Abstract
Introduction
The purpose of the study is to evaluate the effect of medical ozone therapy on fracture healing in rats.
Materials and methods
20 male Wistar-Albino rats were randomly divided into two groups as Control Group (n = 10) and Ozone Group (n = 10). The fracture model was created by bilateral femur transverse osteotomy and fixation with an intramedullary Kirschner wire. No medical treatment was applied to the control group, whereas Ozone gas at a dose of 1 cc/kg at a concentration of 20 µg/ml was administered intraperitoneally to the ozone group for 8 weeks. All groups were sacrificed at the end of the 8th week. Radiological examination was performed by direct radiography of all femurs. The left femurs of both groups were examined histopathologically (Hematoxylin-Eosin), immunohistochemically (BMP-7, Osteocalcin, Osteopontin, TRAP) and histochemically (Masson Trichrome). Biomechanical (3-point bending test) analysis was performed on the right femurs. The liver and kidneys were also examined histopathologically.
Results
Radiographic (p = 0.008) and histopathological (p = 0.001) examinations revealed that fracture healing scores of the Ozone Group were significantly inferior compared to the Control Group. In the immunohistochemical examination, the positivity scores of BMP-7 (p = 0.009), Osteocalcin (p = 0.001) and Osteopontin (p = 0.023) were statistically significantly lower in the Ozone group compared to the control group, while the TRAP (p = 0.016) positivity score was significantly higher. In histochemical examination, Masson Trichrome positivity was found to be significantly lower in the Ozone group compared to the control group (p < 0.001). Biomechanical analysis revealed that fracture healing was lower in the Ozone group compared to the Control group in parameters Yield Force (p = 0.012), Yield at Elongation (p = 0.030), Maximum Force (p = 0.009), Maximum Elongation (p = 0.023), Maximum Stress (p = 0.045). As a result of the examination of possible side effects on liver (p = 1.000) and kidney (p = 0.181), no statistically significant difference was found between the groups.
Conclusion
Medical ozone therapy demonstrated a detrimental effect on fracture union, as evidenced by inferior radiological, histopathological, immunohistochemical, histochemical, and biomechanical outcomes. These findings indicate that systemic ozone treatment may adversely influence bone healing processes.
Keywords: Medical ozone therapy, 3-point bending test, BMP-7, Osteocalcin, Osteopontin, Masson trichrome, Biomechanical, Ozone, Fracture healing, Bone healing
Introduction
Ozone (O3) is an unstable gas molecule naturally found in the atmosphere, consisting of three oxygen atoms. It was first used in bacterial disinfection and industrial areas [1]. The medical use of ozone began in the early 20th century in the treatment of infections in open fractures, serious wounds and gas gangrene. Medical ozone exhibits antimicrobial, anti-inflammatory, antihypoxic, immunostimulatory, regenerative, vasodilatory, angiogenic, and neuroprotective effects [2]. Medical ozone therapy in years of research; It has been applied in musculoskeletal diseases, dental diseases, genitourinary system diseases, gastrointestinal system diseases, cardiovascular diseases, peripheral vascular diseases, neurological diseases and many evidence-based studies have been conducted [3]. It has been observed in many publications that ozone produces very different results in different doses and applications. As a matter of fact, while ozone creates a toxic effect when inhaled into the lungs, it appears to have a positive effect with the appropriate dose and treatment method [4, 5]. While some patient groups benefit from the immune-stimulating effects of ozone, in another patient group, its anti-inflammatory properties are utilized. After dissolving in the plasma, it forms carcinogenic reactive oxygen derivatives in the first reaction, while it has an antioxidant effect in the later stages [6, 7].
