从培养基中吸附蛋白、调节细胞的粘附;可以通过配位键与Ca2+结合,促进钙磷结晶的生长。磷酸化改性的材料在诱导羟基磷灰石生长上非常有效。但是多数含磷物质具有细胞毒性,材料降解后易引起炎症,不适宜作为磷酸来源引入聚合物材料中。此外,常用的化学接枝技术会在生物高分子骨架或侧链中引入无关基团,造成高分子难以降解或者降解产物具有细胞毒性。为解决上述问题,本发明通过前期筛选,选择生物相容性良好的磷酸肌酸(phosphatecreatine, PCr)作为磷源,用以改性生物高分子壳聚糖;同时采用零位接枝技术,在合成过程不会引入任何无关链节。该磷酸肌酸改性壳聚糖材料具有多种应用形式,可制备多孔支架及可注射型水凝胶,可以满足不同骨缺损修复的临床需求。
发明内容
本发明的目的在于克服现有技术遇到的问题,目的是提供一种磷酸肌酸改性壳聚糖材料及其制备方法与应用。该方法基于零位接枝技术,通过接枝改性引入磷酸基团和双键,冷冻-冻干得到磷酸化壳聚糖材料。以生物相容性良好的磷酸肌酸作为磷源,用以引入磷酸基团;以甲基丙烯酸酐作为双键来源,用以引入双键。
本发明的目的通过下述方案实现。
步骤一:磷酸肌酸改性壳聚糖(CS-PCr)材料制备
(1)利用零位接枝技术,在常温和水溶液中将磷酸肌酸接枝到壳聚糖上;
(2)反应液透析除去催化剂和未反应的原料;
(3)冷冻-冻干得到磷酸肌酸改性壳聚糖(CS-PCr);
步骤二:可注射型磷酸肌酸改性壳聚糖(MAC-PCr)材料制备
(1)常温下,磷酸肌酸改性壳聚糖(CS-PCr)与甲基丙烯酸酐反应引入双键;
(2)反应液透析除去未参与反应的原料;
(3)冷冻-冻干得到可甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)材料;
步骤三:磷酸肌酸改性壳聚糖多孔支架制备
(1)磷酸肌酸改性壳聚糖(CS-PCr)溶于去离子水,量取适量溶液倒入模具,超声除去气泡;
(2)模具冷冻-冻干制得多孔支架;
(3)模具中加入京尼平-乙醇水溶液交联2 d;
(4)去离子水洗涤,冷冻-冻干得到磷酸肌酸改性壳聚糖(CS-PCr)多孔支架;
步骤四:磷酸肌酸改性壳聚糖水凝胶制备
(1)甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)与光引发剂溶于去离子水;
(2)取适量体积溶液倒入模具,超声除去气泡;
(3)紫外光固化机光照交联,得到磷酸肌酸改性壳聚糖水凝胶。
具体的,一种磷酸肌酸改性壳聚糖材料的制备方法,包括以下步骤:
(1)将磷酸肌酸溶于MES缓冲液中,再加入活化剂,搅拌均匀;
(2)向步骤(1)所得反应液中加入壳聚糖-乙酸溶液,搅拌反应;
(3)将步骤(2)所得反应液转移至透析袋中透析;
(4)将透析后的溶液冷冻-冻干,得到磷酸肌酸改性壳聚糖(CS-PCr)材料。
优选的,步骤(1)所述MES缓冲液的pH为5-6。
优选的,步骤(1)所述活化剂为1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)/ N-羟基丁二酰亚胺(NHS),所用摩尔比EDC:NHS:磷酸肌酸=1:1:1-1:1:0.1。
优选的,步骤(2)所述壳聚糖-乙酸溶液是将壳聚糖溶于1v/v%乙酸中得到。
优选的,步骤(2)所述反应的温度25℃,时间为24-72 h。
优选的,步骤(3)所述透析袋截留分子量为7000-13000,透析时间为3-7天。
由以上所述的制备方法制得的一种磷酸肌酸改性壳聚糖材料,磷酸肌酸改性壳聚糖材料应用于制备磷酸肌酸改性壳聚糖多孔支架和磷酸肌酸改性壳聚糖可注射型水凝胶。
优选的,所述磷酸肌酸改性壳聚糖多孔支架的制备包括以下步骤:
