RU2385744C1 - Deep wound healing technique - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to medicine, specifically to combustiology and surgery. The technique involves preliminary abrasion of a wound and application of fibroblasts on the wound surface. The fibroblasts are distributed in a gel mixture. Said gel mixture contains a serum-free nutrient medium and methylcellulose in amount 1.0 - 3.0 wt %. ^ EFFECT: method prolongs viability of the cells applied on the wound, prevents occurrence of allergic reaction. ^ 5 cl, 3 ex, 1 tbl, 5 dwg

Description

The present invention relates to medicine, in particular to combustiology and surgery, and can be used to treat deep wounds of various origins, including burns.

A known method of treating wounds using cell cultures (Patent RU2023424, publ. November 30, 1994), which consists in the fact that after surgical treatment, the wound is covered with a cell culture of human fibroblasts, and after the engraftment of fibroblasts, autodermoplasty is performed, for which a split mesh flap is used. The disadvantage of this method is that the cells of the fibroblast culture introduced into the wound quickly (within 24 hours) are destroyed and die under the action of a detachable wound.

There is also known a method involving the use of fibroblasts in a collagen gel (Bell 1983, Journal Inv.Dermatology, v.81, p.2-10), which consists in the fact that the fibroblasts are introduced into the collagen gel, and then placed on the wound. The disadvantage of this method is that such a system is also quickly (within a day) destroyed by wound enzymes.

A number of other methods involve the use of a cell culture of fibroblasts or epidermocytes grown on various types of substrates of synthetic or natural origin (Davydova et al., 2007, Cellular Technologies in Biology and Medicine, No. 4).

Our studies on the fate of cells on wounds showed that cells grown on substrates of biological and synthetic origin, when applied to the wound surface, often die within the first hours after applying them to the wound, which is explained by the insecurity of the introduced cells from the effects of enzymes and cytotoxic substances wounds.

The problem that arose in connection with this circumstance was to develop a method that allows maintaining the viability of the cells applied to the wound for at least 2-3 days, as well as creating a three-dimensional substrate for wounds in the form of a gel that is easy to manufacture and suitable for use as a medium for transferring cells to a wound.

The problem is solved in that the proposed method for the treatment of deep wounds, including applying cells of fibroblasts or epidermocytes to the wound surface, characterized in that, according to the invention, the cells are applied distributed in a gel-like mass, consisting of at least a nutrient medium for the cultivation of animal cells and methyl cellulose in an amount of 1.0-3.0 wt.%.

As a nutrient medium, you can take any nutrient medium suitable for the cultivation of animal cells, including the minimum, for example, the minimum environment of Dalbekko, IGLA, F-12, 199.

In the case of autodermoplasty, including the application of a split reticular skin flap, cells distributed in the aforementioned gel mass are applied over the retinal skin flap.

If necessary, a perforated wound dressing based on plant-derived polysaccharides, for example, Biokol type, or synthetic materials, for example, Parapran type, is additionally applied on top of the applied gel-like mass with cells distributed in it.

In a gel-like mass with cells, serum can be added at a concentration of up to 5 wt.%, Or collagen at a concentration of 0.01-0.05 wt.%, Or pectin at a concentration of 0.1-0.2 wt.%, Or gelatin in concentration of 0.1-0.2 wt.%, or a mixture of these components in any combination and ratio in the total concentration of 0.1-0.2 wt.%,

The essence of the proposed method is based on the following.

Since in gels from collagen and other animal substrates when introduced into the wound, they are destroyed by wound enzymes with subsequent rapid cell death, and allergic reactions are possible, the possibility of using non-protein substrates - various plant-derived polysaccharides: alginate, methylcellulose, to protect cells in the wound has been studied. pectin and carrageenan. In addition, a polyvinyl alcohol gel was used as a control in the experiments.

For the experiments, herbal preparations were taken, forming a gel-like mass (hereinafter gel) in aqueous solutions. The study revealed the effect of these substances on fibroblast cells in culture.

To conduct this experiment, the Dalbekko minimal medium (DMEM) manufactured by Sigma was taken as a basis, into which the studied substances and fibroblasts were added in an amount of 40 thousand cells / ml, which were cultured on Petri dishes for 48 hours.

The research results are shown in the table.

Table Substance Sodium Alginate 2% Cellulose 2% Pectin 2% Carrageenan 1% Polyvinyl alcohol Cell morphology in culture 48 hours after plating Rounded cells without adhesion to the bottom of the culture plastic The cells are flattened, normal shape, without visible changes. Rounded cells, weak growth Cluster growth No cell growth, many destroyed, dying cells

At the same time, it was shown that fibroblasts in DMEM nutrient medium without the content of fetal calf serum (Fetal Bovine Serum characterized, HyClone), which are usually added without fail to the nutrient medium, do not have growth activity. However, if an aqueous solution of methylcellulose is added to the growth medium, the fibroblasts grow and multiply in the same way as when adding growth serum.

