BR102017022237A2 - ORAL REPAIR BANDAGES, PROCESS OF OBTAINING THEM AND USE - Google Patents

Abstract

The present invention describes repair buccal bandages as well as the process for obtaining them. The bandages of the present invention are used in the repair (healing / healing) of buccal, dental and pulp tissues after deep interventions, since these bandages have bioinductive properties, low cost and easy application. By being in close contact with the absorption tissue, the bandages release the drug at the site of action, with the consequent increase in bioavailability, thus promoting local tissue repair effects where it is applied.

Description

ORAL REPAIR BANDAGES, PROCESS OF OBTAINING THE SAME AND USE

Field of the invention:

[001] This invention is part of the field of human needs, more specifically in the area of preparations for dentistry, and describes oral repair bandages, as well as the process of obtaining them.

[002] Also, the use of these bandages is described in the repair (healing / healing) of oral, dental and pulp tissues after deep interventions, since these bandages have bioinductive properties, low cost and easy application.

Basics of the invention:

[003] Research related to biomaterials, as well as their development and applications in the health field, are proving to be increasingly important and instigate the search for polymers with bioactive properties, biodegradability and atoxicity.

[004] Natural polymers, such as chitosan, collagen, alginate, starch, cellulose and carboxymethylcellulose derivatives, acetate and others, have important biophysical and biochemical properties for applications in biotechnological products.

[005] Recent studies suggest the development of materials based on these polymers to replace damaged tissues, stimulating the necessary response for living tissues.

[006] Chitosan is a natural polymer derived from Ndesacetylation of chitin, constituent of the exoskeleton of arthropods, which has hemostatic properties,

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2/27 fungicide, antibacterial and antitumor, in addition to the ability to stimulate cell migration and proliferation, reorganizing the histoarchitecture of cells.

[007] Current scientific literature is well documented with publications that used chitosan in several applications in tissue engineering, in the controlled release of drugs and also as biosensors for clinical diagnosis. Chitosan has promising properties for the formation of scaffolds used in tissue engineering.

[008] In addition to chitosan, another polymer that stands out is alginate, which, being soluble in an aqueous medium, becomes interesting for biomedical applications. The use of this polymer is extremely important for the treatment of wounds that are difficult to heal, as it is considered relatively inert, biocompatible, biodegradable and because it has the ability to form high porosity gel matrices with good mucoadhesive properties.

[009] The union of these polymers (chitosan and alginate) provides the development of a complex in which an electrostatic interaction occurs between the amino groups of chitosan and the carboxyl groups of alginate, promoting greater efficiency in the controlled release of drugs and in the absorption of lesion fluids.

[010] Dentistry has long sought a therapeutic element that can be applied to the oral cavity, which is adhered to the oral mucosa for the treatment of surgical wounds, areas with wounds arising from physical and / or chemical trauma and other types ulcerated oral lesions.

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3/27 [011] This issue is particularly significant when related to the treatment of children with oral trauma, in which post-trauma treatment is often not performed properly, mainly due to difficulties in performing dressings and / or topical application of medications , due to their behavior.

[012] The use of drugs incorporated into such biomaterials can be enhanced through the development of new delivery systems, such as mucoadhesive systems.

[013] Therefore, the present invention provides repair oral bandages, as well as the process of obtaining them. Said bandages are produced from natural polymers, such as chitosan and alginate, incorporated with the drug chlorhexidine or with Portland cement.

[014] The bandages of the present invention are used in the repair (healing / healing) of oral, dental and pulp tissues after deep interventions, since these bandages have bioinductive properties, low cost and easy application.

[015] Because they are in close contact with the absorption tissue, the bandages release the drug at the site of action, with the consequent increase in bioavailability, thus being able to promote local and systemic effects.

State of the art:

[016] Some prior art documents describe oral films as drug delivery systems, such as chlorhexidine.

[017] WO 2012/017469 describes a drug delivery system, which comprises cellulose derivatives, alginates or chitosan. Specifically, the

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4/27 system is useful in topical periodontal treatment when associated with chlorhexidine.

[018] In the case of the product of the present invention, the incorporation of new active ingredients allows a greater diversification of use, in dental, pulp and oral tissues, not only with the objective of controlling microorganisms, but also assisting in the repair and regeneration tissue, acting as a physical barrier and providing protection to the tissues when the bandage is applied.

[019] The drug delivery system presented contains differences in the manufacturing process and in the active principle, Portland cement, the present invention being intended for the repair of other tissues, aiming to act on other cell types and inducing other responses, resembling only to the product of the document cited in relation to the materials used at the base of the system.

[020] JP 2005-263704 refers to an oral film comprising chitosan, sodium alginate and carboxymethylcellulose. The same does not present an application, the use of these active ingredients together being justified to obtain a pH balance and improvement in the delivery of associated drugs, factors which are also observed in the present invention. However, this invention provides for the use of other associated active ingredients and in different concentrations and dissolutions.

