Ozone Therapy Research Articles

In Vitro Investigation of the Antimicrobial Potential and Cell Viability of Different Dosages of Ozone Therapy

ABSTRACT The aim of the study was to evaluate and compare, in vitro, the cytotoxicity of different applied ozone dosages in keratinocyte (NOK) and fibroblast (L929) cell lines, as well as the antimicrobial potential for Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). NOK and L929 cells and strains of gram-positive bacteria S. aureus and gram-negative bacteria E. coli were divided into seven experimental groups: ozone at doses from 0, 0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 mg/L. The effect of ozone on cell viability/cytotoxicity was determined using the alamarBlue® assay, the Live/Dead® assay and the morphometric analysis of the percentage of live and dead cells. The results show that applied ozone dosages of 0.005, 0.01 and 0.02 mg/L showed the highest viability values of NOK and L929 cells, as well as a higher density and percentage of live cells in the 72-hour experimental. However, no difference was observed in the percentage of dead cells between the experimental groups. Furthermore, ozone promoted a reduction in CFUs at all tested dosages. With the results obtained in the present study, it is possible to confirm the restorative potential of ozone therapy in a dose-dependent response, as well as its antimicrobial potential.

Complementary and alternative therapies for managing postoperative pain after lower third molar surgery: a systematic review and network meta-analysis.

The objective of this study was to evaluate the impact of complementary and alternative treatments on postoperative pain following lower third molar surgeries. A comprehensive search of Electronic databases (Embase, MEDLINE via PubMed, and Cochrane Library) and grey literature was conducted up until May 2022. Randomized clinical trials investigating the effect of acupuncture, ozone therapy, laser (LLLT), drainage tube, kinesio-taping, ice therapy, and compressions on pain after LTM surgeries were included. The estimated mean differences (MD) for alternative therapies were pooled using the frequentist approach to random-model network meta-analysis NMA. Eighty-two papers were included in the qualitative analysis; 33 of them were included in the quantitative analyzes. NMA revealed that drainage tube and kinesio-taping were superior in controlling pain 24-hours postoperatively than no-treatment. At 48-hours follow-up, kinesio-taping and LLLT more effective than placebo and drainage tube; and kinesio-taping and LLLT were superior to no treatment. At 72h postoperatively, ozone therapy was superior to placebo; and drainage tube, kinesio-taping, and LLLT were better than no treatment. At 7-days follow-up, ozone and LLLT were superior to placebo; and LLLT and kinesio-taping were superior to no treatment. The SUCRA-ranking placed drainage tube as top-ranking intervention at 48-hours (98.2%) and 72-hours (96%) follow-ups, and ozone (83.5%) at 7-days follow-up. The study findings suggest that these alternative and complementary therapies may be useful in reducing postoperative pain after LTM surgeries, and may offer advantages when combined to traditional pain management methods. Non-pharmacological therapies are gaining popularity among healthcare professionals and patients. This study found that some of these therapies, specifically kinesio-taping and drainage tube were effective in controlling postoperative pain after third molar surgeries. These findings have important implications for clinical practice, as they highlight the potential benefits of incorporating these therapies into postoperative pain management plans.

Impact of ozone therapy on mouse liver mitochondrial function and antioxidant system

Ozone therapy's efficacy might stem from the regulated and mild oxidative stress resulting from ozone's interactions with various biological elements. The present work aimed to characterize the hepatic mitochondrial response to ozone treatment and its relationship with the antioxidant system response. Two groups of mice were used: one control group and another injected intraperitoneally with an O3/O2 mixture (80 ml/kg) for 5 days. Mitochondrial respiration supported by different substrates was significantly inhibited, as well as complexes I and II/III, but not complex IV. The analysis of the electron transport chain complex activity showed significant inhibitions in complexes I and II/III but not in complex IV. These inhibitions can prevent mitochondrial reactive oxygen species (ROS) production. Additionally, there was a decline in glutathione content, unaccompanied by a rise in its oxidized form. The ozone-treated groups showed a significant increase in the activity of superoxide dismutase and glutathione peroxidase, while catalase and glutathione reductase experienced no significant alterations. Adenine nucleotides increased in the ozone group, but only the increase in adenosine diphosphate is significant, so the cell's energy charge is unaffected. This study shows that mitochondria may play a crucial role in ozone treatment. However, it also highlights the need for further studies to understand the molecular mechanism.

