CN118141976A - 一种水凝胶复合物及其应用 - Google Patents
一种水凝胶复合物及其应用 Download PDFInfo
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- CN118141976A CN118141976A CN202211562141.3A CN202211562141A CN118141976A CN 118141976 A CN118141976 A CN 118141976A CN 202211562141 A CN202211562141 A CN 202211562141A CN 118141976 A CN118141976 A CN 118141976A
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- hyaluronic acid
- hydrogel
- aldehyde
- carboxymethyl chitosan
- cells
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Abstract
本发明涉及生物材料和生物医学领域,具体涉及一种水凝胶复合物,所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,本发明的水凝胶可为细胞提供生长分化环境,可满足细胞的局部留存、生长、分化及外泌体的渗透、可降解、可注射、方便临床的微创操作,更好为烫伤和机械损伤及炎症导致的皮肤创伤治疗中发挥较好的效果。
Description
技术领域
本发明涉及生物材料和生物医学领域,具体涉及一种水凝胶复合物。
背景技术
水凝胶,是一类含有大量亲水基团,但不溶于水的三维网络结构聚合物。其中,生物基水凝胶是以天然高分子材料,包括纤维素、淀粉、甲壳素、海藻酸钠、透明质酸为主体通过物理或化学交联制成的水凝胶。生物基水凝胶的成分结构及理化性质类似干细胞外基质,具有良好的生物相容性、可降解性、刺激响应性等,结合其固有的多孔结构和吸水溶胀等特性,使其在药物传递、组织工程、生物传感、环境卫生等领域得到了广泛的研究和应用。近年来,基于水凝胶结构与天然细胞外基质相近,该类水凝胶具有利于细胞存活、生物相容性好、植入创伤小、便于填充等优点,成为最具潜力的组织工程支架材料,被广泛应用于生物医学领域。
据统计,我国每年有2600万人被烧烫伤,平均每天近7万人面对烫伤创面的治疗,严重和广泛的烫伤需要专门的医疗保健和长期住院,导致高昂的社会成本。在严重的病例中,治疗选择是有限的,死亡率可能达到100%;尽管治疗中可以及时恢复体液和电解质平衡、呼吸支持和早期清创,但充分处理伤口本身对于改善患者预后至关重要。适当覆盖伤口不仅可以减少液体流失,还可以降低随后感染的风险;自体皮肤移植被认为是这些伤口的标准护理治疗,因为它提供了快速的覆盖和改善伤口愈合。然而,大面积烫伤的患者缺乏足够的健康皮肤进行移植,对于这些人来说,治疗替代品是有利的。当无法进行自体移植时,带有真皮基质的皮肤替代物可作为大面积创伤的治疗选择。这些皮肤替代物可能是无细胞的或含有分化细胞,通常是自体角质形成细胞和成纤维细胞。尽管这种方法已经取得了成功,但自体细胞的培养非常耗时,异体成纤维细胞或角质形成细胞在放置后不久可能会被排斥。另一方面,采用生物活性物质分泌细胞支架进行治疗时,要求细胞与毛细血管间距在300~400μm以内,以保证支架内装载的细胞存活并完全发挥功能。然而,在烫伤创伤治疗中很难满足此条件的植入部位,其它各种可能的适合植入的部位又都无法达到预期的理想效果,这就导致植入的支架只能部分贴附在人体自身毛细血管表面,而另外未贴附毛细血管的部分由于氧气、营养物质等浓度较低,无法及时交换,致使装载的细胞不能发挥正常功能、存活率降低甚至死亡,产品的持效性较低。
