The efficiency of ozone therapy in the prevention of heterotopic bone formation in an animal model

Abstract

Objectives: In our study, we aimed to evaluate the efficacy of prophylactic ozone therapy (OT) for heterotopic ossification (HO) development after hip surgery in a rat model.

Materials and methods: We randomly divided a total of 20 Wistar albino rats into two equal groups (n=10) as the control group (Group 1) receiving daily intraperitoneal injections of 1 mL of Ringer's lactate solution for a duration of two weeks after undergoing hip surgery and the group (Group 2) receiving OT daily at a dose of 1 mL of an ozone/oxygen combination via intraperitoneal route for a period of two weeks after hip surgery. Hip surgery was performed to mimic the formation of HO. After 12 weeks, the animals were evaluated functionally and sacrificed. Following sacrification, histopathological, and radiological assessments were performed.

Results: The modified Brooker staging method was used for radiological evaluation, and all animals in the control group showed at least Stage 1 HO formation. However, only three animals (30%) in the OT group showed HO formation (p=0.006). In histopathological evaluation, the median HO rates in the high power field were 42.5 (range, 30 to 55) in the control group and 0 (range, 0 to 25) in the OT group (p=0.003), and the median leukocyte count was 5.5 (range, 3 to 7) in the control group and 1 (range, 0 to 3) in the ozone group (p=0.006). Radiological HO, histopathological HO, and inflammation were significantly lower in the ozone group (p<0.05).

Conclusion: Our study results suggest that OT is effective in decreasing HO formation in rats following hip surgery.

Figure 1. Demonstration of the mechanism of heterotopic ossification and our hypothesis that ozone inhibits this mechanism by regulating different pathways.: HO: Heterotopic ossification; VEGF: Vascular endothelial growth factor; BMP: Bone morphogenetic protein Nrf2: Nuclear factor erythroid 2-related factor 2; TLR: Toll-like receptors; NF-κB: Nuclear factor-kappa B; TNF-α: Tumor necrosis factor alpha; IL: Interleukin; HIF1a: Hypoxia-inducible factor 1-alpha; ARNT: Aryl hydrocarbon receptor nuclear translocator; HRE: Hypoxia response element; BMP: Bone morphogenic protein; VEGF: Vascular endothelial growth factor. (Created in BioRender, (2025) https://BioRender.com/cxy7kyj.

Figure 2. (a) Intraoperative image showing blunt dissection of the gluteal muscles after the skin incision. (b) Exposure to the greater trochanter and drilling procedure.

Figure 3. (a) A computed tomography (CT) image of the rat with Stage 1 heterotopic ossification in the coronal pelvis (arrow). (b) The image displays heterotopic ossification (Stage 3) between the major trochanter and acetabulum in a coronal rat pelvic CT scan, as well as a 3D (three-dimensional) rat CT scans.: CT: Computed tomography.

Figure 4. (a) The image shows heterotopic ossification (Stage 1), with hematoxylin and eosin (H&E) at 2× magnification (arrow). (b) Stage 2 heterotopic ossification observed at 2× magnification (arrows).

Figure 5. Radiological degree of heterotopic ossification in different animal groups.

Figure 6. (a) An image of the femoral head and its surrounding area shows no inflammation in the ozone therapy (OT) group. (b, c) The image displays inflammation in the femoral region in the control group.