CN115624655A - 壳聚糖-氧化海藻酸钠水凝胶材料及其制备方法和应用 - Google Patents
壳聚糖-氧化海藻酸钠水凝胶材料及其制备方法和应用 Download PDFInfo
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- CN115624655A CN115624655A CN202211152739.5A CN202211152739A CN115624655A CN 115624655 A CN115624655 A CN 115624655A CN 202211152739 A CN202211152739 A CN 202211152739A CN 115624655 A CN115624655 A CN 115624655A
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- sodium alginate
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Abstract
本发明提供一种壳聚糖‑氧化海藻酸钠水凝胶材料以及制备方法和应用，该水凝胶材料的制备方法包括：配料后先将L‑谷氨酰胺、不含维生素A的B‑27、N2细胞培养添加剂、Insulin、bFGF、EGF、hydrocortisone、Triiodothyronine、肌醇、Y27632、DMEMF12培养基溶于水中；再将氧化海藻酸钠溶于前一步的混合培养基中；用冰醋酸溶解壳聚糖后再与上一步的溶液混合均匀至凝胶形成，即得。本发明的壳聚糖‑氧化海藻酸钠水凝胶材料具有良好的流变性能和完整的三维立体结构，孔径分布均匀，对整形手术创面、烧伤、刮伤、烫伤、各类溃疡等创面都有一定的修复作用，还可直接应用于植发、生发领域。
Description
技术领域
本发明属于生物医学材料技术领域，具体涉及一种壳聚糖-氧化海藻酸钠水凝胶材料及其制备方法和应用。
背景技术
毛囊干细胞是成体干细胞，在体内处于静止状态，在体外培养作用下表现出较强的增殖能力，可以分化成表皮、毛囊、皮脂腺，参与皮肤创伤愈合的过程。毛囊干细胞生活在每个毛囊中，在新的生发周期中快速激活和分裂。因此，毛囊干细胞也常应用于脱发美容领域。随着皮肤组织工程医学研究的深入，毛囊干细胞作为一种新的种子细胞逐渐受到人们的关注，如果能建立一种完善的培养扩增保存体系，可为临床皮肤缺损的修复开辟新的领域和提供更多的选择方法。
基于干细胞的治疗具有相当大的潜力，但在体内研究中，其治疗效果往往不令人满意。其中一个原因是移植的干细胞在移植后失去了显著的生存能力。受伤或受损的组织对细胞生长不利，如活性氧和宿主的免疫反应。此外，移植干细胞周围缺乏细胞支持信号，最终导致移植细胞死亡。因此，目前的研究都将重点放在干细胞移植的物质上，这些物质可以支持细胞存活，诱导其生物活性，并增强细胞在给药位点的滞留。本发明正是要提供一种这样的材料，一方面能够提供干细胞类似组织的生长环境，另一方面还具有新的使用功能。
发明内容
针对现有技术存在的问题和缺陷，本发明提供一种壳聚糖-氧化海藻酸钠水凝胶材料及其制备方法和应用，该材料具有良好的流变性能和完整的三维立体结构，孔径分布均匀，可为干细胞提供类似组织的环境，可应用于毛囊干细胞的培养及保存基质，并且对皮肤创面的治疗和修复有明显作用。本发明的技术方案为：
第一方面，本发明提供一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，包括：