(1)将磷酸肌酸改性壳聚糖(CS-PCr)材料溶于水,再转移至模具中,超声除去气泡,然后冷冻-冻干制得多孔支架;
(2)将多孔支架置于交联剂中交联1-3 d;所述交联剂为浓度为0.01-0.03 g/ml的70-80wt%京尼平-乙醇水溶液;
(3)将交联后的支架用水洗涤,再冷冻-冻干后得到磷酸肌酸改性壳聚糖多孔支架。
进一步优选的,制备孔径均一的多孔支架的冷冻条件:-80℃冰箱冷冻过夜,冻干条件:凝冰器温度-50 ℃,真空度0.4-1 mbar。
优选的,所述磷酸肌酸改性壳聚糖可注射型水凝胶的制备包括以下步骤:
(1)将磷酸肌酸改性壳聚糖(CS-PCr)溶于乙酸溶液中,再加入甲基丙烯酸酐搅拌反应6-12 h;
(2)磷酸肌酸改性壳聚糖和甲基丙烯酸酐质量比为1:2-1:4;
(3)将步骤(1)所得反应液透析除去未参与反应的原料;
(4)将透析后的溶液冷冻-冻干,得到甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr);
(5)将甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)与光引发剂Irgacure 2959溶于水中,得混合液;所述光引发剂在混合液中的浓度为0.0wt5%-0.15wt%
(6)将步骤(4)的混合液倒入模具中,避光超声除去气泡;
(7)将步骤(5)中装有溶液的模具置于紫外光波长为365 nm,强度为5-50 mW的紫外光固化机中光照交联5-30 min,得到磷酸肌酸改性壳聚糖可注射型水凝胶。
进一步优选的,所述模具为圆柱形,模具支架直径为15 mm,高度 3 mm。
与现有技术相比,本发明具有如下优点与效果:
1、本发明有效解决了单纯使用壳聚糖用于骨修复时效果差,形式单一的缺陷,改性壳聚糖材料不仅可以制备多孔支架,而且可以用作可注射型水凝胶,对于拓展壳聚糖材料在骨组织工程中的应用有重大的研究意义。
2、本发明的磷酸肌酸改性壳聚糖多孔支架可用于非承重骨缺损部位的修复。
3、本发明的磷酸肌酸改性壳聚糖可注射型水凝胶有望用于复杂骨损伤创面的微创原位注射修复。
附图说明
图1是实施例1中磷酸肌酸改性壳聚糖材料的核磁磷谱图;
图2是实施例1中磷酸肌酸改性壳聚糖多孔支架照片;
图3是实施例1中磷酸肌酸改性壳聚糖多孔支架电镜图;
图4是实施例1中磷酸肌酸改性壳聚糖水凝胶照片;
图5A是没有磷酸肌酸改性壳聚糖多孔支架用于小鼠颅骨缺损修复CT结果图;
图5B是实施例1中磷酸肌酸改性壳聚糖多孔支架用于小鼠颅骨缺损修复CT结果图。
具体实施方式
为了更好的理解本发明,下面结合实施例对本发明作进一步说明,但是本发明要求保护的范围并不局限于实施例表示的范围。
实施例1
步骤一:
将0.01 mol磷酸肌酸溶于100 ml pH为5的MES缓冲液中,加入活化剂0.01 mol 1-乙基-(3-二甲基氨基丙基碳二亚胺)(EDC)和0.01 mol N-羟基丁二酰亚胺(NHS),常温下反应20 min。将反应后的溶液滴加到100 ml体积浓度为1v/v%的壳聚糖-乙酸溶液中,常温下反应24 h。反应液转移至截留分子量为7 000的透析袋中,每12 h换水一次,透析7 d。透析后的溶液倒入18 cm玻璃培养皿中,-80 ℃冰箱冷冻12 h。将玻璃培养皿置于凝冰器温度为-50 ℃,真空度为0.4的冻干机中冻干2 d得到磷酸肌酸改性壳聚糖材料(CS-PCr),图1为磷酸肌酸改性壳聚糖(CS-PCr)的核磁磷谱结果,表明磷酸肌酸成功接枝到壳聚糖上。
步骤二:
