CN117357702A - 一种原位组织工程支架制备方法及其应用 - Google Patents
一种原位组织工程支架制备方法及其应用 Download PDFInfo
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- CN117357702A CN117357702A CN202311518657.2A CN202311518657A CN117357702A CN 117357702 A CN117357702 A CN 117357702A CN 202311518657 A CN202311518657 A CN 202311518657A CN 117357702 A CN117357702 A CN 117357702A
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- silk fibroin
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Abstract
本发明提供了一种原位组织工程支架制备方法及其应用,涉及医用生物材料技术领域。本发明提供的原位组织工程支架的制备方法,采用静电纺丝技术和超声破碎技术,制得的组织工程支架,立体结构强,具备适宜细胞浸润生长的较大孔径;负载TGF‑β3,可自行募集骨髓间质干细胞迁移至支架移植部位繁殖、分化,实现原位组织工程修复;以胶原包被修饰支架表面,有利于在支架细胞黏附、增殖同时增强支架的可塑性;负载溶菌酶,具有天然的抗菌性,有效降低创伤感染机率。该制备方法简单方便,制备得到的原位组织工程支架生物相容性好,能够用于骨或者软骨组织的修复。
Description
技术领域
本发明涉及医用生物材料技术领域,尤其是涉及一种原位组织工程支架制备方法及其应用。
背景技术
组织工程支架做为种子细胞的临时细胞外基质,是组织工程中关键性材料。理想的工程支架在材料上应具有良好的生物相容性,在结构上应具有完全连通的三维结构,合适的空袭大小,同时能够模拟细胞外基质募集种子细胞,诱导种子细胞迁移、黏附、繁殖、分化,为种子细胞提供适宜生长、繁殖的细胞微环境。原位组织工程同时要求支架具有种子细胞募集作用,避免外源细胞在由体外植入的过程中可能引发的污染及免疫排斥及细胞体外培养成本高等问题。组织工程中通过冷冻干燥法、发泡法和相分离/乳化法、3D打印等方式获取多孔支架材料,这些材料的结构具有一定的仿生性,但普遍存在拉伸力弱,降解速度过快、载药突释等问题,达不到长期修复效应。静电纺丝技术制备的生物支架材料比普通大纤维支架具有更好的拉伸力,纤维直径小、孔隙率高、比表面积大等优点,可以最大程度上模拟细胞外基质,供细胞粘附和增殖。但单纯的静电纺丝制备的纳米膜孔径小,细胞仅能在表面生长,深入不到细胞内部,不利于细胞呈立体式生长。
有鉴于此,特提出本发明。
发明内容
本发明的第一目的在于提供一种原位组织工程支架的制备方法,以克服单纯静电纺丝技术制备纳米纤维支架孔径小,纤维堆叠,细胞不易爬入支架内部生长的问题。
本发明的第二目的在于提供一种原位组织工程支架,其携带生物活性因子,能自主募集种子细胞,以克服种子细胞体外培养成本高、易污染、易排斥等问题。
本发明的第三目的在于提供上述原位组织工程支架在制备软骨或骨组织修复产品中的应用。
为了实现以上目的,提出以下技术方案:
第一方面,本发明提供了一种原位组织工程支架的制备方法,包括以下步骤:
a.将丝素蛋白电纺液进行电纺丝,获得丝素蛋白纳米纤维膜,然后将丝素蛋白纳米纤维膜在交联剂溶液中依次进行交联和超声破碎,获得丝素蛋白纳米纤维悬浮液,再经干燥处理后获得丝素蛋白纳米短纤维;
