CN113713176B - 一种水凝胶及其制备方法与应用 - Google Patents
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Abstract
本发明提供了一种水凝胶及其制备方法与应用,属于生物组织工程技术领域。本发明水凝胶,包括凝胶基质,以及干细胞和外泌体,所述干细胞经缺氧处理,所述外泌体溶于含有生长因子的培养基进行电穿孔处理。本发明提供的水凝胶能够显著提升植入细胞的存活能力,为加快损伤修复提供了可能,外泌体作为无生命的囊泡,为细胞提供了存活需要的营养和存活信号,同时其被细胞内吞后其空间也为细胞生长、迁移等提供了可能。本发明水凝胶可应用于制备修复骨缺损的产品中,在老年、糖尿病和极速修复中有广泛应用前途。
Description
技术领域
本发明属于生物组织工程技术领域,尤其涉及一种水凝胶及其制备方法与应用。
背景技术
由创伤、感染、肿瘤或先天性遗传疾病引起的骨损伤是一种常见的骨科疾病。骨组织本身具有一定的自动修复和再生的能力,但是,大面积的骨缺损需要通过外部干预的方法进行治疗,其再生修复仍然是临床医学面临的一项重大挑战。颌面部缺损是一种常见病,造成颌面缺损的主要原因有先天性和后天性因素两方面:主要有唇腭裂、先天性发育畸形和外伤、颌骨肿瘤或其他疾病所致。对人体的主要影响为咀嚼困难,美观问题和语言功能的障碍。颌面缺损是包括骨与软组织在内颌面部及其器官的缺损缺失,主要包括颌骨、面部软组织、面部器官,这些缺损有些可采取手术整形的方法,但许多情况下还是用口腔修复的方法。修复颌面部骨缺损的主要手段是修补成形术,修补巨大和复杂颌面部骨缺损仍然是颌面外科的挑战。自体骨移植材料受到供体的限制很难得到与骨缺损部位相匹配的外形,因此针对特定的骨缺损部位制备出个性化的骨支架材料具有重要意义。
支架材料是骨组织工程的关键要素之一,寻找一种有良好生物相容性、有较好的促进成骨能力并且能生物降解的支架材料是研究的热点与难点。水凝胶类材料具备类细胞外基质的仿生特性,高度的三维水化网络结构,有利于组织再生和创伤愈合过程中细胞的迁移和生长。在水凝胶中添加细胞能够更好地促进组织损伤修复,但是在水凝胶中植入细胞后,存在细胞存活率低的缺陷,影响水凝胶类材料的使用效果。
发明内容
有鉴于此,本发明的目的在于提供一种能够显著增强植入细胞存活率和骨再生潜能的水凝胶。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种水凝胶,包括凝胶基质,以及干细胞和外泌体,所述干细胞经缺氧处理,所述外泌体溶于含有生长因子的培养基进行电穿孔处理。
优选的,所述凝胶基质为甲基丙烯酸酐化明胶。
优选的,所述缺氧处理的低氧培养条件为1%O2,5%CO2,94%N2。
优选的,所述培养基包括:5-10×DMEM、150-250ng/ml PDGF、150-250ng/ml bFGF和5-10×B27。
优选的,所述电穿孔的电压为600-800V,电容为100-200mF,电击杯的的电极间距为4mm,次数为2-4次。
优选的,所述干细胞和外泌体的用量比为1:104-105。
本发明还提供了一种上述水凝胶的制备方法,包括如下步骤:将所述干细胞和所述外泌体混合于培养基中,所得混合液再与所述凝胶基质混合,添加光引发剂,诱导成胶。
优选的,所述培养基为DMEM培养基。
优选的,所述混合液与所述凝胶基质的体积比为1:3-2:1。
本发明还提供了一种上述水凝胶在制备修复骨缺损产品中的应用。
本发明的有益效果:
在本发明水凝胶中,外泌体作为无生命的囊泡,对其进行工程化处理后,装载细胞存活所需要的高浓度营养成分,为细胞提供存活需要的营养和存活信号,同时外泌体被细胞内吞后其空间也为细胞生长、迁移等提供了可能。干细胞经由缺氧处理,具有更强的耐缺氧能力。本发明设计的水凝胶能够显著提升植入细胞的存活能力和骨再生潜能,为加快骨损伤修复提供了可能。在老年、糖尿病和极速修复中有广泛应用前途。本发明水凝胶可以与3D生物打印技术(bio-3DP)相结合,通过bio-3DP的方式打印理想复杂三维结构的水凝胶支架,为高强度水凝胶修复极限骨缺损提供可能性。
附图说明
