**Background:** Cartilage defects from trauma, tumors, and inflammation are common clinical problems with limited self-repair capacity. Tissue engineering offers a promising approach, but implantation of in vitro engineered cartilage into immunocompetent animals triggers immune responses that lead to extracellular matrix erosion and cartilage resorption. Previous clinical work reported only a 60% success rate for ear reconstruction using engineered cartilage, with the remaining 40% compromised by inflammatory cell infiltration. Existing strategies to mitigate immune reactions—including decellularization, cell sheets, prolonged pre-cultivation, and drug-loaded scaffolds—have limitations in efficacy, cost, or donor site morbidity. This study aimed to develop an immunoisolation strategy using a curcumin-loaded electrospun nanofibrous membrane that would physically block inflammatory cell infiltration while locally releasing an anti-inflammatory agent to suppress cytokine-mediated damage.
**Methods:** PLGA (molecular weight 250,000, L/G 75/25) was dissolved in trichloromethane at 10% w/v. Curcumin and PLGA were mixed at a 1:5 w/w ratio and electrospun into nanofibrous membranes. Characterization included SEM for morphology, FTIR spectroscopy (750–4000 cm⁻¹, resolution 4.0 cm⁻¹, 64 scans), and HPLC (C18 column, detection at 424 nm). In vitro curcumin release kinetics were measured over 14 days in PBS (pH 7.4, 37°C) using UV spectrophotometry at 421 nm. Curcumin stability was assessed by measuring remaining soluble curcumin over 8 hours. Chondrocytes isolated from Sprague Dawley rat articular cartilage (digested with 0.25% type II collagenase for 6 hours) and RAW264.7 macrophages were cultured in DMEM with 10% FBS. Cytocompatibility was evaluated via live/dead staining and CCK-8 assay. Anti-inflammatory effects were assessed by seeding LPS-pretreated (1 μg/mL, 12 h) RAW264.7 macrophages onto membranes for 24 h, followed by Western blotting and ELISA for IL-1β, IL-6, and TNF-α. Porous PLGA scaffolds (12% w/v, freeze-dried at −60°C for 24 h) were seeded with chondrocytes at 50 × 10⁶ cells/mL and cultured for 3 weeks. Engineered cartilage was wrapped with Cur/PLGA or PLGA membranes (edges sealed by hot compression) and subcutaneously implanted into rats (n=6 per group). Samples were harvested at 2 and 4 weeks for gross observation, wet weight measurement, biomechanical testing (Instron-5542, 1 mm/min), biochemical analysis (GAG by Alcian blue, COL II by ELISA), and histological evaluation (H&E, safranin-O, toluidine blue, COL II immunohistochemistry, CD3 and CD68 immunohistochemistry, IL-1β/IL-6/TNF-α immunofluorescence). Statistical analysis used one-way ANOVA and Student's t-test with significance at p < 0.05.
**Key Results:** The Cur/PLGA nanofibrous membrane exhibited nanoscale fibers with 76.13% porosity (vs. 74.26% for PLGA). FTIR confirmed characteristic peaks of both PLGA (1752 cm⁻¹, C=O) and curcumin (1509 cm⁻¹, C=C), with new peaks at 1600 and 1585 cm⁻¹. HPLC confirmed curcumin encapsulation. In vitro release showed sustained curcumin release over 14 days with an initial burst in the first 4 days, accelerated release from days 4–12, and decelerated release on days 12–14. Curcumin stability was markedly improved: approximately 80% of pure curcumin degraded within 8 hours, while 90% of curcumin in the Cur/PLGA membrane remained intact after 8 hours. Live/dead staining showed comparable chondrocyte viability between PLGA and Cur/PLGA groups, with low numbers of dead cells in all groups. SEM confirmed RAW264.7 macrophage adhesion to both membranes, with cell size exceeding pore dimensions. Western blotting and ELISA demonstrated significantly lower expression of IL-1β, IL-6, and TNF-α in the Cur/PLGA group compared to the PLGA group. In vitro engineered cartilage showed typical cartilage appearance with lacunar structure (H&E), abundant GAG deposition (safranin-O), and enhanced COL II secretion (immunohistochemistry). At 2 and 4 weeks post-implantation, Cur/PLGA-packaged samples maintained white cartilage-like appearance, while PLGA and control groups showed red, fibrosis-like appearance. The ranking for wet weight, Young's modulus, GAG content, and COL II content was consistently Cur/PLGA > PLGA > control, with all values decreasing from week 2 to week 4. Histologically, Cur/PLGA samples retained lacunar structure, strongly positive GAG staining (safranin-O and toluidine blue), and COL II deposition at both time points, while cartilaginous phenotype was nearly lost in PLGA and control groups by week 4. Inflammatory cell infiltration (CD3+ and CD68+ cells) and cytokine expression (IL-1β, IL-6, TNF-α) followed the pattern Cur/PLGA < PLGA < control at both time points, with intensities higher at week 4 than week 2. Notably, positive staining for inflammatory markers was lower inside the membrane than outside in both Cur/PLGA and PLGA groups.
**Clinical Implications:** This study presents a practical immunoisolation strategy that combines physical barrier function with local anti-inflammatory drug delivery to protect tissue-engineered cartilage from immune-mediated destruction. The approach addresses a critical barrier to clinical translation—the inflammatory response that has limited the success of engineered cartilage implants in immunocompetent hosts. The Cur/PLGA nanofibrous membrane is simple to prepare, cost-effective, and adaptable to complex tissue shapes (ear, nose, trachea). However, several limitations require attention before clinical application: curcumin was nearly completely released by day 14, which may be insufficient for complete cartilage maturation; only one curcumin concentration was tested; the maximum observation period was 4 weeks; and only a rat model was used. Future work should focus on prolonged curcumin release systems, optimal drug concentration determination, long-term (e.g., 1-year) observations, and validation in large animal models with complex immune systems (sheep or swine).