Natouri Ozra, Barzegar Abolfazl, Nobakht Abbas, Bagheri Afsaneh, Eslami Farhad, Jafarirad Saeed, Tohidifar Nastaran, Nikzad Behzad
Department of Organic and Biochemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran.
Research Center of Biosciences & Biotechnology (RCBB), University of Tabriz, Tabriz, Iran.
Heliyon. 2024 Sep 25;10(19):e38462. doi: 10.1016/j.heliyon.2024.e38462. eCollection 2024 Oct 15.
This study presents biomimetic nanoscaffolds composed of electrospun polycaprolactone-collagen (PCL-Coll) nanofibers, loaded with bioactive Arnebia euchroma (AE) extract and stem cells, to develop cell-based tissue engineering constructs. The incorporation of AE extract, known for its antioxidant and anti-inflammatory properties, into the PCL-Coll nanofibers resulted in nanoscaffolds denoted as PCL-Coll/AE, PCL-Coll/AE, PCL-Coll/AE, and PCL-Coll/AE, corresponding to AE extract concentrations of 0.0, 5.0, 10.0, and 15.0 wt%, respectively. The PCL-Coll/AE nanoscaffolds exhibited high porosity values of 62.81 ± 4.78 %, 59.9 ± 2.10 %, 52.44 ± 2.66 %, and 46.32 ± 1.35 %, respectively, thereby providing an optimal 3D environment for cell attachment and proliferation. Analysis of the AE extract revealed the presence of abundant shikonin, a key bioactive compound, as indicated by its characteristic absorption bands at 490, 520, and 560 nm. FE-SEM data confirmed the homogeneous immobilization of AE compounds within the 3D nanofiber scaffolds, with fibers averaging 316 ± 137 nm in diameter, falling within the range of natural collagen fibers. To evaluate the structural integrity of the fully stem cell-laden scaffolds, we assessed cell growth, cell attachment within the PCL-Coll/AE nanoscaffolds, the cytoprotective effects of AE extract, and the expression levels of stemness-related genes, including Nanog, Rex1, Sox2, Oct4, Klf4, and C-Myc. Real-time PCR assays indicated that key indicators of stemness were upregulated, with average increases from 120 ± 15 % in PCL-Coll/AE to 250 ± 30 % in PCL-Coll/AE over a two-week period. These upregulations promote cell proliferation and facilitate cell cycle progression. Data indicate that increasing the concentration of bioactive AE extract within the scaffolds enhanced cell adhesion on the scaffold surface and improved cell viability after 7 days of culture, with viability rising from 109 ± 6.15 % in PCL-Coll/AE to 145.5 ± 10.11 % in PCL-Coll/AE. Cytoprotective assays against oxidative stress induced by H₂O₂ revealed relative cell viability for PCL-Coll/AE, PCL-Coll/AE, PCL-Coll/AE, and PCL-Coll/AE as 42.78 ± 5.85 %, 49.29 ± 6.79 %, 64.98 ± 3.32 %, and 78.68 ± 3.09 %, respectively. These results correlate with the antioxidant potency of AE extract compounds, particularly shikonin and its derivatives. Therefore, the cell-laden biomimetic PCL-Coll/AE scaffolds, which demonstrate sustained stemness features and a continuous local release of bioactive AE compounds, represent a promising candidate for tissue engineering applications.
本研究提出了一种由电纺聚己内酯 - 胶原蛋白（PCL - Coll）纳米纤维组成的仿生纳米支架，负载生物活性紫草提取物和干细胞，用于开发基于细胞的组织工程构建体。将以其抗氧化和抗炎特性而闻名的紫草提取物掺入PCL - Coll纳米纤维中，得到分别表示为PCL - Coll/AE、PCL - Coll/AE、PCL - Coll/AE和PCL - Coll/AE的纳米支架，对应紫草提取物浓度分别为0.0、5.0、10.0和15.0 wt%。PCL - Coll/AE纳米支架分别表现出62.81±4.78%、59.9±2.10%、52.44±2.66%和46.32±1.35%的高孔隙率值，从而为细胞附着和增殖提供了最佳的三维环境。对紫草提取物的分析表明，存在丰富的紫草素，这是一种关键的生物活性化合物，其在490、520和560 nm处的特征吸收带表明了这一点。场发射扫描电子显微镜（FE - SEM）数据证实了紫草化合物在三维纳米纤维支架内的均匀固定，纤维平均直径为316±137 nm，处于天然胶原纤维的范围内。为了评估完全负载干细胞的支架的结构完整性，我们评估了细胞生长、细胞在PCL - Coll/AE纳米支架内的附着、紫草提取物的细胞保护作用以及干性相关基因（包括Nanog、Rex1、Sox2、Oct4、Klf4和C - Myc）的表达水平。实时聚合酶链反应（PCR）分析表明，干性的关键指标上调，在两周内，从PCL - Coll/AE中的120±15%平均增加到PCL - Coll/AE中的250±30%。这些上调促进细胞增殖并促进细胞周期进程。数据表明，增加支架内生物活性紫草提取物的浓度可增强细胞在支架表面的粘附，并在培养7天后提高细胞活力，活力从PCL - Coll/AE中的109±6.15%上升到PCL - Coll/AE中的145.5±10.11%。针对过氧化氢（H₂O₂）诱导的氧化应激的细胞保护试验表明，PCL - Coll/AE、PCL - Coll/AE、PCL - Coll/AE和PCL - Coll/AE的相对细胞活力分别为42.78±5.85%、49.29±6.79%、64.98±3.32%和78.68±3.09%。这些结果与紫草提取物化合物，特别是紫草素及其衍生物的抗氧化能力相关。因此，负载细胞的仿生PCL - Coll/AE支架表现出持续的干性特征和生物活性紫草化合物的持续局部释放，是组织工程应用的一个有前途的候选者。
