The Effect Of Ozone On Rabbit Knee Osteoarthritis And Its Mechanisms

1. BackgroundThere is an increasing pubilc health problem concerned with osteoarthritis worldwile coming along with life expectancy extending and population aging. It is estimated that 10% of the global health problems came from osteoarthritis, according to the World Health Organization (WHO) report. China, now, is undergoing a rising number of osteoarthritis patients as well, which has become a huge burden on families and society.There is a commun view that osteoarthritis is formed by the interaction of several factors, the specific pathogenesis of which is not fully understood, however. The treatment of osteoarthritis is mainly to relieve the pain and improve joint function in patients, thereby enhance the quality of their life. Prevention and health care is considered to be the simplest and most effective method, the rest of which are only to relieve pain and restore partial joint function, but have a disadvantage of quick relapse as well as side effects that increase the risk of digestive system diseases and vascular diseases, for example, the oral medicines and intra-articular injection. At present, total hip replacement is well accepted treatment for osteoarthritis, but it has the limitation of high expence, short service life and surgical risks which have restricted its popularization in China.Ozone is found to be effective in pain killing, and has been perspribed for osteoarthritis patients, for it has better painkilling effect than that of hormone mixed local anesthetic, and lower side effect rate compared to using steroid. However, basic research lags far behind clinical practice on ozone treatment of osteoarthritis, which greatly restricts the application in the treatment of osteoarthritis.The current study on ozone is focused on its role and mechanism in the blood, but no report was found about the ozone effect on articular cartilage and synovium cells. The blood has a strong buffer system and a variety of antioxidants which reduce the damage under the ozone oxidation, which cannot explain the ozone effect on articular cartilage and synovium cells, because the joint cavity is in a hypoxic environment, and cartilage has no blood supply.There is no international guidiance to regulate the usage of ozone in treatment of osteoarthritis; as a result, different voices on the concentration, dose, period and frequency of ozone usage emerged. As a strong oxidizing substance, it is no doubt that excessive use of ozone can damage the tissue structure. Therefore, further study of effect of ozone therapy on osteoarthritis should be conducted to confirm the concentration and frequency of ozone in the treatment of osteoarthritis with good results and low side effect rate.2. Objectives(1) Study on the effects of different concentrations of ozone on the rabbit knee osteoarthritis'cartilage and synovium and its rule.(2) Study the mechanisms of different concentrations of ozone on rabbit osteoarthritis:through stduy on the inflammatory mediators IL-1, TNF-a and MMP-13 to determine their roles of different concentrations of ozone and their mechanisms; and to explore the concentration of ozone which is most suitable for the treatment of knee osteoarthritis in rabbits.(3) Study on the effects of different frequency of ozone injections in the rabbit knee osteoarthritis'cartilage and synovium, and observe its effect on inflammatory mediators IL-1, TNF-a and MMP-13 and its rule.(4) Study on the effects of different concentrations of ozone in the normal rabbit knee's cartilage and synovium and its rule, and observe its effect on inflammatory mediators IL-1, TNF-a and MMP-13, and to explore whether there is cumulative effect of ozone injection.(5) Study on Effect of ozone on culture of rabbit chondrocytes. (6) Work out the ozone treatment guidline of rabbit knee osteoarthritis; provide more scientific evidence for the development of clinical criterian of ozone therapy for osteoarthritis.3. Materials and Methods(1) The effect of different concentrations of ozone on the rabbit knee osteoarthritis.①Animal ModelThe model of knee osteoarthritis was established by extra-articular mathods that plaster cast fixation was used in the flexed position of rabbit knee for 6 weeks to avoid the interruption of the environment within the knee joint in rabbits.②To determine the concentration of ozone:The general dosage of ozone is 20μg/ml～40μg/ml, and the maximum is 60μg/ml. The ozone generator in this study from Germany Humares Company has a maximal output of 55μg/ml, and the ozone concentration will be set to arithmetic progression of 10μg/ml,20μg/ml,30μg/ml,40μg/ml and 50μg/ml to assess the effect of zone therapy with different dosage on the tissue structure in the joints as precise as possible.