[021] In Fast dlssolving oral films: an innovative drug delivery system and dosage form, reference is made to oral films that comprise a polymer and plasticizers, in addition to drug and other components such as fillers and dyes. Among the polymers, we can mention the

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5/27 carboxymethylcellulose and sodium alginate and, as a plasticizer, glycerol. The materials used here are similar to the family of active ingredients used in the present invention, but they totally differ in the form of preparation and preparation of the final product when compared to the process now described. The ways of dissolving gels and interacting with. the drugs incorporated are complex and different from the proposals of the present invention. Still, the product described here, although it has similarities in the solvents used, its production and applicability are different and innovative in view of the state of the art.

[022] In Chitosan films and hydrogels of chlorhexidine gluconate for oral mucosal delivery, simple or reticulated films of chitosan are shown, which comprise chlorhexidine, which proved to be good prolonged release systems, therefore, very useful for periodontal therapy. The article and the product demonstrated focus on the gingival tissues, while the approach of the product of this invention is focused on the dental and pulp tissues and their repair, in the form of bandages for easy application (and not with hydrogels, as described in the articles ).

[023] In Preparation, in vitro characterization and preliminary in vivo evaluation of buccal polymeric films containing chlorhexidine, alginate or chitosan / alginate monolayer films were prepared for the sustained release of chlorhexidine. Bilayer films were also prepared, using HPMC (derived from cellulose). The films demonstrated good mucoadhesion, comfort and good drug release profiles. The method of preparation and the number of layers, as well as the incorporation of active materials, are

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6/27 different from the processes employed in the present invention.

[024] In contrast, the present invention provides a means of delivering drugs in which carboxymethylcellulose is associated with Portland cement (dental cement), as well as two different radiopacifiers, forming a triple layer membrane aimed at the repair and specific biostimulation of dentin tissues -pulpares.

[025] The bandages produced according to the present invention are unprecedented and meet a specific need in the dental field, showing a material of easy application, containing bioactive and biocompatible materials, with low cost and that, in contact with pulp and dentin tissues, they stimulate repair.

Brief description of the invention:

[026] The present invention describes oral repair bandages, as well as the process for obtaining them. These bandages are used in the repair (healing / healing) of oral, dental and pulp tissues after deep interventions.

[027] In a preferred embodiment of this invention, the bandages of this invention consist of a mucoadhesive drug delivery system and comprise, in their composition, chitosan, carboxymethylcellulose, alginate, glycerol, Portland cement, radiopacifying agents and chlorhexidine, as well as possible substitutes of the same.

[028] Because they are in close contact with the absorption tissue, the bandages release the drug at the site of action, with the consequent increase in bioavailability, thus being able to promote local and systemic effects.

Brief description of the figures:

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7/27 [029] In order to obtain a total and complete visualization of the object of this invention, the figures to which reference is made are presented, as follows.

[030] Figures IA and 1B are photographs of the bandages produced in accordance with the present invention and of the bandage that presented the best result, respectively.

[031] Figures 2A and 2B are Scanning Electron Microscopy (SEM) images of the control bandages in 250X and 1000X magnification.

[032] Figures 3A and 3B are SEM images of the bandages with pure portland cement in 250X and 1500X magnification.

[033] Figures 4A and 4B are SEM images of portland cement and iodoform bandages in 250X and 1500X magnification.

[034] Figures 5A, 5B are SEM images of portland cement and zirconia bandages in 250X, 500X and 1500X magnification.

[035] Figures 6 A-E demonstrate the radiographs of a control material and the bandages made in order to compare and demonstrate its radiopacity.

[036] Figure 7 represents the photographs of the bandages comprising chitosan, portland, portland with iodoform and portland with zirconia, in the dissolution test.

[037] Figure 8 shows cultures of Streptococcus

Mutans, where (A) is a banding group with no cement added, (B) is a banding group with portland cement added; (C) is a banding group with the addition of portland cement with the iodoform radiopacifier and (D) is a banding group with the addition of portland cement with the zirconia radiopacifier.

[038] Figure 9 shows cultures of Candida Albicans, in

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8/27 that (A) is a banding group without addition of cement, (B) is a banding group with addition of Portland cement, (C) is a banding group with addition of portland cement with the radiopacifier iodoform and ( D) is a banding group with the addition of portland cement with the zirconia radiopacifier.

[039] Figure 10 graphically represents the assessment of cell viability by the MTT assay among the different groups tested within 24 hours.

[040] Figure 11 graphically represents the assessment of cell viability by the MTT assay among the different groups tested within 48 hours.

[041] Figure 12 graphically represents the assessment of cell viability by the MTT assay among the different groups tested over the 72 hour period.

[042] Figure 13 graphically represents the comparison between the unlabeled cells that proliferated (red) and the cells labeled at time zero without proliferation (green).

[043] Figure 14 graphically represents the comparison between the control group, cells without treatment (red) and bandage + portland group (green) in 48 hours.

