Bone implant device and pharmaceutical composition containing carbon nanotubes functionalized with hyaluronic acid and use

Abstract

BONE IMPLANT DEVICE COATED WITH HYALURONIC ACID-FUNCTIONALIZED CARBON NANOTUBES AND ITS USE. The present invention relates to the use of hyaluronic acid (HY)-functionalized carbon nanotubes (CNTs) in coating bone implants and in pharmaceutical formulations to promote rapid healing and bone tissue reconstruction. More particularly, the present invention relates to the dental use of a HY-CNT-coated dental implant device with a rapid osseointegration capability.

Description

1 / 30 “BONE IMPLANT DEVICE AND PHARMACEUTICAL COMPOSITION CONTAINING CARBON NANOTUBES FUNCTIONALIZED WITH HYALURONIC ACID AND USE” Field of application The present invention describes a bone implant device coated with hyaluronic acid-functionalized carbon nanotubes (HY-CNTs), capable of promoting rapid osseointegration, and pharmaceutical compositions containing the same, capable of promoting rapid healing and bone tissue reconstruction. State of the art In medicine, bone reconstruction is often an essential prerequisite for the functional rehabilitation of systems. Recovering lost bone structure may require bone grafting surgery or the insertion of biomaterials. Several materials and techniques, which demand a relatively high investment, have been proposed with the aim of optimizing bone repair. This scenario is even more critical where there is a systemic disorder that impairs the repair process, such as in diabetes. Normal bone healing after injury is characterized by the integrated actions of different cells and can be divided into the following phases: inflammation, migration, differentiation, and proliferation of osteogenic cells (SASAKI, T.; WATANABE, C. Stimulation of osteoinduction in bone wound healing by high-molecular hyaluronic acid. Bone, v. 16, n. 1, p. 9-15, 1995; SIQUEIRA JR, JF; DANTAS, CJS Mecanismos Celulares e Moleculares da Inflamação. Rio de Janeiro: Medsi, 238 p., 2000.). Subsequent stages are characterized by extracellular matrix (ECM) synthesis, osteoid mineralization, bone maturation, and remodeling (LALANI, Z.; WONG, M.; BREY, EM; MIKOS, AG, DUKE, PJ; MILLER, MJ; JOHNSTON, C.; MONTUFAR-SOLIS, D. Spatial and temporal localization of FGF-2 and VEGF in healing tooth extraction sockets in a rabbit model. Journal of Oral and Maxillofacial Surgery, v. 63, p. 1500-1508, 2005). 2 / 30 Hyaluronan, also called sodium hyaluronate (HY) or hyaluronic acid, is a high molecular weight glycosaminoglycan (104-107 Da), consisting of repeating non-sulfated disaccharide units of N-acetylglucosamine and D-glucuronic acid (ASLAN, M.; SIMSEK, G.; DAYI, E. The effect of hyaluronic acid-supplemented bone graft in bone healing: experimental study in rabbits. HYAFF®-11 p75, AQUACEL® and hyaluronan towards reactive oxygen species in vitro. Biomaterials, v. 23, p. 15, p. 892-900, 2004). Hyaluronan is widely distributed in tissues such as skin (JUHLIN, L. Hyaluronan in skin).Journal of Internal Medicine, v. 242, p. 61-66, 1997), fluids 15 of synovial junctions, tendon sheaths (ENGSTROM-LAURENT, A. Hyaluronan in joint disease. Journal of Internal Medicine, v. 242, p. 57-60, 1997; FRASER, JRE; LAURENT, TC; LAURENT, UBG Hyaluronan: its nature, distribution, functions and turnover. Journal of Internal Medicine, v. 242, p. 27-33, 1997.), eyes and in most body fluids (MOSELEY, R.; LEAVER, M.;. WALKER, M.; WADDINGTON, RJ; PARSONS, D.; CHEN, WYJ; EMBERY, G. Comparison of the antioxidant properties of HYAFF®-11p75, AQUACEL® and hyaluronan towards reactive oxygen species in vitro. Biomaterials, v. 23, p. 2255-2264, 2002; PRICE, R. L; HABERSTROH, KM; WEBSTER, TJ. Improved osteoblast viability in the presence of smaller-sized carbon fibers. Nanotechnology, v. 15, p. 892-900, 2004), in addition to being present in all extracellular matrices. It acts on tissue morphogenesis, migration, adhesion and cell differentiation (ASLAN, M.; SIMSEK, G.; DAYI, E. The effect of hyaluronic acid-supplemented bone graft in bone healing: experimental study in rabbits. Journal of Biomaterials Applications, v. 20, p. 209-219, 2006; DECHERT, TA; 3 / 30 DUCALE, AE; WARD, SI; YAGER, DR Hyaluronan in human acute and chronic dermal wounds. Wound Repair and Regeneration, v. 14, p. 252-258, 2006.). Exerts influence on cell behavior by direct interaction with cell surface receptors (TURLEY, EA; AUSTEN, L.; VANDELIGT, K., CLARY, C. Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of Ras-transformed cell, Journal of Cell Biology, vol. n. 5, p. 1041-1047, 1991), especially CD44 (LESLEY, J.; HASCALL, VC; TAMMI, M.; HYMAN, R. Hyaluronan binding by cell surface CD44. The Journal of Biological Chemistry, v. 25, p. 26967-26975, 2000; MURAI, T.; KAWASHIMA, H.; Chemistry, v. 278, pp. 32259-32265, 2003;; RUGG, MS; HYMAN, R.; DAY, AJ; MIKECZ, K. TSG-6 modulates the interaction between hyaluronan and cell surface CD44. The Journal of Biological Chemistry, v. 279, n. 24, p. 25745-25754, 2004) and receptors for HA-mediated cell mobility (RHAMM) (TOOLE, BP Hyaluronan in morphogenesis. Journal of Internal Medicine, v. 242, p. 35-40, 1997; SAVANI, RC; CAO, G. A. P. A.; A. P. A. P.; A. P. A. A., v. 242, p. 35-40). ZHOU, Z.; HM Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis The Journal of Biological Chemistry, v. 39, 3677; TURLEY. NOBLE, PW; BOURGUIGNON, LYW Signaling properties of hyaluronan receptors minireview. The Journal of Biological Chemistry,, v. 277, n. 7, p. 4589-4592, 2002, NEDVETZKI, S.; GONEN, E.; ASSAYAG, N.; REICH, R.; WILLIANS, THURMOND, RL; NEUDECKER, F.;RHAMM, a receptor for hyaluronate-mediated motility, compensates for CD44 in CD44-inflated knockout mice: a different interpretation of redundancy. Proceedings of the National Academy of Sciences of the United States of America. 4 / 30 v. 101, η. 52, p. 18081-18086, 2004; GHATAK, S.; MISRA, S.; TOOLE, BP Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells. The Journal of Biological Chemistry, vol. 280, no. 10, p. 8875-8883, 2005). During bone repair processes, hyaluronic acid (HY) can act in the retention of osteoinductive growth factors within the local environment due to its physicochemical properties (SASAKI, T.; WATANABE, C. Stimulation of osteoinduction in bone wound healing by high-molecular hyaluronic acid. Bone·, v. 16, n. 1, p. 9-15, 1995), in addition to mediating osteoclast adhesion to the bone surface (PRINCE, CW. Roles of hyaluronan in bone resorption. BCM). Musculoskeletal Disorders, v. 5, n. 12, 2004) and accelerate revascularization (GIAVARESI, G.; TORRICELLI, P.; FORNASARI, PM; GIARDINO, R.; BARBUCCI, R.; LEONE, G. Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions. Biomaterials, v. 26, p. 3001-3008, 2005) and bone formation in vitro (PILLONI, A.; BERNARD, GW. The effect of hyaluronan on mouse intramembranous osteogenesis in vitro. Cell and Tissue Research, v. 294, p. 323-333, 1998) and in vivo (SASAKI & WATANABE, 1995). In previous studies, we demonstrated that treating rat dental alveoli with HY promotes healing of this structure after extraction of the first upper molars, mainly by accelerating bone deposition and the expression of osteogenic proteins such as osteopontin (OPN) and bone morphogenetic protein type-2 (BMP-2) (MENDES, RM; SILVA, GAB; LIMA, MF; CALLIARI. MV; ALMEIDA, AP; ALVES, JB; FERREIRA, AJSodium hyaluronate accelerates the healing process in tooth sockets of rats. Archives of Oral Biology, v. 53, p. 1155-62, 2008). Carbon nanotubes are metastable forms of carbon resulting from the coiling of a plane of carbon atoms (DRESSELHAUS, MS; DRESSELHAUS, G.; EKLUND, PC Science of Fullerenes and Carmon Nanotubes. Academic Press, San Diego, 1996). They can be single-walled (SWCNTs) or multi-walled (MWCNTs). The diameter of SWCNTs varies 5 / 30 between 0.7 and 1.5 nm (ZHAO, B.; HU, H.; MANDAL, SK; HADDON, RC A bone mimic based on the self-assembly of hydroxyapatite on chemically functionalized single-walled carbon nanotubes. Chemistry of Materials, v.17, p. 3235-3241, 2005), with lengths that are often hundreds or thousands of times greater than their diameter (MACDONALD, RA; LAURENZI, BF; VISWANATHAN, G.; AJAYAN, PM; STEGEMANN, JP Collagen-carbon nanotube composite materials as scaffolds in tissue engineering. Journal of Biomedical Materials Research, v. 74A, p. 489-496, 2005). The strong covalent bonding of its atoms gives CNTs the characteristics of a material with high mechanical strength and, due to their metallic character, they exhibit high thermal and electrical conductivity. Furthermore, the absence of dangling bonds gives this material great chemical inertness, fundamental for its use in biotechnological and biochemical applications (DRESSELHAUS et al., 1996). Bone tissue is a natural compound containing collagen fibrils and hydroxyapatite crystals arranged in a hierarchical organization. At the smallest growth scale, collagen triple helices spontaneously form bundles that act as a nucleation site for the formation of hydroxyapatite nanocrystals. NTCs can control crystal nucleation and the growth of the inorganic component (ZHAO et al., 2005). Furthermore, they support osteoblast growth (PRICE et al., 2004) and bone formation, and therefore represent a huge technological advance in the field of bioengineering (ZANELLO, LP; ZHAO, B.; HU, H.; HADDON, RC. Bone cell proliferation on carbon nanotubes. Nano Letters, v. 14, p. 14, 2006). Previous studies have demonstrated improvements in the biological properties of polymers, such as collagen, after functionalization of CNTs with them (SILVA, EE; DELLA, HHM; FERLAUTO, AS; MOREIRA, RL; RESENDE, RR; OLIVEIRA, S.; KITTEN, GT; LACERDA, RG; LADEIRA, LO Nanostructured 3-D collagen / nanotube biocomposites for future bone regeneration scaffolds. Nano Pesquisa, v. 2, p. 6 / 30 Titanium alloys with aluminum and vanadium (Ti-Al6-V4) and commercially pure titanium (cpTi) are the most widely used materials in the manufacture of implants, given their biomechanical properties, possibility of treatment and finishing of the implant, and ease of sterilization (RAYNHOLT, G. Corrosion current and pH rise around titanium coupled to dental alloys. Scandinavian Journal of Dental Research, v. 96, n. 5, p.466-72, 1988). The basis for the initiation of osseointegration is the recruitment of specific cells and their intimate contact with the implant surface (ADELL, R.; LEKHOM, U.; ROCKLER, B.; BRANEMARK, Pl. A 15-year study of osseointegrated implants in the edentulous jaw. International Journal of Oral Surgery, V. 10, p. 387-416, 1981; ALBREKTSSON, T.; DAHL, E.; ENBOYN, L.; ENGEVALL, S.; ENGQUIST, B.; ERIKSSON, RA A multicenter study of 8139 implants placed in Nobel).; YLIHEIKKILA, P.K.; FELTON, DA Generalizations regarding the process and phenomenon of osseointegration. Part II. In vitro studies. International Journal of Oral and Maxillofacial Surgery, vol. 13, p. 163-74, 1998; SCHNEIDER, GB; PERIN PANAYAGAM, H.; CLEGG, M.; ZAHARIAS, R.; SEABOLD, D.; KELLER, J. Implant surface roughness affects osteoblast gene expression. Journal of Dental Research, vol. 82, p. 372-376, 2003). Undifferentiated mesenchymal cells and pre-osteoblastic cells change their shapes and functions according to the characteristics of this surface, demonstrating that the initial cellular response is critical for achieving osseointegration (DEGASNE, I.; BASLE, MF; DEMAIS, V.; HURE, G.; LESOURD, M.; GROLLEAU, B. Effects of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human osteoblast-like cells (Saos-2) on titanium surfaces. Calcified Tissue International, v. 64, p. 499-507, 1999; COOPER, LFA role for surface topography in creating and maintaining bone at titanium endosseous implants. Journal of Prosthetic Dentistry, vol. 84, no. 5, p. 522-34, 2000; SCHNEIDER et al., 2003, ZHAO eta!., 2005). 7 / 30 Several medical devices that enable osseointegration are described in the prior art. Patent application PI 0419111, for example, describes the use of hyaluronic acid in the coating of dental implants. The titanium screw would be pre-coated with amine through plasma deposition, and then the hyaluronic acid would bind to this coating, allowing its carboxyl portion to be free to bind to calcium, thus enabling osseointegration. Patent application PI 0601751 describes a manufacturing process for a biodegradable biocomposite material using collagen conjugated to carbon nanotubes. Document US2003153965 describes the induction of osteointegration through the use of electrical stimulation of osteoblasts to increase the proliferation of these cells. It suggests using a nanoscale material and a biocompatible polymer and / or ceramic as an electrical conductor. Furthermore, this document suggests the use of nanotubes as a nanoscale electrical conductor. The document PCT / US2008 / 056436 describes the use of prostheses whose surfaces comprise nanotopography of nanofibers, nanotubes, nanochannels, microchannels or microwells, capable of increasing or promoting cell differentiation and viability. Document WO00 / 56377 describes a method for improving medical instruments or devices by coating the surface of said device with carbodiimide-linked hyaluronic acid. Furthermore, the hyaluronic acid can be modified by the presence of Fe3+ or Al3+ cations to prevent calcification of the device and surrounding tissue. Document US2007 / 0026038 describes a method of coating endoprostheses with layers of polysaccharides, particularly hyaluronic acid. Usui Y. and colleagues describe multi-walled carbon nanotubes with high bone compatibility and accelerated regeneration capacity. ♦ 8 / 30 (Carbon nanotubes with high bone-tissue compatibility and bone-formation acceleration effects. Usui Y, Aoki K, Narita N, Murakami N, Nakamura I, Nakamura K, Ishigaki N, Yamazaki H, Horiuchi H, Kato H, Taruta S, Kim YA, Endo M, Saito N. Small, v. 4, n. 2, p. 240-6, 2008). Some articles describe the functionalization of carbon nanotubes with hyaluronic acid: Moulton SE and colleagues studied the interaction kinetics between carbon nanotubes and hyaluronic acid at different concentrations (MOULTON SE, MAUGEY M, POULIN P, WALLACE GG. Liquid crystal behavior of single-walled carbon nanotubes dispersed in biological hyaluronic acid solutions. Journal of the American Chemical Society, v. 129, n. 30, p. 9452-7, . 