Ozone infiltration as an adjunctive treatment to piezoelectric surgery in the management of medication-related osteonecrosis of the jaws: case series of 29 patients.

Medication-related osteonecrosis of the jaws has been reported to be associated with bisphosphonate and RANKL inhibitor medications. This prospective clinical study aimed to assess the outcomes of pre-operative ozone infiltration therapy in patients with established MRONJ. The treatment protocol for ozone applications were designed as 20 applications ozone infiltration therapy followed by surgical interventions of necrotic tissue debridement using piezoelectric surgery instruments. The evaluation of the results based on the clinical and radiologic specifications considering the necrotic lesion reduction and healing. The study included 31 lesions in 29 patients. The mean follow-up was 23.6 months. 25 lesions out of 31 healed totally without any remissions. The outcomes were not affected by any variables such as gender, age, type of pharmacological treatment, lesion location, and MRONJ staging. The statistically significant results were found among the clinical condition of the patients (p = 0.01) and administration route of medications (p = 0.004). Healing was significantly less in patients that received intra-vascular administrations. Clinical conditions of the patients were divided as osteoporosis, oncologic, and arthritis. Significantly better results were obtained in osteoporosis patients. 38% of the population experienced spontaneous sequestration with signs of improvements and the surgical interventions were canceled. According to the results, total healing of MRONJ lesions was seen in 79% patients (81% lesions). Ozone therapy and debridement with Piezoelectric surgery can be considered as a safe and beneficial adjunctive treatment alternative for osteonecrosis lesions in cases of established MRONJ.

The effect of intra-articular ozone injection combined with home-based exercise on pain and function in daily living activities of patients with mild to moderate knee osteoarthritis, a randomized double-blinded controlled clinical trial

BackgroundKnee osteoarthritis is the most common arthritis. Various treatments such as analgesics, exercise therapy, and surgery in high-grade OA have been shown to reduce pain and improve patients' function; however, determining the optimal treatment remains a challenge. Ozone therapy is one of the injection techniques used for symptom relief in these patients. Therefore, this study aimed to evaluate the effect of ozone injection in mild to moderate knee osteoarthritis. MethodsThirty-three patients with grade II-III knee osteoarthritis based on the Kellgren-Lawrence classification were involved in the study, by block randomisation. Totally 42 knees were included. All patients received exercise therapy, 500 mg of acetaminophen tablets (up to 2 g per day as needed), and healthy nutrition. In a double-blinded method, the intervention group received Ozone injections, but the control group received placebo injections. Functional tests, including timed-up-and-go and 6-min walk tests, were assessed at baseline and immediately after the 6-week intervention. In addition, the pain was measured by VAS score, and stiffness and activity of daily living (ADL) were evaluated by KOOS questionnaire before and after a 6-week intervention and then one and six months afterwards. FindingsImprovements in pain and KOOS scores were seen in both groups in the 6th week of injections (p < 0.05), with significant differences between groups. However, the effects on pain and KOOS scores disappeared in the 1st and 6th months of follow-ups in the control group. Nevertheless, the effects persisted in the intervention group compared to the baseline and control group, which means that in the mentioned time points intervention group showed significant improvement compared to the control group (p < 0.05). In addition, functional tests showed significant differences between the two groups in the 6th week of injections (p < 0.001). InterpretationOzone injection is a non-surgical treatment for mild to moderate knee osteoarthritis that could decrease pain and improve function and ADL of patients in the short to mid-term (3–6 months), so it seems that adding Ozone injection to the routine exercise therapy in management of patients with knee OA could improve outcomes.