[044] Figure 15 graphically represents the comparison between the control group, untreated cells (red) and the bandage + portland + iodoform (green) group in 48 hours.

[045] Figure 16 graphically represents the comparison between the control group, untreated cells (red) and the bandage + portland + zirconia (green) group in 48 hours.

[046] Figure 17 graphically represents the comparison between the control group, untreated cells (red) and bandage + portland group (green) in 48 hours.

[047] Figure 18 represents the comparison graphically

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9/27 between the control group, untreated cells (red) and MTA group (green) in 48 hours.

[048] Figure 19 graphically represents the comparison between MTA (red) and pure banding group without adding cement (green) in 48 hours.

[049] Figure 20 graphically represents the comparison between the MTA group (red) and the bandage + portland + iodoform (green) group in 48 hours.

[050] Figure 21 graphically represents the comparison between the MTA group (red) and the bandage + portland + zirconia group (green) in 48 hours.

[051] Figure 22 graphically represents the comparison between the MTA group (red) and the bandage + portland group (green).

[052] Figure 23 graphically represents the expression of mRNA for collagen 1, * VS 12h control group, #VS 24h control group.

[053] Figure 24 plots mRNA expression for FGF2, * VS 12h control group.

[054] Figure 25 graphically represents the expression of mRNA for VEGFa, $ VS 6 and 24h intragroup banding without the addition of cement in comparison with the other periods $ VS 6 and 24h intragroup banding with the addition of portland cement in comparison with the other periods.

[055] Figure 26 are photographs showing cell morphology after contact for 24 hours for the made-up bandage groups, where A is a cementless bandage, B is a portland + bandage, C is a portland + iodoform bandage , D is a bandage + portland + zirconia and 72 hours for the groups of the made bandages, in

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10/27 that E is a bandage without cement, F is a bandage + portland, G is a bandage + portland + iodoform and H is a bandage + portland + zirconia.

Detailed description of the invention:

[056] The present invention describes oral repair bandages, as well as the process for obtaining them. These bandages are used in the repair (healing and / or healing) of oral, dental and pulp tissues after deep interventions.

[057] Mouth repair bandages include:

- chitin and its derivatives or chitosan with a degree of deacetylation greater than or equal to 75%;

- colloidal carbohydrate with emulsifying function or gelling action, such as sodium alginate, agar-agar gel or algae derivatives;

- cellulose derivatives;

- silicate and calcium oxide-based cements, such as Portland cement;

- plasticizing agents, such as glycerol;

radiopacifying agents, such as iodoform, zirconia, barium sulfate and bismuth oxide; and

- therapeutic agent with antimicrobial function, such as chlorhexidine, xylitol; propolis; cetylpyridinium chloride, triclosan and lysozyme enzyme.

[058] Chitin derivatives can be selected from the group consisting of: ethyl-sulfo-chitosan, N, 0-carboxy methyl chitosan, cyanoethyl chitin, chitin xanthogenate, alkali-chitin, alkyl-chitin, hydroxyalkyl chitin carboxymethylquitin and N-carboxymethyl chitosan.

[059] Cellulose derivatives are physiologically

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11/27 inert and has the purpose of interaction with cements, being selected from the group consisting of carboxymethylcellulose, cellulose diacetate and cellulose triacetate, preferably carboxymethylcellulose.

[060] In a preferred embodiment of this invention, the bandages comprise:

- 20 to 60% chitosan in the first and second layers in relation to the final volume, preferably 34% in the first layer and 26% in the second layer;

- 0 to 35% alginate in relation to the final volume, preferably 34%;

- 0 to 35% of carboxymethylcellulose in relation to the final volume, preferably 26%;

- 0 to 6% Portland cement in relation to the final volume, preferably 6%;

- 0.5 to 3% glycerol in relation to the chitosan mass, preferably 1.5%;

- 15 to 30% iodoform or zirconia in relation to the Portland cement mass, preferably 20%; and

- 0 to 3% chlorhexidine, preferably 2%.

[061] The process of obtaining oral repair bandages comprises the steps of:

(A) Preparation of chitosan;

(B) Preparation of alginate;

(C) Preparation of carboxymethylcellulose - CMC;

(D) Preparation of cements;

(E) Incorporation of the drug; and (F) Preparation of the membranes layer by layer.

Step A - Preparation of chitosan:

[062] Chitosan gel is prepared by dissolving the

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12/27 chitosan in a range of 0 to 5% (m / v) in a solution of acetic acid 1 to 5% (w / v). The solution is kept under agitation for a period of time ranging from 8 to 30 hours. After preparing the gel, a volume ranging from 0.5 to 3% of the total volume of gel produced (w / w) of glycerol or another plasticizer is added.

[063] Subsequently, the solution is vacuum filtered and the filtered gel is placed in suitable containers and kept in an ultrasound bath at a temperature between 25 ° C and 30 ° C for a period of time ranging from 1h to a maximum of 3h.

[064] After this step, the gel is stored in a cool place with temperatures between 4 ° C and 6 ° C for later use.