Medical ozone therapy is used in orthopedic diseases such as osteoarthritis, disc herniation, carpal tunnel syndrome, diabetic foot, shoulder pathologies and inflammatory arthritis [8]. Ozone therapy has gained increasing clinical interest in musculoskeletal medicine, especially in degenerative and pain-related conditions. Recent studies have demonstrated that intra-articular ozone injections may provide short-term improvements in pain and function in knee osteoarthritis, although the magnitude and duration of benefit vary among studies [9, 10]. Additionally, ozone therapy has been explored as a minimally invasive option for lumbar disc herniation, with meta-analytic data indicating potential reductions in radicular pain and disability [11]. These findings highlight the growing musculoskeletal applications of ozone therapy and underscore the importance of clarifying its effects on bone regeneration and fracture healing. Although the effect of ozone therapy on bone has been investigated, its effect on fracture healing is still unknown.
In our literature review, we could not find a study in which the effect of ozone on fracture union was examined in detail as radiological, histopathological, immunohistochemical (Osteopontin, Ostecalcin, BMP-7, TRAP), histochemical (Masson Trichrome) and biomechanical (three-point bending test). This study aimed to investigate the effects of medical ozone therapy administered post-surgery on fracture healing in a rat femoral fracture model. Our hypothesis is that medical ozone therapy, which has come to the fore with its regenerative and anti-inflammatory activity in recent years, will delay the union of the fracture.
Material-method
The study protocol was reviewed and approved by the Kütahya Health Sciences University Animal Experiments Local Ethics Committee (Approval No: 2019.04.04, Date: 09.05.2019). All procedures conformed to the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals (NIH, 8th edition). Twenty male Wistar Albino rats (350–400 g) were obtained from the Experimental Animal Research and Application Center of Kütahya Health Sciences University (Kütahya, Türkiye). All animals were bred and maintained under pathogen-free conditions in accordance with institutional and national guidelines for laboratory animal welfare. No privately owned or farm animals were used in this study; therefore, informed consent from external owners was not applicable.
The sample size was determined in accordance with the ARRIVE guidelines to ensure adequate statistical power while minimizing animal use. Based on previous experimental studies evaluating fracture healing in rats treated with biologically active agents, a 30% difference in maximum force between groups was considered biologically relevant, with an expected standard deviation (SD) of approximately 24% of the control mean. Using a two-tailed independent samples t-test with an α level of 0.05 and a desired power (1–β) of 0.80, the required sample size per group was calculated according to the formula n = 2 × (Z_α/2 + Z_β)² / d², where d represents the standardized effect size (Cohen’s d = 1.25). This calculation indicated that 10 rats per group would be sufficient to detect a meaningful difference in the primary outcome with 80% power. Therefore, 20 male Wistar-Albino rats (n = 10 per group) were included in the study. The selected sample size was also consistent with similar studies in the literature and adhered to the 3R principles (Replacement, Reduction, Refinement) by avoiding unnecessary animal use.
Animals were randomly assigned to the Control or Ozone group using a computer-generated random sequence (random number generator in IBM SPSS 27.0). Group allocation was concealed until treatment initiation to avoid selection bias.
Wistar Albino male rats with an average weight of 350–400 g were used. The rats were followed in each separate cage without water and feed restrictions. Animals were housed individually in polycarbonate cages under controlled temperature (22 ± 2 °C), humidity (50 ± 10%), and 12-hour light/dark cycles, with wood-chip bedding and nesting material for enrichment. Water and standard chow were available ad libitum. Humane endpoints were predefined, including early euthanasia criteria for severe distress, infection, or loss of mobility. Rats were observed twice daily for general health, wound integrity, feeding, and activity during the postoperative period.
The rats to be used in the experiment were anesthetized by using Ketamine/Xylazine (90 mg/10 mg) after 4 h of fasting. (90 mg/10 mg). For prophylactic antibiotic therapy, a single dose of 50 mg/kg Cefazolin sodium (was administered intraperitoneally 20–30 min before the operation. Open surgical osteotomy was performed on all rats by creating a transverse fracture in the middle part of the femoral shaft bilaterally. After anatomical reduction, the fracture line was fixed intramedullary with a 1.2 mm Kirschner wire.
Rats were randomly divided into two groups: Control (n = 10) and Ozone (n = 10). Both groups were given 2 doses of 35 mg/kg paracetamol intraperitoneally for postoperative analgesia. No mobilization restrictions were applied to the rats. All rats were mobile and mobile in the cage.
