WO2023113584A1

WO2023113584A1 - Eardrum membrane made of human collagen for repairing eardrum injuries

Info

Publication number: WO2023113584A1
Application number: PCT/MX2022/050118
Authority: WO
Inventors: Juan Pablo AGUILAR ALEMÁN; Grecia Andrea CARDOSO HERNÁNDEZ; Brenda Karen AGUILLON ESTRADA; Beni CAMACHO PÉREZ
Original assignee: Top Health, S.A.P.I. De C.V.
Priority date: 2021-12-14
Filing date: 2022-12-01
Publication date: 2023-06-22
Also published as: MX2021015642A

Abstract

The present invention relates to an eardrum membrane made of human collagen for repairing eardrum injuries, said membrane exhibiting physical, chemical, mechanical and biological properties suitable for its implementation in the treatment of eardrum injuries and/or perforations. The membrane temporarily provides a scaffold and a physical barrier that separates the middle ear from the outer ear, while the tissue is being repaired and the continuity of the eardrum is restored, said membrane characterised in that it comprises a purity > 80% and a concentration of < 30 mg/mL or < 15 mg/cm2, having controlled dimensions of 0.5 to 15 cm in diameter or side, and 0.01 -10 mm in thickness and preferably 1 cm in diameter or side, and 0.03 - 0.3 mm in thickness, with a porosity from 80 to 99% and a pore size between 10 and 200 pm, and in that it further comprises at least one of a tissue glue, an antibiotic solution, drugs, growth factors and/or autologous or allogeneic mesenchymal cells, nanoparticles for the metered release of drugs or the mixtures thereof.

Description

HUMAN COLLAGEN TYMPANIC MEMBRANE FOR THE REPAIR OF EAR DAMAGES

FIELD OF THE INVENTION

The present invention is related to biotechnology and medical science in general, in particular, it is related to methods for repairing tissue lesions and more specifically it refers to a method for obtaining a crosslinked type I human collagen tympanic membrane of single use, used as a scaffold to repair eardrum perforations caused by the insertion of foreign objects into the ear, sudden changes in pressure (barotrauma), acoustic trauma, infections and/or middle ear injuries.

BACKGROUND OF THE INVENTION

The tympanic membrane is a thin, semi-transparent division between the external auditory canal and the middle ear. When sound enters the outer ear, the sound waves reach the tympanic membrane causing it to vibrate, the vibrations are subsequently transferred to the ossicles in the middle ear which transfer the vibratory signals to the inner ear. It is here where the cochlea converts acoustic vibrations into nerve signals that can be interpreted by the nervous system. the membrane The tympanic membrane is composed of a thin connective tissue membrane covered by an epithelial layer on the outer ear and by mucosa on the inner surface.

Faced with a perforation of the tympanic membrane, the patient may present pain, bleeding, hearing loss, tinnitus and vertigo. Diagnosis is based on visual inspection, through otoscopy. In general, no specific treatment is needed to treat perforated eardrums, since, in most cases, the eardrum tends to heal in a period ranging from a few weeks to up to three months without the need for an admixture. treatment. Optionally, oral antibiotics or ear drops may be prescribed to prevent or treat an infection.

However, when it comes to more severe lesions that heal slowly, that become complicated, where the perforation involves the edges of the eardrum or if it represents > 30% of the tympanic membrane, or if a chronic condition develops, treatments to repair the tympanic membrane perforation include everything from placing a tympanic patch, in a process known as myringoplasty, to the surgical procedure (tympanoplasty). Surgery may be necessary for perforations that persist > 2 months, when there is impairment of the ossicular chain, or lesions involving the inner ear.

If the lesion in the eardrum remains, pathogens can penetrate to the middle ear and cause infections (otitis media); in In some cases, chronic otitis media may develop, where infections are recurrent and there is a possibility that the infection may pass into the inner ear, resulting in damage to the auditory ossicles (ear bones) and affecting the patient's hearing.

A persistent ear infection also slows the repair process of the eardrum, by maintaining a constant state of inflammation in response to said infection. On the other hand, and even when the perforation has resolved, infections cause an accumulation of fluids in the middle ear, the presence of these fluids increases the pressure inside the ear, which can result in a ruptured eardrum due to barotrauma.

There are eardrum patches made from different materials. Among the most used are autologous temporal fascia or cartilage grafts, being the reference pattern in the treatment of tympanic perforations; allogeneic grafts, and synthetic and biological materials are also used, depending on the nature of the perforation.

There are cigarette paper patches, latex adhesive tapes (Steri-Strip) or silk patches, which are mainly used for small or sharp traumatic perforations or lacerations. There are also patches on the market made of hyaluronic acid, small intestine submucosa, chitosan, adipose tissue, collagen, acellular dermal matrix grafts, fibrinogen, enriched with growth factors.

In addition to patches and grafts, there are topical or spray suspensions, suspensions which gel or polymerize on the addition of a chemical stimulus, eg UV light, and form a gel at the lesion site.

A search was carried out to determine the closest state of the art, finding the following documents:

Document US2007162119A1 by Johnson Alan J. dated January 08, 2007 was located, which reveals a prefabricated tympanic membrane prosthesis, which includes a piece of collagen sheet processed with a dilute solution of formalin. The piece of collagen foil is molded onto a surface of a tool that is shaped to resemble the natural tympanic membrane of a human subject. The piece of collagen foil is processed with a dilute formalin solution so that the resulting tympanic membrane prosthesis retains its shape. The tympanic membrane prosthesis is semi-rigid, yet flexible, and is packaged in a substantially rigid container to protect its shape during shipping and storage.

However, it is not capable of adhering properly and staying at the lesion site throughout the treatment, and it does not have adequate thickness and porosity to favor the transmission of ambient sound to the inner ear. Document JP2010259767A by Park Chan Hum et al. of June 29, 2009, disclosing an artificial tympanic membrane using silk protein having a film-like structure, obtained by demineralizing and drying silk protein (or silk fibroin) or silk protein complex solution after removing the sericin from the cocoon or silk fiber. The artificial tympanic membrane promotes the reproduction of the tympanic membrane perforated by unexpected disease or accident, provides a thin border of the reproduced tympanic membrane, and has excellent biocompatibility and transparency and effective ease of use. The artificial tympanic membrane is produced by using silk protein or silk protein complex solution obtained from the cocoon or by mixing it with collagen, alginic acid, PEG ( po I ieti I eng I ico I ) or Pluronic F-127, etc.

