WO2022231417A1 - Apparatus and process for the controlled and precise chemical cross-linking of a structure made of human collagen having variable mechanical, chemical and biological features - Google Patents

Apparatus and process for the controlled and precise chemical cross-linking of a structure made of human collagen having variable mechanical, chemical and biological features Download PDF

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Publication number
WO2022231417A1
WO2022231417A1 PCT/MX2021/050070 MX2021050070W WO2022231417A1 WO 2022231417 A1 WO2022231417 A1 WO 2022231417A1 MX 2021050070 W MX2021050070 W MX 2021050070W WO 2022231417 A1 WO2022231417 A1 WO 2022231417A1
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chamber
collagen
crosslinking
vacuum
human collagen
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PCT/MX2021/050070
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Spanish (es)
French (fr)
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Juan Pablo AGUILAR ALEMÁN
Beni CAMACHO PÉREZ
Brenda Karen AGUILLÓN ESTRADA
Grecia Andrea CARDOSO HERNÁNDEZ
Octavio Israel ROJAS GARCÍA
Eduardo MIRAMONTES BEAS
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Top Health, S.A.P.I. De C.V.
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Publication of WO2022231417A1 publication Critical patent/WO2022231417A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J3/00—Processes of utilising sub-atmospheric or super-atmospheric pressure to effect chemical or physical change of matter; Apparatus therefor
    • B01J3/03—Pressure vessels, or vacuum vessels, having closure members or seals specially adapted therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J3/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by one carbon atom

Definitions

  • the present invention is related to the field of mechanics and biotechnology in general; In particular, it is related to an apparatus and process to chemically crosslink in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, which allows reinforcing the bond between fibers to provide better physical properties to the structure.
  • Collagen is one of the most abundant essential proteins in the human body, it represents 25% of the total body protein, which forms part of different tissues; It provides flexibility, elasticity and resistance to pressure. This protein is very abundant in skin, bones, connective tissues and joints (ligaments, tendons and cartilage). It is also found in the cornea and in the walls of blood vessels. Collagen is an essential protein for the proper functioning of the body.
  • Type I collagen is by far the most abundant collagen in the body. It has an unusual amino acid composition, with 33% glycine and 10% proline. It also contains 0.5% 3-hydroxyproline, 10% 4-hydroxyproline, and 1% 5-hydrolysine. These hydroxylated amino acids are post-translationally synthesized from prolyl and lysyl residues in the polypeptide.
  • Collagen contains a small amount of carbohydrates, most of them attached to the hydroxyl group of hydroxylysine in the form of Glu-Gal disaccharide. The carbohydrate content in fibrillar collagens is low (0.5-1% in types I and II), but it is higher in some non-fibrillar types (14% in type IV). Meisenberg, G. and Simmmon, W. Principles of Biochemistry, 4th edition.
  • Type I collagen has the particularity of providing flexibility and elasticity, it is the most abundant in the body, mainly in the bones, tendons, skin and cornea; while type II collagen provides resistance to pressure and is located mainly in cartilage.
  • Collagen is used extensively in cosmetic surgery and in the construction of artificial skin grafts for the treatment of severely burned patients. Collagen products are also used in orthopedic, surgical applications, in a wide variety of dental procedures. Human collagen is derived from cadaveric tissues or debris from surgical procedures, such as the placenta at the time of childbirth; and it has the advantage of significantly reducing the probability of an immune reaction, compared to collagen from other species.
  • stages include a pretreatment of the raw material, hydrolysis and purification of the collagen.
  • stages or operating parameters have been slightly modified to extract collagen from various animal sources, such as cattle, pigs, horses, birds and marine animals; using the skin, scales, bones, tendons, pericardium, cartilage, etc., of said animals.
  • the present invention focuses on a novel process of production of human collagen structures with controlled characteristics, allowing good performance while preserving structural integrity and high purity; but more specifically it focuses on a device developed to be able to carry out chemical cross-linking in a controlled manner of structures made of collagen where they are subjected to a formaldehyde vapor atmosphere, allowing the exposure time to be set between 1 - 90 minutes and concentration of the vapor cloud of the reagent between 0.1 - 100 ppm, resulting in a controlled crosslinking that allows reinforcing the bond between fibers that provides better physical properties to the structure.
  • US patent 10576395B2 by Casali Republic M. and Matthews Michael A. dated November 13, 2017 was located, which discloses a system and method for removing residual glutaraldehyde from a glutaraldehyde-crosslinked natural polymer scaffold.
  • the system includes a cleaning solution comprising carbon dioxide and one or more polar solvents and an environmental chamber which may include a treatment chamber.
  • the environmental chamber is maintained at a temperature greater than 31.1°C and the carbon dioxide is maintained at a pressure greater than 7.38 MPa to form supercritical carbon dioxide.
  • a cross-linked natural polymer scaffold treated by the glutaraldehyde removal system and method may have a glutaraldehyde content of less than 3 ppm.
  • a cross-linked natural polymer frame cleaning solution comprising supercritical carbon dioxide and one or more polar solvents.
  • the above patent does not disclose, nor does it suggest an apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics comprising a cross-linking chamber where the collagen is housed and which is subjected to empty, with a formaldehyde vapor feed duct, which in turn is connected with a connector to a sublimation chamber with a plunger attached to a cap and a stem; with steam flow control means and vacuum generation and control means, offering the possibility of introducing a very precise amount of paraformaldehyde, heating it and bringing it into close contact with the collagen structure under controlled environmental conditions (temperature and pressure). ).
  • Patent US7393437B2 by Chan Barbara P. and So Kwok F. dated September 14, 2005 was also located, which discloses a method for producing scaffolds composed of cross-linked collagen with improved properties such as resistance and stability and maintaining excellent biocompatibility.
  • the method comprises: (a) reconstitution of the three-dimensional collagen extracellular matrix from the collagen monomer solution using methods such as raising the pH of the solution for a certain period of time; (b) crosslinking at least a portion of the matrix by contacting it with a photosensitizing reagent at a particular concentration in the dark for a period of time before or after reconstitution; (c) removing excess photosensitizing reagent; (d) irradiating the scaffolds with a light source of sufficient energy for a period of time to form crosslinked scaffolds; and (e) dehydration of the crosslinked frameworks.
  • the method of the invention may further comprise rolling the rehydrated and crosslinked scaffolds several times to produce large scaffolds.
  • the method of the invention also allows incubation with another component of the extracellular matrix to make composite scaffolds and immobilization of bioactive factors or drugs that can subsequently be released from the scaffolds.
  • the method for producing a cross-linked collagen scaffold or scaffolds disclosed in the referenced patent comprises: (a) providing a solution of extracellular collagen monomers at an acidic pH; (b) raising the pH of the solution to form a reconstituted three-dimensional extracellular collagen matrix; (c) contacting at least a portion of the reconstituted three-dimensional extracellular collagen matrix with a photoactivating reagent; (d) removing excess photoactivating reagent; (e) irradiating the reconstituted three-dimensional extracellular collagen matrix, in a hydrated state without bubbling air or oxygen into the reaction mixture or shaking vigorously, using a light source of sufficient energy to form a lattice frame; and (f) dehydrating the lattice framework.
  • the method used in the above patent uses a photochemical (Rose Bengal) to crosslink.
  • the process involves exposing the collagen to the photochemical and then using ultraviolet (UV) light to crosslink.
  • UV ultraviolet
  • our process exposes the collagen to formaldehyde, which crosslinks the collagen without the need to use any additional additive, such as patent US7393437B2 that uses UV light.
  • collagen is an excellent structural protein and has the advantages of being biocompatible and resorbable, its mechanical properties compared to synthetic polymers are poor.
  • Various methods and techniques have been developed to improve these features and diversify the use of collagen structures; however, these techniques have important disadvantages, such as not being efficient, being processes that require a lot of time, but if they are extended too much, they can denature the collagen; Some techniques or crosslinking agents can cause a crosslinking gradient along the entire collagen structure, generating a structure with heterogeneous mechanical properties; the addition of some chemical components can result in a cytotoxic effect, and said compounds can remain in the collagen structure even after extensive washing; to mention a few.
  • the main objective of the present invention is to make available an apparatus and process for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics.
  • Another objective of the invention is to provide said apparatus and process that also offers the possibility of introducing a very requires paraformaldehyde, heat it and bring it into close contact with the collagen structure under controlled environmental conditions (temperature and pressure).
  • Another objective of the invention is to provide said apparatus and process that also allows controlling the temperature at which the gas is generated, controlling the speed at which the formaldehyde gas passes from one chamber to the other, controlling the time it is in contact with the collagen framework, start and stop the cross-linking process abruptly and ventilate the collagen frameworks continuously.
  • Another objective of the invention is to provide said apparatus and process that also allows support to the collagen structures without producing deformations and at the same time promotes the homogeneous interaction of the collagen structure with the formaldehyde gas.
  • Another objective of the invention is to provide said apparatus and process that also allows gaseous formaldehyde to be produced from powdered paraformaldehyde and used to cross-link collagen structures in a controlled manner.
  • Another objective of the invention is to provide said apparatus and process that also offers the possibility of elaborating collagen structures with structural and dimensional characteristics that are defined and very specific, that contribute to a better integration of the structures in an in vivo environment, influencing cell behavior.
  • Another objective of the invention is to provide said apparatus and process that also offers the possibility of elaborating human collagen structures with controlled characteristics, which also allows the elaboration of collagen structures with physical characteristics suitable for a wide variety of biological applications, for by means of simple and low-cost techniques, which allow the optimization of the concentration of collagen in the structures; such characteristics include the dimensions of the structure, fibrillar density, porosity and pore size, which strongly influence cell behavior.
  • Another objective of the invention is to provide said apparatus and process that also offers the possibility of elaborating human collagen structures, and that allows increasing the mechanical performance of the structures, with a precise and controlled technique, without affecting the integrity, functionality, or biological safety of collagen.
  • the apparatus for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics in accordance with the present invention, consists of a crosslinking chamber with a hermetically sealed lid that integrates a vacuum gauge for vacuum measurement, configured to house a support structure where the collagen to be crosslinked is arranged and placed; said crosslinking chamber is communicated with a sublimation chamber through an interconnection duct with flow control means; said sublimation chamber has a plunger attached to a stem that passes through a hermetically sealed lid, and is configured to house a quantity of paraformaldehyde powder.
  • Said crosslinking chamber also comprises a connected vacuum generating duct with vacuum regulation means connected to a vacuum generating means and means for injecting medical grade air with flow control means for ventilation and expulsion of formaldehyde gas towards a fume hood, once the human collagen is cross-linked.