2007). Bhattacharyya S. and colleagues describe the synthesis of a hydrogel consisting of carbon nanotubes functionalized with hyaluronic acid (BHATTACHARYYA S, GUILLOT S, DABBOUE H, TRANCHANT JF, SALVETAT JP. Carbon nanotubes as structural nanofibers for hyaluronic acid hydrogel scaffolds. Biomacromolecules, v. 9, n. 2, p. 505-9, 2008). Problems in the State of the Art The description of new biomaterials is an extremely important and dynamic area in current biology. The search for a material with favorable characteristics that allow its use in replacing biological tissues, as well as for materials that accelerate / modulate biological repair processes, aims to solve frequent problems in daily clinical practice. Thus, as previously pointed out, several materials and techniques that demand a relatively high investment have been proposed with the aim of optimizing the repair of hard tissues and attenuating bone loss. Furthermore, tissues are environments that make it difficult to use various pharmaceutical formulations, such as ointments and creams. On another note, the population's access to more complex procedures has grown significantly in recent years. Therefore, the 9 / 30 Developing materials that meet this complexity but are low cost is a challenge for modern medicine. Another problem can be attributed to the physicochemical properties of hyaluronic acid, which do not contribute to its use. Its high hygroscopic capacity makes it unsuitable for coating implants and prostheses, because when it comes into contact with serum, blood, or saliva during surgical procedures, it tends to detach from the titanium surface. Thus, there is a great need for new pharmaceutical formulations involving low-cost biomaterials capable of optimizing bone repair and attenuating bone loss. In this context, it is worth noting that formulations containing HY for use in extensive bone defects are already available on the market; however, their low stability hinders and impairs their use. Advantages of the Invention Treating titanium surfaces with HY-functionalized CNTs creates a more attractive contact surface for osteogenic cells, representing a promising strategy for implants. Functionalizing CNTs with HY (HY-CNTs) allows for their availability in more stable pharmaceutical formulations, enabling longer contact between HY and the tissue. Furthermore, formulations based on the functionalization of CNTs with HY promote tissue reconstruction in cases of very extensive bone loss and accelerate healing. These formulations can be presented as biofilms, membranes, flakes, powders, or gels. Thus, products containing CNT-HY can be used in surgical procedures to accelerate healing and promote bone restoration. Furthermore, this functionalization significantly reduces the hygroscopic capacity of HY. This allows the use of the material (NTC-HY) in the form of a biofilm covering titanium implants, since there is a reduction in 10 / 30 swelling of this biofilm in the presence of fluids when compared to a biofilm composed only of HY. Thus, the NTC-HY biofilm remains adhered to titanium for longer, in addition to providing greater structure. The clinical viability of the present invention for bone healing processes and prosthesis coating is confirmed by the biological properties, physical-structural characteristics, and low cost of this biomaterial. Brief description of the Figures Figure 1: Formation of bone trabeculae in the dental alveoli of rats treated with Carbopol, NTC 100pg / mL, HY 1% and NTC-HY 0pg / mL. Figure 2: Representative photomicrographs of histological sections of alveoli from animals treated with different concentrations of HY seven days after tooth extraction. Alveoli from control animals (treated with carbopol) - (A) low magnitude and (B) high magnitude; Alveoli treated with 0.25% HY — (C) low and (D) high magnitude; Alveoli treated with 0.5% HY — (E) low and (F) high magnitude; Alveoli treated with 1% HY - (G) low and (H) high magnitude; Alveoli treated with 2% HY - (I) low and (J) high magnitude; Alveoli treated with 4% HY - (K) low and (L) high magnitude. Figure 3: Representative photomicrographs of histological sections of alveoli from 20 normal and diabetic animals seven days after tooth extraction. Alveoli from normal control animals (treated with carbopol) - (A) low magnitude and (B) high magnitude; Alveoli from diabetic control animals (treated with carbopol) — (C) low and (D) high magnitude; Alveoli from diabetic animals treated with HY 1% (E) low and (F) high magnitude; Alveoli treated with NTC-HY 100 pg / mL - (G) low and (H) high magnitude. Figure 4: Analysis of histological sections of alveoli seven days after tooth extraction. In (A) representative image of the number of cell nuclei in alveoli of 11 / 30 normal control animals, which showed a high number of cell nuclei. In (B) representative image of the number of cell nuclei in alveoli of diabetic control animals and those treated with HY or NTC-HY. Figure 5: Histological analysis of alveoli seven days after tooth extraction. (A) 5 Resorption of the interradicular septum by osteoclasts (indicated by the arrows) was observed in all alveoli (normal control, diabetic control and diabetic treated with HY or with NTC-HY). In (B) cells with the morphological appearance of osteoblasts synthesizing bone matrix inside the alveoli, as indicated by the arrows, and in (C) presence of newly formed blood vessels in the medullary space of the alveoli. Figure 6: Macroscopic analysis of epithelialization of surgical lesions fourteen days after tooth extraction. Reduced epithelialization was observed in diabetic control animals (B) when compared to normal control animals (A). Treatments with 1% HY (C - right side) and NTC-HY (100 pg / mL) (D - right side) accelerated epithelialization of post-surgical lesions, with the NTC-HY treatment standing out. The arrows highlight the lesion areas. Figure 7: Representative photomicrographs of histological sections of alveoli from normal and diabetic animals fourteen days after tooth extraction. Alveoli from normal control animals (treated with carbopol) - (A) low magnitude and (B) high magnitude; Alveoli from diabetic control animals (treated with carbopol) - (C) low and (D) high magnitude; Alveoli from diabetic animals treated with HY 1% - (E) low and (F) high magnitude; Alveoli treated with NTC-HY 100 pg / mL - (G) low and (H) high magnitude. Figure 8: Analysis of histological sections of alveoli fourteen days after 25 tooth extractions. In (A) representative image of alveoli from diabetic control animals; (B) representative image of alveoli from normal control animals and 12 / 30 diabetics treated with HY or NTC-HY, whose histological analyses