METHOD FOR PRODUCING HUMAN COLLAGEN STRUCTURES WITH CONTROLLED CHARACTERISTICS

Abstract

A method for producing human collagen structures with controlled characteristics, having the following stages: a) Tissue conditioning b) Pre-treatment; b1) final washings with distilled water; c) extraction by enzymatic hydrolysis by subjecting the tissue to a solution of acetic acid with pepsin; d) precipitation, where the resulting collagen solution is brought to a high concentration by adding sodium chloride and where the fibers recovered from the sieve are solubilized again in an acetic acid solution; e) dynamic dialysis to purify the solution from the excess of salt present in the collagen solution; f) lyophilization at 40 C. and a vacuum pressure of 0.04 mbar (4 Pa); g) molding or second lyophilization, where a concentration is chosen and collagen is solubilized in an acetic acid solution; once solubilized, collagen is placed in molds to generate the desired structure and once again, the solution is lyophilized; h) crosslinking, where the collagen pieces are subjected to a formaldehyde vapor atmosphere in a crosslinking apparatus and i) pressing, where the collagen structure is subjected to a mechanical force of 400-5000 N compacting to a value of 0.01-10 mm and increasing its fibrillar density.

Claims

1. A method to produce human collagen structures with controlled characteristics, characterized by comprising the following stages: a) tissue conditioning, where tissues or fluids that are not of interest for the method are removed and it is reduced to a particle size of 0.5 to 1 mm in a uniform way, in order to achieve a better interaction between solutions and tissue in the subsequent steps; b) pre-treatment exposure of the previously conditioned tissue to a solution of sodium hydroxide at a mold concentration of 0.05-2 M, using 100 mL per gram of dry tissue, with efficient magnetic agitation and for a time of 2-6 hours, in order to remove surface proteins and leave collagen fibers exposed; b1) carry out a series of final washes with distilled water, which allow neutralizing the pH of the tissue to a pH value between 7-8, before proceeding with the method; c) Extraction where enzymatic hydrolysis is performed by subjecting the tissue to an acid solution of acetic acid at a molar concentration of 0.02-0.5 M and using 450 L per kg of dry tissue, together with 200 g of pepsin per kg of dry tissue (2.27% pepsin in 0.5 M acetic acid) to promote a faster solubilization of collagen and the specific and controlled removal of the terminal carboxyl and amino groups of the molecule; where the extraction is carried out over a period of three days by adding intermediate acid solution, starting with 200 mL of acetic acid per g of dry tissue and after 48 hours 250 mL of acetic acid per g of dry tissue are added, to promote the development of the reaction and where the residues of non-collagenous tissue structures are removed by means of filtration and the method continues; d) precipitation, where the resulting collagen solution (450 mL of solution per g of dry tissue) is brought to a high salt concentration by adding sodium chloride at a rate of 58.44 g of sodium chloride per L of collagen solution, the solution is homogenized with magnetic stirring and the ionic interaction of the salt with the collagen molecules is allowed to generate their precipitation in a time of about 2-6 hours, the resulting solution is sieved to a particle size between 125-850 m; wherein the fibers recovered from the sieve are solubilized again in an acetic acid solution using 350 mL of acetic acid per g of dry tissue; e) dialysis, where in order to purify the solution from the excess of salt present in the collagen solution, a dynamic dialysis system is employed by means of which collagen is placed inside a porous membrane with a pore size of between 12-14 kD that allows the expulsion of impurities when introduced into a dialysis buffer consisting of a solution with a low concentration of acid acetic from 0.02-0.5 M; where the exchange of salt molecules between both solutions originates from their concentration differential, from the collagen solution with a salt concentration of 1-2 M towards the acetic acid solution without the presence of salts and is accelerated by the contact surface and the buffer flow; this stage lasts three to four days, during which the buffer is kept in recirculation and is changed after 48 hours; the conductivity of both solutions is also monitored and the method is stopped when collagen reaches the same conductivity as the buffer at the beginning of the stage of a value between 0.20-0.28 mS/cm; f) lyophilization; where the purified collagen solution is subjected to a lyophilization cycle to concentrate the collagen fibers, favoring their preservation; in order to achieve this, the solution is frozen to 40 C. and subsequently subjected to a vacuum pressure of 0.04-0.2 mbar (4-20 Pa) for a period of two days, during which water and solvents are removed in vapor form drying the collagen without damaging the fibers; where the resulting collagen is weighed and distributed according to what is required in the next step; g) molding or second lyophilization, where depending on the application and expected function, a concentration of between 2.5-10 mg/mL is chosen and collagen is solubilized again in an acetic acid solution; where between 2.5-10 mg of collagen per mL of acetic acid with a molarity of between 0.02-0.5 M are used; once solubilized, collagen is placed in molds that allow to generate the desired structure; once again, the solution is lyophilized at a temperature of 40 C. and a vacuum pressure of between 0.04-0.2 mbar (4-20 Pa), with the exception that a controlled freezing is carried out (which consists of removing heat by gradually lowering the temperature of the plate with which the mold and collagen solution are in contact, allowing the water and acetic acid crystals to be uniform and varied, accommodating the fibers) to generate an estimated average pore size; h) crosslinking, where the collagen pieces are subjected to a formaldehyde vapor atmosphere in a crosslinking apparatus, which allows the exposure time to be set between 1-60 minutes and the concentration of the reagent vapor cloud between 0.2-1.6 ppm resulting in a controlled crosslinking that allows reinforcing the bond between fibers that provides better physical properties to the structure.

