Combined graft of dermal matrix with mesenchymal multipotent stromal cells, method for preparing it and method for wound healing using it

FIELD: medicine.

SUBSTANCE: combined graft (CG) represents a dermal matrix (DM) prepared of a donor layer with self-specific multipotent mesenchymal stromal cells (MMSC) colonised on the surface thereof to the number of not less than 750-1500 thousand per 1 cm2. The group of inventions also refers to a method for preparing the combined graft involving bone marrow sampling from the patient, preparing a MMSC culture, applying a MMSC suspension in the concentration of not less than 1 million/ml on the DM surface at 1 million MMSC per 5 cm2 of the DM.

EFFECT: skin and soft tissue wound healing by accelerating regeneration processes.

11 cl

 

The invention relates to medicine and biotechnology and can be used to treat a wide traumatic defects in surgery, traumatology and combustology.

The main method of treatment of extensive traumatic defects is the primary surgical treatment (PHO) wound with subsequent delayed skin-plastic surgery for closure of the skin defect [Alekseev A.A., Sutugin A.M., Kuznetsov V.A. Modern strategy and tactics of treatment of burns, thermal burns and extensive purulent wounds. Postgraduate education at the present stage. M. - 2000. - c. 277-284].

For wound closure used autodermoplasty (ADP) split flap. However, the results of the ADP in purulent surgery remain unsatisfactory, due to incomplete regeneration of the skin and scar formation [Gubin M.A., Aktov V.N., Lakatos C.O Early reconstructive plastic surgery in the treatment of patients after thermal injury of the head and neck: the Man and his health. - Kursk. - 2000. - Vol, 3. - C-84]. We emphasize that, when ADP is required to get a skin flap from the intact surface of the patient's body, which creates additional difficulties.

Development of cellular technologies opens new possibilities in the treatment of wounds using a variety of cells, including mesenchymal multipotent stromal cells (MSC). In clinical PRA is tick use of allogeneic or autologous cells. The type of cells used is determined by the alleged tactics of treatment, due to the nature of the pathological process. To stimulate regeneration at the expense of the resident progenetor cells used bioactive factors secreted from autologous or allogeneic cells when destruction in situ or in vitro. In particular, the effectiveness of this method was demonstrated in the treatment of borderline burns when you saved the source of regeneration of the skin, to stimulate that use alleyball.

When the deficit resident progenetor cells perform their autotransplantation of intact areas, for example, bone marrow (KM).

The closest technical solution adopted for the prototype, is a method of treating Ozegovich RAS IIIa degree, developed in the Institute of JV to them. N.V. Sklifosovsky, which consists in imposing on burn wound dressings containing alleyball [Patent No. 2320349 "Method of treatment Ozegovich wounds" from 27.03.08]. To speed up the process of regeneration stimulated preserved in the derivatives of the skin progenetor cells. Stimulation of cells perform appliqué on the biological wound dressings consisting of perforated hydrophobic silicone polymer base, coated with human collagen type I, attached to allofibroblasts cell line M-22, polucen the mi at the Institute of poliomyelitis and viral encephalitides. M.P. Chumakov. The dressing is made in terms of cultural block. On button located on the bottom of the Petri dishes hydrophobic transparent perforated silicone film "Carbosil-P coated with human collagen type I, transferred allogeneic diploid fibroblast cell line M-22 passage No. 15-25. The planting density is 5.0 and 7.5×104cells on 1 cm2. Cultivation was performed at 37°C in atmosphere containing 5% CO2and 100% humidity. As a culture medium use medium Needle MEM containing 15% serum of cow embryos and α-glutamine. Within 18-24 hours on the surface of the polymer base fibroblasts form a monolayer. On the organosilicon film "Carbosil-P" transfer of alloparental on the wound.

Transplantation alloparental, according to the method prototype, is from time of receipt until 6 days after injury. Optimal is the imposition of this armband for admission of the patient to the admitting office. Before transplantation conduct thorough washing of burn wounds, remove from the wound surface of a foreign body, desquamated epidermis, the imposition of fibrin. Treat the wound with an antiseptic solution, burns IIIa extent impose a bandage with allofibroblasts that capture the wet-drying gauze bandage.

JV is the FDS prototype treatment of burn wounds has the following disadvantages: limited indications for use only when the edge burns IIIa degree; a small number allowable on 1 cm2headbands and short life expectancy in the wound (no more than 2-3 days), which does not allow efficient enough to stimulate regenerative processes in deep wounds where there is no resident stem cells.

Autologous cells in the wound retain their viability for a long time, being able to replace the resident stem cells and stimulating regeneration. DM is the optimal biocompatible carrier, in comparison with the silicon film.