0.1 g磷酸肌酸改性壳聚糖(CS-PCr)材料加入10 ml体积分数为1v/v%的乙酸溶液中,常温搅拌至溶解。再加入0.2 ml甲基丙烯酸酐,常温下反应6 h。反应液转移至截留分子量为7 000的透析袋中,每12 h换水一次,透析7 d。透析过的溶液倒入18 cm玻璃培养皿中,-80℃冰箱冷冻12 h。将玻璃培养皿置于凝冰器温度为-50 ℃,真空度为0.4 mbar的冻干机中冻干2 d得到甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)。
步骤三:
0.1 g磷酸肌酸改性壳聚糖(CS-PCr)材料加入10 ml去离子水,常温下搅拌至溶解。将溶液倒入模具中,模具尺寸:高3 mm,直径15 mm,超声除去气泡。再将装有溶液的模具置于-80 ℃冰箱冷冻12 h后转移至凝冰器温度为-50 ℃,真空度为0.4 mbar的冻干机中冻干2 d得到磷酸肌酸改性壳聚糖多孔支架。0.1 g京尼平溶于100 ml 70wt%的乙醇水溶液,向每个装有磷酸肌酸改性壳聚糖多孔支架的模具中加入200 μl京尼平-乙醇水溶液,常温下交联3 d,用去离子水洗涤,再置于-80 ℃冰箱冷冻12 h后转移至凝冰器温度为-50 ℃,真空度为0.4 mbar的冻干机中冻干2 d得到交联的磷酸肌酸改性壳聚糖多孔支架,该多孔支架的照片见图2。图3为该多孔支架的SEM电镜图片,由图3可知,支架具有孔径大小均一的连通孔结构。多孔支架用于小鼠颅骨缺损修复CT结果见图5A、图5B。其中,图5A为空白对照组,图5B为植入磷酸肌酸改性壳聚糖多孔支架。植入小鼠颅骨缺损8周后,空白组(图5A)无明显新骨生成,实验组(图5B)缺损中间部位出现明显较大面积的新骨生成,表明磷酸肌酸改性壳聚糖支架具有促进骨再生的作用。
步骤四:
0.1 g甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)加入10 ml去离子水,常温下搅拌至溶解。加入0.005 g光引发剂Irgacure 2959 避光搅拌至引发剂溶解。溶液倒入模具中,模具尺寸:高3 mm,直径15 mm,避光超声除去气泡。将模具置于紫外光交联机紫外光照5min,紫外光波长:365 nm,强度:50 mW,制备得到磷酸肌酸改性壳聚糖水凝胶,该水凝胶的照片见图4。
实施例2
步骤一:
将0.01 mol磷酸肌酸溶于pH为5.5的MES缓冲液中,加入 0.05 mol 1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)和 0.05 mol N-羟基丁二酰亚胺(NHS),常温下反应20 min。将反应后的溶液滴加到100 ml体积分数1v/v%的壳聚糖-乙酸溶液中,常温下反应48 h。反应液转移至截留分子量为10 000的透析袋中,每12 h换水一次,透析5 d。透析后的溶液倒入18 cm玻璃培养皿中,-80 ℃冰箱冷冻12 h。将玻璃培养皿置于凝冰器温度为-50 ℃,真空度为0.7 mbar的冻干机中冻干2 d得到磷酸肌酸改性壳聚糖(CS-PCr)材料,该磷酸肌酸改性壳聚糖(CS-PCr)的核磁磷谱结果与图1相似,表明磷酸肌酸成功接枝到壳聚糖上。
步骤二:
0.1 g磷酸肌酸改性壳聚糖(CS-PCr)材料加入10 ml体积分数为1v/v%的乙酸溶液,常温搅拌至溶解。再加入0.3 ml甲基丙烯酸酐,常温下反应9 h。反应液转移至截留分子量为10 000的透析袋中,每12 h换水一次,透析5 d。透析后的溶液倒入18 cm玻璃培养皿中,-80 ℃冰箱冷冻12 h。将玻璃培养皿置于凝冰器温度为-50 ℃,真空度为0.7 mbar的冻干机中冻干2 d得到甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)。
