CN113577366B - 促进糖尿病难愈性伤口快速愈合的干膜敷料及其制备方法 - Google Patents
促进糖尿病难愈性伤口快速愈合的干膜敷料及其制备方法 Download PDFInfo
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- CN113577366B CN113577366B CN202110923946.5A CN202110923946A CN113577366B CN 113577366 B CN113577366 B CN 113577366B CN 202110923946 A CN202110923946 A CN 202110923946A CN 113577366 B CN113577366 B CN 113577366B
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Abstract
本发明涉及一种促进糖尿病难愈性伤口快速愈合的干膜敷料,主要由干细胞分泌组与壳聚糖基础凝胶按体积比1:(2~6)混匀,在温度25~42℃、湿度10~20%条件下干燥成膜制得。获得的干膜敷料,不但保存、运输条件不受限制,而且方便医患使用,同时还显著提高了敷料对糖尿病难愈性伤口的愈合作用。同时本发明还提供了干膜敷料的制备方法,通过便捷的操作保证了敷料的高活性与稳定性,为其临床应用奠定了基础。
Description
技术领域
本发明涉及膜敷料技术领域,尤其涉及一种促进糖尿病难愈性伤口快速愈合的干膜敷料及其制备方法。
背景技术
糖尿病是一种由于胰岛素分泌缺陷或胰岛素作用障碍所致的以高血糖为特征的代谢性疾病,持续高血糖与长期代谢紊乱可导致全身组织器官的损伤及其功能障碍和衰竭,即常说的并发症。近30年来中国糖尿病的患病率从0.67%飙升到11.6%,糖尿病已成为盛行病。在诸多并发症中,糖尿病难愈性伤口的危险高达25%,全球每年约有400万糖尿病患者发生难愈性伤口。造成糖尿病伤口难愈性高的主要原因有高糖毒性、神经损伤、代谢产物、血管阻塞和生长因子不足等,这也是糖尿病致残的部分缘由。对于糖尿病难愈性伤口,除根据患者情况做一般病因治疗外,对伤口局部外用药物和敷料是非常重要的治疗手段,故开发合适的外用敷料以减轻患者痛苦、缩短病程,具有重要的临床研究意义和应用前景。
凝胶作为伤口敷料是目前国内外的热门研究对象,但凝胶在保存、运输条件方面要求高,并且在临床使用方面操作相对复杂,具有一定的局限性。壳聚糖凝胶具有杀菌、无毒、生物相容性较高和生物可降解等优点,但是壳聚糖凝胶保存不方便,且对糖尿病难愈性伤口的促进作用不够显著,因此制备一种能够显著促进糖尿病难愈性伤口愈合且保存简单方便的敷料成为本发明要解决的技术问题。
发明内容
为解决现有技术存在的不足,本发明提供了一种促进糖尿病难愈性伤口快速愈合的干膜敷料,该敷料将壳聚糖基础凝胶与干细胞分泌组结合后制备成干膜,不但保存、运输条件不受局限,而且方便医患使用,同时还显著提高了敷料对糖尿病难愈性伤口的愈合作用,为其临床应用奠定了基础。
为实现上述目的,本发明提供的促进糖尿病难愈性伤口快速愈合的干膜敷料,主要由干细胞分泌组与壳聚糖基础凝胶按体积比1:(2~6)混匀,在温度25~42℃、湿度10~20%条件下干燥成膜制得;
所述干细胞分泌组主要由汇合度85±5%的脂肪间充质干细胞,经无血清基础培养基培养后,选取能够使细胞活性提高25%以上的分泌组作为合格备用的干细胞分泌组;
所述壳聚糖基础凝胶包括氯化壳聚糖(Chitosan chloride,CSCL)溶液、β-甘油磷酸钠(β-Glycerophosphoric acid disodium salt,β-GP)溶液、明胶(Gelatin)溶液与羟乙基纤维素(Hydroxyethyl Cellulose,HEC)溶液。