2. The method to produce human collagen structures with controlled characteristics, according to claim 1, characterized in that it also comprises the stage of: i) pressing wherein relation to the application and function to be performed by the product, where the collagen structure is subjected to a determined mechanical force of 400-5000 N to compact its dimensions to a desired value from 0.01-10 mm and increase its fibrillar density.

3. The method to produce human collagen structures with controlled characteristics, according to claim 2, characterized in that the pressing generates fine and highly dense collagen structures.

4. The method to produce human collagen structures with controlled characteristics, according claim 1, characterized in that it generates a yield of 35% collagen.

5. The method to produce human collagen structures with controlled characteristics, according to claim 1, characterized in that collagen has a purity 95%.

6. The method to produce human collagen structures with controlled characteristics, according to claim 1, characterized in that the collagen obtained allows the cultivation and co-cultivation of primary and/or line human cells; likewise, it functions as a deposit of growth factors, proteins and exosomes, for its possible use in regenerative therapy.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIG. 1 shows a flow chart of the novel process for the production of human collagen structures with controlled characteristics, in accordance with the present invention.

[0054] For a better understanding of the invention, there will be a detailed description of some of its modalities, shown in the drawings that are attached to this description for illustrative but not limiting purposes.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The characteristic details of the novel process for the production of human collagen structures with controlled characteristics are clearly shown in the following description and in the attached illustrative drawings, which provide reference signs to indicate the same steps.

[0056] During the development of the novel process for the production of human collagen structures with controlled characteristics, a series of problems arose that were gradually resolved until the process was standardized to obtain type I human collagen structures with controlled characteristics, with a yield of 35%, preserving structural integrity and with purity 95%.

[0057] The process to reduce the particle size of tissues was time consuming and involved a lot of manipulation. Besides that irregular pieces were obtained. It was solved by choosing to process the tissue in grinding equipment that would allow obtaining a paste from the tissue, without applying too much mechanical stress that would degrade its structure. In order to maintain a low temperature during the process and to avoid the degradation of the extracted collagen fibers, a refrigerator with internal connection was adapted for the equipment used throughout the process.

[0058] Due to the amount of solution and the size of the fibers obtained after fractional precipitation, a sieving separation was implemented, which reduced the operation time and eliminated the need to acquire a larger and more specialized equipment to carry out separation by centrifugation of larger volumes of solution, resulting in only a 4% loss of fibers.

[0059] To enhance the mechanical performance and resistance to enzymatic degradation of the collagen structures and enable the final structure to adequately perform its intended function, chemical crosslinking of the structure was proposed; this operation falls within the field of bioconjugation.

[0060] In this context, chemical crosslinking should be understood as the creation of covalent bonds between two or more molecules. When carrying out this process, the result should be a structure with greater mechanical resistance, greater resistance to deformation, longer enzymatic degradation time and greater absorption. Different chemical substances that could achieve this effect were investigated and it was decided to try glutaraldehyde and formaldehyde.