步骤三:
0.1 g磷酸肌酸改性壳聚糖(CS-PCr)材料加入10 ml去离子水,常温下搅拌至溶解。将溶液倒入模具中,模具尺寸:高3 mm,直径15 mm,超声除去气泡。再将装有溶液的模具置于-80 ℃冰箱冷冻12 h后转移至凝冰器温度为-50 ℃,真空度为0.7 mbar的冻干机中冻干2 d得到磷酸肌酸改性壳聚糖多孔支架。0.2 g京尼平溶于100 ml 75v/v%的乙醇水溶液,向每个装有磷酸肌酸改性壳聚糖多孔支架的模具中加入200 μl京尼平-乙醇水溶液,常温下交联2 d,用去离子水洗涤,再置于-80 ℃冰箱冷冻12 h后转移至凝冰器温度为-50 ℃,真空度为0.7 mbar的冻干机中冻干2 d得到交联的磷酸肌酸改性壳聚糖多孔支架,该磷酸肌酸改性壳聚糖多孔支架照片与图2相似。多孔支架的SEM电镜图片与图3相似,可见支架具有孔径大小均一的连通孔结构。小鼠颅骨缺损修复效果类似图5B,多孔支架表现出良好的促进骨再生的作用。
步骤四:
0.1 g甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)加入10 ml去离子水,常温下搅拌至溶解。加入0.01 g光引发剂Irgacure 2959 ,避光搅拌至引发剂溶解。溶液倒入模具中,模具尺寸:高3 mm,直径15 mm,避光超声除去气泡。模具置于紫外光交联机紫外光照15min,紫外光波长:365 nm,强度:27.5 mW。制备得到磷酸肌酸改性壳聚糖水凝胶,该磷酸肌酸改性壳聚糖水凝胶的照片与图4相似。
实施例3
步骤一:
将0.01 mol磷酸肌酸溶于100 ml pH为6的MES缓冲液中,加入活化剂0.1 mol 1-乙基-(3-二甲基氨基丙基碳二亚胺(EDC))和0.1 mol N-羟基丁二酰亚胺(NHS),常温下反应20 min。将反应后的溶液滴加到100 ml体积浓度为1v/v%的壳聚糖-乙酸溶液中,常温下反应72 h。反应液转移至截留分子量为13 000的透析袋中,每12 h换水一次,透析3 d。透析后的溶液倒入18 cm玻璃培养皿中,-80 ℃冰箱冷冻12 h。将玻璃培养皿置于凝冰器温度为-50 ℃,真空度为1mbar的冻干机中冻干2 d得到磷酸肌酸改性壳聚糖(CS-PCr)材料,该磷酸肌酸改性壳聚糖(CS-PCr)的核磁磷谱结果与图1相似,表明磷酸肌酸成功接枝到壳聚糖上。
步骤二:
0.1 g磷酸肌酸改性壳聚糖(CS-PCr)加入10 ml体积分数为1v/v%的乙酸溶液中,常温搅拌至溶解。再加入0.4 ml甲基丙烯酸酐,常温下反应12 h。反应液转移至截留分子量为13 000的透析袋中,每12 h换水一次,透析3 d。透析过的溶液倒入18 cm玻璃培养皿中,-80℃冰箱冷冻12 h。将玻璃培养皿置于凝冰器温度为-50 ℃,真空度为1 mbar的冻干机中冻干2 d得到甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)。
步骤三:
0.1 g磷酸肌酸改性壳聚糖(CS-PCr)材料加入10 ml去离子水,常温下搅拌至溶解。将溶液倒入模具中,模具尺寸:高3 mm,直径15 mm,超声除去气泡。再将装有溶液的模具置于-80 ℃冰箱冷冻12 h后转移至凝冰器温度为-50 ℃,真空度为1 mbar的冻干机中冻干2 d得到磷酸肌酸改性壳聚糖多孔支架。0.3 g京尼平溶于100 ml 80v/v%的乙醇水溶液,向每个装有磷酸肌酸改性壳聚糖多孔支架的模具中加入200 μl京尼平-乙醇水溶液,常温下交联1 d,用去离子水洗涤,再置于-80 ℃冰箱冷冻12 h后转移至凝冰器温度为-50 ℃,真空度为1 mbar的冻干机中冻干2 d得到交联的磷酸肌酸改性壳聚糖多孔支架,该磷酸肌酸改性壳聚糖多孔支架照片与图2相似。多孔支架的SEM电镜图片与图3相似,可见支架具有孔径大小均一的连通孔结构。小鼠颅骨缺损修复效果类似图5B,多孔支架表现出良好的促进骨再生的作用。