步骤1，按照水凝胶材料中各成分的终浓度组成配料，包括：壳聚糖0.005～0.05g/mL；氧化海藻酸钠0.01～0.2g/mL；冰醋酸0.005％～0.02％(v/v)；DMEM F12培养基0.2～0.8mL/mL；L-谷氨酰胺0.002～0.02mL/mL；不含维生素A的B-270.001～0.01mL/mL；N2细胞培养添加剂0.5～5μL/mL；Insulin 1～10μg/mL；bFGF 0.02～0.2μg/mL；EGF 0.02～0.2μg/mL；hydrocortisone 0.2～2μg/mL；Triiodothyronine 0.2～2μg/mL；肌醇0.05～0.5mg/mL；Y276320.8～8μg/mL；余量为水；
步骤2，先将L-谷氨酰胺、不含维生素A的B-27、N2细胞培养添加剂、Insulin、bFGF、EGF、hydrocortisone、Triiodothyronine、肌醇、Y 27632、DMEM F12培养基溶于水中；
步骤3，再将氧化海藻酸钠溶于步骤2的混合培养基中；
步骤4，用冰醋酸溶解壳聚糖后再与步骤3的溶液混合均匀至凝胶形成，即得。
进一步地，所述氧化海藻酸钠的制备方法包括：
(1)将海藻酸钠溶于去离子水配制成海藻酸钠溶液；
(2)将高碘酸钠避光加入海藻酸钠溶液中避光搅拌一段时间；
(3)向步骤(2)的反应液中加入乙二醇，继续避光搅拌一段时间；
(4)向步骤(3)的反应液中加入乙醇进行沉淀，过滤出沉淀物；
(5)将沉淀物溶解在去离子水中，进行透析处理，之后将获得的产物冷冻干燥，即得氧化海藻酸钠。
进一步地，所述步骤(1)中海藻酸钠溶液的质量分数为0.5％～5％。
进一步地，所述步骤(2)中高碘酸钠与海藻酸钠的质量配比为0.5～1：1。
进一步地，所述步骤(2)中避光搅拌的控制参数为：搅拌速度400rpm～1000rpm，时间2h～8h。
进一步地，所述步骤(3)中乙二醇的加入量为1～10mL/g海藻酸钠。
进一步地，所述乙二醇替换为丙二醇、丙三醇、丁二醇中的一种。
进一步地，所述步骤(3)中避光搅拌的控制参数为：搅拌速度400rpm～1000rpm，时间0.5h～2h。
进一步地，所述步骤(4)乙醇的加入量为80～100mL/g海藻酸钠。
进一步地，所述透析处理是采用截留分子量为7000的纤维素透析袋，透析液为纯水，室温透析2～5天，每天换液至少3次。
第二方面，本发明提供一种壳聚糖-氧化海藻酸钠水凝胶材料，是采用上述制备方法获得，且所述水凝胶材料冻干后为三维立体结构，具有均匀孔径分布。
第三方面，本发明提供上述水凝胶材料在毛囊干细胞培养及保存中的应用。
进一步地，所述应用包括：将103～106个/mL的毛囊干细胞悬液以体积质量比为1:5～100的比例加入上述水凝胶材料中混匀，进行毛囊干细胞的培养和保存。
第四方面，本发明提供上述水凝胶材料在制备皮肤创面治疗和修复产品上的应用。
进一步地，所述产品还包括毛囊干细胞，所述毛囊干细胞与所述水凝胶材料以一定比例复配。
第五方面，本发明提供上述水凝胶材料在植发或者生发上的应用。
进一步地，所述应用的方法包括：在植发或者生发过程中，将毛囊置于所述水凝胶材料中一定时间。
本发明的有益效果为：。
1.本发明的壳聚糖-氧化海藻酸钠水凝胶材料具有良好的流变性能和完整的三维立体结构，孔径分布均匀。
2.本发明的壳聚糖-氧化海藻酸钠水凝胶可为干细胞提供类似组织的环境，作为干细胞使用和储存的基质，对细胞无毒性，且毛囊干细胞可在此材料中存活5天以上，也为其他干细胞相关研究和体外应用提供更多潜在的科研价值和应用前景。