are represented together. Figure 9: Histological analysis of alveoli fourteen days after tooth extraction. (A) Resorption of the interradicular septum by osteoclasts, indicated by the arrows, was observed in all alveoli (normal control and diabetic control and treated with HY or NTC-HY) and is quite pronounced in this chronology. In (B) the alveolar cavity of a diabetic control animal is illustrated, where little bone formation and a pronounced vascular network were observed, suggesting a delay in the tissue repair process. In (C — diabetic treated with NTC-HY) the alveolar cavities of normal control animals and diabetic animals treated with HY or NTC-HY are represented together, where significant bone neoformation and reduced medullary space were observed. Figure 10: Effects of different concentrations of HY (0.25%, 0.5%, 1%, 2% and 4%) on bone repair of dental alveoli in normal rats, seven days after 15 tooth extractions. (A) Quantification of the percentage of bone trabeculae. (B) Number of cell nuclei. Figure 11: Effects of 1% HY and 100 pg / mL NTC-HY on bone repair in dental alveoli seven days after tooth extraction. (A) Quantification of the percentage of bone trabeculae. (B) Number of cell nuclei. Data are expressed as mean ± SEM. Figure 12: Effects of 1% HY and 100 pg / mL NTC-HY on epithelialization of the external post-surgical lesion fourteen days after tooth extraction. Data are expressed as mean ± SEM * p<0.05 vs. Normal control; # p<0.05 vs. Diabetic control. Figure 13: Effects of HY 1% and NTC-HY 100 pg / mL on bone repair of dental alveoli fourteen days after extractions. (A) Quantification of the percentage of bone trabeculae. (B) Number of cell nuclei. The data are 13 / 30 expressed as mean ± SEM * p<0.05 vs. Normal control; # p<0.05 vs. Diabetic control. Figure 14: Representative images of tibias 14 days after the surgical procedure. Control tibias (clot) — (A); Tibias treated with HY 1% — (B). The 5 tibias treated with HY 1% showed greater healing compared to the control tibias (arrows). Figure 15: Representative images of histological sections of tibias 14 days after the surgical procedure. Control tibias (clot) — (A) low and (B) high magnitude; Tibias treated with HY 1% — (C) low and (D) high magnitude. The 10 tibias treated with HY 1% showed greater formation of bone trabeculae compared to the control tibias (arrows). Figure 16: Scanning electron microscopy of titanium pellets after 14 days of immersion. Figure 17: Scanning electron microscopy of titanium pellets after 28 15 days of immersion. Figure 18: Backscattered electron images. (AE) 14 days; (FJ) 28 days. Detailed description of the invention The present invention relates to the use of carbon nanotubes 20 functionalized with a polysaccharide, such as hyaluronic acid (HY), in the coating of titanium implants and in pharmaceutical formulations to promote rapid healing and bone tissue reconstruction. More particularly, the present invention relates to the use of implants coated with HY-CNTs with a high capacity for osseointegration. The functionalization of CNTs with HY 25 (HY-CNTs) made it possible to obtain a more stable material with a more effective capacity for tissue reconstruction in small and extensive bone defects. 14 / 30 Furthermore, it enabled its use in more stable pharmaceutical formulations, whether in the form of biofilms, membranes, flakes, powders, or gels. Synthesis and purification of carbon nanotubes Single-walled carbon nanotubes (SWNTs) were prepared by the arc discharge method using a Co / Ni catalyst, with a helium atmosphere and a total pressure of 500 Torr (TRIGUEIRO JP, SILVA GG, LAVALL RL, FURTADO CA, OLIVEIRA S, FERLAUTO AS, LACERDA RG, LADEIRA LO, LIU JW, FROST RL, GEORGE GA. Purity evaluation of carbon nanotube materials by thermogravimetric, TEM, and SEM methods). After synthesis, the material was purified by a sequence of thermal oxidation and acid treatments. The purified SWNTs were refluxed in HNO3 (3 mol / L) in a domestic microwave for 15 minutes. Then, they were centrifuged at 7,000 rpm and washed repeatedly with deionized water until complete removal of nitric acid. The final solution, composed of carboxylated SWNTs, was dried for 12 hours at 60°C. The quality of the NTCPS was verified using Raman spectroscopy. Functionalization of carbon nanotubes with hyaluronic acid A solution of carboxylated CNTs (0.5 mg / mL) was placed in a container containing HY (0.5 mg / mL) and mixed until a homogeneous mixture was obtained. This solution was then subjected to ultrasonic treatment for 30 minutes and subsequently washed and filtered through a 0.45 µm millipore filter to remove HY not bound to the CNTs. The material remaining on the filter was transferred to a glass container and placed in an oven at 60°C to dry. The functionalization of CNTs with HY (CNT-HY) was confirmed by infrared spectroscopy. The technology presented can be better understood through the following examples: Example 1 - Surgical procedures 15 / 30 Male Wistar rats were anesthetized with a mixture of 10% ketamine and 2% xylazine (1:1, 0.1 mL / 100 g body weight, IM) and subjected to extraction of the first upper molars. The alveoli were randomly divided and immediately subjected to different treatments. Example 2 - Histological and morphometric analyses The animals were sacrificed by decapitation under anesthesia with 10% ketamine and 2% xylazine (1:1, 0.1 mL / 100 g body weight, IM), 7 days after extractions for histological and morphometric analyses. The maxillae were dissected and fixed in buffered neutral formalin for 48 h at room temperature. After fixation, the maxillae were demineralized in 10% ethylenediaminetetraacetic acid (EDTA) pH 7.2-7.4, dehydrated using alcohol solutions, embedded in paraffin, and serially sectioned to 6 µm thickness in the sagittal plane. The sections were stained with Masson's trichrome for histological and morphometric analyses. The alveoli were anatomically divided into apical, middle, and cervical thirds. Twelve images (40x) were obtained of each third (apical and middle) of the alveoli (distal roots) with well-defined limits (total area of ​​6.4 x 105 µm² per third).The percentage of bone trabeculae in the alveoli was calculated by dividing the trabecular area present by the total area evaluated using the KS300 program (Carl Zeiss). This program 20 was also used to quantify the number of cell nuclei in the total area, as previously described (MENDES ET AL., 2008). Example 3 - Healing of dental alveoli in normal rats. The alveoli were treated with NTC (50, 100, 400, and 800 pg / mL), HY 1% (NIKKOL, Galena, Brazil), NTC-HY (50, 100, 400, and 800 pg / mL), NTC+HY (100-25 pg / mL), or with carbopol (vehicle) (~0.1 mL). The products were injected directly into the alveoli using a syringe (26G), and to maintain them in the alveolar cavity after surgery, the animals were kept in a supine position for approximately 4 hours, the duration of anesthesia. Furthermore, 16 / 30 The animals received a soft diet