[0061] Firstly, a solution of water and glutaraldehyde was used to immerse the structure in it. This method was unfeasible due to the delicate nature of the structure, which prevented the overall shape from being maintained, although it mainly affected the thickness and ductility of the material, making it highly brittle. Glutaraldehyde was simply too aggressive on the collagen structure, collapsing it while crosslinking it. The same technique was tried with a solution of water and formaldehyde, but the results were still unacceptable.

[0062] Afterwards, the decision was made to generate a gaseous medium in which the collagen structures were placed to interact at the molecular level with the formaldehyde gas. This medium or atmosphere was created simply by allowing the mixture of water and formaldehyde to remain for some time in a closed container at room temperature. The aqueous solution, due to the evaporation that naturally occurs at room temperature, eventually saturated the volume of the container. After a specified time, the container was opened and the collagen structures were introduced. Since the structures were inside the container, a specific time began to be counted. This method was equally unfeasible due to several factors: on the one hand, the water and formaldehyde solution did not evaporate in a determinable or controllable manner. In addition, when opening the container to introduce the collagen structures, the equilibrium reached by the atmosphere inside was disturbed.

[0063] This alteration could not be easily determined or measured. Ultimately, this method could not reliably generate repeatable results.

[0064] It was assumed that using mixtures of formaldehyde with some other medium (such as water), would make it more difficult to control the amount of gas generated. Therefore, the decision was made to produce the purest gas possible. It was demonstrated that it was feasible to heat paraformaldehyde until reaching the sublimation temperature and generate formaldehyde gas. Once the gas has been produced, it should be passed to another closed chamber where the collagen structures had previously been placed on a support that would allow homogeneous interaction with the gas. It was also decided that a certain amount of vacuum be produced in the chamber to minimize the effect that atmospheric air might have on the crosslinking process. Another issue that had to be resolved was how to stop the process abruptly in order to control both the start and the end of the crosslinking. To achieve this, a method was conceived to include a medical-grade air intake that would serve to purge the system and remove the formaldehyde.

[0065] By removing the formaldehyde, the interaction between collagen molecules and formaldehyde molecules is stopped, effectively stopping the crosslinking process. All of these design requirements were generated based on observation and experiments as described in the previous paragraphs. So the final design of the apparatus is the result of an experimental process and it is also the solution that allows collagen structures to be achieved with the desired physical, mechanical, chemical and biological compatibility characteristics.

[0066] According to FIG. 1, the novel process for the production of human collagen structures with controlled characteristics, in accordance with the present invention, consists of the following stages: [0067] a) Tissue conditioning, where tissues or fluids that are not of interest for the process are removed and it is reduced to a particle size of 0.5 to 1 mm in a uniform way, in order to achieve a better interaction between solutions and tissue in the subsequent steps. [0068] b) Pre-treatment

