BR102013014155A2 - bone recovery bionanocomposite - Google Patents

Abstract

Bionanocomposite for bone recovery. The present invention describes a bone restorative bionanocomposite with mechanical compression properties ideal for bone grafts of greater mechanical stress. This bionanocomposite can be applied additionally in the reconstruction and filling of the skullcap and filling of bone defects.

Description

Field of the Invention [01] The present invention relates to a bone restorative bionanocomposite related to losses caused or not by trauma. More specifically, the present invention is directed to a polyvinyl alcohol (PVAl), polyurethane (PU) and hydroxyapatite (HA) bionanocomposite.

[02] Bionanocomposite can be used for facial maxillary graft grafting, skullcap filling and filling, and bone gap filling after bone cancer tumors are removed in different skeletal regions.

BACKGROUND OF THE INVENTION [03] In the repair of the human skeleton, the need for defect filler materials is great, for example, in facial cranium reconstruction, orthoplastic implant revision, after bone cyst removal, bone loss due to trauma. , infections or resorption. In macroporous form implants accelerate the healing process, as they allow for the progressive growth of collagen with the following mineralization of bone tissue through open and interconnected pores (Zavaglia, 2003).

[04] Bone is a dynamic tissue as it has a unique ability to self-regenerate or self-reshape to the right extent without leaving a scar, however many bone grafting needs are due to bone defects caused by or not for trauma. The need for synthetic bone grafts depends on the complication of bone defects. For example, if the defect is minor, the bone has its own ability to self-regenerate within a few weeks. Thus, surgery is not required. In the case of severe defects and loss of bone volume that do not heal normally, ie when the tissue is difficult to close, the graft is required to restore function without damaging the tissues. There are several methods available for treating bone defects, which include the traditional methods of autograft and graft transplantation. Although these two therapies are considered clinically good, there are limitations. For example, the supply of autograft material is limited and in grafting from transplantation there is a possibility of pathogen transfer, thus there is a great need for the use of synthetic bone grafts (Murugan and Ramakrishna, 2005).

[05] Numerous single- and multi-phase synthetic bone graft materials are currently available that are capable of ameliorating some of the complications associated with autogenous or allogeneic bone practices. Although good progress has been made in the use of synthetic bone grafts, the manner in which they perform their functions in vivo is very different and most of them differ from natural bone grafts in either composition or structure. Moreover, a single material phase, also called monolithic, does not always provide all the essential characteristics necessary for bone growth, which leads to the ceaseless search for an ideal bone graft. Thus, there is a great need for multiphase or composite materials, with natural bone-like structure and composition, obtained by materials engineering, more specifically tissue engineering.

[06] Nanocomposites based particularly on hydroxyapatite (HA) and collagen have gained immense recognition as bone grafts not only due to their composition, structure and similarity to natural bone, but also due to functional properties such as surface and mechanical strength over of their single phase constituents. In addition, it can be said that bone itself is a natural nanocomposite, with a matrix composed mainly of collagen-rich hydroxyapatite (HA) nanocrystallites, so it is a good choice to opt for a HA / collagen nanocomposite as a bone graft material. An extensive analysis of biomaterials information suggests that HA / collagen is an indicative system for bone regenerative therapy, probably as one of the most appropriate (Murugan and Ramakrishna, 2005; Liu, 2009; Wang, 2009).

[07] When minor bone loss occurs, the graft is often performed with the patient's own bone. When bone crest loss is very large, as in the case of defects left by extensive dental extractions in the anatomy of the jaw bones, grafts with biomaterials are used and thus the preservation of the bone crest is guaranteed, which will favor a subsequent implant or restoration of bone. dental prosthesis (Weiss, 2007).

[08] The initial stability of the mandible compared with the stability of the maxilla makes it possible to consider the immediate implant loading, which in most cases is much more problematic regarding bone quality and quantity. Patients with lower quality and less bone were excluded from implant treatment for a long time. However, the advent of bone reconstruction of deficient areas of both the mandible and the maxilla has improved the possibility of treatment for bone deficient patients. Different bone grafting techniques have been developed, and orthognathic surgical procedures adapted to the special requirements of implant surgery have now caused most bone problems to be resolved (Kahnberg, 2005).

[09] Natural teeth have been replaced by a variety of materials including bone, animal teeth, human teeth, ivory, sea shells, ceramics and metals. Four groups of materials are currently employed in dentistry, which are metals, ceramics, polymers and composite resins. Despite frequent improvements in the physical properties of these materials, none of them are permanent (Anusavice, 2005).

[010] The practice of implant dentistry is currently cited as the most modern method when discussing subjects dealing with oral rehabilitation. Prior to this method the oral rehabilitation problem was solved by the use of conventional removable or fixed prostheses. But the clinical success of implants is related to the phenomenon of osteointegration or osseointegration, which is nothing more than the physical union of the osseointegrated implant with the recipient bone. Branemark and his group of researchers after decades of laboratory and clinical research and development have discovered an implant system that can replace lost natural teeth and achieve this osseointegration (Martins, 2011).

[011] Through the concepts of osseointegration, the craniofacial deformity rehabilitation surgeries presented a great advance. The use of properly made implants and installed with correct surgical techniques is a good alternative for making support pillars of maxillofacial prostheses, giving the patient the possibility of fast and simple placement and removal of these implants. retention, stability and aesthetics. Osteointegrated implants show excellent results, but socializing these benefits is still a major challenge due to the economic issue, and it is not easily accessible to the poor who generally need this type of rehabilitation (Antunes, 2008).

[012] Biomaterials have been presented as an alternative in bone rehabilitation processes. Successful implantation of a biomaterial is associated with the reactions of the patient's organism, such as severity of the inflammatory process triggered, level of patient satisfaction, time required for the restoration of basic activities and time of implantation in the host organism.

[013] Biomaterials should be designed and constructed already specifically programmed to perform in a given application so as to satisfy the fundamental characteristics such as biocompatibility in which the implanted material and its degradation products must be tolerated by the surrounding tissues and should not cause dysfunctions in the environment. organism over time, be chemically inert and stable, easily sterilized and have biofunctionality, ie the material must meet the mechanical characteristics necessary to perform the desired function for as long as necessary.

[014] Many materials are developed for medical applications, such as therapy devices, diagnostic imaging contrast, controlled drug delivery devices (Guo, 2010) and also targeted for nerve and tissue repair over a period of time.