3.本发明的壳聚糖-氧化海藻酸钠水凝胶能促进皮肤细胞和血管的生成，对整形手术创面、烧伤、刮伤、烫伤、各类溃疡等创面都有一定的修复作用，且能减少疤痕的生成，有利于皮肤恢复原有的各类正常组织，如细胞、血管、毛囊、汗腺等；并且使用简单，能缩短伤口暴露时间，避免感染。
4.此材料可直接应用于植发、生发领域，使用方便，能提高毛囊存活率，缩短植发手术时间。
附图说明
图1为本发明实施例1的氧化海藻酸钠(OSA)的核磁共振氢谱表征图谱。
图2为本发明实施例1的水凝胶材料的红外表征图谱。
图3为本发明实施例4的水凝胶材料的外观形态图。
图4为本发明实施例4的水凝胶材料的流变学表征图谱，其中图a为具有不同氨基与醛基摩尔比的水凝胶的流变性能，图b为固定R＝0.5时的流变恢复测试结果，图c为连续阶跃应变测量的应变幅度扫描结果，图d为步骤应变固定为800％时的应变幅度扫描结果。
图5为本发明实施例4的水凝胶材料的SEM测试图谱。
图6为本发明实施例5的水凝胶材料的细胞毒性测试结果，其中，图a为水凝胶与毛囊干细胞培养12、24、48小时后的活/死荧光染色结果，图b为水凝胶与毛囊干细胞培养12、24、48小时后的CCK-8测试结果，图c为水凝胶与毛囊干细胞培养48小时后的细胞状态图。
图7为本发明实施例5的水凝胶材料培养干细胞5天内的活性测试结果。
图8为本发明实施例6中创面愈合进展效果图。
图9为本发明实施例6中创面愈合率统计结果，其中图B表示对应4组小鼠在第3天的伤口面积扫描图，图C表示4组小鼠在不同时间段的伤口面积百分比图(柱状图按照从左到右的顺序依次为对照组、水凝胶组、毛囊干细胞组、水凝胶+毛囊干细胞组)，图D表示4组小鼠的伤口完全愈合时间统计图。
图10为本发明实施例6中皮肤组织切片HE染色结果。
图11为本发明实施例6中皮肤组织切片Masson染色结果。
图12为本发明实施例7中裸鼠养殖1个月的毛发生长情况。
具体实施方式
在本发明的描述中，需要说明的是，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面结合附图和具体的实施例对本发明做进一步详细说明，所述是对本发明的解释而不是限定。
实施例1
本实施例提供一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，包括：
步骤1，制备氧化海藻酸钠，具体包括：(1)将海藻酸钠溶于去离子水配制成质量分数为3％的海藻酸钠溶液；(2)将高碘酸钠按照其与海藻酸钠的质量比为0.5：1避光加入海藻酸钠溶液中，并于600rpm避光搅拌4h；(3)向步骤(2)的反应液中加入乙二醇，乙二醇加入量为5mL/g海藻酸钠，继续于600rpm避光搅拌0.5h；(4)向步骤(3)的反应液中加入乙醇进行沉淀，乙醇加入量为80mL/g海藻酸钠，过滤出沉淀物；(5)将沉淀物溶解在去离子水中，采用截留分子量为7000的纤维素透析袋，透析液为纯水，室温透析2天，每天换液至少3次，之后将获得的产物冷冻干燥，即得氧化海藻酸钠。对获得的氧化海藻酸钠进行核磁共振氢谱表征，仪器为核磁共振波谱仪Ascend 600；结果如图1所示，图1为氧化海藻酸钠(OSA)的核磁共振氢谱，氧化海藻酸钠的核磁共振氢谱中约5.21ppm处的新化学位移归因于氧化海藻酸钠的醛和相邻羟基形成的半缩醛质子分子链，表明氧化海藻酸钠制备成功。
步骤2，按照水凝胶材料中各成分的终浓度组成配料，包括：壳聚糖0.01g/mL；氧化海藻酸钠0.1g/mL；冰醋酸0.01％(v/v)；DMEM F12培养基0.6mL/mL；L-谷氨酰胺0.01mL/mL；不含维生素A的B-270.005mL/mL；N2细胞培养添加剂2μL/mL；Insulin 6μg/mL；bFGF 0.1μg/mL；EGF 0.1μg/mL；hydrocortisone1μg/mL；Triiodothyronine 1μg/mL；肌醇0.2mg/mL；Y276323μg/mL；余量为水。
