界面聚电解质纤维的生物分子在时间控释复合支架

摘要

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

摘要

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

引言

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.¹ For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.² However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.³ Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix.

Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.^4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors^2,7 even cells^8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic² and hydrophilic polymers⁷ to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells.

In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

研究方案

1.准备聚电解质溶液的

1. 纯化脱乙酰壳多糖，如在Liao 等详细说明。简言之，将创建一个1％（重量/体积）壳聚糖在2％的溶液使用（体积/体积）乙酸和真空过滤级93过滤纸。中和使用5M的NaOH将滤液直到pH稳定至7。离心沉淀的脱乙酰壳多糖在1200×g离心10分钟。滗析出上清液，并添加去离子水以洗涤脱乙酰壳多糖。重复离心和洗涤步骤两次以上。冻结的沉淀壳聚糖在-80℃和冷冻干燥O / N以获得纯化形式。存储纯化壳聚糖在除湿柜。
2. 称量1g纯壳聚糖进入无菌组织培养皿。放置在组织培养皿尽可能接近到紫外灯在生物安全柜的脱乙酰壳多糖和暴露于UV光15分钟。使用无菌镊子，将灭菌壳聚糖到玻璃容器中。壳聚糖溶解过滤使用0。15M乙酸至0.5％至1％的终浓度（重量/体积）。
3. 称取0.1克藻酸的钠盐和溶解在蒸馏水去离子（DDI）的10ml水，得到1％（重量/体积）溶液。混合的藻酸的钠盐，至少2小时，在涡流混合器以确保完全溶解。通过过滤0.2微米的注射器过滤器的海藻酸钠溶液。储存在4℃的藻酸盐溶液。
4. 重组人重组生长因子，如血管内皮生长因子（VEGF）或β - 神经生长因子（NGF），如制造商推荐的。

2.绘图IPC纤维

1. 混合蛋白，生长因子或其它生物分子进入电解质溶液具有类似净电荷10-20微升等份。生物分子与净负电荷（例如，牛血清白蛋白〔BSA〕）应与藻酸盐溶液混合。生物分子的净正电荷（如VEGF）应与混合壳聚糖溶液。
2. 将壳聚糖和海藻酸钠的小等份（10-20微升）上覆盖有封口膜平坦光滑的表面。脱乙酰壳多糖和藻酸盐的液滴应该放置在接近但不与彼此相接触。
3. 轻轻浸每个尖端上的一对镊子进入壳聚糖和藻酸盐的微滴。一起捏钳把聚电解质的液滴。当液滴接触到彼此接触，慢慢地将钳子垂直向上从两个液滴（ 图1A）的接口绘制了IPC纤维。
4. 拉伸的IPC纤维上的集电极，所述镊子的末端小心地例如平坦聚合物支架固定在一个旋转的心轴（见第3和4）。旋转心轴以10mm / sec的固定速度以允许形成均匀的和无珠的IPC纤维。增加绘制了IPC的纤维将形成小珠的速度，这将导致结合的爆发释放生化和过早的光纤终端^。10
5. 为了确定掺入的效率，收集所有的剩余液体留在封口膜通过用500μl1X磷酸盐缓冲盐水（PBS）中稀释。测量通过BCA测定法（对BSA），酶联免疫吸附（对VEGF和NGF）或适当的测定法来检测结合的生物分子中的残基的蛋白质或生长因子含量。