[015] PVAI Polyvinyl alcohol is a biodegradable synthetic polymer that has attracted special attention as a biomaterial due to its transparency, strength and biocompatibility. It is water soluble at temperatures above 70 ° C and both liquid and particulate PVAI compositions are currently available on the market and depending on their structure and stereochemical mixture will influence the specific properties of the final product.

PVAI residual acetate groups are essentially hydrophobic, which weaken intra and intermolecular bonds between adjacent hydrogen atoms of hydroxyl groups. When fully hydrolyzed, the melting point is recorded between 210 ° C and 240 ° C and glass transition temperature around 85 ° C (Bavaresco, 2004). It may normally have 20-35% crystallinity, but after heat treatment above its glass transition temperature (Tg), it can be increased by up to 70% and significantly improve its mechanical properties (Pepas, 1987).

Isocyanates are organic compounds which have in their chemical structure the group (-N = C = 0). These NCO groups react easily with compounds that have available active hydrogen atoms with at least two functional groups. The reaction of the isocyanate group occurs when sufficiently active hydrogen is bonded to atoms with an available electron pair, such as nitrogen and oxygen, and when hydrogen is bonded to hydroxyl oxygen, this reaction is called a urethane reaction. In addition to these reactions with active hydrogen-containing compounds, isocyanates can also react with each other to form polymers (Pinto, 2007).

[018] Isocyanates with aliphatic and aromatic chemical structure are currently available. About 95% of all isocyanates consumed are based on toluene diisocyanate (TDI) and methylene diphenyl isocyanate (MDI), and derivatives. However, one of the commercially important reactions is trimerization that leads to the formation of rigid structures called polyisocyanates, such as the HDI trimer HDT used in this work. These groups give rigidity and good thermal stability to the structures of which they are part (De Cario, 2002). HDT is a solvent-free, medium viscosity aliphatic polyisocyanate based on the hexamethene disocyanate trimer (HDI homopolymer).

[019] Polyurethanes (PUs) are from a broad family of polymers that can be potentially useful in tissue engineering in many tissue types for a large number of medical applications.

[020] Biodegradable polyurethane is one of the biocompatible materials used as scaffold matrices in engineered bone tissue (HILL, 2007 and HUANG, 2009). However, the main concern associated with biodegradable polyurethane is the lack of bioactive groups, which limits its application (Huang, 2009). One solution is to mix polyurethanes with bioactive ceramic particles such as tricalcium phosphate or hydroxyapatite (HA) (Huang, 2009; Brovarone and Verne, 2007). A combination of ceramics and polyurethane can improve the bioactivity and mechanical properties of scaffolds (Mathieu, 2001). HA (Ca10 (PO4) 6 (OH) 2) has attracted interest as an implant material for teeth and bones due to the similarity of its crystallography and chemical composition to human tissue (Liu, 2001; Kui, 2001).

[021] Hydroxyapatite has been widely used because of its chemical similarity to bone and good biocompatibility (Cystera, 2005). This biological hydroxyapatite is also composed of ions in various concentrations, such as: Ca2 +, Mg2 +, Na +, C032-, etc., allowing the control of these important ions in body fluids through their release or storage.

[022] One of the important features of the hydroxyapatite structure is that it allows hydroxyl (OH-) groups to be removed relatively easily, generating empty channels between the calcium ion-formed hexagons through which they can be led into the structure. of ceramic material, other ions and molecules (Hench, 1991).

[023] Hydroxyapatite is a bioceramic that has similar composition and structure to the mineral phase of bones and teeth. Depending on its purity, it can withstand heating above 1,200 ° C without decomposition. In addition, it can be modeled like most ceramic materials (Zavaglia, 1993).

[024] Hydroxyapatite, (Ca10 (PO4) 6 (OH) 2) is the major mineral component of bone, and its synthetic form is one of the most widely used biomaterials for skeletal reconstruction due to the lack of local or systemic toxicity in together with its osteoconduction properties (Díaz, 2009). For bone or dental implants lasting for many years, a poorly soluble material consisting of pure hydroxyapatite is used. When the implant is desired to be resorbed by the body, giving way to new bone tissue, a more soluble ceramic is used, generally consisting of a mixture of hydroxyapatite and other phosphates.

[025] Another characteristic of HA is its ability to adsorb, that is, to fix molecules of another substance on its surface. This property allows it to be used in implants, as a support for antibiotics and anticancer drugs, and can also be used for prolonged treatment of infections and bone diseases. In the latter case, gradually releasing the necessary medication in the affected region. The pore size and morphology of highly porous foams can be controlled depending on the process parameters (Cystera, 2005).

[026] A number of related documents describing biocomposites for bone regeneration are found in the literature and the most relevant are cited below.

[027] In studies by Machado (MACHADO, 2010) PU / HA composites are presented. The processability of the polymers allowed the preparation of relatively high load content composites with good homogeneity. However, HA particles at higher proportions were not fully enclosed by the polymer matrix. Future research with these materials involves the study of their chemical stability and evaluation of mechanical performance and in vitro biocompatibility. Eventually, the improved chemical bond between HA and the polymer matrix would be required. The present invention has shown that the proportion of HA was uniformly distributed within the matrix, therefore in a suitable proportion. Moreover, in the work of (MACHADO, 2010), there is no evidence of biocompatibility of the material, since in vivo and in vitro biological tests were not performed.

[028] Dong (DONG, 2009) obtained scaffolds containing 30 wt% nanohydroxyapatite- (n-HA) and 70 wt% polyurethane (PU) by a foaming method. The results showed that n-HA particles were homogeneously dispersed in the PU matrix, with 80% porosity and 271 KPa compressive strength. The porous structure provided a microenvironment with good adhesion for cell growth and proliferation, having the composite basic requirement for tissue engineering and being potential to be applied in the repair and replacement of human knee and articular cartilage menisci. The value of compressive strength is much lower than that of the present invention, which was on the order of 60 to 110 Mpa. The porosity level of the research material mentioned, although it has favored the cellular environment, must have been responsible for the low mechanical resistance, which makes it impossible to repair and replace human knee and articular cartilage menisci because they are impact absorbing sites. that is not the case with the material of the present invention which has an impact absorber in its constitution, the PVAI.

