Bioactive stem cell-laden 3D nanofibrous scaffolds for tissue engineering

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/AE₀, PCL-Coll/AE₅, PCL-Coll/AE₁₀, and PCL-Coll/AE₁₅, 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/AE₀ to 250 ± 30 % in PCL-Coll/AE₁₅ 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/AE₀ to 145.5 ± 10.11 % in PCL-Coll/AE₁₅. Cytoprotective assays against oxidative stress induced by H₂O₂ revealed relative cell viability for PCL-Coll/AE₀, PCL-Coll/AE₅, PCL-Coll/AE₁₀, and PCL-Coll/AE₁₅ 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/AE₁₅ scaffolds, which demonstrate sustained stemness features and a continuous local release of bioactive AE compounds, represent a promising candidate for tissue engineering applications.