RU2526813C1 - Combined graft of dermal matrix with mesenchymal multipotent stromal cells, method for preparing it and method for wound healing using it - Google Patents

Abstract

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

Description

The invention relates to medicine and biotechnology and can be used to treat extensive traumatic defects in surgery, traumatology, and combustiology.

The main method of treating extensive traumatic defects is the primary surgical treatment (PCP) of the wound, followed by a delayed skin-plastic surgery to close the skin defect [Alekseev A.A., Svetukhin A.M., Kuznetsov V.A. The modern strategy and tactics for the treatment of burns, burn disease and extensive purulent wounds. Postgraduate education at the present stage. - M. - 2000. - c. 277-284].

Autodermoplasty (ADP) with a split flap is used to close the wound. However, the results of ADP in purulent surgery remain unsatisfactory, which is associated with incomplete skin regeneration and scar formation [Gubin MA, Ektov VN, Lakatosh K.O. Early reconstructive plastic surgery in the treatment of patients who suffered a thermal injury to the head and neck: Man and his health. - Kursk. - 2000. - Vol. 3. - p. 83-84]. We emphasize that with ADP it is required to obtain a skin flap from the intact surface of the patient’s body, which creates additional difficulties.

The development of cellular technologies opens up new possibilities for treating wounds using various cells, including mesenchymal multipotent stromal cells (MMSCs). In clinical practice, allogeneic or autologous cells are used. The type of cells used is determined by the proposed treatment tactics, due to the nature of the pathological process. To stimulate regeneration due to resident progenitor cells, biologically active factors isolated from autologous or allogeneic cells when they are destroyed in situ or in vitro are used. In particular, the effectiveness of the technique is shown in the treatment of borderline burns, when the source of skin regeneration is saved, allofibroblasts are used to stimulate it.

With a deficit of resident progenitor cells, their autotransplantation from intact regions, for example, bone marrow (CM), is performed.

The closest technical solution adopted for the prototype is a method of treating Ozhegovyh wounds IIIa degree, developed at the Scientific Research Institute of the Joint Venture named after N.V. Sklifosovsky, which consists in applying to the burn wound dressings containing allofibroblasts [Patent No. 2320349 "Method for the treatment of Burn wounds" from 03/27/08]. To accelerate the regeneration process, the progenitor cells preserved in the derivatives of the skin were stimulated. Stimulation of the cells is performed by applying a biological dressing to the wound, consisting of a perforated hydrophobic organosilicon polymer base coated with human type I collagen, with allofibroblasts of the M-22 cell line attached to it, obtained at the Institute of Poliomyelitis and Viral Encephalitis named after M.P. Chumakova. A bandage is made in a culture block. Allogenic diploid fibroblasts of cell line M-22 of passage No. 15-25 are transferred to the hydrophobic transparent perforated silicon carbide film “Carbosil-P” coated with human type I collagen located at the bottom of the Petri dish. The seeding density is 5.0-7.5 × 10 ⁴ cells per 1 cm ² . Cultivation is carried out at 37 ° C in an atmosphere containing 5% CO ₂ and 100% humidity. As the culture medium using the medium Needle MEM containing 15% serum of cow embryos and α-glutamine. Within 18-24 hours, fibroblasts form a monolayer on the surface of the polymer base. On the organosilicon film “Karbosil-P”, allofibroblasts are transferred to the wound.

Allofibroblast transplantation, according to the prototype method, is carried out in the period from the moment of receipt to 6 days after the injury. Optimal is the imposition of this dressing upon admission of the patient to the emergency room. Before transplantation, a thorough toilet of the burn wound is carried out, foreign bodies, desquamated epidermis, fibrin are removed from the wound surface. The wound is treated with an antiseptic solution, dressings with allofibroblasts, which are fixed with a moist-drying gauze bandage, are applied to IIIa degree burns.

The prototype method for the treatment of burn wounds has the following disadvantages: limited indications for use only with borderline burns of IIIa degree; a small number of allofibrobalsts per 1 cm ^{2 of} dressing and a short life expectancy in the wound (no more than 2-3 days), which does not allow stimulating regenerative processes quite effectively in conditions of deep wounds when there are no resident stem cells.

Autologous cells in a wound retain their viability for a long time, possessing the ability to replace resident stem cells and stimulate regeneration. DM is an optimal biocompatible carrier compared to an organosilicon film.

