RU2848597C1 - Method for manufacturing an external agent for healing wounds from human umbilical cord - Google Patents

Abstract

FIELD: external wound treatment agent from human umbilical cord.

SUBSTANCE: invention comprises obtaining and preparing native human umbilical cord by washing it under running water with removal of umbilical cord vessels and blood, weighing, fragmenting, according to the invention, the washed and cleaned umbilical cord is fragmented into particles no larger than 3 mm, it is poured with distilled water at a ratio of 500 ml of water per 50 g of umbilical cord, cooled for 48 hours at a temperature of up to 6°C, filtered and 0.85 g of sodium chloride is added to every 100 ml of solution and brought to a boil, then, during the boiling process, add 0.05 ml of concentrated hydrochloric acid per 100 ml of the resulting mixture, cool to room temperature, filter through sterile filter paper and pour into sterile containers, sterilise the containers in an autoclave at 1.2 atmospheres for 20 minutes, also applies to a method for manufacturing an external agent for treating wounds from human umbilical cord, including obtaining and preparing native human umbilical cord by washing under running water with removal of umbilical cord vessels and blood, weighing, fragmentation, according to the invention, the washed and cleaned umbilical cord is fragmented into particles no larger than 3 mm, pouring distilled water at a ratio of 500 ml of water per 50 g of umbilical cord, cooling for 48 hours at a temperature of up to 6°C, filtering through a filter and adding 0.85 g of sodium chloride per 100 ml of solution and bringing to a boil, then, during the boiling process, add 0.05 ml of concentrated hydrochloric acid per 100 ml of the resulting mixture, cool to room temperature, filter through sterile filter paper and carry out further lyophilisation.

EFFECT: development of an affordable and effective method for obtaining an external agent for wound treatment.

2 cl

Description

The invention relates to the chemical and pharmaceutical industry and is intended for external use for the treatment of both clean and bacterially contaminated superficial and deep wounds in various inflammatory and infectious skin diseases, trophic skin lesions and corneal ulcers.

Over the past 20 years, tissues of perinatal origin have become the object of increasing attention from researchers and practitioners due to their potential for use in cell therapy.

The umbilical cord is an extraembryonic tissue with a fetal phenotype, therefore, regenerative molecules are present in the extracellular matrix (ECM) of its connective tissue.

Cells with stem cell properties derived from amniotic epithelium, Wharton's jelly, and umbilical cord vascular endothelium are already being used in cell therapy in clinical practice. (Analysis of global experience using umbilical cord biomaterials in tissue engineering and 3-D bioprinting. Medicine and Healthcare Organization. Vol. 4, No. 1, 2019. P. 40).

Human placental tissue biomaterial has a wide range of applications in regenerative medicine due to its rich ECM composition and the large number of signaling molecules and growth factors it contains [Roy, A. Placental Tissues as Biomaterials in Regenerative Medicine. / A. Roy, M. Mantay, C. Brannan, S. Griffiths // Biomed Research International. - 2022. - Vol. 21. - 6751456]. This human biomaterial is not subject to aging and is obtained without the use of invasive procedures from donors who have been examined and tested for infectious agents. Another important advantage is that there are no ethical restrictions on its use, since it is disposed of after birth as biological waste.

Human provisional organs have been thoroughly studied as a source of cells with high regenerative potential [I.V. Arutyunyan, A.V. Makarov, A.V. Elchaninov, T.Kh. Fatkhutdinov. Multipotent umbilical cord mesenchymal stromal cells: biological properties and clinical application. Genes and Cells. 2015, Vol. 10, pp. 30-38]. For example, the use of placental mesenchymal stromal cells and Wharton's jelly to stimulate the regeneration of damaged tissues has been described in detail.

The use of the stromal component of umbilical cord Wharton's jelly biomaterial is relatively new. Wharton's jelly consists mainly of collagens, proteoglycans, and glycosaminoglycans (GAGs) immobilized and embedded in a collagen network [E. Bańkowski, K. Sobolewski, L. Romanowicz, L. Chyczewski, and S. Jaworski, “Collagen and glycosaminoglycans of Wharton's jelly and their alterations in EPH-gestosis,” European Journal of Obstetrics, Gynecology, and Reproductive Biology, vol. 66, no. 2, pp. 109–117, 1996]. The umbilical cord ECM contains a variety of different growth factors that are released during its degradation [Sobolewski K, Małkowski A, Bańkowski E, Jaworski S. Wharton's jelly as a reservoir of peptide growth factors. Placenta. 2005; 26(10): 747-52. doi: 10.1016/j.placenta.2004.10.008. PMID: 16226124].