No additional application was made to the control group to measure baseline values. To the ozone group, with a medical ozone generator that applies the electron transfer method from medical oxygen, at a concentration of 20 µg/ml 1 cc/kg intraperitoneally for 8 weeks three times a week (Monday-Wednesday- Friday) was administered as a single dose [12].
At the end of the 8th week, all animals were humanely euthanized under deep anesthesia. Euthanasia was performed by cervical dislocation following intraperitoneal administration of ketamine (90 mg/kg) and xylazine (10 mg/kg), ensuring complete loss of consciousness and absence of reflexes prior to the procedure. This method was selected in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020) and approved by the institutional ethics committee. In addition to the femurs, liver and kidneys of all rats were excised.
The investigators performing radiological, histopathological, immunohistochemical, histochemical, and biomechanical evaluations were blinded to group assignments throughout the analysis phase. Data coding and labeling were handled by an independent researcher not involved in the experimental procedures.
Radiographic assessment
Control and ozone group antero-posterior (AP) direct radiographs were taken on the femurs of the rats. Radiographic images were evaluated using the Lane-Sandhu fracture classification system [13].
Histological assessment
Left femurs removed from rats histopathological, immunohistochemical and histochemical examinations were performed. Only histopathological examination was performed on the liver and kidneys. After the sections were prepared, all tissue sections were prepared under a light microscope and images were viewed under microscope camera photographed and archived.
Histopathological assessment
Femoral bone tissue samples, liver and kidneys stained appropriately with H&E [14]. The tissue sections were scored according to Huo et al.‘s histopathological fracture healing scale [15]. In the histological evaluation of the liver of both groups, scoring was done using the Suzuki classification. Kidney sections belonging to the groups were histologically; The severity and extent of tubular necrosis/atrophy, tubular vacuolar changes, glomerular damage, vascular congestion/thrombosis, and interstitial inflammation were evaluated. Each slice was scored between 0 and 4 [16].
Immunohistochemical assessment
Femoral bone tissue samples stained with bone union and regeneration markers, Osteopontin (Anti- Osteopontin Antibody, ) Osteocalcin (Anti -Osteocalcin Antibody (OC4-30)), BMP (Anti-BMP 7) and TRAP (Anti-TRAP antibody) antibody kits.
In the evaluation of bone tissues showing antibody positivity, immune positive bone tissue scoring was performed for each tissue sections, with 10 different areas at 400x magnification. Staining intensity is classified as 0–3 (0 = negative staining, 1 = low staining intensity, 2 = medium staining intensity, and 3 = high staining intensity), while percentages of positively staining cells are 0–4 (0 = 0%; 1 = 1–25%; 2 = 25–50%; 3 = 50–75%; 4 = 75–100%). Histological scores were calculated by adding these two scores. Immunohistochemical-histochemical positivity scoring system was used for BMP-7, Osteocalcin and Osteopontin immunohistochemical and Masson Trichrome histochemical staining [17].
Osteoclast counts were performed with multinucleated cells with more than three nuclei in TRAP-stained tissue sections. Counting results in ten different areas in four hundred magnifications were recorded and analyzed statistically [18].
Histochemical assessment
Masson Trichrome staining, areas stained with blue indicate regeneration of immature bone tissue, areas stained with red indicate areas without regeneration in mature bone [19]. In the assessment of the tissue sections, the areas painted with blue were counted and scored with the positivity scoring system [17].
Biomechanical assessment
After cleaning the soft tissues around the bone tissue, the intramedullary Kirschner wires were removed to perform the three-point bending test. Three-point bending test was performed with a biomechanical tester (Shimadzu AG -IS 5 KN, Kyoto, Japan). The femoral bones were placed on the horizontal axis, with the fracture line in the centre, at 40 mm loading distance. After the fracture occurred with a constant compression speed of 2 mm/min, it was continued until plastic deformation was observed (Fig. 1).
Fig. 1.
