International Journal of Ozone Therapy 7: 000-000, 2008 Is Medical Ozone Safe when Injected
A Comparative Histological Study in Rat

E. ILIAKIS*, I. PETROPOULOS**, A. ILIAKI***, E. AGAPITOS****, G. AGROGIANNIS**** * Orthopaedic Surgeon Hospital Inspector N.M.Y. Athens; Greece** Orthopaedic Surgeon National Centre for Emergency Aid; Greece*** School of Veterinary Medicine, Aristotle; University of Thessaloniki, Greece**** Laboratory of Anatomy and Pathology; University of Athens, Greece Key words: ozone, safety, cartilage
SUMMARY - The therapeutic use of ozone (O -O ) gas is controversial. Some authors claim the gas
mixture is toxic and therefore out of the question, whereas, others hold that it is useful and effective when used following a certain method to treat degenerative diseases, such as knee osteoarthritis, for which the gas has been used empirically. The present work studied the effects of contact of O -O with healthy knee tissues of Wistar rats and compared them to intra-articular injection of a substance known to damage the cartilage. Thirty-six Wistar rats were studied in two groups. Mono-iodoacetic acid (MIA) was injected in the first group in a single dose και, whereas O -O was injected in the second group in frequent doses three times a week for three weeks. The rats were killed 40 days later and articular cartilage and surrounding tissues were studied histologically. MIA caused degenerative osteoarthritis gradually deteriorating at the knees of the first group whereas no major changes were observed in those of the second group. We conclude that follow-ing the methodology of our study medical ozone appears to be safe for use. Introduction
of corticosteroids in acute pain or hyaluronic acid (when we also seek to improve damage to the car- Knee osteoarthritis (OA) is a common high cost tilage) and have been applied for many years.
disease in orthopaedics and decreases the quality Biochemical changes in the levels C6S, C4S, of life especially in the elderly. The main char- and KS, TN-C 2 chemokine, endothelin and trans- acteristic of OA is degeneration of the articular forming growth factor alpha 3 metalloproteinases cartilage and the subchondral bone frequently fol- (MMP), cytokines such as IL-1 and TNF - alpha 4 lowed by inflammation of the synovial membrane and others appear to influence the development of and aggravation of the supporting structure of the knee. The result of all these is pain and dysfunction For some years intra-articular injection of medi- in the mobility of the knee. Total joint replacement cal ozone (O -O ) has been suggested for the treat- usually constitutes the final suggestion for the ment of knee osteoarthritis symptoms and positive rehabilitation of these patients, but it is not free of results have been reported 5. However, its applica- complications. Thus, when applying this treatment, tion appears to be empirical and no histological or side-effects, age, weight and general physical con- biochemical studies have provided evidence for dition of the patient must be taken into considera- Ozone’s powerful oxidant factor 6 has been Conservative treatment constitutes the first reported to be toxic 7,8,9,10 and hence there is scep- choice and many proposals have been made to ticism concerning its use for medical purposes. alleviate symptoms, expecting at the same time an Other studies have shown that if ozone is used improvement in the histological features. The usual rationally it is safe 11 and therapeutic for certain pharmacological recommendation includes anal- diseases. Medical ozone (O -O mix) appears to gesics, non steroidal anti-inflammatory drugs, diac- behave as a bioregulator 12 when it comes into erein, chondroitin sulphate, glucosamine sulphate, contact with a biological liquid, releasing factors magnesium and others. Intra-articular injections from human endothelial cells 13 and normalizing also constitute a frequent choice and the injection the cellular redox balance 14,15. Studies have shown Is Medical Ozone Safe when Injected Intra-articularly? Figure 1 Sacrifice at 10 days. Initial degeneration of the cartilage with modification of the natural tissue to fibrosis, hyperaemia of the subchondral bone. H.E.×45.
Figure 2 Sacrifice at 40 days. Total destruction of the architecture of the articular cartilage. H.E.×45.
its capacity to alter the levels the cytokines 16 such mechanical techniques 25 or by injection of chemical as TNF-alpha 17, IL 18 and platelet-derived growth substances into the knee joints 26. Monoiodoacetic factor (PDGF), transforming growth factor beta1 acid (MIA) is a material that could cause osteoar- (TGF-beta1), interleukin-8 (IL-8) and other 19,20 thritis in the knees of Wistar rats similar to those of indications of a likely effect of this gas on the humans 27,28. Rodents are the most widely employed articular cartilage when it is injected intra-articu- species for these studies, because their night and larly. Provided that ozone is indicated for use as a day activity has been studied extensively and sev- therapeutic means, due to its well-known antiseptic eral models of osteoarthritis were developed using property 21,22 23,24, it would also succeed in reducing mice, hamsters and Wistars. The present study con- the potential dangers of septic arthritis after intra- stitutes the first stage of comparison of histological articular injection of the gas mixture.
changes and recommended methodology for the Controlled degeneration of the articular carti- proof of action of intra-articular injection of O -O lage for experimental purposes can be caused by International Journal of Ozone Therapy 7: 000-000, 2008 Figure 3 Normal articular cartilage (water, collagen and pro-teoglycans the surface layer thicker than the deeper layers). Tissue sections of the normal articular surface where the cells of the main membrane of histocell fibroblasts fat cells and mast cells are evident.
