Immunosuppressive agents have been recognized as factors that induce changes and modifications in bone metabolism. The purpose of this study was to evaluate the effect of ozonated plant extracts (herein termed ozonated oil) under the influence of Cyclosporin A (CsA) on osseointegration. A total of 20 dental implants were placed in 20 rabbit tibiae assigned to Group A or B. CsA was injected at an immunosuppressive dose in Groups A and B as a single-dose treatment. At the day of surgery, Group A received a single topical ozonated oil treatment (0.55 mL) around dental implants; Group B, the control group, received no ozonated oil. Animals were sacrificed after 8 weeks. Radiographs were obtained at implant surgery and on the day of sacrifice. Bone quality was compared between the 2 groups. Radiographically, osseointegration was microscopically evaluated using scanning electron and light microscopies. In ozonated Group A specimens, light microscopic examination demonstrated evidence of more organized mature bone compared with Group B. Within the limits of this study, the results suggest that short-term administration of CsA, when administered with topical ozonated oil, may influence bone density and the quality of dental implant osseointegration. Therefore, topically applied ozonated oil may influence bone density and the quality of osseointegration around dental implants.
In 1985, Branemark defined contact osteogenesis or osseointegration as “a direct connection between living bone and a load-carrying endosseous implant at the light microscopic level.”1 Today, dental implant therapy has become the ultimate standard for replacing missing teeth. Natural esthetics and optimal function are established with the utilization of dental implants that correlate with patient satisfaction. Despite high success rates in dental implant placement, mechanisms of implant failure remain unclear, especially with so-called early implant failure, or implant loss, within the healing time of such implants. Surgical trauma, acute infection, lack of stability, and insufficient biocompatibility of implant body and systemic conditions are considered as possible causative factors in early implant failure.2
Organ transplantation has increased over the past decade after the successful development of immunosuppressive drug regimens to reduce rejection. Cyclosporin A (CsA) is the most commonly used immunosuppressant agent for preventing graft rejection; it is a fungal cyclic undecapeptide, which has also shown promise in the treatment of autoimmune disorders.3
CsA is a potent immunosuppressive drug used to treat patients who have received organ transplants.4 The complex effects of CsA on bone, including antianabolic effects observed in isolated osteoblastic cells5 and acceleration of bone turnover observed in rat models,6 can lead to bone loss. CsA may contribute to the bone loss associated with immunosuppressant therapy after organ transplantation.7 Published research suggests that this drug could have applications in the treatment of other immunologic diseases.8 Several studies9,10 have reported that CsA increases bone turnover—a process giving rise to a higher resorption than formation rate, which increases the incidence of bone fracture.11–13 Furthermore, use of this compound has been associated with bone mineral loss in animal studies.14,15 However, few studies have reported the relationship between CsA, dental implants, and the quality of osseointegration. Duarte et al16 showed a negative impact on bone density and volume around implants when animals were submitted to short-time CsA administration; similarly, Sakakura et al17 reported a negative influence on bone-to-implant contact when the compound was administered for longer periods. These 2 studies were designed to assess the influence of CsA administration during bone healing immediately after placement of dental implants. Other investigations have reported gingival hyperplasia8,18 and periodontal damage.19
Ozone (O3) has a range of valuable properties that can be exploited for application in medical fields. The ability of O3 to inactivate different microbial species has been reviewed in previously published research.20–24 The bactericidal, virucidal, and fungicidal effects of ozone are known and have been employed in the treatment of infected local lesions. O3 is an extremely powerful oxidative agent (redox potential for the O3/O2 system + 2.07 V), has greater bactericidal properties when compared with chloride or hypochlorite, and has the advantage of presenting lower toxicity.25 Its activity is related to its capacity to interfere with bacterial growth, and it also can inactivate viruses.
A positive effect of ozonated oil in the treatment of infection,24,26,27 herpetic gingivostomatitis, and alveolitis has also been reported.28 The purpose of this study was to histologically evaluate the effects of an ozonated oil formulation on osseointegration around endosseous implants in immunosuppressed rabbits.
Ozone gas reacts with the carbon double bonds in plant oils. This leads to the formation of ozonoids, and the effects of these agents on infection control and wound management have been reported previously.22,24,27 Using a patented ozone reactor, sunflower oil was reacted with ozone gas to produce a thick viscous ozonated oil.