  • Said sublimation chamber is configured to receive heat from a heat transfer medium for the generation of formaldehyde gas that is conducted towards said crosslinking chamber where the collagen is arranged and to where the formaldehyde gas is conducted for the collagen cross-linking process.
  • the invention also contemplates the method for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, which consists of subjecting non-human collagen to an atmosphere of formaldehyde gas in a crosslinking apparatus, with an exposure time of between 1 - 90 minutes and a concentration of a formaldehyde gas cloud of between 0.1 - 100 ppm, resulting in a controlled crosslinking that allows reinforcing the union between fibers that provides better physical properties to the structure.
  • the invention also contemplates a process for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics in a crosslinking apparatus comprising the steps of: a) Placing human collagen to be crosslinked in a support structure and depositing said support structure inside a crosslinking chamber, closing it with a hermetically sealed lid that integrates a vacuum gauge for vacuum measurement and that is interconnected with a sublimation chamber through an interconnection duct with means of flow control, b) Place paraformaldehyde powder in the sublimation chamber by inserting a plunger attached to a stem that passes through a hermetically sealed lid; c) Generate a vacuum between -65 to -75 KPa with a vacuum generating medium connected to a vacuum generating duct with vacuum regulation means in the open position, wherein said vacuum generation duct is connected to said crosslinking chamber and the flow control means of said interconnection duct between the sublimation chamber and the crosslinking chamber in the open position
  • the crosslinked collagen scaffolds are distributed for packaging and storage or shipping for testing.
  • the collagen structures are placed on the support structure specially designed to hold the sponges without deforming them and without allowing them to move.
  • said collagen support structure is made of a very fine anticorrosive steel wire so as to allow complete and homogeneous contact of the collagen structures with the formaldehyde gas.
  • a ratio of between 0.005 mg and 0.01 mg of paraformaldehyde is established for every 1 mg of collagen to be crosslinked; however, it is estimated that the minimum paraformaldehyde needed would be 3.5 mg and a maximum of 20 mg.
  • concentration range of formaldehyde gas in the crosslinking chamber is a minimum of 0.001 mg/L and a maximum of 4 mg/L.
  • Collagen tissue is obtained from cadaveric donors in the case of tendons and fascia, and living donors in the case of amniotic membrane.
  • cadaveric donors the tissue is obtained and transported at a temperature of 2 °C to 8 °C to the facilities, where it is isolated and cleaned using sterile surgical instruments and saline solution 0.9% NaCl (Sodium Chloride).
  • the tissue must come from donors free of infectious diseases such as Hepatitis B/C, HIV, AIDS, Chagas, to name a few.
  • tissue from people who died of metabolic acidosis cannot be accepted.
  • tissue For living donors, the tissue is obtained and transported at a temperature of 2 °C to 8 °C to the facilities, where it is isolated and cleaned using sterile surgical instruments and saline solution 0.9% NaCI (Sodium Chloride).
  • the tissue must come from donors free of infectious diseases such as Hepatitis B/C, HIV, AIDS, Chagas, to name a few.
  • tissue from natural births cannot be accepted, it must be cesarean section without premature delivery ( ⁇ 34 weeks) to avoid contamination.
  • the present description is accompanied, as an integral part thereof, by the drawings with a more non-limiting illustrative character, which They described below.
  • Figure 1 shows a conventional perspective view of the apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention.
  • Figure 2 shows a side view of the apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention.
  • Figure 3 shows a conventional perspective view of the collagen support structure, in accordance with the present invention.
  • Figure 4 shows a schematic diagram of the process for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the preferred embodiment of the invention.
  • the apparatus for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention consists of a crosslinking chamber ( 1) with a hermetic closure cover (2) that integrates a vacuum gauge (3, see fig.
  • said crosslinking chamber (1) is connected to a sublimation chamber (5) through an interconnection duct (6) with flow control means (7).
  • Said sublimation chamber (5) has a plunger (8) attached to a stem (9) that passes through a hermetic sealing lid (10) with hermetic sealing elements (not shown) and that is fixed with fixing means. (not shown) in the upper flange (1 1) of said sublimation chamber (5), and is configured to house a amount of paraformaldehyde powder (FP).
  • Said crosslinking chamber (1) also comprises a connected vacuum generator duct (12) with vacuum regulation means (13) connected to a vacuum generator means (14, see figure 4) and means to inject medical grade air ( 15, see figure 4) with flow control means (16, see figure 4) for ventilation to break the vacuum and expel formaldehyde gas (GF) towards a gas extraction hood (26) once the collagen is crosslinked human (C); where said means for injecting medical grade air (15, see figure 4) with flow control means (16, see figure 4) are mounted on a "T" connector (17, see figures 1 and 2) arranged in the duct interconnection (6) between said crossover chamber (1) and the flow control means (7).
  • Said sublimation chamber (5) is configured to receive heat from a heat transfer medium (18, see section "c" of figure 4) for the generation of formaldehyde vapor that is conducted towards said crosslinking chamber (1 ) where the collagen is arranged and where the formaldehyde vapor is conducted for the crosslinking process of human collagen structures (C).
  • the collagen support structure (4) is defined by a peripheral structure made up of two frames (19, 20) joined with joining means by two sets of posts (21, 22), a plurality of grids (23) with a separation of 5 mm from each other are mounted on support frames (24) that are fixed with attachment means to the set of posts (21, 22); between said grids (23) the collagen sponges (C) to be cross-linked are arranged.
  • the collagen support structure (4) is made on its periphery by aluminum and with a stainless steel mesh, in such a way that the grids (23) define squares of approximately 1 to 5 mm long and 1 to 5 mm wide. Wide. In total, seven grids (23) are attached to the structure with their respective separation spaces (25). The human collagen sponges (C) are manually introduced through the spaces (25) generated by the grids (23), gently sliding them to their desired position within said collagen support structure (4).
  • the process for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics in a crosslinking apparatus comprising the steps of: a) Placing human collagen ( C) to crosslink in a support structure (4) and depositing said support structure (4) inside a crosslinking chamber (1) closing it with a hermetic closure lid (2) that integrates a vacuum gauge (3) for measurement of vacuum and that is interconnected with a sublimation chamber (5) through an interconnection duct (6) with flow control means (7); b) Place paraformaldehyde powder (FP) in the sublimation chamber (5) by inserting a plunger (8, see figures 1 and 2) attached to a stem (9) that passes through a hermetically sealed lid ( 10); c) Generate a vacuum between -65 to -75 KPa with a vacuum generating means (14) connected to a vacuum generation duct (12) with vacuum regulation means (13) in the open position, where said duct of vacuum generation (12) is connected to
  • the human collagen (C) is finally extracted from the crosslinking chamber (1) and released from said support structure (4).
  • Cross-linked human collagen scaffolds are distributed for packaging and storage or shipping for testing.
  • the collagen structures are placed on the support structure specially designed to hold the sponges without deforming them. and without being able to move.
  • a ratio of between 0.005 mg and 0.01 mg of paraformaldehyde per 1 mg of collagen to be crosslinked was established; however, it is estimated that the minimum paraformaldehyde needed would be 2.0 mg and a maximum of 20 mg.
  • the concentration range of formaldehyde gas (GF) in the crosslinking chamber (1) is a minimum of 0.001 mg/L and a maximum of 4 mg/L.

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Abstract

The present invention relates to an apparatus and process for the controlled and precise chemical cross-linking of a structure made of human collagen having variable mechanical, chemical and biological features, characterised in that it comprises a cross-linking chamber having a hermetically sealed lid integrating a vacuum gauge for vacuum measurement, configured to house a support structure where the collagen to be cross-linked is arranged and placed; said cross-linking chamber is connected to a sublimation chamber through an interconnecting duct having flow control means; said sublimation chamber has a plunger attached to a rod that passes through a hermetically sealed lid and is configured to house a quantity of paraformaldehyde powder; wherein said cross-linking chamber further comprises a connected vacuum generator duct having vacuum regulating means connected to vacuum generating means and means for injecting medical air with flow control for venting and expelling the gas into a fume hood, once the human collagen has been cross-linked.

Description

APARATO Y PROCESO PARA ENTRECRUZAR QUÍMICAMENTE DE MANERA CONTROLADA Y PRECISA, UNA ESTRUCTURA HECHA DE COLÁGENO HUMANO CON CARACTERÍSTICAS MECÁNICAS, QUÍMICAS Y BIOLÓGICAS VARIABLES APPARATUS AND PROCESS FOR CHEMICALLY CROSSLINKING, IN A CONTROLLED AND PRECISE MANNER, A STRUCTURE MADE OF HUMAN COLLAGEN WITH VARIABLE MECHANICAL, CHEMICAL AND BIOLOGICAL CHARACTERISTICS
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se relaciona con el campo de la mecánica y la biotecnología en lo general; en lo particular se relaciona con un aparato y proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, que permita reforzar la unión entre fibras para brindar mejores propiedades físicas a la estructura. The present invention is related to the field of mechanics and biotechnology in general; In particular, it is related to an apparatus and process to chemically crosslink in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, which allows reinforcing the bond between fibers to provide better physical properties to the structure.
ANTECEDENTES DE LA INVENCIÓN El colágeno es una de las proteínas esenciales más abundante del cuerpo humano, representa el 25% de la proteína corporal total, que forma parte de diferentes tejidos; aporta flexibilidad, elasticidad y resistencia a la presión. Esta proteína es muy abundante en piel, huesos, tejidos conectivos y articulaciones (ligamentos, tendones y cartílagos). También se encuentra en la córnea y en las paredes de los vasos sanguíneos. El colágeno es una proteína fundamental para el buen funcionamiento del organismo. BACKGROUND OF THE INVENTION Collagen is one of the most abundant essential proteins in the human body, it represents 25% of the total body protein, which forms part of different tissues; It provides flexibility, elasticity and resistance to pressure. This protein is very abundant in skin, bones, connective tissues and joints (ligaments, tendons and cartilage). It is also found in the cornea and in the walls of blood vessels. Collagen is an essential protein for the proper functioning of the body.