Three point bending (Shimadzu AG-IS 5 KN Biomechanical Tester) (Kyoto, Japan)
As a result of the test, maximum strength (N), maximum elongation (mm), maximum stress (N/mm2), maximum percent elongation (%) yield force (N), yield at elongation (mm) and force-time graph for each femur obtained.
Statistical assessment
In the descriptive statistics of the data, mean, standard deviation, median, lowest, highest, frequency and ratio values were used. The distribution of variables was measured with the Kolmogorov Smirnov test. Independent sample t test and Mann-Whitney u test were used in the analysis of quantitative independent data. IBM Statistical Package in Analytics for the social Sciences (SPSS) 27.0 program was used. The results were considered statistically significant for p < 0.05.
Results
In our study, no signs of infection were observed in the surgical procedure and ozone application area of rats belonging to both groups during the 8-week follow-up period. It was observed that there was no neurovascular pathology in all rats and mobilization was uneventful. Implant failure was not observed in any rat. There was no loss of rats during the study.
As a result of the experiment, radiological findings of all femurs, histological findings of left femur, liver and kidneys, and biomechanical examination findings of right femurs were obtained (Tables 1 and 2). The distribution graphs of the findings of the groups are shown in Fig. 2.
Table 1.
Statistical results of studies on fracture union of the groups
| Control group | Ozone group | p | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| mean ± sd | Median | mean ± sd | Median | |||||||
|
Radiological (Lane-Sandhu Fracture Score) |
9.8 ± 3.3 | 12.0 | 6.7 ± 3.8 | 7.0 | 0.008 | m | ||||
|
H&E (Huo Fracture Healing Score) |
7.5 ± 2.1 | 8.0 | 3.1 ± 1.6 | 2.0 | < 0 . 001 | t | ||||
| BMP-7 | ||||||||||
| Intensity | 2.7 ± 0.5 | 3.0 | 1.5 ± 0.7 | 1.0 | 0.002 | m | ||||
| Staining % | 2.6 ± 0.8 | 3.0 | 2.1 ± 0.6 | 2.0 | 0.116 | m | ||||
| Score | 5.3 ± 1.3 | 6.0 | 3.6 ± 1.2 | 3.0 | 0.009 | m | ||||
| Osteocalcin | ||||||||||
| Intensity | 2.6 ± 0.5 | 3.0 | 1.5 ± 0.5 | 1.5 | 0.001 | m | ||||
| Staining % | 2.5 ± 1.1 | 2.5 | 1.2 ± 0.4 | 1.0 | 0.004 | m | ||||
| Score | 5.1 ± 1.4 | 5.0 | 2.7 ± 0.8 | 2.5 | 0.001 | m | ||||
| Osteopontin | ||||||||||
| Intensity | 2.8 ± 0.4 | 3.0 | 2.2 ± 0.6 | 2.0 | 0.025 | m | ||||
| Staining % | 2.9 ± 1.1 | 3.0 | 1.9 ± 1.0 | 1.5 | 0.049 | m | ||||
| Score | 5.7 ± 1.4 | 6.0 | 4.1 ± 1.4 | 4.0 | 0.023 | t | ||||
| TRAP | 4.7 ± 2.7 | 4.0 | 12.7 ± 8.5 | 10.0 | 0.016 | t | ||||
| Masson Trichrome | ||||||||||
| Intensity | 3.0 ± 0.0 | 3.0 | 2.1 ± 0.6 | 2.0 | < 0 . 001 | m | ||||
| Staining % | 3.1 ± 1.0 | 3,5 | 1.5 ± 0.7 | 1.0 | 0.002 | m | ||||
| Score | 6.1 ± 1.0 | 6.5 | 3.6 ± 1.2 | 3.0 | < 0 . 001 | t | ||||
Bold values indicate statistically significant results (p < 0.05)
tindependent sample t test /mMann-whitney u test
Table 2.
