RU2769248C1 - Method for obtaining acellular dermal matrix - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method for obtaining an acellular dermal matrix includes cutting a split pig dermal graft without an epidermal layer with a thickness of 1.0 mm, washing it in distilled water and cutting it into equal squares with dimensions of 3 × 3 cm, followed by holding in a solution of 0.9% sodium chloride for 8-10 minutes, the obtained samples are subjected to ultrasound with a power of 900 Watts for 10-15 seconds and subsequent exposure to isostatic pressure up to 180-200 MPa for 2-3 minutes, after which the samples are dried.

EFFECT: above method makes it possible to eliminate the antigenicity of the dermal matrix while preserving the native three-dimensional protein structure of the dermis, which is important for reproducing the properties of intact skin.

1 cl, 2 dwg, 1 tbl, 1 ex

Description

The invention relates to medicine, namely to surgery, can be used for surgical treatment of deep and extensive burns of the skin in victims.

Mortality in critical (extensive) burns ranges from 60% to 94%. A difficult category is those with deep burns of more than 30% of the body surface due to a shortage of donor areas for skin grafting. An affordable and highly effective resource to overcome this limitation could be the use of donor (cadaveric) skin. In the Russian Federation, the use of cadaveric skin is currently not legally allowed. In this regard, it seems very relevant to develop and test a technology (method) for obtaining and preserving (storing) acellular dermal matrix (ADM) (cell scaffold) from porcine skin for the treatment of deep and extensive burns of the skin in victims. Alternative effective methods of conservative treatment of pathology are hardly can be specified.

In recent decades, in victims of burn injury, due to early surgical treatment, it is possible to quickly remove the burn scab, prevent early sepsis, and the development of burn disease. A serious problem in this case is the temporary replacement of the skin, since the rate of excision of dead tissues always exceeds the possibility of prompt restoration of one's own skin. Despite the widespread use of available xenogenic (lyophilized pigskin - Xenoderm) and synthetic temporary wound dressings, deep burns over 40-50% of the body surface practically predetermine a lethal outcome. Despite the improvement of the applied surgical techniques for closing burn wounds after necrectomy, a consensus on the superiority of one or another approach has not been reached.

Dermal matrices (“scaffolds”) in the context of cellular technologies are a three-dimensional substrate or a temporary scaffold for cells, they prosthetize the dermal layer of the skin and allow a full-fledged skin cover to form on its basis. By origin, scaffolds are classified into biological, synthetic and genetically engineered, the latter being an innovative biotechnical product, the least rejected (more accustomed), safe and effective. Their use will allow at the inpatient stage of providing medical care to victims with burn injuries to reduce the time of treatment and multiply increase the survival rate.

In recent decades, tissue engineering has taken up the solution to this problem. The aim of the work is to create a biologically compatible, having a stable three-dimensional structure, capable of complete engraftment of the dermis substitute. Skin tissue-engineered analogues, both cell-free and cell-based, are examples of technologies that have firmly established themselves in the advanced market. First introduced 30 years ago, these products have already proven to be effective and safe in the treatment of extensive burns and chronic wounds refractory to conventional treatments. Skin substituents can be divided into acellular and containing skin cells.

Many companies use cell-free, variously processed allo or xenografts. Such products are united by the term "accelular dermal matrix" (ADM), they are devoid of the epidermal layer and play the role of matrices that ensure the formation of new tissue at the site of the wound surface and stimulate re-epithelialization. Removal of cellular material significantly reduces the antigenic properties, providing permanent engraftment of such a dressing to the wound bed with vascular germination and gradual filling with the recipient's own cells (Garfein 2003). Due to the bioactive components included in the structure of the native dermis, they have a stimulating effect on angiogenesis and cell migration (Greaves 2015). The presence of its own biological activity distinguishes cell-free dermis preparations from the generation of synthetic dermal equivalents obtained from its individual components: collagen, glycosaminoglycans, hyaluronic acid by chemical crosslinking. The presence of good mechanical properties, resistance to infection, compared with synthetic implants, has led to the widespread use of cell-free dermis preparations in reconstructive surgery of the abdominal wall and breast, rhinoplasty and periodontal surgery. In the future, decellularized dermal matrices are considered as one of the main scaffold options for creating a bioengineered autologous skin equivalent (Shevchenko 2010, Debels 2015).