for 2 days after the surgical procedures. In a recent study, we demonstrated that carbopol does not interfere with alveolar healing 7 days after surgery, thus showing biological inertness (MENDES ET AL, 2008). Figure 1 demonstrates the formation of bone trabeculae in the dental alveoli of rats. Treatment of the alveoli with carbopol-based NTC alone did not significantly alter bone trabeculae formation when compared to carbopol (vehicle) alone (NTC 100 pg / mL: 3.8 ± 1.2% vs. 3.7 ± 1.2% in the carbopol group). On the other hand, both treatment with 1% HY and treatment with NTC-HY showed a significant increase in the percentage of bone trabeculae (NTC-HY 100 pg / mL: 10.9 ± 1.1% vs. 3.7 ± 1.2% in the carbopol group), with no significant difference between them. In this case, the effectiveness of 1% HY was only possible due to the structure of the dental alveolus, which, being a cavity, allows the 1% HY to remain in the application site for a longer time. In cases of extensive bone loss, the instability of HY 1% makes it difficult for it to remain in place for an extended period, thus hindering its effectiveness.Therefore, NTC functionalization with HY is a promising alternative for application in extensive bone loss. Data are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by the Newman-Keuls test with the aid of GraphPad Prism 4 software. Values ​​with p<0.05 were considered significant. (Carbopol — n=11; NTC 100 100pg / ml — n=11, HY 1% - n=9; NTC-HY 100pg / ml - n=7). Example 4 - Effects of HY and HY-functionalized CNTs (HY-CNTs) on bone repair in dental alveoli of type I diabetic rats Experimental groups 1. Rats with type I diabetes mellitus induced according to Frazan et al. 1997 (FRAZAN R, BALLEJO G, SALGADO MC, MORAES MF, SALGADO HC. Heart 17 / 30 variability and baroreceptor function in chronic diabetic rats. Hypertension, 30, 632635, 1997) and normal (non-diabetic) rats were divided into groups: Control group (non-diabetic animals) treated with carbopol, diabetic control treated with carbopol, diabetic treated with 1% HY, and diabetic treated with 5 NTC-HY 100pg / ml, with treatment lasting 7 or 14 days after first molar extractions (Table 1). Carbopol was used as a control because it has a consistency similar to that of HY. In a previous study, it was demonstrated that carbopol does not interfere with bone repair in dental alveoli of rats (MENDES RM, SILVA GA, LIMA MF, CALLIARI MV, ALMEIDA AP, 10 ALVES JB, FERREIRA AJ. Sodium hyaluronate accelerates the healing process in tooth sockets of rats. Archives of Oral Biology, 53, 1155-62, 2008). Table 1 - Experimental groups and treatments administered to the alveoli. Groups Treatments Sacrifice Normal Control Carbopol (vehicle) 7 days Diabetics Control Carbopol (vehicle) Diabetics-HY HY 1% Diabetics-NT C-HY NTC-HY 100 pg / ml of Carbopol Normal Control Carbopol (vehicle) 14 days Diabetics Control Carbopol (vehicle) Diabetics-HY HY 1% Diabetics-NTC-HY NTC-HY 100 pg / ml of Carbopol Surgical exodontia procedures Tooth extraction was performed, as shown in examples 1 and 3. After confirming the integrity of the removed teeth, the alveoli of the control and diabetic animals were randomly divided and immediately treated with 0.5% carbopol (vehicle), 1% HY (NIKKOL, Galena, Brazil), or NTC-HY (100 pg / mL). The HY gel contained less than 2 ppm of heavy metals and arsenic, 0% protein, and 5.2 18 / 30 mg / mL of glucuronic acid and 1% hyaluronic acid (pH=6.1). Sodium hyaluronate, at a concentration of 1%, is presented in gel form due to its high hygroscopic capacity. NTC-HY powder was diluted in carbopol to be subsequently administered into the alveoli. Approximately 0.1 mL of the aforementioned compounds was injected into the alveoli using a small syringe (26G). To ensure that the administered treatments remained in the alveolar cavity for as long as possible after the extractions, the animals were positioned in dorsal recumbency for approximately 4 hours, the duration of anesthesia. No suturing procedures were performed on the palatal and vestibular edges of the gingiva after the surgeries. The rats were fed a soft diet for 48 hours after the extractions to reduce the impaction of hard foods in the operated area. Histological analysis seven days after tooth extraction. Histological analysis was performed as per example 2. Seven days after tooth extraction, the control sockets, treated with carbopol, were filled with dense connective tissue. The presence of blood vessels and numerous cell nuclei was observed, in addition to few newly formed trabeculae (Figures 2A and 2B). Sockets treated with HY, at concentrations of 0.25% and 0.5%, showed a greater number of blood vessels and a smaller number of cell nuclei compared to the control sockets. The reduction in the number of cell nuclei was more evident in the sockets treated with the 0.25% concentration (Figures 2C and 2D - HY 0.25% and Figures 2E and 2F - HY 0.5%).Regarding the neoformation of bone trabeculae, the alveoli treated with HY at concentrations of 0.25% and 0.5% showed a similar formation pattern, with greater trabecular formation inside the alveoli compared to the controls, although this difference was not significant (Figures 2C and 2D - HY 0.25% and Figures 2E and 2F - HY 0.5%). Histological analysis of alveoli treated with HY at a concentration of 1% demonstrated marked bone deposition, mainly in the apical third of the alveoli. These trabeculae... 19 / 30 showed significantly more numerous, organized, and thicker trabeculae compared to the pattern observed in the control alveoli (Figures 2G and 2H). Furthermore, alveoli treated with 1% HY presented a much smaller number of cell nuclei within them than that presented by the control alveoli, 5 inversely mirroring the pattern of bone neoformation (Figures 2G and 2H). Alveoli treated with HY at concentrations of 2% and 4% did not show significant histological differences when compared to the control alveoli. In these alveoli, there was little deposition of newly formed trabeculae and a reduction in the number of cell nuclei that was not histologically significant 10 (Figures 21 and 2J - 2% HY and Figures 2K and 2L - 4% HY). Histological analyses of dental alveoli from normal and diabetic animals treated or not with HY or NTC-HY demonstrated a similar histological pattern. The alveoli of normal control animals, as described above, were filled with dense connective tissue and exhibited scarce bone neoformation, with immature trabeculae located mainly in the apical third of the alveoli, close to the basal bone (Figures 3A and 3B). The alveoli of untreated diabetic animals presented a similar histological pattern (Figures 3C and 3D), with slightly less pronounced bone formation compared to normal control animals. Treatment of the 20 alveoli of diabetic animals with HY (Figures 3E and 3F) or NTC-HY (Figures 3G and 3H) did not alter this pattern, with scarce bone trabeculae observed in the apical third of the alveoli.The number of cell nuclei in the alveoli of