CN114832149A - 一种基于干细胞微包埋复合水凝胶的创面敷料及制备方法 - Google Patents
一种基于干细胞微包埋复合水凝胶的创面敷料及制备方法 Download PDFInfo
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Abstract
本发明以海藻酸盐等海洋生物材料为原料,以微球包埋干细胞技术为核心,搭载水凝胶载体,利用微球的细胞培养和屏障优势,以及干细胞的多向分化特点,构建“微球‑干细胞‑水凝胶”技术体系,建立开发“安全、高效、质量可控”的促进创面愈合、抑制瘢痕、治疗依从性高的医疗器械产品的关键技术,打通开发微球干细胞复合水凝胶创面敷料产品(Ⅲ类医疗器械)产业化技术通道。该水凝胶微球包埋干细胞促进创面愈合技术体系的建立,实现细胞与组织工程等再生医学技术与海洋生物医用材料开发关键技术的高效结合,通过推广可为开发组织工程产品相关单位提供技术支持,并且带动海藻酸盐等海洋生物医药材料的高效利用,具有广泛的开发应用价值。
Description
技术领域
本发明属于创伤敷料技术领域,具体涉及一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法。
背景技术
干细胞(stem cell)是一类具有自我更新(self-renewing)和多向分化潜能的原始未分化细胞。干细胞可以通过分裂和分化产生单一功能细胞:生理情况下替代衰老或凋亡细胞,维持机体组织结构的完整和正常生理机能的发挥;病理情况下修复、替代病变缺失的组织细胞,改善或恢复受损组织的结构和功能。干细胞在组织修复、器官重建和基因治疗中发挥重要作用,是继手术治疗和药物治疗后最有临床应用价值医疗手段,对人类健康产生重要影响。以干细胞为核心的再生医学成为当今医学研究的热点和焦点。
自1957年,Chang等受生物细胞膜的启发,提出了“人工细胞”的概念,微球包埋技术越来越受到人们的重视和关注。九十年代微球包埋的研究扩展到脑垂体、肾、甲状腺等器官移植领域和治疗中枢神经系统混乱等领域。近年来微球包埋的应用领域也从医学领域扩展到了其它学科,随着酶固定化,微生物细胞固定化以及动植物细胞固定化等研究的飞速发展,已经形成了一个范围广泛的固定化生物催化剂的研究领域。微球包埋的应用是一个正在发展的研究方向,作为一种具有分离功能的细胞载体,实际上是融固定化培养与产品分离于一体的微反应器。包埋后的细胞生长在条件适宜的微环境中,免受外部搅拌产生的剪切和大分子免疫蛋白的影响,从而达到催化、培养、生产免疫物质的目的。微球具有保护物质免受环境条件影响,掩盖异味和颜色,降低毒性,改善物质的可加工性和稳定性,延缓或控制被包埋材料的释放,将不可混的化合物隔离等功能。广义地说,微球具有改善和提高物质表观及其性质的能力。更确切的说,微球是能够储存微细状态的物质,并在需要时释放该物质。
水凝胶是一种高度交联、以水为分散介质的亲水性网络结构,在过去60年间得到广泛研究,作为一种生物相容性材料,其在许多临床应用中显现出巨大潜力。Wichterle等提出创面保持湿润有助于愈合,并推荐使用亲水性凝胶覆盖创面。由于水凝胶具有生物相容性、软组织样含水量和类似于天然细胞外基质的三维多孔网络结构等独特性质,它成为一种理想的伤口敷料材料。水凝胶除了能有效保持创面愈合所需的湿润环境外,还是一种舒适且易于更换的材料,能有效缓解创面疼痛,降低伤口温度。水凝胶被广泛认为适用于各种创面愈合过程的所有阶段,具有无刺激性,不与生物组织发生反应,且对代谢物具有良好渗透性。目前已有多种天然或人工聚合物材料制成的水凝胶敷料产品进入临床使用,它们具有止血、保湿、吸收渗出液、允许气体交换等多种功能。但目前慢性创面存在感染、缺血、缺氧等复杂情况,结构功能单一的普通水凝胶敷料不能满足实现需求。
发明内容
本发明的目的在于提供一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法。
本发明首先提供一种基于干细胞微包埋复合水凝胶的创面敷料的制备方法,该方法包括:
步骤一:在培养基中培养人脐带来源的间充质干细胞(MSCs),将原代培养的MSCs在液氮罐中保存。取液氮罐中保存的MSCs冻存管,复苏,在二氧化碳培养箱中培养,传代培养至第五代后用于后续的步骤。
步骤二:含干细胞微球的制备。采用静电喷雾法,海藻酸钠-MSCs混合液经喷雾、固化后形成内部包埋有MSCs的海藻酸钠微球,将经过静电喷雾形成的微球收集于氯化钙溶液中进行交联,得到包埋有MSCs的海藻酸钠微球。微球粒径可通过粒度仪检测。
步骤三:复合胶原蛋白水凝胶的制备。在一定条件下中,调节胶原蛋白溶液为中性。加入步骤一的包埋有MSCs的海藻酸钠微球搅拌均匀后得到初级复合胶原蛋白水凝胶,对初级复合胶原蛋白水凝胶进行培养,得到复合胶原蛋白水凝胶。复合水凝胶的机械强度通过拉力机检测,空隙大小通过扫描电镜观察,降解时间通过酶解实验检测。
优选的是,所述步骤一中的培养基为①α-MEM 5%-10%FBS或②人间充质干细胞无血清基础培养基5%-10%人间充质干细胞无血清添加物。
优选的是,所述步骤一的培养温度为37℃,二氧化碳浓度为5%。
优选的是,所述步骤二的海藻酸钠溶液的浓度为1-3%(w/v),粘度为100-300CPS。
优选的是,所述步骤二的氯化钙溶液的浓度为5%-15%(w/v)。
优选的是,所述步骤二的所有材料经过高压蒸汽灭菌(121℃,20min)。
优选的是,所述步骤三的一定条件为冰水水浴。
优选的是,所述步骤三的初级复合胶原蛋白水凝胶培养条件为:在二氧化碳培养箱中初次培养成胶后,加培养基,37℃,5%CO2培养箱中再次培养。
本发明还提供上述制备方法得到的基于干细胞微包埋复合水凝胶的创面敷料。
本发明的有益效果:
本发明首次提出以“微球-干细胞-水凝胶”的创新形式制备创面敷料的理念,并建立相应的技术体系,开展相关产品,是对创面修复临床治疗技术的一种有益探索和创新性补充;
以海洋来源的海藻酸盐为原料,具有生物相容性好、安全无毒等天然优势;
创新性的采用微球包埋技术固定培养干细胞,可持续稳定释放细胞生长因子,发挥其在烧烫伤创面愈合和皮肤修复方面的独特作用;
采用水凝胶作为覆盖微球的载体和物理屏障,隔绝外界污染,具有制备工艺成熟,能有效提高产业化转化效率的优势。
附图说明
图1海藻酸钠微球光学显微镜照片
图2胶原蛋白水凝胶照片
图3内部包埋有MSCs的海藻酸钠微球荧光照片
图4伤口愈合率结果图
具体实施方式
以下结合实施例对本发明作进一步详细描述:
实施例1海藻酸钠微球的制备
1、1%海藻酸钠溶液:称取1.0g海藻酸钠粉末置于蓝盖瓶中,并在瓶中放置一磁力转子,经121℃,15min灭菌。灭菌后在无菌条件下加入生理盐水溶解到100mL,4℃冰箱内保存。5%CaCl2溶液:称取50g CaCl2,加入去离子水溶解定容到1L,用0.22μm滤膜过滤除菌后备用。
2、将小型喷雾干燥仪器(BUCHI Mini Spray Dryer B-290)的进样管插入盛有1%海藻酸钠溶液的容器中。在喷头的下方放置一个盛有5%氯化钙溶液的15cm细胞培养皿,并通过磁力搅拌器转子的搅拌使液体处于运动状态。
3、参数设定:设定仪器参数(雾化气体流量20cfm、海藻酸钠溶液进料功率10%)制备微球观察对微球的影响。
4、微球制备:海藻酸钠溶液在蠕动泵的推动下到达喷头在压缩空气的作用下形成球形的细小液滴,液滴喷洒到流动的含有氯化钙的交联池中,使海藻酸钠液滴迅速固化成微球。
5、微球收集:将含有微球的氯化钙溶液转移到70μm细胞筛中,过滤液体、收集微球,4℃保存,得到海藻酸钠微球(如图1)。
实施例2胶原蛋白水凝胶的制备
1、胶原蛋白溶剂(0.01M PBS溶液):称取8g NaCl,0.2g KCl,1.44g Na2HPO4,0.24gKH2PO4,去离子水溶解并定容到1L,并在每1L PBS溶液中加入10mg酚红、10mL冰醋酸,0.22μm滤膜过滤除菌。5mg/mL胶原蛋白溶液:称取0.5g胶原蛋白,加入到上述溶液中使胶原蛋白溶解,并定容到100mL。2M NaOH溶液:称取8g NaOH,溶解定容到100mL,0.22μm滤膜过滤除菌。