Requirements for dermal matrices: dermal substitutes must reproduce the main properties and functions of the dermis, namely:

1. Creation of protection against moisture loss and infection;

2. Restoration of the anatomical structure and mechanical properties of the skin;

3. Biocompatibility - dermal matrices should not cause an immune response, promote cell adhesion, migration and vascularization;

4. Bioresorbability - over time, such matrices should gradually degrade with replacement by newly formed tissue.

Benefits of cell-free natural dermal matrices:

1. Preservation of the native 3D structure of collagen-elastin components and porosity ensures water-gas permeability and mechanical strength;

2. Preservation of the basement membrane ensures the adhesion of keratinocytes and the formation of the epidermis;

3. Elimination of cells provides a decrease in the antigenic properties of the skin.

Disadvantages of cell-free natural dermal matrices:

1. Risk of transmission of infections;

2. Partial preservation of antigenic properties;

3. Often complex procedures for obtaining and high cost.

Commercial products based on decellularized dermal matrices:

Allogeneic - based on donor human skin

- AlloDerm® (Life Corporation, TX, US) - the most widely used product is chemically treated donor skin (Ralston 1999). Fresh donor skin is treated with saline to remove the epidermal layer and extract cells, then subjected to a freeze-dry procedure. It has been used to treat burns since 1992. The product has been shown to take root well, colonize the patient's own cells and reduce scarring (Jones 2002).

- GLYADERM® - Gliaderm is produced by EuroSkin Bank (Netherland) from glycerinized leather by long-term treatment in alkali (Richters2008). Glycerin is used as a preservative. Also, glycerol has a bactericidal property and the procedure of glycerinization in high concentration glycerol solutions (up to 85%) leads to a gradual inactivation of microorganisms. Glycerinized skin loses its elasticity and becomes more rigid, during this procedure all cells die, but the immunogenicity of the product decreases along with them, which prolongs the engraftment period. When treated with alkali, all cellular components of the hair and, accordingly, antigenic structures are removed, all growth factors associated with the matrix are removed, the physicochemical properties of the dermis deteriorate sharply, but collagen and elastic fibers are preserved, providing the mechanical properties of the dermis. Gliaderm can be used as a substitute for the dermis.

Xenogenic - based on animal skin.

Pigskin dermal matrices are very similar in properties to allogeneic matrices and represent the first example of a natural tissue substitute based on animal material. The availability of raw materials makes it possible to significantly simplify and reduce the cost of manufacturing products based on it, and the risk of transmission of viral agents such as HIV and NerV is also reduced. Despite all the disadvantages associated with the use of xenogenic material, many researchers suggest that in the future such products will dominate the markets for biomaterials (Ge 2009, Shahrokhi 2014).

To date, there are several xenogenic matrices on the commercial market:

- Permacol® - decellularized porcine dermis, consisting of chemically cross-linked collagen and elastin fibers (Parker 2006). Designed for abdominal plasty, has reduced properties of biointegration and vascularization.

- MatriDerm® (Medskin solutions Dr. Suwelack AG, Germany) - a product of bovine origin, is a collagen matrix coated with elastin hydrolyzate, lyophilized (Kolokythas 2008, Haslik 2010).

- Strattice® (Kinetic Concept Inc) decellularized porcine dermis, designed for reconstructive breast surgery.

- Xenoderm® - lyophilized pigskin.

- Xenoderm (CJSC AB Medical) is a domestic biological coating made of lyophilized pigskin, developed and clinically tested in Russia. For its manufacture, the split skin of young individuals that have passed veterinary control for the absence of infectious and parasitic diseases is used. The coating is sterilized by ionizing radiation and freeze-dried by freezing and drying in a vacuum. It is made in the form of solid or perforated plates of white or yellowish “ivory” color, 10 by 10 cm and 10 by 15 cm in size. , cleared of necrosis during the period of their preparation for auto dermoplasty. In addition, Xenoderm coatings are used to protect transplanted split perforated autodermal grafts and donor wounds.