The proposed invention is based on the fact that resident mesenchymal cells of umbilical cord Wharton's jelly are capable of differentiating into various human tissues, namely vascular muscle cells and skin cells. Consequently, products from Wharton's jelly can be used to restore defects in skin, cartilage, bone and muscle tissue, and to heal wounds.

The use of human umbilical cord tissue as a medicinal raw material must exclude the risk of infection or other adverse effects. Currently, numerous methods are known for processing and obtaining medicinal raw materials and matrices from the human placenta and umbilical cord.

The decellularization process is described in the article "Biotechnological Cell-Free Non-Immunogenic Product Preserves the Key Regenerative Structural Components of the Human Umbilical Cord" (Biotechnology, 2023, Vol. 9, No. 1, pp. 49-59). Prior to decellularization, human umbilical cord samples were rinsed several times with deionized water (Aquaphor, Russia) to remove blood components, sterilized with a 6% hydrogen peroxide solution, and dissected in a laminar flow hood under aseptic conditions, removing the vein and arteries. The human umbilical cord Wharton's jelly, along with its covering membrane, was then mechanically fragmented and homogenized (gentleMACS™ Dissociator, Milteniy Biotech, Germany). Decellularization was performed by the detergent method using sterile solutions containing 0.01 and 0.05% sodium dodecyl sulfate (LenReaktiv, Russia) for 24 h at room temperature in an ES-20/60 shaker (Biosan, Latvia) at a rotation speed of 180 rpm. Upon completion of the process, the samples were repeatedly washed with deionized water and a phosphate buffer solution (pH 7.4) in order to remove the detergent and detached cells. The obtained material was lyophilized (VaCo 5-II, ZIRBUS Technology, Germany), disinfected with UV irradiation in a BMB-II-Laminar-S-1.5 NEOTERIC microbiological safety cabinet (Lamsystems, Russia) at a UV-C radiation flux power of 12 W for 15 min and stored hermetically sealed at a temperature of -20°C.

Patent RU2795904 describes a method for producing an acellular matrix, including umbilical cord preparation steps including sterilization with a 10% hydrogen peroxide solution, fragmentation and homogenization, decellularization, washing of the reagent and detached cells, neutralization with sodium hydroxide solution, centrifugation, and lyophilization. A 0.01% sodium dodecyl sulfate solution is used to remove cells from the ECM during decellularization.

Patent RU2816638 proposes obtaining a decellularized matrix by fragmentation, freezing of biological material, its subsequent grinding, and decellularization.

A method for obtaining biologically active substances from the placenta is known (patent RU2033797, application No. 5068585, published on April 30, 1995).

The preparation of placenta specimens involves obtaining an oxidizing reagent, washing the placenta in a neutral barrier solution, separating the amnion, umbilical cord, smooth chorion, and villous chorion, coarsely mincing the villous chorion before hemolysis, hemolysis of the resulting tissues in a neutral barrier solution, tissue denaturation in chlorine dioxide, washing from the oxidizing solution, coarsely mincing the denatured amnion, umbilical cord, and smooth chorion, and tissue dispersion. Hemolysis and tissue denaturation occur separately, denaturation modes vary for different tissues, and dispersion is sterilizing. Oxidation is performed with a chlorine dioxide solution.

A disadvantage of known methods is that most manufacturing techniques are labor-intensive, requiring specialized and expensive equipment, reagents, and highly qualified personnel. They also require special conditions for the preparation and storage of the drug.

A method for producing a cell-free hydrogel from Vartan jelly of human umbilical cord for intra-articular use is known (patent RU2745995 application No. 2020130589, published on 05.04.2021, bulletin No. 10).

The known method describes the stages of processing human umbilical cord for the purpose of producing a cell-free hydrogel.

According to the known method, after obtaining the native umbilical cord, it is sterilized with hydrogen peroxide solution, prepared, fragmented, and homogenized. It is decellularized with sodium hydroxide, then neutralized with hydrochloric acid. The reagent and detached cells are washed away, neutralized with sodium hydroxide, and centrifuged again, followed by lyophilization. The resulting lyophilized matrix is then solubilized by treating it with pepsin in a hydrochloric acid solution, after which the medium is neutralized to physiological pH values with sodium hydroxide under specific conditions.

