CN116196467A - 一种纳米纤维增强负载ADSCs自修复水凝胶及其制备方法 - Google Patents
一种纳米纤维增强负载ADSCs自修复水凝胶及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种纳米纤维增强负载ADSCs自修复水凝胶及其制备方法,首先合成氧化瓜尔胶,在瓜尔胶溶液中滴加氧化剂,避光剧烈搅拌反应后,终止氧化;然后将终止氧化的溶液进行透析,透析后冷冻干燥得到氧化瓜尔胶;然后制备纳米纤维:利用静电纺丝技术将高分子聚合物溶液进行静电纺丝,静电纺丝后经真空干燥形成纳米纤维;最后制备负载纳米纤维和ADSCs水凝胶:制备ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液,灌注到纳米纤维中,得到纳米纤维增强负载ADSCs自修复水凝胶。该纳米纤维增强负载ADSCs自修复水凝胶具有较好的组织粘合能力、延展性及持久性,并促进创面的修复,可用于组织工程及再生医学等技术领域。
Description
技术领域
本发明涉及生物材料领域,具体涉及一种纳米纤维增强负载ADSCs自修复水凝胶及其制备方法。
背景技术
伤口在我们的日常生活中经常发生,近年来,伤口的治疗已经逐渐演变成全世界的一个基本医疗问题。多种材料被应用于伤口修复,包括天然生物材料,如壳聚糖、胶原蛋白、海藻酸盐,以及合成生物材料,如聚乙烯醇、聚己内酯、聚乙二醇。这些生物材料通常以水凝胶、电纺纳米纤维支架、微流控微粒子、多孔泡沫和微针贴片的形式存在。其中,水凝胶被广泛采用,并在伤口愈合中表现出验证的价值,因为其固有的高湿度和多孔结构,能够吸收组织渗出物,促进氧气和营养物质的渗透,保持伤口区域的湿润状态,以及提供细胞外基质的生物模拟微环境,改善细胞的增殖和组织的修复。
虽然有很多成功的案例,但目前用于伤口愈合的水凝胶大多缺乏组织粘合能力,不能保证材料在伤口部位的长期维持。此外,大多数水凝胶的力学强度比较差,在伤口部位的延展性及整体保持的持久性较差,容易破碎。
发明内容
本发明的目的是提供一种纳米纤维增强负载ADSCs自修复水凝胶及其制备方法,具有较好的组织粘合能力、延展性及持久性,并促进创面的修复。
为实现上述目的,本发明提供的技术方案是:
一种纳米纤维增强负载ADSCs自修复水凝胶的制备方法,包括以下步骤:
步骤一、合成氧化瓜尔胶:
在瓜尔胶溶液中滴加氧化剂,避光剧烈搅拌反应后,终止氧化;然后将终止氧化的溶液进行透析,透析后冷冻干燥得到氧化瓜尔胶;
步骤二、制备纳米纤维:
利用静电纺丝技术将高分子聚合物溶液进行静电纺丝,静电纺丝后经真空干燥形成纳米纤维;
步骤三、制备负载纳米纤维和脂肪干细胞(ADSCs)的水凝胶:
制备含有ADSCs的细胞完全培养基;取一含有ADSCs的细胞完全培养基,将步骤一得到的氧化瓜尔胶溶解于该含有ADSCs的细胞完全培养基中,得到ADSCs氧化瓜尔胶溶液;另取一含有ADSCs的细胞完全培养基,将明胶溶解于该含有ADSCs的细胞完全培养基中,得到ADSCs明胶溶液;将ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液混合均匀,灌注到步骤二制备的纳米纤维中,得到纳米纤维增强负载ADSCs自修复水凝胶。
为优化上述技术方案,采取的具体措施还包括:
步骤一中,瓜尔胶溶液为瓜尔胶粉末在水中溶解后,85℃-95℃下磁力搅拌0.5-1.5小时配制,瓜尔胶溶液的浓度范围为5-10mg/ml;氧化剂为高碘酸钠溶液,高碘酸钠溶液的浓度为20-30mg/ml;瓜尔胶粉末与高碘酸钠的反应质量比为0.5-1:0.2-0.3;滴加高碘酸钠溶液后避光剧烈搅拌反应20-28小时。
步骤一中,加入乙二醇终止氧化,瓜尔胶溶液与乙二醇的体积比为100:2-4。
步骤一中,透析为放置在透析袋(10000Da)中在水中透析4-6天,冷冻干燥为在温度-45℃至-40℃下冷冻干燥3-4天。