步骤3，先将L-谷氨酰胺、不含维生素A的B-27、N2细胞培养添加剂、Insulin、bFGF、EGF、hydrocortisone、Triiodothyronine、肌醇、Y 27632、DMEM F12培养基溶于水中。
步骤4，再将氧化海藻酸钠溶于步骤2的混合培养基中。
步骤5，用冰醋酸溶解壳聚糖后再与步骤3的溶液混合均匀至凝胶形成，获得壳聚糖-氧化海藻酸钠水凝胶材料。
对本实施例获得的氧化海藻酸钠、壳聚糖-氧化海藻酸钠水凝胶材料等进行红外表征，具体如下：
仪器：傅里叶变换红外光谱仪VERTEX 70；
程序参数：扫描波数4000-600cm-1，分辨率4cm-1；
表征材料：海藻酸钠、氧化海藻酸钠、壳聚糖-氧化海藻酸钠水凝胶材料。
结果如图2所示，图2中展示了海藻酸钠(SA)、氧化海藻酸钠(OSA)和壳聚糖-氧化海藻酸钠水凝胶材料(ASCS-6)的红外光谱。与SA的红外光谱相比，OSA在1732cm-1处有一个新的吸收峰；这可以归因于醛基的伸缩振动，这表明发生了氧化反应。ASCS-6的红外光谱峰消失，表明OSA的醛基在交联反应中被消耗。
实施例2
本实施例提供一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，与实施例1的区别在于：步骤2中，按照水凝胶材料中各成分的终浓度组成配料，包括：壳聚糖0.05g/mL；氧化海藻酸钠0.01g/mL；冰醋酸0.02％(v/v)；DMEM F12培养基0.2mL/mL；L-谷氨酰胺0.02mL/mL；不含维生素A的B-270.001mL/mL；N2细胞培养添加剂5μL/mL；Insulin 1μg/mL；bFGF 0.2μg/mL；EGF 0.2μg/mL；hydrocortisone 0.2μg/mL；Triiodothyronine 2μg/mL；肌醇0.05mg/mL；Y 276320.8μg/mL；余量为水。获得壳聚糖-氧化海藻酸钠水凝胶材料。
实施例3
本实施例提供一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，与实施例1的区别在于：步骤2中，按照水凝胶材料中各成分的终浓度组成配料，包括：壳聚糖0.005g/mL；氧化海藻酸钠0.2g/mL；冰醋酸0.005％mL/mL；DMEM F12培养基0.8mL/mL；L-谷氨酰胺0.002mL/mL；不含维生素A的B-270.01mL/mL；N2细胞培养添加剂0.5μL/mL；Insulin 10μg/mL；bFGF 0.02μg/mL；EGF 0.02μg/mL；hydrocortisone 2μg/mL；Triiodothyronine 0.2μg/mL；肌醇0.5mg/mL；Y 276328μg/mL；余量为水。获得壳聚糖-氧化海藻酸钠水凝胶材料。
实施例4
本实施例考察水凝胶的体外材料学表征。
1)水凝胶的成胶时间及水凝胶形态
步骤1～4的操作同实施例1，步骤5的考察采用小管倒置法，具体如下：
将壳聚糖的醋酸溶液与氧化海藻酸钠的混合培养基溶液在EP管中混合均匀后，每间隔10秒将管倒置一次，观察管内液体是否沿管壁流下。当液体不再流动时记录的时间即为成胶时间。水凝胶外观形态：如图3所示，壳聚糖的醋酸溶液与氧化海藻酸钠的混合培养基溶液以固定比例混合初期为液态，室温下经20s后交联完成，形成水凝胶，倒置后不从瓶内流下。