③Animal groupsThe experimental animals were divided into normal group, model group, oxygen group and 5 ozone groups:group ozone 10μg/ml, group of ozone 20μg/ml, group of ozone 30μg/ml, group of ozone 40μg/ml ozone and group of ozone 50μg/ml. All the rabbits were made osteoarthritis models expect the normal group. The oxygen group was given an injection of 2ml pure oxygen in the knee joint cavity weekly for 3 weeks. And the ozone groups were injected the concentration of ozone as they were marked weekly for 3 weeks.④Pathological examination and the Mankin score:After the cartilage specimens decalcified, embedded in paraffin, sliced, HE staining made into pathology, index Mankin score according to the cartilage surface structure, cell times, cell cloning and matrix staining,0 point for normal,10 points being the most serious injury. After synovium embedded in paraffin, sliced, HE staining made into pathology to observed synovium complete or not, with or without hyperplasia, whether inflammatory cells have immersed, whether apoptosis or necrosis, then evaluation the effect of different concentrations of ozone on synovium.⑤Detection of IL-1, TNF-αand MMP-13 in cartilage and synoviumIL-1, TNF-αand MMP-13 was detected using enzyme-linked immunosorbent assay (ELISA) method. Purified antibody specific for IL-1, TNF-αand MMP-13 is separate linked chemically to an enzyme. The samples to be tested are coated onto the surface of plastic wells to which they bind nonspecifically; residual sticky sites on the plastic are blocked by adding irrelevant proteins. The labeled antibody is then added to the wells under conditions where nonspecific binding is prevented, so that only binding to IL-1, TNF-αand MMP-13 separate causes the labeled antibody to be retained on the surface. Unbound labeled antibody is removed from all wells by washing, and bound antibody is detected by an enzyme-dependent color-change reaction. This assay allows arrays of wells known as microtiter plates to be read in fiberoptic multichannel spectrometers, greatly speeding the assay.⑥Data analysisThe experimental data was analyzed by the software of SPSS 13.0. Differences were considered statistically significant at P<0.05.(2) The effect of different times of ozone injection on the experimental knee①Animal ModelNew Zealand white rabbits had right knee for osteoarthritis modeling by the previous mathod.②To determine the concentration of ozoneAbove experimental results sμggest that the optimum concentration of ozone in rabbit knee osteoarthritis is 20μg/ml, so in this experiment, the ozone concentration using 20μg/ml.③Animal groups:According to the frequency of ozone injections were divided into 4 groups: group 0 time, group 1 time, group 3 times, group 5 times were treated with 0 (that is, not to puncture the ozone but injection only),1,3 and 5 times of ozone injection. The concentration of ozone injection is 20μg/ml, in the multiple injections groups, two injections interval of 1 week.④Pathological examination and the Mankin score:The same as described before.⑤Cartilage and synovium tissue of IL-1, TNF-a and MMP-13 test:The same as described before.⑥Data statistical analysis:The same as described before.(3) the times of ozone in different effect on the normal rabbit knee①Animal Model:This part of experiment use normal rabbit knee joint use, no modeling.②To determine the concentration of ozone:In this part, the ozone concentration is 20μg/ml.③Animal groups:According to the frequency of ozone injections were divided into 4 groups: group 0 time, group 1 time, group 3 times, group 5 times were treated with 0 (that is, not to puncture the ozone but injection only),1,3 and 5 times of ozone injection. The concentration of ozone injection is 20μg/ml, in the multiple injections groups, two injections interval of 1 week.④Pathological examination and the Mankin score:The same as described before.⑤Cartilage and synovium tissue of IL-1, TNF-a and MMP-13 test:The same as described before.⑥Data statistical analysis:The same as described before.(4) Effect of ozone on culture of rabbit chondrocytes①Cartilage derived cells, isolated and culturedNew Zealand white rabbits (either sex) weight 2～2.5kg, sterile blades cut to take the thickness of the cartilage of about 0.1～0.2mm, filled with PBS solution into the dish, cut into pieces, put into 25ml culture flask and washed cartilage with PBS twice to remove residual blood, add 0.1% II typecollagenase 2ml digestion for 20 minutes, the supernatant was discarded after gentle pipetting, and repeated once more; then add 0.1%Ⅱcollagenase 5ml, under 37℃for 6 to 8 hours to digest shocks, 1500r/min centrifugal cell pellet for 3 min, abandoned after the clear digestive fluid, washed three times with culture medium to wash away the cell surface of the digestive enzymes; re-suspended into the cell suspension. Sterile 200-mesh strainer filter cell suspension, then counting.By 5×105/hole density of cells seeded in 6 well plate suspension, choose low-sμgar medium DMEM,20%fetal bovine serum, 1%(v/v) double-resistant, 0.69mM L-ascorbic acid and 2mM Valley ammonia amide, buffer selected HEPES. The 6-well placed in PCO2 5% of the incubation chamber for 72 hours. Under the microscope showed that most cells adhered to the solid growth into Asia and the integration of state, given intervention.