As can be seen, the process for obtaining the artificial tympanic membrane of said document is very different from our invention and uses silk protein as raw material which, optionally, can be mixed with collagen, alginic acid, PEG ( p o I i eti I e n g I i c o I ) or Pluronic F-127. Despite the fact that it is mentioned that it has excellent biocompatibility, said membrane does not provide the benefits of the human collagen tympanic membrane of the present invention, it is not capable of adhering properly and staying in the lesion site throughout the treatment, it does not It has a suitable thickness and porosity to favor the transmission of ambient sound to the inner ear.

Some of the products described commercially or in other patents are not able to adhere and stay at the lesion site throughout the treatment. An example is cigarette paper or rice paper patches that tend to move along the surface of the eardrum or detach from it, exposing the area of the lesion and, therefore, failing to fulfill the function of a patch. Due to the above, it is common in these cases that a second medical intervention is required to reposition the patch in the appropriate area, which can cause inconveniences for the patient.

Other commercial or patented products are made from synthetic materials, so they cannot be broken down by the body, and a second medical intervention is necessary to remove the patch. In addition to the above, due to their synthetic nature, this type of product tends to provoke a more noticeable inflammatory response, which slows down the healing process and can cause discomfort in the patient.

Some of the products described commercially or in other patents use autologous tissue grafts that, although they represent the gold standard in the treatment of tympanic perforations, involve a surgical procedure on a donor area, and the surgical procedure to implant the graft on the eardrum, which prolongs patient recovery times, increases treatment costs, and involves a greater risk of complications of surgery.

Some of the products described commercially or in other patents, use biological materials, derived from the extracellular matrix, such as hyaluronic acid, which can promote the formation of granular tissue which, although necessary for the healing process, if generated in an uncontrolled manner, can lead to the formation of fibrotic tissue that it would affect the mobility of the tympanic membrane and, therefore, its function in the transmission of ambient sound to the inner ear. In addition, the production of the membrane with a controlled porosity in this type of products is a sophisticated and complex process that requires specialized equipment, which represents an increase in production costs.

Some of the products described commercially or in other patents use biological materials derived from the extracellular matrix, such as collagen; however, it is xenogeneic collagen that presents a higher risk of provoking an inflammatory response, because it comes from a different species from that of the recipient (patient).

Other products described commercially or in other patents use high concentrations of collagen, while other processes describe specialized and more complex methods for making the patch.

Some of the products described commercially or in other patents contain human collagen; however, the product has not been treated with proteases, so some regions of the collagen chain, responsible for provoking an antigenic response, may still be present, and generate an inflammatory response in the patient.

Given the need for a single-use, cross-linked type I human collagen tympanic membrane, used as a scaffold in the repair of perforated eardrums caused by the insertion of foreign objects into the ear, sudden pressure changes (barotraumas), acoustic traumas, infections and/or injuries of the middle ear, or as part of a surgical procedure, it was that the present invention was developed.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to make available a human collagen tympanic membrane for the repair of tympanic lesions that presents appropriate physical, chemical, mechanical and biological properties for its implementation in the treatment of tympanic lesions and/or perforations.

Another objective of the invention is to provide said human collagen tympanic membrane for the repair of tympanic lesions that, in addition, is made from a biological, degradable, highly biocompatible material and whose obtaining does not involve performing a surgical procedure on the patient, unlike autologous grafts. Another objective of the invention is to provide said human collagen tympanic membrane for the repair of tympanic lesions that, in addition, temporarily provides a scaffold and a physical barrier that separates the middle ear from the external ear, while the tissue repair is carried out. and the continuity of the eardrum is reestablished.

Another objective of the invention is to provide said human collagen tympanic membrane for the repair of tympanic lesions that, moreover, can be offered at a lower cost than currently available products and with greater effectiveness, due to its allogeneic protein nature.

Another objective of the invention is to provide said human collagen tympanic membrane for the repair of tympanic lesions that, in addition, allows it to be incorporated on the damaged tympanic membrane tissue and that offers a greater retention capacity allowing the addition of substances, factors or additives to the product that enhances its repair function, or that serve to broaden the purpose of use of the collagen membrane.

Another objective of the invention is to provide said human collagen tympanic membrane for the repair of eardrum injuries that, in addition, offers greater mechanical performance so that it resembles the resistance and flexibility of the human eardrum and can remain in the application site without breaking, detaching. , move, degrade prematurely or generate a thickening in the tissue or abnormality in the eardrum that harms the patient.

Another objective of the invention is to provide a standardized and optimized process for the manufacture of a human collagen tympanic membrane for the repair of tympanic lesions, which allows dispensing with more sophisticated techniques and a lower production cost than similar products in the market, without compromising quality.

Another objective of the invention is to provide a standardized and optimized process for the manufacture of a human collagen tympanic membrane for the repair of tympanic lesions, which also reduces the risk of infection by external pathogenic agents and accelerates the closure of the perforation of the eardrum. eardrum.

And all those qualities and objectives that will become apparent when carrying out a general and detailed description of the present invention supported by the illustrated modalities.

BRIEF DESCRIPTION OF THE INVENTION

During the design and development of the tympanic membrane for the repair of tympanic lesions of the present invention, various problems arose to be solved in order to build a functional prototype. The main problem was to develop a highly biocompatible product that did not generate a heightened immune response and that was made up of a bioactive material that promoted the healing process. In this regard, it was surprisingly found that collagen of human origin complied with these requirements, since intraspecific biocompatibility represents an advantage over materials used in other existing products.

The second problem to solve was that the dimensions of the product should be similar to the human tympanic membrane, especially in thickness, since a patch that is too thick could hinder and slow down the healing of the lesion, in addition to the risk of decreased hearing capacity of the patient during treatment due to the thickening of the tissue and consequent loss of mobility of the tympanic membrane. This was resolved by testing different I i o fi I i z a t i o n conditions and incorporating a pressing process to produce human collagen structures with controlled characteristics.

Similarly, it had to be guaranteed that the product, despite being a sheet, presented a porous structure with the appropriate pore size to promote cell adhesion and migration. This point was resolved by optimizing the concentration of the collagen solution and, therefore, the amount of collagen contained in each membrane. The next problem to solve was related to the functionality of the product. In order for the membrane to perform its function correctly, it is necessary that it be adaptable and resistant to the natural environment of the application site. The collagen membrane has to be able to adhere to the eardrum and withstand pressure changes between the outer ear and the inner ear, without detaching from the eardrum. In addition, it must be flexible so as not to interfere with the vibrations of the eardrum and not affect the hearing capacity of the patient. This was solved by increasing the mechanical properties of the collagen membrane through the controlled system of collagen crosslinking. Once a correlation was established between the degree of crosslinking and the characterization of the mechanical performance, the membrane was tested in a simulated environment where the ear conditions were mimicked. At this stage, the membrane met the requirements for flexibility, adhesion, resistance and cell migration, in addition to observing a liquid retention capacity despite its collapsed structure.