这就急需一种新的基质来作为体外细胞的载体,解决目前伤口敷料中细胞存活的关键问题,实现烫伤等创面治疗,免除患者由于传统敷料反复更换和干细胞注射治疗带来的精神、肉体负担。
发明内容
脐血单核细胞(CordBloodMononuclearCell,CB-MNC)已被研究作为烫伤创面的治疗替代物。在过去几年中,在实验模型和人类中,脐血单核细胞已被有效地用于治疗多种疾病,如血液和免疫介导疾病、心脏功能障碍、骨损伤和皮肤溃疡。脐血单核细胞被认为适合于治疗皮肤溃疡和烫伤创面,不仅因为它们能够产生不同类型的细胞,还因为它们的旁分泌潜能、分泌细胞因子(TGF-b、IL-10、IL-6)、趋化因子(CCL2、CCL5、CXCL12)和生长因子(VEGF、IGF、bFGF、SDF、HGF),这可能有助于愈合过程。脐血单核细胞的另一个有趣特性是免疫调节,它可能直接或间接影响免疫系统的不同组成部分,从而控制炎症。此外,脐血单核细胞几乎没有免疫原性,因此可以使用现成的同种异体细胞,研究指出沿着溃疡周围注射绿色荧光蛋白标记的CB-MNC能够加速上皮化,促进血管生成,并增加血管和内皮生长因子的表达。研究还发现脐血单核细胞可以成功治愈实验模型和患者的急性和慢性伤口。最近的研究表明,CB-MNC治疗可以促进大鼠和猪烫伤的愈合;此外,CB-MNC促进血管生成并加速肉芽组织的发育。脐血单核细胞也被证明对常规治疗无效的烫伤患者有效。Rasulov在临床治疗中验证反复局部应用自体脐血单核细胞能够促进伤口愈合,并使烫伤患者的血浆水平正常化,临床和实验支持使用自体和异体脐血单核细胞治疗烫伤创面。采用脐血单核细胞治疗同种和异种烫伤创面时,脐血单核细胞扩增需要较长时间,自体细胞可能无法立即用于治疗;基于脐血单核细胞呈现MHC分子的低表达,可使同种和异种应用成为可能,因此,来自健康供体的异基因脐血单核细胞,可能是广泛烫伤患者初期和急性护理的更合适的替代方案。
综合上述脐血单核细胞在创伤治疗中的优势和利用脐血单核细胞治疗同种和异种烫伤创面中存在的制约因素,本研究团队通过制备、筛选出生物安全性好的水凝胶,进一步将成纤维细胞和脐血单核细胞分别与水凝胶(NOCC/A-HA)共培养,发现本研究中水凝胶共培养存活细胞的存活率得到大幅度提升;同时研究也证实本发明的水凝胶(NOCC/A-HA)可以防止伤口组织受损时发生的组织粘连,并提高伤口愈合的效率;在体外小鼠烫伤模型治疗实验中,负载脐血单核细胞的水凝胶复合物对小鼠烫伤创伤的愈合更快,愈后无疤痕,治疗21天后,观察到伤口完全闭合,同时该治疗小鼠的毛发在伤口区的再生比其他组更明显;新皮肤与周围皮肤无明显差异;
根据水凝胶(NOCC/A-HA)共培养细胞的数据,完成了本发明。
第一方面,本发明提供一种水凝胶复合物,所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,其中羧甲基壳聚糖和醛透明质酸的体积比为1-10:1;所述醛透明质酸由透明质酸通过官能团醛基化修饰获得,所述透明质酸选自天然透明质酸或者人工合成透明质酸;
进一步的,所述细胞选自成纤维细胞、肌细胞、上皮细胞、粘膜细胞和/或干细胞等;
更进一步的,所述干细胞选自脐带干细胞、亚全能干细胞、神经干细胞、脐带间充质干细胞、肝干细胞、心肌干细胞、血管内皮祖细胞、表皮成纤维干细胞和软骨干细胞中的一种或多种;
进一步的,所述羧甲基壳聚糖和醛透明质酸的体积比优选为2:1、4:1、6:1或8:1;