2、将所有溶液放入4℃冰箱内预冷。用注射器分别抽取胶原蛋白溶液30mL到使用冰水水浴的小烧杯中,缓慢滴加NaOH溶液并不断搅拌。直至在酚红指示剂的显示下溶液由黄变红。此时溶液的pH值接近中性。
3、迅速将上述溶液使用注射器转移到6孔板中,每孔5mL。放于37℃培养箱中使胶原纤维析出形成水凝胶(如图2)。
实施例3含干细胞微球的胶原蛋白水凝胶的制备
1、干细胞的培养:所用细胞培养基为人间充质干细胞无血清基础培养基(产品货号:SC2013-G-A)10%人间充质干细胞无血清添加物(产品货号:SC2013-G-B)。所用干细胞为人脐带来源的间充质干细胞(MSCs)。经原代培养的MSCs冻存在液氮罐中保存。所用10cm细胞培养皿提前加入5mL促贴壁试剂(1×,产品货号:2012-G-C)放入37℃,5%CO2培养箱中孵育3-4h。取液氮罐中保存的MSCs冻存管,在预热至37℃的水浴锅中温育,不断晃动使管中细胞快速解冻。将液体转移到离心管中,并加入10mL培养基。之后1000rpm,5min离心,倒掉上清液,补加培养基吹打至无肉眼可见颗粒物。弃去培养皿中促贴壁试剂,将细胞悬液转移到细胞培养皿中,并补加培养基至总体积为10mL。轻轻晃动使皿中细胞分布均匀。在37℃,5%CO2培养箱中培养,传代培养至第五代后用于后续的步骤。
2、取第5代培养至汇合的MSCs,弃去培养皿中原培养基,加入5mL PBS(0.01M,pH=7.2-7.4)洗涤贴壁细胞,弃去PBS,重复两次。每皿加入1mL胰蛋白酶替代物(产品货号:SC2013-G-D),放入37℃培养箱中对贴壁MSCs消化,2min后取出培养皿,加入等体积无血清胰蛋白酶终止液(产品货号:TBD20180080)终止消化,轻轻吹打使贴壁细胞脱落、混匀。使用细胞计数板计算细胞密度,对细胞悬液1000rpm,5min离心,离心后弃去上层胰酶以及终止液。之后加入定量培养基吹打重悬MSCs,并调整细胞密度为4×106个/mL,使用1mL无菌注射器吸取等体积的2%海藻酸钠溶液与MSCs细胞悬液混合均匀。此时MSCs细胞密度为2×106个/mL,海藻酸钠的浓度为1%。
3、对小型喷雾干燥仪器的样品管道使用75%酒精、生理盐水冲洗、排干液体后将进样管插入细胞海藻酸钠混合液中。准备一个15cm的细胞培养皿,将皿放置在喷雾干燥器的喷头下方,倒入30mL无菌CaCl2溶液,并在皿中放置一个经过酒精消毒、CaCl2溶液润洗的磁力转子。
4、打开喷雾干燥器的雾化气体、蠕动进料泵,海藻酸钠-MSCs混合液经喷雾、固化后形成成内部包埋有MSCs的海藻酸钠微球(如图3)。经70μm细胞筛过滤除去CaCl2溶液,将收集到的微球使用生理盐水洗涤3次、PBS洗涤2次、培养基润洗3次,之后重悬并平均转移到24孔细胞培养板中,使每孔微球、细胞含量大致相同。隔天换液。
5、按照上述水凝胶的制备过程,在6孔板中将胶原蛋白溶液的pH值使用NaOH溶液(1M)调整至中性后、迅速将包埋有MSCs的海藻酸钠微球添加到胶原蛋白溶液中,通过搅拌使微球在胶原蛋白溶液中分布均匀。待放入37℃培养箱中5min成胶后,补加培养基,37℃,5%CO2培养箱中培养。
实施例4复合水凝胶创面敷料用于烧烫伤模型的研究
1、烧烫伤模型的构建。采用热水烫伤法制备烧烫伤模型。
1.1模具的制作:取50mL离心管,将下部剪短尖端用利器切下,使其下部开口为直径10mm的圆。水浴锅预热至95℃。
1.2所用动物为SD大鼠,在实验前一天晚上大鼠禁食,称量大鼠体重,按照每100g体重0.3mL的剂量腹腔注射10%的水合氯醛。
1.3麻醉满意后,将大鼠背部朝上放置于解剖台上,使用动物脱毛剂除去大鼠背面毛发,并用生理盐水湿润的纱布擦拭除去残留的脱毛剂。
1.4在大鼠背部备皮区域选择合适的位置,制造四个烫伤区域。具体步骤如下:将模具开口处紧贴于大鼠背部皮肤,模具竖直放置,向模具内注入95℃热水至离心管刻度线35mL处,停留15s,迅速移去模具并使用生理盐水冲洗,小心操作防止烫伤。
2、水凝胶对大鼠伤口的处理。
2.1每只大鼠4个创面分别使用干细胞微球复合水凝胶、空白水凝胶、干细胞、纱布进行治疗处理。