The human collagen tympanic membrane of the present invention was made with allogeneic bioactive compounds in order to lessen the patient's inflammatory response and contribute to a faster healing process and decrease the risk of possible complications. This was resolved by previously implementing a standardized and optimized production line for obtaining human collagen with high yields, and its conditioning to meet specific dimensional requirements, to through a simple and low-cost process.

Firstly, it was sought to achieve the dimensions through, only, the lyophilization process. By modifying the amount of collagen solution to be lyophilized and its concentration, it was sought that, upon removing the moisture, a collagen membrane would be generated. However, these adjustments resulted in membranes that were incomplete, very fragile, with an uneven surface, or that were not manipulable. In other cases, only dispersed collagen fibers were obtained. At the highest concentrations and solution volumes, a three-dimensional structure was obtained, resembling a sponge.

Due to the above, it was decided to find the combination of minimum collagen concentration and minimum volume of the solution, with which the scaffold with the least possible thickness was obtained, ensuring that a complete surface structure was formed, that it was uniform and that was resistant to manipulation. Simultaneously, experiments were carried out to manufacture a biofilm, generating a hydrogel from the collagen solution, and allowing it to dry until obtaining a thin layer of collagen that resembled a plastic sheet. This last option was discarded due to the absence of pores and the preparation of a collagen sponge continued.

Once the relationships between the concentration of the collagen solution and the volume to be used were determined, with Once the scaffoldings that best met the requirements were obtained, the possibility of collapsing the structure mechanically, through pressing, was discussed. Different pressing parameters were tested, including the applied force and the compression time. Some scaffolds did not withstand the pressure and broke, either because of their low collagen content or because the pressing parameters were excessive; On the other hand, some scaffolds did show a reduction in thickness, but recovered their original shape after a few seconds, this due to their high collagen content or because the pressing parameters, contrary to the previous case, were not effective. In the same way, we proceeded to determine the applied force-time relationship in which integral membranes were obtained and that they maintained the reduction in thickness, even if they were rehydrated.

Finally, and as previously mentioned, to ensure that the product fulfills its purpose of use in the real environment of application, a correlation was made between the degree of collagen crosslinking, addressed through an in vitro digestibility test, in set with TNBS (2,4,6-trinitrobenzenesulfonic acid) colorimetric technique; and the characterization of the mechanical properties exhibited, through a special universal machine for tissues and biological materials. The membrane was hydrated with physiological solution and/or blood, observing preservation of the structure and retention capacity with a maximum increase of 20 times its original weight, and without losing the ease of handling of the product. Subsequently, the wet membrane was placed in a flexible spherical body, remaining attached to the surface, expanding and contracting as the material inflated. During this elongation effort, the membrane did not rupture or tear and, during retraction, it recovered its original dimensions, it did not fold, nor did it form excessive grooves or wrinkles.

In general, the present invention refers to a single-use crosslinked type I human collagen tympanic membrane, used as a scaffold in the repair of perforated eardrums caused by the insertion of foreign objects into the ear, sudden changes in pressure ( barotraumas), acoustic traumas, infections and/or lesions of the middle ear.

The allogeneic nature of the type I human collagen tympanic membrane guarantees a more efficient repair process, since intraspecific biocompatibility allows a more controlled inflammatory response, unlike products manufactured from bio-materials from other animals. .

In relation to the above, the human collagen tympanic membrane represents an alternative to current treatments, contributing to a more dynamic repair and in less time that prevents possible future complications such as infections, recurrent perforations or extensive damage to the middle ear and/or internal, and avoiding, in some cases, the surgical procedure. The human collagen tympanic membrane of the present invention consists of a human collagen membrane, with physical, chemical, mechanical and biological characteristics that establish it as an appropriate alternative for the repair of tympanic injuries and/or perforations.

In general terms, the process for producing human collagen tympanic membrane in accordance with the present invention consists of the following stages, carried out in a temperature range of 2 - 25°C, reaching temperatures of up to -80°C in the stages of I i of i I i zation . a) Condition the tissue obtained from human amniotic membrane, tendon and fascia, removing tissues or fluids that are not of interest to the process and reducing the particle size in a uniform manner to a size between 0.5 mm and 2 mm with a mill. drum or rotor, to achieve a better interaction between solutions and tissue in subsequent steps. b) Pre-treat the previously conditioned tissue, exposing it to a 0.05 - 2 M sodium hydroxide solution, using 50 - 1000 mL per gram of dry tissue, with efficient magnetic stirring at 100 - 500 RPM and for a period of 1 hour to 24 hours, in order to remove surface proteins and leave the collagen fibers exposed; b 1) Wash with distilled water to neutralize the pH of the tissue until it reaches a pH between 7 and 8, before the next step; c) Extract the collagen by enzymatic hydrolysis subjecting the tissue to an acid solution of acetic acid at a concentration of 0.02 - 0.5 M and using 50 - 1000 L per kg of dry tissue, together with 50 - 1000 g of pepsin per kg of dry tissue, to promote more rapid collagen solubilization and promote a specific and controlled elimination of the terminal carboxyl and amino groups of the molecule; where the extraction is carried out in a period of three days making additions of intermediate acid solution (begins with 200 mL of acetic acid and 0.05 - 0.5 g of pepsin per g of dry tissue and after 48 hours 250 mL of acid are added acetic acid per g of dry tissue to promote the development of the reaction and where residues of non-collagenous tissue structures are removed by means of filtration with a 50 - 900 m sieve and the process is continued d) Precipitate the collagen bringing the resulting collagen solution (450 mL of solution per g of dry tissue) to a high salt concentration, adding 20 - 100 g of sodium chloride per 1 L of collagen solution, homogenizing the solution with magnetic stirring and leaving that the ionic interaction of the salt with the collagen molecules generates their precipitation, for a period of between 2 to 6 hours; subsequently, sieve the resulting solution to a particle size of 50 to 900 µm; where the fibers recovered from the screen are solubilized again in an acetic acid solution, using 50 - 1000 mL of acetic acid per g of dry fabric; e) Dialyze the collagen solution in order to purify the solution from excess salt present in the collagen solution, in a dynamic dialysis system in which collagen is placed within a 12-14 kDa porous membrane to expel impurities by introducing it into a dialysis buffer consisting of a low concentration 0.02-0.5 M acetic acid solution; where the exchange of salt molecules between both solutions originates from their concentration differential, from the collagen solution with a salt concentration of 0.5 -2 M to the acetic acid solution without the presence of salts, and is accelerated by contact surface and buffer flow; this stage lasts from two to five days, during which the buffer is kept in recirculation and is changed after 6 to 48 hours; The conductivity of both solutions is also monitored and the process is stopped when the collagen reaches the same conductivity as the buffer at the beginning of the stage (0.10 -0.5 mS/cm). f) Freeze-drying the purified collagen solution by subjecting it to a freeze-drying cycle to concentrate the collagen fibers, favoring their preservation; For this, the solution is frozen at a temperature between -10°C and -80°C and is subsequently subjected to a vacuum pressure of 0.02 - 0.2 mbar (2 - 20 Pa) for a period of one to four days. , during which water and solvents are removed in the form of steam, drying the collagen without damaging the fibers; where the resulting collagen is weighed and distributed according to what is required in the next step. g) Mold and lyophilize for the second time, where, depending on the application and expected function, a concentration of 0.5 to 30 mg of collagen per mL of acetic acid with a molarity of 0.02 to 0.5 M is chosen; once solubilized, the collagen is placed in molds that allow to generate the structure with the desired dimensions; again, the lyophilization of the solution is carried out at a temperature between -10°C and -80°C and a vacuum pressure of 0.02 - 0.2 mbar (2 -20 Pa), with the exception that a controlled freezing is carried out that It consists of removing the heat by gradually decreasing the temperature of the plate with which the mold and the collagen solution are in contact, allowing the crystals of water and acetic acid to be uniform and varied, accommodating the fibers to generate a size of estimated average pore; h) Press the collagen structure that, considering the application and function that the product must perform, is subjected to a determined mechanical force of between 100 - 5000 N to compact its dimensions to a desired value from 0.001 - 10 mm and increase its density. fibrillar. i) Crosslink the collagen pieces by subjecting them to a formaldehyde vapor atmosphere in a crosslinking apparatus, at an exposure time of between 1 to 90 minutes and a concentration of the reagent vapor cloud of 0.1 to 100 ppm, resulting in a Controlled crosslinking that allows to reinforce the union between fibers to provide better physical properties to the structure.