The ozonation of unsaturated fatty acids (UFAs), including polyunsaturated fatty acids present in such ozonated oil products, is a complex reaction system that proceeds via the previous generation of more polar ozonides (ie, insertion of O3 into 1 or more >C = C< bonds). Degradation of primary ozonides via scission of the C-C and one of the O-O bonds generated gives rise to the production of a Criegee (biradical) intermediate and UFA-specific aldehydes, ketones, or carboxylic acids.29 For example, a major product arising from the interaction of O3 with oleoylglycerol species (and the consequent fragmentation of their corresponding ozonides) is a 9 carbon–length acyl chain with a terminal aldehydic functional group, which remains glycerol-bound in the sn-1 or -2 position.30
When generated by the direct reaction of O3 with UFAs present in bacterial cytoplasmic membranes, these oxidation products are at least partially responsible for the microbicidal activity of this oxidant in view of their enhanced polarity (ie, they have the capacity to cause the disruption of such membranes).30
Materials and Methods
A total of 20 white adult New Zealand male rabbits aged 9–12 months weighing between 3 and 3.5 kg were used in this study. The animals were kept in individual cages with free access to food and tap water.
All animals were submitted to a daily subcutaneous immunosuppressive dose31 of 10 mg CsA/kg body weight (CsA Sandimmune, Novartis Pharma AG, Basel, Switzerland) for a period of 14 days. Animals were randomly assigned to 2 treatment groups (10 animals/group). Group A received a topical ozonated oil gel (ozonated sunflower plant oil, Dr Julian Holmes, Cape Town, South Africa). The osteotomy site volume was calculated to be 0.274 mL3/cm3, and 0.550 mL of ozonated sunflower oil was applied directly into each implant osteotomy site to fill the site; excess ozonated oil was allowed to flood over surrounding bone and soft tissues. Group B acted as the control group. A total of 20 FRIOS IMZ (Friatec, Friedrischsfeld, Germany) cylinder titanium plasma–sprayed (TPS) implants with diameter 3.33 mm and length 8.00 mm were inserted into the right tibia of each rabbit.
Animals were fasted for 12 hours before surgery. Ketamine (50 mg/kg body weight [ketamine HCl, 50 mg/mL]; EIPICO, Cairo, Egypt) and Xylazine (20 mg; MH Reg No 1373/99 Vet, ADWIA, El-Oubor, Egypt) were administered intramuscularly. In addition, 2% (wt/vol) lidocaine was injected locally at the surgical sites (Mepecaine, Alexandria Company for Pharmaceuticals, Alexandria, Egypt).
Surgical procedures were conducted in compliance with the ethical principles for animal research, as approved by institutional guidelines. Once general anesthesia had been established, the tibia region of each animal was shaved and thereafter washed with iodine. A 33-cm incision then was made along the medial aspect of the proximal tibia.
Under continuous irrigation with sterile saline, the implant installation procedure was carried out according to the manufacturer's instructions. A marker drill was used to create a saucer shape depression for the round head bur. A round head bur was used to create a purchase point in the compact bone to facilitate positioning of the spiral and cannon drills used later. The round head bur was used with exterior irrigation because it penetrates the bone only by a small degree.
Drilling the Pilot Site
This site establishes the implantation depth and aligns the implant axis. The site was prepared with a D2-mm spiral drill at a speed of approximately 1000 rpm. The site has been and was drilled to the correct depth for the implant. The initial site was prepared for all implants using a D2.8-mm cannon drill at a speed of approximately 2000 rpm. The final receptor site for D3.3 cylindrical screws and cylindrical implants was prepared with a D3.3 canon drill. Finally, once the implant had been placed, the implant-seating instrument was placed over the placement head, and the implant was gently tapped into position with a surgical mallet. The placement head was then simply snapped off the sealing screw.
