RU2768156C1 - Method for preparing porcine dermis for creating acellular dermal matrix in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine, namely to regenerative medicine, plastic surgery. On the side surface of a pig at age of 3.5–6 months, section of 20–25 × 60–70 cm is marked. Single sample of a full-thickness skin flap is cut. Sample is uniformly placed on the roller; the flap is stretched with Kocher clamps. Epidermal layer is removed using a dermatome. Then, using a disk knife with diameter of 100 mm with the condition of its perpendicular position relative to the flap surface, 1–3 derma samples are cut out for a matrix with size of 10±0.5 × 20 cm and thickness of 0.7 mm.

EFFECT: method enables to obtain samples without epidermis, with precisely specified thickness and dimensions, which provides further production of high-quality acellular dermal matrix (ADM) with optimal characteristics for use as a fixing material in reconstructive plastic surgery.

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Description

The present invention relates to experimental medicine, namely to regenerative medicine, and can be used to create a porcine acellular dermal matrix (ADM) as a fixing material in reconstructive plastic surgery for breast cancer.

Breast cancer is one of the most common oncological diseases of the female population, occupying 20.7% in the structure of the incidence of malignant neoplasms in Russia [Kaprin A.D., Starinsky V.V., Petrova G.V. Malignant neoplasms in Russia in 2013 (morbidity and mortality) M.: MNIOI im. P.A. Herzen branch of the Federal State Budgetary Institution "NMIRC" of the Ministry of Health of Russia, 2015. P. 250].

Reconstructive plastic surgery using a cell-free fixing biomatrix will solve the problem of soft tissue deficiency after mastectomy. The use of innovative low-cost biomatrices will make it possible to preserve the pectoralis major muscle and its function, which will improve the quality of life of patients and accelerate social rehabilitation [Ermoshchenkova M.V. et al. The use of biological and synthetic materials in reconstructive plastic surgery in patients with breast cancer //Research'n Practical Medicine Journal. - 2017. - Vol. 4. - No. one].

To perform mammoplasty of only one breast, the required matrix size is 10 × 20 cm, therefore, in surgical practice, commercially available matrices have to be stitched together, which reduces their mechanical strength and, accordingly, the clinical effectiveness of their use [Lanier S.T. et al. The effect of acellular dermal matrix use on complication rates in tissue expander/implant breast reconstruction //Annals of plastic surgery. - 2010. - T. 64. - no. 5. - C. 674-678]. In addition, preoperative preparation of a matrix of the required size can lead to bacterial contamination of materials.

The result of the project will be a cell-free biomatrix of the required size, which is a perforated flap of porcine dermis treated with a detergent-enzymatic method, suitable for implantation during experimental restorative plasty in order to firmly fix the model prosthesis. Thus, to achieve this goal, it is necessary to develop a technique for sampling the dermis with a flap of at least 10 × 20 cm in size and 0.7 mm thick.

Currently, protocols have been developed for taking donor skin using only flaps limited in size. To create a dermal matrix necessary for use in reconstructive plastic surgery, a larger size of the dermal matrix is required [Glasberg S.V., Light D. AlloDerm and Strattice in breast reconstruction: a comparison and techniques for optimizing outcomes //Plastic and reconstructive surgery. - 2012. - T. 129. - No. 6. - C. 1223-1233.; Macadam S.A., Lennox P.A. Acellular dermal matrices: economic considerations in reconstructive and aesthetic breast surgery //Clinics in plastic surgery. - 2012. - T. 39. - no. 2. - C. 187-216]

In particular, a split skin autograft (RCA) sampling method is known. RCA is a part of the tissue consisting of the epidermis and dermis. They can vary in thickness - from 0.02 cm to 0.5 cm. The method consists in incising the skin along the perimeter of the autograft form of the required size, transferring the incision plane to the plane of dissection parallel to the skin surface, directing the tip of the dissecting instrument towards the skin area to be taken at the required depth until the formation of the free edge of the skin area to be taken, after which this edge is wrapped with the inner surface outward and shifted over the skin surface with the formation of tension between the skin area to be taken and the deep layers of the skin, then the stretched layers are gradually incised, adjusting the thickness of the taken split skin autograft by the level of incision , and shift its free edge to obtain an autograft of the required shape and size [Adamyan R.T. et al. RF patent for the invention No. 2234261 C1 dated 20.08.2004 "Method of harvesting a split skin autograft"].