El colágeno tipo I es, con diferencia, el colágeno más abundante en el cuerpo. Tiene una composición de aminoácidos inusual, con un 33% de glicina y un 10% de pro lina. También contiene un 0.5% de 3-hidroxiprolina, un 10% de 4-hidroxiproli na y u n 1 % de 5- hidrolisina. Estos aminoácidos hidroxilados son sintetizados postrad uccionalmente a partir de residuos prolilo y lisilo en el polipéptido. El colágeno contiene una pequeña cantidad de hidratos de carbono, la mayoría de ellos unidos al grupo hidroxilo de la hidroxilisina en forma de disacárido Glu -Gal. El contenido en hidratos de carbono en los colágenos fibri lares es bajo (0.5-1 % en los tipos I y I I), pero es mayor en algu nos tipos no fibrilares (14% en el tipo IV). Meisen berg, G. y Simmmon, W. Principios de bioquímica, 4a edición. Type I collagen is by far the most abundant collagen in the body. It has an unusual amino acid composition, with 33% glycine and 10% proline. It also contains 0.5% 3-hydroxyproline, 10% 4-hydroxyproline, and 1% 5-hydrolysine. These hydroxylated amino acids are post-translationally synthesized from prolyl and lysyl residues in the polypeptide. Collagen contains a small amount of carbohydrates, most of them attached to the hydroxyl group of hydroxylysine in the form of Glu-Gal disaccharide. The carbohydrate content in fibrillar collagens is low (0.5-1% in types I and II), but it is higher in some non-fibrillar types (14% in type IV). Meisenberg, G. and Simmmon, W. Principles of Biochemistry, 4th edition.
El colágeno tipo I tiene la particularidad de aportar flexibilidad y elasticidad, es el más abu ndante en el cuerpo, principalmente en los huesos, tendones, la piel y la córnea; en tanto el colágeno tipo I I aporta resistencia a la presión y se localiza principalmente en los cartílagos. Type I collagen has the particularity of providing flexibility and elasticity, it is the most abundant in the body, mainly in the bones, tendons, skin and cornea; while type II collagen provides resistance to pressure and is located mainly in cartilage.
El colágeno se utiliza extensivamente en cirugí a cosmética y en la construcción de injertos de piel artificial para el tratamiento de pacientes con quemaduras severas. Los productos de colágeno también se emplean en aplicaciones ortopédicas, quirúrgicas, en una amplia variedad de procedimientos odontol ógicos. El colágeno humano se deriva de tejidos cadavéricos o de despojos de procedimientos quirúrgicos, tales como la placenta al momento del parto; y presenta la ventaja de reducir significativamente la probabilidad de una reacción inmune, en comparación con el colágeno proveniente de otras especies. Collagen is used extensively in cosmetic surgery and in the construction of artificial skin grafts for the treatment of severely burned patients. Collagen products are also used in orthopedic, surgical applications, in a wide variety of dental procedures. Human collagen is derived from cadaveric tissues or debris from surgical procedures, such as the placenta at the time of childbirth; and it has the advantage of significantly reducing the probability of an immune reaction, compared to collagen from other species.
Debido a la importancia del colágeno en diversas aplicaciones médicas e industriales, se han desarrollado procesos para producir estructuras de colágeno humano. Due to the importance of collagen in various medical and industrial applications, processes have been developed to produce human collagen structures.
Existen procesos industriales q ue pueden obte ner resultados similares en cuanto a la obtención de colágeno. Si n embargo, no se ha encontrado un proceso q ue pueda obtener específicamente colágeno humano tipo I, con un rendimiento del 35%. Tampoco se ha encontrado un proceso que p ueda transformar el co lágeno humano obtenido en estructuras bidimensionales o tridimensionales variadas y con características controlables. Tampoco se ha encontrado u n proceso que pueda controlar de manera precisa las características del colágeno. There are industrial processes that can obtain similar results in terms of obtaining collagen. However, no process has been found that can specifically obtain type I human collagen, with a yield of 35%. Neither has a process been found that can transform the human collagen obtained into varied two-dimensional or three-dimensional structures with controllable characteristics. Neither has a process been found that can precisely control the characteristics of collagen.
También se conocen otros procesos de obtención de colágeno que comprenden etapas definidas ya conocidas ampliamente por aquellos que están inmersos en esta área. Las etapas, en forma general, incl uyen un pretratamiento de la materia prima, hidrólisis y p urificación del colágeno. Existen procesos en los cuales ciertas etapas o parámetros de operación, han sido ligeramente modificados para extraer colágeno de diversas fuentes animales, tales como bovi nos, porcinos, equinos, aves y animales marinos; utilizando la piel, escamas, huesos, tendones, pericardio, cartílago, etcétera, de dichos animales. Other processes for obtaining collagen are also known that comprise defined steps already widely known by those who are immersed in this area. The stages, in general, include a pretreatment of the raw material, hydrolysis and purification of the collagen. There are processes in which certain stages or operating parameters have been slightly modified to extract collagen from various animal sources, such as cattle, pigs, horses, birds and marine animals; using the skin, scales, bones, tendons, pericardium, cartilage, etc., of said animals.
También se conocen métodos de producción de colágeno in vitro, por medio del uso de li neas celulares. Methods of collagen production in vitro, through the use of cell lines, are also known.
Otras metodologías que se conocen descri ben un pretratamiento ácido o básico de la materia prima, por ejemplo, con NaOH a diferentes concentraciones, temperaturas y tiempos de exposición. También existen técnicas de hidrólisis para obtener colágeno soluble en diversos ácidos, o a través de tratamientos enzimáticos. Para la hidrólisis química se han empleado ácidos orgánicos como el ácido acético, ácido láctico, ácido cítrico y similares; o ácidos inorgánicos tales como el ácido clorhídrico; mientras que para la hidrólisis enzimática se reportan enzimas proteoliticas tales como ficina, pepsina, entre otras. De igual forma, dichas técnicas se operan en diversos rangos de parámetros de operació n de temperatura, tiempo, pH y concentración de la solución y enzima. Otros procesos son aquellos diseñados para la obtención y purificación de diversos tipos de colágeno a partir de u na misma fuente, por medio de técnicas de precipitación fraccionada, a diferentes concentraciones de sales como NaCI, sulfato de amonio y similares, o con etanol . Other methodologies that are known describe an acidic or basic pretreatment of the raw material, for example, with NaOH at different concentrations, temperatures and exposure times. There are also hydrolysis techniques to obtain soluble collagen in various acids, or through enzymatic treatments. Organic acids such as acetic acid, lactic acid, citric acid and the like have been used for chemical hydrolysis; or inorganic acids such as hydrochloric acid; while for enzymatic hydrolysis, proteolytic enzymes such as ficin, pepsin, among others, are reported. Similarly, these techniques are operated in various ranges of operating parameters of temperature, time, pH and concentration of the solution and enzyme. Other processes are those designed to obtain and purify various types of collagen from the same source, by means of fractional precipitation techniques, different concentrations of salts such as NaCl, ammonium sulfate and the like, or with ethanol.
También se conocen procedimientos para la p urificación y concentración de colágeno, tales como diálisis y liofilización . Methods for the purification and concentration of collagen, such as dialysis and lyophilization, are also known.
Existen procedimientos para elaborar estructuras defi nidas de colágeno, tales como andamios, esponjas, membranas, recubrimientos, hidrogeles, etcétera, a través de la liofilización, la gelificación, el “electrospinning” o electrohilado, la bioimpresión, entre otros. There are procedures to make defined collagen structures, such as scaffolds, sponges, membranes, coatings, hydrogels, etc., through lyophilization, gelation, "electrospinning" or electrospinning, bioprinting, among others.
Se han descrito métodos físicos y químicos para el reforzamiento de las cadenas del biopolimero y mejora de la estabilidad y rigidez de la estructura, asi como para controlar la tasa de degradación in vivo e in vitro. Respecto a estos métodos, se pueden mencionar el tratamiento deshid rotérmico, la radiación ultravioleta, el entrecruzamiento químico con aldehidos como el glutaraldehido y el formaldehido en solución, el uso de carbodiimidas y transglutaminasa microbiana. Physical and chemical methods have been described for reinforcing the biopolymer chains and improving the stability and rigidity of the structure, as well as for controlling the degradation rate in vivo and in vitro. Regarding these methods, we can mention dehydrothermal treatment, ultraviolet radiation, chemical crosslinking with aldehydes such as glutaraldehyde and formaldehyde in solution, the use of carbodiimides and microbial transglutaminase.
De igual forma, existen procesos de compresión de las estructuras de colágeno, particularmente los hidrogeles, para modificar las propiedades mecánicas y densidad fibrilar de la estructura de colágeno. Similarly, there are compression processes for collagen structures, particularly hydrogels, to modify the mechanical properties and fibrillar density of the collagen structure.
La presente invención se centra en un proceso novedoso de producción de estructuras de colágeno humano con característi cas controladas, que permita u n buen rendimiento preservando la integridad estructural y p ureza elevada; pero más específicamente se centra en un aparato desarrollado para poder llevar acabo un entrecruzamiento q uímico de manera controlada de estructuras hechas de colágeno donde se someten a una atmósfera de vapor de formaldehldo, permitiendo establecer el tie mpo de exposición de entre 1 - 90 minutos y concentración de la nube de vapor del reactivo de entre 0.1 - 100 ppm, resultando en un entrecruzamiento controlado que permita reforzar la unión entre fibras q ue brinde mejores propiedades físicas a la estructura. The present invention focuses on a novel process of production of human collagen structures with controlled characteristics, allowing good performance while preserving structural integrity and high purity; but more specifically it focuses on a device developed to be able to carry out chemical cross-linking in a controlled manner of structures made of collagen where they are subjected to a formaldehyde vapor atmosphere, allowing the exposure time to be set between 1 - 90 minutes and concentration of the vapor cloud of the reagent between 0.1 - 100 ppm, resulting in a controlled crosslinking that allows reinforcing the bond between fibers that provides better physical properties to the structure.
Se realizó una búsqueda para determinar el estado de la técnica más cercano, encontrándose los sig uientes documentos. A search was carried out to determine the closest state of the art, finding the following documents.