在诸多间充质干细胞中选择脂肪间充质干细胞作为本发明干膜敷料的主成分来源,利用脂肪间充质干细胞在培养过程产生的干细胞分泌组,与具有特定组成的壳聚糖基础凝胶相结合,通过干细胞分泌组含有的多种炎症调节因子和丰富的生长因子,与氯化壳聚糖、β-GP、明胶以及HEC的功能性协同,多方面调节伤口环境,对受损细胞起到修复作用,促进伤口愈合。一方面利用氯化壳聚糖能够促进细胞对营养成分或者药物分子的吸收作用,以及β-GP和明胶与氯化壳聚糖相结合利于凝胶保持稳定且利于装载和释放蛋白药物的优势,助益干细胞分泌组的炎性因子和生长因子充分发挥功效,在炎性因子调节伤口处的炎症反应、氯化壳聚糖对伤口处常见菌发挥良好抑制作用、生长因子促进伤口处细胞的增殖和迁移等多方面作用下,加速伤口愈合;另一方面,借助HEC的保水、成膜特性以及干细胞分泌组免疫原性低的优势,使敷料经过干燥制备成干膜后,依然能保持对伤口高活性愈合作用,从而显著提高对糖尿病难愈性伤口的愈合功效。另外,壳聚糖基础凝胶的良好生物降解性、生物相容性,以及氯化壳聚糖可溶于水的特性,极大方便了干膜敷料的应用。
作为对上述技术方案的限定,所述脂肪间充质干细胞选用体外培养12代以内的脂肪间充质干细胞。
作为对上述技术方案的限定,所述脂肪间充质干细胞经无血清培养基培养达到汇合度要求。
作为对上述技术方案的限定,所述壳聚糖基础凝胶主要由质量分数1%~3%的氯化壳聚糖溶液、质量分数10%~13%的β-甘油磷酸钠溶液和质量分数2%~5%的明胶溶液与质量分数2%~4%的羟乙基纤维素溶液按照体积比8:(2~6):(3~7):(3~6)混合得到。
进一步限定脂肪间充质干细胞的培养代数与富集条件,使干细胞分泌组具有多种高活性炎性因子和生长因子,增强干膜敷料的愈合作用;限定壳聚糖基础凝胶的有效组分配比,提高干膜敷料性能。
同时,本发明还提供了如上所述促进糖尿病难愈性伤口快速愈合的干膜敷料的制备方法,包括以下步骤:
a、制备干细胞分泌组:将体外培养12代以内的脂肪间充质干细胞进行检测,通过流式细胞仪筛选出表面特异性标志蛋白符合标准的脂肪间充质干细胞,然后经由无血清培养基培养,当脂肪间充质干细胞汇合度达到85±5%时,弃上清、洗涤,再加入无血清基础培养基培养,收集培养液,去除细胞碎片、浓缩、除菌,得到干细胞分泌组,检测其提高细胞活性能力,当活性提高25%以上时,方为合格备用的干细胞分泌组,将其置于-20℃保存待用;
b、配制壳聚糖基础凝胶:将质量分数1%~3%的氯化壳聚糖溶液、10%~13%的β-GP溶液、2%~5%的明胶溶液与质量分数2%~4%的羟乙基纤维素溶液按照体积比8:(2~6):(3~7):(3~6)的比例混合均匀;
c、制备干膜敷料:将干细胞分泌组与壳聚糖基础凝胶按照体积比为1:(2~6)的比例混合均匀,灌装到模具中,在温度25~42℃、湿度10~20%条件下干燥成膜,得到可常温保存使用的干膜敷料。
作为对上述技术方案的限定,步骤a中所述表面特异性标志蛋白的筛选标准为阳性蛋白CD90、CD44、CD73表达率均大于95%,阴性蛋白CD45表达率小于5%。
作为对上述技术方案的限定,步骤a中所述无血清培养基选用Mesenchy StemCells Basal Medium或者成人脂肪间充质干细胞无血清培养基。
作为对上述技术方案的限定,步骤a中所述无血清基础培养基选用无血清RPMI1640培养基或无血清DMEM培养基,培养环境为37℃、体积分数5%CO2的细胞培养箱。
本发明干膜敷料的制备操作简单、可重复性高,能够保持敷料的高活性与稳定性,为干膜敷料的规模化制备和推广应用提供保障。