The disadvantages of this method is the source of raw materials - human skin, which requires careful research to exclude potential infections in donors. Such a source of raw materials limits the possibility of obtaining large flaps. Also, the disadvantage is the sampling of the dermis simultaneously with the epidermis, the removal of which from the material in order to create a dermal acellular matrix is a rather laborious procedure.

As the closest analogue, the method of skin grafting with combined skin autografts for deep skin burns was taken, where an epidermal flap 0.3 mm thick is cut from the donor site with a disk electrodermatome. Combined grafts are formed by placing an electrodermatome disk in such a way that half of its diameter is located above the previous donor wound, and the other half is above the healthy skin adjacent to the donor wound, thereby obtaining a combined graft consisting of 2 parts: half of the width is represented by the dermal component, and the other half is epidermal, and the ratio of the width of the epidermal and dermal parts is 1:1 [Loginov L.P. et al. RF patent for the invention No. 2434595 C1 dated November 27, 2011 “Method of skin grafting with combined skin autografts for deep skin burns”].

The main disadvantages of this method are the adaptation of the method to the collection of flaps with a width of no more than 4-5 cm, taking samples of different histological composition: half of the width is represented by the dermal component (devoid of the epidermis), and the other half is epidermal (covered with the epidermis).

Tasks:

- exclude the use of human skin;

- exclude the presence of the epidermis in the resulting dermis sample, thereby reducing the cost of obtaining the dermal matrix;

- improve the quality of the sample for the experiment;

- ensure uniform sample thickness (0.7 mm) throughout;

- obtain a sample of the porcine dermis with dimensions not less than 10±0.5 × 20 cm;

- expand the possibilities of obtaining the dermis to create an ADM with optimal characteristics for use as a fixing material in reconstructive plastic surgery.

The essence of the proposed invention is that in order to create an acellular dermal matrix (ADM), a section of 20-25 × 60-70 cm in size is preliminarily marked on the lateral surface of the pig, a single sample of a full-thickness skin flap is cut out, the sample is evenly placed on a roller, the flap is stretched with Kocher clamps, after which the epidermal layer is removed using a dermatome; then, using a circular knife with a diameter of 100 mm, with the condition of its perpendicular position relative to the surface of the flap, 1-3 samples of the dermis for the matrix are cut out with dimensions of 10 ± 0.5 × 20 cm and a thickness of 0.7 mm.

The technical result is to obtain 1-3 samples of the dermis with dimensions not less than 10 ± 0.5 × 20 cm and a thickness of 0.7 mm. The use of a previously removed skin flap instead of sampling the dermis directly from the lateral surface of the animal's body makes it possible to obtain samples without epidermis, with precisely specified thickness and dimensions, which ensures further obtaining of ADM with optimal characteristics, as well as at a lower cost for using it as a fixing material in reconstructive plastic surgery.

The method has been tested in laboratory conditions for 1.5 years on biological material (dermis) of experimental animals (Landrace pigs). The results fully confirmed the tasks to be solved. Dermis samples were obtained that did not contain epidermis, with a uniform sample thickness of 0.7 mm throughout, dimensions of at least 10 ± 0.5 × 20 cm.

The method is carried out as follows: under operating conditions, the animal in a state of anesthesia is laid on its side, the donor site is marked with a size of 20-25 × 60-70 cm, including the areas of the buttocks, back, lateral surface of the abdomen and neck. With the help of a syringe, injection impregnation of subcutaneous adipose tissue with saline is performed. Then, a single skin incision is made along the intended contour and with uniform sawing movements of the scalpel along the plane, a full-thickness skin flap is obtained over the entire sampling area with a single sample, including the epidermis, dermis, and mosaic areas of adipose tissue. Next, the collected skin flap is placed on a roller, stretched with Kocher clamps, creating a uniform tension over the entire area, treated with vaseline oil, and the epidermis is cut off from the donor site with a disk electrodermatome, on which a gap of 0.7 mm is preliminarily set, preparing the flap for dermal sampling. Next, using an electrodermatome with a disk knife diameter of 100 mm, dermal samples 0.7 mm thick are taken in a direction perpendicular to the direction of the flap.