进一步的,所述水凝胶复合物还包括DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等中的一种或多种培养液。
第二方面,本发明提供一种含有水凝胶的细胞培养基,所述细胞培养基含有羧甲基壳聚糖、醛透明质酸和细胞培养液,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,其中羧甲基壳聚糖和醛透明质酸的体积比为1-10:1;所述醛透明质酸由透明质酸通过官能团醛基化修饰获得,所述透明质酸选自天然透明质酸或者人工合成透明质酸;
进一步,所述培养液选自DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等中的一种或多种。
第三方面,本发明提供一种水凝胶复合物在制备皮肤创伤物质中的用途。
进一步的,所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1;所述醛透明质酸由透明质酸通过官能团醛基化修饰获得,所述透明质酸选自天然透明质酸或者人工合成透明质酸;
进一步的,所述水凝胶复合物还包括DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等的一种或多种培养液。
进一步的,所述创伤是指烧伤、机械损伤或炎症导致的皮肤或粘膜的创伤,所述粘膜包括口腔粘膜或鼻黏膜等。
进一步,所述修复创伤的物质可以是药物或医疗器械。
第四方面,本发明提供一种制备水凝胶复合物的方法,所述制备方法包括如下步骤:
S01醛透明质酸的合成:透明质酸钠进行醛基化修饰;
S02水凝胶复合物合成:先将需要的细胞培养液注入灭菌后的醛透明质酸溶液中,再将细胞注入含有细胞培养液的醛透明质酸溶液中,最后按比例加入羧甲基壳聚糖,形成水凝胶复合物。
进一步的,所述羧甲基壳聚糖和醛透明质酸的比例为1-10:1。
第五方面,本发明提供一种含有水凝胶的培养基的制备方法,所述制备方法包括如下步骤:
S01醛透明质酸的合成:透明质酸钠进行醛基化修饰;
S02水凝胶合成:将细胞培养液注入灭菌后的醛透明质酸溶液中,再按比例加入羧甲基壳聚糖,其中羧甲基壳聚糖和醛透明质酸的比例选自1-10:1,最终形成水凝胶培养基。
进一步的,所述细胞培养液包括DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等中的一种或多种。
附图说明
图1透明质酸氧化为醛透明质酸的示意图。
图2 A-HA的1HNMR光谱。
图3通过席夫碱反应制备NOCC/A-HA水凝胶的示意图。
图4NOCC/A-HA水凝胶的形态扫描电镜图。
图5NOCC/A-HA水凝胶的可注射性和自愈合性图。
图6NOCC/A-HA水凝胶的流变性。
图7NOCC/A-HA水凝胶的平衡溶胀性。
图8NOCC/A-HA水凝胶与细胞相容性
图9NOCC/A-HA水凝胶的溶血性实验
图10(a)CB-MNC在PBS单纯水凝胶中存活1-21天的荧光染色图;
(b)CB-MNC在PBS单纯水凝胶中存活1-21天的平均存活率趋势图;
(c)CB-MNC在含1%人血白蛋白生理盐水制备的水凝胶中存活1-21天的荧光染色图;
(d)CB-MNC在含1%人血白蛋白生理盐水制备的水凝胶中存活1-21天的平均存活率趋势图;(e)CB-MNC在DMEM水凝胶中存活1-21天的荧光染色图;
(f)CB-MNC在DMEM水凝胶中存活1-21天的平均存活率趋势图。
图11(a)体外培养干细胞在DMEM培养基中0-96h的活死细胞染色;
(b)干细胞在DMEM培养基中0-96h的平均存活率趋势图;
