生物活性，PCL为基础的“自我装配”形状记忆聚合物支架的制备
Summary
Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.
Abstract
Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect “self-fitting.” In this way, following exposure to warm saline (~60 ºC), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 ºC) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.
Introduction
目前考虑的颅颌面（CMF）骨缺损治疗的金标准，收获的自体移植的移植是由复杂的移植手术，供体部位发病率和有限1阻碍。一个特别困难被整形并紧紧固定在刚性移植到缺陷，以获得骨整合和预防移植物吸收。组织工程已被调查作为一种替代战略，以自体移植和人工合成骨替代品（ 如骨水泥）2,3。关键的组织工程方法的成功是一组特定属性的支架。第一，为了实现骨整合，支架必须形成与相邻的骨组织4紧密接触。该支架也应该是骨传导，允许细胞迁移，营养扩散和neotissue沉积4,5。这种行为通常是实现了与可生物降解的SCAffolds表现出高度互联的孔结构。最后，该支架应的生物活性，以促进融合和粘接与周围骨组织5。
在这里，我们提出了一个协议，准备组织工程支架具有这些属性。重要的是，这种支架显示能力的“自我适应”到，由于其形状记忆行为6不规则的CMF缺陷。温敏形状记忆聚合物（SMP系统）是已知的在暴露于经受形状改变以加热7,8。中小事务所是由“netpoints”（即化学或物理交联），这决定了永久的形状和“转换段”，它保持暂时形状，并恢复永久的形状。开关段表现出相应于任一所述的玻璃化转变（T 克 ）热转变温度（T 反式）或熔融聚合物的转变（Tm值）。由于其结果，污水收集可以顺序变形为暂时形状在T> T 反式，在T <T 反式固定在暂时形状，并恢复到在T> T 反式的永久形状。因此，SMP支架可以实现“自我装配”中的CMF缺陷如下6。曝光后，以温盐水（T> T 转 ），一个SMP支架将成为延展性，允许一个一般制造的圆筒状的支架是用手压成不规则的缺陷，具有形状恢复促进扩大脚手架的缺陷边界。在冷却（T <T 反式），脚手架将回到其相对更为刚性的状态，具有形状固定性保持缺陷在其新的临时形状。在这个协议中，将SMP支架选自聚己内酯（PCL）中制备，可生物降解的聚合物广泛研究的组织再生和其它生物医学应用9-11。对于形状记忆，日PCL电子Tm值用作经t 反式和43之后60°C之间变化，取决于在PCL 12的分子量。在这个协议中，支架的Ť 反式 （即Tm值）是56.6±0.3ºC6。
为了达到骨传导性，协议的开发，使PCL基的SMP支架与基于溶剂的铸造颗粒淋（SCPL）方法6,13,14高度互连的孔。聚己内酯丙烯酸酯（PCL-DA）（Mn为=〜10,000g / mol的）得到使用，以允许快速，光化学交联和溶解于二氯甲烷（DCM），以允许溶剂浇铸在盐模板。以下光化学固化和溶剂蒸发，盐模板除去浸出到水中。平均盐大小调节支架孔径。重要的是，该盐模板融合用水之前溶剂浇铸实现孔隙interconnectiviTY。
生物活性传授给了SMP的支架原位形成聚多巴胺涂到孔壁6。生物活性通常通过包含玻璃或玻璃-陶瓷填料15引入到支架。但是，这些可能引起不必要的易碎的机械性能。多巴胺已经显示，以形成对各种基材16-19中粘附，薄聚多巴胺层。在这个协议中，将SMP支架进行多巴胺的弱碱性溶液（pH = 8.5），以形成聚多巴胺的所有孔壁表面6 nanothick涂层。除了 增强表面的亲水性以改进细胞粘附和传播，聚多巴胺已被证明是生物活性在形成的羟基磷灰石（HAP）在暴露于模拟体液（SBF）18,20,21的条款。在最后的步骤，将涂覆的支架被暴露在85℃（T>Ť 反式）的wh热处理ICH导致脚手架致密化。热处理是前面提到的是为支架的形状记忆行为所必需的，或许是由于PCL结晶 区域重组，以更接近14。
我们另外描述的方法中的不规则缺陷模型的自装配行为表征，形状记忆行为而言 应变控制环状热机械压缩试验（即形状恢复和形状固定性），孔的形态，并在体外生物活性 。策略定制支架性能也提出了。
Protocol
Representative Results
Discussion
这个协议描述了一种聚多巴胺涂覆的，PCL-基于支架，其自入行为的制备，以及骨诱导性和生物活性，使得在不规则CMF骨缺损的治疗兴趣它。该协议的各方面可以被改变，以改变各种支架功能。
该协议开始于PCL二醇，允许UV固化的丙烯酸酯化。在所报告的实例中，PCL二醇Mn为是〜10,000g / mol的。然而，通过适当地调整丙烯酰氯量和的Et 3 N的PCL-DA了PCL二醇可以被用?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢得克萨斯州A与M大学工程试验站（TEES）对这项研究的财政支持。林赛指甲非常感谢来自得克萨斯州A与M大学路易斯·斯托克斯联盟少数人参与（LSAMP）和美国国家科学基金会（NSF）研究生研究奖学金计划（GRFP）的支持。张大伟感谢得克萨斯州A与M大学论文奖学金。
Materials
| Polycaprolactone-diol (Mn ~ 10,000 g/mol) | Sigma-Aldrich | 440752 | |
| Dichloromethane (DCM) | Sigma-Aldrich | D65100 | Dried over 4A molecular sieves |
| 4-dimethylaminopyridine (DMAP) | Sigma-Aldrich | D5640 | |
| Triethylamine (Et3N) | Sigma-Aldrich | T0886 | |
| Acryloyl chloride | Sigma-Aldrich | A24109 | |
| Ethyl Acetate | Sigma-Aldrich | 319902 | |
| Potassium Carbonate (K2CO3) | Sigma-Aldrich | 209619 | |
| Anhydrous magnesium sulfate (MgSO4) | Fisher | M65 | |
| Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | |
| 2,2-dimethoxy-2-phenyl acetophenone (DMP) | Sigma-Aldrich | 196118 | |
| 1-vinyl-2-pyrrolidinone (NVP) | Sigma-Aldrich | V3409 | |
| Ethanol | Sigma-Aldrich | 459844 | |
| Dopamine Hydrochloride | Sigma-Aldrich | H8502 | |
| Tris buffer (2mol/L) | Fisher | BP1759 | Used at 10 mM concentration, pH = 8.5 |
| Sieve | VWR | 47729-972 | |
| UV-Transilluminator (365 nm, 25 W) | UVP | 95-0426-02 | |
| Centrifuge | Eppendorf | 5810 R | |
| Dynamic Mechanical Analyzer (DMA) | TA Instruments | Q800 | |
| High Resolution Sputter Coater | Cressington | 208HR | |
| Scanning Electron Microscope (SEM) | FEI | Quanta 600 |
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