Step B - Preparation of alginate:

[065] The alginate gel was prepared by dissolving 0.5 to 10% (w / v) of sodium alginate in a volume ranging from 10 to 200 ml of distilled water. The solution is kept under stirring for a period of time ranging from 8-30 hours. After preparing the gel, a volume ranging from 0.5 to 3% of the total volume of gel produced (w / w) of glycerol or other plasticizer is added.

[066] Subsequently, the solution is vacuum filtered and the filtered gel is placed in suitable containers and kept in an ultrasound bath at a temperature between 25 ° C and 30 ° C for a period of time ranging from 1h to a maximum of 3h .

[067] After this step, the gel is stored in a cool place with temperatures between 4 ° C and 6 ° C for later use.

Step C - Preparation of carboxymethyl cellulose - CMC:

[068] The carboxymethyl cellulose gel was prepared

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13/27 by dissolving 0.5 to 5% (m / v) of carboxymethylcellulose in a volume ranging from 25 to 250 ml of distilled water (m / v). The solution is kept under stirring for a period of time ranging from 8 to 30 hours. The filtered gel is placed in suitable containers and kept in an ultrasound bath at a temperature ranging from 25 ° C to 30 ° C for an interval of 1h to a maximum of 3h.

[069] After this step, the gel is stored in a cool place with temperatures between 4 ° C to 6 ° C for later use.

Step D - Cement preparation:

[070] Cements with radiopacifying substances are prepared following a mass / mass ratio of 80% cement and 20% radiopacifiers. The powders are taken to a vibration device for a period of time ranging from 5 to 10 minutes of agitation for mixing and homogenization of the components.

[071] Then, the mixture obtained is sieved with fine-grained mesh, preferably mesh between 0.60 mm to 0.40 mm, aiming at the removal of the coarsest grains and homogenization, being stored in a suitable container at room temperature between 25 ° to 30 ° C for later use.

[072] Portland cement presents itself as a viable pulp capping material and inducer of dentin-pulp tissue repair. It has calcium minerals as constituents, which assist in the process of repair and remineralization of dentinal tissue.

[073] Still, it has antimicrobial activity, which helps in the elimination of contaminants from the region where the

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14/27 is applied, collaborating to obtain a contaminant-free zone and favorable to dentin-pulp repair.

[074] The radiopacifiers used in this invention are iodoform, which has antimicrobial and radiopacifying properties, and zirconia oxide, an inert material used in prophetic implants in the medical field of orthopedics.

Step E - Incorporation of the drug:

[075] Chlorhexidine (20% chlorhexidine digluconate) was incorporated into the alginate gel in a concentration ranging from 0.5 to 3% (w / w).

[076] In this stage, inert dyes and flavorings can also be added to oral tissues

Step F - Preparation of the membranes:

[077] After preparing the gels, they are packed in containers that facilitate their handling. Before preparing the membranes, the containers must receive a new ultrasonic bath for an interval that varies from 1 to 3 hours to remove bubbles and homogenize the prepared gels.

[078] Each bandage unit is prepared with three layers, according to its active principle, which are: chitosan / alginate-chlorhexidine / chitosan or chitosan / CMC cement / chitosan.

[079] Any container that resists a temperature of 50 ° C and has high edges is used as a mold to prevent the gels from draining out of the container.

[080] An initial layer of chitosan gel is spent on the mold container, weighing between 1.5 g and

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15/27

1.7 g of chitosan gel, approximately 34% of the final volume of the bandage. The gel is spread evenly over the entire surface, observing that no air bubbles form in the layer. The container is then placed in a drying oven with a temperature ranging from 25 ° C to 30 ° C for a period of 5 to 15 minutes.

[081] The second layer of carboxymethylcellulose is added, with a mass between 1.0 g and 1.3 g of the gel, approximately 26% of the final volume of the bandage. The homogenization process of the layer is repeated, and the plates are again dried in an oven, with temperature varying between 25 ° C to 30 ° C for a period of 3 to 5 minutes.

[082] Alternatively, for the production of the membrane containing chlorhexidine, the second layer of the bandage consists of a layer of alginate gel with a mass between 1.0 g and 1.3 g alginate gel containing the drug. In this step, flavorings and dyes can be added to the bandage. The homogenization process is repeated and the plates are again dried in an oven with a temperature ranging from 25 ° C to 30 ° C for a period of 1 to 3 minutes.

[083] Approximately 0.3 g to 0.5 g of cement or its preparation with radiopacifier, making up about 6% of the final volume of the bandage, is incorporated by sprinkling, with the aid of extra fine granulation sieves (pores between 0.30 mm and 0.40 mm), on the second layer of carboxymethylcellulose of the bandage. In this step, dyes can also be added to make the product more visible when used in deep cavities.

[084] At the end of the process, the third layer of chitosan is added to the membranes, the process being

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16/27 deposition, mass, homogenization and drying similar to that described initially for the first layer of this same material.