图1为不同处理水凝胶中相对存活细胞数。
具体实施方式
本发明提供了一种水凝胶,包括凝胶基质,以及干细胞和外泌体,所述干细胞经缺氧处理,所述外泌体溶于含有生长因子的培养基进行电穿孔处理。
在本发明中,所述凝胶基质优选为甲基丙烯酸酐化明胶(GelMA),GelMA是一种光敏性生物水凝胶材料,具有优异的生物相容性,可由紫外光或可见光激发固化反应,形成适于细胞生长与分化且有一定强度的三维结构。
本发明对于干细胞的种类没有特殊限定,只要是能够参与骨再生的干细胞种类均可。在本发明具体实施例中,使用的是间充质干细胞。在本发明中,所述缺氧处理优选为将干细胞在低糖培养基中进行低氧培养,其中低氧培养的条件优选为1%O2,5%CO2,94%N2,低糖培养指的是采用比普通干细胞培养基含糖量低的培养基进行培养,在本发明具体实施例中,使用的是DMEM培养基,其含糖量为5.6mmol/L。在本发明中,所述缺氧处理的时间优选为5-9天,更优选为6-8天。本发明对干细胞进行缺氧处理,使干细胞具有更强的耐缺氧能力,使其更加适应水凝胶中的微环境。
本发明对于外泌体的来源没有特殊要求,采用本领域公知的外泌体获取途径获取得到均可,在本发明具体实施例中,收集的是血浆外泌体。本发明对于生长因子的种类没有特殊限定,只要是能使外泌体富含营养以及细胞生长、存活所需要的关键生长因子均可,在本发明中,所述培养基优选的包括5-10×DMEM、150-250ng/ml PDGF、150-250ng/ml bFGF和5-10×B27,更优选的包括6-9×DMEM、180-220ng/ml PDGF、180-220ng/ml bFGF和6-9×B27,其中B27指的是一种抗氧化、无血清培养基添加成分,是神经元培养基常用的添加物。在本发明中,对溶于含有生长因子培养基的外泌体进行电穿孔处理时,所述电穿孔的电压优选为600-800V,更优选为650-750V,所述电穿孔的电容优选为100-200mF,更优选为130-170mF,所述电穿孔电击杯的的电极间距优选为4mm,所述电穿孔的次数优选为2-4次,更优选为3次。外泌体经本发明方法进行工程化处理后,使外泌体富含营养和细胞生长和存活所需要的关键生长因子。
在本发明水凝胶中,所述干细胞和外泌体的用量比优选为1:104-105。
本发明还提供了一种上述水凝胶的制备方法,包括如下步骤:将所述干细胞和所述外泌体混合于培养基中,所得混合液再与所述凝胶基质混合,添加光引发剂,诱导成胶。
在本发明制备方法中,用于所述干细胞和所述外泌体混合的培养基优选为DMEM培养基,本发明对于DMEM的具体配方没有特殊限定,采用本领域常规DMEM培养基配方均可。在本发明中,所述混合液与所述凝胶基质的体积比优选为1:3-2:1,更优选为1:1。本发明对于光引发剂的种类没有特殊限定,采用本领域常规引发剂种类均可,在本发明具体实施例中,所述光引发剂选用的是LAP光引发剂(苯基-2,4,6-三甲基苯甲酰基亚磷酸锂),LAP光引发剂交联更迅速,对细胞损害更小,所述LAP光引发剂的添加量优选为0.5%(w/v)。本发明对于诱导成胶的方式没有特殊限定,在本发明具体实施例中,选用蓝光诱导成胶,诱导成胶的波长优选为400nm,诱导成胶的时间优选为10S。
本发明还提供了一种上述水凝胶在制备修复骨缺损产品中的应用。
本发明对于产品的种类没有特殊限定,包括医疗药品等。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
将间充质干细胞(MSC)置于商品化的低糖DMEM培养基中,维持低氧条件1%O2,5%CO2,94%N2培养7天,使其更加适应在水凝胶中的微环境,得预处理后MSC。
收集血浆外泌体,溶于10×DMEM+200ng/ml PDGF+200ng/mLbFGF+10×B27培养基中,在电压为700V,电容为150mF,电击杯的电极间距为4mm的条件下电穿孔处理3次,使外泌体富含营养和细胞生长、存活所需要的关键生长因子,得工程化处理后外泌体。
将上述预处理后MSC和工程化处理后外泌体按照MSC细胞数量:外泌体数量为1:104的比例混合于DMEM培养基中,得混合液,将混合液与浓度为20%的gelMA(选苏州智能制造研究院,GM-60型号)等体积混合,添加0.5%(w/v)LAP光引发剂,400nm波长蓝光诱导10S成胶。