Thus, for the treatment of patients with extensive traumatic soft tissue defects there is a need to develop technologies create a new composite graft with autologous MSC applicable at different stages of treatment.

The technical result is achieved by the use of technology for combined transplant: dermal matrix with autologous MSC providing wound healing of skin and soft tissues, due to the acceleration of regenerative processes.

The method of obtaining a combined transplant (KT)

The technology of CT involves several steps and is carried out in conditions of cultural block to ensuring sterility and biological security of the obtained graft.

1 this is the Receipt of the dermal matrix

Dermal matrix made of split human skin, harvested from a donor cadaver, thickness up to 1 mm Fence of the skin flap is made in the operating room using a disk dermatome in compliance with the rules of asepsis and antisepsis.

The manufacturer of the dermal matrix is divided into 3 phases: separation of dermis and epidermis, devitalizate dermis, ensuring the biocompatibility of the matrix. DM is a plate of white or light beige color, consisting of collagen and elastin fibers without cells and cellular structures.

stage 2. Obtaining autologous MSC bone marrow (KM).

The patient under local anesthesia or General anesthesia in the operating conditions in compliance with the rules of asepsis and antisepsis of the wing of the Ilium prepare KM. The trocar to pierce the soft tissue, the dotted line in the bone. The trocar attach a syringe with a volume of 20.0 ml, containing 5,000 IU of heparin, which gather Malaspina. Prepare 20-40 ml KM. Not later than 2 hours Malaspina take in the culture unit. In the culture unit of KM on the gradient density Linfoot p=1.077 distinguish nucleated cells. Cells are placed in culture flasks to obtain pesticidesin cells. Adherent to plastic cells cultured 3-4 passage in the environment DMEM with the addition of 10% bovine fetal the first serum to obtain culture MMSC. After the third passage in suspension MSC determine the phenotype and the number of damaged cells. The cellular material suitable for the manufacture of CT if he biobazaar contains 80-95% intact cells and more than 80% of cells with the phenotype CD105+CD90+CD45-CD34-. In case of recognition of culture MMSC suitable for the manufacture of CT, from preparing the suspension in the culture medium of DMEM containing 10% fetal bovine serum to cell concentration not less than 1 million/ml For the manufacture of CT use 1 million MMSC 5 cm2EBM.

stage 3. To create a combined transplant DM with MMSC KM.

In terms of the culture unit and ensuring sterility of the transplant DM area of 25-30 cm2placed in a sterile Petri dish, and the appropriate size. In a Petri dish on DM pipette cause cell suspension (MSC) with a concentration of 1 million cells/ml, based on 1 million MMSC 5 cm2DM (for example, 5 ml of suspension containing at least 5-6 million MMSC). For colonization DM MMSC the graft is placed in CO2-incubator at 37°C and 5% concentrations of CO2in the air for 24 hours. Cellular component CT appreciate the vital staining with a fluorescent dye. Preserving sterility CT dropper put sterile fluorescent dye, krasivaya the living cells without damaging them. Using a fluorescent microscope, count the number of adherent cells. CT must contain on its surface 750-1500 th cells on 1 cm2.

Description CT.

Combined transplant is a dermal matrix derived from the donor layer of the dermis by removing cells, fixed to its surface autologous MSC at least 750-1500 thousand on 1 cm2. In appearance CT is a skin plate area of 25-30 cm2thickness of 0.4-0.6 mm In microscopy on the surface of the DM visualized attached cells.

Methods of use.

The method is used in the treatment of wounds requiring plasticity in the deferred period.

Contraindications to the use of the method are:

- blood borne infections

- malignant blood diseases

- reducing the compliance of the patient (dementia and other mental illnesses)

- purulent-septic complications

- fractures of the pelvis.

To calculate required for deferred plastics in the number of CT in PHO, assess the area of the wound. CT is produced in sufficient quantity to complete wound closure.

In the dressing room under sterile conditions in compliance with the rules of asepsis and antisepsis, the patient removes the bandage from the wound. The wound triple handle sterile gauze t is Nanami, soaked in 5% aqueous solution of chlorhexidine, get wet sterile dry gauze pad. To prevent contamination of the wound, the skin around the wound three-treated with 0.5% alcoholic chlorhexidine, avoiding the wound. The wound sterile CT in sterile tray perforined across the surface with a scalpel with a frequency of 1 perforation 5 mm 1 cm2to create ways of outflow of wound, lay on the bottom of the wound cells down model on the wound so that the edges of the CT scan performed at the wound edges is not more than 0.1 to 0.3 see then fixed with a sterile gauze bandage.