(c)体外培养干细胞在含1%人血白蛋白生理盐水中0-96h的活死细胞染色;
(d)干细胞在含1%人血白蛋白生理盐水中0-96h的平均存活率趋势图。
图12单纯水凝胶治疗21天后的创面愈合情况和水凝胶负载CB-MNC体系治疗21天后的创面愈合情况。
图13Control group,Gel group,Gel+CB-MNC组小鼠0-21天皮肤的HE染色切片。
具体实施方式
醛透明质酸(A-HA):醛基化修饰的透明质酸既醛透明质酸,具有自交联和原位交联形成水凝胶的特性,也可在加入小分子交联剂的情况下实现交联,这种醛基化透明质酸及其交联后形成的水凝胶可被用于生物医药和医学美容等领域。在本发明中,醛透明质酸由透明质酸(HA)中的邻位羟基在强氧化剂氧化条件下形成醛基后,才能与羧甲基壳聚糖(NOCC)发生席夫碱反应,合成水凝胶。
本文所述术语“培养基”和“培养液”,依据细胞培养的需求,可以选择固体形式的培养基或者液体形式的培养液,同一种培养液或培养基除形态不同外,其所含成分是一致的。
实施例1羧甲基壳聚糖醛透明质酸(NOCC/A-HA)水凝胶的合成
1.1醛透明质酸(A-HA)的合成
(1)将透明质酸钠1.0g,2.5mmol溶于100mL双重蒸馏水中,浓度为10mg/mL;
(2)当HA完全溶解后,添加高碘酸钠水溶液2.5mmol,5mL,在黑暗中室温下反应24h;
(3)加入1mL乙二醇以淬灭未反应的高碘酸钠。将反应在室温下在搅拌1小时,将所得溶液通过蒸馏水进行彻底透析(MWCO 10000)3天来纯化溶液,在透析过程中每天至少换水三次;
(4)通过冷冻干燥获得干燥产物,并通过核磁进行结构表征。
1.2羧甲基壳聚糖醛透明质酸NOCC/A-HA水凝胶的制备
(1)将NOCC和A-HA以30mg/mL的浓度溶于磷酸盐缓冲液(PBS)中;
(2)通过将NOCC和A-HA溶液以2:1、4:1、6:1、8:1的体积比混合来制备原位交联的水凝胶,并通过FTIR近红外广谱进行结构表征。
1.3形态学研究
NOCC/A-HA水凝胶的形态通过扫描电子显微镜(SEM)表征。NOCC和A-HA在室温下交联形成水凝胶,然后将水凝胶的冻干产物在液氮中降温,取出后迅速脆断,在观察之前在横截面上涂上一层薄薄的金,测试在VEGA3 TESCAN电子显微镜上进行,使用20KV的加速电压下观察水凝胶的表面和横截面的形貌。
1.4流变性质研究
NOCC/A-HA水凝胶的流变特性通过25mm平板的ARES-G2旋转流变仪测量。将在PBS中制备的水凝胶置于平板上原位成胶进行测试,分析储能模量G′和损耗模量G″随时间、应变及频率的变化。
1.5平衡溶胀性质
用重量分析法测定冻干水凝胶的溶胀率。将冷冻干燥的水凝胶称重,并在37℃(PBS,pH=7.4)中。在设定的时间间隔,取出水凝胶样品用滤纸擦拭表面水后称重。当样品的重量保持恒定时,达到溶胀平衡,所有实验均重复三次。使用以下公式计算平衡溶胀率和质量损失率。
M0是干凝胶的初始质量,Ms是溶胀的水凝胶的湿质量,Md是溶胀后再冻干的水凝胶的干燥质量。
1.6NOCC/A-HA的可注射性和自愈性
将NOCC/A-HA水凝胶前体溶液与中性红溶液均匀混合,然后装入注射器中进行凝胶化,将形成的水凝胶注入水中或在培养皿上书写以测试其可注射性。
水凝胶的自愈合行为是在PBS中制备水凝胶样品,其中一些用中性红染成红色,将样品分成两块,然后立即在37℃下将红色的胶块和透明的胶块放在一起,以测试其自愈合行为。
1.7实验结果
(1)醛透明质酸(A-HA)的合成流程图(见图1);
(2)制得的醛透明质酸在通过核磁进行结构表征(见图2a-2b);