2)流变学表征
将壳聚糖和氧化海藻酸钠按照氨基和醛基的摩尔比为0、0.2、0.5、0.8、1配料，然后按照实施例1步骤2～5的方式制备水凝胶。将形成的水凝胶材料进行流变学表征，仪器：Malvern Kinexus Pro+，结果如图4所示，图4a为具有不同氨基与醛基摩尔比的水凝胶的流变性能，R＝M-NH2:M-CHO(0≤R≤1)。氨基与醛基的摩尔比在0.8时具有最高的储能模量。R从0变化到0.5，水凝胶的储能模量(G')从717±113Pa急剧增加到5814±166Pa，当R从0.5持续增加到1时，G'达到6044±224Pa的平台，然后略微下降到5316±318Pa。这可能是因为更具动态性的亚胺键(与酰基腙键相比)的浓度随R增加，导致聚合物网络不稳定。固定R＝0.5的流变恢复测试，水凝胶的应变幅扫描结果显示，G′和损耗模量(G″)曲线在80％的应变处相交，表明水凝胶的状态介于固体和该临界点附近的流体。随着应变进一步增加到800％，由于水凝胶网络的坍塌，G'从≈5880Pa急剧下降到≈79Pa(图4b)。基于应变幅度扫描结果，进行连续阶跃应变测量以测试水凝胶的流变恢复行为。当振荡剪切应变从1％增加到80％并保持100秒时，G'和G”重叠，而它们在应变回1％后立即恢复其原始值(图4c)。同样，当较大应变(300％和800％)和小应变(1％)稍后交替应用时，G′也迅速恢复了初始值。此外，步骤应变固定为800％，但改变为
断裂应变的持续时间上被彻底调查流变恢复行为的影响(图4d)。数据表明，在去除断裂应变后，G'立即恢复，而与加载时间无关。这些结果表明，当水凝胶受到振荡剪切应变时，水凝胶的聚合物网络表现出快速恢复。
3)孔径分布
将实施例1的水凝胶材料进行孔径分布测试，先将水凝胶制成圆柱形，冷冻干燥后切去表面，利用扫描电镜观察冻干后水凝胶的孔径分布，结果如图5所示，水凝胶冻干后表现出完整的三维立体结构，具有均匀的孔径分布。
实施例5
本实施例考察水凝胶材料的体外细胞学表征，采用的是实施例1获得的水凝胶材料。
1)水凝胶的细胞毒性考察
将水凝胶材料与约105个/mL的毛囊干细胞以10:1的质量体积比混合后，分别在37℃的培养箱培养12、24、48小时后，取10g混合物加100mL生理盐水混合，离心。混合离心2次后收集毛囊干细胞。采用活/死细胞染色试剂盒进行荧光染色，在荧光显微镜下观察测试结果。同时将部分离心收集的毛囊干细胞用毛囊干细胞培养基稀释，并接种于96孔板中，在37℃培养箱中培养48h后，观察细胞形态，并进行CCK-8染色。实验以新复苏的毛囊干细胞作为对照。
水凝胶与毛囊干细胞培养12、24、48小时后的活/死荧光染色结果和对应的CCK-8测试结果、细胞形态结果如图6所示，表明毛囊干细胞的细胞活力均高于80％，进一步表明水凝胶无细胞毒性。
2)培养5天内的干细胞活性
按上述水凝胶的细胞毒性考察方法重新进行了毛囊干细胞包封在水凝胶内进行1天、3天和5天的培养实验，活/死荧光染色结果如图7所示，其中水凝胶Ⅰ是水凝胶与毛囊干细胞以10:1的质量体积比混合，水凝胶Ⅱ是水凝胶与毛囊干细胞以20:1的质量体积比混合。实验结果显示毛囊干细胞在水凝胶内生长状态良好，无明显死细胞出现。
实施例6
本实施例考察水凝胶材料对C57小鼠全层损伤创面修复评价，采用实施例2获得的水凝胶材料，具体过程如下：
1)手术流程：
a)手术前对手术器械进行消毒和灭菌操作，小鼠禁食一晚，提供饮水；
b)将C57小鼠随机分为4组，分别为空白组、水凝胶组、毛囊干细胞组、水凝胶+毛囊干细胞组；
