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WO2023113584A1 - Membrane tympanique au collagène humain pour la réparation de lésions du tympan - Google Patents

Membrane tympanique au collagène humain pour la réparation de lésions du tympan Download PDF

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Publication number
WO2023113584A1
WO2023113584A1 PCT/MX2022/050118 MX2022050118W WO2023113584A1 WO 2023113584 A1 WO2023113584 A1 WO 2023113584A1 MX 2022050118 W MX2022050118 W MX 2022050118W WO 2023113584 A1 WO2023113584 A1 WO 2023113584A1
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collagen
repair
eardrum
membrane
solution
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Spanish (es)
Inventor
Juan Pablo AGUILAR ALEMÁN
Grecia Andrea CARDOSO HERNÁNDEZ
Brenda Karen AGUILLON ESTRADA
Beni CAMACHO PÉREZ
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Top Health SAPI De CV
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Top Health SAPI De CV
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/18—Internal ear or nose parts, e.g. ear-drums
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen

Definitions

  • 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.
  • 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.
  • 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.
  • oral antibiotics or ear drops may be prescribed to prevent or treat an infection.
  • 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.
  • otitis media infections
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • biological materials derived from the extracellular matrix
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the collagen membrane 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • DAPI 4',6-diamidino-2-pheni I indo I
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m; 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
  • the membrane is subjected to evaluation.
  • Figure 2 shows the electrophoresis gel with the collagens extracted from human amniotic membrane, tendon and fascia.
  • 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).
  • the absorbance of the samples was 0.1 ⁇ 0.02, being below the reference value of 0.15.
  • concentration of formaldehyde in the collagen structures is less than 0.001% and meets the requirements stipulated by the FEUM.
  • 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 ⁇ m.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • 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).
  • Cell migration is a crucial property that the graft must fulfill, since this is the principle for tissue repair.
  • the healing mechanism of an eardrum injury is different from that of other parts of the body, for example, the skin.
  • 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.
  • 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.
  • 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.
  • DAPI 4',6-diamidino-2-phenytoindol
  • 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:
  • 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;
  • 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.

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Abstract

La présente invention se rapporte à une membrane tympanique au collagène humain pour la réparation de lésions du tympan qui présente des propriétés physiques, chimiques, mécaniques et biologiques appropriées pour être utilisée dans le traitement de lésions et/ou de perforations du tympan, qui fournit temporairement un échafaudage et une barrière physique qui sépare l'oreille moyenne de l'oreille externe, tandis que s'effectue la réparation du tissu et se rétablit la continuité du tympan, ladite membrane tympanique selon l'invention étant caractérisée en ce qu'elle a une pureté > 80% et une concentration < 30 mg/mL ou < 15 mg/cm2, avec des dimensions contrôlées de 0,5 à 15 cm de diamètre ou de côté, et 0,01 -10 mm d'épaisseur et de préférence 1 cm de diamètre ou de côté et une épaisseur de 0,03 - 0,3 mm), avec une porosité de 80 à 99% et une taille de pore comprise entre 10 et 200 pm, et en ce qu'elle comprend au moins un des éléments suivants: une colle tissulaire, une solution d'antibiotique, des substances pharmaceutiques, des facteurs de croissance et/ou des cellules mésenchymateuses autologues ou allogéniques, des nanoparticules pour la libération dosée de substances pharmaceutiques ou de mélanges de celles-ci.
PCT/MX2022/050118 2021-12-14 2022-12-01 Membrane tympanique au collagène humain pour la réparation de lésions du tympan Ceased WO2023113584A1 (fr)

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MXMX/A/2021/015642 2021-12-14
MX2021015642A MX2021015642A (es) 2021-12-14 2021-12-14 Membrana timpanica de colageno humano para la reparacion de lesiones de timpano.

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025073993A1 (fr) * 2023-10-05 2025-04-10 Royal College Of Surgeons In Ireland Implant en couches et système pour réparer une perforation dans une membrane tympanique

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025073993A1 (fr) * 2023-10-05 2025-04-10 Royal College Of Surgeons In Ireland Implant en couches et système pour réparer une perforation dans une membrane tympanique

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