Thus, for the treatment of patients with extensive traumatic soft tissue defects, there is a need to develop a technology for creating a new combined graft with autologous MMSCs applicable at different stages of treatment.

The technical result is achieved by applying the technology of obtaining a combined transplant: a dermal matrix with autologous MMSCs, which provides healing of skin and soft tissue wounds due to the acceleration of regeneration processes.

A method of obtaining a combined transplant (CT)

The technology for obtaining CT involves several stages and is carried out in a culture block with the provision of sterility and biological safety of the resulting transplant.

Stage 1 Obtaining dermal matrix

The dermal matrix is made from split human skin harvested from a corpse donor, up to 1 mm thick. A skin flap is taken in the operating room using a disk dermatome in compliance with aseptic and antiseptic rules.

The manufacture of the dermal matrix is divided into 3 stages: the separation of the dermis and epidermis, dermalization of the 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.

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

In a patient under local anesthesia or anesthesia in the operating room, observing the rules of asepsis and antiseptics, the ilium is harvested from the iliac wing. The soft tissue is pierced with a trocar, the bone is punctured. A 20.0 ml syringe containing 5000 IU of heparin is attached to the trocar, with which a myeloaspirate is collected. Harvest 20-40 ml of KM. Not later than 2 hours, the myeloaspirate is delivered to the culture block. In the culture block from CM on the density gradient Lympholite p = 1.077, nucleated cells are isolated. Cells are placed in culture bottles to obtain plastic adhesive cells. Cells attached to the plastic are cultured 3-4 passages in DMEM medium with the addition of 10% bovine fetal serum to obtain MMSC culture. After the third passage in the MMSC suspension, the phenotype and the number of damaged cells are determined. Cellular material is suitable for the production of CT if it is biosafety, contains 80-95% of intact cells and more than 80% of cells with the phenotype CD105 ⁺ CD90 ⁺ CD45 ^- CD34 ^- . If the MMSC culture is recognized as suitable for the production of CT, a suspension is prepared from it in a DMEM culture medium containing 10% fetal bovine serum with a cell concentration of at least 1 million / ml. For the production of CT, 1 million MMSCs per 5 cm ² DM are used.

3 stage. Creation of a combined DM transplant with MMSC KM.

Under the conditions of the culture block, ensuring sterility, the DM transplant with an area of 25-30 cm ^{2 is} placed in a sterile Petri dish of the appropriate size. A cell suspension (MMSC) with a cell concentration of 1 million / ml is applied to a Petri dish on a DM with a pipette, based on 1 million MMSCs per 5 cm ² DM (for example, 5 ml of a suspension containing at least 5-6 million MMSCs). For the colonization of DM MMSC, the transplant is placed in a CO ₂ incubator at a temperature of 37 ° C and 5% concentration of CO ₂ in air for 24 hours. The cellular component of CT is evaluated by vital staining with a fluorescent dye. While maintaining sterility, a sterile fluorescent dye is applied to the CT using a pipette, which stains living cells without damaging them. Using a fluorescence microscope, the number of adherent cells is counted. CT should contain on its surface 750-1500 thousand cells per 1 cm ² .

Description of CT.

A combined graft is a dermal matrix obtained from the donor layer of the dermis by removing cells with autologous MMSCs fixed to its surface in an amount of at least 750-1500 thousand per 1 cm ² . In appearance, the CT is a skin plate with an area of 25-30 cm ² , a thickness of 0.4-0.6 mm. By microscopy, attached cells are visualized on the surface of the DM.

The method of use.

The method is used in the treatment of wounds requiring plastic surgery in the delayed period.

Contraindications to the use of the method are:

- blood contact infections

- malignant blood diseases

- reduction in patient compliance (dementia and other mental illnesses)

purulent-septic complications

- pelvic fractures.

For the calculation required for delayed plastic surgery, the number of CT scans for PEC, the area of the wound is estimated. CT is made in an amount sufficient to completely close the wound.