[029] Vital (VITAL, 2006) evaluated composites of synthetic and carbon hydroxyapatite (HAC) and synthetic hydroxyapatite, carbon and sodium bicarbonate phosphate (HACF), both in solid form as bone substitute in 36 adult rabbits. At 150 days it was found that in both treated and control groups, there was already bone marrow and almost no longer the presence of cartilaginous tissue. In both groups there was mature bone tissue in the failure region, showing very advanced bone repair processes and concluded that both types of hydroxyapatites used were biocompatible because there was no evidence of implant rejection in either animal. or inflammatory reaction. In treated animals, the bone regeneration process occurred earlier than in the control group, which proves the osteoconductive capacity of hydroxyapatite. The research cited used biphasic composites of synthetic hydroxyapatite and carbon (HAC) and synthetic hydroxyapatite, carbon and sodium acid phosphate (HACF) and in the daily clinical evaluations of the wound were observed presence of inflammatory reaction, pain sensitivity and wound dehiscence. Already in the present invention, due to the polyurethane phase with antibacterial action, no such reactions were observed.

[030] In studies by Liu (UU, 2009) it was found that loading of aliphatic PU with up to 50 wt% HA showed results indicating the average size of macropores and micropores from 100pm to 510pm, which demonstrates a good Perspective to be used as scaffold in bone tissue. The search compressive strength results for 30% HA were much lower, on the order of 227 KPa, than those presented in the present invention. The bionanocomposite of this invention showed, for 25% and 33% HA, compressive strength values of the order of 60 MPa and 111 MPa respectively. Furthermore, the research did not perform in vivo testing, so there is no evidence of biocompatibility of the material obtained, unlike the results presented in the present invention.

[031] Chetty (CHETTY, 2007) studied a HA-coated PU-based auricular cartilage implant and observed that after 24 and 72 h HA surfaces exhibited significantly more metabolically active cells compared with virgin PU surfaces. This indicates that HA surfaces are cytocompatible to fibroblasts and can potentially be applied to cartilage tissue replacement. However, disadvantageously, it was revealed by microscopic examination that the specimens had a coating defect. The present invention, on the other hand, shows a chemical interaction between HA and PU that prevents the registration of bionanocomposite failures.

[032] Dou (DOU, 2011) obtained antibacterial HA-gelatin minocycline nanocomposites and concluded that the nanocomposite has good bioactivity and may have antimicrobial activity, and may be a promising biomaterial for use as bone tissue repair and regeneration. The researchers only performed in vitro tests for a nanocyte material, with minocycline, which is an antibiotic drug, which already guarantees the material antibacterial action. Already in the present invention the good results are proven by in vitro and in vivo tests, without the need to use any kind of drug, because the antibacterial action of the polyurethane is efficient so as not to cause inflammation and infections. Furthermore, unlike the bionanocomposite presented in this invention, the material developed by Dou has no mechanical strength.

[033] Huang (HUANG, 2009) used a bioactive factor to improve this property in polyurethane and obtained high porosity HA scaffolds of 61 to 65% and suitable macropore sizes of 200 to 600pm and compressive strength of. 4.0 to 5.8MPa. The present invention associates the polyurethane to the hydroxyapatite bioactivity and the bionanocomposite presents mechanical properties much higher than the properties of the mentioned material, by employing PVAI in its composition.

Hill (HILL, 2007) describe biodegradable polyurethane (PU) scaffolds in tissue engineering to create new bone. PU discs were seeded with osteoblasts and implanted in nude mice. One group contained pure PU deployed disks while the other group contained deployed PU / HA disks. After 5 weeks both groups showed radiographic and histological evidence of significant bone formation, with a tendency for greater bone formation in the group that contained PU / HA composite implants. Already present the proposed bionanocomposite in addition to the presence of PU and HA, the PVAI, also present, has the function of absorbing impacts and resisting the efforts caused by chewing. It is admitted that the jaw works like a lever of the third kind. The fulcrum is the temporomandibular joint itself (TMJ) which together with the teeth receives a force load during the chewing movement. The developed force can be more or less absorbed by the fulcrum according not only to the amount generated, but also to the size of the distance between the resistance elements, in this case the teeth and the fulcrum (TMJ). In this case, chewing with the incisors increases the resistance arm and the load on the fulcrum is also increased. The maxillary bones and TMJ are adapted for molar chewing. Mechanical forces developed at molar level are better absorbed and drained. In incisive chewing, the load transferred to the TMJ is almost twice as high. Thus, for a composition focused on a maxillary facial graft, the scaffold developed by the author mentioned above could not be indicated because it does not have the material with impact-absorbing properties, important for chewing action, such as Poii (vinyl alcohol). which exists in the bionanocomposite composition of the present invention. Furthermore, in the present invention the possible inflammatory effect after implantation in subcutaneous tissue was tested, which showed greater biocompatibility, unlike the work by Hill (Hill, 2007) which does not prove the absence of this effect. Therefore the invention is a biomaterial with the proven antibacterial action of polyurethane associated with the bioactivity of nanohydroxyapatite, the dispersiveness and impact absorber property of polyvinyl alcohol, which remains in the composition after the synthesis of polyurethane forming with it. a blends, features of great importance for facial maxillary graft that are not verified in the scaffold described above.

[035] Wang (Wang, 2009) also worked with HA / PU scaffolds and concluded that they had good affinity for cells and are suitable for regeneration or replacement of bone defects. These scaffolds had 20-60% by weight HA. . However, disadvantageously, polyurethane was specifically used as a pore modeler without effective action on scaffold. And the cytocompatibility of the scaffold HA / PU from the study was evaluated by culturing a single cell type, MG63, which are derived from human bone sarcoma, with characteristic features of osteoblasts. Unlike Wang's proposal, the present invention not only keeps the PU in its final structure but also has an additional component, PVAI, which guarantees the absorption of impacts. The mechanical compression properties of the present invention, which is important for bone grafts of greater mechanical stress, as is the case with jaw bones, were of the order of 60 to 111 MPa, much higher than 114 kPa from the work cited above. Furthermore, the present invention demonstrates the cytocompatibility and antibacterial action of the proposed composition by employing CHO Chinese rat ovarian cells, NIH3T3 rat embryonic fibroblast cells and VERO cells, a recommended fibroblast cell line for cytotoxicity testing and proved its antibacterial action, since the animals did not show any pain, inflammation and / or infection during the postoperative period when they were observed.

