Transcutaneous CO₂ application combined with low-intensity pulsed ultrasound accelerates bone fracture healing in rats

Abstract Background Low-intensity pulsed ultrasound (LIPUS) is a non-invasive therapy that accelerates fracture healing by promoting endochondral ossification, osteoblast differentiation, and angiogenesis. We recently reported that the transcutaneous application of CO 2 using a hydrogel accelerated fracture healing in association with the promotion of angiogenesis, blood flow, and endochondral ossification. We hypothesized that transcutaneous CO₂ application, combined with LIPUS, would promote bone fracture healing more than the single treatment with either of them. Methods Femoral shaft fractures were produced using drop weight in twelve-week-old rats. Animals were randomly divided into four groups: the combination of CO 2 and LIPUS, CO 2 , LIPUS, and control groups. After the CO 2 absorption-enhancing hydrogel was applied, the limb was sealed in a CO 2 -filled bag for 20 min. LIPUS was applied for 20 minutes. At weeks 1, 2, 3, and 4 after the fractures, we assessed the bone union rate and fracture healing score using radiography. We histologically assessed the degree of fracture healing and evaluated gene expression by real-time PCR to assess chondrogenic differentiation, osteogenic differentiation, and angiogenesis at weeks 1, 2, 3, and 4. For the biomechanical assessment, a three-point bending test was performed at week 4. 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 2 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 gene expression assessment, significant upregulation of Runx2 and VEGF was detected in the combination group compared to the LIPUS and CO 2 monotherapy groups. In the biomechanical assessment, the ultimate stress was significantly higher in the combination group than in the LIPUS and CO 2 groups. Conclusions The combination therapy of transcutaneous CO 2 application and LIPUS had a superior effect in promoting fracture healing through the promotion of angiogenesis and osteoblast differentiation compared to monotherapy. In clinical practice, combination therapy may be more beneficial to patients than monotherapy.