[085] After the preparation of the three layers of the bandages, the containers containing the bandages are again dried, with temperature varying between 25 ° C and 35 ° C for a period of 12 to 24 hours for the final drying of the product. After the final drying, the bandages are removed from the containers.

[086] The bandages are then wrapped in parchment paper and kept in a desiccator containing silica sealed with airtight lids until use.

[087] The bandages of the present invention are used in the repair (healing / healing) of oral, dental and pulp tissues after deep interventions, since these bandages have bioinductive properties, low cost and easy application.

[088] Because they are in close contact with the absorption tissue, the bandages release the drug at the site of action, with the consequent increase in bioavailability, thus being able to promote local and systemic effects.

Tests:

- Tactile and Visual Evaluation of Bandages:

[089] Bandages with different concentrations of chitosan and carboxymethylcellulose gels in each layer were evaluated, visually and in a tactile way, resulting in bandages with different thicknesses and malleability for a study of the best concentration range of gels in the layers aiming at their clinical use. Also, the bandages had different amounts of cement and the way the bandages were added. Starting

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17/27 of the result of this test, the ranges of each component were defined to obtain an ideal product.

[090] According to Figure IA, it was observed:

- Bandage 1: it seems acceptable and with good malleability, however the cement is not evenly distributed.

- Bandage 2: it is within the expected standard for the product, with homogeneous distribution of the cement by the bandage and good malleability (shown in Figure 1B).

- Bandage 3: very porous, the cement loosening to the touch.

- Bandage 4: very brittle consistency, not feasible for clinical use, poor handling material.

- Bandage 5: good malleability, but very porous, the cement loosens on contact with the bandage, in addition to being very thin.

- Bandage 6: a little hard, with poor handling and the cement was not evenly distributed.

- Bandage 7: cement loose to the touch and very thin.

[091] After the tactile and visual evaluation of the different bandages made from the different concentration ranges, it was agreed that, for the other tests, the concentration considered ideal for the final material would be used, being this:

- 1.7 g of chitosan in the first layer;

- 1.3 g of carboxymethylcellulose in the second layer;

- 1.0 g of pulverized cement (being tested pure and associated with two radiopacifiers);

- 1.3 g of final chitosan layer.

- Scanning Electron Microscopy [092] Scanning electron microscopy (SEM) was performed with the aim of analyzing the distribution of the

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18/27 cement in the bandages, as well as the surface texture of them.

[093] To this end, the following were analyzed:

- Control bandage (without cement);

- Bandage with pure portland cement;

- Bandage with portland cement and iodoform; and

- Bandage with portland cement and zirconia.

- Control bandage (without cement) (figures 2A and B): the images revealed a homogeneous surface, without any irregularities. The image is clear due to the transparency of the analyzed material, so the beam of light passes through the entire sample.

- Bandage with pure portland cement (figures 3A and B): the images showed a good distribution of the cement by the bandage, the surface being quite uniform, similar to what is observed in the control group. In the images at higher magnification, larger particles of cement may be noticed, which may not have broken in the cement dusting process.

- Bandage with portland cement and iodoform (figures 4A and B): there is a good distribution of the cement along the bandage surface and the crystals that are observed in the greatest increases are iodoform that did not break in the process of homogenization with the cement. In the image at the lowest magnification (250 X), there are also some bubbles of the polymer, which are not possible with the naked eye to be detected.

- Banding with portland cement and zirconia (figures 5A, B and C): they showed good distribution of the cement and, in the images in greater magnification, the zirconia crystals are noted

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19/27 on the bandage surface (normal, according to the literature).

- Radiographic Test:

[094] Radiographs of the bandages were taken to assess their radiopacity. As a standard for comparison, gutta-percha cones, material for routine use in endodontics and with good radiopacity (figure 6A) were used.

[095] The bandage without cement (figure 6B) does not have any material added, so the radiography is veiled, without image, since the materials that make up the bandage do not have radiopacity.

[096] The bandage only with Portland cement (figure 6C) also does not present radiopacity and the veiled radiography, without image.

[097] In the banding with portland cement and iodoform radiopacifier (figure 6D), there are, quite discreetly, inside the circle, some crystals of the iodoform, which are radiopaque.

[098] In the banding with portland cement and zirconia radiopacifier (figure 6E), it is possible to observe the entire bandage extension with a uniform distribution of the radiopacifying agent.

- Absorption and dissolution test:

[099] The bandages were cut into squares and immersed in DMEM solution (neutral pH 7.0 solution used for cell culture) and kept in a 5% CO2 oven at 37 ° C.

[100] After 24 hours of incubation, it was possible to observe that the three formulations containing Portland cement did not present any type of dissolution or loss of

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20/27 integrity in contact with the environment.

[101] Ά bandage without addition of cement, with pure chitosan, presented with the appearance of a viscous gel, but without becoming soluble in the DMEM culture medium.