normal control animals was high, with this cell population concentrated mainly in the middle third of these alveoli (Figure 4A). The 25 alveoli of diabetic control animals showed a similar histological pattern, and treatment of diabetic animals with HY or NTC-HY did not alter this pattern either (Figure 4B). Qualitative analyses of the alveoli demonstrated, in all groups, bone resorption of the interradicular septum by osteoclasts (Figure 5A), in addition to 20 / 30 presence of cells with the morphological appearance of osteoblasts depositing bone matrix inside the alveoli (Figure 5B). In addition, the presence of blood vessels inside the alveoli of normal and diabetic animals was observed (Figure 5C). Histological analysis fourteen days after tooth extraction. Macroscopic analysis of the epithelialization of surgical lesions demonstrated a much more pronounced and accelerated epithelialization in normal control animals (Figure 6A) when compared to diabetic control animals (Figure 6B). Treatment of diabetic animals with HY (Figure 6C - right side) or NTC-HY (Figure 6D - right side) was able to accelerate the epithelialization of the surgical lesion when compared to diabetic control animals, with the animals treated with NTC-HY standing out. The epithelialization effect can be observed through the closure of the lesion edges and consequent reduction of its area. Microscopically, it was observed that the alveoli of normal control animals were filled with a large network of mature, thick, and well-organized bone trabeculae, with restricted medullary space observed inside these alveoli (Figures 7A and 7B). On the other hand, the alveoli of diabetic control animals showed a severe reduction in bone neoformation inside, with immature trabeculae observed, preferentially concentrated in the apical third of these alveoli, close to the interdental and interradicular septa (Figures 7C and 7D). Treatment of the alveoli of diabetic animals with HY (Figures 7E and 7F) or NTC-HY (Figures 7G and 7H) was able to significantly accelerate bone repair, with a large quantity of mature, thick, and well-organized bone trabeculae observed inside these alveoli.This bone neoformation in treated alveoli occurred preferentially in the apical third of the alveoli, although both treatments induced considerable bone formation in the middle third of the alveoli as well, especially the treatment with NTC-HY. 21 / 30 The alveoli of diabetic control animals exhibited a large cell population inside (Figure 8A), mainly in the middle third, when compared to the alveoli of normal control animals (Figure 8B). Treatment of the alveoli with HY or NTC-HY significantly reduced the cell population inside them (Figure 8B), demonstrating a histological pattern similar to that of alveoli from normal control animals. These histological analyses suggest that the tissue repair process is more delayed in untreated diabetic animals and that treatment with HY or NTC-HY is able to significantly accelerate this process.Corroborating the initial histological observations described above, it was observed that alveoli from diabetic control animals presented a pronounced network of newly formed vessels within them (Figure 9B), suggesting a significant delay in the tissue repair process, since normal control animals and diabetic animals treated with HY or NTC-HY presented, fourteen days after tooth extraction, a smaller quantity of blood vessels and restricted medullary space within the alveoli, resulting from the pronounced bone neoformation in this location (Figure 9C). However, these were histological observations of a qualitative nature, since no quantification of blood vessels within the alveoli was performed. Furthermore, significant resorption of the interradicular septum by osteoclasts was also observed qualitatively in all alveoli (Figure 9A). Morphometric Analysis Prior to conducting experiments with diabetic animals, an evaluation of the effects of different concentrations of HY (0.25%, 0.5%, 1%, 2%, and 4%) on bone repair in dental alveoli 7 days after tooth extractions was performed on normal animals. An average percentage of bone trabeculae neoformation of 6.15 ± 1.96% was observed in alveoli treated with carbopol (control), and treatments with concentrations of 0.25% (9.74 ± 1.90%), 0.5% (9.60 ± 3.26%), and 1% (15.21 ± 2.00%) of HY were able to... 22 / 30 progressively increase the formation of new trabeculae in the alveoli. However, only the alveoli treated with the 1% concentration showed a statistically significant difference compared to those treated with carbopol. The alveoli treated with concentrations of 2% (6.72 ± 1.78%) and 4% (8.31 ± 1.49% of HY showed similar values ​​for the formation of new bone trabeculae when compared to the control alveoli (Figure 10A). Regarding the number of cell nuclei, the average value in the control alveoli was 8,243 ± 0.58 nuclei, and treatment with different concentrations of HY was able to reduce the number of cell nuclei inside the alveoli, with the concentrations of 0.25% (5,943 ± 0.31 nuclei) and 1% (5,671 ± 0.38 nuclei) standing out, showing statistically significant reductions compared to the control alveoli (Figure 10B). When analyzing the apical third of the alveoli, an average percentage of bone trabeculae neoformation of 8.21 ± 2.99% was observed in the alveoli treated with carbopol, and treatments with concentrations of 0.25% (12.53 ± 3.18%) and 0.5% (12.85 ± 3.42%) of HY slightly increased bone neoformation compared to the control alveoli.The 1% HY concentration (21.07 ± 2.90%) was able to significantly increase bone formation in the apical third of the alveoli. Regarding the number of cell nuclei, the average value in the control alveoli was 8,130 ± 20 0.47 nuclei, and treatment with different HY concentrations reduced the number of nuclei inside the alveoli, especially the 1% concentration (5,530 ± 0.50 nuclei), which showed statistically lower values ​​than those observed in the control alveoli (Table 2). In the analysis of the middle third, an average percentage of newly formed trabeculae of 4.10 ± 2.11% was observed in the control alveoli, and the treatments with different HY concentrations were not able to significantly increase bone formation inside the alveoli. The average number of cell nuclei in the middle third of control alveoli was 8.390 ± 0.82 nuclei and treatments with the aforementioned HY concentrations reduced the number of nuclei inside the alveoli. 