The Effect of Ozone Therapy Stimulation on Diabetes Wound Healing Process

&lt;p&gt;Health problems that are currently developing include diabetes mellitus (DM) with various complications including diabetic ulcers. Diabetic ulcers are the main cause of prolonged pain and even amputation in DM patients. Diabetic wounds are a complication of diabetes that often occurs in the extremities, increasing morbidity and lessens the patient's personal satisfaction. Ozone therapy is one method used to speed up the healing process for diabetic ulcers. This study plans to examine the impact of ozone treatment feeling on diabetic injury mending. The exploration strategy utilizes a semi exploratory technique with a pretest and posttest non-identical benchmark group plan. The testing strategy is a non-likelihood inspecting method.The population in this study were all patients suffering from diabetic wounds who received wound care at the Edwcare clinic in Langsa city. The respondents of this study were patients suffering from diabetic wounds who received wound care at the Edwacare wound care center in Langsa City, Indonesia consisting of 28 respondents who received modern wound care dressings and 28 respondents received wound care combined with modern dressings and ozone therapy. The research instrument for assessing wound severity used the Bates-Jensen Wound Assessment Tool. Data analysis used the Friedman and Kruskal Wallis tests. Based on the research results, it was found that there was a more significant effect between wound care using a combination of modern dressings and ozone therapy with a Mean Rank value of 39.75 compared to wound care that only received modern dressing wound care with a Mean Rank value of 17.25 with a value of p -value &amp;lt;0.05. The conclusion is that the effect of ozone stimulation significantly influences the healing process of diabetic wounds and is recommended as a nursing intervention that can be applied in treating diabetic wounds.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;Keywords: Diabetes mellitus, diabetic wounds, ozone therapy, stimulation&lt;/em&gt;&lt;/strong&gt;&lt;/p&gt;

Examining the Proliferative Effect of Ozonated Olive Oil and Ozonated Distilled Water on Healthy Colon Fibroblast Cells and Colon Cancer Cells

Ozone therapy is an alternative form of treatment based on the administration of ozone gas to the body in the treatment of diseases and different medical conditions, and ozone applications are recommended in some studies. In this study, it was aimed to determine the possible anti-cancer activity by enriching extra virgin olive oil and distilled water with ozone gas and determining its cytotoxic effect on colon cancer and normal colon fibroblast cells. The effect of ozone enriched extra virgin olive oil and distilled water on cell viability was determined by MTT assay on DLD1 (colon cancer) and CCD-18Co (healthy colon fibroblast) cell lines. In the DLD-1 cell line, ozonated distilled water and olive oil decreased in vitro cell viability at all concentrations and this decrease was most pronounced at higher concentrations (5 and 10 ppm). In the CCD-18Co cell line, ozonized distilled water and ozonized olive oil increased in vitro cell viability at all concentrations, but this increase was not significant compared to the control. The results of this study are consistent with the results of other studies in the literature. Therefore, ozone therapy is thought to be promising in cancer treatment.

Ozone Therapy on Fungi that Cause Onychomycosis

ABSTRACT Onychomycosis is a common fungal infection of the nails, predominantly caused by dermatophytes and is a notoriously difficult condition to treat. Thus, the objective of the present study was to evaluate and compare, in vitro, the efficacy of different applied ozone dosages against the main fungi that cause onychomycosis. Dermatophyte fungi Trichophyton rubrum (ATCC 28,188) and Microsporum gypseum (ATCC 24,102) were incubated at 28 °C for 14 days. Treatments were divided into nine experimental groups according to applied ozone dosages: Control Group 0 mg/m2: fungi were cultured but received no treatment; cultured fungi that received ozone therapy for 157, 314, 471, 628, 785 943, 1100 and 1257 mg/m2. The main results indicate that topical ozone therapy effectively reduced the germination percentage of T. rubrum and M. gypseum in all experimental periods, achieving a complete eradication with a treatment time of 628 and 1257 mg/m2, respectively. In conclusion, ozone therapy used topically with an applied ozone dosage of 628 mg/m2 for T. rubrum and 1257 mg/m2 for M. gypseum promoted antifungal action on the main dermatophytes responsible for critical complications of onychomycosis. Therefore, ozone therapy can be proposed as an adjuvant for dermatological treatments

Comparing Fluoride Varnish, Fissure Sealant and Ozone for Children Caries Prevention

