Analysis of biomechanical properties of tibias after bone failure and ozone treatment in rats

Introduction: Ozone is a potent antioxidant that acts as a precursor of various radicals, being indicated as a powerful therapy, assisting in the process of tissue healing. Objectives: The proposal of this study was to analyze material and structural properties via mechanical testing in tibias after application of ozone in bone defects produced surgically. Methods: Ten male 40-day old albino Wistar rats have been used, divided in two groups: control group and ozone group, this last one being treated with ozonized water in 25 μg/mL of concentration until the day of euthanasia. Trichotomy and longitudinal incision was conducted in the animals’ leg skin, exposing the tibia’s diaphysis of both antimers, and with help of a high rotation pen a flaw has been produced on the bone. After 60 days of surgery the animals were euthanized, and tibias were collected for biomechanical analysis. Results: The results of the biomechanical properties – structural and material – evidenced significant interactions through exposure to ozone, showing a diminished bone resistance in animals from the control group, observed by the decrease of the maximum force (N) needed to rupture the bone when compared to the value needed to break the bones of the animals from the ozone group, and the analysis of the morphometrical properties did not show any difference between both experimental groups. Conclusion: The use of ozone did not alter the morphological structures of the tibias, and the group which used ozone presented more resistance during mechanical testings, because the maximum force for the rupture of tibia was greater in this group.


Introduction
In 1840, Christian Friedrich Schönsein discovered a more allotropic and active variant of oxygen (O2): ozone (O3) (Grootveld M, et al., 2004) (Veranes XG, et al., 1999). The first ozone generator was developed by Werner von Siemens in Germany around 1854, and the first report of its therapeutical use was made by C. Lender in 1870, with the proposal of blood purification. Ozone is a potent antioxidant that can act as a precursor of a series of other radicals, with both in vitro and in vivo actions (Grootveld M, et al., 2004) .
Ozone is a gas that stirs controversies because even though it has great potential of therapeutic utilization, it is also a highly toxic gas, very useful in the stratosphere as an absorber of ultraviolet radiation (Grootveld M, et al., 2004) (Bocci VA, 2005) (Buliés JCE, et al., 1997).
Numerous indication possibilities exist, with well proved indicatives for the treatment of some clinical situations. Furthermore, many of these therapies are referred to in literature, i.g. potent antimicrobial action, easy local or systemic application, low cost, no collateral effect, intolerance, or contraindication (Martínez-Sánchez G, et al., 2005) (Bocci VA, 2006).
There are many ways to administer ozone therapy. Besides the endovenous path that should be avoided due to the high risk of embolia (Bocci VA, 2005), literature describes four main means of administration: autohemotherapy, rectal insufflation, hermetical bag of ozone, and topic application with ozonized water and oil (Bocci VA, 2006). The most used ozone therapy method is a autohemotherapy. (MAH), in which a predetermined volume of blood is drawn from the patient (200-270 mL), mixed with the proportional gas volume (O3 + O) and reinfused endovenously.
Ozone concentration must respect the therapeutic window between 10 and 80 µg/mL (Bocci VA, 2006). Due to high instability and ozone toxicity, its gaseous form is the safest to be incorporated to fluids like blood, water, isotonic solutions, and oils for clinical application (Cardoso CC, et al., 2000). It can be mixed with oilse.g. sunflower, which has high affinity due to being rich in unsaturated acidsthrough electric discharges. When ozonized, these acids form ozonides and, with hydrolysis, aldehydes, ketones, and hydrogen peroxides can be formed, which are responsible for triggering biochemical reactions (Lincheta LF, et al., 2000) ( Siqueira JF, et al., 2000).
Tissue healing consists in progressive and ordered events, characterized by various concomitant phases regulated by specific biological events, that initiate in the moment of trauma and remain carious for a period (Kandler B, et al., 2005). All the healing phases are coordinated by cytokines and specific growth factors. For example, prostaglandins, interleukins (IL), and nitric oxide actuate in inflammations; epithelial growth factor (EGF) actuates in re-epithelization; fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) actuate in angiogenesis; and platelet derived growth factor (PDGF), FGF, Research, Society and Development, v. 9, n. 9, e530997474, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7474 6 tumor growth factor β (TGF-β), IL-1, and IL-4 (Kwon YB, et al., 2006) actuate in migration and proliferation of fibroblasts, and in deposition and remodeling of extracellular matrix (collagens type I, II, and III).