②Experimental groups:Cells were divided into six groups(group A-F):group A for control (normal), B for NF-κB inhibitor group (NF-Ⅰ), group C for ozone (O3), D for IL-1β(IL), E means IL-1β+NF-κB inhibitor group (IL+NF-I), F for IL-1β+ozone group (IL+O3). Each group had 5 samples.According to the results of preliminary experiments, the use of ozone concentration is 20μg/ml, the amount of ozone added to is about 0.5ml, that each culture's ozone hole by adding a dose of 10μg. The concentration of NF-κB inhibitors diluted to 100μg/ml, each hole by adding 100μL, then 10μg/holes. IL-1βconcentration is 100ng/ml, each hole by adding 100μL, that means 10ng/holes.③Intervention and at different time points to obtain the culture mediumThe 6-well culture plate in PCO2 5% of the incubation chamber for 72 hours, and prepared to intervene. First, abandoning the original 6-well culture plates in medium and added to each well in the new medium 1000μL. Add into group B, group E the corresponding holes 100μL NF-κB inhibitors with concentration of 100μg/ml, in group D, group E, group F the corresponding holes in the culture add 100μL IL-1βwith concentration of 100ng/ml; then each group added to the 2000μL at total; then in group C, group F corresponds to the concentration of holes injected of 0.5ml ozone with 20μg/ml, from access to the interval between injection of ozone to control in less than 15s, the whole process of injection not less than lmin, and the syringe needle is always below the corresponding culture medium.After all interventions, within 5 minutes take 500μL of culture medium of each hole, placed in vials stored in-20℃; 24h after each hole take 500μL of culture medium again, placed in vials in the same-20℃preservation; 48h later, the culture medium remaining in the holes was placed in vials stored-20℃. Its replacement with new medium,had no intervention, cartilage cells prepare to be detected.④Observe the content and the detection indexDetected in the supernatant of each group at different time points levels of T-SOD, MDA, NO and iNOS, using immunohistochemistry of cartilage cells to observed NF-κB, COX-2 expression, Western Blot detection of NF-κB that chondrocytes expression, flow cytometry in each group to detect chondrocyte apoptosis.⑤Statistical analysisThe experimental data was analyzed by the software of SPSS 13.0. Differences were considered statistically significant at P<0.05.4. Results(1) Effect of different concentrations of ozone on cartilagethe is different, and was effect correlated with concentration.It can be observed in the experiment that different concentrations of ozone can aggravate or mitigate the rabbit knee cartilage damage in osteoarthritis. Determine the extent of cartilage damage by Mankin score usually,0 point for normal, the higher the score the more serious injury. The distribution of Mankin score was:ozone 50μg/ml group (7.8 points)> control group (6.8 points) and the oxygen group (6.8 points)> Ozone 40μg/ml group (5.40 points)> Ozone 30μg/ml (4.6 points)> Ozone 10μg/ml group (4.4 points)> Ozone 20μg/ml group (3.4 points)> control group (0.20 points). When ozone concentration increased from 10μg/ml to 20μg/ml, cartilage damage is reduce, and in the range from 20μg/ml to 50μg/ml, with the increase of ozone concentration, chondrocytes'injury aggravated, once reaching 50μg/ml, whice damage is higher than model group and oxygen group, not only can not play a therapeutic role, but increased articular cartilage damage.(2) Ozone inhibits synovium hyperplasia significantly in a dose-effect relationship.In the groups of ozone injection, the times of rabbit knee synovium membrane is far less than model group and oxygen group, also less than the normal group; with the increase of ozone concentration, synovium decrease increasingly apparent. Since ozone can inhibit the proliferation of synovium, the higher the concentration of ozone, the stronger the inhibition. Under microscope, model group and oxygen group with marked hyperplasia of synovium tissue, normal group's synovium cells were normal arrangement, each ozone group's synovium more atrophy with the concentration of ozone increase. Concentration up to 40μg/ml and above, compared with normal, synovium cells significantly reduced, the integrity of the outer synovium structure loss. The higher the concentration of ozone, oxidative damage to the synovium tissue more severe.