[036] US2011 / 0313538 of 08/09/2007 describes a bone repair scaffold, where PU is used for forced pore formation and evaporated at processing temperature, so it does not remain in the final composition . Already in the present invention the presence of the PU phase, which is preferably obtained from polyvinyl alcohol, proves excellent blood compatibility and antibacterial properties, which guarantees a promising proposal of the present bionanocomposite to repair bone loss and orthodontic applications. Micropore and macropore formation is naturally obtained at the time of bionanocomposite processing by the reaction of carbon with oxygen other than US2011 / 0313538 A1.

US2012 / 0029653 of 16/11/2010 describes a material for bone regeneration, composed of resorbable materials and collagen. Unlike this application the present invention is applied to facial maxillary graft and promotes collagen production when implanted in the living organism after seven days, demonstrating its superiority as it is not necessary to add another product to the reaction, different from the US2012 / 0029653 patent biomaterial which has a collagen phase in its constitution. In addition this patent application US2012 / 002965 relates to a material for treating bone tissue defects and injuries while the physical characteristics of the present bionanocomposite, due to the presence of polyvinyl alcohol, promote a high absorption rate of impact, which specifies it for the mandibular area where the bones in this region receive major impact daily.

In view of the foregoing, the present invention has advantages in several respects. Firstly, it proposes a new bionanocomposite suitable for facial maxillary bone grafts that allows the flow of nutrients and ions where bone regeneration is promoted in the area around the scaffoid in bone implants. Furthermore, said bionanocomposite has a three-phase constitution based on polyurethane (PU), polyvinyl alcohol (PVAI) and hydroxyapatite (HA). The presence of PU enables excellent blood compatibility and antibacterial properties, which guarantees a promising proposal of the present bionanocomposite to repair bone loss and orthodontic applications, as well as the formation of micropores and macropores in the structure. The present invention advantageously employs PVAI which, due to its physical properties, ensures high impact elasticity and abrasion resistance as well as high biocompatibility and high barrier properties. PVAI itself acts as a dispersant in the mixture, requiring no other resources for this function. Another crucial point is the presence of nanostructured HA that has contributed to the control, amount, random geometry and pore and micropore interconnection that are excellent characteristics for osteoconduction. Another advantage is that this bionanocomposite promotes collagen production when implanted in the living organism after 7 days.

Brief Description of the Invention The present invention relates to a bone restorative bionanocomposite composed of polyvinyl alcohol (PVAI), polyurethane (PU) and hydroxyapatite (HA).

[040] The polyurethane employed is a reaction product of an isocyanate and a polyvinyl alcohol polyol, the isocyanate being selected from aliphatic isocyanates, 4-diisocyanate butane (BDI), 1,6-diisocyanate hexane (HDI), 4,4-Methylene dicyclohexyl diisocyanate (HMDI), preferably HDT polyisocyanate, hexamethylene diisocyanate trimer (HDI), employed between 1 and 2 g, preferably 2 g. And hydroxyapatite is added in a ratio of 25% to 33%, preferably 25%.

It is a further object of the present invention said bone restorative bionanocomposite with optional components to provide some desirable feature not achieved with the major components.

[042] It is an additional object to use said bionanocomposite for facial maxillary graft grafting, skullcap filling and filling and bone failure filling after bone cancer tumors removed in different regions of the skeleton.

Brief Description of the Figures: The structure of the present invention, along with additional advantages thereof can be better understood by reference to the annexes and the following description: Figure 1 shows the PVAI-PU / HA biomaterial spectra in the monitored FTIR from 25 ° C to 150 ° C. - Figure 2 shows the PVAI-PU / HA biomaterial spectra with FTIR heating. - Figure 3 shows the rheology curves of PVA-PU formation. - Figure 4 shows rheology curves of the PVAIPU / HA biomaterial. - Figure 5 shows the X-ray diffractogram of PVAI-PU / HA biomaterial with 25% HA. - Figure 6 shows the X-ray diffractogram of PVAI-PU / HA biomaterial with 33% HA. - Figure 7 shows a graph indicating the average compressive strength of PVAI-PU blend and PVAI-PU / HA bionanocomposites with 25% and 33% HA. - Figure 8 shows the TG-DSC curve of biomaterial with 25% HA. - Figure 9 shows the TG-DSC curve of the biomaterial with 33% HA.

Brief Description of Attachments: [044] Annex 1 presents the bionanocomposite of the present invention for characterization and biological testing in dimensions, 0 4mm and height equal to 60mm (1) 0 4mm and height equal to 1mm (2) 0 6mm and height equal 3mm (3) and 2cmx2cm (4).

[045] Annex 2 shows an image of the surface of the PVAI-PU blend observed by SEM.

[046] Annex 3 shows an image of the inner surface of the 25% HA biomaterial observed by SEM.

Annex 4 presents EDS-analyzed bionanocomposite spectra.

[048] Annex 5 shows the slides after sectioning for histological analysis of the region filled with the PVA-PU / HA biomaterial.

[049] Appendix 6 shows the blades of control material.

[050] Annex 7 presents an image of the biomaterial implanted in the subcutaneous tissue of the back of the mouse for 14 days, observed by scanning electron microscopy, where C: fibrous material; BM: biomaterial; CL: fibrous layer; CC: corneal layer.

Detailed Description of the Invention The present invention describes a bone restoration bionanocomposite comprising the following major components: polyvinyl alcohol (PVAI), polyurethane (PU) and hydroxyapatite (HA).

[052] The most commonly used process in the production of polyurethanes is that which involves the reaction of a compound with two or more alcohol functional groups, such as a polyether polyol or polyester polyol with an isocyanate, di or polyfunctional (Cordeiro, 2007), ( Zhang, 2011) forming urethane bonds. In addition to isocyanate and polyol, a chain extender may also be used in the reaction, with molar ratio proportions.

More specifically, the preparation of the bionanocomposite of the present invention may be initiated by employing an isocyanate, being selected from aliphatic isocyanates such as 1,4-diisocyanate butane (BDI), 1,6-diisocyanate hexane (HDI), 4,4-Methylene dicyclohexyl diisocyanate (HMDI), preferably HDT polyisocyanate, hexamethylene diisocyanate trimer (HDI) and a polyvinyl alcohol polyol, by weight and without the use of the chain extender.

