CN111821515A - 壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架及制备方法 - Google Patents

壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架及制备方法 Download PDF

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CN111821515A
CN111821515A CN202010797121.9A CN202010797121A CN111821515A CN 111821515 A CN111821515 A CN 111821515A CN 202010797121 A CN202010797121 A CN 202010797121A CN 111821515 A CN111821515 A CN 111821515A
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chitosan
recombinant collagen
electrostatic spinning
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莫秀梅
冯文浩
朱世辉
盛嘉隽
樊璐露
高佩
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Qinghai Chuangming Medical Equipment Co ltd
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Abstract

本发明涉及组织工程和生物制造技术领域，尤其是一种壳聚糖‑人源重组胶原蛋白静电纺丝纳米纤维支架及制备方法，具体制备方法为：将人源重组胶原蛋白、壳聚糖和聚己内酯三种组分按照一定配比配制成一定浓度的六氟异丙醇纺丝溶液，在一定环境条件下，将配制所得的溶液通过静电纺丝技术制备成壳聚糖、人源重组胶原蛋白和聚己内酯静电纺丝纳米纤维支架，然后通过交联剂将其交联，并将交联后的支架用于软骨组织再生。本发明所制备的壳聚糖‑人源重组胶原蛋白静电纺丝纳米纤维支架具有良好的纤维形貌，良好的生物相容性和生物可降解性，且具有引导软骨再生的能力，效果显著，可以用于软骨组织缺损修复。

Description

壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架及制备 方法
技术领域
本发明涉及组织工程和生物制造领域，具体领域为一种纳米纤维支架。
背景技术
每年我国骨缺损或功能障碍患者超过300万人。骨损伤和骨缺损临床上十分常见，目前一般采用自体骨或异体骨移植修复治疗。虽然自体骨移植的修复效果优于异体骨移植，但是自体骨移植在材料来源方面存在着严重的缺陷。从自体异位取骨，病人易患手术后并发症，失败率高达10～30％，严重制约了其在临床的广泛应用；异体骨移植在材料筛选、储存方面相当困难，比较昂贵，并容易产生免疫排斥反应、可能携带一些病原体，引起机体疾病的发生，造成机体一定的损伤等，失败率更高。同时，异体骨被取代缓慢，新生骨体积偏小。为了克服自体骨和异体骨移植存在的种种问题，人们试图通过天然的或合成途径，取得理想的骨修复材料。
对于一种理想的骨修复材料首先应该具备的特性有：1)生物相容性：可与骨直接进行化学结合，不阻止骨细胞在其表面的正常活性或干扰其周围骨细胞的自然再生过程，对骨组织的分解吸收具有传导性。2)机械耐受性：以小梁骨为准，抗压强度应大于5MPa，抗压模量在45～100MPa之间。3)生物降解性：在一定时间内被宿主骨替代，不影响骨组织的修复，无毒副作用。4)诱导再生性：通过自身或添加骨诱导因素，刺激或诱导骨骼生长。简言之，移植物的生物特性应与自然骨相似。
近年来，对骨替代物的寻找集中于人工骨修复材料，例如金属材料、生物陶瓷、天然高分子材料等，但这些材料在生物相容性、生物降解性、诱导成骨性和生物机械强度方面有着不同程度的缺陷。
发明内容
本发明的目的在于提供一种壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架及制备方法。
为实现上述目的，本发明提供如下技术方案：
一种壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，具体为：将人源重组胶原蛋白、壳聚糖和聚己内酯三种组分按照一定配比配制成一定浓度的六氟异丙醇纺丝溶液，在一定环境条件下，将配制所得的溶液通过静电纺丝技术制备成静电纺丝纳米纤维支架，然后通过交联剂将其交联，即得壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架。
其中，以质量百分比计算，人源重组胶原蛋白5～15％，壳聚糖2～5％，聚己内酯20～40％，六氟异丙醇40～70％。
其中，所述壳聚糖为低分子量壳聚糖，分子量为30kDa～80kDa。
其中，所述聚己内酯的分子量为60kDa～90kDa。
其中，所述交联剂为戊二醛，采用蒸汽交联3h。
其中，所述静电纺丝方法为，将纺丝溶液吸入注射器中，将注射器放到静电纺丝机上进行静电纺丝，纺丝电压为11-13kV，针头与接收板之间的距离为18-23cm，推进泵的推进速度为0.5-0.8ml/h，纺丝环境温度为35-40℃。
与现有技术相比，本发明的有益效果是：本发明所制备的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架具有良好的纤维形貌，良好的生物相容性和生物可降解性，且具有引导软骨再生的能力，效果显著，可以用于软骨组织缺损修复。
附图说明
图1是实施例1中壳聚糖-人源重组胶原蛋白纳米纤维支架的SEM图；
图2是实施例2中L929细胞在壳聚糖-人源重组胶原蛋白纳米纤维支架上的细胞电镜照片；
图3是实施例4中不同试验组的基因CollagenⅡ和Aggrecan的表达结果。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，具体为：
以质量百分比计算，人源重组胶原蛋白8％，壳聚糖2.5％，聚己内酯30％，六氟异丙醇59.5％。将人源重组胶原蛋白、壳聚糖和聚己内酯三种组分配制成六氟异丙醇纺丝溶液，将纺丝溶液吸入注射器中，将注射器放到静电纺丝机上进行静电纺丝，纺丝电压为11kV，针头与接收板之间的距离为18cm，推进泵的推进速度为0.5ml/h，纺丝环境温度为35℃，制备成静电纺丝纳米纤维支架，然后通过戊二醛蒸汽交联3h，即得壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架。
实验中根据所需支架的大小制作接收装置，并进行静电纺丝，可以得到纤维形貌良好的壳聚糖-人源重组胶原蛋白纳米纤维支架。图1所示为该纤维支架的扫描电镜图片，可以观察到光滑连续的纳米纤维结构。
其中，所述壳聚糖的分子量为80kDa。其中，所述聚己内酯的分子量为60kDa。
实施例2
一种壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，具体为：
以质量百分比计算，人源重组胶原蛋白10％，壳聚糖4％，聚己内酯25％，六氟异丙醇61％。将人源重组胶原蛋白、壳聚糖和聚己内酯三种组分配制成六氟异丙醇纺丝溶液，将纺丝溶液吸入注射器中，将注射器放到静电纺丝机上进行静电纺丝，纺丝电压为12kV，针头与接收板之间的距离为20cm，推进泵的推进速度为0.6ml/h，纺丝环境温度为38℃。通过静电纺丝技术制备成静电纺丝纳米纤维支架，然后通过戊二醛蒸汽交联3h，即得壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架。
图2为该支架上面所增殖细胞的扫描电镜图片，同样可以观察到光滑连续的纳米纤维结构，同时可以观察到L929细胞在纤维材料上生长粘附情况良好，说明壳聚糖-人源胶原蛋白纳米纤维支架具有优异的生物相容性。
其中，所述壳聚糖的分子量为50kDa。其中，所述聚己内酯的分子量为70kDa。
实施例3
一种壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，具体为：
以质量百分比计算，人源重组胶原蛋白10％，壳聚糖3％，聚己内酯30％，六氟异丙醇57％。将人源重组胶原蛋白、壳聚糖和聚己内酯三种组分配制成六氟异丙醇纺丝溶液，将纺丝溶液吸入注射器中，将注射器放到静电纺丝机上进行静电纺丝，纺丝电压为13kV，针头与接收板之间的距离为23cm，推进泵的推进速度为0.8ml/h，纺丝环境温度为40℃。通过静电纺丝技术制备成静电纺丝纳米纤维支架，然后通过戊二醛蒸汽交联3h，即得壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架。
其中，所述壳聚糖的分子量为60kDa。其中，所述聚己内酯的分子量为90kDa。
实施例4
取上述实施例中的纤维支架制成6孔板直径的圆片状并置入六孔板中，加入DMEM/F12完全培养基浸没支架，放入培养箱中12h。不含纤维支架为空白对照组，含有纤维支架为试验组，两组中分别包含了诱导组和非诱导组。加入骨髓间充质干细胞(BSC),再加入800微升的DMEM/F12完全培养基，培养24h。然后去除诱导组的培养基，加入成骨分化培养基培养，非诱导组继续用DMEM/F12培养基培养。
RNA提取和定量PCR。检测培养在两种纤维支架上的BMSC在诱导培养基和非诱导培养基的培养条件下，细胞中的Collagen Ⅱ和Aggrecan两种目的基因的表达情况，其中以GAPDH作为内标基因。
具体操作步骤：(1)总RNA的提取，将细胞培养在纤维支架上21天后，总细胞RNA用Trizol裂解。(2)反转录，mRNA在反转录酶的作用下反转录为互补DNA(cDNA)。(3)定量PCR,cDNA作为模板，在PCR仪中进行40次循环扩增。(4)结果处理，每个目的基因的相对表达水平按CT法来计算：
A＝CT(目的基因，待测样本)-CT(内标基因，待测样本)
B＝CT(目的基因，对照样本)-CT(内标基因，对照样本)
K＝A-B
表达倍数＝2-K
其检测结果如图3所示含有壳聚糖的试验组中Collagen Ⅱ和Aggrecan的表达结果要明显高于空白对照组。可以发现纤维支架可以明显促进BMSC向软骨分化的能力，具有诱导软骨再生的能力。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (7)