(3) The changes of three inflammatory mediators expression in cartilage and synovium.After injection into the joint cavity of ozone can cause cartilage and synovium tissue IL-1, TNF-a and MMP-13 expression levels changed, the consistent trends in the cartilage and synovium tissue are consistent, whice are:ozone 50μg/ml group> oxygen group and model group> Ozone 40μg/ml group> of ozone and ozone 30μg/ml 10μg/ml group Groups> Ozone 20μg/ml group> normal group.10μg/ml～30μg/ml of ozone concentration in the range, the expression of three inflammatory mediators in cartilage and synovium showed a "U"-shaped distribution, in 20μg/ml or more, as the concentration increased, expression of three inflammatory mediators were significantly increased, once reaching 50μg/ml, its expression more than the model group and the oxygen group. The ozone concentration of 50μg/ml significantly increased joint tissue damage.(4) Effects of different times of ozone injection on rabbit knee cartilage and synovium are different, and there are times-effect relationshipThis part of experiment in order to observe the results of different times injections in osteoarthritis. The results showed that after injection of ozone in different times, different effects on cartilage, Mankin score was:0 times group (6.75 points) and 5 group (6.75 points)> 1 group (4.63 points)> 3 group (3.38 points), but 1 time group and 3times group showed no significant difference (P=0.32). Injection of ozone can be prompted to some extent reduce the cartilage damage, but the times of injections is greater than or equal to 5 times, may aggravate cartilage damage. With the times of ozone injection increase, less synovium tissue.The distributions of IL-1, TNF-a and MMP-13 in cartilage and synovium are similar, which were:0 time group> 5 times group> 1 time group> 3 times group. Clew that injected ozone 3 times or less can reduce joint damage, while the times of injections up to 5 times, caused damage.(5) Different times of ozone injection on normal rabbit articular cartilage and synovium affected and there are times-effect relationshipIn order to evaluate the the security of t different imes of ozone articular injection, whether it is reasonable that two ozone injection interval is lweek, the injection of ozone at different times in normal rabbit articular, results show that different times after the injection of ozone, the impact of cartilage different, Mankin score was:5 times high to low group (3.75 points) and 3 groups (2.38 points)> 1 group (1.38 points)> 0 times group (0.13 points). Display that the 20μg/ml ozone injection in the normal knee will cause cartilage mild damage, and with the times of injections increases, the degree of injury gradually increase. With the increase in the times of ozone injection, less the number of synovium tissue.The distributions of IL-1, TNF-a and MMP-13 in cartilage and synovium are similar, both increased with the injection times increase. Normal articular injection of 20μg/ml ozone may cause cartilage and synovium damage, and the injury has a cumulative effect of the injection interval of 1 week, more the times of injections, The more serious the damage caused.(6) Ozone impact on the cultivation of cartilage cellsWith IL-1βpresence, NF-κB inhibitors and ozone can inhibit the expression of NF-κB, and NF-κB inhibitors inhibition was stronger than ozone; ozone can significantly reduce the IL-1βinduced chondrocyte apoptosis and necrosis; ozone can increase chondrocyte content of T-SOD in the culture medium and reduce the level of MDA, also reduce the level of iNOS and NO by a certain degree.5. Conclusions①Low concentration of ozone could possibly delay the progression of osteoarthritis, while the higher concentration of ozone can aggravate joint damage. 20μg/ml is the best concentrations of ozone in five different concentrations while intra-articular injection for 3 times.②Different times of ozone injection on rabbit knee osteoarthritis made different results of cartilage and synovium,3 times injection can reduce degree of knee injury in rabbits maximum, and when injected up to 5 times could increase the degree of joint damage. In the use of ozone treatment of osteoarthritis, we should pay attention to the total does of ozone injection, if necessary, appropriate to reduce the frequency of ozone injections to prevent joint damage aggravated.③Ozone injected into normal rabbit knee joint, it will cause certain degree of cartilage and synovial tissue damage, with the increase in the number of injection, damage aggravated. Ozone injection into the joint cavity might had certain cumulative effect in a short time after injected, the use of ozone treatment of osteoarthritis should be appropriate to extend the interval between two treatments.④With IL-1βpresence, NF-κB inhibitors and ozone can inhibit the expression of NF-κB, and NF-κB inhibitors inhibition was stronger than ozone; ozone can significantly reduce the IL-1(3 induced chondrocyte apoptosis and necrosis; ozone can increase chondrocyte content of T-SOD in the culture medium and reduce the level of MDA, also reduce the level of iNOS and NO by a certain degree.⑤Using ozone articular injection for treatment of osteoarthritis, it should follow the "low concentration, low frequency, long interval" principle.