NOCC的核磁共振氢谱(图2a);A-HA的核磁共振氢谱(图2b);NOCC在3.1ppm(H-2)、3.5-4.0ppm(H-3至H-6)处呈现特征峰,在2.2ppm处呈现小单峰,分配给N-乙酰基的甲基质子;在4.2和4.4ppm处检测到两个小峰,分别分配给NOCC的C2的N位和C6的O位的-CH2COO-质子,与氨基(N位)和伯羟基(O位)上的羧甲基取代一致;A-HA在3.2和3.7ppm之间的宽信号对应于糖环中的质子;HA的N-乙酰基的甲基质子在2.0ppm检测到;在4.9、5.0和5.1ppm下观察到的信号为醛基;通过比较HA主链中N-乙酰基的醛和甲基的积分来量化氧化程度得到的氧化度为56%,氧化度=(A醛基/2)/(甲基/3)A:峰面积。
(3)NOCC/A-HA水凝胶通过FTIR近红外广谱进行结构表征(见图2c);
NOCC/A-HA水凝胶通过NOCC的氨基和A-HA的醛基之间的席夫碱反应交联;HA在3400、1616和1078cm-1处呈现特征峰,分别与-OH吸收、-COOH的反对称拉伸振动和C-O键的拉伸吸收有关;A-HA的光谱与HA的光谱非常相似,只是在1730cm-1处出现一个属于醛基的小带;在NOCC的光谱中,1599和1411cm-1处的谱带分别与羧酸盐(-COO)的不对称和对称拉伸有关;水凝胶的光谱显示了NOCC和A-HA的所有特征谱带;3200-3500和1100cm-1处的谱带分别归因于游离OH和NH2基团以及CO拉伸;醛基1730cm-1处的小条带消失,1640cm-1的条带属于羧酸盐和亚胺基团,这证明水凝胶的形成。
(4)通过席夫碱反应制备水凝胶(NOCC/A-HA)(见图3);
醛透明质酸上的醛基与羧甲基壳聚糖上的氨基发生席夫碱反应形成亚胺键(图3圆球)。亚胺键之间的交联形成了网状交联结构。
(5)NOCC/A-HA水凝胶的形态通过扫描电子显微镜(SEM)表征,使用20KV的加速电压下观察水凝胶的表面和横截面的形貌(见图4),并评估其应用场景;
NOCC和A-HA的体积比直接影响到凝胶的流变性能、均匀性和应用范围。当NOCC体积增加时,孔壁会得到加强,而NOCC体积过大,没有足够的交联键位点,其内部结构也会出现不均匀的现象。不同比例的水凝胶的表面形态,由扫描电镜拍摄,所有比例尺都是100μm。具体来说,2:1比例水凝胶孔径较大,孔隙相互连接较少,孔隙之间的连接更薄,更脆弱(图4a),流动性较其他各比例较强,可用于复杂创面的敷料;4:1比例水凝胶孔径较小,孔洞分布均匀,孔隙率高,孔壁则更强更厚(图4b),凝胶的粘弹性强,具有均匀的相互连接的孔隙内部结构,同时凝胶材料可以为细胞提供营养物质,并支持细胞的附着和增殖,最有利于应用于伤口敷料或细胞附着支持。6:1比例水凝胶孔径分布不均匀,孔壁较厚(图4c),凝胶吸水率较高,粘弹性较强可用于防止手术后粘连。而8:1比例水凝胶孔径较小,孔隙率高孔隙连接紧密(图4d),凝胶的吸水性弱,粘弹性较稳定,可用于局部注射作为基质使用。
综上所述,NOCC/A-HA水凝胶的4:1比例被证明是一种适合作为生物支架的水凝胶,它具有优越的形态,具有高度的均匀性,有利的孔径大小和适当的密度,以及适当的润湿性。具有合适的物理特性和良好的生物相容性。最后,将NOCC/A-HA凝胶基质作为组织工程应用中的一种有效的生物支架材料来评价干细胞在体外的存活率。
(6)NOCC/A-HA的可注射性和自愈性(见图5);