The use of cell-free matrices based on donor skin in tissue engineering and regenerative medicine is growing every year; currently, methods have been developed for obtaining such products both for individual tissues (dermis, mesothelium, pericardium) and for entire organs (heart, kidney, trachea, bladder) (Atala 2012, Ashkan and Atala 2017). Cell-free matrices, due to the preservation of the three-dimensional architectonics of structural components and related growth factors, adhesion molecules, and membrane components, are able to stimulate various biological processes: proliferation, migration, and remodeling. Despite the fact that ECM significantly stimulate tissue repair, the success of their use is highly dependent on the method of their preparation (decellularization method). The methods used for decellularization act in parallel on the elements of the extracellular matrix and lead to a change in the mechanical and biological properties of the tissue. There is a direct relationship between these two properties, so it is important to find a balance in which the preservation of the main mechanical and biological properties would be combined with the maximum removal of antigenic determinants.

The methods used to obtain tissue, including dermal matrices, by their nature are divided into:

- chemical

- biological

- physical

Each of the methods has its advantages and limitations; in practice, depending on the type of tissue (thickness, composition), combined methods are used.

Table 1 shows the characteristics of the main methods of tissue decellularization:

based on http://www.regmedgrant.com/files/review2_rus.pdf

There is also known a method for obtaining an acellular dermal matrix, in which a porcine dermis 3 mm thick is taken after preliminary removal of the epidermis by a dermatome, then the samples are frozen to a temperature of -80 ° C, after defrosting, the samples are poured with a Trypsin-Versen solution and placed in a shaker-incubator in a mode of 100 rpm at 37°C for 18 hours; at the second stage, the samples are placed on a rotating platform at 170 rpm and subjected to sequential cyclic exposure to detergent solutions: 1% Triton XI00 solution for 2 hours and 4% sodium deoxycholate solution in combination with 0.002 M Na2-EDTA for 2 hours, the total number of treatment cycles is 5. After each detergent, the samples are washed in deionized water for 10 minutes. At the third stage, the samples are treated with a solution of porcine pancreatic DNase I (2000 IU was dissolved in 200 ml of phosphate buffer with calcium and magnesium) in a shaker incubator at 100 rpm at 37°C for 4 hours. Decellularization is completed by exposure to a 10% solution of chlorhexidine bigluconate in phosphate buffer for 24 hours with a change of solution every 6 hours. After that, the quality of decellularization is confirmed by histological examination, molecular biological analysis and cultural methods. (Patent for IZ RF No. 2717088 dated October 28, 2019, published on March 18, 20 Bull. No. 8)

The method makes it possible to reduce the time of exposure to decellularizing solutions, to reduce the level of residual DNA in the tissue to 60 ng/mg of tissue in a wet sample.

The closest analogues of the declared from the field of technology have not been identified.

The technical result achieved by using the present invention is the elimination of the antigenicity of the dermal matrix, leading to an immune response and transplant rejection as a result of the complex effect of physical and chemical factors on the dermis, leading to the most complete elimination of antigenic determinants while maintaining the native three-dimensional protein structure of the dermis, which is important for reproducing the properties of intact skin.

The technical result is achieved by the fact that in the method for obtaining an acellular dermal matrix, a split porcine dermal graft without an epidermal layer 1.0 mm thick is cut, then the sample is washed in distilled water, cut into equal squares 3 × 3 cm in size, followed by exposure to sodium chloride solution 0.9% for 8-10 minutes. The resulting samples are subjected to ultrasonic treatment for 10-15 seconds and subsequent exposure to isostatic pressure from 180 to 200 MPa for 2-3 minutes, after which the samples are dried.

Cutting a split porcine dermal graft without a 1.0 mm thick epidermal layer is selected to ensure that only the dermal layer of the skin is used.

The choice of the size of the transplant squares 3×3 cm is due to the technical characteristics of the apparatus for cutting the skin (electric dermatome) and the convenience of using it as a surgical implant for closing wound defects of the skin.

The time and magnitude of exposure to ultrasound and isostatic pressure are due to experimental studies of the evaluation and analysis of the relationship between the time and magnitude of ultrasound and isostatic pressure and the 1.0 mm thick porcine skin dermis, which allows you to remove the cellular elements of the dermis without destroying collagen and elastin fibers, thus preserving the matrix ADM (scaffold).

The invention is illustrated by drawings, where in Fig. 1 is a schematic representation of an isostatic pressure treatment plant, FIG. 2 - obtained porcine skin dermis.

To carry out the processing of pig skin to obtain a cellular dermal matrix, installations for creating isostatic pressure up to 200 MPa were manufactured and tested. The installation works as follows. Pressed samples 1 are placed in the working chamber of the multiplier cylinder 3, which is filled with the working fluid 2, closed by the piston 4 and placed in the autoclave 5 and closed with a retaining ring 6. The unit is equipped with a pressure gauge 7.