To obtain the PU prepolymer 1 to 2 g, preferably 2 g of the viscous HDT material with preferably 1 g of poly (vinyl alcohol) particles are used at each reaction. Hydroxyapatite is added in proportions ranging from 25 to 33%, preferably 25% of the bionanocomposite, resulting in a 1: 2 to 1 ratio preferred mixture of PVAI, HDT and HA starting materials respectively.

In addition to these components, the bionanocomposite of the present invention may further comprise optional components to provide some desirable feature not achieved with the aforementioned components. Some of these compounds that may be added to the biocomposite are as follows: Anti-inflammatory agents; - growth factors; - cytokines; - hormones; - Stem cells; - bone marrow cells; - cartilage cells; - bone cells; - blood cells.

[056] Bionanocomposite can be potted in different molds according to the region in which it will be applied, placed in a greenhouse for curing and then removed and cooled naturally to room temperature, demoidized and referred for morphological, mechanical and biological characterization. .

[057] The bionanocomposite of the present invention has mechanical compression properties which are important for bone grafts of greater mechanical stress, such as maxillary bones ranging from 60 to 111 MPa. Besides being a material with water absorption capacity ranging from 2.2% to 6.3%, with apparent density ranging from p = 1.1 g / cm3 and p = 1.3 g / cm3 and with porosity ranging from p = 21.5% and 38.2%.

Example 1: Obtaining PVAI-PU / HA Bionanocomposite

[058] Initially, reactions for the synthesis of polyurethane (PU) were performed. Two grams of the HDT viscous material was mixed with one gram of Polyvinyl alcohol particles at each reaction in a polypropylene becker under magnetic stirring with heating at ambient temperature to 80 ° C. to obtain the PU prepolymer. Then hydroxyapatite was added to the mixture in three distinct proportions of 25%, 33% and 40% in each reaction, slowly and for 15 minutes, time that according to FTIR the materials already started reaction in the mixture, even with the temperature control at 80 ° C. The mixture was removed from the becker and filled into three different molds. The first closed stainless steel mold with a size for cylindrical samples with a diameter of 6 mm and a height of 3 mm. The second enclosed polypropylene mold with an internal diameter of 4 mm and a height of 60 mm and, third, an open Teflon mold for prism-shaped samples with a size of 20x20x2 mm3. After filling with the mixture, the material molds were placed in an oven for curing at 120 ° C for 30 minutes and then naturally removed and cooled to room temperature and demolded.

[059] Annex 1 shows the biomaterial obtained in three distinct forms.

Example 2: Fourier Transform Infrared Spectroscopy -FTIR

[060] Infrared absorption spectra were recorded by a THERMO SCIENTIFIC NICOLET IR10Q spectrophotometer. Reaction kinetics were monitored by FTIR and biomaterial wavelength measurements from ambient temperature of 25 ° C to 150 ° C were considered.

[061] After an 18-day room temperature cure experiment, FTIR monitoring was performed under the same conditions on both the starting materials and the PVAI-PU / HA biomaterial. For the analysis measurements, the materials were wrapped in polyethylene (PE) film for better reading in a paper sample holder adapted to the equipment. Material analyzes were performed by subtracting the PE bands from 4000 to 500 cm -1.

[062] PVAI-PU / HA infrared spectra that have been monitored from ambient temperature of 25 ° C to 150 ° C are shown in Figure 1. It is noted that typical absorptions of hydroxyapatite OH ion movements are difficult to see right after the start of mixing (point 1) and visible at the end of the reaction (point 2); Apatite phosphate vibrations (point 3) are changed during PU matrix formation and recovered at the final instants (point 4). Release of 002 during the reaction is responsible for the formation of pores in the structure (point 5).

[063] Figure 2 shows biomaterial spectra monitored during FTIR formation kinetics with heating from 15 to 75 minutes, where points of modification of hydroxyl ions and NH group formation of PU and modifications to phosphate groups (PO) are highlighted. ) of hydroxyapatite.

Example 3: Rheological Testing [064] Rheology is responsible for obtaining information on the effect of various factors that influence the processing of polymeric materials. Rheological studies were performed through tests to obtain variables such as processing temperature for hot cure and viscosity, both for the reaction of the PVAI and HDT mixture that formed PVAI-PU and in the PVAI-PU / HA biomaterial mixture.

[065] Both PVAI-PU and PVAI-PU / HA materials still in the viscous state were subjected to rheological tests on HAAKE Rheo Stress 6000, rheometer model, PP35 sensor, PP35 thermal controller type RS 6000, up to 150 ° C, same temperature adopted in FTIR. Storage modulus (G '{u>)) and dissipation modulus (Θ "(ω)) were verified as a function of temperature as well as temperature in PVAI / HDT and PVAI-PU / HA composite materials and processing temperature for hot cure of composite material.

[066] Figures 3 and 4 show the rheology curves of PU formation and PVAI-PU / HA biomaterial, respectively.

From determining the gel points of PVAI-PU formation at 130 ° C and the PVAI-PU / HA formation at 115 ° C, the ambient temperature range up to 120 ° C was used as the temperature of cure of PVAI-PU / HA biomaterial for 30 minutes at this last temperature. Such conditions showed excellent result for cure processing. The presence of nanocrystals may restrict polyol relaxation, leading to viscoelastic behavior, (Wik, 2011).

[068] The material cured on the rheometer at 115 ° showed white color and showed no signs of degradation, this visual morphology was determinant for the further processing to obtain the bionanocomposites with the appropriate characteristics.

Example 4: X-ray Diffraction (DR-X) [069] X-ray measurements of PVAI and PVAI + HDT were performed by the PHILIPS-binary (scan) (RD) and goniometer PW1800 X-ray technique using the powder method, with copper Ka-ray source Ka and scanning in the range of 5o to 60 ° (2θ).

The phases present in biomaterials and HA were determined by X-ray powder diffraction (XRD) in total sample. A PHILIPS-binary X-ray diffractometer with a normal focus PW 1800 Goniometer and a copper anode X-ray tube were used. Data acquisition of the records was obtained through an interphase and software, and the treatment of the data with the APD software, Automated Powder Diffaction.

