Low-Intensity Pulsed Ultrasound Enhances Intramembranous Bone Healing in a Critical-Size Bone Defect of the Rat Calvaria

Objectives: Low-intensity pulsed ultrasound (LIPUS) has been widely utilized as a biophysical modality accelerating fracture healing, particularly in bones undergoing endochondral ossification. However, its efficacy in facilitating intramembranous ossification remains unclear. This study aimed to evaluate the effects of LIPUS and autologous bone (AB) on bone healing in a critical-size bone defect (CSBD) model of the rat calvaria. Methods: We performed micro-computed tomography (micro-CT) and immunohistochemical TNF-α analysis on bone specimens to assess osteogenesis. Results: Micro-CT demonstrated significant increases in newly formed bone on day 30 compared with days 7 and 15 across all groups (p < 0.001). The highest bone volume was observed in the AB group (26.83%), followed by the LIPUS group (23.74%), and the lowest in the control (15.85%). Immunohistochemical analysis revealed significantly higher TNF-α expression on day 7 in the control group (172.0 ± 1.1) than in the AB (133.8 ± 0.9) and LIPUS (125.2 ± 0.8) groups (p < 0.001). On day 15, TNF-α expression was significantly higher in the LIPUS group (137.7 ± 1.3) than in both the AB (134.2 ± 1.8) and control (126.6 ± 2.2) groups (p < 0.001). At day 30, TNF-α levels in the LIPUS group (147.6 ± 1.9) remained significantly higher than in the control group (115.8 ± 0.9) (p < 0.001), with no significant difference compared to the AB group (146.3 ± 0.8). Conclusions: Although AB grafting achieved the greatest bone volume, LIPUS demonstrated considerable regenerative potential and may represent a promising non-invasive therapeutic approach to enhance intramembranous bone regeneration.