According to the process for producing human collagen tympanic membrane in accordance with the present invention described above, a type I human collagen tympanic membrane is obtained, with a purity of > 80% and concentration of < 30 mg/mL or < 15 mg/cm ² , of controlled dimensions of 0.5 to 15 cm in diameter or side, and 0.01 -10 mm thick and preferably 1 cm in diameter or side, and thickness of 0.03 - 0.3 mm), with a porosity of 80 to 99% and a pore size of between 10 and 200 pm.

The membrane is flexible, absorbable, adherent, resistant to the internal pressures of the ear and biocompatible.

The membrane is cut, according to the dimensions of the lesion observed, and later, it is hydrated with physiological solution, antibiotic solution or any other that the doctor considers appropriate. The moist membrane is placed over the eardrum, accessing through the ear canal and completely covering the lesion site. The membrane adheres to the eardrum due to its hydrophilic nature. In this way, the collagen membrane provides a provisional support that heals the discontinuity of the eardrum, allowing some transmissibility of sound waves, and on which the cells of the eardrum can migrate from the edge of the wound to the center, and will generate the epithelial and fibrous layers that will close the wound.

The average porosity of the elaborated membranes was 83.81 ± 7.09.

In the membrane manufactured according to the present invention, the smallest pores have a size ranging from 10 to 13.81 ± 4.94 pm, while the largest ones range from 100-200 pm, especially in the cross section of the membrane, which contributes to cell adhesion and migration on the membrane, as described later.

The thickness of the membrane is 234.79 ± 47.44 m.

The membrane presents an average moisture retention percentage of 1304.07 ± 166.81 %, and the mass increase was 14.04 ± 1.67 times the original weight (Ps). Noting that, despite the fact that the membrane absorbed liquids, there was no turgor of it, and the dimensions of the membrane were not affected.

Various substances can be added to the membrane, such as: Tissue glue to improve adherence of the membrane to the implant site; an antibiotic solution to treat or prevent an ear infection while the eardrum is being repaired; growth factors and/or autologous or allogeneic mesenchymal cells that, due to their signaling capacity, can promote or enhance biochemical pathways related to wound healing and tissue repair; nanoparticles for the dosed release of drugs or any other substance that can be used to treat any symptom or condition of the wound; In general, any synthetic or biological substance, compound or molecule that, due to its bioactive or therapeutic nature, can be used to improve or accelerate tissue repair processes, or as a treatment for any deterioration, alteration or damage to health. In order to better understand the characteristics of the invention, the present description is accompanied, as an integral part thereof, by the drawings with an illustrative but non-limiting nature, which are described below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a block diagram of the process for obtaining the human collagen tympanic membrane for the repair of tympanic lesions, in accordance with the present invention.

Figure 2 shows a gel graph of eIectrophoresis with the collagens extracted from human amniotic membrane, tendon and fascia, in accordance with the process of the present invention.

Figure 3 shows an image of a prototype human collagen tympanic membrane, in one of its presentations, in accordance with the present invention.

Figures 4A and 4B show an image that corresponds to the superficial and transversal analysis, respectively, of the collagen membrane, presenting a hierarchical porosity.

Figures 5A and 5B show two images obtained by cytochemical staining with 4',6-diamidino-2-pheni I indo I (DAPI) showing the cell migration in a human collagen tympanic membrane in accordance with the present invention, after 15 days of culture, with 4X and 20X objectives, respectively.

Figure 6A shows an image of a human collagen tympanic membrane for tympanic injury repair, in accordance with the present invention, which has been subjected to contact with blood and is subjected to manipulation with forceps showing their resistance to manipulation. for placement in the damaged tympanic membrane.

Figure 6B shows an image of a human collagen tympanic membrane for tympanic injury repair, in accordance with the present invention, which has been adhered onto the surface of a balloon to demonstrate its ability to adhere to surfaces.

Figure 6C shows an image of a human collagen tympanic membrane for tympanic injury repair, in accordance with the present invention, that has been adhered onto the surface of a balloon and stretched to demonstrate its resistance to tearing. expansion.

Figure 6D shows an image of a human collagen tympanic membrane for tympanic injury repair, in accordance with the present invention, which has been adhered onto the surface of a balloon and stretched showing its ability to preserve shape and properties when returning to the original shape.

