优化结构静电纺丝血管移植在大鼠模型中的植入
Summary
在此, 我们提出了一种改进的静电纺丝方法, 以制造粗纤维和大孔隙的 PCL 血管移植, 并描述了一个协议, 以评估在体内的性能在大鼠腹主动脉置换模型。
Abstract
在这里, 我们提出了一个制备大孔 PCL 血管移植的协议, 并描述了一个模型的应用腹主动脉替代模式的评估协议。静电纺丝血管移植通常具有相对较小的孔隙, 限制细胞浸润到移植物中, 阻碍新动脉的再生和重塑。本研究采用改良的工艺制备了较厚纤维 (5-6 µm) 和较大孔隙 (~ 30 µm) 的 PCL 血管移植。采用大鼠腹主动脉模型植入法对移植物的远期表现进行评价。超声分析显示, 即使植入12月后, 移植物仍未发生动脉瘤或狭窄。大孔结构改善细胞肉芽, 从而促进组织再生3月。更重要的是, 没有出现不良重塑的迹象, 例如12月后移植壁内的钙化。因此, 经改良大孔处理的静电纺丝 PCL 血管移植可作为长期植入的动脉替代物。
Introduction
合成高分子的血管移植广泛应用于心血管疾病 (心血管病) 的临床治疗。不幸的是, 在小直径血管移植 (D < 6 毫米) 的情况下, 没有成功的产品, 由于低通畅触发的血液流速降低, 这往往导致血栓形成, 内膜增生, 和其他并发症1。
组织工程提供了一个替代策略, 以实现长期通畅和动态平衡的基础上的支架引导血管再生和重建。详细来说, 血管移植作为一个三维模板, 可以在血管组织再生过程中提供机械支持和结构指导, 并影响细胞功能, 包括细胞黏附、迁移、增殖和分泌细胞外基质2。到目前为止, 各种合成聚合物已被评价为应用于血管组织工程。在这些聚合物中, 聚 (己内酯) (PCL) 由于良好的细胞相容性和缓慢的降解, 从几个月到两年不等, 已被深入研究。用静电纺丝处理的大鼠主动脉模型4、5、6、PCL 血管移植表现出优良的结构完整性和通畅性, 并不断增加细胞侵袭和新生血管移植壁长达6月。然而, 不良组织重塑, 包括细胞的回归和毛细血管和钙化, 也被观察到更长的 timepoints, 长达18月。
Cellularization 血管移植是决定组织再生和重塑7的关键因素。静电纺丝作为一种多功能技术, 已广泛应用于纳米纤维结构8的血管移植的制备。不幸的是, 相对较小的孔隙结构往往导致细胞浸润不足的静电纺丝血管移植, 这限制了随后的组织再生。为了解决这一问题, 尝试了各种技术来提高孔径和整体孔隙率, 包括盐/聚合物浸出9,10, 集热器的修改, 激光辐照后处理11等。事实上, 静电纺丝的结构 (包括纤维直径、孔隙大小和孔隙度) 与加工条件12、13密切相关。在静电纺丝过程中, 通过改变聚合物溶液的浓度、流量、电压等参数, 可以很容易地控制纤维的直径. 14,15, 因此, 孔隙大小和孔隙度得到了相应的增强。
我们最近报告了一个改良的 PCL 静电纺丝移植与大孔结构 (纤维直径 5-7 µm 和毛孔 30-40 µm)。在活体移植中, 取代大鼠腹主动脉显示出高的通畅率, 以及良好的内皮化和平滑肌再生3月后手术16。更重要的是, 即使在植入一年后, 也不会有钙化和细胞回归等不良组织重塑。
Protocol
Representative Results
Discussion
细胞浸润对16体内血管移植的再生和重塑至关重要。有限细胞浸润通常与移植的相对较小的毛孔有关, 阻碍细胞向移植壁迁移。为了解决这一困难, 我们开发了一种改进的方法来制备静电纺丝 PCL 血管移植的大孔结构。详细地说, 孔隙大小随纤维厚度的增加而增大, 其加工参数可以很容易地控制。结果表明,在体外植入后, 宿主细胞很容易渗入到大孔移植的壁内, 细胞构?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了自然科学基金项目 (81522023、81530059、91639113、81772000、81371699和 81401534) 的财政支持。
Materials
| Poly(ε-caprolactone) (PCL) pellets (Mn=80,000) | Sigma | 704067 | |
| Methanol | Tianjin Chemical Reagent Company | 1060 | |
| Alcohol | Tianjin Chemical Reagent Company | 1083 | |
| Chloroform | Tianjin Chemical Reagent Company | A1007 | |
| Sucrose | Tianjin Fengchuan Company | 2296 | |
| Triton X-100 | Alfa Aesar | A16046 | |
| Sprague Dawley rats | Laboratory Animal Center of the Academy of Military Medical Sciences | ||
| Normal saline | Hebei Tiancheng Pharmaceutical company | ||
| Chloral hydrate | Tianjin Ruijinte chemical company | 2223 | |
| Heparin sodium Injection | Tianjin Biochem Pharmaceutical company | ||
| Gentamycin Sulfate Injection | Jiangsu Lianshui Pharmaceutical company | ||
| Mouse anti-α-SMA primary antibody | Abcam | ab7817 | |
| Mouse anti-smooth MYH primary antibody | Abcam | ab683 | |
| Rabbit polyclonal anti-rat elastin antibody | Abcam | ab23748 | |
| Rabbit anti-von Willebrand factor primary antibody | Abcam | ab6994 | |
| Goat anti-mouse IgG (Alexa Fluor 488) | Invitrogen | ab150117 | |
| Goat anti-rabbit IgG (Alexa Fluor 488) | Invitrogen | ab150077 | |
| 5% normal goat serum | Zhongshan Golden bridge | ZLI9022 | |
| Hematoxylin and eosin (H&E) | Beijing leagene biotech | DH0006 | |
| Masson's trichrome | Beijing leagene biotech | DC0032 | |
| Verhoeff-van Gieson (VVG) | Beijing leagene biotech | DC0059 | |
| Von Kossa | Beijing leagene biotech | DS0003 | |
| Surgical sutures needles with thread,3-0 silk | Shanghai Jinhuan medical supplies company | G3002b | |
| Surgical sutures needles with thread,9-0 silk | Shanghai Jinhuan medical supplies company | H901 |
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