- pH test:

[102] After the absorption and dissolution test of the bandages in DMEM medium, a rapid test was performed to measure the pH of the solution obtained at the end of the 24 hours of contact between the medium and the bandages.

[103] For this measurement, a concentration of 1 cm2 / mL was used for the bandages and for the MTA 0.1 g / mL material. As previously described, the materials to be

tested were immersed in DMEM medium and left in greenhouse in 5% CO2, at 37 ° C for 24 hours. [104] The results can be seen in the table 1 below: Table 1 - pH measurement result

GROUP MEASURE 1 MEASURE 2 AVERAGE Pure Bandage 7.24 7.50 7.37 Bandage + Portland 7.60 7.40 7.50 Bandage + Portland + Iodoform 7.36 7.35 7.36 Bandage + Portland + Zirconia 7.27 7.41 7.34 MTA 8.84 8.60 8.72

[105] The results obtained allow us to conclude that the bandages do not abruptly alter the pH of the DMEM solution, and remain within the acceptable standard for performing cytotoxicity tests by means of cell culture. The gold standard MTA, changes the pH of the medium, and at the end of the experiments may cause a change in the results obtained,

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21/27 related to cell death.

- Microbiological tests:

[106] In vitro tests were performed to assess the antimicrobial efficiency of the bandages against two strains of microorganisms commonly found in the oral cavity and associated with common diseases in the mouth, Streptococcus mutans and Candida albicans.

[107] To measure the zone of inhibition, the distance in a straight line between one edge and the other of the inhibition zone was considered, passing through the center of the bandage.

[108] To this end, the groups were divided as follows:

Group I - banding without cement (pure chitosan)

Group II - banding with portland cement

Group III - banding with Portland cement + iodoform;

Group IV - banding with Portland cement + zirconia;

Group VI - gold standard MTA.

[109] The microbiological tests did not show the formation of inhibition halos, however, the observation of the culture plates with the bandages allowed the visualization that there was no growth of the microorganisms on the tested bandages.

[110] These findings allow us to conclude that the bandages made with and without cement do not have bactericidal properties for the strains of microorganisms tested, however they have a bacteriostatic capacity, since the growth of microorganisms on the bandages was not observed (see Figures 8 and 9).

- Cell viability test using the MTT method:

[111] The MTT assay (3- (4,5-dimethyl-2 bromide) Petition 870170078557, 10/16/2017, page 32/72

22/27 thiazolyl) -2,5-diphenyl-2H-tetrazolium) is used to determine cell viability, quantified by reducing this salt to formazan crystals (blue / purple) by the cellular metabolic activity of mitochondrial enzymes.

[112] The averages of the absorbance values obtained by reading on the spectrophotometer, referring to the MTT tests, were grouped according to the different groups and periods of the study, as shown in Table 2.

Table 2 - Absorbance values (mean and standard deviation) of the groups after the MTT test with a cell density of 1 x 10 ³ in the periods of 24, 48 and 12 hours. Periods (h) Groups 24 48 72 Bandage 0.153 + 0.05 0.261 + 0.06 0.435 + 0.10 Bandage + portland 0.126 + 0.04 0.210 + 0.07 0.238 + 0.07 Bandage + portland / iodoform 0.138 + 0.05 0.243 + 0.09 0.319 + 0.09 Bandage + portland / zirconia 0.144 + 0.05 0.237 + 0.09 0.264 + 0.15 MTA 0.252 + 0.22 0.103 + 0.07 0.171 + 0.12 Positive Control 0.187 + 0.07 0.261 ± 0.10 0.373 +0.14 Negative Control 0.134 + 0.06 0.164 + 0.08 0.173 + 0.06

[113] In the MTT trial, there was a statistically significant difference between the periods (P = 0.02), in which the period

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23/27 of 48 hours showed greater cell viability among the three evaluated periods (P = 0.02). When comparing the tested groups, there was a statistically significant difference (P = 0.031) of all the groups tested compared to the MTA group in the period of 48 hours, in which this group presented lower values related to cell viability. In the period of 24 and 72 hours, there was no statistically significant difference between the groups (Figures 10, 11 and 12).

[114] According to the results obtained so far, it was found that the bandages developed with and without the addition of portland cement do not induce negative effects on the cell viability of pulp fibroblasts from human primary teeth, showing good biocompatibility.

- Cell proliferation analysis:

[115] Cell proliferation analysis was performed by counting cells by flow cytometry, using CFSE labeling. The purpose of this analysis is to prove that the tested materials allow normal cell proliferation when in contact with cells.

[116] The analysis revealed that the banding groups with the addition of cement plus radiopacifiers showed a more evident cell proliferation when compared to the control group treated only with the culture medium.

[117] Figure 13 shows the comparison at time zero and after the 48 hours of experiment, in which it is possible to observe, in red, cell proliferation and, in green, the marked cells.

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24/27 [118] The comparison between the tested groups of the fabricated bandages and the MTA compared to the control group revealed an evident cell proliferation for the groups bandage (Figure 14), bandage + portland + iodoform (Figure 15) and bandage + portland + zirconia (Figure 16).