Thus, in the method for obtaining an acellular dermal matrix, a combined approach was used, including the stages of graft processing: physical - exposure to ultrasound, chemical treatment - in a saline solution (NaCl) followed by exposure to excess isostatic pressure.

The inventive method for obtaining acellular dermal matrix is implemented as follows:

Step 1. A split porcine dermal graft without a 1.0 mm thick epidermal layer is cut with an electric dermatome.

Step 2. Rinse thoroughly in distilled water.

Step 3. Pig dermis 1 mm thick is cut into equal squares 3 × 3 cm in size.

Step 4. Pig dermis 1 mm thick, 3 × 3 cm in size (sample) is kept in a 0.9% sodium chloride solution. Within 8-10 minutes

Step 5 The samples are subjected to isostatic pressure from 180 to 200 MPa for 2-3 minutes to ensure the compaction of the samples.

Step 6 Samples are freeze-dried.

Thus, the proposed method for obtaining an acellular dermal matrix in comparison with other known approaches provides the following advantages:

• application of an ultrasound source,

• combined approach (ultrasound + hypertonic solution),

• compact installation,

• availability,

• the process of exposure to isostatic pressure (pressing) can be organized in any place equipped with an air line with a pressure of 0.6 MPa for the operation of a hydraulic pump. A hand pump can be used, in which case no air is required.

• obtaining an acellular dermal matrix without antigenic properties.

The conducted experiments on exposure to ultrasound showed a high degree of purification of the material from antigens while maintaining the protein framework of the dermis, i.e. obtaining the target properties of the acellular dermal matrix. In the first series of experiments of the combined approach, a maximum ultrasound power of 900 watts was used and the time of processing samples at a constant power varied from one to ten minutes. Results of the second series of experiments:

The use of ultrasound (US) leads to varying degrees of destruction of cells of intradermal structures (blood vessels, glands, hair, nerves). The degree of destruction does not correlate in any way with the time of exposure to ultrasound and is determined by the characteristics of the skin sample itself (thickness, presence of appendages). A sample of the acellular dermis is shown in Fig. 2. - dermis of pig skin. Collagen fibres. Absence of other structural components of the dermis. Stained with hematoxylin and eosin. Magnification 100.

The claimed method for obtaining an acellular dermal matrix can be widely used in combustiology, general surgery, plastic surgery, urology, maxillofacial surgery, bariatric surgery. The method is intended for specialists who have the skills of reconstructive interventions on the skin, pelvic floor, rectocele, hernioplasty, i.e. specialists of various surgical profiles of departments of health care facilities of the city, regional and federal levels.

The claimed method for obtaining an acellular dermal matrix ensures the achievement of a new technological medical result in solving the problem, in increasing the effectiveness of treatment, while being universal, easy to use and accessible to a wide range of surgeons.

The proposed method is illustrated by an example implementation.

Example.

The sample (pig skin 1 mm thick) is placed in a glass beaker with a 0.9% sodium chloride solution. Three beakers with samples are mounted on a grid placed in an ultrasonic bath at a given distance from its radiating surface. The bath is filled with water in such a way that the beakers with the samples are immersed in water to a third of their height and the samples are kept for 8-10 minutes. After processing, the samples are taken out and placed in an isostatic pressure unit to ensure compaction.

Pressed samples 1 are placed in the working chamber of the cylinder 3 of the multiplier, which is filled with the working fluid 2, closed by the piston 4 and placed in the autoclave 5 and closed with the retaining ring 6. The unit is equipped with a pressure gauge 7. When the pressure reaches 100-200 MPa, the pressing process is considered complete. Next, the samples are removed and placed in freeze-drying. Process completed.

Claims (1)

A method for obtaining an acellular dermal matrix, which includes cutting a split porcine dermal graft without an epidermal layer 1.0 mm thick, washing it in distilled water and cutting it into equal squares 3 × 3 cm in size, followed by keeping it in a sodium chloride solution of 0.9% for 8-10 minutes, the resulting samples are subjected to ultrasonic treatment with a power of 900 watts for 10-15 seconds and subsequent exposure to isostatic pressure up to 180-200 MPa for 2-3 minutes, after which the samples are dried.

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