步骤二中,高分子聚合物选自聚乳酸羟基乙酸(PLGA)、聚己内酯(PCL)、聚乳酸(PLA)、聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)、聚苯乙烯(PS)中的一种或多种;步骤二中,静电纺丝的方法为将高分子聚合物配制为高分子聚合物溶液,然后加入静电纺丝装置的注射器中进行静电纺丝,收集静电纺丝材料;其中,调控静电纺丝装置的挤出流速为0.01-1mL/h,电压为8-20kV,喷头与收集板之间的距离为10-20cm;其中,高分子聚合物溶液的浓度为0.1-0.3g/mL,溶剂为三氯甲烷、二氯甲烷、六氟异丙醇、二甲基甲酰胺N,N-Dimethylformamide(DMF)、水中的一种或多种。
步骤三中,含有ADSCs的细胞完全培养基的组成为浓度为10^6/mL的ADSCs、Dulbecco's modified eagle medium(DMEM)培养基、质量分数为10%的胎牛血清及质量分数为1%的双抗。
步骤三中,氧化瓜尔胶与含有ADSCs的细胞完全培养基的质量/体积比为0.01-0.03:1g/mL;明胶与含有ADSCs的细胞完全培养基的质量/体积比为0.1-0.3:1g/mL。
步骤三中,ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液的混合体积比例为1:1。
步骤三中,纳米纤维中ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液的混合溶液的灌注量为100-500uL。
本发明还保护所述方法制备的纳米纤维增强负载ADSCs自修复水凝胶。
与现有技术相比,本发明的有益效果是:
本发明的纳米纤维增强负载ADSCs自修复水凝胶通过瓜尔胶动态可逆的席夫碱连接合成,被赋予了出色的自修复能力和良好的剪切稀化行为,使得该纳米纤维增强负载ADSCs自修复水凝胶能轻松方便地通过从注射器中挤至伤口处。此外,基于水凝胶上的醛基和组织上的氨基之间的席夫基,水凝胶可以在生理温度下与组织达到很强的粘附力,可良好的贴合粘附在创面处。通过负载纳米纤维,可以显著提高水凝胶的力学性能,在伤口部位的延展性及整体保持的持久性较好。通过负载ADSCs,可以进行免疫调节,促进创面的修复。该多功能水凝胶可用于组织工程及再生医学等技术领域中,具有广阔的临床应用前景。
附图说明
图1瓜尔胶氧化前后的结构式。
图2实施例1氧化瓜尔胶的一维核磁共振氢谱图。
图3实施例1中得到的氧化瓜尔胶/明胶水凝胶的扫描电镜图片。
图4实施例1中得到的纳米纤维的扫描电镜图片。
图5实施例1中得到的纳米纤维增强负载ADSCs氧化瓜尔胶/明胶在损伤表面粘附7天后,组织修复效果相比于其他两组更加显著。
具体实施方式
以下通过实施例的形式对本发明的上述内容再作进一步的详细说明,但不应将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容所实现的技术均属于本发明的范围。
下述实施例中所使用的实验方法,如无特殊说明均为常规方法,所用的试剂、方法和设备,如无特殊说明均为本技术领域常规试剂、方法和设备。
实施例中,含有ADSCs的细胞完全培养基的组成为浓度为10^6/mL的ADSCs、Dulbecco's modified eagle medium(DMEM)培养基、质量分数为10%的胎牛血清及质量分数为1%的双抗。
实施例1PLGA纳米纤维增强负载ADSCs自修复水凝胶的制备,用于皮肤创面损伤修复
步骤一、合成氧化瓜尔胶:
将1g瓜尔胶粉末溶于100mL dH2O中,90℃下磁力搅拌1小时配制瓜尔胶水溶液,之后将200mg的高碘酸钠溶于10mL dH2O中滴加到瓜尔胶溶液中。避光剧烈搅拌反应24小时后,加入3mL的乙二醇终止氧化。最后,将溶液在dH2O中透析5天,-45℃冷冻干燥3天,得到氧化瓜尔胶,如图1所示。一维核磁共振氢谱中特征峰的出现表明氧化瓜尔胶成功合成,如图2所示。
步骤二、制备纳米纤维:
将PLGA以0.1g/mL的浓度溶于三氯甲烷中,磁力搅拌过夜后配制PLGA溶液。将PLGA溶液加入到注射器中,并安装于注射泵上。调控溶液的挤出流速为0.04mL/h、电压10kV及喷头与收集板之间的距离12cm,将收集到的材料真空干燥得到PLGA纳米纤维,扫描电镜如图3所示。
步骤三、制备负载纳米纤维和ADSCs水凝胶:
将0.02g的氧化瓜尔胶溶于1mL细胞完全培养中,90℃下磁力搅拌1小时配制氧化瓜尔胶溶液。将2g明胶溶于10mL细胞完全培养基中配制明胶溶液,细胞完全培养基含有10^6/mL ADSCs。将相同体积的氧化瓜尔胶溶液与明胶溶液混合后灌注到PLGA纳米纤维膜中,灌注量为200uL,几分钟内即可得到PLGA纳米纤维增强负载ADSCs自修复水凝胶。扫描电镜结果显示该水凝胶具有多孔结构,如图4所示。
步骤四、大鼠皮肤创面损伤粘附
用手术刀在大鼠皮肤上产生一个直径为10毫米的缺损,在缺损部位分别加入PBS、PLGA纳米纤维增强负载ADSCs自修复水凝胶。不做处理的缺损区作为对照组。所有手术都是在无菌环境下进行的。术后3天、5天和7天,用数码相机对缺损区进行观察和拍照,并处死大鼠以获得缺损区周围的组织进行进一步评估。图5a为修复7天后的HE染色图片,5b为修复7天后的马森染色图片,i为空白对照组,ii为PBS组,iii为PLGA纳米纤维增强负载ADSCs自修复水凝胶。染色结果表明PLGA纳米纤维增强负载ADSCs自修复水凝胶可以促进创面的修复。
实施例2PVP纳米纤维增强负载ADSCs自修复水凝胶的制备,用于角膜损伤修复
步骤一、合成氧化瓜尔胶:
将0.5g瓜尔胶粉末溶于100mL dH2O中,90℃下磁力搅拌1小时配制瓜尔胶水溶液,之后将300mg的高碘酸钠溶于10mL dH2O中滴加到瓜尔胶溶液中。避光剧烈搅拌反应24小时后,加入3mL的乙二醇终止氧化。最后,将溶液在dH2O中透析5天,-45℃冷冻干燥3天,得到氧化瓜尔胶。
步骤二、制备纳米纤维:
将PVP以0.3g/mL的浓度溶于六氟异丙醇中,磁力搅拌过夜后配制PVP溶液。将PVP溶液加入到注射器中,并安装于注射泵上。调控溶液的挤出流速为0.7mL/h、电压15kV及喷头与收集板之间的距离18cm,将收集到的材料真空干燥得到PVP纳米纤维。
步骤三、制备负载纳米纤维和ADSCs水凝胶:
将0.03g的氧化瓜尔胶溶于1mL细胞完全培养中,90℃下磁力搅拌1小时配制氧化瓜尔胶溶液。将2.5g明胶溶于10mL细胞完全培养基中配制明胶溶液,细胞完全培养基含有10^5/mL ADSCs。将相同体积的氧化瓜尔胶溶液与明胶溶液混合后灌注到PLGA纳米纤维膜中,灌注量为400uL,几分钟内即可得到PLGA纳米纤维增强负载ADSCs自修复水凝胶。
步骤四、大鼠角膜损伤粘附
用手术刀在大鼠角膜上产生一个直径为5毫米的缺损,在缺损部位分别加入PBS、PVP纳米纤维增强负载ADSCs自修复水凝胶。不做处理的缺损区作为对照组。所有手术都是在无菌环境下进行的。术后3天、5天和7天,用数码相机对缺损区进行观察和拍照,并处死大鼠以获得缺损区周围的组织进行进一步评估。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何形式上的限制,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,依据本发明的技术实质,对以上实施例所作的任何简单的修改、等同替换与改进等,均仍属于本发明技术方案的保护范围之内。
Claims (10)
1.一种纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:包括以下步骤:
步骤一、合成氧化瓜尔胶:
在瓜尔胶溶液中滴加氧化剂,避光剧烈搅拌反应后,终止氧化;然后将终止氧化的溶液进行透析,透析后冷冻干燥得到氧化瓜尔胶;
步骤二、制备纳米纤维:
利用静电纺丝技术将高分子聚合物溶液进行静电纺丝,静电纺丝后经真空干燥形成纳米纤维;