通过自愈合试验评估水凝胶的自修复性能,水凝胶(NOCC/A-HA)选择4:1。将染色的红色水凝胶和透明水凝胶切成两块,并在37℃下将切好的两块放在一起。样品在30秒后完全整合。愈合的水凝胶可以剥离并支撑其自身重量(图5a)。水凝胶的自修复能力可归因于可逆亚胺键交联的重建,以及水凝胶两部分之间组分或组分交换的迁移。对自修复水凝胶进行了流变学实验,如图5b所示,自愈合的水凝胶呈现出与原始水凝胶非常接近的G'和G”值(800、500),从而证实了出色的自愈能力。水凝胶的自愈能力能够确保其在临床治疗中应用于伤口时的结构完整性,从而促进复杂伤口的修复和治疗。
通过注射器注射实验评估水凝胶的可注射性(图5c)。对于生物学应用的可注射水凝胶,胶凝时间起着重要的作用,因为较短的胶凝时间可能导致针头堵塞,而较长的胶凝时间则可导致封装细胞不可逆转的损失。研究发现NOCC/A-HA水凝胶可以通过26G针头连续注入,并且在注入后保持其完整性。
(7)NOCC/A-HA的流变性(见图6);
各比例水凝胶的流变特性:在27℃和不同振荡应变条件下,各比例水凝胶的储能模量变化(见图6a)。通过存储模量(G')和损失模量(G”)随时间的变化,在流变仪板上原位监测混合NOCC和A-HA水溶液的凝胶化过程。对于所有样品,储能模量最初低于损耗模量2:1比例水凝胶施加同等应力条件下粘弹性最小,4:1比例水凝胶施加同等应力条件下粘弹性较高且随着应力增加粘弹性无明显变化较为稳定,6:1比例水凝胶施加同等应力条件下粘弹性较4:1小,8:1比例水凝胶施加同等应力条件下粘弹性较小但随着应力增加粘弹性无明显变化较为稳定。
各比例水凝胶在不同频率条件下的流变特性:在27℃和0.1-100Hz频率下,各比例水凝胶随应变的变化(见图6b)。在同等频率下2:1,4:1,6:1,8:1比例水凝胶都能稳定保持凝胶状态。
(8)NOCC/A-HA的平衡溶胀性。
在生理盐水(pH=7.4,37℃)中24小时,各比例水凝胶的溶胀率(见图7)。
水凝胶的溶胀特性对于作为细胞载体或支架的应用具有重要意义。不同比例的水凝胶在生理盐水中37℃下24小时的溶胀率。4:1比例水凝胶表现出最高的膨胀率,这是因为均匀的多孔结构有利于吸水和保水性能。
实施例2水凝胶封装细胞的合成与评估
2.1水凝胶与细胞相容性实验
为了评估水凝胶与细胞共培养的相容性,以此判断水凝胶是否拥有细胞毒性,将2:1,4:1,6:1,8:1比例水凝胶与L929细胞进行共培养,通过MTT染色实验确定细胞的存活率。
实验发现,所有样品的细胞增殖率在72小时内均高于75%(见图8)。因此,不同比例的水凝胶对L929小鼠成纤维细胞没有毒性,可以按照ISO 10993安全的在临床应用中使用。
2.2水凝胶的溶血性实验
取200μL重悬好的人血红细胞悬液与不同比例的凝胶浸提液混合,吹打混匀后置于37℃中培养1h。设置空白对照PBS、阴性对照BSA溶液(20μM)和阳性对照0.1% TritonX-100。1h后,将孵育完毕的细胞取出,室温下1000g离心10min。取无菌干净的96孔板,将离心所得上清注入各孔中,每孔200μL。置于酶标仪下读取各孔吸光值,读数波长为540nm。
实验发现与阳性对照组相比,不同比例的水凝胶在与血细胞共孵育过程中不具有显著溶血作用;因此可以认为此水凝胶对哺乳动物血细胞无溶血活性(见图9)。
2.3水凝胶封装细胞的合成与细胞存活实验
为了评估水凝胶培养细胞的情况,首先测试了脐带血单个核细胞(CB-MNC)在无菌条件下在水凝胶中的封装情况。即将NOCC和A-HA溶液通过紫外灭菌,然后将A-HA溶液与脐带血单个核细胞(CB-MNC)溶液等体积混合均匀后,分别将NOCC和A-HA溶液以2:1、4:1、6:1、8:1的体积混合在灭菌后的孔板中原位形成水凝胶,凝胶中的细胞密度固定为1.0×106个细胞/ml,培养观察。
2.4体外细胞存活实验