Figure 7 shows an image of a human collagen tympanic membrane for tympanic injury repair, in accordance with the present invention, subjected to absorption tests to determine the percentage moisture retention that the structure allowed.

For a better understanding of the invention, a detailed description of some of its modalities will be made, shown in the drawings that, for illustrative but not limiting purposes, are attached to this description.

DETAILED DESCRIPTION OF THE INVENTION

The characteristic details of the human collagen tympanic membrane for tympanic injury repair are clearly shown in the following description and the attached illustrative drawings, the same reference numerals serving to denote the same parts.

The manufacturing process of the human collagen tympanic membrane for the repair of tympanic lesions has been standardized and optimized, disregarding more sophisticated techniques that allow, without compromising quality, a cost of production lower than that of similar products on the market. In this way, a human collagen tympanic membrane is presented for the repair of tympanic lesions, which can be offered at a lower cost than currently available products and with greater effectiveness, due to its protein nature.

The process to produce the human collagen tympanic membrane for the repair of tympanic lesions ranges from the isolation of the tissue to obtaining the collagen, as well as its subsequent transformation into bi- or three-dimensional structures. It consists of a total of nine main steps: tissue conditioning, pre-treatment, extraction, precipitation, dialysis, lyophilization, molding, pressing, and cross-linking, as well as an optional compression step.

This process makes it possible to extract allogeneic type I collagen, and with it, adjust it to the appropriate dimensions and chemically condition it (crosslink it) to achieve functional characteristics in vivo, in the intended application. Finally, this last process allows adaptation to be able to get up to speed and thus reach the desirable in vitro and in vivo degradation conditions.

According to figure 1, in general terms the process for producing human collagen tympanic membrane in accordance with the present invention consists of the following stages, carried out in a temperature range of 2 - 25°C, reaching temperatures down to -80°C in the I i of i I i zation stages. a) Condition the tissue obtained from human amniotic membrane, tendon and fascia, removing tissues or fluids that are not of interest to the process and reducing the particle size in a uniform manner to a size between 0.5 mm and 2 mm with a mill. drum or rotor, to achieve a better interaction between solutions and tissue in subsequent steps. b) Pre-treat the previously conditioned tissue, exposing it to a 0.05 - 2 M sodium hydroxide solution, using 50 - 1000 mL per gram of dry tissue, with efficient magnetic stirring at 100 - 500 RPM and for a period of 1 hour to 24 hours, in order to remove surface proteins and leave the collagen fibers exposed; b 1) Wash with distilled water to neutralize the pH of the tissue until it reaches a pH between 7 and 8, before the next step; c) Extract the collagen by enzymatic hydrolysis by subjecting the tissue to an acid solution of acetic acid at a concentration of 0.02 - 0.5 M and using 50 - 1000 L per kg of dry tissue, together with 50 - 1000 g of pepsin per kg of tissue. dry, to promote a more rapid so I ubi I ization of the collagen and promote a specific and controlled elimination of the terminal carboxyl and amino groups of the molecule; where the extraction is carried out in a period of three days making additions of intermediate acid solution (begins with 200 mL of acetic acid and 0.05 - 0.5 g of pepsin per g of dry tissue and after 48 hours 250 mL of acid are added acetic acid per g of dry tissue to propitiate the development of the reaction and where the residues of non-collagenous tissue structures are eliminated by means of a filtration with a 50 - 900 m sieve and the process is continued. d) Precipitate the collagen by bringing the resulting collagen solution (450 mL of solution per g of dry tissue) to a high salt concentration, adding 20 - 100 g of sodium chloride per 1 L of collagen solution, homogenizing the solution with magnetic stirring and allowing the ionic interaction of the salt with the collagen molecules to generate their precipitation, for a period of between 2 to 6 hours; subsequently, sieve the resulting solution to a particle size of 50 to 900 µm; where the fibers recovered from the sieve are solubilized again in an acetic acid solution, using 50 - 1000 mL of acetic acid per g of dry tissue); e) Dialyze the collagen solution in order to purify the solution of excess salt present in the collagen solution, in a dynamic dialysis system in which the collagen is placed inside a 12 to 14 kDa porous membrane to expel impurities when introduced to a dialysis buffer consisting of a solution with a low concentration of 0.02 - 0.5 M acetic acid; where the exchange of salt molecules between both solutions originates from their concentration differential, from the collagen solution with a salt concentration of 0.5 -2 M to the acetic acid solution without the presence of salts, and is accelerated by contact surface and buffer flow; This stage lasts from two to five days, during which the buffer is kept in recirculation and is changed after 6 to 48 hours; The conductivity of both solutions is also monitored and the process is stopped when the collagen reaches the same conductivity as the buffer at the beginning of the stage (0.10 -0.5 mS/cm). f) Freeze-drying the purified collagen solution by subjecting it to a freeze-drying cycle to concentrate the collagen fibers, favoring their preservation; For this, the solution is frozen at a temperature between -10°C and -80°C and is subsequently subjected to a vacuum pressure of 0.02 - 0.2 mbar (2 - 20 Pa) for a period of one to four days. , during which water and solvents are removed in the form of steam, drying the collagen without damaging the fibers; where the resulting collagen is weighed and distributed according to what is required in the next step. g) Mold and lyophilize for the second time, where, depending on the application and expected function, a concentration of 0.5 to 30 mg of collagen per mL of acetic acid with a molarity of 0.02 to 0.5 M is chosen; once solubilized, the collagen is placed in molds that make it possible to generate the structure with the desired dimensions; again, the lyophilization of the solution is carried out at a temperature between -10°C and -80°C and a vacuum pressure of 0.02 - 0.2 mbar (2 -20 Pa), with the exception that a controlled freezing is carried out that It consists of removing the heat by gradually decreasing the temperature of the plate with which the mold and the collagen solution are in contact, allowing the crystals of water and acetic acid to be uniform and varied, accommodating the fibers to generate a size of estimated average pore; h) Press the collagen structure that, considering the application and function that the product must perform, is subjected to a determined mechanical force of between 100 - 5000 N to compact its dimensions to a desired value from 0.001 - 10 mm and increase its density. fibrillar. i) Crosslink the collagen pieces by subjecting them to a formaldehyde vapor atmosphere in a crosslinking apparatus, at an exposure time of between 1 to 90 minutes and a concentration of the reagent vapor cloud of 0.1 to 100 ppm, resulting in a Controlled crosslinking that allows to reinforce the union between fibers to provide better physical properties to the structure.

At the end of the process the membrane is subjected to evaluation.