Background: Caries is the most prevalent oral health disease in children globally. Well-known method such as fluoride varnish and fissure sealant has been used to prevent caries overtime. Ozone therapy considered as a new innovation in caries prevention for children which has not been studied much for its effectiveness compared to the other popular method. Objective: This review aimed to compare caries preventive methods with fluoride varnish (FV), fissure sealant (FS) and ozone (O) for children and decide which method is the most effective. Literature Study: PubMed, SpringerLink, EBSCOhost and Science direct were searched from December 2021 to January 2022. The criteria included all randomized controlled trials and clinical trials comparing the caries preventive effect between FV, FS and O on children’s permanent teeth. Discussion: A total of 12 articles were included. Regarding dentin caries prevention, 2 studies found resin FS in favour of no FS and 2 studies found FV in favor of controls. 1 study agreed glass ionomer FS and no FS comparable. 3 studies agreed FV and FS were comparable. 1 study agreed resin FS and glass ionomer FS comparable. 1 study also found FV, FS and O comparable. Regarding dentin remineralization, 2 studies found O were comparable with FS dan calcium hydroxide base. These methods are safe for children. Conclusion: Fluoride varnish, fissure sealant and ozone are effective in preventing children’s caries with their own benefits and drawbacks. None of the methods is superior over another as there were a variety of the data reported.

Use of Ozone Therapy and Thymoquinone in the Prevention of Formaldehyde Toxicity by Inhalation: An Experimental Study.

The study determined the damage caused by formaldehyde (FA) exposure in blood and liver samples using biochemical markers. Histopathological analysis was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method and measurement of CD68 cell density. To what extent the antioxidant molecules thymoquinone (TQ) and ozone (O3) reversed the damage caused by FA exposure was investigated, both when used alone and combined. Fifty-six Sprague-Dawley male rats of eight to ten weeks of age were used in the experiment. The rats were divided into eight groups, with seven rats in each group: the untreated control group, the group treated with TQ (10 mg/kg/day), the group treated with O3 (150 μg/kg/day), the group treated with TQ+O3, the group exposed to FA (10 ppm 8 h/day), the group receiving FA+TQ, the group receiving FA+O3, and the group receiving FA+TQ+O3.Serum aspartate transaminase (AST), alanine transaminase (ALT), total antioxidant (TAS, U/mL), and total oxidant (TOS, nmol/mL) levels were analyzed. TAS and TOS levels, CD68 cell density, and apoptotic cells were determined in liver tissues. FA exposure caused an increase in serum AST and ALT levels of (p<0.05) experimental animals, a decrease in TAS levels in serum (p=0.03) and liver (p>0.05) and an increase in TOS levels (p>0.05), TUNEL positivity (p<0.001), and CD68 cell density (p=0.004). Administration of TQ and O3as antioxidants significantlyreversed biochemical and histopathological alterations in the serum and liver. TQ and ozone therapy suppressed oxidative stress caused by FA exposure and reversed the emerging histopathological deteriorations. Ozone therapy did not suppress the effects of TQ. Therefore, ozone therapy can be given as a supportive therapy along with the main therapeutic agents. We think TQ and ozone therapy may be useful to protect individuals exposed to FA.

Efficacy and safety of ozone therapy for knee osteoarthritis: an umbrella review of systematic reviews.

The objective of this study is to evaluate the effectiveness and safety of ozone therapy (OT) in the treatment of knee osteoarthritis (KOA), which is the most common form of the disease. We analysed systematic reviews (SRs) of randomised controlled trials (RCTs) using the "A MeaSurement Tool to Assess systematic Reviews" (AMSTAR2) instrument to evaluate their quality. We developed a narrative synthesis report with eight SRs (15 RCTs/3,685 patients) to summarise the findings. The AMSTAR2 analysis indicated that all reviews had critically low confidence ratings. Statistically significant effects in pain reduction using OT compared to placebo groups were reported in three SRs. OT was shown to be comparable to other therapies in one SR and not superior in the other five. Six SRs highlighted the need for additional RCTs with improved methodological quality to confirm the efficacy of OT for KOA. SRs found fewer consistent effects for improving joint function. Regarding safety, seven SRs reported a low prevalence of minor adverse events linked with OT. Finally, this umbrella review highlights the beneficial effects and safety of OT in the treatment of KOA, particularly in pain control. The low methodological quality of RCTs and SRs limits the possibility of drawing conclusions on the effectiveness of the procedure in comparison to other therapies. Ensure adequate compliance with guidelines such as Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and AMSTAR2 has the ability to improve the quality of SRs in this area.