[0069] Exposure of the previously conditioned tissue to a solution of sodium hydroxide at a mold concentration of 0.05-2 M, using 100 mL per gram of dry tissue, with efficient magnetic agitation and for a time of 2-6 hours, in order to remove surface proteins and leave collagen fibers exposed; [0070] b1) carry out a series of final washes with distilled water, which allow neutralizing the pH of the tissue to a pH value between 7-8, before proceeding with the process. [0071] c) Extraction where enzymatic hydrolysis is performed by subjecting the tissue to an acid solution of acetic acid at a molar concentration of 0.02-0.5 M and using 450 L per kg of dry tissue, together with 200 g of pepsin per kg of dry tissue (2.27% pepsin in 0.5 M acetic acid) to promote a faster solubilization of collagen and the specific and controlled removal of the terminal carboxyl and amino groups of the molecule; where the extraction is carried out over a period of three days by adding intermediate acid solution, starting with 200 mL of acetic acid per g of dry tissue and after 48 hours 250 mL of acetic acid per g of dry tissue are added, to promote the development of the reaction and where the residues of non-collagenous tissue structures are removed by means of filtration and the process continues. [0072] d) Precipitation, where the resulting collagen solution (450 mL of solution per g of dry tissue) is brought to a high salt concentration by adding sodium chloride at a rate of 58.44 g of sodium chloride per L of collagen solution, the solution is homogenized with magnetic stirring and the ionic interaction of the salt with the collagen molecules is allowed to generate their precipitation in a time of about 2-6 hours. The resulting solution is sieved to a particle size between 125-850 m; wherein the fibers recovered from the sieve are solubilized again in an acetic acid solution using 350 mL of acetic acid per g of dry tissue; [0073] e) Dialysis, where in order to purify the solution from the excess of salt present in the collagen solution, a dynamic dialysis system is employed by means of which collagen is placed inside a porous membrane with a pore size of between 12-14 kD that allows the expulsion of impurities when introduced into a dialysis buffer consisting of a solution with a low concentration of acid acetic from 0.02-0.5 M; where the exchange of salt molecules between both solutions originates from their concentration differential, from the collagen solution with a salt concentration of 1-2 M towards the acetic acid solution without the presence of salts and is accelerated by the contact surface and the buffer flow; this stage lasts three to four days, during which the buffer is kept in recirculation and is changed after 48 hours; the conductivity of both solutions is also monitored and the process is stopped when collagen reaches the same conductivity as the buffer at the beginning of the stage of a value between 0.20-0.28 mS/cm. [0074] f) Lyophilization; where the purified collagen solution is subjected to a lyophilization cycle to concentrate the collagen fibers, favoring their preservation; in order to achieve this, the solution is frozen to 40 C. and subsequently subjected to a vacuum pressure of 0.04-0.2 mbar (4-20 Pa) for a period of two days, during which water and solvents are removed in vapor form drying the collagen without damaging the fibers; where the resulting collagen is weighed and distributed according to what is required in the next step. [0075] g) Molding or second lyophilization, where depending on the application and expected function, a concentration of between 2.5-10 mg/mL is chosen and collagen is solubilized again in an acetic acid solution; where between 2.5-10 mg of collagen per mL of acetic acid with a molarity of between 0.02-0.5 M are used; once solubilized, collagen is placed in molds that allow to generate the desired structure; once again, the solution is lyophilized at a temperature of 40 C. and a vacuum pressure of between 0.04-0.2 mbar (4-20 Pa), with the exception that a controlled freezing is carried out (which consists of removing heat by gradually lowering the temperature of the plate with which the mold and collagen solution are in contact, allowing the water and acetic acid crystals to be uniform and varied, accommodating the fibers) to generate an estimated average pore size; [0076] h) crosslinking, where the collagen pieces are subjected to a formaldehyde vapor atmosphere in a crosslinking apparatus, which allows the exposure time to be set between 1-60 minutes and the concentration of the reagent vapor cloud between 0.2-1.6 ppm resulting in a controlled crosslinking that allows reinforcing the bond between fibers that provides better physical properties to the structure.

[0077] In one of the modalities of the invention, the process also includes the stage of: [0078] i) Pressing (or additional stage according to the application), where, in relation to the application and function to be performed by the product, where the collagen structure is subjected to a determined mechanical force of 400-5000 N to compact its dimensions to a desired value from 0.01-10 mm and increase its fibrillar density.

[0079] With the production process, it has been possible to use of an available, accessible raw material with the highest degree of biocompatibility; obtaining functional and structurally intact collagen, with profitable extraction yields through a compatible process for industrial production conditions. In addition, said process allows the elaboration of collagen structures with adjustable physical characteristics for a wide range of biological applications, by means of simple and low-cost techniques that allow the optimization of collagen concentration in the structures; such characteristics include the dimensions of the structure, fibrillar density, porosity and pore size, which strongly influence cell behavior.

[0080] Likewise, it was possible to increase the mechanical performance of the structures, with a precise and controlled technique, without affecting the integrity, functionality, or biological safety of collagen.

[0081] The human collagen structures obtained by the described process are also characterized in that they allow the cultivation and co-cultivation of primary and/or line human cells, and they function as a deposit of growth factors, proteins and exosomes, for their possible use in regenerative therapy.

[0082] The invention has been sufficiently described so that a person with average knowledge in the field can reproduce and obtain the results mentioned in the present invention. However, any person skilled in the relevant technical field of this invention may be capable of making modifications not described in the present application; nevertheless, if the matter claimed in the following claims is required for the implementation of these modifications in a specific structure or in the manufacturing process thereof, such structures must be included within the scope of the invention.