本发明的干膜敷料具有以下优点:①能够高效持续释放蛋白,修复受损细胞,加速糖尿病难愈性伤口愈合;②为伤口处皮肤细胞的生长提供支撑,更有利于创伤的愈合;③具有吸收渗液、防水透气和杀菌等特点,可以防止伤口感染;④具有取材方便、分离过程简单、保存简单的优点,克服了传统水凝胶保存条件高和临床使用难度大等缺点,方便患者和医务人员使用,为其临床应用奠定了基础。
附图说明
图1为本发明干膜敷料与未形成干膜的凝胶敷料照片;
图2为本发明干膜敷料与未形成干膜的凝胶敷料、文献对照凝胶的蛋白释放试验结果图;
图3为本发明干膜敷料的动物实验结果图。
具体实施方式
下面将结合实施例,对本发明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
实施例一
本实施例涉及促进糖尿病难愈性伤口快速愈合的干膜敷料及其制备。
实施例1.1
按下述操作制备促进糖尿病难愈性伤口快速愈合的干膜敷料。
a、制备干细胞分泌组
a1、获取脂肪间充质干细胞,可参照下述处理方法:
组织的处理:
①脂肪组织转移至50mL离心管,每管40mL,以800g离心力,4℃离心8min,离心后的脂肪组织转移至新的离心管中,等体积加入0.9%生理盐水,以800g离心力,4℃离心8min;重复洗涤一次;
②等体积向脂肪沉淀中加0.1%I型胶原酶,充分混匀,封口后放入37℃摇床,消化45min,取出消化好的脂肪组织,混匀,以800g离心力,离心10min;
③弃掉上层脂肪,获得基质细胞,用RPMI 1640培养基重悬细胞沉淀,以800g离心力,离心10min;重复本步骤一次,洗涤细胞沉淀;
④弃掉离心后的上清,RPMI 1640培养基重悬细胞沉淀并混匀,用100目尼龙筛网过滤器过滤细胞,将过滤后的细胞以800g离心力,离心10min,弃上清,1mL RPMI 1640培养基重悬细胞沉淀,混匀后加入4mL预冷的红细胞裂解液,室温静置4min,裂红,以400g离心力,离心5min弃上清。
干细胞原代培养:用无血清RPMI 1640基础培养基重悬细胞沉淀,对细胞计数后调整其密度为7.0×105/mL,向T75培养瓶中先加入14mL培养基,再加入1mL细胞悬液,混匀,置于37℃、5%CO2细胞培养箱中培养,24h后,细胞换液,去除未贴壁细胞。
脂肪间充质干细胞的传代培养:细胞生长密度达到85%左右时,弃上清,PBS洗涤细胞,加入0.25%胰酶消化,1min后加入培养基终止消化,1000rpm/min离心5min,弃上清,加入无血清细胞培养基重悬细胞,将细胞悬液接种至细胞培养皿,将其置于37℃、5%CO2细胞培养箱中培养。
a2、获取干细胞分泌组:将实验室保存的体外培养12代以内的脂肪间充质干细胞,通过流式细胞仪检测干细胞表面的特异性标志蛋白,符合阳性蛋白CD90、CD44、CD73表达率均大于95%,阴性蛋白CD45表达率小于5%的脂肪间充质干细胞,通过Mesenchy StemCells Basal Medium-北京银丰鼎诚或者成人脂肪间充质干细胞无血清培养基-Cyagen培养进行汇合,当脂肪间充质干细胞汇合度达到85±5%左右时,弃上清,加入PBS缓冲液(pH7.4)洗涤三次,加入无血清RPMI 1640培养基或无血清DMEM培养基,培养三天后收集培养基,离心去除细胞碎片,0.45μm滤器过滤,然后通过超滤管进行10倍浓缩,最后通过0.22μm滤器进行过滤除菌,得到干细胞分泌组,检测其提高细胞活性能力,当活性提高25%以上时,方为合格备用的干细胞分泌组,-20℃保存待用。
b、制备壳聚糖基础凝胶:
2%氯化壳聚糖溶液:称取0.8g氯化壳聚糖粉末,加入ddH2O,定容至40ml,摇床摇晃均匀,制备成2%的壳聚糖溶液,4℃放置待用;
11.5%β-GP溶液:称取2.3gβ-GP粉末于50ml离心管中,加入ddH2O,定容至20ml,混匀,制备成11.5%的β-GP溶液,4℃放置待用;
