Transcutaneous CO application combined with low-intensity pulsed ultrasound accelerates bone fracture healing in rats.

Background: Low-intensity pulsed ultrasound (LIPUS) is a non-invasive therapy that accelerates fracture healing. As a new treatment method for fracture, we recently reported that the transcutaneous application of CO accelerated fracture healing in association with promoting angiogenesis, blood flow, and endochondral ossification. We hypothesized that transcutaneous CO application, combined with LIPUS, would promote bone fracture healing more than the single treatment with either of them.

Methods: Femoral shaft fractures were produced in 12-week-old rats. Animals were randomly divided into four groups: the combination of CO and LIPUS, CO, LIPUS, and control groups. As the transcutaneous CO application, the limb was sealed in a CO-filled bag after applying hydrogel that promotes CO absorption. Transcutaneous CO application and LIPUS irradiation were performed for 20 min/day, 5 days/week. At weeks 1, 2, 3, and 4 after the fractures, we assessed the fracture healing process using radiography, histology, immunohistochemistry, real-time PCR, and biomechanical assessment.

Results: The fracture healing score using radiographs in the combination group was significantly higher than that in the control group at all time points and those in both the LIPUS and CO groups at weeks 1, 2, and 4. The degree of bone fracture healing in the histological assessment was significantly higher in the combination group than that in the control group at weeks 2, 3, and 4. In the immunohistochemical assessment, the vascular densities of CD31- and endomucin-positive microvessels in the combination group were significantly higher than those in the control and LIPUS groups at week 2. In the gene expression assessment, significant upregulation of runt-related transcription factor 2 (Runx2) and vascular endothelial growth factor (VEGF) was detected in the combination group compared to the LIPUS and CO monotherapy groups. In the biomechanical assessment, the ultimate stress was significantly higher in the combination group than in the LIPUS and CO groups.

Conclusion: The combination therapy of transcutaneous CO application and LIPUS had a superior effect in promoting fracture healing through the promotion of angiogenesis and osteoblast differentiation compared to monotherapy.