In a dressing room under sterile conditions, observing the rules of asepsis and antiseptic, the patient removes the bandage from the wound. The wound is treated three times with sterile gauze swabs moistened with a 5% aqueous solution of chlorhexidine, soaked with a sterile dry gauze swab. In order to prevent wound contamination, the skin around the wound is treated three times with a 0.5% alcohol solution of chlorhexidine, avoiding its entry into the wound. After treatment of the wound, a sterile CT in a sterile tray is perforated over the entire surface with a scalpel with a frequency of 1 perforation of 5 mm per 1 cm ² , to create paths for the outflow of wound, put the cells down on the bottom of the wound, model along the wound so that the edges of the CT protrude beyond the edges wounds no more than 0.1-0.3 cm. Then fix with a sterile gauze bandage.

Comparative clinical and experimental confirmation of the effectiveness of CT.

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

- does not require frequent changes, which avoids daily dressings, subjectively, patients noted a decrease in pain;

- under DM, a microenvironment favorable for healing is created, the matrix performs a barrier function and prevents wound contamination;

- after removal, the bottom of the wound is covered with bright fine-grained granulations, which prevents the formation of hypertrophic scars.

However, there was no objective difference in the timing of wound healing when using DM and in the treatment by the traditional method.

To confirm the effectiveness of CT, a series of experiments on mice was performed.

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

The animals were divided into 3 groups depending on the treatment method used. Under anesthesia, a skin fragment about 10 mm in diameter to the fascia was dissected with scissors on mice freed from the hair of the back. To prevent wound contraction, the edges were sutured to a polyvinyl chloride (PVC) ring with a diameter of 12 mm. Thus, the area of the wound defect was about 2% of the body surface.

In group 1, a sterile gauze swab dipped in a 5% aqueous solution of chlorhexidine was placed on the bottom of the wound.

In group 2, DM was placed at the bottom of the wound.

In group 3, DM was placed at the bottom of the wound with autologous MMSC (CT) attached to its surface.

Dressings to improve contact with the wound were sutured with separate sutures, covered with a gauze swab, which was sutured to a PVC ring.

The animals were withdrawn from the experiment on days 3, 7, and 14. The wound with the dressing was dissected, evaluated by the macroscopic and histological method (degree of granulation tissue maturity, completeness of epithelization of the wound surface).

Macroscopic evaluation

When withdrawing from the experiment in all individuals suppuration in the wound area was not observed. In animals of the first group there is no healing; 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. In animals of the third group, complete wound healing was noted.

Histological evaluation

Histological specimens stained with hemotoxylin-eosin and the Van Gieson method were examined at a magnification of × 100.

In the 1st group, when using a sterile gauze swab moistened with a 5% aqueous solution of chlorhexidine, necrotic changes are observed at the bottom of the wound for 3 days, the vessels of the underlying tissues are dilated, full-blooded, with leukostasis and leukodiadedesis, marginal epithelization is weakly expressed. On day 7, a massive scab forms on the surface of the wound, an overgrowth of the marginal epithelial layer with partial growth under the scab is observed, cell-tissue detritus is found in the bottom of the wound, immature granulation tissue is formed on the periphery. By 14 days, the entire bottom of the wound is filled with immature granulation tissue, epithelization of the wound surface is incomplete. The granulation tissue contains a large number of polymorphonuclear leukocytes (PNL), macrophages and thin-walled vessels.

Thus, by 14 days in this group, complete healing of the wound defect was not observed. Pronounced inflammatory infiltration persists, the formed granulation tissue is immature. Wound healing continues due to marginal epithelization.

In group 2, on the 3rd day, DM completely closes the wound and turns into a homogeneous oxyphilic mass. DM infiltrated by neutrophils at the site of contact with the wound. In the bottom of the wound there is a weak inflammatory infiltration by neutrophils and macrophages, isolated fibroblasts appear, and there are no newly formed vessels. Weak growth of the marginal epithelial layer with hypertrophy of keratinocytes is observed. On the 7th day, the DM turns into a scab. The bottom of the wound is filled with immature granulation tissue with a large number of fibroblasts and a moderate amount of PMNs and macrophages. There is a large number of newly formed thin-walled vessels. Slight growth of the marginal epithelial layer under the scab is noted. On day 14, marked growth of the epithelial layer under the scab is noted. A large number of thin-walled vessels are preserved in granulation tissue. Weak infiltration of PNL and macrophages is noted, fibroblasts predominate.

Thus, complete healing of the wound defect is not observed. The formed granulation tissue is moderately mature. Wound healing continues due to pronounced marginal epithelization.