4%明胶溶液:称取1.00g明胶粉末于50ml离心管中,加入ddH2O,定容至25ml,混匀,制备成4%的β-GP溶液,4℃保存;
2.5%HEC溶液:称取0.75g HEC粉末,加入ddH2O,定容至30ml,摇床中摇晃均匀,制备成2.5%的HEC溶液,4℃保存;
将2%氯化壳聚糖溶液、11.5%β-GP溶液和4%明胶溶液以及2.5%HEC溶液按照体积比8:4:4:3的比例混合,摇床摇匀,得到壳聚糖基础凝胶。
c、制备干膜敷料:将步骤a制得的干细胞分泌组、与步骤b获得的壳聚糖基础凝胶按照体积比1:4的比例混合均匀,灌装到模具中,温度37℃、湿度10%条件下放置24h,干燥成膜得到干膜敷料,常温保存待用。
实施例1.2
促进糖尿病难愈性伤口快速愈合的干膜敷料的制备:将制得的干细胞分泌组、壳聚糖基础凝胶按照体积比为1:4的比例混合均匀,灌装到模具中,24℃放置44h,干燥成膜,常温保存待用。
脂肪间充质干细胞分泌组的制备参照实施例1.1中干细胞分泌组的制备步骤,基础凝胶的制备是将3%氯化壳聚糖溶液和10%β-GP溶液和4%明胶溶液以及4%HEC溶液按照体积比8:4:4:3的比例混合,摇床摇匀。
实施例二
本实施例涉及制备条件对干膜敷料的影响。
实施例2.1
下面实施例给出壳聚糖基础凝胶不同组成配比与不同成膜条件下对于敷料制备影响,见表1,表中未写明的条件均与实施例1.1一致。
上表实施例2.1.2形成的干膜与实施例2.1.5形成的凝胶照片见附图图1,由上表结果可见,当2%氯化壳聚糖溶液、11.5%β-GP溶液和4%明胶溶液以及2.5%HEC溶液的体积比为8:4:4:3时,37℃、24h条件下所制得干膜的韧性最好,24℃、24h条件下所制得的凝胶的粘稠性和流动性最好,即实施例2.1.2和实施例2.1.5的配方制备的干膜和凝胶物理性能最好。
实施例2.2
下面试验在壳聚糖基础凝胶组成不变的条件下,干燥成膜不同温、湿度条件对于敷料的影响。
壳聚糖基础凝胶组成为2%CSCL(Chitosan chloride)12mL、11.5%β-GP6mL、4%Gelatin 6mL和2.5%HEC 4.5mL,混匀后在37℃摇床20min,加入模具中,每孔加3ml,观察不同的温度、湿度对凝胶干燥成膜的影响。
(1)恒温25℃:
当湿度在10%-20%,温度为25℃时,成膜时间约为44h。
(2)恒温37℃:
(3)恒温42℃:
(4)恒温42℃、湿度高于20%:
当湿度在10%~20%,温度为37~42℃时,成膜时间约为24h。
结论:当温度低于25℃时,不便于成膜,当温度高于42℃时,对干膜中装载的蛋白性能产生影响,因此温度控制在25~42℃。
实施例三
本实施例用于验证本发明干膜敷料的愈合功效。
实施例3.1
通过凝胶蛋白释放实验,研究干膜敷料对于干细胞分泌组有效成分释放性能的影响。
将实施例2.1.2制备的装载干细胞分泌组的干膜敷料、实施例2.1.5制备的装载干细胞分泌组的凝胶、以及文献对照凝胶(将壳聚糖溶于盐酸中制备成2%壳聚糖溶液,然后将壳聚糖、10%β-GP和2%HEC按照体积比16:4:5的比例混合制备成凝胶)分别转移至12孔板中,每孔加入4ml PBS,24℃放置,在预定的时间点每孔吸取50μL释放液,同时补加相同体积的PBS,预定的时间点为0h、2h、4h、8h、10h、15h、20h、24h。将每个时间点的释放液放置于4℃,取完全部时间点后,通过BCA检测释放液中的蛋白浓度。
结果显示,在相同时间点,实施例2.1.2的干膜敷料对蛋白的释放量明显高于实施例2.1.5凝胶和文献对照凝胶对蛋白的释放量,见附图图2。故本发明的干膜敷料对其中装载的干细胞分泌组具有较高的释放量,干细胞分泌组的高效释放更有利于伤口愈合。
实施例3.2
通过动物实验,研究装载干细胞分泌组的干膜敷料对糖尿病鼠创伤的修复功能。