3.制作普鲁兰多糖葡聚糖复合水凝胶支架（PD）多糖和IPC纤维

1. 制造牺牲普鲁兰框架IPC纤维集合
   1. 称出支链淀粉的多糖并用蒸馏水去离子（DDI）水混合，以创建一个20％（重量/体积）的水溶液。混合普鲁兰多糖溶液O / N，以保证均匀性。
   2. 投将15克的支链淀粉溶液倒入一个直径10厘米的组织培养聚苯乙烯（TCPS）菜。干普鲁兰多糖溶液O / N在37℃。切普鲁兰多糖薄膜7成毫米×7毫米的方形框架。
2. 准备普鲁兰葡聚糖多糖溶液
   1. 创建一个30％（重量/体积）的多糖类的溶液支链淀粉和葡聚糖:在DDI水（3比1的比例）。混合O / N，以确保多糖溶液的均匀性。慢慢在碳酸氢钠添加到多糖溶液以达到20％的终浓度（重量/体积）。混合O / N，以确保溶液的均匀性。储存在4℃的多糖溶液。
3. 收集普鲁兰框架IPC纤维
   1. 贴上使用鳄鱼夹牺牲普鲁兰框架（第3.1节）。粘鳄鱼夹和支链淀粉帧上使用塑料涂覆粘合带的转动心轴的末端。旋转与贴帧芯棒以10毫米/秒的恒定速度。普鲁兰多糖架可贴到所希望的方向旋转芯棒。
4. 用画一双镊子（第1节）和ATTAC的IPC纤维小时IPC纤维的结束画上旋转普鲁兰框架。绘制了IPC纤维以恒定的速度。一旦到达了IPC纤维的末端，干燥纤维上帧构建O / N在室温。
5. IPC嵌入纤维进入PD水凝胶支架
6. 以交联每克支链淀粉葡聚糖溶液中，添加11％的100微升（重量/体积）三偏磷酸钠水溶液和氢氧化钠将100μl10M钠⁷使用stirplate为1至2分钟，混合溶液在60rpm。添加的三偏磷酸钠和氢氧化钠之后，将多糖溶液将几乎立即交联。倾粘性多糖溶液到纤维上帧构造完全嵌入的IPC纤维。孵育结合支链淀粉葡聚糖的IPC纤维（PD-IPC），在60℃进行30分钟，形成化学交联的复合支架（ 图1B）。
7. 注意:在通风柜执行步骤3.3.2，并使用适当的保护相等pment乙酸是腐蚀性和易燃。
8. 为了诱导孔形成在PD-IPC脚手架，淹没整个支架在20％（重量/体积）乙酸20分钟。
9. 为5分钟，在1×PBS中洗涤的PD-IPC支架除去未反应的试剂以100rpm摇动。重复此步骤2次。
10. 除去多余的PBS，并立即冻结PD-IPC支架在-80°CO / N。冻干支架的任何控制释放生物活性或使用试验前至少24小时。

4.制作PCL和IPC纤维复合支架

注意:二氯甲烷是一种有害物质。二氯甲烷处理时，使用通风橱和个人防护装备。

1. 创建原始和图案的PDMS基板
   1. 创建一个纯净的聚二甲基硅氧烷（PDMS）的弹性基板采用了一块所需尺寸的TCPS使用软光刻工艺。创建patterned的PDMS衬底 ​​通过使用标准软光刻方法对聚（甲基丙烯酸甲酯）与所需的形貌的模板^。12
2. 制造牺牲PCL框架IPC纤维集合
   1. 称取PCL和溶解在二氯甲烷中以创建一个0.9％（重量/体积）溶液。每1 ^平方厘米面积的PDMS衬底 ​​的，滴加入500μl的0.9％的PCL溶液。允许所有的二氯甲烷溶剂完全蒸发，在通风橱。重复铸造0.9％PCL到薄膜变厚至期望的厚度的过程。从PDMS基板取下干燥PCL电影。创建在使用适宜尺寸的冲床在PCL帧的孔^。2
3. 在PCL框架上收集IPC纤维
   1. 上贴上鳄鱼夹与孔（从4.2.1）牺牲PCL框架。通过使用塑料涂层胶带粘鳄鱼夹到旋转心轴。附加IPC纤维的结束画上PCL FRAM开始旋转，速度为10mm /秒（第2节）以恒定的速度前即IPC纤维拉伸结束后，干燥纤维上的帧构建O / N在4℃。
4. 嵌入光纤的框架构建成图案PCL基板
   1. 降500微升0.9％PCL解决方案到PDMS基板打造一个纯净的或有图案的PCL基地，根据需要。施放的0.9％的PCL溶液多层以获得具有所需厚度的脚手架。允许所有的二氯甲烷溶剂完全蒸发，在通风橱。
   2. 放置在纤维上的帧结构（第4.3.1节）在PCL基座的顶部。加在纤维上帧构造多次0.9％的PCL溶液，以获得所需的厚度和完全嵌入的IPC纤维，制造的PCL-IPC复合支架（ 图1C）。

5.测量生物制剂的释放从复合IPC支架

1. 将复合PD-IPC或PCL-IPC支架和单机IPC纤维分别在24孔板。
2. 沉浸脚手架和单机IPC纤维用500μl1X PBS的。孵育样品在37℃。收集的PBS在不同时间点（释放介质）和替换用500μl1X PBS中。
3. 测量在使用BCA测定法（BSA），酶联免疫吸附（VEGF和NGF）或其他适当的测定法来计算掺入的生物分子的累积释放曲线的释放介质蛋白或生长因子的量。