c)剔除C57小鼠背部毛发，用安尔碘对小鼠背部皮肤进行消毒后，用手术剪剪除皮肤，在小鼠背部皮肤造成4个直径为12毫米的全层皮肤损伤创面；
d)将水凝胶与毛囊干细胞悬液以一定的比例混合，灌装于注射器内，注射在创面处，每个创面注射量为200微升；其余组加对应样品各200微升。
e)用3M Tegaderm-Film 1624W对手术处理后的创面进行覆盖，小鼠分笼饲养。
2)评价指标：
a)创面愈合进展如图8所示，在所有时间点，用水凝胶+毛囊干细胞治疗的伤口都显示出比其他组明显更快的愈合过程。第7天所有创面均显示创面面积减少一半，水凝胶+毛囊干细胞治疗的创面几乎愈合并被新生上皮覆盖，而其他组在第14天仍出现开放性创面。第21天完全愈合，水凝胶+毛囊干细胞治疗的组几乎没有可见瘢痕组织，而对照组创面仍未愈合。
如图9所示，图B表示对应4组小鼠在第3天的伤口面积扫描图，图C表示4组小鼠在不同时间段的伤口面积百分比图，图D表示4组小鼠的伤口完全愈合时间统计图。图B结果显示，在第3天的早期愈合阶段，水凝胶+毛囊干细胞处理的组伤口面积为53.09±2.97％，毛囊干细胞组伤口面积为58.40±3.15％，对照组伤口面积为78.50±5.23％，表明水凝胶+毛囊干细胞治疗显着减少的伤口面积。图C结果显示，随着愈合时间的延长，凝胶+毛囊干细胞处理的创面在各组中保持最佳愈合状态，第14天水凝胶+毛囊干细胞治疗组、毛囊干细胞治疗组、水凝胶治疗组和空白对照组的开放创面面积分别为9.88±2.43％、21.31±5.15％、24.22±5.00％和43.02±6.69％。在第21天，水凝胶+毛囊干细胞治疗组组几乎完全愈合，而Control仍然显示出统计学上显着的更大伤口面积。图D结果中的平均完全伤口愈合时间也证实了水凝胶+毛囊干细胞治疗组的促进愈合效果。
b)皮肤组织切片HE染色结果，如图10所示，从第7天到第21天，各组创面长度缩短，肉芽组织厚度增加。水凝胶+毛囊干细胞治疗组的创面长度在统计学上最短，肉芽组织最厚。在第7天，水凝胶+毛囊干细胞治疗组中可以观察到大量新形成的肉芽组织，然而，Control和水凝胶组的样本仅显示少量再生组织。第21天，水凝胶+毛囊干细胞治疗组创面完全被新生上皮覆盖，疤痕极少，水凝胶+毛囊干细胞治疗组创面长度最小，新生肉芽组织最大。此外，所有组中水凝胶+毛囊干细胞治疗的伤口中具有最厚的新生表皮。
c)皮肤组织切片Masson染色结果，如图11所示，从第7天到第21天，所有组的胶原蛋白密度都大大增加，而水凝胶+毛囊干细胞治疗组在所有组中显示出最高的胶原蛋白密度，其次是毛囊干细胞、水凝胶和空白对照组。具体而言，水凝胶组和对照组在第7天观察到的胶原纤维很少，在第21天发现的胶原蛋白少于水凝胶+毛囊干细胞和毛囊干细胞组，而水凝胶+毛囊干细胞组在第21天的伤口面积也最小，胶原纤维丰富。
实施例7
本实施例考察水凝胶材料对毛囊裸鼠体表毛发移植评价，采用实施例2获得的水凝胶材料，具体过程如下：
1)将新取出的毛囊置于水凝胶中，在37±1℃条件下储存30天。
2)在裸鼠背部用消毒后的注射器针头制造一定数量的针孔创面。将储存30天后毛囊移植至针孔创面。
3)裸鼠养殖1个月，观察毛发生长情况。结果如图12所示，实验结果表明，在37±1℃条件下储存30天后的毛囊，移植至裸鼠后仍能正常生长。
综上所述，本发明的水凝胶材料的优势在于：
1.可为干细胞提供类似组织的环境，作为干细胞使用和储存的基质，对细胞无毒性，且毛囊干细胞可在此材料中存活5天以上，为其他干细胞相关研究和体外应用提供更多潜在的科研价值和应用前景。