b.将含有TGF-β3和溶菌酶的胶原溶液包裹a步骤获得的丝素蛋白纳米短纤维,经塑形和干燥后制备得到原位组织工程支架。
作为进一步技术方案,所述丝素蛋白电纺液中丝素蛋白的浓度为0.2-0.25g/mL;
和/或,所述丝素蛋白电纺液还包括助仿剂聚氧化乙烯和甲酸;所述丝素蛋白电纺液中聚氧化乙烯的浓度为0.30-0.40g/mL;所述丝素蛋白电纺液中甲酸的体积浓度为95%-98%。
作为进一步技术方案,所述电纺丝的参数包括:喷丝头直径为0.5-0.6mm、压力为12-15kv、流速为0.2-0.5mL/h、接收距离为10-12cm。
作为进一步技术方案,所述交联剂溶液为体积百分比为70%-80%的乙醇溶液;
和/或,所述交联的时间为25-30min。
作为进一步技术方案,所述超声的功率为600-700W;
所述超声的时间为25-30min。
作为进一步技术方案,所述干燥为冷冻干燥。
作为进一步技术方案,所述胶原溶液中,胶原的浓度为0.4-0.6mg/mL;
和/或,所述胶原溶液中,TGF-β3的浓度为5-15μg/mL;
和/或,所述胶原溶液中,溶菌酶的浓度为5-15mg/mL;
和/或,所述胶原溶液的pH为7.2-7.4。
作为进一步技术方案,所述丝素蛋白纳米短纤维与胶原溶液的料液比为5-8mg/mL。
第二方面,本发明提供了一种原位组织工程支架,采用所述的制备方法制备得到。
第三方面,本发明提供了上述原位组织工程支架在制备软骨或骨组织修复产品中的应用。
与现有技术相比,本发明具有如下有益效果:
(1)采用静电纺丝技术和超声破碎技术,制得的原位组织工程支架,立体结构强,具备适宜细胞浸润生长的较大孔径。
(2)负载TGF-β3,可自行募集骨髓间质干细胞迁移至支架移植部位繁殖、分化,实现原位组织工程修复。
(3)以胶原包被修饰支架表面,有利于在支架细胞黏附、增殖同时增强支架的可塑性。
(4)负载溶菌酶,具有天然的抗菌性,有效降低创伤感染机率。
(5)材料均采用天然生物材料,生物相容性好,支架无毒,可负载多种活性物质,不局限于原位组织工程支架也可做为药物载体或敷料、敷贴,仿生细胞外基质,长时间持续缓释负载药物或活性分子,达到持续修复、治疗的效果。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1原位组织工程支架的外观;
图2为实施例1原位组织工程支架的扫描电镜图;
图3为支架的抑菌性结果;
图4为支架的生物相容性检测结果;
图5为细胞划痕实验结果;
图6为Transwell小室实验结果;
图7为支架对兔软骨修复结果。
具体实施方式
下面将结合实施方式和实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施方式和实施例仅用于说明本发明,而不应视为限制本发明的范围。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。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。
第一方面,本发明提供了一种原位组织工程支架的制备方法,包括以下步骤:
a.将丝素蛋白电纺液进行电纺丝,获得丝素蛋白纳米纤维膜,然后将丝素蛋白纳米纤维膜在交联剂溶液中依次进行交联和超声破碎,获得丝素蛋白纳米纤维悬浮液,再经干燥处理后获得丝素蛋白纳米短纤维;
b.将含有TGF-β3和溶菌酶的胶原溶液包裹a步骤获得的丝素蛋白纳米短纤维,经塑形和干燥后制备得到原位组织工程支架。