Groups belonging biomechanics three point bending test results and statistics
| Control group | Ozone group | p | ||||
|---|---|---|---|---|---|---|
| Mean ± sd | Median | Mean ± sd | Median | |||
| Three Point Bending | ||||||
| Yield force | 67.5 ± 21.1 | 74.1 | 44.9 ± 14.3 | 41.0 | 0.012 | t |
| Yield at elongation | 0.6 ± 0.3 | 0.5 | 0.8 ± 0.2 | 0.8 | 0.030 | t |
| Max force | 71.7 ± 21.4 | 78.8 | 47.8 ± 14.8 | 44.8 | 0.009 | t |
| Max elongation | 0.6 ± 0.3 | 0.5 | 0.9 ± 0.2 | 0.9 | 0.023 | t |
| Max stress | 33.0 ± 14.2 | 30.0 | 21.7 ± 8.6 | 21.1 | 0.045 | t |
| Max percent elongation | 4.4 ± 2.1 | 3.5 | 5.1 ± 1.5 | 5.5 | 0.326 | m |
Bold values indicate statistically significant results (p < 0.05)
tindependent sample t test /mMann-Whitney u test
Fig. 2.
Distribution frequencies of the groups a Lane-sandhu radiological fracture healing score, b Huo histopathological fracture healing score, c BMP-7 positivity score, d Osteocalcin positivity score, e Osteopontin positivity score, f TRAP osteoclast count score, g Masson trichrome positivity score, h Three point bending maximum force (N)
The Lane-Sandhu Fracture Scores of the Control and Ozone Groups is shown in Fig. 3. In the radiographic analysis, fracture union was found to be statistically significantly lower in the Ozone group than in the Control group (p = 0.008). The radiological evaluation results of the groups are shown in Table 1; Fig. 2a.
Fig. 3.
The lane-sandhu fracture scores (a. Control, b. Ozone)
In the histopathological examination, fracture union was found to be statistically significantly lower in the Ozone group than in the Control group (p = 0.001) (Table 1; Fig. 2(b)). Fracture healing was significantly less in the Ozone group compared to the control group, with extensive fibrous tissue and little cartilage tissue observed (Fig. 4(a)). Only cartilage tissue formations were observed instead of bone tissue (Fig. 4(a2)).
Fig. 4.
a1 Significant fracture healing in the control group, mature bone tissue image (star). a2 Decreased fracture healing in ozone group compared to control group, cartilage tissue image only (arrow), H&E x 10. b1 Significant BMP-7 expression in the control group (Arrow). b2 Decreased BMP-7 expression in the ozone group (arrow), BMP-7 × 10. c1 Significant osteocalcin expression in the control group (Arrow). c2 Decreased osteocalcin expression in the ozone group (Arrow) Osteocalcin x 10. d1 Significant osteopontin expression in the control group (Arrow). d2 Decreased osteopontin expression in the ozone group (Arrow), Osteopontin x 10. e1 Few osteoclasts expressing TRAP in the control group (arrow). e2 Increased number of osteoclasts expressing TRAP in the ozone group (arrow), TRAP x 10. f1 Diffuse new shaped callus structure in blue color is observed in the control group (Arrow), f2 Decreased callus structure in the ozone group (Arrow), Masson Trichrome x 10
BMP-7, Osteocalcin and Osteopontin expression was significantly increased in the Control group and decreased in the Ozone group (Fig. 4 (b), (c), (d)). In the immunohistochemical examination, BMP-7 (p = 0.009), Osteocalcin (p = 0.001) and Osteopontin (p = 0.023) positivity score was found to be statistically significantly lower in the Ozone group than in the Control group (Table 1; Fig. 2 (c), (d), (e)).
There were fewer TRAP-expressing osteoclasts in the control group compared to the Ozone group (Fig. (4e)). The number of multinucleated osteoclasts in TRAP staining was found to be statistically significantly higher in the Ozone group than in the Control group (p = 0.016) (Table 1, Fig. (2f)).
Blue reshaped callus tissue was observed in Masson Trichrome staining in the control group; A small amount of callus tissue was seen in the ozone group (Fig. (4f)). Masson Trichrome positivity score was found to be statistically significantly lower in the Ozone group than in the Control group (p < 0.001) (Table 1, Fig. (2 g)).