An experiment was conducted to study the dependence of the growth and proliferative activity of fibroblasts in serum-free DMEM supplemented with methyl cellulose in various concentrations. Test concentrations of methyl cellulose were: 0.001%; 0.01%; 0.1%; 0%; 0.5%; one%; 1.5%; 2% Cells were seeded in 9.9 cm ² Petri dishes. The inoculum density of the cells was 50 thousand / cm ² . Cell counting was performed after 48 hours of cultivation using a Goryaev chamber, after staining the cells with trypan blue.

The results of the dependence of the number of fibroblast cells on the concentration of methylcellulose in serum-free DMEM are presented in the form of diagrams in Fig. 1.

Figure 2 presents a graph of the diameter of the contraction collagen disk versus the concentration of methylcellulose in the presence of fibroblasts.

As a contraction (compressible) disk used collagen gel at a concentration of 0.5 mg / ml, prepared on DMEM, in which methyl cellulose was added at a concentration of 0.3 mg / ml; 0.5 mg / ml; 0.8 mg / ml; 1 mg / ml and 1.2 mg / ml and a culture of human fibroblast cells, which were added to the finished gel at a concentration of 50 thousand / cm ² Petri dishes. Collagen gel with cells was placed in Petri dishes with a diameter of 3.5 cm. The diameter of the contraction disc was measured in centimeters. Readings were taken every 24 hours. A graph of the contraction of the collagen disc is presented in figure 2.

As can be seen from the data presented in figure 2, the degree of contraction of the disk depends on the amount of methylcellulose.

Thus, the fact of the multiplication of fibroblast cells in serum-free DMEM in the presence of methylcellulose and the fact of preventing the contraction of collagen gel in vitro was established.

To study the survival in the wound of the cells introduced into it, experiments were performed on male mice (16 individuals) of the SHK line, weighing ~ 35 g, in which, after treatment of the surgical field under general anesthesia (intraperitoneal injection of 1% propofol emulsion, the average dose of 0.5 ml per 35 g of animal weight), 2 wounds 1 × 1 cm in size were applied symmetrically to each other on the back. Fibroblasts (180-200 thousand / cm ² ) grown on a substrate made of fluororubber (80%) and methylcellulose (20%) (together with this substrate) were applied to one wound, and a gel prepared on DMEM medium containing embryo fibroblasts was applied to another wound mice carrying the gene for green fluorescent protein (GFP), in an amount of 180-200 thousand / ml and having the following composition:

a) methyl cellulose at a concentration of 15 mg / ml;

or b) collagen at a final concentration of 0.05 mg / ml, mixed with methyl cellulose at a concentration of 15 mg / ml.

To prevent evaporation, the wound was covered with a film coating of the Biokol type (TU 939370 = 001-23431671). The observation was carried out 3-5 days.

The fate of the fibroblasts placed in the wound was observed visually using an Axiovert-200 microscope, because the green protein they synthesize gives bright green fluorescence when using ultraviolet light.

The experiment showed long-term (up to 3 days) luminescence intensity of a green fluorescent protein produced by fibroblasts placed in a wound in a gel containing both methyl cellulose and collagen and methyl cellulose, which confirms the long-term preservation of fibroblast viability. At the same time, in a wound covered with a substrate with fibroblasts, on the contrary, only traces of cells were found without preserving the glow, which confirms their death under the action of the contents of the wound during the first hours.

This is confirmed by photographs (at a magnification of × 400) of prints on the glass of the wound contents 24 hours after the gel with fibroblasts based on methylcellulose and the Biokol film on top of the gel was applied to the wound (Fig. 3).

As a control, a photograph is presented (Fig. 4) (at 400 × magnification) of the prints on the glass of the wound contents 24 hours after the application of a substrate made of fluororubber (80%) and methyl cellulose (20%) with fibroblasts (180-200 thousand / cm ² ).

As can be seen from figure 3, there is a glow of many cells, which confirms the preservation of their viability, while in figure 4 only single cells shine.

Figure 5 presents a photograph (at × 400 magnification) of fibroblasts placed in a wound in a gel containing collagen and methyl cellulose, which also confirms the preservation of cell viability one day after their placement in the wound.

In limited clinical observations in patients with burn and traumatic wounds, the advantage of using fibroblasts placed in a gel from nutrient medium and methyl cellulose was also established over the use of cells placed on a substrate made of fluorinated rubber latex (80%) and methyl cellulose (20%) or collagen (0 ,one%). This was expressed, first of all, in saving time and the possibility of applying procedures for applying fibroblasts to a wound in any conditions, as well as in improving regenerative processes in a wound, which is associated with a longer duration of fibroblast cells in the gel mass and their long-term stimulating effect on reparative processes .