[119] In these comparisons, it is possible to observe a proximity of the proliferation curves, indicating that the materials did not cause cell death and did not alter the cell proliferation process.

[120] The bandage + portland (Graph 17) and MTA (Graph 18) groups showed a distance from the proliferation control curve, which indicates that these treatments showed less cell proliferation when compared to the control, therefore, these materials modulated negatively the cell proliferation process, decreasing it in the studied period.

[121] Still with the data obtained in flow cytometry, a direct comparison of the experimental groups of the banding against the MTA was carried out, a commercial material used as a parameter for the banding.

[122] The results obtained demonstrated that the pure bandage without addition of cements and the bandages with addition of portland cement and radiopacifiers showed greater cell proliferation when compared to MTA (Figures 19, 20 and 21) and the group of bandages with addition of only Portland cement showed proliferation results similar to those of the MTA group (Graph 22).

[123] These results indicate that the addition of radiopacifiers to portland cement, as well as their incorporation into chitosan banding, formed a

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25/27 more biocompatible product in several aspects, demonstrated here by visualizing greater cell proliferation when compared to the product (MTA) already on the market.

- Real-time PCR:

[124] Real-time PCR analyzes were performed in order to verify the expression of some proteins related to mineralization and growth factors, being the targets of choice DMP-1 and DSPP for mineralization and growth factors FGF2, VEGFa and Collagen 1. In this experiment, the B2M gene was used as a reference gene.

[125] The analyzes revealed that the fibroblasts of primary teeth, after contact with the materials tested in the periods of 6, 12 and 24 hours, do not express DMP-1 and express a small amount of DSPP, both proteins related to mineralization. These results indicate the need for new tests against other cell types, such as odontoblasts or odontoblasts-like, in order to study the expression of these proteins.

[126] The data obtained from the readings showed little or insufficient expression. Regarding the other proteins analyzed, there was a significant expression of them in the studied periods.

[127] For the expression of collagen 1, the results obtained demonstrated that there was no significant difference between the groups after 6 hours of contact with the materials. In the 12-hour period, a peak of collagen expression is observed in the banding group without the addition of cement, showing a difference when compared with the others

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26/27 groups studied.

[128] After 24 hours of contact of the materials with the cells, a decrease in the expression of collagen is observed in the test groups when compared with the control group, however the comparison between them does not present a statistically significant difference (Figure 23). These results allow us to conclude that the tested materials do not alter the cellular function of fibroblasts, which remain active and secrete collagen in the three studied periods.

[129] In the analysis of FGF2 expression, which is a fibroblast growth factor, it was possible to observe an equity in the expression of this protein between the groups and the periods analyzed. Only in the 12-hour period, the banding group with the addition of portland cement and iodoform showed a peak of expression surpassing the control group (Figure 24).

[130] These results corroborate with other results of cell viability and proliferation indicating that the bandages and the tested control material do not alter the cell function and growth of the fibroblasts used in the research, which indicates the biocompatibility of the materials tested for this cell type.

[131] For the expression of the endothelial growth factor VEGFa, the results obtained demonstrated that VEGFa expression is present in all periods studied for all groups, and a peak of expression can be observed for the banding group without the addition of cement when compared to control group within 12 hours, this same peak can also be observed in the banding group with the addition of portland cement.

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27/27 [132] The intra-group analysis showed a difference between the periods studied for the banding group without the addition of cement, which showed a very expressive peak in the period of 12 hours, differing from the other periods, the same being observed for the group bandage with the addition of portland cement.

[133] The other groups showed no differences between them and intra-group in the studied periods (Figure 25). These results are encouraging, as they demonstrate the ability of VEGFa expression for all groups, indicating the ability of the developed bandages to act on endothelial growth, assisting in revascularization.

- Cell morphology:

[134] In all tests involving cell cultures, microscopic monitoring of cells was performed in the proposed experimental periods in order to observe the presence or absence of changes in the cell morphology of fibroblasts before and after treatments with the tested materials.

[135] Throughout all experiments, no changes in cell morphology were observed (Figure 26), which suggests that the bandages developed do not cause cellular changes or mutations.

[136] Those skilled in the art will value the knowledge presented here and may reproduce the invention in the modalities presented and in other variants, covered by the scope of the attached claims.