Ozone therapy effect in medication-related osteonecrosis of the jaw as prevention or treatment: microtomographic, confocal laser microscopy and histomorphometric analysis.

This study aimed to evaluate the efficacy of ozone therapy in the preoperative (prevention) and/or postoperative (treatment) of MRONJ. Forty male Wistar rats were caudally treated with zoledronic acid (ZOL) and to ozone therapy before extraction (prevention, POG), after extraction (treatment, TOG), or both (prevention and treatment, TPOG), and treated with saline (SAL). The animals received intramuscular fluorochrome (calcein and alizarin), and 28 days postoperatively, they were euthanized, and the tissues were subjected to microtomographic computed tomography (microCT), LASER confocal, and histomorphometric analyses. Micro-CT showed a higher bone volume fraction average in all groups than that in the ZOL group (P < 0.001), the ZOL group showed high porosity (P = 0.03), and trabecular separation was greater in the TOG group than in the POG group (P < 0.05). The mineral apposition rate of the POG group was high (20.46 ± 6.31) (P < 0.001), followed by the TOG group (20.32 ± 7.4). The TOG group presented the highest mean newly formed bone area (68.322 ± 25.296) compared with the ZOL group (P < 0.05), followed by the SAL group (66.039 ± 28.379) and ZOL groups (60.856 ± 28.425). Ozone therapy modulated alveolar bone repair in animals treated with ZOL, mainly after surgery trauma, leading to bone formation as healing tissue. Osteonecrosis has been a challenge in dentistry, and owing to the lack of a consensus regarding therapy, studies presenting new therapies are important, and ozone has been one of the therapies explored empirically.

Efficiency of ozone (O3) therapy on experimental acidic skin burns in rats

In this study, it was aimed to investigate the efficacy of O3 therapy in experimentally induced HF skin burns in rats. A total of 20 healthy male Wistar Albino rats (weighing 250-300 g, aged 16 weeks) were used as the material of the study. They were divided into two groups (as experimental and control groups) of 10 rats which were housed individually and fed ad libitum. Hydrofluoric acid (HF) skin burns were induced in all animals. The ozonized (20 µg O3/mL) liquid vaseline was applied topically for seven days to the experiment group as well as parallelly, a saline solution was applied to the control group. In the histopathologic evaluation, inflammation, vascularization, epithelial regeneration and fibrosis were evaluated.&#x0D; The epithelial proliferation and collagenization were higher and statistically significant in the experimental group, while the infiltration was higher and statistically significant in the control group. As a result of this study, it was observed that ozone plays an important role in the tissue repair process in chemical burns. The increase of fibroblast activation and re-epithelization in the experimental group demonstrated that ozone therapy could be suggested as first aid in HF skin burns. It was concluded that more studies on ozone are needed to shed light on the subject.

Ozone therapy as a method of prevention and treatment in surgery and oncology (a literature review)

The effectiveness of ozone therapy in the treatment of cancer pathology lies in the proven process of destruction of atypical cells due to the inhibition of vicious anaerobic metabolism. Ozone selectively causes the death of cancer cells, since healthy cells have their own antioxidant defense system, while atypical cells do not. Ozone therapy for cancer pathology is an auxiliary treatment method that involves the effect of the oxygen-ozone layer on cancerous cellular structures. Numerous clinical studies confirm that ozone therapy is well tolerated and is extremely rarely accompanied by side effects and does not cause negative reactions in the human body. The advantages of ozone therapy include: safety for human health, minimal side effects, proven effectiveness, low cost of equipment and the possibility of use as cancer prevention. In addition, this treatment method is indicated for people suffering from chronic diseases and disorders that increase the risk of cancer, such as atherosclerosis, hypertension, type 2 diabetes, obesity, immunosuppression, menstrual disorders, etc. The use of ozone in medicine is very promising due to its anti-inflammatory, immunomodulatory and antitumor effects, and its hepatoprotective effect makes it possible to reduce the patient’s time in the hospital due to the rapid restoration of liver function in the postoperative period.