2.能促进皮肤细胞和血管的生成，对整形手术创面、烧伤、刮伤、烫伤、各类溃疡等创面都有一定的修复作用，且能减少疤痕的生成。
3.可直接应用于植发领域，使用方便，能提高毛囊存活率，缩短植发手术时间。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，其特征在于：包括：
步骤1，按照水凝胶材料中各成分的终浓度组成配料，包括：壳聚糖0.005～0.05g/mL；氧化海藻酸钠0.01～0.2g/mL；冰醋酸0.005％～0.02％(v/v)；DMEM F12培养基0.2～0.8mL/mL；L-谷氨酰胺0.002～0.02mL/mL；不含维生素A的B-270.001～0.01mL/mL；N2细胞培养添加剂0.5～5μL/mL；Insulin 1～10μg/mL；bFGF 0.02～0.2μg/mL；EGF 0.02～0.2μg/mL；hydrocortisone 0.2～2μg/mL；Triiodothyronine 0.2～2μg/mL；肌醇0.05～0.5mg/mL；Y276320.8～8μg/mL；余量为水；
步骤2，先将L-谷氨酰胺、不含维生素A的B-27、N2细胞培养添加剂、Insulin、bFGF、EGF、hydrocortisone、Triiodothyronine、肌醇、Y 27632、DMEM F12培养基溶于水中；
步骤3，再将氧化海藻酸钠溶于步骤2的混合培养基中；
步骤4，用冰醋酸溶解壳聚糖后再与步骤3的溶液混合均匀至凝胶形成，即得。
2.根据权利要求1所述的一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，其特征在于：所述氧化海藻酸钠的制备方法包括：
(1)将海藻酸钠溶于去离子水配制成海藻酸钠溶液；
(2)将高碘酸钠避光加入海藻酸钠溶液中避光搅拌一段时间；
(3)向步骤(2)的反应液中加入乙二醇，继续避光搅拌一段时间；
(4)向步骤(3)的反应液中加入乙醇进行沉淀，过滤出沉淀物；
(5)将沉淀物溶解在去离子水中，进行透析处理，之后将获得的产物冷冻干燥，即得氧化海藻酸钠。
3.根据权利要求2所述的一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，其特征在于：所述步骤(1)中海藻酸钠溶液的质量分数为0.5％～5％。
4.根据权利要求2或3所述的一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，其特征在于：所述步骤(2)中高碘酸钠与海藻酸钠的质量配比为0.5～1：1。
5.根据权利要求2所述的一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，其特征在于：所述步骤(3)中乙二醇的加入量为1～10mL/g海藻酸钠。
6.根据权利要求2所述的一种壳聚糖-氧化海藻酸钠水凝胶材料的制备方法，其特征在于：所述步骤(4)乙醇的加入量为80～100mL/g海藻酸钠。
7.一种壳聚糖-氧化海藻酸钠水凝胶材料，其特征在于：是采用权利要求1～6任意一项所述的制备方法获得，且所述水凝胶材料冻干后为三维立体结构，具有均匀孔径分布。
8.权利要求7所述的壳聚糖-氧化海藻酸钠水凝胶材料在毛囊干细胞培养及保存中的应用。
9.权利要求7所述的壳聚糖-氧化海藻酸钠水凝胶材料在制备皮肤创面治疗和修复产品上的应用。
10.权利要求7所述的壳聚糖-氧化海藻酸钠水凝胶材料在植发或者生发上的应用。
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