纳米纤维凝胶材料IKVAV多肽的自组装及其与骨髓基质干细胞的相容性研究

研究纳米纤维凝胶材料IKVAV多肽的自组装及其与骨髓基质干细胞的相容性，为其应用于神经组织工程提供实验依据。合成IKVAV多肽两亲性分子，进行自组装，用透射电镜检测。将IKVAV多肽纳米纤维凝胶与BMSCs复合培养，倒置显微镜下观察细胞生长情况，Calcein—AM／PI染色计数活细胞比例，检测IKVAV多肽对BMSCs增殖和粘附的影响。IKVAV多肽可成功自组装成为纳米纤维凝胶，其与BMSCs复合培养细胞生长良好，活细胞数达90％以上，IKVAV多肽对BMSCs增殖没有影响，并可促进BMSCs的粘附。IKVAV多肽可自组装形成纳米纤维凝胶，并且与BMSCs有着良好的生物相容性，是一种很有前景的神经组织工程支架材料。     

自组装纳米纤维凝胶培养基中神经干细胞的增殖与分化

背景：异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸具有良好的生物活性,在特定条件下可自组装成含IKVAV纳米纤维凝胶支架,是一种天然的脊髓基质仿生材料。 目的：进一步观察体外自组装异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸纳米纤维凝胶对骨髓源性神经干细胞增殖及向神经元分化的影响。 方法：取第2代新生SD大鼠骨髓源性神经干细胞与自组装异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸纳米纤维凝胶共培养作为实验组,设置神经干细胞单独培养为对照组、单独自组装异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸纳米纤维凝胶为空白对照组,CCK-8法及流式细胞仪检测培养1,3,5,7,10, 14 d的细胞增殖与神经干细胞向神经元分化的比例;MTT法检测自组装异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸纳米纤维凝胶对第2代新生SD大鼠骨髓源性神经干细胞的毒性。 结果与结论：实验组不同时间点细胞增殖率及向神经元分化的细胞比例均高于对照组(P < 0.05),两组细胞增殖均于第7天达峰值。MTT法检测结果显示自组装异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸纳米纤维凝胶无细胞毒性。表明异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸纳米纤维凝胶有较好的生物活性,可促进神经干细胞的增殖,提高神经干细胞向神经元分化的比例。     

体外自组装IKVAV纳米纤维凝胶在二维体系中对神经祖细胞分化的影响

背景：大量实验证明体外自组装的纳米支架材料可以促进神经祖细胞增殖与分化。 目的：观察体外自组装含IKVAV纳米纤维凝胶在二维体系中对神经祖细胞分化的影响。 方法：培养SD大鼠乳鼠神经祖细胞并用免疫荧光方法鉴定。在DMEM/F12触发下,含IKVAV的两亲性多肽溶液形成三维多孔凝胶,于透射电镜下观察其结构。将神经祖细胞分别接种到1%含IKVAV的两亲性多肽溶液及0.1 g/L多聚赖氨酸包被的盖玻片上,培养1,3,7 d后采用神经丝蛋白、胶质纤维酸性蛋白检测其分化情况。 结果与结论：培养出巢蛋白阳性细胞,并且能分化为神经丝蛋白阳性的神经元与胶质纤维酸性蛋白阳性的胶质细胞,证实为神经祖细胞;含IKVAV的两亲性多肽溶液自组形成凝胶,并在透射电镜下显示为纳米纤维,直径为7.0~8.0 nm,长度为100~1 500 nm; IKVAV的两亲性多肽溶液组向神经元分化得能力明显优于多聚赖氨酸组。说明体外自组装IKVAV纳米纤维凝胶在二维培养体系中对神经祖细胞分化有一定的促进作用。     