23 / 30 with emphasis on concentrations of 0.25% (5,843 ± 0.32 nuclei) and 1% (5,800 ± 0.42 nuclei). However, there were no significant differences between the groups (Table 2). Table 2: Percentage of bone trabeculae and number of cell nuclei in the apical and middle 5 thirds of control animals (treated with carbopol) and animals treated with different concentrations of HY. Apical Third Middle Third Trabeculae (%) Cell Nuclei (x103) Trabeculae (%) Cell Nuclei (x103) Carbopol 8.21 ±2.99 8.13 ±0.47 4.10 ±2.11 8.39 ± 0.82 HY 0.25% 12.53 ±3.18 6.06 ± 0.38 6.95 ± 2.23 5.84 ± 0.32 HY 0.5% 12.85 ±3.42 6.21 ± 0.86 6.85 ± 3.09 6.64 ± 1.03 HY 1% 21.07 ±2.90* 5.53 ± 0.50 * 9.35 ± 4.86 5.80 ± 0.42 HY 2% 9.15 ±2.01 6.65 ± 0.81 4.29 ± 2.22 7.25 ± 0.49 HY 4% 10.08 ± 1.93 6.54 ± 0.45 6.55 ± 1.91 6.30 ± 0.57 Data are expressed as mean ± SEM * p<0.05 vs. carbopol, HY 2% and HY 4%, p<0.05 vs. Carbopol. Repair of dental alveoli in diabetic rats seven days after tooth extraction. Seven days after tooth extraction, no significant differences were observed between the alveoli of normal control and diabetic control animals, both treated with carbopol, in the neoformation of bone trabeculae (5.97 ± 15 2.34% in normal control vs. 3.24 ± 0.86% in diabetic control, Figure 11A) and in the number of cell nuclei (7,633 ± 0.75 nuclei in normal control vs. 7,133 ± 0.28 nuclei in diabetic control, Figure 11B). Treatment of the alveoli of diabetic animals with HY or with NTC-HY was not able to alter the formation of trabeculae (1.64 ± 0.55% in diabetics treated with HY and 5.02 ± 3.01% in diabetics treated with NTC-HY, Figure 11A) and the number of nuclei. 24 / 30 cells (7,217 ± 0.43 nuclei in diabetic animals treated with HY and 7,550 ± 0.41 nuclei in diabetic animals treated with NTC-HY, Figure 11B) compared to the alveoli of diabetic control animals 7 days after first molar extractions. When analyzing the apical third of the alveoli, normal control animals showed an average percentage of 8.81 ± 3.48% of bone trabeculae neoformation within the alveoli. Diabetic control animals showed a slight reduction in bone formation (5.87 ± 1.48%) when compared to normal animals. Treatment of the alveoli of diabetic animals with HY (2.72 ± 0.94%) or NTC-HY (3.99 ± 1.63%) was not able to increase bone formation parameters in the apical third. The average number of cell nuclei in the apical third of the alveoli of control animals was 7,733 ± 0.75 nuclei, and diabetic animals in control (7,350 ± 0.35 nuclei) or treated with HY (7,717 ± 0.45 nuclei) or with NTC-HY (7,717 ± 0.45 nuclei) had an average number of nuclei.825 ± 0.27 nuclei) showed similar values ​​(Table 3). In the middle third of the alveoli, the average percentage of bone neoformation in normal control animals was 3.12 ± 2.22%, and diabetic control animals (0.62 ± 0.40%) or those treated with HY (0.55 ± 0.34%) showed significantly reduced bone formation values ​​in this region of the alveolus. Diabetic animals treated with NTC-HY showed a slight increase (6.06 ± 4.60%) in bone formation in this region, even exceeding the formation values ​​presented by normal control animals. The average number of cell nuclei in the alveoli of normal control animals was 7,517 ± 0.80 nuclei, and diabetic control animals (6,917 ± 0.20 nuclei) or those treated with HY (6,733 ± 0.39 nuclei) or with NTC-HY (7,225 ± 0.66 nuclei) showed similar values ​​(Table 3). 25 / 30 Table 3: Percentage of bone trabeculae and number of cell nuclei in the apical and middle thirds of normal control and diabetic control animals or those treated with HY or NTC-HY, seven days after tooth extractions. Apical Third Middle Third Trabeculae (%) Cell Nuclei (x103) Trabeculae (%) Cell Nuclei (x103) Normal Control 8.81 ± 3.48 7.73 ± 0.75 3.12 ± 2.22 7.51 ± 0.80 Diabetic Control 5.87 ± 1.48 7.35 ± 0.35 0.62 ± 0.40 6.91 ± 0.20 Treated Diabetic (HY) 2.72 ± 0.94 7.71 ± 0.45 0.55 ± 0.34 6.73 ± 0.39 Treated Diabetic (NTC-HY) 3.99 ± 1.63 7.82 ± 0.27 6.06 ±4.60 7.22 ±0.66 The data are expressed as mean ± SEM. Repair of dental alveoli in diabetic rats fourteen days after tooth extraction. Macroscopic analysis of the external post-surgical lesion area demonstrated a large, statistically significant difference in epithelialization between normal control animals and diabetic control animals (0.79 ± 0.40 mm in normal control animals vs. 6.49 ± 0.62 mm2 in diabetic control animals, Figure 12) 14 days after tooth extraction. Only treatment of diabetic rats with NTC-HY was able to significantly accelerate epithelialization of the lesion areas (2.63 ± 0.94 mm2) compared to diabetic control rats. Although treatment of diabetic rats with HY (4.00 ± 1.24 mm2) also stimulated the acceleration of epithelialization, this effect did not reach a statistically significant difference (Figure 12). Microscopically, significant differences were observed between the alveoli of normal control animals and diabetic control animals in the neoformation of bone trabeculae (41.92 ± 6.34% in normal control vs. 11.16 ± 5.10% in diabetic control, Figure 13A) and in the number of cell nuclei (5,033 ± 0.46 26 / 30 nuclei in normal control vs. 7,540 ± 0.78 nuclei in diabetic control, Figure 13B) 14 days after tooth extraction. Treatment of the alveoli of diabetic animals with HY or with NTC-HY significantly accelerated trabecular formation (29.43 ± 3.29% in diabetics treated with HY and 36.90 ± 3.07% in diabetics treated with NTC-HY, Figure 13A) and also significantly reduced the number of cell nuclei (5,740 ± 0.42 nuclei in diabetics treated with HY and 5,460 ± 0.33 nuclei in diabetics treated with NTC-HY, Figure 13B) compared to the alveoli of diabetic control animals 14 days after tooth extraction. When analyzing the apical third of the alveoli in isolation, a significant difference was observed in the percentage of bone trabeculae between the alveoli of normal control animals and diabetic control animals (40.60 ± 4.86% in normal control vs. 16.56 ± 7.19% in diabetic control) and in the number of cell nuclei (5,000 ± 0.44 nuclei in normal control vs. 7,000 ± 0.44 nuclei in normal control vs. 7,000 ± 0.44 nuclei in diabetic control).580 ± 0.75 nuclei in diabetic control). Treatment of diabetic animals with HY or NTC-HY significantly accelerated bone trabeculae neoformation (34.89 ± 5.33% in diabetics treated with HY and 38.20 ± 1.13% in diabetics treated with NTC-HY) and also significantly reduced the number of cell nuclei (5,320 ± 0.53 nuclei in diabetics treated with HY and 5,440 ± 0.26 nuclei in diabetics treated with NTC-HY) (Table 4). In the middle third, a significant difference was also observed in the percentage of bone neoformation between the alveoli of normal control animals and diabetic control animals (43.25 ± 8.38% in normal control animals vs. 5.76 ± 3.45% in diabetic control animals) and in the number of cell nuclei (5,033 ± 0.54 nuclei in normal control animals vs. 7,520 ± 0.88 nuclei in diabetic control animals).Treatment of diabetic animals with HY or NTC-HY was able to increase the formation of bone trabeculae (23.96 ± 4.32% in diabetic animals treated with HY and 35.59 ± 6.30% in diabetic animals treated with NTC-HY), with only treatment with NTC-HY being able to significantly increase this parameter compared to control diabetic animals. Furthermore, treatment of diabetic animals with... 27 / 30 HY or NTC-HY was able to reduce the number of cell nuclei (6,220 ± 0.52 nuclei in diabetics treated with HY and 5,480 ± 0.45 nuclei in diabetics treated with NTC-HY), although these values ​​did not show a