2.2在制造创面之后,分别将相应的材料处理到伤口上,之后使用3M TegadermFilm覆盖以固定各组材料。再以医用透气无纺布胶带包扎。术后各大鼠单笼饲养以防止饲养。
2.3为防止感染,每只大鼠每天肌肉注射青霉素2次,4万U每次,连续注射3天。
3、伤口愈合率评价及结果分析
在相应的时间点对创面进行观察、拍照,使用image J统计创面面积,计算伤口愈合率(如图4)。
从烫伤治疗后7d、14d和21d各组的伤口愈合率来看,与使用纱布(40.71%,62.52%,92.38%)相比,使用干细胞微球复合水凝胶(62.51%,88.87%,99.68%)、空白水凝胶(45.25%,72.28%,98.35%)和干细胞治疗(49.65%,83.53%,98.12%)的创面愈合效果更好,并且使用复合水凝胶的治疗效果要明显优于空白水凝胶和干细胞组。
Claims (11)
1.一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,包括以下步骤:
步骤一:在培养基中培养人脐带来源的间充质干细胞(MSCs),将原代培养的MSCs在液氮罐中保存,取液氮罐中保存的MSCs冻存管,复苏,在二氧化碳培养箱中培养,传代培养至第五代后用于后续的步骤;
步骤二:含干细胞微球的制备,采用静电喷雾法,海藻酸钠-MSCs混合液经喷雾、固化后形成内部包埋有MSCs的海藻酸钠微球,将经过静电喷雾形成的微球收集于氯化钙溶液中进行交联,得到包埋有MSCs的海藻酸钠微球;
步骤三:复合胶原蛋白水凝胶的制备,在一定条件下,调节胶原蛋白溶液为中性,加入步骤一的包埋有MSCs的海藻酸钠微球搅拌均匀后得到初级复合胶原蛋白水凝胶,对初级复合胶原蛋白水凝胶进行培养,得到复合胶原蛋白水凝胶。
2.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤一中的培养基为①α-MEM 5%-10%FBS或②人间充质干细胞无血清基础培养基5%-10%人间充质干细胞无血清添加物。
3.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤一的培养温度为37℃,二氧化碳浓度为5%。
4.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤一的培养后的干细胞通过微球包埋,检测生长状态,细胞活性,物质分泌,存活时间等。
5.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤二的海藻酸钠溶液的浓度为1-3%(w/v),粘度为100-300CPS。
6.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤二的氯化钙溶液的浓度为5-15%(w/v)。
7.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤二的所有材料经过高压蒸汽灭菌(121℃,20min)。
8.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤二的含干细胞微球粒径通过粒度仪检测,包埋干细胞生长状态评价通过荧光显微镜观察检测。
9.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤二微球包埋干细胞后,需搭载水凝胶作为载体和物理屏障,制备完成后,检测机械强度、孔隙大小、降解时间等。
10.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤三的一定条件为冰水水浴。
11.根据权利要求1所述的一种基于干细胞微包埋复合水凝胶的创面敷料及其制备方法,其特征在于,所述步骤三的初级复合胶原蛋白水凝胶培养条件为:在二氧化碳培养箱中初次培养成胶后,加培养基,37℃,5%CO2培养箱中再次培养。
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