1.一种壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，其特征在于：将人源重组胶原蛋白、壳聚糖和聚己内酯三种组分按照一定配比配制成一定浓度的六氟异丙醇纺丝溶液，在一定环境条件下，将配制所得的溶液通过静电纺丝技术制备成静电纺丝纳米纤维支架，然后通过交联剂将其交联，即得壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架。
2.根据权利要求1所述的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，其特征在于：以质量百分比计算，人源重组胶原蛋白5～15％，壳聚糖2～5％，聚己内酯20～40％，六氟异丙醇40～70％。
3.根据权利要求1所述的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，其特征在于：所述壳聚糖为低分子量壳聚糖，分子量为30kDa～80kDa。
4.根据权利要求1所述的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，其特征在于：所述聚己内酯的分子量为60kDa～90kDa。
5.根据权利要求1所述的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，其特征在于：所述交联剂为戊二醛，采用蒸汽交联3h。
6.根据权利要求1所述的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架的制备方法，其特征在于：所述静电纺丝方法为，将纺丝溶液吸入注射器中，将注射器放到静电纺丝机上进行静电纺丝，纺丝电压为11-13kV，针头与接收板之间的距离为18-23cm，推进泵的推进速度为0.5-0.8ml/h，纺丝环境温度为35-40℃。
7.采用权利要求1-6任一所述的制备方法制得的壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架。
CN202010797121.9A 2020-08-10 2020-08-10 壳聚糖-人源重组胶原蛋白静电纺丝纳米纤维支架及制备方法 Pending CN111821515A (zh)

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