By means of SDS-PAGE electrophoresis, the identity of the collagen types obtained with the collagen extraction process described above was determined. Figure 2 shows the electrophoresis gel with the collagens extracted from human amniotic membrane, tendon and fascia. SDS-PAGE gel for collagen samples. M: Molecular weight marker. Lane 1: Collagen type I standard (0.5 mg/mL). Lane 2: Collagen from tendon (1 mg/mL). Lane 2: Amniotic membrane collagen (1 mg/mL). Lane 4: Fascia collagen (1 mg/mL).

For all samples, two bands of higher intensity with an approximate size of 116 and 130 kDa were observed.

The foregoing demonstrates that with the process of obtaining and Collagen purification Type I collagen was isolated, since the visualized bands are close to 112 and 95 kDa in size, corresponding to the two a1 chains and the a2 chain, respectively (Santos, et al., 2013); in addition to coinciding with the band pattern exhibited by the commercial type I collagen standard (see figure 2).

Once the type of collagen was identified, its purity was determined by quantifying, colorimetrically, the hydroxyproline content. Table 1 shows the concentration of hydroxyproline and the percentage of collagen.

Table 1.- Hydroxyproline concentration and the percentage of collagen obtained by the process of the present invention.

Concentration of Concentration of

Percentage of

Sample Hydroxyproline collagen collagen (%)

(mg/mg) (mg/mL)

Tendon 0.112 0.8371 83.7

Membrane

0.145 1.0732 107.3

amniotic

This coincides with the hydroxyproline content of 0.135 mg/mg in type I collagen from mammals (Capella-Monsonis, et al., 2018). In addition, it is demonstrated that the collagen obtained from human tendon and amniotic membrane corresponds to a collagen of great purity and that more than 80% of the protein content of the samples is translated into collagen proteins. Finally, because the crosslinking process is carried out with formaldehyde, the residual formaldehyde content in the collagen membranes was determined, according to the Pharmacopeia of the United Mexican States (FEUM), in the Supplement for Medical Devices. Table 2 summarizes the results obtained. The absorbance value obtained for the 0.001% (m/v) formaldehyde reference solution was 0.15.

Table 2.- Absorbance of the crosslinked collagen structures, obtained by the process of the present invention.

Water Weight (mL) Chromotropic Acid Absorbance

Structures (mg) (mL)

0.8 0.16 3.2 O.1O

0.8 0.16 3.2 0.08

0.8 0.16 3.2 0.12

According to the previous results, it is obtained that, on average, the absorbance of the samples was 0.1 ± 0.02, being below the reference value of 0.15. The above indicates that the concentration of formaldehyde in the collagen structures is less than 0.001% and meets the requirements stipulated by the FEUM.

According to the process for producing human collagen tympanic membrane in accordance with the present invention described above, a type I human collagen tympanic membrane is obtained, with a purity of > 80% and a concentration of < 30 mg/mL or < 15 mg/cm ² , of controlled dimensions (1 cm in diameter or side, and thickness of 0.03 - 0.3 mm), with a porosity of 80 to 99% and a pore size of between 10 and 200 µm.

In the development of scaffolds for tissue engineering, both the porosity and permeability are important aspects, since porosity provides the necessary space for cell migration through the scaffold and for the formation of the extracellular matrix; while the permeability allows the influx of nutrients and the elution of metabolic waste, for which it is sought that the devices have high porosity and high permeability.

To determine the porosity of the collagen membrane, the dimensions of the structures were measured and the volume of the structures (Vm) was determined. The membranes were weighed individually, prior to experimentation (Wo). Subsequently, they were immersed in 5 mL of absolute ethanol and left for 24 h at room temperature. After time, the collagen structures were removed and the superficial excess of liquid was removed with a filter paper. The membranes were immediately weighed (W2 ) and the percentage of porosity was determined, according to the following formula, where p is the density of absolute ethanol:

(3.30 mg — 1.20 mg)/(789.7 mg/cm ³

Pl% x 100 = 91.91%

0.00289 ^cm3

(3.50 mg — 0.8 mg)/(789.7 mg/cm ³ )

P2 _% x 100 = 80.76% 0.00423 ^cm3

(3.10 mg — 1.10 mg)/(789.7 mg/cm ³ ')

P3 _% x 100 = 78.75%

0.00322 ^cm3 The average porosity of the elaborated membranes was 83.81 ± 7.09.

In relation to the above, an analysis by SEM microscopy was performed to determine the pore size. Figure 4A shows the collapsed structure of the scaffold, creating smooth surfaces, but maintaining a porous structure with heterogeneity in pore size, as well as in their morphology. A heterogeneous microstructure is desirable in tissue engineering, since it resembles the complex hierarchical structure found in biological systems; different pore sizes influence different cellular processes. Nanopores (<100 nm) are important for the formation of collagen fibers and extracellular matrix, while macropores (100 m-mm) influence cell seeding, distribution, migration, and neovascularization in vivo.

The scaffolds must allow the formation of functional gap junctions, and the appropriate interaction with other cells and/or with the extracellular matrix. For indirect cell-cell contact, the pore size should be large enough to ensure cell nutrition but not too large to prevent cell migration. Cell migration through the membrane requires a balance between cell size, adhesion, pore size, and surface topography. Scaffolds with pore sizes from 50 nm to 12 µm regulate cell adhesion, cell-cell interaction, and migration across the membrane. scaffolding with size pore > 100 pm have a greater number of functional units necessary for the regeneration of various tissues.

For skin regeneration, a pore size range of 20-120 pm had been established, optimal for cell viability and activity. It was shown that cell adhesion decreased with increasing pore size, attributing to the fact that smaller pores (120 pm) offer a greater surface area to which cells can adhere, after inoculation. However, after 7 days of culture, a higher cell count was observed in scaffolds with larger pore sizes (150-200 pm and 300-350 pm). On the other hand, 325 pm pore sizes facilitated greater cell infiltration through the scaffold, exhibiting a uniform cell distribution, migrating completely from the edge to the center of the scaffold.

In the membrane manufactured according to the present invention, the smallest pores have a size of 13.81 ± 4.94 pm, while the largest ones range between 100-200 pm, especially, in the cross section of the membrane (see Figure 4B). , which contributes to cell adhesion and migration in the membrane, as described later.

Likewise, it can be observed in figure 4B that the thickness of the membrane has values of 168.92 pm, 201.25 pm and 268.34 pm, being able to establish the average thickness of 234.79 ± 47.44 pm. The thickness of the tympanic membrane varies depending on the different zones that compose it, but in general, most authors have agreed on a thickness of between 30-150 m (Van der Jeught, 2013). It has been observed that, during repair of the tympanic membrane, there is the possibility of a hyperproliferation of the epithelial layer, which generates a thickening of the tympanic membrane and, therefore, extends the healing process, in addition to the fact that it can affect temporarily the hearing ability of the patient (Kim et al., 2017). For this reason, reducing the thickness of the collagen structures is important, so as not to add volume to the injured area and contribute to a more controlled proliferation.