两种新型功能化自组装多肽水凝胶的制备和体外研究

目的研究自组装多肽水凝胶的表征及其生物相容性、促轴突再生的作用。方法本文制备两种分别模拟神经生长因子(NGF)和脑源性神经营养因子(BDNF)的功能化自组装多肽,通过圆二色谱分析、扫描电镜和原子力显微镜观察其表征,并将其用于培养雪旺细胞和PC12细胞。结果扫描电镜结果表明这种纳米纤维水凝胶具有类似细胞外基质的三维网络结构,圆二色谱分析和原子力显微镜结果显示该水凝胶具有典型的β折叠的色谱。体外结果表明雪旺细胞能很好地在多肽水凝胶中生长、铺展,同时PC12细胞能在多肽水凝胶中长出不同长度的轴突。结论自组装多肽水凝胶具有类似细胞外基质的纳米纤维结构,与神经类细胞的生物相容性良好,并且能够促进轴突生长。     

骨髓基质干细胞与功能化自组装多肽的生物相容性研究*

目的检测骨髓基质干细胞(BMSCs)在功能化自组装多肽水凝胶中分化及生物相容性。方法自组装多肽RADA16与多肽RADA16-IKVAV委托上海吉尔生物科技公司合成,将两者混合制备功能化自组装多肽水凝胶,用原子力显微镜(AFM)观察其形态学特征。体外分离、扩增、纯化鼠BMSCs并流式分析其表面抗原标志,将BMSCs分别接种到接种到RADA16自组装多肽水凝胶(对照组)与功能化自组装多肽水凝胶(实验组)表面。激光扫描共聚焦显微镜(LSCM)观察细胞迁移情况。应用Nestin、MAP2、GFAP免疫荧光染色,检测骨髓基质干细胞分化情况。结果 AFM显示功能化自组装多肽水凝胶由纳米纤维组成,其直径为7.0~8.0 nm。分离的BMSCs细胞表面标志CD90、CD44、CD34和CD45的阳性率分别为93.71%、92.5%、1.1%、2.18%。共培养后实验组中BMSCs的增殖及迁移能力均明显高于对照组(0.05);实验组中MAP2阳性细胞百分率较对照组显著提高(0.05),GFAP阳性细胞百分率较对照组显著降低(0.05)。结论功能化自组装多肽水凝胶具有良好的细胞生物相容性,从而为神经组织工程提供了一个有前景的生物材料。     

天然氨基酸生物材料的自组装与细胞相容性研究

天然氨基酸(NAA)是一种具有良好生物相容性的材料,对其进行功能化设计是近年来的热门研究领域。本文中将基于蚕丝蛋白的特征氨基酸序列(Gly-Ala)与离子互补多肽序列(Arg-Ala-Asp-Ala)混编,设计了多肽RAG-16。采用原子力显微镜、旋转流变仪、傅立叶变换红外光谱仪、倒置荧光显微镜等技术对多肽RAG-16的自组装结构、流变学性能以及细胞相容性等性质进行了表征。结果表明:多肽RAG-16在溶液中具有自组装特性,能够形成纳米级三维网络结构,所形成的水凝胶力学性能较佳。通过分析得知,多肽二级结构中Silk I结构比例增加是其力学性能增强的主要原因。荧光染色显示,绝大多数接种于多肽水凝胶中的MC3T3-E1细胞能够存活,且能在不同的三维平面上生长增殖,表明该材料具有良好的细胞相容性。综上所述,本实验中设计的NAA材料在生物医学领域有较大的应用潜力。     

自组装多肽纳米纤维水凝胶在细胞三维培养中的应用及特征

背景：自组装多肽类材料因其独特的设计及良好的生物相容性和可降解性在众多三维支架材料中脱颖而出。 目的：综述RADA类离子互补型自组装多肽支架材料的结构和功能化设计,从细胞三维培养方面探讨多肽类材料作为细胞载体材料在细胞治疗中的应用前景。 方法：由作者通过PubMed、Web of science数据库及CNKI数据库检索有关自组装多肽水凝胶的相关文献,检索词为“self-assembly peptide, tissue engineering;自组装多肽,组织工程”,检索文献量总计224篇,纳入包含多肽材料设计、功能化多肽材料、多肽材料用于细胞三维培养方面的研究,最终纳入48篇。 结果与结论：从物理结构角度讲,多肽材料可以在生理环境中自组装成具有纳米级纤维和较高孔隙率的水凝胶,最大程度上模拟细胞外基质的结构,保障细胞生存在一个真正的三维环境中。从生物功能角度讲,多肽材料可以根据不同需求复合特异性的生物活性短肽片断,赋予材料一定的细胞特异性,可以促进细胞的黏附、增殖或分化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