Figure 4 The tissues in and around the knee (apart from the cartilage) did not show any major alterations in the long run.
Material and Method
terior joint of the left knee (placebo) was injected with NaCl solution.
Thirty-six (36) male wistar rats weighing 150- 2. Second group (18 Wistars): O -O mix was inject- 180g were employed in our study. A variable vol- ed into the posterior joint of the right knee while in ume (5 to 50 µL ) automatic pipette was required. the left knee joint (placebo) was injected with O .
An ozone generator (Multiossigen mod. PM 93, The animals remained in plastic cages through- Italy). Biochemical reactors (Iodoacetic acid I out the experiment. They were exposed to light 8136 amp 5×500 mg, Sigma-Aldrich). The animals every twelve hours (6-18) and the temperature were divided into two groups. After anesthesia and was maintained at 20°C. The subjects received a disinfection a pre-set quantity was administered short anesthesia with the use of ether, long enough for the action, and after the treatment they were 1. First group (18 Wistars): MIA was injected into returned to their cages. The rats were killed by the posterior joint of the right knee while the pos- Is Medical Ozone Safe when Injected Intra-articularly? Figure 5 A microscopic image of the right knee joint treated with infusion of medical ozone (O -O mix) H.E.×45. Note that the natu- ral architecture is maintained. Inflammatory evidence on the soft tissues of the joint was absent.
Figure 6 Microscopic images of left knees infused with oxy-gen (O ). Note that the natural architecture of the tissues is maintained and articular inflammation is absent. H.E.×45.
The Wistars in the first group (MIA) were killed at two different times. Nine (9) rats ten days later, and nine rats 40 days later. The quantity of MIA had been set at 0.3 mg (corresponding to 0.05 ml of substance) per knee.
A) Macroscopically, the tissues in and around the For the Wistars in the second group (O -O mix) knee (except for the cartilage) did not show major the O and the O -O mix injections were made three times a week for nine sessions in total. The B) Macroscopically and microscopically, the artic- administered quantity had been set at 0.05 ml in ular cartilage presented considerable degenera- concentrations of 20 µg O /ml O concentration tive alterations which gradually worsened in two empirically used for the treatment of osteoarthritis phases with characteristics of non-specific arthritis The cartilage samples and the synovial mem- C) Knee joints infused with 0.9% NaCl (placebo) brane were studied histologically after being put in test tubes containing formalin 1/10.
D) During the study there were no side effects International Journal of Ozone Therapy 7: 000-000, 2008 or animal losses except for the reduction of the the activation of the immunogenic system by the mobility of the right knee in the animals of the first ozone, which has an anti-inflammatory action due to lipid peroxidation products with a consequent inhibition of cyclooxygenase-2 44,45.
A crucial factor, which accompanied the moni- A) During the histological testing, no significant toring of results, was the strengthened constancy change occurred in the cartilage or synovial mem- of the studied gas with the construction of ozone brane of the healthy knee animals, either after generators which continuously monitor the con- ozone administration (figures 4 and 5) at the pre- centration of ozone in real time by a photometer defined concentration for our experiment, or after An initial histological and biochemical study B) No undesired event or animal loss.