Cell migration is a crucial property that the graft must fulfill, since this is the principle for tissue repair. In particular, the healing mechanism of an eardrum injury is different from that of other parts of the body, for example, the skin. Normally, when faced with an injury, the response of the tissues is to begin a series of phases of hemostasis, inflammation, cell proliferation and, finally, cell migration; in the case of the tympanic membrane, cell migration precedes proliferation. In addition, granulation tissue usually develops and serves as the basis for re-epithelialization; in the case of the eardrum, the squamous epithelium develops first, followed by the rest of the epithelial layer, and concludes with the fibrous layer. Likewise, it has been observed that the points of greatest proliferation in the tympanic membrane are the ring and the handle of the malleus. On the other hand, the particularity with the eardrum is that the edge of the wound is suspended, there is no underlying tissue matrix that can support the regenerating epithelium and migratory vessels that, by necessity, must be derived from the surrounding lamina propria (Teh et al., 2013).

In relation to the above, in a study the interaction of human fibroblasts with the extracellular matrix of decellularized porcine and human dermis was compared, finding that the decellularized dermis of human origin presented a greater infiltration of fibroblasts and a greater distance traveled from the inoculation surface (Armour et al., 2006).

Cell migration studies were performed by culturing human fibroblasts on the collagen tympanic membrane. In these studies, it was observed that fibroblasts proliferated and infiltrated the porous structure of the membrane (see figures 5A to 5B), which demonstrates that the membrane would allow cells to migrate from the edges of the lesion to the center of the lesion. itself, where the cells begin the deposition of protein components that repair the continuity of the layers of the tympanic membrane.

To evaluate the capacity of the collagen membrane to promote cell migration, fibroblasts were cultured on its surface and the culture was kept incubated for 15 days (37°C, 5% CO2). By cytochemical staining with 4',6-diamidino-2-phenytoindol (DAPI), fibroblast nuclei were visualized, According to figures 5A and 5B it was possible to observe that the collagen membrane supported the growth of fibroblasts and the cells were able to migrate through the entire structure, despite its compaction. Cells can be distinguished individually by staining of the nuclei (see arrows), on top of the collagen scaffold; or as areas of light (see circles) due to the high cell density present, in the lower planes of the membrane, evidencing excellent cell infiltration. Additionally, the collagen membranes remained intact and were easily handled, even after being in culture.

To evaluate the mechanical resistance of the membrane, wet behavior tests were carried out. First, the collagen membrane was immersed in freshly drawn blood, and placed on an inflated balloon, to assess adhesion and resistance to expansion, since pressure changes in the ear are close to atmospheric pressure.

The collagen membranes showed resistance to manipulation, after being in contact with blood. They maintained the defined membrane shape, adhered to surfaces, and withstood the force of extension as the balloon was inflated. Figures 6A, 6B, 6C and 6D show evidence of this.

As part of the physical characterization of the membrane, absorption tests were performed to determine the percentage of retention allowed by the structure. For this, the method described by the Pharmacopoeia of the United Mexican States, in the Supplement for Medical Devices, was followed.

The initial dry weight (Ws) of a membrane is recorded. Subsequently, the membrane is completely immersed in 20 meters of MiliQ water and allowed to settle for 72 h at room temperature. After the period, the membrane is removed from the water and the excess water is allowed to drain for 1 minute. Next, it is weighed, noting the final wet weight (Ph). The moisture retention percentage (R%) is determined, according to the following formula:

(11 — 0.9)mg

/?lo/ _o — - - - — -X 100 = 1122.22% 0.9 mg

(21.7 - 1.4)mg

/?2o _/o — - - — - - — - x 100 = 1450%

1.4mg

(21.6 - 1.5)mg

/?3o _/o — - - - - - — - x 100 = 1340%

1.5mg

In accordance with the above, it was determined that the membrane presents an average moisture retention percentage of 1304.07 ± 166.81 %, and the mass increase was 14.04 ± 1.67 times the original weight (Ps). Noting that, despite the fact that the membrane absorbed liquids, there was no turgor of it, and the dimensions of the membrane were not affected (see figure 7). Various substances can be added to the membrane, such as: Tissue glue to improve adherence of the membrane to the implant site; an antibiotic solution to treat or prevent an ear infection while the eardrum is being repaired; growth factors and/or autologous or allogeneic mesenchymal cells that, due to their signaling capacity, can promote or enhance biochemical pathways related to wound healing and tissue repair; nanoparticles for the dosed release of drugs or any other substance that can be used to treat any symptom or condition of the wound; In general, any synthetic or biological substance, compound or molecule that, due to its bioactive or therapeutic nature, can be used to improve or accelerate tissue repair processes, or as a treatment for any deterioration, alteration or damage to health.

The invention has been sufficiently described so that a person with average knowledge in the matter can reproduce and obtain the results that we mention in the present invention.

Claims

CLAIMS Having sufficiently described the invention, the content of the following claim clauses is claimed as property.

1.- A human collagen tympanic membrane for the repair of tympanic lesions and/or epithelial lesions, characterized by comprising a purity of > 80% and a concentration of < 30 mg/mL or < 15 mg/cm ² , of dimensions controlled that go from 0.5 to 15 cm in diameter or side, and 0.01 -10 mm thick, with a porosity of 80 to 99% and pore size of between 10 to 200 µm.

2.- The human collagen tympanic membrane for the repair of tympanic lesions according to claim 1, characterized in that it is made of allogeneic collagen.

3. The human collagen tympanic membrane for the repair of eardrum lesions according to claim 1, characterized in that it is made of type I collagen.

4. The human collagen tympanic membrane for the repair of eardrum lesions according to claim 1, characterized in that it has a porosity of 80 to 99%.

5. The human collagen tympanic membrane for the repair of eardrum lesions according to claim 1, characterized in that it has a thickness of 0.03 - 0.3 mm.

6.- The human collagen tympanic membrane for the repair of eardrum lesions according to claim 1, characterized in that it has an average moisture retention percentage of 1304.07 ± 166.81% and the mass increase of <30 times the original weight .

7. The human collagen tympanic membrane for the repair of eardrum lesions according to claim 1, characterized in that it also comprises at least one synthetic or biological substance, compound or molecule that, due to its bioactive or therapeutic nature, improves o accelerate the tissue repair processes of the eardrum lesion.