[071] In Figures 5 and 6 the intensity of HA peaks becomes weaker in the presence of PU attesting a decrease in HA crystallinity in the lower HA PVAI-PU / HA biomaterial, a situation also found by (Wang, 2009). The three most intense HA peaks had a small displacement in the PVAI-PU / HA biomaterial with a 25% HA ratio, meaning that after the PU formation reaction there may have been a change in the nature of HA by the reaction of the hydroxyls of its structure.

[072] The PVAI-PU / HA biomaterial still has overlapping peaks at 20 near 20 ° (crystalline) and 20 near 40 ° (amorphous) characteristic of semicrystalline PVAI (ZHANG, 2010) confirming PVAI remnant analysis by FTIR.

Example 5: Water Absorption [073] Water absorption after immersion was calculated using Equation 1 (Asefnejad, 2011; Martinez-Valencia, 2011).

Ab (%) = (nri2-m1) / ith x 100 Equation. 1 [074] Where mi is the dry mass before immersion and iri2 is the wet mass after immersion.

[075] Five samples from each scaffold were weighed to within ± 0.0001 g on an analytical balance and their weights recorded. After weighing the samples were immersed in a glass becker with 10 ml of distilled water. After 24 hours the samples were removed from the water and then the surface water was removed with paper and the samples were reweighed. The assay was performed at 22 ° C.

[076] The water absorption values found in this survey 6.3% and 2.2% were inversely proportional to the amounts of hydroxyapatite, which shows that the hydrophilic nature of the biomaterial was higher with a lower proportion of HA.

Example 6: Density The pycnometer was washed with alcohol, dried at room temperature and filled with distilled water, which was checked. The mass of each biomaterial sample was duly measured as well as the mass of the pycnometer and the combined mass of the sample and pycnometer with distilled water three times.

[078] The apparent density of the biomaterial was calculated as a function of the pycnometer mass with water, the solid body in the watch glass and the pycnometer mass with the solid body in it. The assay was performed at 22 ° C.

The values for biomaterial apparent density were p = 1.3 g / cm3 and p = 1.1 g / cm3 which are directly proportional to the amounts of hydroxyapatite contained in the biomaterial.

Example 7: Porosity [080] Scaffold porosity was determined using liquid displacement or Archimedes principle, a method similar to that reported by (Guan, 2005; Liu, 2010) and five samples were evaluated for each scaffold in the USP Lorraine laboratory. with a temperature of 18 ° C.

Each sample of biomaterial with two different proportions of hydroxyapatite was immersed in a cylinder containing a volume of 10 ml of V1 ethanol. The sample was kept in ethanol for 5 minutes to allow ethanol to fill the sample pores. Total ethanol volume and scaffold with impregnated ethanol were recorded as V2. Ethanol-impregnated scaffold was removed from the cylinder and the residual ethanol volume was recorded as V3. The assay was performed at 18 ° C. The scaffold porosity “p” was calculated according to Equation 2. p = (Vi - V3) / (V2 -V3) x100% Equation. 2 [082] Porosity values were p = 21.5% for the material with the highest amount of hydroxyapatite and 38.2% for the biomaterial with the lowest proportion of hydroxyapatite.

Example 8: Mechanical Compression Test Mechanical tests are performed according to normative guidelines, among them ASTM D 695-96 which contains compression test guidelines. This test method determines the mechanical properties of rigid, unreinforced and reinforced plastics, including high modulus composites, when loaded under compression at relatively low stress rates or even loading. Standard specimens are used. This process is applicable to a composite module up to 41,370 MPa (6,000,000 psi) (ASTM D 695-96).

[084] Compression test is the application of a uniaxia compressive load! in a specimen and whose response of this test is obtained by the linear deformation measured between the distance of the plates that compress that specimen.

[085] The sample for the standard test shall be in the form of a straight cylinder or prism whose length is twice its main width or diameter (ASTM D 695-96).

[086] A total of 30 0 6 mm and 3 mm high cylindrical specimens according to ASTM D695-96 were tested under compression. The tests were performed on an Emic DL500 universal machine at the FEM-UFPA mechanical testing laboratory at 30 specimens distributed in 10 of each of the materials as follows, PVAI-PU, PVAi-PU / HA with 25% HA and PVAI-PU / HA with 33% HA.

[087] Because these specimens are small in size, the loading was interrupted prior to the rupture of approximately 40% of the original length deformation (Wang, 2009; Liu, 2009; Liu, 2010), to prevent compression plates had contact, which could compromise machine performance.

[088] Figure 7 shows the values of each mean strength and standard deviation of PVAI-PU blend and bionanocomposites with 25% and 33% HA under compression. These values are the result of bionanocomposite tests until deformation of up to 40% of the total length of the specimen. It can be seen that the result with 33% HA content in the PU matrix composite makes the compressive strength higher.

Despite the higher proportion with higher proportion of hydroxyapatite, the material selected for biological assays contained the lowest proportion of hydroxyapatite because other important properties for a biomaterial were considered, such as porosity, density and absorption that were more appropriate. in the bionanocomposite with lower HA ratio.

Example 9: Scanning Electron Microscopy (SEM) [090] Characterization of the normal and fracture surfaces of both starting materials and bionanocomposites was obtained using a LEO Scanning Electron Microscope model 1450 VP and a ZEISS Modello Scanning Electron Microscope, EVO MA-15.

[091] Annex 2 shows an image of the surface of PVAI-PU where mainly the formed PU is observed and PVAI particles are not discernible. And Annex 3 shows an image of the inner surface of the biomaterial as used in biological assays, where PVAI microparticles are observed in the domain of bionanocomposite, polyurethane and hydroxyapatite phase, pores and micropores of varying sizes, which have adequate architecture for biomaterial with application in tissue engineering.

Example 10: Dispersive Energy Spectroscopic Analysis (EDS) [092] EDS analysis was performed on both polyvinyl alcohol, hydroxyapatite and biomaterial starting materials to verify how the constituent particles of these materials were chemically altered. After obtaining the SEM material images, images were selected for EDS analysis. Four (4) points were randomly determined in each image for analysis.

[093] PVAI-PU / HA biomaterial images, Annex 4, shows EDS-analyzed bionanocomposite spectra on the left and determination of points on the PVAI-PU / HA material on the right attesting that the structure contains the constituent elements of the starting materials. and the Ca / P ratio ranged from 2.3 to 3.1. The uniform distribution of these materials will actively influence the ionic balance between biological fluid and biomaterial. Table 1 shows the concentration of the elements that make up the bionanocomposite analyzed by EDS.