建立小鼠糖尿病模型,然后将正常鼠和糖尿病鼠分为三组,分别为对照组(Ctrl)、糖尿病组(DM)和糖尿病干膜敷料(DM-CM)组,每组5只。对糖尿病鼠和正常鼠背部两侧分别进行直径8mm的全层皮肤切除,建立糖尿病鼠创伤模型;然后用弹性绷带处理对照组和糖尿病组小鼠伤口,用装载干细胞分泌组的干膜敷料处理DM-CM组小鼠伤口,并定期更换,不断观察和测量评估伤口愈合情况。
结果显示,糖尿病组(DM)小鼠的伤口愈合速度明显慢于对照组(Ctrl),糖尿病干膜敷料组(DM-CM)小鼠的伤口面积明显小于糖尿病组(DM),见附图图3。由此说明装载脂肪间充质干细胞分泌组的干膜敷料可以显著促进糖尿病难愈性伤口的愈合。
Claims (7)
1.一种促进糖尿病难愈性伤口快速愈合的干膜敷料,其特征在于:该干膜敷料主要由干细胞分泌组与壳聚糖基础凝胶按体积比1:(2~6)混匀,在温度25~42℃、湿度10~25%条件下干燥成膜制得;
所述干细胞分泌组主要由汇合度85±5%的脂肪间充质干细胞,经无血清基础培养基培养后,选取细胞活性提高25%以上的分泌组作为合格备用的干细胞分泌组;
所述壳聚糖基础凝胶包括氯化壳聚糖溶液、β-甘油磷酸钠溶液、明胶溶液和羟乙基纤维素溶液,主要由质量分数1%~3%的氯化壳聚糖溶液、质量分数10%~13%的β-甘油磷酸钠溶液、质量分数2%~5%的明胶溶液与质量分数2%~4%的羟乙基纤维素溶液按照体积比8:(2~6):(3~7):(3~6)混合得到。
2.根据权利要求1所述促进糖尿病难愈性伤口快速愈合的干膜敷料,其特征在于:所述脂肪间充质干细胞选用体外培养12代以内的脂肪间充质干细胞。
3.根据权利要求1所述促进糖尿病难愈性伤口快速愈合的干膜敷料,其特征在于:所述脂肪间充质干细胞经无血清培养基培养达到汇合度要求。
4.一种如权利要求1~3中任一项所述促进糖尿病难愈性伤口快速愈合的干膜敷料的制备方法,其特征在于,该制备方法包括以下步骤:
a、制备干细胞分泌组:将体外培养12代以内的脂肪间充质干细胞进行检测,通过流式细胞仪筛选出表面特异性标志蛋白符合标准的脂肪间充质干细胞,然后经由无血清培养基培养,当脂肪间充质干细胞汇合度达到85±5%时,弃上清、洗涤,再加入无血清基础培养基培养,收集培养液,去除细胞碎片、浓缩、除菌,得到干细胞分泌组,检测其提高细胞活性能力,当活性提高25%以上时,方为合格备用的干细胞分泌组,将其置于-20℃保存待用;
b、配制壳聚糖基础凝胶:将质量分数1%~3%的氯化壳聚糖溶液、10%~13%的β~GP溶液、2%~5%的明胶溶液与质量分数2%~4%的羟乙基纤维素溶液按照体积比8:(2~6):(3~7):(3~6)的比例混合均匀;
c、制备干膜敷料:将干细胞分泌组与壳聚糖基础凝胶按照体积比为1: (2~6)的比例混合均匀,灌装到模具中,在温度25~42℃、湿度10~20%条件下干燥成膜,得到可常温保存使用的干膜敷料。
5.根据权利要求4所述促进糖尿病难愈性伤口快速愈合的干膜敷料的制备方法,其特征在于:步骤a中所述表面特异性标志蛋白的筛选标准为阳性蛋白CD90、CD44、CD73表达率均大于95%,阴性蛋白CD45表达率小于5%。
6.根据权利要求4所述促进糖尿病难愈性伤口快速愈合的干膜敷料的制备方法,其特征在于:步骤a中所述无血清培养基选用Mesenchy Stem Cells Basal Medium或者成人脂肪间充质干细胞无血清培养基。
7.根据权利要求4所述促进糖尿病难愈性伤口快速愈合的干膜敷料的制备方法,其特征在于:步骤a中所述无血清基础培养基选用无血清RPMI 1640培养基或无血清DMEM培养基,培养环境为37℃、体积分数5%CO2的细胞培养箱。
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