经皮 CO 应用联合低强度脉冲超声加速大鼠骨折愈合。

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.

背景：低强度脉冲超声（LIPUS）是一种促进骨折愈合的非侵入性治疗方法。作为骨折的一种新的治疗方法，我们最近报道经皮 CO 应用可促进血管生成、血流和软骨内骨化，从而加速骨折愈合。我们假设经皮 CO 应用联合 LIPUS 比单独应用其中任何一种治疗方法更能促进骨骨折愈合。

方法：在 12 周龄大鼠中产生股骨干骨折。动物随机分为四组：CO 和 LIPUS 联合组、CO 组、LIPUS 组和对照组。作为经皮 CO 应用，在应用促进 CO 吸收的水凝胶后，将肢体密封在充满 CO 的袋子中。经皮 CO 应用和 LIPUS 照射每天进行 20 分钟，每周 5 天。在骨折后 1、2、3 和 4 周，我们使用放射照相术、组织学、免疫组织化学、实时 PCR 和生物力学评估来评估骨折愈合过程。

结果：在所有时间点，联合组的 X 线骨折愈合评分均明显高于对照组，在第 1、2 和 4 周时均明显高于 LIPUS 和 CO 组。在组织学评估中，联合组的骨折愈合程度在第 2、3 和 4 周时明显高于对照组。在免疫组织化学评估中，第 2 周时，联合组 CD31 和内粘蛋白阳性微血管的血管密度明显高于对照组和 LIPUS 组。在基因表达评估中，与 LIPUS 和 CO 单药治疗组相比，联合组 runt 相关转录因子 2（Runx2）和血管内皮生长因子（VEGF）的表达显著上调。在生物力学评估中，联合组的极限应力明显高于 LIPUS 和 CO 组。

结论：与单独治疗相比，经皮 CO 应用联合 LIPUS 治疗在促进血管生成和成骨细胞分化方面具有更优的促进骨折愈合效果。