Comparative clinical and experimental confirmation of the effectiveness of CT.

In clinical practice we have used DM as a temporary biological coating 7 patients with extensive traumatic defects of the skin. When using DM noted the following positive aspects:

- does not require frequent changes, which avoids daily dressings, subjectively, patients noted decreased pain syndrome;

under the DM creates conducive to healing the microenvironment, the matrix performs a barrier function and prevents contamination of the wound;

- after removing the bottom of the wound covered with bright fine granulations, which prevents the formation of hypertrophic shall ubzow.

But was not observed objective difference in terms of wound healing when using DM and in the treatment of the traditional method.

To confirm the effectiveness of CT was conducted series of experiments on mice.

An experimental study was conducted on 27 outbred white mice. All animals were of the same age (3-5) months. The weight of the specimens was 25-30, Mice were kept on a standard diet of the vivarium. After application of the wound each mouse was kept in a separate cage.

Animals were divided into 3 groups depending on the applied methods of treatment. Anesthetized mice liberated from wool area of the back with scissors was dissected fragment of the skin with a diameter of about 10 mm to fascia. To prevent contraction of the wound edges were added to polyvinyl chloride (PVC) ring with a diameter of 12 mm, Thus, the area of the wound defect was approximately 2% of the body surface.

In group 1 at the bottom of the wound was Packed with sterile gauze swab, moistened with a 5% aqueous solution of chlorhexidine.

In group 2 at the bottom of the wounds were laid DM.

In group 3 at the bottom of the wounds were laid DM attached to its surface autologous MSC (CT).

Headbands to improve contact with the wound was added separate seams, covered with a gauze pad that was attached to the PVC ring.

Conclusion animals from the experiment was performed 3, 7, 14 days. The wound from the surface is scoy was dissected, were evaluated by macroscopic and histological method (degree of maturation of granulation tissue, full epithelialization of the wound surface).

Macroscopic evaluation

At the conclusion of the experiment in all individuals of a festering wound was not observed. Animals of the first group healing no, the bottom of the wound is covered with granulations. In the second group the bottom of the wound is covered with a scab, tightly fixed on the wound. Animals of the third group showed complete healing of the wound.

Histological evaluation

Painted hematoxylin-eosin and by the method of van Gison histological specimens were examined at magnification ×100.

In the 1st group using sterile gauze swab, moistened with a 5% aqueous solution of chlorhexidine on 3 days in the bottom of the wound observed necrotic lesions, vascular underlying tissues expanded, full-blooded, with the phenomena of leukostasis and leukodepleted, marginal epithelization poorly expressed. On day 7, on the surface of the wound formed a massive scab, there has been a proliferation of regional epithelial layer with partial ingrowth of scab in the bottom of the wound cell-tissue debris, is formed on the periphery of immature granulation tissue. For 14 days the bottom of the wound filled with immature granulation tissue, epithelialization of the wound surface is incomplete. In granulation tissue, a large number of polim is FNO-nuclear leukocytes (PAL), macrophages and thin-walled vessels.

Thus, to 14 days in this group complete healing of the wound defect is not observed. Remains severe inflammatory infiltration, formed granulation tissue immature. Continues the healing of wounds due to epithelialization.

In group 2 to 3 day DM completely covers the wound and is converted into a homogeneous oxytelinae mass. DM infiltrated by neutrophils at the site of contact with the wound. In the bottom of the wound weak inflammatory infiltration of neutrophils and macrophages, appear isolated fibroblasts, the newly formed vessels are absent. There has been little expansion of the regional epithelial layer with hypertrophy of keratinocytes. On day 7 DM turns into a scab. The bottom of the wound filled with immature granulation tissue with a large number of fibroblasts and moderate amount of PAL and macrophages. There are a large number of newly-formed thin-walled vessels. Small growth boundary epithelial layer of scab. 14 day marked increase in epithelial layer of scab. In granulation tissue remains a large number of thin-walled vessels. There is a slight infiltration of PAL and macrophages, is dominated by fibroblasts.

Thus, the complete healing of the wound defect is not observed. is formed granulation tissue is moderately Mature. Continues the healing of wounds due to severe epithelialization.