The use of a gel containing methyl cellulose allows you to include various substances that affect healing (polysaccharides, antiseptics, growth factors).

The method was tested in clinical conditions at the Department of thermal lesions, wounds and wound infections of the Russian Medical Academy of Postgraduate Education (RMAPO) on the basis of the burn center of the 36th City Clinical Hospital of Moscow for patients with deep burn wounds requiring surgical treatment to restore the integrity of the skin . In practice, the treatment is as follows.

Before applying a gel-like mass with cells (fibroblasts or epidermocytes), the wound is surgically treated in the amount of necrectomy and autodermoplasty is performed with a mesh skin flap.

After that, a gel-like mass with fibroblasts containing methyl cellulose is applied, which, if necessary, is coated with a film of the Parapran type (TU 9393-011 = 52708501) or Biokol (TU 939370 = 001-23431671).

EXAMPLE 1

Patient Trushkov K.V. 28 years. Diagnosis: Thermal (flame) burn of both upper limbs, posterior surface of the trunk II-III AB degree 32% of the body surface (IIIB 16%). Injury from 06/08/08. He entered the department on 06/08/08. On the 9th day, in order to reduce intoxication, surgical necrectomy was performed in the area of burn wounds on an area of 16% of the body surface. After 7 days, a tangential wound excision was performed on the same area with a simultaneous autodermoplasty with a perforated autoloskot with a 1: 4 magnification factor and application of a gel containing minimal Dalbekko medium and methylcellulose in an amount of 1.0% over which autoclaved fibroblasts were distributed, and over gel - wound dressing "Parapran".

On the ligation three days after the operation, active epithelization of transplant cells began, which was completed on the 10th day.

EXAMPLE 2

Patient Pautov K.G. 49 years old. Diagnosis: Thermal (flame) burn of the head, neck, trunk of the upper and lower extremities of I-II-III AB, degree 41% of the body surface, IIIB - up to 2% of the body surface. Injury from 06/22/08. He entered the department on 06/23/08. On the 2nd day after admission, a toilet (debrizing) of burn wounds was made with removal of epidermal scraps on the entire wound surface. A gel containing a minimum IgLA medium and methylcellulose in an amount of 1.5%, in which cultured fibroblasts were distributed, and a “Biokol” wound cover, was applied to grade IIIAB wounds on an area of 25% of the body surface. 3 days during the first dressing after removal of the wound cover on the wound surface, signs of incipient wound epithelization were noted. The wound was again sheltered by the Biokol wound cover to prevent infection and trauma, and create a moist environment.

During subsequent dressings, no signs of suppuration of the wounds were noted. Complete epithelialization of wounds was completed on the 18th day after the injury.

EXAMPLE 3

Sick Novikova T.A. 54 years old. Diagnosis: Thermal (boiling water) burn of the left thigh and lower leg of II-III AB degree 10% of the body surface (IIIB - 5% bp). Injury from 09/30/07. Admitted to the department on 10/06/07. On the 5th day after admission, a tangential excision of granulating wounds with simultaneous autodermoplasty was performed on an area of 5% of the body surface (perforated autoslap with a 1: 4 magnification factor).

A gel containing minimal DMEM medium and methyl cellulose in an amount of 2.5% was applied on top of the skin flap placed on the wound, in which cultured fibroblasts were distributed, and on top of the gel, the Biokol wound cover.

The postoperative course is smooth. The beginning of epithelialization in cells was noted on the 3rd day. Complete epithelization of cells on day 10. Discharged on the 18th day after receipt in satisfactory condition.

The examples of specific performance confirm that the proposed method is feasible. It was used to treat 23 patients with deep, mainly burn wounds of various localization, requiring surgical treatment to restore the integrity of the skin.

Claims (5)

1. A method of treating deep wounds, including pre-cleaning the wound and applying to the wound surface fibroblasts distributed in a gel mass containing a nutrient medium, characterized in that the gel mass contains serum-free nutrient medium and methyl cellulose in an amount of 1.0-3.0 wt. % 2. The method according to claim 1, characterized in that as a serum-free nutrient medium, a minimal medium is used, for example, Dalbeko, or NEEDL, or F-12, or 199. 3. The method according to claim 1, characterized in that in the case of using an autodermoplast, a gel-like mass with cells is applied to the wound surface through a mesh flap of an autodermoplast. 4. The method according to claim 1, characterized in that in addition to the gel-like mass, a non-adhesive wound dressing is applied based on plant-derived polysaccharides, for example, Biokol, or based on synthetic materials, for example, Parapran. 5. The method according to claim 1, characterized in that the gel-like mass further includes collagen in an amount of 0.01-0.005 wt.%.

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