The site of implantation was then irrigated profusely with normal saline, and the soft tissue was repositioned and approximated with black silk 1.0 suture. The area was washed with a mixture of iodine and 70% (vol/vol) ethanol. After surgery, the topical antibiotic Bivacyn aerosol powder (Néomycine, Bacitracine, Lek Pharmaceutical and Chemical Company d.d., VerošKova57, Ljubljana, Slovenia) was applied to the sutured area to protect against postoperative infection. Each animal received intramuscular PAN-Terramycin antibiotic at a dose of 1.0 cm3/10 kg (oxytetracycline HCl, Pfizer, Giza, Egypt), and an analgesic at 0.05 mg/kg for 3 successive days and ozonated sunflower oil were applied over the sutured healing incisions. Animals were allowed full weight bearing with no mobility restrictions immediately postoperatively.
Assessment of direct bone contact with titanium
Radiographic evaluation was performed to assess bone density on the day of surgery (T0) and at the time of sacrifice (T1).
Radiographs were taken immediately after implant surgery (T0) and at sacrifice (T1) using the Digora Digital Images System (Orion Company, Soredex, Finland). Radiographic exposure was carried out using a Trophy X-ray machine (Trophy Radiologie, Marne La Vallee Cedex, France) (Figure 1).
Digora Imaging software was used to evaluate bone density changes. Three linear density measurements were made lateral to the implant, a line was then drawn parallel to the implant, and 2 other lines were created parallel to the first line, 1 mm apart. Finally, bone density along the 3 lines was recorded, and the mean value of the 3 readings was calculated for the lateral side.
Histologic Examination of the Tibia
At the end of the eighth week after implant placement (T1), all rabbits were sacrificed by an overdose of intravenous nembutal (NYSE:LLY, Indianapolis, Ind). The bone containing the implants was immediately immersed in 10% (vol/vol) buffer formalin solution for fixation of the specimens for 48 hours. At sacrifice, no indication of inflammation or gross infection was noted around the implants. Each specimen was dehydrated in a graded alcohol series for 10 hours and embedded in methyl methacrylate without decalcification. After polymerization, sections were made through the longitudinal axis of the implants and through the surrounding nondecalcified bone. The embedded tissue was cut into 150-µm-thick sections with a low-speed diamond wheel using tap water lubrication. The sections were sanded on an abrasive paper under tap water to obtain a uniform surface finish.
For histologic study, specimens were gold-sputtered and examined with a scanning electron microscope (SEM; Philips XL30, 5600 MD, Philips Research, Eindhoven, Holland) at 10 × 10 spot magnification. Sections were further ground to about 2020 µm thickness and then were observed under a light microscope.
Data sets were expressed as the mean ± standard deviation (SD). Analysis of variance (ANOVA) and Student's t tests were used. Untransformed data were employed to assess the significance of differences: mean bone density values at the P < .05 level were considered significant.
Radiographic analysis of the tibia
No significant differences were noted between the ozonated and control groups at T0 and T1; indeed, mean bone density in Group A and group (±SD) values for Groups A and B, respectively, were 126.7 ± 3.3 and 117.7 ± 8.4 at baseline (T0), and 134.1 ± 5.0 and 124.0 ± 5.6 after 2 months (T1). At the end of the study period, no statistically significant differences were found in the percent change of bone density measurement data at T0 and at T1 in both groups (Tables 1 through 3 and Figures 2 and 3). Indeed, no significant differences were seen between the mean values of each of the 4 study groups, although that observed between Groups A and B at the T1 time point was close to statistical significance (P = .08).
Overall Radiographic Characteristics
The baseline images showed healthy bone without disturbance. At sacrifice, the bone close to the implants in all groups was characterized by the formation of new bone along the implant threads. Regarding the area of the peri-implant bone, differences observed among the 2 groups also were found not to be statistically significant.
Scanning electron microscopic examination
SEM revealed that close bone implant contact was achieved in both studied groups at the end of the study period. Implants had almost engaged the bone in the ozonated specimens, and a wide space was still observed in those from control specimens. These observations were further confirmed histologically via light microscopic examination (Figures 4 and 5).
Light microscopic examination
Light microscopic examination demonstrated evidence of new bone formation next to the implant in Groups A and B. The newly formed bone assumed morphology complementary to the threads and showed a tendency to migrate to the space formed between the implants and the bone surfaces. In Group A, intimate contact between the implant surface and the newly formed mature bone was observed. The new bone showed numerous Haversian systems (Figure 6), which represent a feature of mature bone formation. Bone surfaces facing the implants illustrated cellular proliferation and differentiation, promoting a stronger and more organized type of mature bone, or lamellar bone tissue. For Group B, formation of the primary osteons, a characteristic of immature bone tissue, was observed (Figure 7).