In group 3 on 3 day CT completely covers the wound becomes homogeneous oxytelinae mass. CT infiltrated by neutrophils at the site of contact with the wound, it begins to grow regional epithelium at the wound there is a slight inflammatory infiltration, there are newly-formed thin-walled vessels, there is an active migration of fibroblasts. On day 7, significant growth in regenerating epithelial layer under CT, turned into a scab, granulation tissue is moderately Mature with a large number of full-blooded vertically oriented thin-walled vessels of cellular elements is dominated by fibroblasts, there is a moderate amount of leukocytes. 14 day wound covering is missing, has been full epithelialization of the wound surface, the epithelium is only loosely attached to the underlying tissue on its surface begins to form micro, in granulation tissue reduces the number of cellular elements and thin-walled vessels, although they are still vertically oriented, saved a small amount of PAL, the thickness of the formed hem is much less than in the first group.

Thus, when using CT observed acceleration of the maturation of granulation tissue than the Oia with other groups. Inflammatory infiltration of the weak. There is a complete closure of the defect of the newly formed epithelium.

In all groups the healing of the wound defect was due to epithelialization. The best result was observed in the third group, where all mice were epithelialization, which indicates a higher rate of regeneration compared to 1 and 2 groups.

The use of CT allows to stimulate the regeneration of wounds and shortens the period of epithelialization. When using CT were observed reduction phase of inflammation, however, was accelerated maturation of granulation tissue, which indicates the allocation of growth factors, autologous MSC contained in CT. Thus, the reduction phase of inflammation and excretion of growth factors, autologous cells allows to accelerate epithelialization.

The proposed method of obtaining and using CT promising for application in clinical practice, in particular for the recovery of the skin in patients with extensive traumatic wounds with soft tissue defect.

1. Composite graft for repair of the skin, consisting of dermal matrix (DM)obtained from the donor layer of the dermis, with colonized on the surface of autologous mesenchymal multipotent stromal cells is (MMSC), where MMSC contained on the surface of the composite graft in an amount not less 750-1500 th cells on 1 cm2.

2. Composite graft according to claim 1, in which the thickness DM is up to 1 mm.

3. Composite graft according to claim 1, which is designed to treat patients with extensive traumatic soft tissue defects.

4. A method of obtaining a composite graft according to claim 1, including the bone marrow (KM) in patients receiving culture of mesenchymal multipotent stromal cells (MSC), application of a suspension MMSC with cell concentration not less than 1 million/ml on the surface of the dermal matrix (DM) at the rate of 1 million MMSC 5 cm2DM and colonization of the dermal matrix mesenchymal multipotent stromal cells to achieve a number MMSC not less 750-1500 thousand on 1 cm2.

5. The method according to claim 4, in which for getting MMSC of KM on the gradient density Linfoot p=1,077 distinguish nucleated cells, the cells are placed in culture flasks to obtain pesticidesin cells adherent to plastic cells cultured 3-4 passage in the environment DMEM with the addition of 10% fetal bovine serum, culture MMSC, after 3-4 passage assess the suitability of crops MMSC for the manufacture of a combined transplant (KT), and in the case of prirodno and, from preparing the suspension.

6. The method according to claim 5, whereby the cellular material suitable for the manufacture of CT if it contains 80-95% intact cells and more than 80% of cells with the phenotype CD105+CD90+CD45-CD34-.

7. The method according to claim 4, whereby the suspension is prepared in culture medium, DMEM containing 10% fetal bovine serum to cell concentration not less than 1 million/ml

8. The method according to claim 4, in which use dermal matrix (DM)obtained by sampling the skin flap thickness up to 1 mm from the donor-cadaver, separation of the dermis and epidermis, the implementation of the devitalisation of the dermis with getting ready the dermal matrix.

9. Method of recovery of the skin in patients with extensive traumatic wounds with soft tissue defects, including modeling of the shape of the wound composite graft according to claim 1, obtained by the method according to claim 4, after which the combined transplant perforined, lay on the bottom of the wound surface, populated cells down and fix.

10. The method according to claim 8, in which the combined transplant perforined over the entire surface with a frequency of one perforation 5 mm 1 cm2.

11. The method according to claim 8, in which the combined transplant (KT) model on the wound so that the edges of the CT scan performed at the wound edges is not more than 0.1 to 0.3 see



 

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2 ex, 6 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical industry, in particular to the application of the lanocholesterol fraction of wool grease as a bioemulsifier for cosmetic anti-aging preparations. Application of the lanocholesterol fraction of wool grease as the bioemulsifier for cosmetic anti-aging preparations is obtained by alkaline hydrolysis of wool grease with a mixture of ethanol, sodium hydroxide, pyrogallol and water, or with a mixture of ethanol, sodium hydroxide, pyrogallol, toluene and water, taken in a specified ratio, with further separation and purification under certain conditions.

EFFECT: application of the described above fraction from wool grease contributes to long-lasting moistening and enhancement of the water-retaining ability of skin.

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