Preliminary Experimental Study Into The Safety Related To Using Medical Ozone In Orthopeadic Clinical Application

BackgroundOzone (O₃) with strong oxidation and instability, in the air at normal temperature and pressure easily reduce to oxygen (has half-life about 45 minutes). Medical ozone (O₂-O₃) is the mixture of ozone and pure oxygen. Different from the daily used and industrial used ozone, medical ozone must have high purity and the concentration of O₃ must be controllable. With high concentration of O₃, it may destroy the cell with strong oxidation damage; but when the concentration is too low, there is not a pharmacological effect. In common, the concentrations of O₃ for clinical application are about 20 to 50μg/ml respectively. In recent years, medical ozone generations general used in abroad has been approved by SFDA [SFDA (I) 20042540177] introduction of domestic clinical application in our country. Medical ozone therapy developed in the past 50 years, but it still strange for our clinicians. In Europe, medical ozone are widely used for the treatment of diabetic foot wide, trauma and the healing of skin ulcers, stroke, ulcerative colitis, arthritis, prolapse of intervertebral disc, et al, its clinical efficacy has been fully affirmed.Compared with the traditional therapy in the application of orthopaedic surgery, particular in the treatments of pain, ozone therapy has its unique advantages:â‘ minor trauma: use only 19 to 21G needle, cause little damage, no hematoma formation , no root or large blood vessels or abdominal injuries have been reported;â‘¡have small adverse reaction after operation, except a few patients in the short term pain;â‘¢simple, compare with other therapy, ozone therapy can ease patients' pain, save time and reduce operations are subject of X-ray radiation;â‘£each operation consumes only case of a needle, medical oxygen and power consumption almost negligible, and extremely well-suited to China's national conditions;⑤indications of a wider scope of age: This technique is safe for elderly patients;â‘¥treatment can be repeated: ozone in vivo metabolism quickly, no cumulative effect of repeat treatment. Clinical results show that ozone therapy has significant advantages, but why it has not really promoted, and even be refused by many doctors and patients? The most fundamental reason is that the basic research of ozone therapy is lagging far behind in clinical practice, many roles and mechanisms of the therapy still unknown, what limit its expansion of the field of clinical treatment. In particular, the lack of basic research on safety of the ozone therapy blocks its development. So the safety of medical ozone therapy studies appears to be every important.At present, the related research focuses on the ozone in the blood cells, in particular the impact of erythrocytes. Ozone has a strong sense of oxidation, contact a certain concentration and duration of ozone will cause the body damage. Most research on the ozone focus on the affection on blood erythrocytes.Although plasma has series of buffer system, such as antioxidant enzymes and glutathione, to prevent accumulation of the superoxide anion and hydrogen peroxide, which may produce alterations of tissue structure secondary to cell membrane degradation, but there is still part of the ozone in the plasma generates reactive oxygen species (ROS). In vitro experiments, the ROS can trigger a number of biochemical pathways. ROS degradation in 0.5 to 1.0 minutes by antioxidant system in the plasma. As soon as contact and dissolved in the plasma, ozone react with anti and polyunsaturated fatty acids, and cascade generated a series of complexes which had been definited, such as hydrogen peroxide and lipid peroxidation products. Studies by Bocci, etc, suggested that when concentration of medical ozone was less than 80μg/ml, buffer systems and anti-oxidation system in the blood and red blood cell, can resistant the ozone oxidization and avoid cells and tissue damage, thus ozone can play a role as drug. When ozone concentrations more than 80μg/ml, it causes tissue damage easily, with the concentration of ozone increasing, there is more obvious damage.In subarachnoid cavity, joint cavity and the abdominal cavity all have a certain amount of buffer substances and antioxidant substances, which can be quickly activated to play antioxidant effect in contact with ozone, and their roles of resistance ozone injury are strong.Muscle and nerve tissue have little tissue fluid surrounding. Therefore, the antioxidant capacity of such tissue needs to be examined. When administration ozone therapy in these tissue, whether the safe concentration of ozone are the same as using in blood , joint cavity or subarachnoid space still uncertain, and need experimental confirmation.Almost all doctors using ozone for treatment declared that ozone itself is a powerful disinfectant, antibiotics and therefore almost no ozone treatment exist the risk of infection. But there were some reports in foreign that fulminating septicemia secondary to oxygen-ozone therapy for Lumbar Disc Herniation. What happened? We know that ozone is a powerful antibacterial, and widely used for industrial and daily disinfection treatment, in particular water disinfection, compared with the traditional method, ozone has a high efficiency, no residues of harmful substances, and economic advantages. Ozone's powerful antibacterial capability relies on its strong oxidation; the magnitude and capacity of antibacterial of ozone depend on concentration and action time. Medical ozone is made by medical ozonizer use the medical oxygen through various channels and connections, which may polluted the medical ozone, various aspects of the manufacturing process and its components also can not guarantee that its sterile; moreover, clinical application of ozone concentration was strictly limited within a scope (medical ozone concentration <80μg/ml, usually 20 to 50μg/ml), the volume of gas is also restricted, so medical