[094] Table 4.7 - Concentration of elements obtained by EDS for the PVAI-PU / HA biomaterial.

Example 11: X-ray Fluorescence Spectrophotometry (XRF) [095] For biomaterial analysis, an X-ray fluorescence spectrophotometry (XRF) assay was performed. The elements of the PVAI-PU / HA biomaterial content were determined on a Rigaku brand, Rix 3100 model.

[096] The results of the fluorescence assay show that the Ca / P ratio of hydroxyapatite is greater than the original value, this confirms the excess calcium in the biomaterial hydroxyapatite structure. The amounts values are shown in Table 2.

[097] Table 2 - Average biomaterial composition by X-ray fluorescence.

Example 12: Simultaneous Differential Exploratory Calorimetry (TG-DSC) [098] The analyzes were performed at a heating rate of 10 ° C per minute in a nitrogen atmosphere in the ambient temperature range at 600 ° C on NETZSCH equipment, STA409C model. Analyzes were performed on the biomaterial PVAI-PU / HA with proportions of 25% and 33% hydroxyapatite.

[099] TG-DSC curves shown in Figures 8 and 9 provide information on the thermal stability of biomaterials with 25% and 33% HA, respectively.

[0100] The first endothermic peak near 225 ° C refers to the melt temperature of the biomaterial. The second endothermic peak is due to the combustion of organic material when material loss occurs. And the third peak corresponds to the reaction of hydroxyapatite in the biomaterial.

Significant changes in onset of mass loss were not observed between the curves as both begin at approximately 250 ° C. This loss is a consequence of the decomposition of biomaterials. However, a 25% loss is observed for the 25% HA composition and 17% for the 33% HA composition. Thus it can be said that with the increase in the amount of HA, there is, however small, an increase in the thermal stability of the material.

From approximately 300 ° C an intense decay occurs and this may be related to the breakdown of rigid segments of the biomaterial, producing free radicals that attack the soft segments of the biomaterial, resulting in chain breakage process.

Example 13: In vitro Biological Assays Chinese rat ovary (CHO) cells were cultured in monolayer and kept in a greenhouse with 5% CO2 and 95% atmospheric air and supplemented in cells (Roswell Park Memorial Institute) medium RPMI 1640, containing 10% fetal bovine serum (SFB), after tripinization, 3,000 cells were seeded in a 96-well cell culture microplate. Then increasing dilutions of the PVAI-PU / HA biomaterial extract (50pl / well, 4 wells / per dilution) were added, after which the plate was equilibrated at 37 ° C. The total volume in each well should be 10ΟμΙ. Four-well control columns are prepared with medium without cell placement (blank) and medium, rather than extract, with cells (negative control = 100%). After 72 h, 20μΙ of a (20: 1) mixture of 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyi) -2- (4-sulfophenyl) 2H tetrazolium) (MTS) a 0.2% and 0.09% phenazine methosulfate (PMS) in Phosphate buffer saline (PBS) was added to the test wells and allowed to incubate for 2 hours. Dye incorporation was measured by a 490nm microplate reader against white. The cytotoxicity index (IC) 50% is estimated by the interpolation curve, as the concentration of the biomaterial extract resulting from the 50% inhibition of MTS incorporation, correlating the average percentage of viable cells in relation to the concentration of the extracts from the biomass. graphic.

[0104] In the tests carried out through the positive and negative controls it can be verified that both the polyvinyl alcohol components that in reaction with the diisocyanate formed the polyurethane and hydroxyapatite and the composites obtained by the mixture showed no toxicity.

[0105] A mouse embryonicfibroblast cell line (NIH3T3) was cultured on the biomaterial. NIH3T3 cells were plated at concentrations 5x105 and 106 cells per biomaterial. The biomaterial was kept in 300μΙ of modified Dulbecco's medium (modified Dulbecco's Eagle's medium - DMEM) with 10% fetal bovine serum and also poly-l-lysine for three days in an oven at 37 ° C with 5% CO2. Cell proliferation analysis was performed by cell viability testing with tetrazoyl-3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl bromide (MTT).

VERO cells, a fibroblast-type cell line recommended for cytotoxicity testing, were used. Cells were cultured in DMEM medium (Gibco) supplemented with 10% Bovine Fetal Serum (SFB, Gibco) at 37 ° C with 5% CO2 in the atmosphere.

Extracts of the analyzed materials were obtained by incubating them in a 24-well culture plate at a ratio of 0.2 g of material per ml of 10% SFB DMEM medium at 37 ° C for 48 hours without shaking. After the incubation period, the medium was used to grow VERO cells, thus allowing to evaluate the possible release of toxic substances in the culture medium. Indirect cytotoxicity testing and extract extraction were performed according to international recommendations (ISO-10993-5, 1992; ISO-10993, 1997, NBR-ISO10993, 1999; SJOGREN, 2000).

For the cell viability test by the MTT method (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) a cell suspension at a concentration of 3x10 6 cells / ml was inoculated into 96-well culture plate (Corning Costar Corporation, Cambridge, MA, USA) and grown for 24 hours at 37 ° C.

After the 24 hour incubation period, the culture medium contained in the plate was replaced by the extract of the materials. The positive toxicity control (CPT) was a DMEM solution containing 10% SFB and 10% phenol and the negative toxicity control (CNT) polystyrene extract.

After the 24-hour cultivation period the material extract was removed and the wells were washed with 200μΙ saline phosphate buffer solution (PBS). After this procedure 200μΙ DMEM medium with 10mM Hepes buffer and 50μΙ MTT Sigma (5pg / ml in PBS) was added and the culture plate was incubated in the dark for 4 hours at 37 ° C. After this time the MTT medium was replaced by 200μΙ Dimethyl Sulphoxide (DMSO) and the plate was stirred for 30 minutes.

Absorbance was read on a Microplate reader at 540nm wavelength. Wells where no cells were cultured (blank) were used as reaction control, in which the same reagents as described above were added. [0112] Test results performed by the MTT method demonstrate that NIH3T3 - murine fibroblast cells , cultured on the biomaterial are viable since the cells plated in the presence of the reagent formed, which can be visualized due to the turquoise coloration observed in the images. It was also observed that the use of adhesion protein is of great necessity for cell adhesion. Different cell concentrations were also used on the biomaterial, but regardless of concentration the adhesion was efficient. Example 14: In vivo Biological Assays Assay 1 Four four-year-old Wistar rats with an average weight of 400 grams were kept and monitored in individual cages with food and water ad libitum and controlled temperature. The experiments were approved by the Animal Ethics Committee.