examined: a) histologically, discs from patients C) On the contrary, a shorter recovery period was operated while treated with O -O , and b) histo- observed after the anesthesia and an increased logically and bio-chemically, discs and their soft tissues on animals (rabbits) after intradiscal or paravertebral infusion. Findings showed the close connection between ozone concentration in the Discussion
change of the volume of the δι σκου and the levels of cytokines (IL,IF,TNF) 46,47. Ozone concentra- Ozone in the form of an O -O gas mix has been tion within the O -O mixture injected is the most recommended for many years as a treatment for crucial factor because it is what controls the move- many diseases 23,29,30. Initially, its application was ment of blood vessels and other elements of the limited to the positive nutritional effects on tissues blood. The concentration and quantity of O -O 31 due to the improvement the gas induces on the chosen in this study was the one which has long circulation 32,33,34,35,36,37,38,39,40. Today, the knowledge and been used empirically, adjusted to the weight of wide use of medical ozone as a treatment in the form of major AHT and/or AHTs constitutes a This is the first of a series of studies designed challenging, safe and effective method if applied to show whether the medical ozone, empirically used for the treatment of knee osteoarthritis, is Today’s widest use of O -O is mainly with the experimentally safe, and whether it promotes the treatment of herniated disc and its symptoms. re-establishment of the damaged cartilage. The Its administration in the form of a gas mixture is methodology followed was the same for both direct (intradiscal or intraforaminal) 41,42 or indirect groups, with the only modification being the route by intramuscular injection into the paravertebral of administration. Multiple infusions of medical ozone were chosen (three times a week for three Ozone is a disputed gas. According to some weeks) so that the results will go to extremes, authors, it is a powerful oxidant harmful to the if they are destructive. The O -O concentration human body. Scepticism concerning its admin- selected was the one which has been used empiri- istration and doubts over its therapeutic indica- cally by man for patients suffering from knee tions in any of its forms constituted a conviction osteoarthritis: 20 µg O /ml O . The schedule of the for a considerable number of doctors. Ozone histological and clinical effects is also described in application, which was a personal choice of the this study. Based on this data concerning the ani- consulting physician. Although the gas was admin- mals (Wistar rats their general clinical condition istered empirically, physicians ascertained daily before, during and after MIA and O -O infusion remarkable therapeutic results, absence of side- was simultaneously monitored. Special emphasis effects and their patients’ appreciation for their was placed to the mobility of the knees front and method. Its acknowledgement and acceptance in rear (treatable). It was deemed possible that for a the broader medical discipline has changed since further comparison to demonstrate the heterosid- Bocci’s research became known and a set of rules ed knee of the study 0.9% NaCl be administered and conditions under which O , once considered potentially toxic, can be therapeutically useful, was The histological samples were O -O by a spe- introduced. Today we know why and how the result cialized histopathologist and the Mankin 48,49 (table is achieved in a series of diseases 23,29,30 and how this 1) method was employed for its evaluation (tables low-cost and manageable element can be used.
2-7). The intra-articular infusion of O -O did not Studies on orthopaedic diseases are mentioned cause apparent changes in the mobility of the in clinical findings, dealing mainly with disc hernia- knees and has not caused histological damage to tion. The satisfactory outcome has been ascribed to the articular cartilage or the synovial membrane Is Medical Ozone Safe when Injected Intra-articularly? Table 1 Histological and histochemical grading system for evalu-
ation of articular cartilage degeneration (Mankin et Al).
treatment. This is possibly due to the development of some kind of metabolic acceleration mechanism of the narcotic substance (ether) or directly due to oxygen-ozone which could have entered the cir-culation during the filtering through the synovial The increase in life expectancy means a greater frequency of knee osteoarthritis of the elderly (tables 2 and 3). The clinical condition that mainly which in conjunction with negative working prac- concern the mobility of the studied knees is com- tices that cause cartilage disorders even at younger parable to the one where the knees were infused ages make new methods for the treatment and alleviation of the symptoms even more imperative. Medical ozone is already used empirically by many erative findings it caused proved the correctness physicians as a supplementary or single therapy. If (tables 4-7) of the employed technique and the fact the histological results confirm these findings this that it always constitutes a sufficient method for will give a crucial boost to this method for treat- causing experimental osteoarthritis and compari- son of therapeutic material when needed. All the Leaving aside the ozone production machine, materials used were bearable for all the animals the cost of the materials and the gas mix is mini- judging from the observation of the behaviour of mal. The already extensive bibliography on ozone our samples (fatality, aggressiveness, claudication, behaviour in the body and especially in the immune system and herniated disc, and its local antiseptic The animals whose knees were infused with activity, gave a starting point and provided scien- O -O recovered faster after the completion of the tific grounds for this therapeutic method.
International Journal of Ozone Therapy 7: 000-000, 2008 It is imperative that this be proven experimen- Conclusion
tally before any extensive use. The present study shows us that there was no histological damage This research shows that the intra-articular infu- and the next study will investigate the therapeutic sion of MIA caused osteoarthritis in the animals very soon after injection. This step is considered Is Medical Ozone Safe when Injected Intra-articularly? necessary for the study of treatment techniques shorter recovery period after the anesthesia and for knee maintenance or operation, initially on more rigorous activity of the Wistars was observed compared to the rats treated with MIA. These The intra-articular infusion of medical ozone findings could constitute the grounds for a study to the healthy tissues of the knees of Wistar rats of pathological knees and the first step towards does not cause local damage or any undesired the dictum “primum non nuocere” for a potential systematic or local events. On the contrary, the References
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