Treatment Strategies for Incisors of Children Affected by Molar Incisor Hypomineralization: A Narrative Review

Today, molar incisor hypomineralization (MIH), which affects approximately one in seven children, is defined as a hypomineralized developmental enamel defect that often impacts at least one permanent first molar and frequently affects permanent incisors as well. Symptoms and signs include demarcated opacities of various colors, post-eruptive enamel deterioration, atypical caries and restorations, hypersensitivity, tooth loss due to MIH, and difficulty in achieving anesthesia. A detailed review of the scientific literature shows that there are many studies evaluating different treatment approaches for permanent first molars affected by MIH. On the other hand, there are very few scientific studies evaluating treatment approaches for affected incisors in patients with MIH. Most of these studies consist of case reports or series. White/creamy and/or yellow/brown demarcated opacities are commonly observed in affected incisors in patients with MIH. While these opacities increase the susceptibility of enamel to deterioration and dental caries, they also cause aesthetic problems and related psychosocial consequences. Treatment methods, such as resin infiltration, microabrasion, and/or dental bleaching, have been proposed for aesthetic and restorative purposes in affected incisors in patients with MIH. Additionally, various approaches to increase mineral content and relieve hypersensitivity have been recommended. The number of randomized controlled and prospective studies is quite low, but many case reports and case series have been encountered. The purpose of this review was to provide a comprehensive overview of the different treatment management modalities for permanent incisors affected by MIH. As a result, while resin infiltration, dental bleaching, microabrasion, and/or etch–bleach–seal techniques are preferred for aesthetic and restorative purposes in these teeth, it has been observed that agents containing casein phosphopeptide amorphous calcium phosphate, casein phosphopeptide amorphous calcium fluoride phosphate, fluoride, and calcium glycerophosphate increase the mineral content. Additionally, studies have reported that ozone and low-level laser therapy, in addition to these remineralizing agents, reduce hypersensitivity in these teeth. Although the findings of this review indicate that the level of evidence for current approaches is not high, clinicians may prefer one or more of the treatment approaches mentioned in this article based on experience and patient expectations.

Effect of ozoniotherapy in the treatment of necrosis after hair transplantation: case report

Introduction: Nowadays, ozone plays an important role in wound healing and tissue repair as a therapy and as antimicrobial, bactericidal and fungicidal agent. It attains recognition in hair transplantation as a treatment for necrosis due to hypoxic-ischemic local syndrome. Objective: to demonstrate the therapeutic evolution of ischemia in a hair transplant after ozone therapy sessions. Methods: The patient was evaluation and gave consent to photographic records. He went through 30 topic applications of ozonated oil, with a 10 drop dosage daily; 12 bag ozone sessions for 10 minutes; besides subcutaneous applications, with 30% ozone concentration and a very small gas volume (1-2 ml) with 30G needle. Results: Evolution of the case was registered with images and tissue coloring and changes evidenced. Conclusion: It is clear that ozone therapy made wound healing and tissue repair faster, since there was an increase of epithelial cells and neoangiogenesis due to therapy, resulting in almost complete repair of the patient’s hair transplant at the end of the sessions.

American Academy of Orthopaedic Surgeons Technology Overview Summary: Platelet-Rich Plasma (PRP) for Knee Osteoarthritis.