为了观察CB-MNC在生理盐水、含1%人白蛋白的盐水中制备的水凝胶中的存活时间,用前面提到的细胞封装方法进行细胞封装,培养后所有细胞用AO-PI试剂盒染色。
为了证明水凝胶对细胞存活的有益作用,将脐带血单个核细胞(CB-MNC)培养在液体培养基中,即DMEM以及含1%人白蛋白的盐水中。培养后所有细胞用AO-PI试剂盒染色。
利用AO-PI进行活/死细胞的染色。在荧光显微镜下观察荧光时,可调节分辨率对比度等得到更美观的图片。活细胞为绿色荧光,死细胞为红色荧光。负载CB-MNC体系通过荧光活死细胞染色法,检测细胞的存活率。
2.5实验结果
通过活细胞/死细胞染色进行CB-MNC细胞相容性测试以确定细胞活力。在生理盐水制备的水凝胶中,细胞呈圆形,在培养21天后均匀分布在水凝胶中(图10a);第21天,细胞存活率为51%(图10b)。CB-MNC也在含有1%人白蛋白的盐水中制备的水凝胶中培养(图10c),并在DMEM培养基中培养(见图10e)。细胞形态与生理盐水中制备的水凝胶非常相似。相反,在1%人白蛋白的盐水中制备的水凝胶(图10d)和DMEM(图10f)中培养的细胞在第21天的存活率分别为56%和55%,即略高于生理盐水中制备细胞的存活率。这些发现表明,人白蛋白对细胞生长有益。
无水凝胶存在时,在DMEM中培养12小时后,细胞活力为47%,96小时后所有细胞均凋亡(图11a和b)。在含1%人白蛋白的盐水中培养细胞的情况下(图11c,d),12小时后细胞活力为55%,96小时之后所有细胞都凋亡。因此,尽管两种培养基都可以为细胞生长提供营养,由于缺乏良好的生存环境,细胞最终都无法生存。因此,我们确定水凝胶网络可以有利于细胞附着的粘附配体偶联,从而提供有利于细胞生长的环境。
脐血单核干细胞在体外培养基培养,在96小时内全部死亡。在水凝胶生物支架中干细胞的存活率得到了大幅度提升(数值比较),在PBS单纯水凝胶/含1%人血白蛋白生理盐水制备的水凝胶/DMEM水凝胶中,包载后的第15天还有55%以上的干细胞存活。结果表明,NOCC/A-HA水凝胶具有良好的细胞相容性和无毒性;NOCC/A-HA水凝胶相互连通的多孔结构使营养物质和氧气得以渗透,为细胞提供了适宜的环境。
实施例3体外小鼠烫伤模型的建立与治疗
建立小鼠烫伤模型,使用水凝胶负载CB-MNC体系治疗烫伤,观察治疗效果,愈后瘢痕情况。开发了载有CB-MNC的水凝胶系统,以评估其在治疗烫伤并预防疤痕形成的潜力。在小鼠背侧皮肤建立深Ⅱ度烫伤模型,分别用不处理的烫伤组(Control group)作为对照,单纯水凝胶组(Gel group)和载有CB-MNC的水凝胶组(Gel+CB-MNC group)治疗伤口并观察伤口愈合情况。
Control group和Gel group在烫伤后的第一天都有伤口肿胀,而Gel+CB-MNCgroup没有出现伤口肿胀。这可能归因于CB-MNCs减少伤口炎症反应的能力。Control group烫伤愈合缓慢伴有表皮颜色加深,创面结痂和皮下炎症等现象,愈后疤痕较大。而且第3~7天创面出现结痂,表皮颜色加深,颜色不均匀,皮下有炎症反应。Gel组愈合速度较对照组快,愈后瘢痕较对照组小。Gel+CB-MNC组与其他两组相比愈合更快,愈后无疤痕,治疗21天后,观察到伤口完全闭合。此外,该组的毛发在伤口区的再生比其他组更明显。新皮肤与周围皮肤无明显差异(见图12a)。
伤口愈合区域图形和数据结果表明,单纯水凝胶具有一定的促进烫伤修复的能力。负载CB-MNCs的水凝胶可促进烫伤愈合,在愈合过程中具有促进创面毛发生长,避免炎症反应和减少愈后瘢痕形成(见图12b)。
实施例4愈合伤口的组织生理学