TGF-β3对骨髓间质干细胞具有良好的诱导作用和趋向作用,能够诱导骨髓间质干细胞分泌多种旁分泌生长因子促进其分化和肉芽组织形成、血管生成、细胞外基质生成的反应细胞,可作为原位组织工程重要的趋化因子。本发明以TGF-β3为原料制备原位组织工程支架。
胶原是结缔组织和皮肤的主要结构成分,其粘性高、生物相容性好、可降解,本发明以胶原为原料制备敷料,用于纤连蛋白和溶菌酶的负载。
丝素蛋白具有良好的亲水性、吸水能力、生物降解性、生物相容性、细胞黏附能力和优异的机械性能,本发明以丝素蛋白作为敷料的骨架。
本发明提供的原位组织工程支架的制备方法,采用静电纺丝技术和超声破碎技术,制得的原位组织工程支架,立体结构强,具备适宜细胞浸润生长的较大孔径;负载TGF-β3,可自行募集骨髓间质干细胞迁移至支架移植部位繁殖、分化,实现原位组织工程修复;以胶原包被修饰支架表面,有利于在支架细胞黏附、增殖同时增强支架的可塑性;负载溶菌酶,具有天然的抗菌性,有效降低创伤感染机率。该制备方法简单方便,制备得到的原位组织工程支架生物相容性好,能够用于骨或者软骨组织的修复。
在一些可选的实施方式中,所述丝素蛋白电纺液中丝素蛋白的浓度例如可以为,但不限于0.2g/mL、0.21g/mL、0.22g/mL、0.23g/mL、0.24g/mL或0.25g/mL。
在一些可选的实施方式中,所述丝素蛋白电纺液还包括助仿剂聚氧化乙烯和甲酸;
所述丝素蛋白电纺液中聚氧化乙烯的浓度例如可以为,但不限于0.30-0.40g/mL、0.32g/mL、0.34g/mL、0.36g/mL、0.38g/mL或0.40g/mL;
所述丝素蛋白电纺液中甲酸的体积浓度例如可以为,但不限于95%、96%、97%或98%。
在一些可选的实施方式中,所述电纺丝的参数包括:喷丝头直径为0.5-0.6mm、压力为12-15kv、流速为0.2-0.5mL/h、接收距离为10-12cm。
在一些可选的实施方式中,所述交联剂溶液为体积百分比为70%-80%的乙醇溶液。
本发明中以乙醇为交联剂对丝素蛋白纳米纤维膜进行交联。
在一些可选的实施方式中,所述交联的时间例如可以为,但不限于25min、26min、27min、28min、29min或30min。
在一些可选的实施方式中,所述超声的功率例如可以为,但不限于600W、620W、640W、660W、680W或700W;
所述超声的时间例如可以为,但不限于25min、26min、27min、28min、29min或30min。
本发明中,通过超声以破碎丝素蛋白纳米纤维膜,进而获得丝素蛋白纳米短纤维。
在一些可选的实施方式中,所述干燥为冷冻干燥。
在一些可选的实施方式中,所述胶原溶液中,胶原的浓度为0.4mg/mL、0.5mg/mL或0.6mg/mL。
在一些可选的实施方式中,所述胶原溶液中,TGF-β3的浓度例如可以为,但不限于5μg/mL、7μg/mL、9μg/mL、11μg/mL、13μg/mL或15μg/mL。
在一些可选的实施方式中,所述胶原溶液中,溶菌酶的浓度例如可以为,但不限于5mg/mL、7mg/mL、9mg/mL、11mg/mL、13mg/mL或15mg/mL。
在一些可选的实施方式中,所述胶原溶液的pH为7.2-7.4。
通过对胶原溶液组分的进一步优化和调整,使得制备得到的原位组织工程支架的效果更好。
在一些可选的实施方式中,所述丝素蛋白纳米短纤维与胶原溶液的料液比例如可以为,但不限于5mg/mL、6mg/mL、7mg/mL或8mg/mL。
通过对胶原溶液和丝素蛋白纳米短纤维配比的进一步优化和调整,进一步提高原位组织工程支架的组织修复效果。
第二方面,本发明提供了一种原位组织工程支架,采用所述的制备方法制备得到。
