Vandegrift Meredith T, Szpalski Caroline, Knobel Denis, Weinstein Andrew, Ham Maria, Ezeamuzie Obinna, Warren Stephen M, Saadeh Pierre B
The Institute of Reconstructive Plastic Surgery, New York University Langone Medical Center, New York, New York.
The Institute of Reconstructive Plastic Surgery, New York University Langone Medical Center, New York, New York.
J Surg Res. 2015 May 1;195(1):360-7. doi: 10.1016/j.jss.2015.01.003. Epub 2015 Jan 13.
Acellular dermal matrix (ADM) is widely used for structural or dermal replacement purposes. Given its innate biocompatibility and its potential to vascularize, we explored the possibility of ADM to function as a small interfering RNA (siRNA) delivery system. Specifically, we sought to improve ADM vascularization by siRNA-mediated inhibition of prolyl hydroxylase domain-2 (PHD2), a cytoplasmic protein that regulates hypoxia inducible factor-1α, and improve neovascularization.
Fluorescently labeled siRNA was used to rehydrate thin implantable ADM. Pharmacokinetic release of siRNA was determined. Twelve millimeter sections of ADM reconstituted with PHD2 siRNA (nonsense siRNA as control) and applied to dorsal wounds of 40 FVB mice. Grafts were sewn in, bolstered, and covered with occlusive dressings. Photographs were taken at 0, 7, and 14 d. Wounds were harvested at 7 and 14 d and analyzed (messenger RNA, protein, histology, and immunohistochemistry).
Release kinetics was first-order with 80% release by 12 h. By day 14, PHD2-containing ADM appeared viable and adherent, whereas controls appeared nonviable and nonadherent. Real-time reverse transcription-polymerase chain reaction demonstrated near-complete knockdown of PHD2, whereas vascular endothelial growth factor and FGF-2 were increased 2.3- and 4.7-fold. On enzyme-linked immunosorbent assay, vascular endothelial growth factor was increased more than fourfold and stromal cell-derived factor doubled. Histology demonstrated improved graft incorporation in treated groups. Immunohistochemical demonstrated increased vascularity measured by CD31 staining and increased new cell proliferation by denser proliferating cell nuclear antigen staining in treated versus controls.
We concluded that ADM is an effective matrix for local delivery of siRNA. Strategies to improve the matrix and/or genetically alter the local tissue environment can be envisioned.
脱细胞真皮基质（ADM）被广泛用于结构或真皮替代目的。鉴于其固有的生物相容性及其血管化的潜力，我们探索了ADM作为小干扰RNA（siRNA）递送系统的可能性。具体而言，我们试图通过siRNA介导的脯氨酰羟化酶结构域2（PHD2）抑制来改善ADM血管化，PHD2是一种调节缺氧诱导因子-1α的细胞质蛋白，并改善新生血管形成。
使用荧光标记的siRNA对可植入的薄ADM进行复水。测定siRNA的药代动力学释放。用PHD2 siRNA（无义siRNA作为对照）重构的12毫米ADM切片应用于40只FVB小鼠的背部伤口。将移植物缝合、支撑并用封闭敷料覆盖。在第0、7和14天拍照。在第7和14天收获伤口并进行分析（信使RNA、蛋白质、组织学和免疫组织化学）。
释放动力学为一级，12小时释放80%。到第14天，含PHD2的ADM看起来存活且附着，而对照看起来不存活且不附着。实时逆转录-聚合酶链反应显示PHD2几乎完全敲低，而血管内皮生长因子和FGF-2分别增加了2.3倍和4.7倍。在酶联免疫吸附测定中，血管内皮生长因子增加了四倍多，基质细胞衍生因子增加了一倍。组织学显示治疗组的移植物整合得到改善。免疫组织化学显示，与对照组相比，通过CD31染色测量的血管生成增加，通过更密集的增殖细胞核抗原染色测量的新细胞增殖增加。
我们得出结论，ADM是局部递送siRNA的有效基质。可以设想改善基质和/或基因改变局部组织环境的策略。