The animals were anesthetized with intramuscular injection of ketamine (85 mg / kg; im) and xylazine (12 mg / kg; im). The hair was shaved and a midline incision (20 mm) was made in the skin along the sagittal suture. The musculature and periosteum were knocked out exposing the parietal bone. Two 5.0 mm bone defects were created with a trephine in the rat calvary. The 4 mm diameter and 1 mm thick PVA-PU / HA disc-shaped implants sterilized with ethylene oxide (Dias, 2010) were inserted into the right bone defect and the left defect was not filled and was Used for control. After surgery, the musculature and periosteum were repositioned. Postoperatively, Dipyrone analgesic (500 mg / ml; vo) was used for the subsequent 24-hour period. All animals were closely monitored and received pelleted feed and ad libitum water.

The animals were anesthetized and sacrificed after 30 days of implantation by cervical dislocation. The implant discs were harvested and fixed in 10% formalin, decaffeinated (DONG, 2009), dehydrated and paraffin embedded. Each explant was then processed for histological analysis where 5pm sections were obtained which were stained with Hematoxylin & Eosin (Hill, 2007; Dong, 2009; Kim, 2011). The biocompatibility of the PU / PVA implant was quantified by the severity of the fibrosis capsule, foreign body reaction and nonspecific inflammatory process. Osteoconduction properties were determined by bone regeneration capacity.

In the implant slide, Annex 5, there is the presence of thick basophilic, acellular, compact solid masses resembling synthetic bone tissue, interspersed with phbrins, dilated and congested capillaries, sparse giant cells called osteoclasts.

[0117] In the control slide, Annex 6, we noted the presence of prominent osteocytes in the injured tissue and would need some time to close what does not happen with the implant blade where tissue closure with the bionanocomposite is visible confirming biointegration and biocompatibility between bionanocomposite and recipient bone. There are also some fibroses on both slides.

Assay 2 Six Swiss mice (30-35g) were divided into three groups of two animals and kept in 12-hour light / dark cycle plastic boxes (Jovanovic, 2010) and room temperature (22 ± 1) ° C. , with water and food ad libitium. The experiment was developed according to the animal research ethics committee.

Mice were anesthetized with ketamine / xylazine 2: 1 diluted in saline. After shaving, a 10 mm incision was made in the dorsal midline in the animal and the autoclave disc-shaped PVA-PU / HA biomaterial for 0 4 mm and 1 mm thick thermolabile materials was implanted in the subcutaneous space. dorsal spine (Hiil, 2007). After surgery the tissues were repositioned and sutured. Postoperatively no medication was used. All animals were closely monitored and received pelleted feed and ad libitum water. The animals were sacrificed in CO2 chamber for the removal of implanted material 1, 7 and 14 days after implantation. (Hafeman, 2012).

[0120] The biomaterial was taken together with the mouse tissue and fixed in a solution containing 2.5% 25% glutaraldehyde, 4% paraformaldehyde 2.5% sucrose in sodium cacodylate buffer (Khandwekar, 2010). 0.1 M, pH 7.2 for 2 hours at room temperature. After fixation, cells were washed 3 times in 0.1 M cacodylate buffer and then post-spun in solution containing: 1% osmium tethoxide and 0.8% potassium ferrocyanide for 1 hour at room temperature. The material was washed and then dehydrated in increasing series of ethanol (50, 70, 90% and 100%) (Kim, 2011; Dias, 2010) for 20 minutes at room temperature. The samples were dried by the critical point method (Model K 850 - Emitech Brand) using CO2. The material was mounted on an appropriate support (stub) and metallized with a gold film approximately 2pm thick using the Emitech K550-England apparatus. The analysis was performed under LEO 1450VP scanning electron microscope.

[0121] Annex 7 shows an image of the biomaterial implanted in the subcutaneous tissue of the back of the mouse for 14 days, obtained by scanning electron microscopy. Overview of the interaction of biomaterial (BM) with subcutaneous tissue layers; B- detail of the region highlighted in A. The intimate contact of the cellular fibrous layer formed around the biomaterial (arrow) and the extensions of the fibrous material that make up the layer are observed; C- Overview of the interaction of the biomaterial with the skin layers. The formation of the fibrous layer is observed around the entire biomaterial; D- detail of the region highlighted in C. The process of cell invasion in the biomaterial is observed.

Claims (9)

1. Bionanocomposite comprising polyurethane, polyvinyl alcohol polyol, and hydroxyapatite. Bionanocomposite according to Claim 1, characterized in that the polyurethane is a reaction product of an isocyanate and a polyvinyl alcohol polyol. Bionanocomposite according to Claim 2, characterized in that the isocyanate is selected from aliphatic isocyanates, 4-diisocyanate butane (BDI), 1,6-diisocyanate hexane (HDI), 4,4-methylene dicyclohexyl diisocyanate (HMDI), preferably HDT polyisocyanate, hexamethylene diisocyanate (HDI) trimer. Bionanocomposite according to Claim 3, characterized in that the HDT polyisocyanate is employed between 1 and 2 g, preferably 2 g. Bionanocomposite according to Claim 1, characterized in that the polyvinyl alcohol is preferably employed 1g. Bionanocomposite according to Claim 1, characterized in that it comprises hydroxyapatite in proportions ranging from 25 to 33%, preferably 25% of the bionanocomposite. Bionanocomposite according to claim 1, characterized in that it comprises optional components selected from anti-inflammatory agents, growth factors, cytokines, hormones, stem cells, bone marrow cells, cartilage cells, bone cells, blood cells. Bionanocomposite according to claim 1, characterized in that it has mechanical compression properties ranging from 60 to 111 Mpa, water absorption capacity ranging from 2.2% to 6.3%, bulk density ranging from p = 1 , 1 g / cm3 and p = 1.3 g / cm3 and porosity ranging from p = 21.5% to 38.2%. Use of the bionanocomposite described in claims 1 to 7, characterized in that it is applied in the preparation of a buccal maxillary facial graft composition, reconstruction and filling of the skullcap and filling of bone defects in different regions of the skeleton.