步骤四:
0.1 g甲基丙烯酰化磷酸肌酸改性壳聚糖(MAC-PCr)加入10 ml去离子水,常温下搅拌至溶解。加入0.015 g光引发剂Irgacure 2959 避光搅拌至引发剂溶解。溶液倒入模具中,模具尺寸:高3 mm,直径15 mm,避光超声除去气泡。将模具置于紫外光交联机紫外光照30 min,紫外光波长:365 nm,强度:5 mW,制备得到磷酸肌酸改性壳聚糖水凝胶,该磷酸肌酸改性壳聚糖水凝胶的照片与图4相似。
Claims (10)
1.一种磷酸肌酸改性壳聚糖材料的制备方法,其特征在于,包括以下步骤:
(1)将磷酸肌酸溶于MES缓冲液中,再加入活化剂,搅拌均匀;
(2)向步骤(1)所得反应液中加入壳聚糖溶液,搅拌反应;
(3)将步骤(2)所得反应液转移至透析袋中透析;
(4)将透析后的溶液冷冻-冻干,得到磷酸肌酸改性壳聚糖材料。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)所述MES缓冲液的pH为5-6。
3.根据权利要求1所述的制备方法,其特征在于,步骤(1)所述活化剂为EDC/NHS,所述EDC:NHS:磷酸肌酸的摩尔比为1:1:1-1:1:0.1。
4.根据权利要求1所述的制备方法,其特征在于,步骤(2)所述壳聚糖溶液为壳聚糖-乙酸溶液。
5.根据权利要求1所述的制备方法,其特征在于,步骤(2)所述反应的时间为24-72 h。
6.根据权利要求1所述的制备方法,其特征在于,步骤(3)所述透析袋截留分子量为7000-13000,透析时间为3-7天。
7.由权利要求1-6任一项所述的制备方法制得的一种磷酸肌酸改性壳聚糖材料。
8.权利要求7所述的一种磷酸肌酸改性壳聚糖材料应用于制备磷酸肌酸改性壳聚糖多孔支架或磷酸肌酸改性壳聚糖可注射型水凝胶。
9.根据权利要求8所述的应用,其特征在于,所述磷酸肌酸改性壳聚糖多孔支架的制备包括以下步骤:
(1)将磷酸肌酸改性壳聚糖材料溶于水,再转移至模具中,超声除去气泡,然后冷冻-冻干制得多孔支架;
(2)将多孔支架置于交联剂中交联1-3 d;所述交联剂为浓度为0.01-0.03 g/ml的京尼平-乙醇水溶液;
(3)将交联后的支架用水洗涤,再冷冻-冻干后得到磷酸肌酸改性壳聚糖多孔支架。
10.根据权利要求8所述的应用,其特征在于,所述磷酸肌酸改性壳聚糖可注射型水凝胶的制备包括以下步骤:
(1)将磷酸肌酸改性壳聚糖材料溶于乙酸溶液中,再加入甲基丙烯酸酐搅拌反应6-12h;所述磷酸肌酸改性壳聚糖和甲基丙烯酸酐的质量比为1:2-1:4;
(2)将步骤(1)所得反应液透析除去未参与反应的原料;
(3)将透析后的溶液冷冻-冻干,得到甲基丙烯酰化磷酸肌酸改性壳聚糖;
(4)将甲基丙烯酰化磷酸肌酸改性壳聚糖与光引发剂Irgacure 2959溶于水中,得混合液;所述光引发剂在混合液中的浓度为0.05wt%-0.15wt%;
(5)将步骤(4)的混合液倒入模具中,避光超声除去气泡;
(6)将步骤(5)中装有溶液的模具置于紫外光波长为365 nm,强度为5-50 mW的紫外光固化机中光照交联5-30 min,得到磷酸肌酸改性壳聚糖可注射型水凝胶。
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