Se ubicó la patente US 10576395B2 de Casali Dominic M. y Matthews Michael A. del 13 de noviembre de 2017, la cual revela un sistema y método para eliminar el glutaraldehido residual de un armazón de polímero natural reticulado con gl utaraldehido. El sistema incl uye una solución de limpieza que comprende dióxido de carbono y uno o más disolventes polares y una cámara ambiental que puede incluir una cámara de tratamiento. La cámara ambiental se mantiene a una temperatura superior a 31.1 °C y el dióxido de carbono se mantiene a una presión superior a 7.38 MPa para formar dióxido de carbono supercritico. Un armazón de polímero natural reticulado tratado mediante el sistema y método de eliminación de glutaraldehido puede tener un contenido de glutaraldehido de menos de 3 ppm. También se proporciona una solución de limpieza de armazones de polímero natural reticulado q ue comprende dióxido de carbono supercritico y uno o más disolventes polares. La patente anterior no revela, ni sugiere un aparato para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables que comprenda u na cámara de entrecruzamiento donde se aloja el colágeno y que se somete a vacio, con un ducto de alimentación de vapor d e formaldehido, que a su vez esté conectado con un conector a una cámara de sublimación con un émbolo unido a una tapa y a un vástago; con medios de control de flujo de vapor y medios de generación y control de vacio, que ofrezca la posibilidad de introducir una cantidad muy precisa de paraformaldehido, calentarlo y ponerlo en un contacto estrecho con la estructura de colágeno bajo condiciones ambientales controladas (temperatura y presión). US patent 10576395B2 by Casali Dominic M. and Matthews Michael A. dated November 13, 2017 was located, which discloses a system and method for removing residual glutaraldehyde from a glutaraldehyde-crosslinked natural polymer scaffold. The system includes a cleaning solution comprising carbon dioxide and one or more polar solvents and an environmental chamber which may include a treatment chamber. The environmental chamber is maintained at a temperature greater than 31.1°C and the carbon dioxide is maintained at a pressure greater than 7.38 MPa to form supercritical carbon dioxide. A cross-linked natural polymer scaffold treated by the glutaraldehyde removal system and method may have a glutaraldehyde content of less than 3 ppm. Also provided is a cross-linked natural polymer frame cleaning solution comprising supercritical carbon dioxide and one or more polar solvents. The above patent does not disclose, nor does it suggest an apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics comprising a cross-linking chamber where the collagen is housed and which is subjected to empty, with a formaldehyde vapor feed duct, which in turn is connected with a connector to a sublimation chamber with a plunger attached to a cap and a stem; with steam flow control means and vacuum generation and control means, offering the possibility of introducing a very precise amount of paraformaldehyde, heating it and bringing it into close contact with the collagen structure under controlled environmental conditions (temperature and pressure). ).
Se ubicó también la patente US7393437B2 de Chan Barbara P. y So Kwok F. del 14 de septiembre de 2005, la cual divulga un método para producir andamios compuestos de colágeno reticulado con propiedades mejoradas tales como resistencia y estabilidad y mantiene una excelente biocompatibilidad. El método comprende: (a) la reconstitución de la matriz extracelular de colágeno tridimensional a partir de la solución de monómero de colágeno usando métodos tales como elevar el p H de la solución durante cierto periodo de tiempo; (b) reticular al menos u na parte de la matriz poniéndola en contacto con un reactivo fotosensibilizante a una concentración particular en la oscuridad durante cierto período de tiempo antes o después de la reconstitución; (c) eliminar el exceso de reactivo fotosensibilizante; (d) irradiación de los armazones con u na fuente de luz de energía suficiente durante u n cierto período de tiempo para formar armazones reticulados; y (e) deshidratación de los armazones reticulados. El método de la invención puede comprender, además, laminar los armazones rehidratados y reticulados varias veces para producir armazones grandes. El método de la invención también permite la incubación con otro componente de la matriz extracelular para fabricar armazones de comp uestos y la inmovilización de factores bioactivos o fármacos que se pueden liberar posteriormente de los armazones. El método para producir un armazón o andamios de colágeno reticulado divulgado en la patente referida comprende: (a) proporcionar u na solución de monómeros de colágeno extracelulares a un pH ácido; (b) elevar el pH de la solución para formar una matriz de colágeno extracelular tridimensional reconstituida; (c) poner en contacto al menos una parte de la matriz de colágeno extracelular tridimensional reconstituida con u n reactivo fotoacti vante; (d) eliminar el exceso de reactivo fotoacti vante; (e) irradiar la matriz de colágeno extracelular tridimensional reconstituida, en un estado hidratado si n burbujear aire u oxígeno en la mezcla de reacción o agitar vigorosamente, usando una fuente de luz de energía suficiente para formar u n armazón reticulado; y (f) desh idratar el armazón reticulado. Patent US7393437B2 by Chan Barbara P. and So Kwok F. dated September 14, 2005 was also located, which discloses a method for producing scaffolds composed of cross-linked collagen with improved properties such as resistance and stability and maintaining excellent biocompatibility. The method comprises: (a) reconstitution of the three-dimensional collagen extracellular matrix from the collagen monomer solution using methods such as raising the pH of the solution for a certain period of time; (b) crosslinking at least a portion of the matrix by contacting it with a photosensitizing reagent at a particular concentration in the dark for a period of time before or after reconstitution; (c) removing excess photosensitizing reagent; (d) irradiating the scaffolds with a light source of sufficient energy for a period of time to form crosslinked scaffolds; and (e) dehydration of the crosslinked frameworks. The method of the invention may further comprise rolling the rehydrated and crosslinked scaffolds several times to produce large scaffolds. The method of the invention also allows incubation with another component of the extracellular matrix to make composite scaffolds and immobilization of bioactive factors or drugs that can subsequently be released from the scaffolds. The method for producing a cross-linked collagen scaffold or scaffolds disclosed in the referenced patent comprises: (a) providing a solution of extracellular collagen monomers at an acidic pH; (b) raising the pH of the solution to form a reconstituted three-dimensional extracellular collagen matrix; (c) contacting at least a portion of the reconstituted three-dimensional extracellular collagen matrix with a photoactivating reagent; (d) removing excess photoactivating reagent; (e) irradiating the reconstituted three-dimensional extracellular collagen matrix, in a hydrated state without bubbling air or oxygen into the reaction mixture or shaking vigorously, using a light source of sufficient energy to form a lattice frame; and (f) dehydrating the lattice framework.
El método utilizado en la patente anterior usa un fotoqulmico (Rosa de Bengala) para entrecruzar. El proceso consiste en exponer el colágeno al fotoqulmico y después utilizar luz ultravioleta (UV) para entrecruzar. Por otro lado, nuestro proceso expone el colágeno a formaldehldo, el cuál entrecruza el colágeno sin necesidad de utilizar algún aditamento adicional, como la patente US7393437B2 que utiliza la luz UV. The method used in the above patent uses a photochemical (Rose Bengal) to crosslink. The process involves exposing the collagen to the photochemical and then using ultraviolet (UV) light to crosslink. On the other hand, our process exposes the collagen to formaldehyde, which crosslinks the collagen without the need to use any additional additive, such as patent US7393437B2 that uses UV light.
La patente anterior no revela, ni sugiere un aparato para entrecruzar q uímicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables que comprenda u na cámara de entrecruzamiento donde se aloja el colágeno y que se somete a vacio, con un ducto de alimentación de vapor de formaldehldo, que a su vez esté conectado con un conector a una cámara de sublimación con un émbolo unido a una tapa y a un vástago; con medios de control de flujo de vapor y medios de generación y control de vacio, q ue ofrezca la posi bilidad de introducir una cantid ad muy precisa de paraformaldehl do, calentarlo y ponerlo en un contacto estrecho con la estructura de colágeno bajo condiciones ambientales controladas (temperatura y presión). The above patent does not disclose, nor does it suggest an apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics comprising a cross-linking chamber where the collagen is housed and which is subjected to vacuum, with a formaldehyde vapor feed duct, which in turn is connected with a connector to a sublimation chamber with a plunger attached to a cap and a stem; with steam flow control means and vacuum generation and control means, offering the possibility of introducing a very precise amount of paraformaldehyde, heating it and bringing it into close contact with the collagen structure under controlled environmental conditions (temperature and pressure).
La posibilidad de elaborar estructuras de colágeno con características estructurales y dimensionales definidas y muy específicas, que contribuyan a una mejor integración de las estructuras en un ambiente in vivo, influenciando el comportamiento celular, se ha buscado abordar por med io de técnicas que pueden llegar a ser muy costosas, poco accesibles o adaptables a gran escala, o q ue requieren de equipos especializados, tales como el “electrospinning” o electrohilado. The possibility of elaborating collagen structures with defined structural and dimensional characteristics and very specific, which contribute to a better integration of structures in an in vivo environment, influencing cellular behavior, has been sought to address through techniques that can become very expensive, inaccessible or adaptable on a large scale, or that require of specialized equipment, such as “electrospinning” or electrospinning.
De igual forma, a pesar de que el colágeno es una excelente proteí na estructural y presenta las ventajas de ser biocompatible y reabsorbible, sus propiedades mecánicas respecto a polímeros sintéticos son pobres. Diversos métodos y técnicas han sido desarrolladas para mejorar estas prestaciones y diversificar el uso de las estructuras de colágeno; sin embargo, dichas técnicas presentan desventajas importantes, tales como no ser eficientes, ser procesos que requieren de mucho tiempo, pero que si se extienden demasiado p ueden llegar a desnaturalizar el colágeno; alg unas técnicas o agentes entrecruzantes pueden causar un gradiente de entrecruzamiento a lo largo de toda la estructura de colágeno, generando una estructura con propiedades mecánicas heterogéneas; la adición de algunos componentes químicos puede resultar en un efecto citotóxico, y dichos compuestos pueden permanecer en la estructura de colágeno aún después de lavados extensivos; por mencionar algunas. Ante la necesidad de contar con un aparato para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, que permita reforzar la unión entre fi bras para brindar mejores propiedades físicas a la estructura, fue que se desarrolló la presente invención. Similarly, despite the fact that collagen is an excellent structural protein and has the advantages of being biocompatible and resorbable, its mechanical properties compared to synthetic polymers are poor. Various methods and techniques have been developed to improve these features and diversify the use of collagen structures; however, these techniques have important disadvantages, such as not being efficient, being processes that require a lot of time, but if they are extended too much, they can denature the collagen; Some techniques or crosslinking agents can cause a crosslinking gradient along the entire collagen structure, generating a structure with heterogeneous mechanical properties; the addition of some chemical components can result in a cytotoxic effect, and said compounds can remain in the collagen structure even after extensive washing; to mention a few. Faced with the need to have an apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with mechanical, chemical and biological variables, which allows reinforcing the bond between fibers to provide better physical properties to the structure, was that the present invention was developed.