本发明提供的原位组织工程支架,立体结构强,生物相容性好,安全性好,具备适宜细胞浸润生长的较大孔径,能够长时间持续缓释负载药物或活性分子,达到持续募集种子细胞增殖分化、修复、治疗的效果。
第三方面,本发明提供了上述原位组织工程支架在制备软骨或骨组织修复产品中的应用。
本发明提供的原位组织工程支架,立体结构强,生物相容性好,安全性好,能够用于软骨或骨组织修复。
下面通过具体的实施例和对比例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细地说明之用,而不应理解为用于以任何形式限制本发明。
需要说明的是,以下实施例及试验例中,“SF”是指丝素蛋白纳米短纤维,“COL”是指胶原,“FN”是指纤连蛋白,“LYZ”是指溶菌酶。
实施例1
一种原位组织工程支架(SF(短纤维)-COL-LYZ-TGF3),制备方法如下:
首先,提取鼠尾胶原:
抽取大鼠尾腱置于含双抗的PBS溶液中清洗至洗脱液清澈。将洁净大鼠尾腱:0.1%醋酸溶液浴比3∶400于4℃浸泡48h后,7000rpm离心30min,后取上清液进行BCA法检测胶原浓度,上清液-20℃保存备用。
其次提取丝素蛋白:
将洁净蚕茧:碳酸钠溶液浴比1∶200煮沸30min后,去离子水清洗、65℃干燥2h得到脱胶蚕丝。按脱胶蚕丝:三元溶液浴比1:50在75℃水浴中溶解24h后离心,得到的上清液即丝素蛋白溶液。将离心后的丝素蛋白溶液转移至透析袋中,超纯水持续透析4d,后置于4℃冰箱冷藏透析3d。冷冻干燥3d后得到白色絮状丝素蛋白即丝素蛋白。
然后,制备丝素蛋白纳米短纤维:
配制电纺液,电纺液中含有0.2g/mL的丝素蛋白、0.30g/mL的聚氧化乙烯(PEO)和体积浓度为98%的甲酸,将电纺液装入直径为0.55mm的注射器,在电压12-15kv,流速0.2-0.5ml/h,接收距离11cm参数下电纺,获得丝素蛋白纳米纤维膜。将上述丝素蛋白纳米纤维膜在75%乙醇中交联30min,在功率650w,冰浴超声破碎30min条件下制成纳米纤维悬浮液,7000rpm/min,离心15min除去乙醇,冷冻干燥12h以上获得丝素蛋白冻干纳米短纤维。
接着制备负载TGF-β3的原位组织工程支架:
用0.1%NaOH将冰浴上的胶原溶液pH调节在7.2-7.4范围内,胶原浓度调节为0.5mg/ml,得到胶原溶液。将10μg/ml TGF-β3和10mg/ml溶菌酶负载于上述胶原溶液中,后以此胶原溶液混匀包裹丝素蛋白纳米短纤维(丝素蛋白纳米短纤维与胶原溶液的料液比为6mg/mL),后加入合适的细胞孔板塑形室温放置下形成凝胶,后-80℃过夜冷冻,进行过夜冷冻干燥后构建出负载TGF-β3纳米短纤维原位组织工程支架,如图1所示。
对比例1
一种支架(SF(短纤维)-COL),与实施例1的区别在于,未负载溶菌酶和TGF-β3。
对比例2
一种支架(SF-COL纳米膜),与对比例1的区别在于,电纺获得丝素蛋白纳米纤维膜后,未进行破碎直接进行胶原包覆。
对比例3
一种支架(SF(短纤维)-COL-LYZ),与对比例1的区别在于,未负载TGF-β3。
试验例1
对上述制备的支架材料进行各项性能的测试,具体过程如下:
(1)SF-COL-LYZ-TGF3原位组织工程支架形貌表征
取一小块制得的SF-COL-LYZ-TGF3支架贴于导电胶并固定在样品台上,在金箔中喷金约10s,于扫描电压15.0kV条件下扫描电镜观察其形貌。结果如图2所示,SEM观察,具有大孔结构,孔径大小适中且孔道相互连通,孔隙较多。
(2)支架抑菌能力的测试