A disadvantage of the known method is that exposure to strong oxidizing agents and rough mechanical processing causes changes in the biochemical composition of the tissues. Furthermore, the presence of finely dispersed tissue residues requires additional methods for purification and stabilization of the resulting product, as well as restoration of physiological pH values.

The technical result is the development of an accessible and effective method for obtaining an external agent for the treatment of wounds.

Fragmentation is the process of breaking something down into many small, disjointed pieces.

Decellularization is a procedure for removing the cellular component from allografts (tissues) using various methods (physical, enzymatic, and chemical) to produce a non-immunogenic, effective, and safe construct based on the natural extracellular matrix. Decellularization is a process used in biomedical engineering to separate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving behind the ECM scaffold of the original tissue, which can be used to create artificial organs and tissue regeneration.

There are two options for preparing an external remedy for wound treatment: 1) in liquid form and 2) in dry form.

A method is proposed for the production of an external agent for the treatment of wounds from the human umbilical cord, which includes obtaining and preparing native human umbilical cord by rinsing it under running water with the removal of umbilical cord vessels and blood, weighing it, and fragmenting it.

The fragmentation process is carried out until particles no larger than 3 mm are obtained, resulting in a homogeneous mixture. The particle size after fragmentation is essential for further separation of liquid mixtures by filtration through filters, which in their simplest form can be made of sterile gauze. Furthermore, fragmentation is used to facilitate the penetration and action of decellularizing agents.

The washed and cleaned umbilical cord, fragmented into particles no larger than 3 mm, is poured with distilled water (hydromodule 1;10), cooled for 48 hours at a temperature of up to 6°C, filtered through a filter and 0.85 g of sodium chloride is added to every 100 ml of solution and brought to a boil, then during the boiling process 0.05 ml of concentrated hydrochloric acid is added per 100 ml of the resulting mixture, cooled to room temperature, filtered through sterile filter paper and poured into sterile containers, which are sterilized in an autoclave at 1.2 atmospheres for 20 minutes.

The temperature should not exceed 6°C to prevent spoilage of the material. Temperatures should not fall below freezing, as freezing umbilical cord tissue will cause undesirable changes in the tissue, including physical, histological, colloidal-chemical, biochemical, and biological changes, which are critical to the quality of the material. Cooling at the specified temperature for 48 hours prepares the mixture for subsequent filtration. The fragmented particles swell, and the solution takes on the appearance of a gelatinous colloidal solution.

The resulting mixture is then filtered. Sterile medical gauze is used as a filter. Table salt is added to the resulting mixture at a ratio of 0.85 g of sodium chloride per 100 ml of mixture. The use of table salt in a similar process is known from a published source (Patent No. 2714327, "Method for Accelerated Decellularization of Biological Tissues or Organs").

The filtered mixture is brought to a boil, and the boiling process is immediately stopped, preserving the biologically active substances in the solution.

A process of chemical decellularization of the resulting mixture is used. 0.05 ml of concentrated hydrochloric acid is added to 100 ml of the solution to neutralize it. The use of chemical decellularization using acids is described in a well-known published source (the dissertation "Tissue-specific matrices from decellularized liver and articular cartilage fragments for tissue engineering").

https://transpl.ru/upload/medialibrary/934/934dde2164fbec24f1a96bf3d7a54c19.pdf?ysclid=m7ofukljpb70087766.

The resulting mixture is filtered through sterile filter paper and poured into sterile containers.

The solution-containing containers are placed in an autoclave and sterilized at 1.2 atmospheres for 20 minutes. Then, random laboratory tests are performed on the prepared material, including microbial culture.

The proposed method proposes the development of an affordable and effective method of bioactive external agent for wound treatment.

Currently, the use of materials identical in properties to native human tissues is a pressing issue. Collagen is a natural material whose advantages are well-known. The use of collagen-containing matrices is currently known from various published sources. Decellularized materials lack immunogenic properties and are beneficial for human tissues. (Decellularized human umbilical arteries as a basis for small-caliber tissue-engineered blood vessels. Cell Transplantology and Tissue Engineering. Vol. 8, No. 1, 2013.)

When applied topically, the present invention's topical treatment intervenes in the regenerative process, exerting a noticeable effect on the tissue cells themselves and providing a favorable environment for activating cellular processes. Wound healing occurs significantly faster.

The resulting product, which is intended for use in the treatment of thrombophlebitis, joint diseases, colloid scars, and rough post-operative wounds, was studied in the laboratory.