O BJ ETIVOS D E LA I NVE N CI Ó N La presente invención tiene como objetivo principal hacer disponible un aparato y proceso para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológi cas variables. OBJECTIVES OF THE INVENTION The main objective of the present invention is to make available an apparatus and process for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, permita generar estructuras de colágeno humano con características mecánicas, químicas y biológicas variables. Otro objetivo de la invención es proveer dicho aparato y proceso que además, permita generar estructuras basadas en colágeno humano con diferentes formas, funciones, tamaños, densidades, características mecánicas, resistencias a degradación enzimática y biocompatibilidad . Another objective of the invention is to provide said apparatus and process that also allows the generation of human collagen structures with variable mechanical, chemical and biological characteristics. Another objective of the invention is to provide said apparatus and process that also allows generating structures based on human collagen with different shapes, functions, sizes, densities, mechanical characteristics, resistance to enzymatic degradation and biocompatibility.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, ofrezca la posibilidad de introducir una cantidad muy precisa de paraformaldehí do, calentarlo y ponerlo en un contacto estrecho con la estructura de colágeno bajo condiciones ambientales controladas (temperatura y presión). Otro objetivo de la invención es proveer dicho aparato y proceso que además, permita controlar la temperatura a la que se genera el gas, controlar la velocidad a la que pasa el gas formaldehldo de una cámara a la otra, controlar el tiempo que está en contacto con la estructura de colágeno, iniciar y detener el proceso de entrecruzamiento de manera abrupta y ventilar las estructuras de colágeno de manera conti nua. Another objective of the invention is to provide said apparatus and process that also offers the possibility of introducing a very requires paraformaldehyde, heat it and bring it into close contact with the collagen structure under controlled environmental conditions (temperature and pressure). Another objective of the invention is to provide said apparatus and process that also allows controlling the temperature at which the gas is generated, controlling the speed at which the formaldehyde gas passes from one chamber to the other, controlling the time it is in contact with the collagen framework, start and stop the cross-linking process abruptly and ventilate the collagen frameworks continuously.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, permita dar soporte a las estructuras de colágeno sin producir deformaciones y al mismo tiempo promover la interacción homogénea de la estructura de colágeno con el gas formaldehldo. Another objective of the invention is to provide said apparatus and process that also allows support to the collagen structures without producing deformations and at the same time promotes the homogeneous interaction of the collagen structure with the formaldehyde gas.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, permita producir formaldehldo gaseoso a partir de paraformaldehldo en polvo y usarlo para entrecruzar de manera controlada las estructuras de colágeno. Another objective of the invention is to provide said apparatus and process that also allows gaseous formaldehyde to be produced from powdered paraformaldehyde and used to cross-link collagen structures in a controlled manner.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, ofrezca la posibilidad de elaborar estructuras de colágeno con características estructurales y dimensional es definidas y muy especificas, que contribuyan a una mejor integración de las estructuras en un ambiente in vivo, influenciando el comportamiento celular. Another objective of the invention is to provide said apparatus and process that also offers the possibility of elaborating collagen structures with structural and dimensional characteristics that are defined and very specific, that contribute to a better integration of the structures in an in vivo environment, influencing cell behavior.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, ofrezca la posibilidad de elaborar estructuras de colágeno humano con características controladas, que además permita la elaboración de estructuras de colágeno con características físicas ajusfadles para u na amplia variedad de aplicaciones biológicas, por medio de técnicas sencillas y de bajo costo, que permiten la optimización de la concentración de colágeno en las estructuras; tales características incluyen las dimensiones de la estructura, densidad fibrilar, porosidad y tamaño de poro, que influyen fuertemente en el comportamiento celular. Another objective of the invention is to provide said apparatus and process that also offers the possibility of elaborating human collagen structures with controlled characteristics, which also allows the elaboration of collagen structures with physical characteristics suitable for a wide variety of biological applications, for by means of simple and low-cost techniques, which allow the optimization of the concentration of collagen in the structures; such characteristics include the dimensions of the structure, fibrillar density, porosity and pore size, which strongly influence cell behavior.
Otro objetivo de la invención es proveer dicho aparato y proceso que además, ofrezca la posibilidad de elaborar estructuras de colágeno humano, y q ue permita aumentar las prestaciones mecánicas de las estructuras, con una técnica p recisa y controlada, sin afectar la integridad, funcionalidad, o seguridad biológica del colágeno. Another objective of the invention is to provide said apparatus and process that also offers the possibility of elaborating human collagen structures, and that allows increasing the mechanical performance of the structures, with a precise and controlled technique, without affecting the integrity, functionality, or biological safety of collagen.
Y todas aquellas cualidades y objetivos que se harán aparentes al realizar una descripción general y detallada de la presente invención apoyados en las modalidades ilustradas. And all those qualities and objectives that will become apparent when making a general and detailed description of the present invention supported by the illustrated modalities.
B REVE D E SC RI PCI Ó N D E L I NVE N TO De manera general el aparato para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de conformidad con la presente invención consiste en una cámara de entrecruzamiento con una tapa de cierre hermético que integra un vacuómetro para la medición de vacio, configurada para alojar una estructura de soporte donde se dispone y se coloca el colágeno a entrecruzar; dicha cámara de entrecruzamiento está comu nicada con u na cámara de sublimación a través de un ducto de interconexión con medios de control de flujo; dicha cámara de sublimación presenta un émbolo unido a un vástago que pasa a través de una tapa de sellado hermético, y está configurada para alojar una cantidad de paraformaldehi do en polvo. Dicha cámara de entrecruzamiento además comprende conectado un d ucto generador de vacio con medios de regulación de vacio conectado a un medio generador de vacio y medios para inyectar aire grado médico con medios de control de fl ujo para ventilación y expulsión del gas de formaldehido hacia una campana de extracción, una vez entrecruzado el colágeno humano. BRIEF SCRIPT OF THE DELETION In general, the apparatus for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention, consists of a crosslinking chamber with a hermetically sealed lid that integrates a vacuum gauge for vacuum measurement, configured to house a support structure where the collagen to be crosslinked is arranged and placed; said crosslinking chamber is communicated with a sublimation chamber through an interconnection duct with flow control means; said sublimation chamber has a plunger attached to a stem that passes through a hermetically sealed lid, and is configured to house a quantity of paraformaldehyde powder. Said crosslinking chamber also comprises a connected vacuum generating duct with vacuum regulation means connected to a vacuum generating means and means for injecting medical grade air with flow control means for ventilation and expulsion of formaldehyde gas towards a fume hood, once the human collagen is cross-linked.
Dicha cámara de sublimación está configurada para reci bir calor desde un medio de transferencia de calor para la generación de gas de formaldehido que se conduce hacia dicha cámara de entrecruzamiento donde se dispone el colágeno y hacia d onde se conduce el gas de formaldehi do para el proceso de entrecruzamiento de colágeno. La invención también contempla el método para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, que consiste en someter coláge no humano a una atmósfera de gas de formaldehido en un aparato de entrecruzamiento, con un tiempo de exposición de entre 1 - 90 mi nutos y concentración de una nube de gas de formaldehi do de entre 0.1 - 100 ppm resultando en un entrecruzamiento controlado que permita reforzar la u nión entre fibras que brinda mejores propiedades físicas a la estructura. Said sublimation chamber is configured to receive heat from a heat transfer medium for the generation of formaldehyde gas that is conducted towards said crosslinking chamber where the collagen is arranged and to where the formaldehyde gas is conducted for the collagen cross-linking process. The invention also contemplates the method for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, which consists of subjecting non-human collagen to an atmosphere of formaldehyde gas in a crosslinking apparatus, with an exposure time of between 1 - 90 minutes and a concentration of a formaldehyde gas cloud of between 0.1 - 100 ppm, resulting in a controlled crosslinking that allows reinforcing the union between fibers that provides better physical properties to the structure.
La invención también contempla un proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con caracterí sticas mecánicas, químicas y biológicas variables en un aparato de entrecruzamiento que comprende las etapas de: a) Colocar colágeno humano a entrecruzar en una estructura de soporte y depositando dicha estructura de soporte dentro de una cámara de entrecruzamiento cerrándola con una tapa de cierre hermético que integra u n vacuómetro para la medición de vacio y que está interconectada con una cámara de sublimación a través de un ducto de interconexión con medios de control de flujo, b) Colocar paraformaldehi do en polvo en la cámara de sublimación insertando un émbolo unido a un vástago que pasa a través de una tapa de sellado hermético; c) Generar un vacio de entre - 65 a - 75 KPa con u n medio generador de vacio conectado a un ducto de generació n de vacio con medios de regulación de vacio en posición abierta, en donde dicho ducto de generación de vacio está conectado a dicha cámara de entrecruzamiento y los medios de control de flujo de dicho ducto de interconexión entre la cámara de sublimación y la cámara de entrecruzamiento en posición abierta; d) Calentar la cámara de sublimación a una temperatura de entre 120 °C a 160 °C mediante un medio de calentamiento para producir el gas de formaldehido que es contenido ahí durante un tiempo de entre 8 a 20 minutos; estando dichos medios de control de fl ujo de dicho ducto de interconexión de la cámara de sublimación y la cámara de entrecruzamiento en posición cerrada, habiendo desactivado el medio generador de vacio y estando en posición cerrada dichos medios de regulación de vacio; e) Colocar en posición abierta los medios de control de flujo de dicho ducto de i nterconexión de la cámara de sublimación y la cámara de entrecruzamiento, y se acciona el émbolo de la cámara de sublimación forzando el paso del gas de formaldehido hacia la cámara de entrecruzamiento; f) Dejar expuesto el coláge no por un periodo de entre 1 a 90 mi nutos con el gas de formaldehido dentro de la cámara de entrecruzamiento y luego expulsar el gas de formaldehido desde la cámara de entrecruzamiento mediante un flujo de aire grado médico alimentado desde una fuente de sumi nistro de aire médico controlado por medios de control de flujo en posición abierta dispuesta en un conector del ducto de interconexión que conecta dicha cámara de sublimación y la cámara de entrecruzamiento, para detener el proceso de entrecruzamiento, estando en posición abierta los medios de control de flujo dicho ducto de interconexión y medios de regulación de vacio para el rompimiento de vacio y expulsión del gas de formaldehido hacia una campana de extracción de gases. El colágeno finalmente es extraído de la cámara de entrecruzamiento y liberado de dicha estructura de soporte. The invention also contemplates a process for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics in a crosslinking apparatus comprising the steps of: a) Placing human collagen to be crosslinked in a support structure and depositing said support structure inside a crosslinking chamber, closing it with a hermetically sealed lid that integrates a vacuum gauge for vacuum measurement and that is interconnected with a sublimation chamber through an interconnection duct with means of flow control, b) Place paraformaldehyde powder in the sublimation chamber by inserting a plunger attached to a stem that passes through a hermetically sealed lid; c) Generate a vacuum between -65 to -75 KPa with a vacuum generating medium connected to a vacuum generating duct with vacuum regulation means in the open position, wherein said vacuum generation duct is connected to said crosslinking chamber and the flow control means of said interconnection duct between the sublimation chamber and the crosslinking chamber in the open position ; d) Heat the sublimation chamber to a temperature of between 120 °C to 160 °C by means of a heating medium to produce the formaldehyde gas that is contained therein for a time of between 8 to 20 minutes; said flow control means of said interconnection duct of the sublimation chamber and the crosslinking chamber being in the closed position, the vacuum generating means having been deactivated and said vacuum regulation means being in the closed position; e) Place the flow control means of said interconnection duct of the sublimation chamber and the crosslinking chamber in the open position, and the plunger of the sublimation chamber is activated, forcing the passage of formaldehyde gas towards the chamber of crossover; f) Leave the collagen exposed for a period of between 1 to 90 minutes with the formaldehyde gas inside the crosslinking chamber and then expel the formaldehyde gas from the crosslinking chamber by means of a flow of medical grade air fed from a source of medical air supply controlled by flow control means in the open position arranged in a connector of the interconnection duct that connects said sublimation chamber and the crosslinking chamber, to stop the crosslinking process, being in position open the flow control means said interconnection duct and vacuum regulation means for breaking the vacuum and expelling the formaldehyde gas towards a gas extraction hood. The collagen is finally extracted from the crosslinking chamber and released from said support structure.