Claims (16)

1. Mouth repair bandages characterized by the fact that they comprise: - chitin and its derivatives or chitosan with a degree of deacetylation greater than or equal to 75%, preferably 30 to 60% of chitosan in relation to the final volume, more preferably 34%; - colloidal carbohydrate with emulsifying function or gelling action, such as sodium alginate, agar-agar gel or algae derivatives, preferably 0 to 35% alginate in relation to the final volume, more preferably 34%; - cellulose derivatives, preferably 0 to 35% carboxymethylcellulose in relation to the final volume, more preferably 26%; - silicate and calcium oxide-based cements, such as portland cement, preferably 0 to 6% Portland cement in relation to the final volume, more preferably 6%; - plasticizing agents, such as glycerol, preferably 0.5 to 3% glycerol in relation to the chitosan mass, more preferably 1.5%; radiopacifying agents, such as iodoform, zirconia, barium sulfate and bismuth oxide, preferably 15 to 30% iodoform or zirconia in relation to the Portland cement mass, more preferably 20%; and - therapeutic agent with antimicrobial function, such as chlorhexidine, xylitol; propolis; cetylpyridinium chloride, triclosan and lysozyme enzyme, preferably 0 to 3% chlorhexidine, more preferably 2%. Petition 870170078557, of 10/16/2017, p. 39/72 2. Bandages, according to claim 1, characterized by the fact that chitin derivatives are selected from the group consisting of: ethyl-sulfo-chitosan, Ν, Ο-carboxy methyl chitosan, cyanoethyl chitin, chitin xanthogenate, alkali- chitin, alkyl-chitin, carboxymethyl chitin hydroxyalkyl of chitin and N-carboxymethyl chitosan. 2/4 3/4 according to the fact that, in step (b), the alginate gel is prepared by dissolving 0.5 to 10% (w / v) of sodium alginate in a volume ranging from 10 to 200 mL of distilled water. 3. Bandages, according to claim 1, characterized by the fact that cellulose derivatives are selected from the group consisting of carboxymethylcellulose, cellulose diacetate and cellulose triacetate, preferably carboxymethylcellulose. 4/4 characterized by the fact that, in step (E), the therapeutic agent is incorporated into the alginate gel, together with dyes and flavorings inert to the oral tissues, if present. 4. Process for obtaining repair oral bandages, as defined in claims 1 to 3, characterized by comprising the steps of: (A) Preparation of chitosan; (B) Preparation of alginate; (C) Preparation of carboxymethylcellulose - CMC; (D) Preparation of cements; (E) Incorporation of the drug; and (F) Preparation of the membranes layer by layer. 5. Process according to claim 4, characterized in that, in step (a), the chitosan gel is prepared by dissolving the chitosan in a range of 0 to 5% (m / v) in an acid solution acetic 1 to 5% (m / v). 6. Process according to claim 4 or 5, characterized in that the solution is kept under agitation for a period of time ranging from 8 to 30 hours to obtain the gel, followed by the addition of a volume ranging from 0, 5 to 3% of the total volume of gel produced (m / m) of the plasticizer, followed by vacuum filtration. Petition 870170078557, of 10/16/2017, p. 40/72 7. Process, claim 4, 8. Process according to claim 4 or 7, characterized in that the solution is kept under agitation for a period of time ranging from 8 to 30 hours to obtain the gel, followed by the addition of a volume ranging from 0, 5 to 3% of the total volume of gel produced (m / m) of plasticizer, followed by vacuum filtration. 9. Process according to claim 4, characterized in that, in step (c), the carboxymethylcellulose gel is prepared by dissolving 0.5 to 5% (w / v) of carboxymethylcellulose in a volume ranging from 25 to 250 mL of distilled water (m / v). 10. Process according to claim 4 or 9, characterized by the fact that the solution is kept under agitation for a period of time ranging from 8 to 30 hours to obtain the gel, followed by filtration and ultrasound bath at a temperature that varies between 25 ° C and 30 ° C for a time interval of 1h to a maximum of 3h. 11. Process, according to claim 4, characterized by the fact that, in step (D), the cements are prepared following a proportion w / w of 80% cement and 20% radiopacifiers, with the mixture stirred for an interval time ranging from 5 to 10 minutes and sieved with fine-grained mesh, preferably mesh between 0.60 mm to 0.40 mm. 12. Process according to claim 4, Petition 870170078557, of 10/16/2017, p. 41/72 13. Process, in wake up with The claim 4, characterized by fact of, in stage (F) , the gels receive ultrasonic bath per a break what It varies from 1 to 3 am prior to the assembly of the bandages, which can be chitosan / alginate-chlorhexidine / chitosan or chitosan / CMC cement / chitosan. 14. Process according to claim 13, characterized in that the initial layer of chitosan gel has a mass between 1.5 g and 1.7 g, the second layer of carboxymethylcellulose gel has a mass between 1.0 g and 1, 3 g, the cement or its preparation with radiopacifier has a mass between 0.3ga0.5g and the last layer of chitosan has a mass of 1.5g and 1.7g. 15. Process according to claim 13, characterized in that, alternatively, the initial layer of chitosan gel has a mass between 1.5 g and 1.7 g, the alginate - chlorhexidine gel layer has a mass between 1, The gel, 3g and the last layer of chitosan has a mass of 1.5 g and 1.7 g. 16. Use of bandages, as defined in claims 1 to 3 and obtained according to the process of claims 4 to 15, characterized by the fact that it is in the repair, healing and / or healing of oral, dental and pulp tissues after deep interventions.