Kandler et al. emphasized that in the whole healing process, including in bone tissue
and systemic diseases, needs of an equilibrium between the production of oxygen reactive species (ORS) and its neutralization to avoid toxicity. The hydrogen peroxides neutralization by platelets, protecting the tissue from granulation during bone remodeling, has been exemplified.
Osseous consolidation is composed by coordinated interaction among different types of cells and the liberation of various cytokines (Samee M, et al., 2008). Histomorphological evaluations have shown that ozonized water can alter the process of osseous repair, improving and amplifying vascular neoformation, and expanding the number of osteoclasts next to the injured region, but was not able to stimulate neoformation of osseous trabeculae (Kim HS, et al., 2009).
Considering the great number of accidents that involve extensions bone fractures and the corrective surgical interventions, the study of substances that can assist in bone repair is high relevance for orthopedics and odontological clinics. This study evaluated structuralmaximum break up force, displacement at maximum force, and extrinsic bone rigidityand material biomechanical propertieselasticity module, maximum tension, and deformation at maximum tensionby mechanical testings in tibias after application of ozone in bone defects surgically produced.

Material and Methods
From the point of view of the problem approach, this is a qualitative and quantitative research, exploratory and experimental, regarding the objective of data collection respectively. Garcia, J.A.D., et al, 2015 was used for the methodology of scientific research Animals: there have been used 10 male 40-day old albino rats given by the animal house of the Federal University of Alfenas (UNIFAL-MG). The animals have been divided into two groups of five animals each, one being a control group (CT) and the other ozone group (OZ). The animals were maintained in conventional cages and sawdust. All animals were given the same solid feed (Autoclavable Nuvilab CR-1) and ad libitum water. The animals' weight was checked weekly and every three days solid and liquid consumption by the animals were measured. Research, Society and Development, v. 9, n. 9, e530997474, 2020 (CC BY 4.   Source: Collection of article. Research, Society and Development, v. 9, n. 9, e530997474, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7474 8 In Figure 1 it observed the photo of the ossea failure performed in the right tibia to illustrate how the procedure was performed.
Mechanical test: Animals' right tibias were removed and stored in a freezer (-20 °C) until the day prior to mechanical test. For mechanical test, tibias (n=10 per group) were submitted to three-point bending test until complete fracture at a velocity of 1.3 mm/min. An TA.XT plus (Texture Analyser) set with a load cell of 50 Kgf was used. Each tibia was tested in the anterior-posterior plane (concave-up position), with the anterior surface of the bone facing upwards. The distance between the two bone ends was 50 mm, and in order to obtain the resistance value, a load was applied to the middle third of the bone (Soares EV, et al., 2010) (diaphysis at the site of the surgical intervention) by means of a tip, coupled to the equipment. The load and displacement data were obtained directly from the TA.XT system and recorded with a computer coupled to the testing machine (Figure 2a e 2b). These data were used for the acquisition and calculation of the structural properties: maximum load, displacement at maximum load, and extrinsic stiffness. The extrinsic stiffness was calculated as the slope of the most linear portion of the elastic region of the load-displacement curve.  The material properties were obtained from the structural properties (Soares, EA, et al., 2015) (Weisbroth SH, et al., 1977. The following material properties were evaluated: maximum stress, strain at maximum stress and elastic modulus. On the basis of the loaddisplacement data, these parameters were calculated using the following equations: Where σ is the stress, L is the distance between the two lower supports, c is the maximum distance from pixels to the line that crosses the center of the mass, Є is the strain, d is the displacement, and E is the elastic modulus. The mechanical tests were carried out at the Department of Anatomy of Federal University of Alfenas. Statistical analysis -Final weight (g), daily liquid intake (ml), daily solid intake (g), biomechanical tibias analysis, and morphological and morphometric comparison were statistically compared between the two groups by analysis of variance followed by Tukey's test, with the level of significance set at 1 and 5%, respectively. Means with different letters were significantly different (5%) from each other.
Anatomical dimensions of the tibia -After removal of all soft tissue from the tibia, the following four measurements were obtained with a digital caliper and magnifying glass: 1) tibia length; 2) tibia diaphysis width (measured at the narrowest point of the mid-tibia); 3) width of proximal tibia; 4) width of distal tibia.