新型自组装水凝胶NBD/RADA16可促进前成骨细胞的分化

背景：RADA16是较成熟的自组装纳米短肽材料,在亲水面往复形成互补离子键,可组装为纳米纤维,并且能够促MC3T3 E1细胞的黏附、伸展和增殖。 目的：观察新型自组装多肽水凝胶NBD/RADA16对小鼠前成骨细胞MC3T3 E1成骨分化能力的影响。 方法：将MC3T3 E1细胞分别接种于自组装多肽水凝胶NBD/RADA16与RADA16水凝胶中,进行成骨诱导培养,以单纯成骨诱导培养的细胞为对照。诱导培养1,3,6 d检测细胞碱性磷酸酶活性;诱导培养7 d后,Western Blot检测细胞骨形态发生蛋白2的表达;诱导培养21 d后,茜素红染色观察细胞钙化结节。 结果与结论：MC3T3 E1细胞在NBD/RADA16多肽水凝胶上生长状态良好,优于在RADA16上生长的细胞。自组装多肽水凝胶NBD/RADA16上MC3T3 E1细胞的碱性磷酸酶活性高于RADA16水凝胶上及单纯成骨诱导培养的细胞(P < 0.01)。自组装多肽水凝胶NBD/RADA16上MC3T3 E1细胞的矿化基质沉积、骨形态发生蛋白2表达高于RADA16水凝胶上的细胞(P < 0.01)。结果提示NBD/RADA16自组装多肽水凝胶较RADA16水凝胶更能促进MC3T3 E1细胞的成骨分化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

自组装多肽纳米纤维支架的结构特点及应用优势

背景：3D自组装肽纳米纤维凝胶支架能很好模拟体内的微环境,提供一种能促进细胞生长、改善细胞功能、具有合理构成细胞外基质的结构模式。 目的：综述自组装多肽纳米纤维支架的基础研究及其在神经组织工程中的研究现状。 方法：检索2000至2013年PubMed数据库、维普数据库有关自组装多肽纳米纤维支架研究进展的文献,关键词为“自组装多肽,纳米纤维支架,神经组织工程,神经干细胞;self-assembling peptide,nanofiber scaffold,RADA16,Nerve tissue engineering,Neural stem cell”。 结果与结论：自组装多肽纳米纤维支架作为新型组织工程支架材料不仅解决了材料与细胞相容性差的问题,而且在维持材料的三维特性、促进细胞活性、模仿细胞外基质等方面均优于其他支架材料,是一种理想的组织工程材料,为神经损伤修复研究提供了新的方法。但自组装多肽领域内仍面临一些挑战,短期的问题包括自组装多肽与新型生物高分子的整合,以及与相对成熟传统移植物的整合;长期问题涉及如何更好地应对体内免疫系统,如何对细胞内的靶点进行治疗,以及如何预测未来高整合支架的发展方向等。     

自聚肽纳米纤维支架对骨髓源性心肌干细胞的作用研究

目的　研究自聚肽纳米纤维支架对骨髓源性心肌干细胞（MCSCs）生长和存活的作用。 方法　设计和固相合成自聚肽,扫描电镜和原子力显微镜下观察其自组装后的形态结构和与MCSCs的相互关系;通过CCK-8法和免疫荧光染色检测纳米纤维支架对MCSCs增殖和分化情况。 结果　1%多肽溶液自组装成直径约10 nm,长度为100 ~ 300 nm纳米纤维,并相互交织成孔径为50 ~ 200 nm的网状结构;MCSCs在纤维支架中培养1 d后,可见细胞呈长梭形,位于三维纳米纤维支架的网孔中,生长状态良好;生长曲线显示细胞在纤维支架中的增殖能力明显高于对照组;MCSCs在纤维支架中诱导培养4 周时,形态和排列方式均发生明显改变。 结论　纤维支架与MCSCs有良好生物相容性,纤维支架有利于MCSCs的生长和定向分化。     