The force-time graph obtained in the 3-point bending test of rats in both groups is shown in Fig. 5. In the biomechanical examination, Yield Force (p = 0.012), Yield at Elongation (p = 0.030), Maximum Force (p = 0.009), Maximum Elongation (p = 0.023), Maximum Stress (p = 0.045) obtained by the three-point bending test were significantly lower in the Ozone group than in the Control group (Table 2; Fig. 2 (h)).
Fig. 5.
Force-time graph of the 3-point bending tests of the control and ozone groups
Statistically significant difference wasn’t observed between the groups in the histopathological examination of the liver (p = 1.000) and kidneys (p = 0.181).
Discussion
Medical ozone exhibits antimicrobial, anti-inflammatory, antihypoxic, immunostimulatory, regenerative, vasodilatory, angiogenic, and neuroprotective effects [2]. In our study, we found that medical ozone therapy had negative effects on fracture union histologically, biomechanically, and radiologically in the femur fracture model.
The strong local and systemic anti- inflammatory efficacy of ozone therapy has been proven by studies. Although important information about the mechanism by which the anti-inflammatory activity occurs in these studies, the mechanism of action has not been clearly resolved yet. TNF-R2 level is a receptor specific to the fracture healing phase [20]. Chen et al. found that it decreased TNF-alpha and TNF-R2 levels and increased TNF-R1 levels after intra-articular ozone injection in rats with rheumatoid arthritis [21]. The decrease in the levels of TNF-R2 in ozone treatment gives rise to the logic that it will delay fracture healing. IL-10 is an anti-inflammatory cytokine. Tartarini et al. conducted a similar study in 2020; compared the inflammatory cytokine levels in the synovial fluid of animals treated with ozone and showed that proinflammatory cytokines decreased and IL-10 levels increased [22]. It shows anti-inflammatory activity by suppressing proinflammatory cytokines with Nrf-2 phosphorylation. Roche et al. In a study conducted on 28 multiple sclerosis patients in 2017, he applied ozone with rectal insufflation at a dose of 20 µg/ml three times a week for one month and measured antioxidant and proinflammatory cytokine levels. He reported that the proinflammatory cytokine TNF-alpha and IL-1β levels decreased after treatment and increased CK-2 expression with Nrf-2 phosphorylation in mononuclear cells [23]. Zeng et al. In his clinical study on both experimental animals and humans, research was conducted on lesions treated with local ozone therapy for psoriasis. He discovered that Toll like receptor 2 (TLR2) / nuclear factor-κB (NF-κB) signaling pathway was suppressed and initiated the anti-inflammatory process. It also confirmed significant suppression of spleen T helper 17 (Th-17) cells in a mouse model [24]. Previous studies have demonstrated the anti-inflammatory efficacy of ozone therapy in other models. In our study, although cytokines were not measured, the observed delayed healing may hypothetically be associated with suppression of inflammatory mediators.