The Platelet-Rich Plasma (PRP) for Knee Osteoarthritis Technology Overview is based on a systematic review of current scientific and clinical research. Through analysis of the current best evidence, this technology overview seeks to evaluate the efficacy of PRP for patients with knee osteoarthritis. The systematic literature review resulted in 54 articles: 36 high-quality and 18 moderate-quality. The findings of these studies were summarized to present findings on PRP versus control/placebo, acetaminophen, non-steroidal anti-inflammatory drugs, corticosteroids, exercise, prolotherapy, autologous conditioned serum, bone marrow aspirate concentrate, hyaluronic acid, and ozone therapy. In addition, the work group highlighted areas that needed additional research when evidence proved lacking on the topic and carefully noted the potential harms associated with an intervention, required resource utilization, acceptability, and feasibility.

Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Evaluation of the healing potential of short-term ozone therapy for the treatment of diabetic foot ulcers.

The availability of research on short-term ozone therapy for diabetic foot ulcers (DFUs) is limited, and even when it is accessible, it mainly comprises of basic analysis conducted during long-term ozone therapy. This study was to evaluate the efficacy of short-term ozone therapy in promoting wound healing in DFUs. A retrospective analysis was conducted on 89 patients with type 2 diabetes complicated by DFUs. The patients were divided into two groups: ozone therapy group (n=41) and control group (n=48). Wound condition, change of bacterial types, changes in inflammatory indicators (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], and procalcitonin [PCT]), vascular endothelial growth factor (VEGF), cytokines [Interleukin 6 (IL-6) and tumor necrosis factor-α(TNF-α)], and oxidative stress levels (superoxide dismutase [SOD], malondialdehyde [MDA], and total antioxidant capacity [T-AOC]) were observed pre-treatment and after 1 week. After a 12-week of follow-up, wound healing rate, amputation rate, inpatient day, duration of antibiotics, reinfection rate, incidence of new ulcers, readmission rate, and reoperation rate, and cumulative wound healing rate using Kaplan-Meier curves were assessed. After 1 week of treatment, the ozone therapy group showed higher VEGF, SOD, and T-AOC levels compared to the control group (P<0.05), while CRP, PCT, ESR, IL-6, TNF-α, MDA levels and bacterial types were lower (P<0.05). The ozone therapy group had a higher wound healing rate after a 12-week follow-up (P<0.05). Kaplan-Meier curves indicated a higher cumulative wound healing rate in the ozone therapy group (P<0.05). Additionally, the ozone therapy group had lower inpatient day, duration of antibiotics, reinfection rate, and readmission rate compared to the control group (P<0.05). Short-term ozone therapy is effective in promoting wound healing in DFUs by reducing inflammation, increasing growth factor levels, improving oxidative stress status, shortening healing time, and improving long-term prognosis. These findings suggest the potential of short-term ozone therapy as a valuable treatment modality for DFUs.

Nociceptive and histomorphometric evaluation of the effects of ozone therapy on the rat masseter muscle in a carrageenan model of myofascial pain

ObjectiveThis study evaluated the effects of intramuscular ozone therapy on nociception, inflammation, and tissue damage caused by the injection of carrageenan in the masseter muscle of rats. DesignRat masseter muscles were injected with saline or carrageenan. Seventy-seven adult male rats were divided into six groups: Sal, saline; Car, carrageenan; Ibup + Sal, ibuprofen and saline; Ibup + Car, ibuprofen and carrageenan; O3 + Sal, ozone and saline; and O3 + Car, ozone and carrageenan. The mixture of 5% ozone and 95% oxygen (20 µg/mL) was administered three times in the course of a week. Nociceptive responses in the masseter muscles were measured using a head withdrawal threshold, determined by an electronic von Frey anesthesiometer. The animals were euthanized one or eight days after the carrageenan injection, and the masseters were submitted to histological and histomorphometric analyses. ResultsMechanical allodynia and inflammation levels were reduced in the Ibup + Car group compared to the other groups. Myonecrosis was similar among carrageenan-treated groups. Picrosirius red stained sections showed more collagen fibers and more regenerating myofibers in the O3 + Car group compared to the other groups. Eight days after carrageenan injection, the O3 + Car group showed neutrophils close to the regenerating myofibers. ConclusionsIntramuscular ozone therapy did not alleviate mechanical allodynia, and it did not protect the masseter muscle against the deleterious effects produced by carrageenan, probably due to the mode of administration of this therapeutic agent.