3D自组装肽纳米纤维支架对胰岛细胞分泌功能的影响

背景：3D自组装肽纳米纤维支架能很好模拟体内微环境,给予细胞必要的结构模式,促进细胞外基质的正确组成及细胞的生长,改善细胞功能。 目的：体外观察3D自组装肽纳米纤维水凝胶支架对胰岛细胞分泌功能的影响。 方法：将3D自组装肽纳米纤维水凝胶支架与成年大鼠胰岛细胞共培养,AO-PI荧光染色法检测胰岛细胞的活性及生存率;放射免疫法测定胰岛细胞的分泌功能;扫描电镜观察胰岛细胞包裹在3D纳米支架中成三维立体的生长状态。 结果与结论：在3D纳米支架培养环境中胰岛细胞纯度≥80%;3D纳米支架组胰岛生存率及胰岛细胞分泌功能明显高于无支架组(2D培养组)(P < 0.05);扫描电镜显示自组装肽纳米纤维支架形成了具有几何形状的纳米级薄层,将胰岛细胞包裹在3D纳米支架中,胰岛细胞成三维立体生长。表明3D自组装肽纳米纤维支架可为胰岛细胞体外生存提供3D培养环境,改善胰岛细胞的活性、分泌功能及形态,延长胰岛细胞体外生存期。     

Design of a RADA16-based self-assembling peptide nanofiber scaffold for biomedical applications

RADA16 (RADARADARADARADA) is an amphiphilic polypeptide composed of 16 amino acids, which is composed of alternating positively charged arginine (R), hydrophobic alanine (A) and negatively charged aspartic acid (D) that repeat periodically throughout the composition. This structure allows RADA16 to form an extremely stable and highly ordered β-sheet structure by noncovalent bonding (ionic bonds, hydrogen bonds, hydrophobic action, π-π bonds, etc.). Moreover, it can form a three-dimensional (3D) nanofiber hydrogel scaffold in neutral pH with water content higher than 99% and with a physiological saline solution, having excellent biocompatibility and low immunogenicity, etc. Its degradation products are amino acids, which can reduce the possibility of an inflammatory reaction and have little effect on the normal healing process of damaged tissue. In addition, the special 3D structure of RADA16 facilitates the proliferation and differentiation of cells, making it widely used in cell culture scaffolds. Subsequent studies have found that the C-terminus or N-terminus of RADA16 is modified by a specific functional peptide, which not only retains the original function of RADA16 but also gives the RADA16 self-assembling hydrogel a more powerful function. In recent years, RADA16 and RADA16-based fusion peptides have been applied in biomedical fields, such as 3D cell culture, tissue repair, rapid hemostasis, and delivery systems, which have broad prospects. This review focuses on recent research and applications of RADA16 and RADA16-based self-assembling peptide nanofiber scaffold (SAPNS) in biomedicine.     

Neural Progenitor Cells Survival and Neuronal Differentiation in Peptide-Based Hydrogels

We designed nanofibrous hydrogels as 2-D and 3-D scaffolds for anchorage-dependent cells. The IKVAV-containing peptide amphiphile molecules spontaneously self-assembled into higher-order nanofiber hydrogels under cell-containing media. Neural progenitor cells (NPCs) were incubated in peptide-based hydrogels. Effects of self-assembling hydrogels on survival and neural differentiation of NPCs were observed. Peptide was synthesized using a solid-phase method. TEM study of the hydrogel revealed a network of nanofibers. Phase-contrast light micrographs showed that the described hydrogel had no observable cytotoxicity to NPCs. Additionally this hydrogel could induce cells to differentiate into neuron-like cells and glial-like cells. Moreover, the cells encapsulated within hydrogel had a higher neuronal differentiation rate than in the surface of the hydrogel. This self-assembled hydrogel might serve as nerve tissue-engineering scaffold.     