实施例2
将造血干细胞(后续分化成破骨细胞)置于含糖量为4.8mmol/L的DMEM培养基中,维持低氧条件1%O2,5%CO2,94%N2培养6天,使其更加适应在水凝胶中的微环境,得预处理后造血干细胞。
收集血浆外泌体,溶于5×DMEM+150ng/ml PDGF+150ng/mLbFGF+5×B27培养基中,在电压为600V,电容为100mF,电击杯的电极间距为4mm的条件下电穿孔处理2次,使外泌体富含营养和细胞生长、存活所需要的关键生长因子,得工程化处理后外泌体。
将上述预处理后造血干细胞和工程化处理后外泌体按照造血干细胞数量:外泌体数量为1:105的比例混合于DMEM培养基中,得混合液,将混合液与浓度为20%的gelMA(选苏州智能制造研究院,GM-60型号)按照体积比为1:3的比例混合,添加0.3%(w/v)LAP光引发剂,400nm波长蓝光诱导10S成胶。
实施例3
将MSC置于含糖量为4.0mmol/L的DMEM培养基中,维持低氧条件1%O2,5%CO2,94%N2培养9天,使其更加适应在水凝胶中的微环境,得预处理后造血干细胞。
收集血浆外泌体,溶于9×DMEM+250ng/ml PDGF+250ng/mLbFGF+9×B27培养基中,在电压为800V,电容为200mF,电击杯的电极间距为4mm的条件下电穿孔处理4次,使外泌体富含营养和细胞生长、存活所需要的关键生长因子,得工程化处理后外泌体。
将上述预处理后MSC和工程化处理后外泌体按照MSC细胞数量:外泌体数量为1:105的比例混合于DMEM培养基中,得混合液,将混合液与浓度为20%的gelMA(选苏州智能制造研究院,GM-60型号)按照体积比为2:1的比例等体积混合,添加0.4%(w/v)LAP光引发剂,400nm波长蓝光诱导10S成胶。
对比例1
与实施例1的区别在于不含有MSC和外泌体,其余均同实施例1,为无细胞组。
对比例2
与实施例1的区别在于将MSC置于常规DMEM培养基中常规培养,未进行低糖低氧处理,不含有外泌体,其余均同实施例1,为常氧细胞组。
对比例3
与实施例1的区别在于不含有外泌体,其余均同实施例1。记为缺氧预处理细胞组。
对比例4
与实施例1的区别在于外泌体未进行电穿孔处理,其余均同实施例1,为缺氧预处理细胞+对照外泌体组。
以实施例1为缺氧预处理细胞+工程化外泌体组,将实施例1、对比例1-4所得的凝胶分别加入1×PBS,细胞培养箱常规培养48h后,将水凝胶取出后,进行冰冻包埋,切片后,Hoechst染色,进行细胞计数。结果如图1所示。
由图1可以看出,对细胞进行缺氧处理和添加外泌体均能有效提高细胞的存活率,对外泌体进行工程化处理后,其提高存活率的效果更佳,本发明提供的水凝胶能够显著提升细胞存活率。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种水凝胶,其特征在于,包括凝胶基质,以及干细胞和外泌体,所述干细胞经缺氧处理,所述外泌体溶于含有生长因子的培养基进行电穿孔处理;
所述培养基包括:5-10×DMEM、150-250 ng/ml PDGF、150-250 ng/ml bFGF和5-10×B27;
所述缺氧处理的低氧培养条件为1%O2,5%CO2,94%N2;
所述电穿孔的电压为600-800V,电容为100-200mF,电击杯的电极间距为4mm,次数为2-4次;
所述干细胞和外泌体的数量比为1:104-105。
2.根据权利要求1所述的水凝胶,其特征在于,所述凝胶基质为甲基丙烯酸酐化明胶。
3.权利要求1~2任意一项所述水凝胶的制备方法,其特征在于,包括如下步骤:将所述干细胞和所述外泌体混合于培养基中,所得混合液再与所述凝胶基质混合,添加光引发剂,诱导成胶。
4.根据权利要求3所述的制备方法,其特征在于,所述混合液与所述凝胶基质的体积比为1:3-2:1。
5.权利要求1-2任意一项所述水凝胶在制备修复骨缺损产品中的应用。
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