Las estructuras de colágeno entrecruzadas son distribuidas para ser empacadas y almacenadas o enviarse para la realización de pruebas. The crosslinked collagen scaffolds are distributed for packaging and storage or shipping for testing.
Las estructuras de colágeno se colocan en la estructura de soporte especialmente diseñado para sujetar las esponjas sin deformarlas y sin que se p uedan mover. The collagen structures are placed on the support structure specially designed to hold the sponges without deforming them and without allowing them to move.
En la modalidad preferida de la i nvención dicha estructura de soporte de colágeno está hecha de un alambre de acero anticorrosivo muy fi no de modo que permite el contacto completo y homogéneo de las estructuras de colágeno con el gas de formaldehido. In the preferred embodiment of the invention, said collagen support structure is made of a very fine anticorrosive steel wire so as to allow complete and homogeneous contact of the collagen structures with the formaldehyde gas.
En la modalidad preferida de la invención se establece una relación de entre 0.005 mg y .01 mg de paraformaldehido por cada 1 mg de colágeno por entrecruzar; sin embargo, se tiene estimado que lo mí nimo de paraformaldehido necesario seria de 3.5 mg y un máximo de 20 mg . Para un volumen de la cámara de entrecruzamiento de 5 L; guardado esa correlación en base al volumen de dicha cámara de entrecruzamiento. El rango de concentración del gas de formaldehido en la cámara de entrecruzamiento es mí nimo de 0.001 mg/L y máximo de 4 mg/L. In the preferred embodiment of the invention, a ratio of between 0.005 mg and 0.01 mg of paraformaldehyde is established for every 1 mg of collagen to be crosslinked; however, it is estimated that the minimum paraformaldehyde needed would be 3.5 mg and a maximum of 20 mg. For a crosslinking chamber volume of 5 L; saved that correlation based on the volume of said chamber of crossover The concentration range of formaldehyde gas in the crosslinking chamber is a minimum of 0.001 mg/L and a maximum of 4 mg/L.
El tejido de colágeno es obtenido de donadores cadavéricos en el caso de tendones y fascia, y donadores vivos en caso de membrana amniótica. Para donadores cadavéricos, el tejido es obtenido y transportado a una temperatura de 2 °C a 8 °C a las instalaciones, donde es aislado y limpiado utilizando instrumental quirúrgico estéril y solución salina 0.9% NaCI (Cloruro de Sodio). El tejido debe de provenir de donadores libres de enfermedades infecciosas tales como Hepatitis B/C, VI H, SI DA, Chagas, por mencionar alg unas. Además, no se puede aceptar tejido proveniente de personas fallecidas por acidosis metabólica. Para donadores vivos, el tejido es obten ido y transportado a una temperatura de 2 °C a 8 °C a las instalaciones, donde es aislado y limpiado utilizando instrumental q uirúrgico estéril y sol ución salina 0.9% NaCI (Cloruro de Sodio). El tejido debe de provenir de donadores libres de enfermedades i nfecciosas tales como Hepatitis B/C, VI H, SI DA, Chagas, por mencionar algunas. Además, no se puede aceptar tejido proveniente de partos naturales, debe ser cesárea sin parto prematuro (<34 semanas ) para evitar contaminación. Para comprender mejor las características de la presente invención se acompaña a la presente descripción, como parte integrante de la misma, los dibujos con carácter ilustrativo más no limitativo, que se describen a conti nuación. Collagen tissue is obtained from cadaveric donors in the case of tendons and fascia, and living donors in the case of amniotic membrane. For cadaveric donors, the tissue is obtained and transported at a temperature of 2 °C to 8 °C to the facilities, where it is isolated and cleaned using sterile surgical instruments and saline solution 0.9% NaCl (Sodium Chloride). The tissue must come from donors free of infectious diseases such as Hepatitis B/C, HIV, AIDS, Chagas, to name a few. In addition, tissue from people who died of metabolic acidosis cannot be accepted. For living donors, the tissue is obtained and transported at a temperature of 2 °C to 8 °C to the facilities, where it is isolated and cleaned using sterile surgical instruments and saline solution 0.9% NaCI (Sodium Chloride). The tissue must come from donors free of infectious diseases such as Hepatitis B/C, HIV, AIDS, Chagas, to name a few. In addition, tissue from natural births cannot be accepted, it must be cesarean section without premature delivery (<34 weeks) to avoid contamination. In order to better understand the characteristics of the present invention, the present description is accompanied, as an integral part thereof, by the drawings with a more non-limiting illustrative character, which They described below.
B REVE D E SC RI PCI Ó N D E LAS FI G U RAS B RIEF DESCRIPTION OF THE FIGURES
La figura 1 muestra u na vista en perspectiva convencional del aparato para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de conformidad con la presente i nvención. La figura 2 muestra una vista lateral del aparato para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de conformidad con la presente invención. La figura 3 muestra una vista en perspectiva convencional de la estructura de soporte del colágeno, de conformidad con la presente invención. Figure 1 shows a conventional perspective view of the apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention. Figure 2 shows a side view of the apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention. Figure 3 shows a conventional perspective view of the collagen support structure, in accordance with the present invention.
La figura 4 muestra un diagrama esquemático del proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de conformidad con la modalidad preferida de la i nvención. Figure 4 shows a schematic diagram of the process for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the preferred embodiment of the invention.
Para una mejor comprensión del invento, se pasará a hacer la descripción detallada de alguna de las modalidades del mismo, mostrada en los dibujos que con fines il ustrativos mas no limitativos se anexan a la presente descripción. D E SC RI PCI Ó N DETALLADA D E L I NVE N TO For a better understanding of the invention, a detailed description will be made of some of its modalities, shown in the drawings that are attached to this description for illustrative but not limiting purposes. DETAILED DESCRIPTION OF CRIME
Los detalles característicos del aparato y proceso para entrecruza r químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de conformidad con la presente invención, se muestran claramente en la siguiente descripción y en los dib ujos il ustrativos que se anexan, sirviendo los mismos signos de referencia para señalar los mismos pasos. Haciendo referencia a las figuras 1 , 2 y 4, el aparato para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de conformidad con la presente invención consiste en una cámara de entrecruzamiento (1 ) con una tapa de cierre hermético (2) que i ntegra un vacuómetro (3, ver fig. 4) para la medición de vacío, la cual se fija con medios de fijación y empaques (no mostrados) para cierre hermético, configurada para alojar una estructura de soporte de colágeno (4, ver figuras 3, 4) donde se dispone y se coloca el colágeno (C, ver figura 4) a entrecruzar; dicha cámara de entrecruzamiento (1 ) está comu nicada con una cámara de sublimación (5) a través de un ducto de interconexión (6) con medios de control de flujo (7). Dicha cámara de sublimación (5) presenta un émbolo (8) unido a un vástago (9) que pasa a través de una tapa de sellado hermético (10) con elementos de sellado hermético (no mostrados) y que se fija con medios de fijación (no mostrados) en la ceja superior (1 1 ) de dicha cámara de sublimación (5), y está configurada para aloja r una cantidad de paraformaldehí do en polvo (FP). The characteristic details of the apparatus and process for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention, are clearly shown in the following description and drawings. illustrative ows that are annexed, serving the same reference signs to indicate the same steps. Referring to figures 1, 2 and 4, the apparatus for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in accordance with the present invention consists of a crosslinking chamber ( 1) with a hermetic closure cover (2) that integrates a vacuum gauge (3, see fig. 4) for vacuum measurement, which is fixed with fixing means and gaskets (not shown) for hermetic closure, configured for accommodate a collagen support structure (4, see figures 3, 4) where the collagen (C, see figure 4) to crosslink is arranged and placed; said crosslinking chamber (1) is connected to a sublimation chamber (5) through an interconnection duct (6) with flow control means (7). Said sublimation chamber (5) has a plunger (8) attached to a stem (9) that passes through a hermetic sealing lid (10) with hermetic sealing elements (not shown) and that is fixed with fixing means. (not shown) in the upper flange (1 1) of said sublimation chamber (5), and is configured to house a amount of paraformaldehyde powder (FP).
Dicha cámara de entrecruzamiento (1 ) además comprende conectado u n ducto generador de vacio (12) con medios de regulación de vacio (13) conectado a un medio generador de vacio (14, ver fig ura 4) y medios para inyectar aire grado médico (15, ver figura 4) con medios de control de flujo (16, ver figura 4) para ventilación para el rompimiento de vacio y expulsión del gas de formaldehido (GF) hacia una campana de extracción de gases (26) una vez entrecruzado el colágeno humano (C); en donde dichos medios para inyectar aire grado médico (15, ver figura 4) con medios de control de flujo (16, ver figura 4) se montan en u n conector “T” (17, ver figuras 1 y 2) dispuesto en el ducto de interconexión (6) entre dicha cámara de entrecruzamiento (1 ) y los medios de control de fl ujo (7). Said crosslinking chamber (1) also comprises a connected vacuum generator duct (12) with vacuum regulation means (13) connected to a vacuum generator means (14, see figure 4) and means to inject medical grade air ( 15, see figure 4) with flow control means (16, see figure 4) for ventilation to break the vacuum and expel formaldehyde gas (GF) towards a gas extraction hood (26) once the collagen is crosslinked human (C); where said means for injecting medical grade air (15, see figure 4) with flow control means (16, see figure 4) are mounted on a "T" connector (17, see figures 1 and 2) arranged in the duct interconnection (6) between said crossover chamber (1) and the flow control means (7).