Ozone therapy can be applied in different treatment doses and methods in clinical and preclinical applications. Systemic effects are produced by both rectal infusion and intraperitoneal methods related to ozone therapy in experimental animals [6]. Recent studies have provided clearer insight into the dose-dependent biological behavior of ozone therapy in musculoskeletal and bone tissues. Jeyaraman et al. highlighted that ozone exhibits a narrow therapeutic window, where low, well-controlled doses promote beneficial oxidative preconditioning, whereas higher doses induce mitochondrial stress, increase oxidative burden, and interfere with regenerative pathways critical for tissue repair [25]. Malatesta et al. further demonstrated that low-dose ozone acts as a eustress stimulus by activating the Nrf2–ARE antioxidant defense system, while escalating doses disrupt cellular redox homeostasis and may suppress the inflammatory signaling required during early phases of bone healing [26]. Chen et al. In his study on 5 rats treated with intraperitoneal medical ozone therapy at concentrations of 10, 20, 30, 40, 50 µg/ml, TNF and TNF receptors were investigated in rats treated with an osteoarthritis model. It was found that anti-inflammatory mediators were low in the group treated with ozone treatment up to 40 µg/ml [21]. In our study, regenerative dose selection was made, and intraperitoneal ozone application was chosen with repeated low-dose therapy. Also, the range of 20–40 µg/ml is the optimum concentration to activate the immune system [27, 28]. In accordance with previous studies, we applied 20 µg/ml concentration and 1 ml/kg ozone dose. In the preclinical experiments, it has been seen that the application of ozone as three doses per week is safe [23]. At the same time, the concentration and dose of ozone we used in our study are compatible with current clinical treatments [6]. When determining the ozone treatment dose, it is important to choose an effective dose that does not cause toxic effects on tissues and organs. When determining the ozone treatment dose, it is important to choose an effective dose that does not cause toxic effects on tissues and organs. We performed histopathological examination to determine whether the treatment applied in our study had any pathological effects on the liver and kidneys. As a result of the investigation, we found that applying low-dose ozone therapy for a long time did not cause toxic effects on the liver and kidneys of the rats.
This study was designed to evaluate the biological effect of a single systemic ozone dose that falls within the clinically accepted therapeutic range. Although this ensured methodological consistency and animal welfare (3R ethical principles (Replacement, Reduction, and Refinement), the absence of a dose–response comparison limits the generalizability of the findings. Although this approach ensured methodological consistency and animal welfare, even at this regenerative dose, ozone therapy resulted in a delayed fracture-healing response, suggesting that its biological effects may vary depending on tissue type and inflammatory phase. Future studies including multiple concentrations and administration routes would be valuable to clarify potential dose-dependent effects of ozone on bone healing.
The maximum load found during the biomechanical test gives information about the qualitative and quantitative status of the callus tissue. Various methods have been used for biomechanical analysis in experimental animal studies. Traction, compression, three-point bending and rotation are a few of them [29]. Three-point bending test was used in our study. In our study, we interpreted the low values obtained in the ozone group as ozone therapy delaying the fracture healing.
There are different studies in the literature that include the effects of ozone therapy on bone healing. In the study conducted by Duman et al. in a unilateral fracture model on 60 rats, a total of 6 ozone and control groups were formed, which were terminated at the 2nd, 4th and 6th weeks. After the fracture model, ozone treatment was applied to the ozone group by the rectal infusion method with a dose of 1 ml/kg. In the study, histopathological and biomechanical assessment were performed on the groups. According to the results of the study, they suggested that ozone treatment had a positive effect on fracture healing. In the histopathological analysis of the study, the scores of the Modified Lane-Sandhu scoring system were evaluated between the groups for each criterion. The total Lane-Sandhu fracture score of the groups was not determined. The union and bone marrow organization were found to be higher in the Ozone group, which was terminated in the 2nd week, compared to the Control group. No significant difference was found in the Ozone group, which was terminated in the 4th week, compared to the control group. Bone marrow organization at 6 weeks was higher than the control group. The sum of the values of these criteria belonging to the groups was taken into statistical analysis. Among these criteria, union, cortical bone formation and bone marrow organization were found to be significantly higher in the Ozone group. There was no significant difference in spongy bone formation between the two groups. In the biomechanical examination part of the study, no significant difference was observed in the groups terminated in the 2nd and 4th weeks. Biomechanical examination results were found to be significantly higher in the Ozone group, which was terminated at the 6th week, compared to the control group [30]. Although it was reported in this study that ozone had a positive effect on fracture healing, it was proven in our study that it had a negative effect, supported by many parameters.