8. The human collagen tympanic membrane for the repair of tympanic lesions according to claim 1, characterized in that said synthetic or biological substance, compound or molecule is selected from at least one of tissue glue, an antibiotic solution, drugs, growth factors and/or autologous or allogeneic mesenchymal cells, nanoparticles for the dosed release of drugs or their mixtures.

9.- A process for producing a human collagen tympanic membrane for the repair of tympanic lesions, characterized in that it comprises the steps of: a) Conditioning the tissue obtained from the human amniotic membrane, tendon and fascia; b) Pre-treat the fabric previously conditioning it with a sodium hydroxide solution; b1) Wash with distilled water to neutralize the pH of the tissue; c) Extracting the collagen by enzymatic hydrolysis; d) Precipitate the collagen at a high salt concentration, adding sodium chloride; e) Dialyze the collagen solution in a dialysis buffer to expel impurities; f) Lyophilize the collagen solution; g) Mold and lyophilize a second time to generate the structure with the desired dimensions; h) Pressing the collagen structure; and i) Crosslinking the collagen pieces by subjecting them to a formaldehyde vapor atmosphere in a crosslinking apparatus.

10.- The process for producing a human collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that it is carried out in a temperature range of 2 - 25°C, reaching temperatures of up to -80 °C in the lyophilization stages.

11.- The process for producing a human collagen tympanic membrane for the repair of tympanic lesions according to claim 9, characterized in that in the step of conditioning the tissue obtained from the human amniotic membrane, tendon and fascia, the tissues are removed or fluids that are not of interest to the process and the particle size is reduced in a uniform way to a size between 0.5 mm to 2 mm with a drum or rotor mill, to achieve a better interaction between the solutions and the tissue in the subsequent steps.

12. The process for producing a human collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that in the tissue pretreatment stage, it is exposed to a sodium hydroxide solution at 0.05 - 2 M, using 50 - 1000 mL per gram of dry tissue, with efficient magnetic agitation of 100 - 500 RPM and for a period of 1 hour to 24 hours, in order to remove surface proteins and leave the collagen fibers exposed;

13. The process for producing a human collagen tympanic membrane for the repair of tympanic lesions according to claim 9, characterized in that in the washing step the pH is adjusted to a pH between 7 and 8.

14.- The process for producing a human collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that in the collagen extraction step by enzymatic hydrolysis, the tissue is subjected to an acid solution of acid acetic acid at a concentration of 0.02 - 0.5 M and using 50 - 1000 L per kg of dry tissue, together with 50 - 1000 g of pepsin per kg of dry tissue, to promote a more rapid solubilization of collagen and promote a specific and controlled elimination of the carboxyl and amino terminal groups of the molecule.

15.- The process for producing a human collagen tympanic membrane for the repair of tympanic lesions according to Claim 14, characterized in that the extraction is carried out over a period of three days making intermediate acid solution additions, starting with 200 mL of acetic acid and 0.05 - 0.5 g of pepsin per g of dry tissue and after 48 hours 250 are added. mL of acetic acid per g of dry tissue to promote the development of the reaction and where residues of non-collagenous tissue structures are removed by filtration with a 50-900 µm sieve.

16.- The process for producing a human collagen tympanic membrane for the repair of tympanic lesions according to claim 9, characterized in that the collagen precipitation step is the resulting collagen solution (450 mL of solution per g of dry tissue ) is brought to a high salt concentration, adding 20 - 100 g of sodium chloride per 1 L of collagen solution, homogenizing the solution with magnetic stirring and allowing the ionic interaction of the salt with the collagen molecules to generate the precipitation of the same, during a time between 2 to 6 hours; subsequently, sieve the resulting solution to a particle size of 50 to 900 µm; where the fibers recovered from the screen are solubilized again in an acetic acid solution, using 50 - 1000 mL of acetic acid per g of dry fabric;

17.- The process for producing a human collagen tympanic membrane for the repair of tympanic lesions according to Claim 9, characterized in that the step of dialysing the collagen solution to purify the solution of excess salt present in the collagen solution, is performed in a dynamic dialysis system in which the collagen is placed inside a porous membrane of 12 to 14 kDa to expel impurities when introduced to a dialysis buffer consisting of a low concentration solution of 0.02 - 0.5 M acetic acid; where the exchange of salt molecules between both solutions originates from their concentration differential, from the collagen solution with a salt concentration of 0.5 -2 M to the acetic acid solution without the presence of salts, and is accelerated by contact surface and buffer flow; this stage lasts from two to five days, during which the buffer is kept in recirculation and is changed after 6 to 48 hours; monitoring the conductivity of both solutions and the process is stopped when the collagen reaches the same conductivity as the buffer at the beginning of the stage (0.10 -0.5 mS/cm).

18.- The process for producing a human collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that in the step of lyophilizing the purified collagen solution, it is frozen at a temperature between -10 °C and -80 °C and subsequently subjected to a vacuum pressure of 0.02 - 0.2 mbar (2 - 20 Pa) for a period of one to four days, during which water and solvents are removed in the form of steam, drying the collagen without damaging the fibers.

19.- The process to produce a collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that in the step of molding and lyophilizing for the second time, a concentration of 0.5 to 30 mg of collagen is chosen per mL of acetic acid with a molarity of 0.02 to 0.5 m; once solubilized, the collagen is placed in molds to generate the structure with the desired dimensions; again, the lyophilization of the solution is carried out at a temperature between -10°C and -80°C and a vacuum pressure of 0.02 - 0.2 mbar (2 -20 Pa), with the exception that a controlled freezing is carried out that It consists of removing the heat by gradually decreasing the temperature of the plate with which the mold and the collagen solution are in contact, allowing the crystals of water and acetic acid to be uniform and varied, accommodating the fibers to generate a size of estimated average pore.

20.- The process for producing a human collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that in the step of pressing the collagen structure, it is subjected to a determined mechanical force of between 100 - 5000 N to compact its dimensions to a desired value from 0.001 - 10 mm and increase its fibrillar density.

21.- The process for producing a human collagen tympanic membrane for the repair of eardrum lesions according to claim 9, characterized in that in the step of crosslinking the pieces of collagen, they are subjected to an atmosphere of formaldehyde vapor in an apparatus of crosslinking, at an exposure time of between 1 to 90 minutes and concentration of the reagent vapor cloud from 0.1 to 100 ppm, resulting in a controlled crosslinking that allows reinforcing the union between fibers to provide better physical properties to the structure.