分别将已分离纯化的金黄色葡萄球菌敏感株、耐药标准株、耐药临床株在M-H培养基中扩增后用0.5麦氏比浊管校正。校正后立即用无菌棉拭子将三种菌液分别均匀涂布于M-H琼脂表面,涂布三次,每次平板旋转60°,最后棉拭子沿平板内缘涂抹一周。盖上盖后干燥5min。分别裁剪、塑型SF-COL纳米膜、SF(短纤维)-COL、SF(短纤维)-COL-LYZ、SF(短纤维)-COL-LYZ-TGF3四组材料,直径为5mm圆片,紫外灭菌后,按一定间隔贴于上述M-H琼脂表面,并轻轻按压贴紧。而后放入37℃恒温生化培养箱中培养18h后拍照记录,结果如图3所示。含有溶菌酶的SF(短纤维)-COL-LYZ、SF(短纤维)-COL-LYZ-TGF3两组支架均出现抑菌圈,SF(短纤维)-COL-LYZ-TGF3更具有杀菌功效。
(3)支架生物相容性
将兔骨髓间充质干细胞传至P2代并调整细胞浓度为4×105。按材料成分不同,设置空白组(Control)、SF-COL纳米膜、SF(短纤维)-COL、SF(短纤维)-COL-LYZ-TGF3、将150μl细胞悬液接种至不同组分组别支架上,置37℃、5%CO2的培养箱。不含支架兔骨髓间充质干细胞为空白对照组。于第3、5、7、9天进行CCK-8实验,结果如图4所示。结果显示,对照组细胞生长增殖曲线呈“S”形,前7天细胞数较多,但增殖速度弱于其他组;SF(短纤维)-COL组细胞数高于SF-COL纳米膜且在第9天高于空白组,表明细胞在SF(短纤维)-COL组三维立体结构较SF-COL纳米膜二维平面具有更大的增殖潜力;与SF(短纤维)-COL-LYZ-TGF3支架共培养组兔骨髓间充质干细胞在连续培养9d后,较其他两处理组,生长增殖呈明显上升趋势,未观察到接触抑制效应,而空白组出现大量细胞崩解,接触抑制较为明显。实验结果表明制备的SF(短纤维)-COL-LYZ-TGF3支架能够为种子细胞提供良好的生长微环境。
(4)支架对骨髓间质干细胞诱导迁移
细胞划痕实验
吸取对数生长期兔骨髓间充质干细胞,接种于6孔板,细胞密度为4×105,置37℃、5%CO2的培养箱培养。待细胞融合密度达到70%~80%时,用200ul的移液器尖头。
沿直尺垂直于孔底划痕,PBS冲洗2~3遍,实验组SF(短纤维)-COL-LYZ-TGF3覆盖细胞表面,加入2ml无血清F12培养液,于37℃、5%CO2恒温培养。于划痕后0h、24h、48h进行观察,由图5结果可见,在24h内SF(短纤维)-COL-LYZ-TGF3划痕宽度内充满了细胞,而空白对照组划痕宽度孔隙较大,边缘仅少量细胞侵入。细胞划痕实验结果表明负载SF(短纤维)-COL-LYZ-TGF3支架对骨髓间质干细胞有诱导、迁移作用。
Transwell小室实验
在Transwell小室下室加入SF(短纤维)-COL-LYZ-TGF3,在上室加入100μl细胞密度为1*104的兔骨髓间充质干细胞悬液,置37℃、5%CO2培养箱中培养5d后,结晶紫染色,3%醋酸脱色,显微镜下观察细胞数目并拍照,后用100ul 0.025%胰酶洗脱小室上细胞并用酶标仪分别测定两组细胞洗脱液在570nm处吸光度。Transwell小室实验结果(图6)显示实验组穿过上室的骨髓间质干细胞数量高于对照组。此结果说明负载TGF3生长因子的SF(短纤维)-COL-LYZ-TGF3可诱导骨髓间充质干细胞纵向迁移,具有募集骨髓间充质干细胞作用。
(5)兔软骨修复实验