statistically significant difference compared to the control diabetics (Table 4). Table 4: Percentage of bone trabeculae and number of cell nuclei in the apical and middle thirds of normal control and diabetic control animals or those treated with HY or NTC-HY, fourteen days after tooth extraction. Apical Third Middle Third Core Centers Trabeculae (%) Trabeculae (%) Cell phones (xl O3) Cell phones (xl 03) Normal Control 40.60 ± 4.86 ' 5.00 ± 0.44 43.25 ± 8.38 ' 5.03 ± 0.54 ' Diabetic Control 16.56 + 7.19 7.58 ± 0.75 5.76 ± 3.45 7.52 ± 0.88 Treated Diabetic (HY) 34.89 + 5.33 ' 5.32 ± 0.53 1 23.96 ± 4.32 6.22 ± 0.52 Treated diabetic (NTC-KY) 38.20 ± 1.13' 5.44 ± 0.26 ' 35.59 ± 6.30 ' 5.48 ± 0.45 Data are expressed as mean ± SEM * p<0.05 vs. control diabetics. Example 5 - Effects of Sodium Hyaluronate and Sodium Hyaluronate-Functionalized Carbon Nanotubes on Rat Tibia Repair, Surgical Procedure Male Wistar rats, with an average age of 3 months and weighing between 200 and 250 g, were used. Two days before the surgical procedure, the 15 animals received a booster dose of multibiotic (0.84 mg / 100 g of body weight) via intramuscular injection. For the surgeries, the animals were anesthetized with an intramuscular injection of a combination of 2% xylazine hydrochloride (0.1 mL / 100 g) and 10% ketamine hydrochloride (0.1 mL / 100 g). After immobilizing the 20 animals, the area was shaved and disinfected. An incision was made longitudinally with a scalpel blade on the ventral region of the left paw. 28 / 30 tibia. The musculature was separated from the periosteum and a perforation was made in the bone with a 1.6 mm drill bit at low speed. The region was irrigated with sodium chloride and the area was washed with the same solution. Different treatments were performed according to the following experimental groups: (i) control (clot), (ii) ~0.1 mL of 1% HY gel, (iii) -0.1 mL of NTCPS gel, (iv) -0.1 mL of NTCPS-HY gel, (v) -0.1 mL of NTCPM gel and (vi) -0.1 mL of NTCPM-HY gel in the left tibias so that the entire cavity was filled with the gel. The skin was sutured. At the end of the surgical procedure, the animals received another dose of reinforced multibiotic 10 (0.84 mg / 100 g of body weight) and a dose of banamine (0.22 mg / 100 g of body weight), intramuscularly. After 7 days, the animals were sacrificed by decapitation and the tibias were immediately fixed by immersion in 10% buffered neutral formalin (NTF) at room temperature for 48 h. Table 5: Schematic arrangement of the experimental groups. TREATMENT GROUP I Clot II HY 1% III NTCPS IV NTCPS-HY V NTCPM VI NTCPM-HY Histological processing After fixation, the tibias were immersed in running water to remove excess fixative and neutralize the formic acid through four 15-minute baths. They were then demineralized in a 10% nitric acid solution for approximately 2 days (BANCROFT, JD; STEVES, A.; TURNER, DR. Theory and practice of histological techniques. 4th ed. Churchill Livingstone. New York. 315 p., 1996). After demineralization, the tibias 29 / 30 were immersed in running water to remove excess acid in four 15-minute baths prior to the paraffin embedding process. Approximately one hundred and twenty semi-serial sections, 6 µm thick, were obtained from each sample and mounted on histological slides previously silanized with 2% silane. The slides from each sample were stored for processing with Masson's trichrome and hematoxylin and eosin. Four alternating sections from each sample were selected for staining with Masson's trichrome, and another four sections were selected for staining with hematoxylin and eosin. Twelve images of each defect were obtained, visualized using a 40X objective and digitized using a JVC TK1270 / RGB microcamera. The methodology employed for microscopic imaging, image segmentation, and definition of morphometry conditions has been previously described in the literature (CALIARI, M. V. Basic Principles of Digital Morphometry: KS300 for beginners. Belo Horizonte: UFMG, 149 p., 1997). For morphometric analysis, the following parameters were considered: percentage of bone trabeculae present in relation to the analyzed area and number of cell nuclei. The area occupied by the bone trabeculae was obtained using the KS300 program contained in the Carl Zeiss image analyzer. The pixels of the bone trabeculae were selected from the real image, followed by the creation of a binary image and obtaining the area in pm². The nuclei of all cell types present were counted by selecting the pixels and creating another binary image. Samples with inadequate inclusion and staining, and lack of integrity of the histological sections, were excluded. Histological analysis Macroscopic analysis of the tibias showed that the bone defect filled with 1% HY was almost completely repaired compared to the defect filled only with a clot (Figure 14). Microscopically, greater formation of bone trabeculae was evident with less... 30 / 30 number of cell nuclei in histological sections of animals treated with 1% HY compared to controls (Figure 15). Example 6 - Titanium tile coating Sterilized titanium pellets were used, with each pellet immersed in a different material (n=3 per group), as described below: • Carbopol (vehicle) • NTC • NTC-HY (100 pg / mL in carbopol) . HY 1% • HY 1% + NTC (100 pg of NTC / mL of HY) The pellets were dried at room temperature (~30°C) in a laminar flow hood for approximately 12 hours and stored in a sterile environment for use in salt deposition analysis experiments and cell cultures. It was observed that after 14 (Figure 16) and 28 (Figure 17) days of immersion in a subsaturated solution of CaCh and Na2HPO4, the pellets coated with NTCHY showed greater calcium and phosphate deposition than those coated only with HY and HY+NTC. After immersion of the pellets in the solution, due to their high hygroscopic capacity, those coated with HY showed greater biofilm swelling, which may have hindered salt deposition. The CNTs, once functionalized with HY, conferred greater structure to the HY; consequently, the biofilm remained adhered to the titanium for a longer time. The dark spots observed in Figure 18 depict differences in the phases or chemical groups present. More intense agglomerates were observed after 14 and 28 days in the pellets treated with CNT-HY (Figure 18C, 14 days and Figure 18H, 28 days), indicating more intense deposits in these groups. Bar = 150 µm.

Claims

1 / 1 CLAIMS 1. BONE IMPLANT DEVICE, characterized by being made of titanium coated with single-walled carbon nanotubes functionalized with hyaluronic acid, wherein the concentration of said coating in pharmaceutical formulations is 50 to 800 μg / mL in excipient.

2. BONE IMPLANT DEVICE, according to claim 1, characterized by the hyaluronic acid-functionalized carbon nanotubes being comprised in pharmaceutical formulations in the form of biofilms, membranes, flakes, powders or gels. Petition 870260069849, dated 07 / 14 / 2026, page 7 / 7

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