Dicha cámara de sublimación (5) está configurada para recibir calor desde un medio de transferencia de calor (18, ver inciso “c” de la figura 4) para la generación de vapor de formaldehi do que se conduce hacia dicha cámara de entrecruzamiento (1 ) donde se dispone el colágeno y hacia donde se conduce el vapor de formaldehido para el proceso de entrecruzamiento de estructuras de colágeno humano (C). De acuerdo con la figura 3, la estructura de soporte de colágeno (4) está definida por u na estructura periférica conformada de dos marcos (19, 20) unidos con medios de unión por dos j uegos de postes (21 , 22), una pluralidad de rejillas (23) con una separación de 5 mm entre sí se montan en marcos de soporte (24) que se fijan con medios de unión al juego de postes (21 , 22); entre dichas rejillas (23) se disponen las esponjas de colágeno (C) a entrecruzar. Said sublimation chamber (5) is configured to receive heat from a heat transfer medium (18, see section "c" of figure 4) for the generation of formaldehyde vapor that is conducted towards said crosslinking chamber (1 ) where the collagen is arranged and where the formaldehyde vapor is conducted for the crosslinking process of human collagen structures (C). According to figure 3, the collagen support structure (4) is defined by a peripheral structure made up of two frames (19, 20) joined with joining means by two sets of posts (21, 22), a plurality of grids (23) with a separation of 5 mm from each other are mounted on support frames (24) that are fixed with attachment means to the set of posts (21, 22); between said grids (23) the collagen sponges (C) to be cross-linked are arranged.
La estructura de soporte de colágeno (4) está hecha en su periferia por aluminio y con u n mallada de acero inoxidable, de tal manera que las rejillas (23) definen cuadrados de aproximadamente de 1 a 5 mm de largo y de 1 a 5 mm de ancho. En total, se colocan siete rejillas (23) a la estructura con sus respectivos espacios de separación (25). Las esponjas de colágeno humano (C) es introducido manualmente a través de los espacios (25) generados por las rejillas (23), deslizándolas suavemente hasta su posición deseada dentro de dicha estructura de soporte de colágeno (4). The collagen support structure (4) is made on its periphery by aluminum and with a stainless steel mesh, in such a way that the grids (23) define squares of approximately 1 to 5 mm long and 1 to 5 mm wide. Wide. In total, seven grids (23) are attached to the structure with their respective separation spaces (25). The human collagen sponges (C) are manually introduced through the spaces (25) generated by the grids (23), gently sliding them to their desired position within said collagen support structure (4).
De acuerdo con la fig ura 4, el proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables en un aparato de entrecruzamiento que comprende las etapas de: a) Colocar colágeno humano (C) a entrecruzar en una estructura de soporte (4) y depositando dicha estructura de soporte (4) dentro de una cámara de entrecruzamiento (1 ) cerrándola con una tapa de cierre hermético (2) que integra un vacuómetro (3 ) para la medición de vacío y que está i nterconectada con u na cámara de sublimación (5) a través de un ducto de i nterconexión (6) con medios de control de fl ujo (7); b) Colocar paraformaldehl do en polvo (FP) en la cámara de sublimación (5) insertando un ém bolo (8, ver figuras 1 y 2) u nido a un vástago (9) que pasa a través de una tapa de sellado hermético (10); c) Generar un vacio de entre -65 a -75 KPa con un medio generador de vacio (14) conectado a u n ducto de generación de vacio (12) con medios de regul ación de vacio (13) en posición abierta, en donde dicho ducto de generación de vacio (12) está conectado a dicha cámara de entrecruzamiento (1 ) y estando los medios de control de flujo (7) de dicho ducto de i nterconexión (6) entre la cámara de sublimación (5) y la cámara de entrecru zamiento (1 ) en posición abierta; d) Calentar la cámara de sublimación (5) a u na temperatura de entre 120 °C a 160 °C mediante un medio de transferencia de calor (18) para producir el gas de formaldehido (GF) q ue es contenido ahí durante un tiempo de entre 8 a 20 minutos; estando dichos medios de control de flujo (7) de dicho ducto de interconexión (6) de la cámara de sublimación (5) y la cámara de entrecruzamiento (1 ) en posición cerrada, habiendo desactivado o desconectado el medio generador de vacio (14) y estando en posición cerrada dichos medios de regulación de vacio (13); e) Colocar en posición abierta los medios de control de flujo (7) de dicho ducto de interconexión (6) de la cámara de sublimación (5) y la cámara de entrecruzamiento (1 ), y accionar el émbolo (8) de la cámara de sublimación (5) forzando el paso del gas de formaldehido (GF) hacia la cámara de entrecruzamiento (1 ); f) Dejar expuesto el colágeno humano (C) soportado en la estructura de soporte (4) por un periodo de entre 1 a 90 minutos con el gas de formaldehido (GF) dentro de la cámara de entrecruzamiento (1 ) y luego exp ulsar el gas de formaldehido (GF) desde la cámara de entrecruzamiento (1 ) mediante un flujo de aire grado médico con medios para i nyectar aire medico (15) alimentado desde una fuente de suministro de aire grado médico controlado por medios de control de flujo (16) en posición abierta dispuesta en u n conector “T” (17, ver figuras 1 y 2) del ducto de interconexión (6) que conecta dicha cámara de sublimación (5) y la cámara de entrecruzamiento (1 ), para detener el proceso de entrecruzamiento, estando en posición abierta los medios de control de flujo (7) de dicho ducto de i nterconexión (6) y medios de regulación de vacío (13) para el rompimiento de vacío y expulsión del gas de formaldehido (GF) hacia una campana de extracción de gases (26). According to figure 4, the process for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics in a crosslinking apparatus comprising the steps of: a) Placing human collagen ( C) to crosslink in a support structure (4) and depositing said support structure (4) inside a crosslinking chamber (1) closing it with a hermetic closure lid (2) that integrates a vacuum gauge (3) for measurement of vacuum and that is interconnected with a sublimation chamber (5) through an interconnection duct (6) with flow control means (7); b) Place paraformaldehyde powder (FP) in the sublimation chamber (5) by inserting a plunger (8, see figures 1 and 2) attached to a stem (9) that passes through a hermetically sealed lid ( 10); c) Generate a vacuum between -65 to -75 KPa with a vacuum generating means (14) connected to a vacuum generation duct (12) with vacuum regulation means (13) in the open position, where said duct of vacuum generation (12) is connected to said crosslinking chamber (1) and being the flow control means (7) of said interconnection duct (6) between the sublimation chamber (5) and the crosslinking chamber opening (1) in open position; d) Heat the sublimation chamber (5) to a temperature between 120 °C and 160 °C by means of a heat transfer medium (18) to produce the formaldehyde gas (GF) that is contained there for a time of between 8 to 20 minutes; said flow control means (7) of said interconnection duct (6) of the sublimation chamber (5) and the crosslinking chamber (1) being in the closed position, having deactivated or disconnected the vacuum generating means (14) and said vacuum regulation means (13) being in the closed position; e) Place the flow control means (7) of said interconnection duct (6) of the sublimation chamber (5) and the crosslinking chamber (1) in the open position, and actuate the plunger (8) of the chamber sublimation (5) forcing the passage of formaldehyde gas (GF) towards the crosslinking chamber (1); f) Leave the human collagen (C) supported on the support structure (4) exposed for a period of between 1 to 90 minutes with formaldehyde gas (GF) inside the crosslinking chamber (1) and then expel the formaldehyde gas (GF) from the crosslinking chamber (1) by means of a medical grade air flow with means to inject medical air (15) fed from a medical grade air supply source controlled by flow control means (16 ) in open position arranged in a "T" connector (17, see figures 1 and 2) of the interconnection duct (6) that connects said sublimation chamber (5) and the crosslinking chamber (1), to stop the process of crossover, being in the open position the flow control means (7) of said interconnection duct (6) and vacuum regulation means (13) for breaking the vacuum and expelling the formaldehyde gas (GF) towards a hood gas extraction (26).
Las etapas “a” y “b” puede invertirse, sin q ue éste cambio sea relevante para el proceso. El colágeno h umano (C) finalmente es extraído de la cámara de entrecruzamiento (1 ) y liberado de dicha estructura de soporte (4). Las estructuras de colágeno humano entrecruzadas son distribuidas para ser empacadas y almacenadas o enviarse para la realización de pruebas. Stages "a" and "b" can be reversed, without this change being relevant to the process. The human collagen (C) is finally extracted from the crosslinking chamber (1) and released from said support structure (4). Cross-linked human collagen scaffolds are distributed for packaging and storage or shipping for testing.
Las estructuras de colágeno se colocan en la estructura de soporte especialmente diseñado para sujetar las esponjas sin deformarlas y sin que se p uedan mover. The collagen structures are placed on the support structure specially designed to hold the sponges without deforming them. and without being able to move.
Se estableció una relación de entre 0.005 mg y 0.01 mg de paraformaldehido por cada 1 mg de colágeno por entrecruzar; sin embargo, se tiene estimado que lo mí nimo de paraformaldehido necesario seria de 2.0 mg y un máximo de 20 mg. Para un volumen de la cámara de entrecruzamiento (1 ) de 5 L; guardado esa correlación en base al volumen de dicha cámara de entrecruzamiento (1 ). El rango de concentración del gas de formaldehido (GF) en la cámara de entrecruzamiento (1 ) es mí nimo de 0.001 mg/L y máximo de 4 mg/L. A ratio of between 0.005 mg and 0.01 mg of paraformaldehyde per 1 mg of collagen to be crosslinked was established; however, it is estimated that the minimum paraformaldehyde needed would be 2.0 mg and a maximum of 20 mg. For a volume of the crosslinking chamber (1) of 5 L; saved that correlation based on the volume of said crosslinking chamber (1). The concentration range of formaldehyde gas (GF) in the crosslinking chamber (1) is a minimum of 0.001 mg/L and a maximum of 4 mg/L.
El invento ha sido descrito suficientemente como para que una persona con conocimientos medios en la materia pueda reproducir y obtener los resultados que mencionamos en la presente i nvención. Sin embargo, cualquier persona hábil en el campo de la técnica que compete el presente invento puede ser capaz de hacer modificaciones no descritas en la presente solicitud, sin embargo, si para la aplicación de estas modificaciones en una estructura determinada o en el proceso de man ufactura del mismo, se req uiere de la materia reclamada en las siguientes reivindicaciones, dichas estructuras deberán ser comprendidas dentro del alcance de la invención. 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. However, any skilled person in the field of the technique covered by the present invention may be able to make modifications not described in this application, however, if for the application of these modifications in a certain structure or in the maintenance process ufacture of the same, the matter claimed in the following claims is required, said structures must be included within the scope of the invention.

Claims

RE IVI N DI CACI O N E S Habiendo descrito suficientemente la invención, se reclama como propiedad lo contenido en las siguientes cláusulas reivindicatorías. CLAIMS Having sufficiently described the invention, what is contained in the following claim clauses is claimed as property.