Results and Discussion
Laboratory animals help in the study of various humane diseases and their treatments, because they surpass the clinical studies limitation. Knowledge regarding the process of osseous cicatrization and osseous biomechanics are majorly obtained from experimental studies, which have helped to comprehend osseous reparation (Soares EV, et al., 2010).
In this study the animals have been submitted to surgical procedure in order to generate an osseous defect in the tibia and in order to guarantee the animals' health during the experiment, the animals' weight, liquid intake (water), and solid intake (feed) were all controlled. Weisbroth et al. wrote that variations in the amount of solid and liquid intake can provoke modifications on biological responses in animal experimentation.
Solid intake inferior to 25 g/day and significant losses of mass during the experiment characterized malnutrition in rodents (Palencia G, et al., 1994). However, our results demonstrated that the rats from the CT group (299,12+8,5) and OZ group (313,37,12+8,5) gained weight during the experiment and there was no significant differences between these groups.
According to Svendsen and Hau (Svendsen P, 1984), the rats should be fed approximately 25 g of feed and from 15 to 80 mL of water. In our experiment, all the animals had solid and liquid intake between ideal limits, not characterizing protein malnutrition or dehydration.
Animals from group CT and OZ had adequate solid and liquid intake according to Table 1, and this data corroborate the confirmation that during the experiment the animals didn't present malnutrition or dehydration. Our study demonstrated that the maximum force to break the tibias of the animals from the group OZ was greater than the force needed to cause the rupture of the bones of the animals from group CT.
Gain in weight (g), solid intake (g) and liquid intake (mL) were satisfactory, with no occurrence of significant interactions in experimental groups (Table 1). All animals showed weight gain, solid and liquid intake within the ideal limits not characterizing protein malnutrition or dehydration. The animals of the CT and OZ group, according to table 1, presented adequate feed and liquid intake, data that corroborate the confirmation that during the experiment the animals did not present malnutrition or dehydration. This could leave doubt as to whether the results found were due to the use of ozonio.
A secure and correct ozone dose represents a non-deleterious acute oxidative stress that induces an antioxidant cellular response which is able to revert a chronic oxidative stress, i.e. helps normalizing the unstable redox balance in many diseases. This act can help improving circulation (local vasodilatation and angiogenesis) and oxygen support, favoring metabolism and cytokines liberation, autacoids and growth factors, that, alongside antimicrobial activity, represent crucial elements in the treatment of metabolic, inflammatory, infectious and neoplastic diseases. The action of ozone therapy can be interpreted as a nontoxic "therapeutic shock", capable of restoring homeostasis from being a modifier of the physiologic response (Buliés JCE, 1996) (Bocci VA, 2006). Bucci, 1997, points  Research, Society and Development, v. 9, n. 9, e530997474, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7474

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The analyzes of morphometric properties did not reveal differences among experimental groups. As illustrated in Table 2, we can observe that the variables in question (Tibia length, proximal width of tibia and diaphesys width of tibia) did not show significant differences when comparing the groups of animals studied.
Results of the biomechanical properties (structural and material) showed significant interactions mediant the exposure to ozone, with osseous resistence decrease in animals from the control group, observed by the decrease of the necessary maximum force (N) to rupture the bone compared to the needed value to break the bones of the animals from the ozone group (Table 3). Although there is literature that associates the use of ozone and osseous repair, the work developed by Kim et al. revealed in histomorphological analysis that the use of ozonized water can alter the osseous repair process, improving and expanding vascular neoformation and the number of osteoclasts next to the injured region, but is not able to stimulate the neoformation of osseous trabeculae.

Conclusion
Analysis of the maximum force of rupture demonstrated itself different between the groups, as the force needed to rupture the tibias of the group treated with ozone was greater.
Biomechanical analysis evaluated diverse parameters and even though the force parameter was the only one that presented significant different results, it indicates that the bone tissue is more resistant. We believe these data reveal the need of future studies that histologically evaluate the tissue in terms of calcium and collagen deposition.
As a proposal of a new study on the effects of ozõnio we will seek the analysis of the neoformed bone and the use of immunohistochemistry to corroborate the beneficial findings of biomechanics presented in this study. Furthermore the study with ozone for biomechanics in repair in other types of tissues such as ligaments and tendons can be expanded.