Pulsed Electromagnetic Field-Stimulated Alginate–Chitosan/Hydroxyapatite Nanocomposite Scaffolds for Accelerated Repair of Critical-Size Femoral Bone Defects

Critical-size bone defects remain a major clinical challenge, as impaired endogenous healing and an unfavorable local microenvironment prevent spontaneous bridging of the defect. Here, we report a bio-physical regenerative strategy that combines a three-dimensional, porous alginate–chitosan/hydroxyapatite (Alg-CS/HA) nanocomposite scaffold with targeted pulsed electromagnetic field (PEMF) stimulation. Rod-like hydroxyapatite (HA) nanoparticles (~12 × 86 nm, Ca/P = 1.64) were synthesized and incorporated at 30 wt% into an ionically crosslinked Alg-CS polyelectrolyte matrix. HA incorporation significantly increased the compressive strength (22.41 ± 1.35 vs. 14.53 ± 1.81 kPa) and modulus (156.51 ± 2.15 vs. 74.34 ± 1.43 kPa) of the scaffold, while preserving an interconnected porous architecture (75.97 ± 7.8% porosity) and slowing enzymatic biodegradation (76.70 ± 3.82% mass loss at 28 days). MTT assays on human skin fibroblasts confirmed that both formulations were highly cytocompatible, with viability exceeding 85% at concentrations up to 50 mg/mL. In vivo, bilateral critical-size femoral defects (6 mm diameter) were created in New Zealand rabbits to evaluate bone regeneration under two PEMF regimes (5 and 50 Hz; 0.22 and 0.5 mT) applied for 3 weeks. Untreated defects showed essentially no repair (1.0 ± 2.4%), whereas scaffold implantation alone yielded 20.0 ± 4.3% new bone formation. PEMF stimulation produced a clear frequency- and intensity-dependent enhancement of osteogenesis, culminating in 90.0 ± 5.2% defect bridging in the low-frequency, high-intensity group (5 Hz, 0.5 mT), a 4.5-fold increase over the scaffold-only control. Histological analysis showed dense, mature trabecular bone with abundant osteocytes, active osteoid deposition, and histological features consistent with vascularization in this group. These preliminary results were obtained with n = 3 defect sites per group. Together, they suggest that coupling an Alg-CS/HA nanocomposite scaffold with low-frequency, high-intensity PEMF stimulation is a promising, non-invasive strategy for enhancing the repair of critical-size bone defects, warranting confirmation in larger studies.