将兔麻醉备皮消毒后,随机取左或右膝胫骨上端内侧纵切口,长约3cm,逐层切开皮肤和皮下组织,直至关节腔,切断翼状皱襞和脂肪垫,屈膝暴露股骨关节软骨面,电钻造成两个膝关节胫骨近端关节面软骨直径4mm、深3mm的缺损。生理盐水冲洗缺损部位,随机在某一缺损处植入SF(短纤维)-COL-LYZ-TGF3,另一处缺损不做处理作为空白对照组。再次用无菌生理盐水冲洗,清除血渍,逐层缝合,碘伏内外全面消毒后缝合伤口,用无菌医用纱布包扎,避免伤口感染。4周后再次打开关节腔,取出兔膝关节,结果显示(图7),正常软骨面光滑,有弹性;空白对照组软骨未再生,色泽灰暗,软骨下骨外露明显;SF(短纤维)-COL-LYZ-TGF3治疗组软骨欠光滑,有光泽,未见软骨下骨外露现象,表明SF(短纤维)-COL-LYZ-TGF3对软骨缺损具有良好的修复效果。
综上所述,本发明实施例能够制得负载TGF-β3和溶菌酶并包裹鼠尾胶原凝胶的负载TGF-β3的原位组织工程支架,且制得的支架有以下优点:
(1)采用静电纺丝技术和超声破碎技术,制得的原位组织工程支架,立体结构强,具备适宜细胞浸润生长的较大孔径。
(2)负载TGF-β3,可自行募集骨髓间质干细胞迁移至支架移植部位繁殖、分化,实现原位组织工程修复。
(3)以胶原包被修饰支架表面,有利于在支架细胞黏附、增殖同时增强支架的可塑性。
(4)负载溶菌酶,具有天然的抗菌性,有效降低创伤感染机率。
(5)材料均采用天然生物材料,生物相容性好,支架无毒,可负载多种活性物质,不局限于原位组织工程支架也可做为药物载体或敷料、敷贴,仿生细胞外基质,长时间持续缓释负载药物或活性分子,达到持续修复、治疗的效果。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
1.一种原位组织工程支架的制备方法,其特征在于,包括以下步骤:
a.将丝素蛋白电纺液进行电纺丝,获得丝素蛋白纳米纤维膜,然后将丝素蛋白纳米纤维膜在交联剂溶液中依次进行交联和超声破碎,获得丝素蛋白纳米纤维悬浮液,再经干燥处理后获得丝素蛋白纳米短纤维;
b.将含有TGF-β3和溶菌酶的胶原溶液包裹a步骤获得的丝素蛋白纳米短纤维,经塑形和干燥后制备得到原位组织工程支架。
2.根据权利要求1所述的制备方法,其特征在于,所述丝素蛋白电纺液中丝素蛋白的浓度为0.2-0.25g/mL;
和/或,所述丝素蛋白电纺液还包括助仿剂聚氧化乙烯和甲酸;所述丝素蛋白电纺液中聚氧化乙烯的浓度为0.30-0.40g/mL;所述丝素蛋白电纺液中甲酸的体积浓度为95%-98%。
3.根据权利要求1所述的制备方法,其特征在于,所述电纺丝的参数包括:喷丝头直径为0.5-0.6mm、压力为12-15kv、流速为0.2-0.5mL/h、接收距离为10-12cm。
4.根据权利要求1所述的制备方法,其特征在于,所述交联剂溶液为体积百分比为70%-80%的乙醇溶液;
和/或,所述交联的时间为25-30min。
5.根据权利要求1所述的制备方法,其特征在于,所述超声的功率为600-700W;
所述超声的时间为25-30min。
6.根据权利要求1所述的制备方法,其特征在于,所述干燥为冷冻干燥。
7.根据权利要求1所述的制备方法,其特征在于,所述胶原溶液中,胶原的浓度为0.4-0.6mg/mL;
和/或,所述胶原溶液中,TGF-β3的浓度为5-15μg/mL;
和/或,所述胶原溶液中,溶菌酶的浓度为5-15mg/mL;
和/或,所述胶原溶液的pH为7.2-7.4。
8.根据权利要求7所述的制备方法,其特征在于,所述丝素蛋白纳米短纤维与胶原溶液的料液比为5-8mg/mL。
9.一种原位组织工程支架,其特征在于,采用权利要求1-8任一项所述的制备方法制备得到。
10.权利要求9所述的原位组织工程支架在制备软骨或骨组织修复产品中的应用。
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