Engineering a collagen/kappa-carrageenan membrane functionalized with extracellular vesicles for enhanced bone regeneration.

Bone regeneration is essential for functional rehabilitation and remains a major challenge in regenerative medicine, driving the development of advanced biomaterials. This study aimed to evaluate collagen/κ-carrageenan (κ-carr) membranes incorporating medium-derived extracellular vesicles (mEVs) as a bioactive strategy for bone regeneration. Membranes were fabricated using collagen extracted from rat tail tendons combined with κ-carr to improve mechanical stability and biocompatibility. mEVs were isolated from MC3T3-E1 cell culture supernatants at a concentration of 20 μg/mL and incubated with the membranes under agitation to promote vesicle absorption with collagen fibrils. Vesicles were characterized by nanoparticle tracking analysis (NTA), zeta potential, atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The membranes were evaluated for mechanical properties, morphology, wettability, and mEV release over 100 h by NTA. In vitro assays assessed cell viability and cytotoxicity, as well as matrix mineralization by Alizarin Red staining after 21 days. For in vivo evaluation, 5-mm critical-size calvarial defects were created in Sprague-Dawley rats and treated with blood clot (Control), collagen/κ-carr membrane (Membrane), or collagen/κ-carr membrane incorporating mEVs (Membrane + mEV). Bone regeneration was evaluated by in vivo micro-computed tomography (μCT) immediately after surgery (T0) and at 14 (T1) and 28 (T2) days. Statistical analysis used two-way ANOVA followed by Tukey’s post hoc test. The mEVs exhibited a particle size of 80–100 nm, negative zeta potential (−12.6 mV), characteristic nanoscale morphology, and TNAP activity. In vitro assays demonstrated low cytotoxicity and increased cell viability after 48 h. mEV release from the membranes remained stable for up to 100 h, followed by a decrease. Alizarin Red staining indicated enhanced mineralization in the presence of the membranes compared with the control. μCT analysis revealed significantly greater bone formation at 28 days in the Membrane + mEV group, with increased bone volume (BV), bone volume fraction (BV/TV), and bone surface (BS) compared with the other groups. Overall, incorporation of mEVs into collagen/κ-carr membranes enhanced bone regeneration, highlighting their potential as an innovative strategy for bone tissue engineering and regenerative medicine.

This work was supported by FAPESP (2024/12254-1; 2024/02130-3; 2019/08568-2; CNPq 318127/2025-3).