The effect of a self-assembling peptide nanofiber scaffold (peptide) when used as a wound dressing for the treatment of deep second degree burns in rats

RADARADARADARADA (RADA16-I) peptide, consisting of 16 alternating hydrophobic and hydrophilic (also alternating negative and positive charges) amino acids, forms extremely stable beta-pleated sheet structure and then self-assembles into nanofibers to produce high-order interwoven nanofiber scaffold hydrogel. To investigate its therapeutic effects, a burn model of partial thickness-deep dermal injury (the deep second degree burns) was performed at the dorsal skin of female Sprague-Dawley rats with an electrical scalding machine. The wounds treated with either RADA16-I or control materials were carefully examined at morphological, histological and cellular levels. We found that RADA16-I can advance the time of eschar appearance and the time of eschar disappearance both by 3-5 days, and speed up wound contraction by 20-30% compared with contrast groups (chitosan, poly(DL)-lactic acid (PDLA), collagen I and the blank) without obvious edema. Immunohistochemical studies showed that both FGF and EGF were obviously expressed in nascent tissue such as epidermis and glands when wounds were treated with the RADA16-I after injury. When peptide stock solution was diluted from 10 to 0.17 mg/mL, atomic force microscopy (AFM) observation showed that the shape of peptide nanofibers changed from the globular-pieces-clustered filaments with 4.8 +/- 0.38 nm in height, 61.6 +/- 6.10 nm in width and 708 +/- 80.2 nm in length, to general filaments with 1.4 +/- 0.36 nm, 17.5 +/- 1.13 nm and 1108 +/- 184 nm. The nanofiber surface porosity gradually decreased from 49-70% to 12-28%. These characteristics contribute to wound healing by offering an "ideal dressing" moist healing microenvironment and a nanofiber 3D scaffold. These results suggest that the self-assembling peptide might be a promising wound dressing with being simple, effective, and affordable.     

Functionalized self-assembling peptide nanofiber hydrogel as a scaffold for rabbit nucleus pulposus cells

In this study, a new functionalized peptide RLN was designed containing the bioactive motif link N, the amino terminal peptide of link protein. A link N nanofiber scaffold (LN-NS) was self-assembled by mixing peptide solution of RLN and RADA16. The characterization of LN-NS was tested using atomic force microscopy (AFM). The biocompatibility and bioactivity of this nanofiber scaffold for rabbit nucleus pulposus cells (NPCs) were also evaluated. This designer functionalized nanofiber scaffold exhibited little cytotoxicity and promoted NPCs adhesion obviously. In three-dimensional cell culture experiments, confocal reconstructed images testified that the functionalized LN-NS-guided NPCs migration from the surface into the hydrogel considerably, in which the RADA16 scaffold did not. Moreover, the functionalized LN-NS significantly stimulated the biosynthesis of extracelluar matrices (ECM) by NPCs. Our findings demonstrate that the functionalized nanofiber scaffold containing link N had excellent biocompatibility and bioactivity with rabbit NPCs and could be useful in the nucleus pulposus regeneration.     

The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel

The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.     

促血管内皮细胞生长的功能化自组装多肽框架材料的筛选和制备

背景：功能化多肽框架材料由于良好的生物相容性及生物活性,可以用来促进血管生成的研究。 目的：设计并筛选出能够促进内皮细胞血管生成的功能化多肽框架材料。 方法：将设计和筛选出的功能多肽片断通过固相合成法复合在自组装多肽RADA16-I的C末端,将RADA16-I与功能化自主装多肽以1∶1的比例混合,加入无菌培养板在37 ℃下孵育过夜以促进其凝胶,通过换培养基调节pH值。在凝胶上对内皮细胞进行2D培养。观察功能化自组装多肽框架材料的圆二色谱、原子力显微镜照像,内皮细胞黏附、增殖情况。 结果与结论：功能化自组装多肽框架材料与Matrigel的形貌相似且是均一的纳米纤维材料。其中RAD/KLT和RAD/PRG具有促进内皮细胞黏附及增殖的功能。表明,功能化多肽框架材料RAD/KLT和RAD/PRG具有用于促内皮细胞血管生成的进一步研究的潜力。