步骤三、制备负载纳米纤维和脂肪干细胞ADSCs的水凝胶:
制备含有ADSCs的细胞完全培养基;取一含有ADSCs的细胞完全培养基,将步骤一得到的氧化瓜尔胶溶解于该含有ADSCs的细胞完全培养基中,得到ADSCs氧化瓜尔胶溶液;另取一含有ADSCs的细胞完全培养基,将明胶溶解于该含有ADSCs的细胞完全培养基中,得到ADSCs明胶溶液;将ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液混合均匀,灌注到步骤二制备的纳米纤维中,得到纳米纤维增强负载ADSCs自修复水凝胶。
2.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤一中,瓜尔胶溶液为瓜尔胶粉末在水中溶解后,85℃-95℃下磁力搅拌0.5-1.5小时配制,瓜尔胶溶液的浓度范围为5-10mg/ml;氧化剂为高碘酸钠溶液,高碘酸钠溶液的浓度为20-30mg/ml;瓜尔胶粉末与高碘酸钠的反应质量比为0.5-1:0.2-0.3;滴加高碘酸钠溶液后避光剧烈搅拌反应20-28小时。
3.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤一中,加入乙二醇终止氧化,瓜尔胶溶液与乙二醇的体积比为100:2-4。
4.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤一中,透析为放置在10000Da透析袋中在水中透析4-6天,冷冻干燥为在温度-45℃至-40℃下冷冻干燥3-4天。
5.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤二中,高分子聚合物选自聚乳酸羟基乙酸PLGA、聚己内酯PCL、聚乳酸PLA、聚乙烯醇Poly(vinyl alcohol)(PVA)、聚乙烯吡咯烷酮Polyvinyl pyrrolidone(PVP)、聚苯乙烯Polystyrene(PS)中的一种或多种;步骤二中,静电纺丝的方法为将高分子聚合物配制为高分子聚合物溶液,然后加入静电纺丝装置的注射器中进行静电纺丝,收集静电纺丝材料;其中,调控静电纺丝装置的挤出流速为0.01-1mL/h,电压为8-20kV,喷头与收集板之间的距离为10-20cm;其中,高分子聚合物溶液的浓度为0.1-0.3g/mL,溶剂为三氯甲烷、二氯甲烷、六氟异丙醇、二甲基甲酰胺N,N-Dimethylformamide(DMF)、水中的一种或多种。
6.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤三中,含有ADSCs的细胞完全培养基的组成为浓度为10^6/mL的ADSCs、Dulbecco'smodified eagle medium(DMEM)培养基、质量分数为10%的胎牛血清及质量分数为1%的双抗。
7.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤三中,氧化瓜尔胶与含有ADSCs的细胞完全培养基的质量/体积比为0.01-0.03:1g/mL;
明胶与含有ADSCs的细胞完全培养基的质量/体积比为0.1-0.3:1g/mL。
8.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤三中,ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液的混合体积比例为1:1。
9.根据权利要求1所述的纳米纤维增强负载ADSCs自修复水凝胶的制备方法,其特征在于:步骤三中,纳米纤维中ADSCs氧化瓜尔胶溶液和ADSCs明胶溶液的混合溶液的灌注量为100-500uL。
10.权利要求1-9任一项所述方法制备的纳米纤维增强负载ADSCs自修复水凝胶。
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