1.- Un aparato para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, caracterizado por comprender una cámara de entrecruzamiento con una tapa de cierre hermético q ue i ntegra un vacuómetro para la medición de vacio, configurada para alojar una estructura de soporte donde se dispone y se coloca el colágeno a entrecruzar; dicha cámara de entrecruzamiento está comunicada con una cámara de sublimación a través de un ducto de interconexión con medios de control de flujo; dicha cámara de sublimación presenta un émbolo unido a un vástago que pasa a través de una tapa de sellado hermético, y está configurada para alojar una cantidad de paraformaldehido en polvo; en donde dicha cámara de entrecruzamiento además comprende conectado un d ucto generador de vacio con medios de regulación de vacio conectado a un medio generador de vacio y medios para inyectar aire médico con medios de control de fl ujo para ventilación y expulsión del gas hacia u na campana de extracción de gases, una vez entrecruzado el colágeno humano. 1.- An apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, characterized by comprising a cross-linking chamber with a hermetically sealed lid that integrates a vacuum gauge for the vacuum measurement, configured to accommodate a support structure where the collagen to be crosslinked is arranged and placed; said crosslinking chamber is in communication with a sublimation chamber through an interconnection duct with flow control means; said sublimation chamber has a plunger attached to a stem that passes through a hermetically sealed lid, and is configured to house a quantity of paraformaldehyde powder; wherein said crosslinking chamber also comprises a vacuum generating duct connected with vacuum regulation means connected to a vacuum generating means and means for injecting medical air with flow control means for ventilation and expulsion of the gas towards a fume hood, once the human collagen has been cross-linked.
2.- El aparato para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de acuerdo con la reivindicación 1 , caracterizado porque dicha cámara de sublimación está configurada para recibir calor desde un medio de transferencia de calor para la generación de vapor de formaldehído dentro de la misma para el tratamiento de entrecruzamiento de estructuras de colágeno humano. 2.- The apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, according to claim 1, characterized in that said sublimation chamber is configured to receive heat from a heat transfer medium for the generation of formaldehyde vapor within it for the crosslinking treatment of human collagen structures.
3.- El aparato para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de acuerdo con la reivindicación 1 , caracterizado porq ue dicha estructura de soporte de colágeno está definida por una estructura periférica conformada de dos marcos unidos con medios de unió n por dos juegos de postes; en donde una pluralidad de rejillas con una separación entre si se montan en marcos de soporte que se fijan con medios de unión al j uego de postes, y en donde entre dichas rejillas se disponen las esponjas de colágeno a entrecru zar. 3. The apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, according to claim 1, characterized in that said collagen support structure is defined by a peripheral structure made up of two frames joined with means of union by two sets of posts; where a plurality of grids with a separation between them are mounted on support frames that are fixed with joining means to the set of posts, and where the collagen sponges to be cross-linked are arranged between said grids.
4.- El aparato para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de acuerdo con la reivindicación 3, caracterizado porque dichos soportes están hechos de aluminio y las rejillas de acero inoxidable. 4. The apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, according to claim 3, characterized in that said supports are made of aluminum and steel grids stainless.
5.- El aparato para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, de acuerdo con la reivindicación 3, caracterizado porque dichas rejillas definen cuadrados de aproximadamente de 1 a 5 mm de largo y de 1 a 5 mm de ancho. 5.- The apparatus for chemically cross-linking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, of according to claim 3, characterized in that said grids define squares approximately 1 to 5 mm long and 1 to 5 mm wide.
6.- Un proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, en un aparato de entrecruzamiento, caracterizado por comprender las etapas de: a) Colocar colágeno humano a entrecruzar en una estructura de soporte y depositando dicha estructura de soporte dentro de una cámara de entrecruzamiento cerrándola con una tapa de cierre hermético que integra u n vacuómetro para la medición de vacío y que está interconectada con una cámara de sublimación a través de un ducto de interconexión con medios de control de flujo, b) Colocar paraformaldehí do en polvo en la cámara de sublimación insertando un émbolo unido a un vástago que pasa a través de una tapa de sellado hermético; c) Generar un vacío de entre -65 a -75 KPa con un medio generador de vacío conectado a un ducto de generación de vacío con medios de regulación de vacío en posición abierta, en donde dicho ducto de generación de vacío está conectado a dicha cámara de entrecruzamiento y los medios de control de flujo de dicho ducto de interconexión entre la cámara de sublimación y la cámara de entrecruzamiento en posición abierta; d) Calentar la cámara de sublimación a una temperatura de entre 120 °C a 160 °C mediante un medio de calentamiento para producir el gas de formaldehído que es contenido ahí durante un tiempo de entre 8 a 20 minutos; estando dichos medios de control de fl ujo de dicho ducto de interconexión de la cámara de sublimación y la cámara de entrecruzamiento en posición cerrada, habiendo desactivado el medio generador de vacio y estando en posición cerrada dichos medios de regulación de vacio; e) Colocar en posición abierta los medios de control de flujo de dicho ducto de i nterconexión de la cámara de sublimación y la cámara de entrecruzamiento, y se acciona el émbolo de la cámara de sublimación forzando el paso del gas de formaldehído hacia la cámara de entrecruzamiento; f) Dejar expuesto el colágeno por un periodo de entre 1 a 90 mi nutos con el gas de formaldehído dentro de la cámara de entrecruzamiento y luego expulsar el gas de formaldehído desde la cámara de entrecruzamiento mediante un flujo de aire médico alimentado desde una fuente de sumi nistro de aire médico controlado por medios de control de flujo en posición abierta dispuesta en un conector del ducto de interconexión que conecta dicha cámara de sublimación y la cámara de entrecruzamiento, para detener el proceso de entrecruzamiento, estando en posición abierta los medios de control de flujo dicho ducto de interconexión y medios de regulación de vacio para el rompimiento de vacio y expulsión del gas hacia una campana de extracción de gases. 6.- A process for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, in a crosslinking apparatus, characterized by comprising the steps of: a) Placing human collagen to be crosslinked in a support structure and depositing said support structure inside a crosslinking chamber, closing it with a hermetically sealed lid that integrates a vacuum gauge for vacuum measurement and that is interconnected with a sublimation chamber through an interconnection duct with media flow control, b) Place paraformaldehyde powder in the sublimation chamber by inserting a plunger attached to a stem that passes through a hermetically sealed lid; c) Generate a vacuum of between -65 to -75 KPa with a vacuum generating means connected to a vacuum generation duct with vacuum regulation means in the open position, wherein said vacuum generation duct is connected to said chamber crosslinking and the flow control means of said interconnection duct between the sublimation chamber and the crosslinking chamber in the open position; d) Heat the sublimation chamber to a temperature between 120 °C to 160 °C using a heating medium to produce the formaldehyde gas that is contained therein for a time between 8 to 20 minutes; said flow control means of said interconnection duct of the sublimation chamber and the crosslinking chamber being in the closed position, the vacuum generating means having been deactivated and said vacuum regulation means being in the closed position; e) Place the flow control means of said interconnection duct of the sublimation chamber and the crosslinking chamber in the open position, and the plunger of the sublimation chamber is activated, forcing the passage of formaldehyde gas towards the chamber of crossover; f) Expose the collagen for a period of between 1 to 90 minutes with the formaldehyde gas inside the crosslinking chamber and then expel the formaldehyde gas from the crosslinking chamber by means of a flow of medical air fed from a power source. medical air supply controlled by flow control means in the open position arranged in a connector of the interconnection duct connecting said sublimation chamber and the crosslinking chamber, to stop the crosslinking process, the control means being in the open position of flow said interconnection duct and vacuum regulation means for breaking the vacuum and expelling the gas towards a gas extraction hood.
7.- El proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano de acuerdo con la reivindicación 6, caracterizado porque la relación de paraformaldehí do con respectos al colágeno es de entre 0.005 mg a 20 mg de paraformaldehido por cada 1 mg de colágeno a entrecruzar. 7. The process for chemically cross-linking in a controlled and precise manner, a structure made of human collagen according to claim 6, characterized in that the ratio of paraformaldehyde with respect to collagen is between 0.005 mg to 20 mg of paraformaldehyde for every 1 mg of collagen to be crosslinked.
8.- El proceso para entrecruzar químicamente de manera controlada y precisa, una estructura hecha de colágeno humano de acuerdo con la reivindicación 6, caracterizado porque el rango de concentración del gas de formaldehido en la cámara de entrecruzamiento es mí nimo de 0.001 mg/L y máximo de 4 mg/L. 8. The process for chemically crosslinking in a controlled and precise manner, a structure made of human collagen according to claim 6, characterized in that the concentration range of formaldehyde gas in the crosslinking chamber is a minimum of 0.001 mg/L and maximum of 4 mg/L.
9.- Un método para entrecruzar químicamente de manera controlada y precisa, u na estructura hecha de colágeno humano con características mecánicas, químicas y biológicas variables, caracterizado porque consiste en someter colágeno h umano a u na atmósfera de vapor de formaldehido en un aparato de entrecruzamiento, con un tiempo de exposición de entre 1 - 90 mi nutos y concentración de una nube de gas de formaldehi do de entre 0.1 - 100 ppm. 9.- A method for chemically crosslinking in a controlled and precise manner, a structure made of human collagen with variable mechanical, chemical and biological characteristics, characterized in that it consists of subjecting human collagen to an atmosphere of formaldehyde vapor in a crosslinking apparatus , with an exposure time of between 1 - 90 minutes and a concentration of a formaldehyde gas cloud of between 0.1 - 100 ppm.
PCT/MX2021/050070 2021-04-29 2021-10-28 Apparatus and process for the controlled and precise chemical cross-linking of a structure made of human collagen having variable mechanical, chemical and biological features WO2022231417A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5147514A (en) * 1987-08-02 1992-09-15 University Of North Carolina Process for cross-linking collagenous material and resulting product
US20030153638A1 (en) * 2001-08-16 2003-08-14 Industrial Technology Research Institute Method for crosslinking porous biodegradable polymers
US7393437B2 (en) * 2004-09-14 2008-07-01 The University Of Hong Kong Photochemically crosslinked collagen scaffolds and methods for their preparation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5147514A (en) * 1987-08-02 1992-09-15 University Of North Carolina Process for cross-linking collagenous material and resulting product
US20030153638A1 (en) * 2001-08-16 2003-08-14 Industrial Technology Research Institute Method for crosslinking porous biodegradable polymers
US7393437B2 (en) * 2004-09-14 2008-07-01 The University Of Hong Kong Photochemically crosslinked collagen scaffolds and methods for their preparation

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