ozone dose be restricted. The ozone reacts with antioxidant system in vivo rapidly, and its oxidation ability reduces, as a result, maintain its strong antibacterial ability is questionable. So there are two questions: First, whether medical ozone contains pathogens such as bacteria? Second, if there are bacteria or other pathogens in medical ozone, when it comes to clinical application, dose medical ozone itself has the ability to kill the pathogens? Clinical physicians use ozone for treatments common concern these questions and the latter affect patients on the acceptability of the ozone treatment in a certain extent, and block the development of ozone therapy. But we have not yet seen relevant reports.Purpose1. To explore whether the medical ozone include bacteria, and whether it contain its ability of antibiosis in vivo;2. To evaluate the influence of medical ozone on rat peripheral nerve structure and function;3. To evaluated the pathological changes of skeletalmuscle in normal rabbit by different concentrations of ozone injection.Method 1. First part: Bacterial Culture Experiments of Medical Ozone(1)16 Wistar rats were randomly divided into four groups (n=4 in each group). Medical ozone in different concentrations (10μg/ml, 30μg/ml, 50μg/ml, 80μg/ml) were respectively injected into both knee cavities (0.5ml in each knee cavity) according to four groups; using disposable gas filter needl when obtained medical ozone to inject into the left sides, while without using when it comes to the left sides for control. 24 hours after injection, douche fluid of the joints was obtained for bacterial culture.(2) Medical ozone in different concentrations (10μg/ml, 30μg/ml, 50μg/ml, 80μg/ml, using or without using disposable gas filter needl) from ozonizer 20cm upper puff towards culture capsules for 20 minutes.2. Second part: Effects of Medical Ozone on Peripheral Nerve in Rat30 wistar rats were randomly divided into six groups (n = 5): Groupâ… ozone 10, groupâ…¡ozone 30, groupâ…¢ozone 50, groupâ…£ozone 80, groupâ…¤pure O₂ and groupâ…¥control. In groupâ… ,â…¡,â…¢andâ…£, 1ml of medical ozone (O₂-O₃) 10μg/ml,30μg/ml,50μg/ml,80μg/ml was injected at the junction of Gluteus maximus margin and lateral edge of the long head of biceps femoris respectively, in groupâ…¤, 1 ml of pure O₂ was injected at the same point, and in group VI, punctura without any injection. Ozone was manufactured by E80 ozone generator (Ozoneline Co, Italy). The rats were investigated by both gross measurement and behavioral changes. 1 day, 1 week and 3 week after injection, rat hindlimb footprints were measured and calculated sciatic nerve function index (SFI), and after three weeks, all right sciatic nerves were exposured under anesthesia, Near Neural Stimulation of the rat sciatic nerve and calculated and record nerve conduction velocity, latency and maximum amplitude; Animals were sacrificed for pathology, and ipsilateral triceps surae were talen for wet weight.3. Third part: Impact of Different concentration of Ozone Injection on Pathological Changes of Rat Skeletal Muscle定义30 wistar rats were randomly divided into 5 groups (n=6). Groupâ… for ozone 10, groupâ…¡ozone 30, groupâ…¢ozone 50, groupâ…£ozone 80, and groupâ…¥control. In groupâ… ,â…¡,â…¢andâ…£, 1ml of ozone 10μg/ml,30μg/ml,50μg/ml,80μg/ml was injected into right ectogluteus respectively, and in groupâ…¤, punctura at the same point without any injection.. Biopsy was performed after 10min, 30min and 3 weeks with the lightmicroscope, selecting the injection area.4. Statistics analysisThe experimental data was analyzed by the software of SPSS 13.0, and an associated probability of 5% (P<0.05) was considered significant.Results1. First part: Bacterial Culture Experiments of Medical Ozone(1) Douche fluid culture of the joints, the control group and the 80μg/ml experimental subgroup have no colony, 10μg/ml, 30μg/ml and 50μg/ml subgroup of the experimental group had one blood agar plate with colonies, the number of colonies reduced as medical ozone concentrations increased.(2) All blood agar plates for medical ozone gas directly cultured have no colony.2. Second part: Effects of Medical Ozone on Peripheral Nerve in RatNo serious behavior abnormalities were observed in any animals.; SFI Comparison in the various time and various groups have no significant difference (P<0.05), nerve conduction velocity, latency and maximum amplitude difference amongst the groups was not significant (P<0.05) ; There were no abnormalities in peripheral nerves pathologically after injection.3. Third part: Impact of different concertration of ozone injection on pathological changes of rat skeletal muscleCompared to the control, light microscope of groupâ…¡,â…¢andâ…£showed myocyte swelling, myocyte's bouncary became unclear, transverse striation became disorder, myocyte necrosis, dissolve and vacuolus degeneration partly after 10min both in two groups. Inflammation cells infiltrated in muscle interspace and blood vessel based on myocyte necrosis and dissolve in the injection area after 30min. Myocyte atrophy obviously, fibrous tissue accrementition ponderosus, hyalinization partly could be seen after 3 weeks. There were no abnormal pathological changes in groupâ… andâ…¤.Conclusion1. Medical ozone may contain bacteria, the disinfection of medical ozone after injection may weaken or even disappear, and in the application of medical ozone therapy, should used something as one-time gas filters when connect to ozone generator.2. Concentration from 10μg/ml to 80μg/ml of medical ozone injection around rat's peripheral nerve will not cause serious sequelae, the stucture and function of peripheral nerve have no serious damage, and this finding provides evidence of the safety of ozone injected around peripheral nerve.3. Myocyte damage occurs after injection of ozone with concentration of 30μg/ml,50μg/ml and 80μg/ml, fibrous tissue repairs the damage after 3 weeks. Ozone with concentration of 10μg/ml does little harm to skeletal mucsle.