Example 1. Under operating conditions, an animal aged 3.5 months in a state of anesthesia was laid on its side, a donor area 25 × 60 cm in size was marked, including the buttocks, back, lateral surface of the abdomen and neck. Using a syringe, the subcutaneous adipose tissue was impregnated with physiological saline. Then, a single skin incision was made along the intended contour and with uniform sawing movements of the scalpel along the plane, a full-thickness skin flap was obtained over the entire sampling area in a single sample, including the epidermis, dermis, and mosaic areas of adipose tissue. Next, the collected skin flap was placed on a roller, stretched with Kocher clamps, creating a uniform tension over the entire area, treated with vaseline oil, and the epidermis was cut from the donor site with a disc electrodermatome, preparing the flap for dermis sampling. Then, using an electrodermatome with a disk knife diameter of 100 mm, 3 dermal samples 0.7 mm thick were taken in the direction perpendicular to the direction of the flap, 10.5 × 20 cm in size.

Example 2. Under the conditions of the operating room, a 4-month-old animal in a state of anesthesia was laid on its side, a donor site 20 × 70 cm in size was marked, including the buttocks, back, lateral surface of the abdomen and neck. Using a syringe, the subcutaneous adipose tissue was impregnated with physiological saline. Then, a single skin incision was made along the intended contour and with uniform sawing movements of the scalpel along the plane, a full-thickness skin flap was obtained over the entire sampling area in a single sample, including the epidermis, dermis, and mosaic areas of adipose tissue. Next, the collected skin flap was placed on a roller, the flap was stretched with Kocher clamps, creating a uniform tension over the entire area, treated with vaseline oil, and the epidermis was cut from the donor site with a disk electrodedermatome, preparing the flap for dermal sampling. Then, using an electrodermatome with a disk knife diameter of 100 mm, 3 dermal samples 0.7 mm thick were taken in the direction perpendicular to the direction of the 9.5 × 20 cm flap.

Example 3. Under the conditions of the operating room, a 5-month-old animal in a state of anesthesia was laid on its side, a donor site measuring 24 × 65 cm was marked, including the buttocks, back, lateral surface of the abdomen and neck. Using a syringe, the subcutaneous adipose tissue was impregnated with physiological saline. Then, a single skin incision was made along the intended contour and with uniform sawing movements of the scalpel along the plane, a full-thickness skin flap was obtained over the entire sampling area in a single sample, including the epidermis, dermis, and mosaic areas of adipose tissue. Next, the collected skin flap was placed on a roller, the flap was stretched with Kocher clamps, creating a uniform tension over the entire area, treated with vaseline oil, and the epidermis was cut from the donor site with a disk electrodedermatome, preparing the flap for dermal sampling. After that, 3 dermal samples 0.7 mm thick were taken with an electrodermatome with a disc knife diameter of 100 mm in the direction perpendicular to the direction of the 10.2 × 20 cm flap.

Example 4. Under the conditions of the operating room, a 4.5-month-old animal in a state of anesthesia was laid on its side, a donor area 25 × 70 cm in size was marked, including the buttocks, back, lateral surface of the abdomen and neck. Using a syringe, the subcutaneous adipose tissue was impregnated with physiological saline. Then, a single skin incision was made along the intended contour and with uniform sawing movements of the scalpel along the plane, a full-thickness skin flap was obtained over the entire sampling area in a single sample, including the epidermis, dermis, and mosaic areas of adipose tissue. Next, the collected skin flap was placed on a roller, the flap was stretched with Kocher clamps, creating a uniform tension over the entire area, treated with vaseline oil, and the epidermis was cut from the donor site with a disk electrodedermatome, preparing the flap for dermal sampling. After that, 3 dermal samples 0.7 mm thick were taken with an electrodermatome with a disk knife diameter of 100 mm in the direction perpendicular to the direction of the 10 × 20 cm flap.

Claims (1)

A method for preparing the dermis of a pig for creating an acellular dermal matrix (ADM) in an experiment, which consists in preliminarily marking an area of 20-25 × 60-70 cm in size on the lateral surface of a pig, at the age of 3.5-6 months, and cutting out a single sample a full-thickness skin flap, the sample is evenly placed on the roller, the flap is stretched with Kocher clamps, after which the epidermal layer is removed using a dermatome; then, using a disc knife with a diameter of 100 mm, with the condition of its perpendicular position relative to the surface of the flap, 1-3 samples of the dermis for the matrix are cut out with dimensions of 10 ± 0.5 × 20 cm and a thickness of 0.7 mm.