In group 3, on day 3, CT completely closes the wound, turns into a homogeneous oxyphilic mass. CT is infiltrated by neutrophils at the site of contact with the wound, marginal epithelium begins to grow under it, weak inflammatory infiltration is noted in the wound, newly formed thin-walled vessels appear, active migration of fibroblasts is observed. On day 7, a significant growth of the regenerative epithelial layer under CT was noted, which turned into a scab, the granulation tissue is moderately mature with a large number of full-blooded vertically oriented thin-walled vessels, fibroblasts predominate from the cellular elements, a moderate number of leukocytes is present. There is no wound coverage on day 14, there was complete epithelization of the wound surface, the epithelium is loosely attached to the underlying tissue, a microrelief begins to form on its surface, the number of cellular elements and thin-walled vessels in the granulation tissue decreases, although they are still vertically oriented, a small number of PNLs remain, the thickness of the forming scar is much less than in the first group.

Thus, when using CT, accelerated maturation of granulation tissue is observed in comparison with other groups. Inflammatory infiltration is weak. Complete closure of the defect by the newly formed epithelium is observed.

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

The use of CT allows you to stimulate the regeneration of wounds and reduces the time of epithelization. When using CT, a reduction in the phase of inflammation was noted, however, the maturation of granulation tissue was accelerated, which indicates the isolation of growth factors by autologous MMSCs contained in CT. Thus, the reduction of the phase of inflammation and the release of growth factors by autologous cells allows acceleration of epithelization.

The proposed method for the production and use of CT is promising for use in clinical practice, in particular for the restoration of the skin in patients with extensive traumatic wounds with a soft tissue defect.

Claims (11)

1. A combined graft for restoration of the skin, consisting of a dermal matrix (DM), obtained from the donor layer of the dermis, with autologous mesenchymal multipotent stromal cells (MMSC) colonized on its surface, where MMSCs are contained on the surface of the combined graft in an amount of at least 750- 1500 thousand cells per 1 cm ² . 2. The combined graft according to claim 1, in which the thickness of the DM is up to 1 mm 3. The combined transplant according to claim 1, which is intended for the treatment of patients with extensive traumatic defects of soft tissues. 4. The method of obtaining a combined transplant according to claim 1, including collecting bone marrow (CM) from a patient, obtaining a culture of mesenchymal multipotent stromal cells (MMSC), applying a suspension of MMSC with a cell concentration of at least 1 million / ml on the surface of the dermal matrix (DM) based on 1 million MMSCs per 5 cm ² DM and colonization of the dermal matrix with mesenchymal multipotent stromal cells until the number of MMSCs is at least 750-1500 thousand per 1 cm ² . 5. The method according to claim 4, in which to obtain MMSCs from CM on a density gradient Lympholite p = 1.077, nucleated cells are isolated, cells are placed in culture bottles to obtain plastic adhesive cells, cells attached to the plastic are cultured 3-4 passages in DMEM medium with addition 10% bovine fetal serum, to obtain the MMSC culture, after 3-4 passage, the suitability of MMSC cultures for the manufacture of a combined graft (CT) is assessed, and if appropriate, a suspension is prepared from it. 6. The method according to claim 5, according to which the cellular material is suitable for the manufacture of CT if it contains 80-95% of intact cells and more than 80% of cells with the phenotype CD105 ⁺ CD90 ⁺ CD45 ^- CD34 ^- . 7. The method according to claim 4, according to which the suspension is prepared in a DMEM culture medium containing 10% fetal bovine serum with a cell concentration of at least 1 million / ml. 8. The method according to claim 4, in which a dermal matrix (DM) is used, obtained by collecting a skin flap up to 1 mm thick from the donor corpse, separating the dermis and epidermis, performing dermal dermalization to produce a finished dermal matrix. 9. A method of restoring the skin in patients with extensive traumatic wounds with a soft tissue defect, including modeling according to the wound shape of the combined graft according to claim 1, obtained by the method according to claim 4, after which the combined graft is perforated, laid on the bottom of the wound with a surface inhabited by cells down and fix. 10. The method of claim 8, in which the combined graft is perforated over the entire surface with a frequency of one perforation of 5 mm per 1 cm ² . 11. The method of claim 8, in which the combined graft (CT) is modeled on the wound so that the edges of the CT protrude beyond the edges of the wound by no more than 0.1-0.3 cm.