High success rates have been reported for implant-supported prostheses in fully and partially edentulous patients. However, among other factors, the medical status of the patient has been associated with biological failure of dental implants.32,33
The immunosuppressive drug Cyclosporin A (CsA) is widely used after organ transplantation and has shown promising results in the treatment of various autoimmune diseases.3 However, the adverse effects of CsA associated with this treatment have been observed frequently34 and reported in the published literature.35
Recently, the significance of CsA with regard to alveolar bone has received some attention. Patients undergoing CsA medication may not be considered ideal candidates for implant therapy because of their suspected compromised general health and immune status. Although human case reports and animal studies indicate that implant therapy is successful in osteoporotic subjects, the success rates are variable. In vivo studies indicate that CsA accelerates bone remodeling and results in bone loss.18 In CsA-treated rabbits, a significant decrease in the bone area next to the implant has been recorded, whereas the degree of bone-to-implant contact observed was found to be comparable in test and control groups.16
Increased bone remodeling and trabecular bone loss have been observed in CsA-exposed animals.16 Increased osteoblastic and decreased osteoclastic activity in patients undergoing CsA treatment has also been reported.36 The precise mechanisms underlying the effects of CsA on bone metabolism are still unknown. However, Orcel et al37 have suggested that CsA affects the immune system by selectively influencing the lymphokine-monokine cascade, and it has been recognized that cytokines, such as interleukin-1, are among a number of products of the lymphokine-monokine cascade that affect bone metabolism.38 Although in vitro studies indicate that CsA inhibits bone resorption,37 it has been observed that in vivo, CsA administration results in a severe high turnover osteopenic state,39 which is dependent on both dose and duration of therapy.6
Thus the use of immunosuppressive therapy before or during dental implant placement should be considered because the possibility that the prognosis of an implant-supported prosthesis may be influenced by the bone density around the implant.32
New Zealand rabbits were selected for use as experimental animals in this study because they are comparatively easy to handle and maintain; healthy animals of this species are readily available from local laboratories. In addition, this species is known to maintain uniformity in its genetic characteristics; therefore, there is very little difference in anatomic, histologic, and physiologic characteristics among animals.40,41
Animal sacrifice was carried out 8 weeks after implant insertion because the bone healing response in such animals starts during the first week, peaks around 3–4 weeks, and arrives at a relative steady state with only minor bone remodeling 6–8 weeks after implant insertion. In fact, follow-up longer than 1 year later has revealed that the general histologic picture of bone and vascular architecture remained largely unchanged.42,43
The subcutaneous route of CsA administration used in this study has been suggested as the route of choice to provide a more consistent plasma concentration of CsA than that derived through any other route. Blood levels of CsA between 100 and 400 ng/mL have been shown to be sufficient to promote immunosuppression in humans.44 Likewise, in animals, blood levels between 100 and 400 ng/mL have been considered to be effective. The dosage of CsA used in the present study (10 mg/kg SQ) has been shown to produce blood plasma levels sufficient to induce immunosuppression in rodents.19
Extraoral use of ozone in dentistry has shown promising results, and Murakami et al45 have reported that ozone is effective against bacteria and viruses. However, some of the uses of ozone in medicine remain controversial.20
Ozone can exert protective effects by oxidative preconditioning, via its abilities to oxidatively precondition, stimulate, and preserve endogenous antioxidant systems in the physiologic environments to which it is administered, and by blocking the pathway/or the blockage of pathways for reactive oxygen species generation.27
Traditionally, ozone has been dissolved in olive oil. However, the use of ozonated sunflower and other plant oils has been reported.22,24,27 The antimicrobial actions of ozonated oils represent a novel pharmaceutical approach to the management of a variety of medical and dental problems.22
Topical application of ozonated oils allows rapid disinfection, and this process enhances the healing of wounds. Unfortunately, only a small amount of documented information is currently available in the scientific and clinical literature regarding the use of ozonated oils and these novel therapeutic agents. It has been reported that when ozonated oil is layered over the exudates of an ulcer, the ozonoid at the oil-water interface moves slowly into the wound; by reacting with biomolecules, it generates a steady soluble UFA. Derived ozonides are slowly transported into the aqueous phase, and subsequent reaction of ozonide-derived carbonyl oxide species with water can give rise to a perpetual flow of microbicidal aldehydes and hydrogen peroxide (H2O2) via a hydroxyhydroperoxide intermediate.46 The effects of sterilization and improved oxygenation are responsible for the observed accelerated rate of healing. Moreover, local application of ozonated oil in aphthous ulcers occurring on the tongue, lips, and cheeks of many people allows extremely rapid healing and disappearance of these ulcers, and the ozonoids alleviate pain.47
So far no studies have observed the effect of topical ozonated oil in enhancing osseointegration. The present study aimed to evaluate the influence of topical ozonated oil preparation in conjunction with immunosuppressive therapy on osseointegration. The results of the present study demonstrate that short-term coadministration of CsA with topical ozonated oil may not affect preexisting bone adjacent to the implant surface, and may result in accelerated formation of mature bone around the dental implants.