复合IPC支架6.种子细胞来测试释放生物制剂的生物活性

1. 消毒使用UV光在生物安全柜为至少20分钟的冷冻干燥的PD-IPC或PCL-IPC复合支架。
2. 使用标准的细胞培养技术，以获得在200μl生长培养基2×10 ⁵个细胞的细胞悬浮液。种子的浓缩细胞悬浮液上的复合支架。 AFTER 20分钟，生长培养基补足体积至完全淹没支架。
3. 通过测量如Alamar蓝代谢活性测定，PC12轴突生长法或免疫荧光技术标准的细胞活性。

结果

在这篇文章中，我们试图建立复合支架与IPC纤维各种生物分子的持续释放。在此研究中使用的生物分子的特征见于表1。的IPC纤维第一嵌入到亲水的PD水凝胶来创建的PD-IPC复合支架（ 图1B）。模型分子BSA的第一测试，以确定使用复合支架用于控制生物分子释放的可行性。 BSA的掺入的PD-IPC支架为45±0.97％的效率。 BSA的从PD-瞳距释放呈接近线性的动力学具有初始减毒突释之后?...

讨论

IPC纤维通过两个带相反电荷的聚电解质的相互作用形成。该方法使用从聚电解质的界面的复杂的提取，便利了一种用于纤维形成稳定的自组装过程。 IPC的纤维形成的机制可确保任何生物分子加入到类似电荷的聚电解质可以在络合过程中引入^。10,11相反，除了生物分子的成带相反电荷的聚电解质将导致瞬间沉淀。用于IPC纤维的简单的制造方法借给多功能性在将各种生物材料如细胞，生长因?...

材料

Name	Company	Catalog Number	Comments
Pullulan	Hayashibara Inc Okayama Japan		Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.
Dextran	Sigma Aldrich	D1037	MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.
Sodium Bicarbonate	Sigma Aldrich	S5761	Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.
Sodium Trimetaphosphate	Sigma Aldrich	T5508	This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.
Sodium Hydroxide	Sigma Aldrich	S5881	This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.
Chitosan	Sigma Aldrich	448877	MW 190-310 kDa. Acetylation degree of 75% to 85%. Purification of chitosan is required to create stable IPC fibers.
Acetic Acid	Merck		This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.
Alginic acid sodium salt from brown algae, low viscosity	Sigma Aldrich	A2158	Dissolve in water overnight. Filter through sterile 0.2 µm syringe filter before use. Store at 4 °C.
Bovine Serum Albumin	Sinopharm Chemical Reagent		Dissolve in sterile PBS and filter using 0.2 µm syringe filter before use.
BCA assay kit	Pierce	23225	This kit was used to measure BSA release from PD-IPC scaffolds.
Human Recombinant Vascular Endothelial Growth Factor	R&D systems	293-VE	Dissolve growth factor in 0.2% heparin solution to a final concentration of 5 mg/ml.
Heparin Sodium Salt From Porcine	Sigma Aldrich	H3393	This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 µm syringe filter before use.
Human Umbilical Vein Endothelial Cells (HUVEC)	Lonza	C2517A	This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.
Alamar blue	Life Technologies	DAL1025	This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.
ScanVac Coolsafe Lyophilizer	Labogene	7.001.200.060	This is a non-programmable freeze dryer that operates at -105 to -110 °C. This can be replaced by other standard lab lyophilizers.
Polycaprolactone (PCL)	Sigma Aldrich	181609	MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.
Dichloromethane	Sigma Aldrich	V800151	This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.
Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)	Dow Corning	(240)4019862	The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.
Human Recombinant Beta-Nerve Growth Factor (NGF)	R&D systems	256-GF	Reconstituted in sterile DI water to a final concentration of 100 µg⁠/⁠ml. Aliquot and store in -20 °C until use.
Human Mesenchymal Stem Cells (hMSC)	Cambrex		This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.
Rat PC12 Pheochromocytoma Cells	ATCC		This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.
Grade 93 filter paper	Whatman	Z699675	This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH 7.
Swing bucket centrifuge	Eppendorf	5810R	To be used during the purification of chitosan using 1,200 x g speed.
Motor with mandrel rotating at constant speed	Rhymebus	RM5E	The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.
Phosphate buffered saline	FirstBase		Sterilize through filtration (0.2 µm filter) and autoclave.
10-mm diameter Tissue Culture Polystyrene Dish (TCPS)	Greiner		The TCPS dish is used for casting of pullulan frame.
Human VEGF ELISA kit	R&D systems	DVE00	The ELISA kit is used for detection of VEGF in the release medium.
Human NGF ELISA kit	R&D systems	DY256	The ELISA kit is used for detection of NGF in the release medium.
Plastic Coated Adhesive Tape	Bel-Art	9040336	The adhesive tape is used to securely stick the alligator clip to the rotating mandrel