通过使用苏木精和伊红(H&E)染色指示愈合伤口的表皮和组织再生。观察愈伤组织的情况。
烫伤后第一天,观察到烫伤皮肤表皮细胞核固缩,真皮深层受损,胶原纤维融合,毛囊残留,这些观察符合深二度烫伤的标准。第7天,Gel+CB-MNC组表皮再生,棘细胞层水肿减少,炎症浸润减少;Gel组表皮再生,真皮排列松弛,炎症浸润中度;对照组显示不完全角化和高炎症浸润。第14天,Gel+CB-MNC组皮肤附件出现新生血管和再生;Gel组血管再生量少,炎症浸润减少;对照组表现出不完美角化和表皮再生的减少。第21天,Gel+CB-MNC组表皮及真皮恢复状态良好,无瘢痕形成;Gel组创面恢复后表皮增厚;对照组创面恢复后皮肤附肢再生、表皮增厚和瘢痕形成较少(见图13)。
Claims (10)
1.一种水凝胶复合物,所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1;所述醛透明质酸由透明质酸通过官能团醛基化修饰获得,所述透明质酸选自天然透明质酸或者人工合成透明质酸。
2.由权利要求1所述一种水凝胶复合物,其特征在于所述细胞选自成纤维细胞、肌细胞、上皮细胞、粘膜细胞和/或干细胞。
3.由权利要求1所述一种水凝胶复合物,其特征在于所述羧甲基壳聚糖和醛透明质酸的体积比选自2:1、4:1、6:1或8:1。
4.一种含有水凝胶的细胞培养基,所述培养基含有羧甲基壳聚糖、醛透明质酸和细胞培养液,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1;所述醛透明质酸由透明质酸通过官能团醛基化修饰获得,所述透明质酸选自天然透明质酸或者人工合成透明质酸。
5.如权利要求4所述一种含有水凝胶的细胞培养基进一步,其特征在于所述培养液选自DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS和平衡盐溶液(BSS)中的一种或多种。
6.一种水凝胶复合物在制备皮肤创伤物质中的用途,所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞,羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶,其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1;所述醛透明质酸由透明质酸通过官能团醛基化修饰获得,所述透明质酸选自天然透明质酸或者人工合成透明质酸。
7.如权利要求6所述一种水凝胶复合物在制备皮肤创伤物质中的用途,其特征在于所述创伤是指烧伤、机械损伤或炎症导致的皮肤或粘膜的创伤,所述粘膜包括口腔粘膜或鼻黏膜等。
8.如权利要求一种水凝胶复合物在制备皮肤创伤物质中的用途,其特征在于所述所述修复创伤的物质可以是药物或医疗器械。
9.一种制备水凝胶复合物的方法,所述制备方法包括如下步骤:
S01醛透明质酸的合成:透明质酸钠进行醛基化修饰;
S02水凝胶复合物合成:先将需要的细胞培养液注入灭菌后的醛透明质酸溶液中,再将细胞注入含有细胞培养液的醛透明质酸溶液中,最后按比例加入羧甲基壳聚糖,其中羧甲基壳聚糖和醛透明质酸的比例选自1-10:1。
10.一种含有水凝胶的培养基的制备方法,所述制备方法包括如下步骤:
S01醛透明质酸的合成:透明质酸钠进行醛基化修饰;
S02水凝胶合成:将细胞培养液注入灭菌后的醛透明质酸溶液中,再按比例加入羧甲基壳聚糖,其中羧甲基壳聚糖和醛透明质酸的比例选自1-10:1,最终形成水凝胶培养基。
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