Radiographic evaluation showed that there were no significant differences between the 2 groups at baseline. On completion of the study period, both groups revealed a significant increase in bone density when compared with their baseline values. However, no statistically significant difference in bone density was observed in both groups at the end of the study period.
Gotfredsen et al48 proposed the use of SEM images obtained with back-scattered electrons in this study. This method allows for the use of thick sections and the acquisition of high-contrast images because back-scattered electron contrast is highly dependent on the atomic number of chemical metals/metal ions detectable in such specimens. Consequently, bone ions appear darker than those of titanium.49
In this study, SEM results revealed direct contact between the bone and implant structures in both Groups A and B at the end of the study period. Examination revealed that the implant had almost engaged the bone in the ozonated specimens collected from the sampling group treated with both CSA and ozonated oil, while a readily observable, lengthy space remained between the bone-implant surface in control specimens.
In the present study, light microscopic examination demonstrated evidence of new bone formation in both Groups A and B. In ozonated specimens collected from the ozonated oil-treated group (Group A), intimate contact between the surface of the implant and the newly formed mature bone was observed. The new bone showed numerous Haversian systems and interstitial bone, with bone surfaces at the implant surface showing cellular proliferation and differentiation, promoting a stronger and more organized type of mature bone, or mature lamellar bone tissue. Control group specimens demonstrated the formation of primary osteons, which represent a characteristic of immature bone tissue.
Finally, observed differences between the radiographic and microscopic evaluation in this study highlight the need for dental practitioners and health care professionals who place implants to be aware that the quality of osseointegration is difficult to assess by X rays alone. Currently, X-ray evaluation is considered to be the standard of care. This study showed that X rays can be misleading in terms of discriminating between immature and mature bone tissue around implants. Alternative technologies such as ultrasonics should be considered for the clinical evaluation of osseointegration.50
Results of the present study suggest that short-term administration of CsA in conjunction with topically applied ozonated oil may exert a significant influence on bone density. Ozone therapy appears to act as a biological response modifier. However, additional studies should be considered to address the long-term consequences of maintenance dosage schedules of CsA and topically applied ozonated oils for bone mineral metabolism around titanium implants.
Amany A. El Hadary, Hala H. Yassin, and Sameh T. Mekhemer claim to have no financial interest in any company or any of the products mentioned in this article. Julian Holmes discloses that he is the Clinical Director for Grey Cell Enterprises (http://www.greycellenterprises.com), a company that manufactures ozonated plant oils. The ozonated plant oils were donated by Dr Julian Holmes for this study. This study was self-funded, and no other funding was received for the study.
Note on Clinical Relevance
Scientific Rationale for Study: Cyclosporin A (CsA) is known to interfere with bone density and may lead to bone density loss and fracture. Ozone has been shown in previous studies to accelerate the healing process. To date, no studies have examined the role of ozonated plant oils in implant osseointegration.
Principal Findings: Ozonated plant oils applied at the implant placement stage and during the healing phase accelerate the healing of soft tissues, reduce the risk of infection, and lead to more mature bone formation around the implant, resulting in accelerated osseointegration of the implant.
Practical Implications: Ozonated plant oils offer a simple application and predictable end result of advanced osseointegration. These ozonated oils are cheap and 100% organic and carry no risk of bacterial resistance or host sensitivity.