Frascino et al. In 2013, he investigated the effect of ozonized water on monocortical bone defect in hyperglycemic rats on 48 rats. Rats were divided into hyperglycemic and control groups and cortical defect was created on the anterolateral aspect of the femur in rats. The bone defect of the ozone group was washed with 100 ml of ozonized distilled water (0.004 mg/ml), and with 100 ml of distilled water in the control group. On the 14th and 21st days, the animals were sacrificed, and histological examination was performed. Although an increase in blood vascularity was observed in the histomorphological examination, no change in trabecular regeneration was detected. An increase in osteoclasts was observed in TRAP staining. He suggested that ozonized water activates the increase of osteoclasts with the increase of reactive oxygen radicals (ROS) [31]. In our study, osteoclast activity was found to be significantly higher in ozone group TRAP staining.
Erdemci et al. investigated the effects of local and systemic ozone therapy in rats in their study on alveolar bone formation after tooth extraction. They reported that local ozone did not cause any difference in the alveolar bone healing process, while long-term application of systemic ozone before and after surgery could increase trabecular bone density. However, they did not find a statistically significant difference in both groups with the control group [32].
Kan et al. In their study on 60 rats, after creating a 5 mm bone defect in the skull, groups treated with hyperbaric oxygen (2.8 atm) and intraperitoneal ozone (0.7 mg/kg) for five postoperative days were formed and examined histomorphologically. Rats were sacrificed on the 5th, 15th and 30th days. It has been reported that it significantly increased bone healing in the Hyperbaric Oxygen and Ozone groups compared to the control group, but the application of both did not make a significant difference [33].
Early inflammation plays a critical regulatory role in initiating fracture healing, and suppression of this inflammatory cascade has been shown to impair the transition from hematoma formation to callus development. Pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are essential for recruiting mesenchymal progenitor cells and promoting angiogenesis during the early stages of bone repair. Kon et al. demonstrated that interruption or downregulation of these cytokines leads to delayed callus formation and impaired fracture healing kinetics in vivo, highlighting the necessity of a tightly regulated early inflammatory response for normal bone regeneration [20]. In this context, the strong anti-inflammatory effect of ozone therapy may suppress these early signals and thereby contribute to the delayed fracture union observed in our study. Although our findings suggest that the pronounced anti-inflammatory activity of ozone may contribute to delayed fracture healing, this remains speculative since inflammatory cytokines (e.g., TNF-α, IL-6, IL-10) and redox markers were not measured in this study. The study was initially designed under the hypothesis that ozone would have a regenerative effect; therefore, no cytokine profiling was planned. As the results demonstrated an opposite trend, future studies should include cytokine assays and molecular analyses to clarify the mechanistic relationship between ozone’s anti-inflammatory properties and bone regeneration.
Conclusion
In our study, it was our first goal to examine the effect of medical ozone therapy, which has come to the forefront with its regenerative and anti-inflammatory activity in recent years, on fracture union. In our study, radiological, histopathological, immunohistochemical, histochemical and biomechanical examinations were performed. With the results of the study, we found that medical ozone therapy delayed fracture healing and affected it negatively. We believe that the methods, results and interpretations we used will be illuminating for preclinical and clinical studies in the field of orthopedics and traumatology.
Acknowledgements
The authors would like to thank the Kütahya Health Sciences University Experimental Animal Application and Research Center for their valuable assistance during the study process.
Author contributions
Conceptualization: S.K., S.N.K. Methodology: S.K. Validation: S.K.Ö., N.D.D., S.K. Formal analysis: S.K., N.D.D. Investigation: S.K., S.İ., T.C.D. Resources: S.K. Data curation: S.K., A.N.D. Writing – original draft: S.K. Writing – review & editing: S.K.Ö., N.D.D., S.N.K. Visualization: S.K. Supervision: S.K. Project administration: S.K. All authors read and approved the final manuscript.
Funding
This study was supported by the Kütahya Health Sciences University Scientific Research Projects Coordination Unit as a medical specialization thesis study (Project code: TTU-2020-44, Meeting No: 2020/8, Date: 12.08.2020).
Data availability
The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
This study was approved by the Kütahya Health Sciences University Animal Experiments Local Ethics Committee (Approval No: 2019.04.04, Date: 16.05.2019; and Approval No: 2020.06.01, Date: 27.05.2020). All procedures were conducted in accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals (NIH, 8th edition).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.