The resulting liquid product, according to the first independent claim, has the following organoleptic characteristics: a colorless, transparent, odorless liquid. Specific qualitative tests revealed the presence of polysaccharides and hyaluronic acid. The main components of umbilical cord connective tissue—collagen and elastin fibers—were also preserved. Cells, nuclei, and debris were successfully removed. No morphologically intact cells were observed in the product.

According to the second claim of the invention, a lyophilization process was carried out. A slightly yellowish powder with particles of varying sizes was obtained.

The proposed method for obtaining an external remedy is accessible, does not require expensive equipment, and, due to the fact that valuable natural raw materials are used, it has proven its effectiveness.

A clinical example demonstrates the effectiveness of the product obtained using the stated method.

Studies of the effect of the product on the dynamics of wound healing were conducted on adult outbred male mice weighing 18-25 g. The experiments were conducted in compliance with the ethical principles of animal experimentation in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental Purposes and the Order of the Ministry of Health of the Russian Federation dated June 19, 2003, No. 267.

The day before the experiment, the mice were plucked from the site of the intended wounds using Deep depil depilatory cream. Under inhalation anesthesia, a circular flap of skin measuring 60 ^mm2 was cut from the back of the neck using a stencil and surgical scissors with rounded tips. The wounds were left open until the end of the experiment.

The mice were divided into three groups of 10 animals each. The first group served as a control; the wound was left open without treatment and healed spontaneously. In the second group, Solcoseryl gel was applied to the wound. In the third group, the wound was treated with the treatment. The experimental groups of mice received 0.2 g of the treatment immediately after surgery and then daily for 14 days. Changes in wound area were recorded once a day by measuring the wound diameter with calipers. Wound area was calculated using the formula: The effectiveness of the drug was assessed based on observations of the dynamics of wound healing processes, the general condition of the animals, the presence or absence of an inflammatory process in comparison with the above-mentioned groups.

Statistical data processing was performed using the Statgraphics software package. The significance of differences between control and experimental results was assessed using Student's t-test.

The study results showed that mice tolerated the developed product well compared to the control group. No irritation or allergic reactions were observed in the test animals. During the observation period, no cases of wound suppuration or complications were observed in the animals. The product was easily applied to the wounds, absorbing wound fluid without drying out the wound bed.

An analysis of the study results showed that the product creates favorable conditions for post-traumatic regeneration, reducing wound healing time by 4 days compared to the control group. The wound-healing effect of the product was evident from the first day after wounding and was characterized by a 6.3% reduction in wound area. From days 1 to 10, a significant reduction of 48.07% compared to the control group was noted (p < 0.05). By day 14 of the experiment, the wound scar was virtually invisible, indicating the product's beneficial effect on wound healing.

Thus, studies conducted on a full-thickness wound model in mice demonstrated high wound healing efficacy after application of the product. When a thin layer of the wound-healing agent is applied to the wound surface, the active ingredients are released, promoting rapid wound healing. The effect of topical application of the product in both liquid and dry form is similar.

Claims (2)

1. A method for producing an external agent for treating wounds from the human umbilical cord, including obtaining and preparing a native human umbilical cord by washing it under running water with the removal of the umbilical cord vessels and blood, weighing it, fragmenting it, characterized in that the washed and cleaned umbilical cord is fragmented into particles no more than 3 mm in size, filled with distilled water at the rate of 500 ml of water per 50 g of umbilical cord, cooled for 48 hours at a temperature of up to 6 ° C, filtered through a filter and 0.85 g of sodium chloride is added to every 100 ml of solution and brought to a boil, then during the boiling process 0.05 ml of concentrated hydrochloric acid is added to 100 ml of the resulting mixture, cooled to room temperature, filtered through sterile filter paper and poured into sterile containers, the containers are sterilized in an autoclave at a mode of 1.2 atmospheres for 20 min. 2. A method for producing an external agent for treating wounds from the human umbilical cord, which includes obtaining and preparing a native human umbilical cord by washing it under running water with the removal of the umbilical cord vessels and blood, weighing it, fragmenting it, characterized in that the washed and cleaned umbilical cord is fragmented into particles no more than 3 mm in size, filled with distilled water at the rate of 500 ml of water per 50 g of umbilical cord, cooled for 48 hours at a temperature of up to 6 °C, filtered through a filter and 0.85 g of sodium chloride is added to every 100 ml of the solution and brought to a boil, then during the boiling process 0.05 ml of concentrated hydrochloric acid is added per 100 ml of the resulting mixture, cooled to room temperature, filtered through sterile filter paper and further lyophilized.