Method for skin restoration in patients with vast wounds with soft tissue defects (versions)

FIELD: medicine.

SUBSTANCE: waste skin is incised on the periphery of the wound. The incised skin is prepared from the residual subcutaneous fat and milled to fragments having an area of 1-2 mm2. A dermal matrix made of dead donor's tissue is simulated after the wound shape. The dermal matrix (DM) is perforated to create traumatic discharge outflow tracts and laid on a wound bed so that to form a diastasis between perforation edges. Patient's waste skin fragments are introduced into the perforations in a staggered order. The wound is closed with a sterile gauze dressing. The other invention is allogenic mesenchymal multipotent stromal cell (MMSC) transplantation onto the wound bed in the form of the suspension by means of injections at a depth of 0.1-0.3 mm at a rate of 10 injections per 1 cm2. A volume of each injection makes 0.05-0.1 ml. The dermal matrix made of dead donor's tissue is simulated after the wound shape. That is perforated to form the traumatic discharge outflow tracts and laid on the wound bed. The wound is closed with the sterile gauze dressing. The effect is ensured by using the DM as an optimum biocompatible cell carrier and creating a required micromedium in the wound, stimulating the regeneration processes ensured by a combination with the homogenised autologous skin or allogenic MMSC injections into the wound.

EFFECT: inventions provide optimising skin and soft tissue wound healing ensured by stimulating the regeneration processes by means of stromal cells, including allo- and auto-MMSC and resident stem cells.

5 cl, 2 ex

 

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. -s-284].

For wound closure used autodermoplasty (ADP) split flap. However, the results of the ADP in purulent surgery are not satisfactory, 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 practice is the IR 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.

Allogeneic and autologous MSC stimulate the regeneration of wounds. There is a method of treatment of burn wounds IIIA degree developed at the research Institute of the JV to them. N.V. Sklifosovsky, which consists in imposing on burn wound dressings containing alleyball [Patent No. 2320349 "Method of treatment of burn wounds". 27.03.08; No. 2373944 "Method of treatment of burn wounds. 27.11.09]. However, the method is effective to promote healing of only the surface of thermal skin defect (for example, burn IIIA degree), which preserves progenitor cell in the derivatives of the skin (hair follicles). While deep skin defects and, consequently, the absence of resident progenitor cells method is inefficient. Transplantation in RAS is autologous MSC from other sources allows you to stimulate the regeneration of epithelial tissues [Clinical and experimental rationale for the use of allogeneic fibroblasts for the treatment of burn wounds III And class. / Zhirkova EJ Discard Sciences 2011. P.57]

The closest technical solution adopted for the prototype, is the method of obtaining and using dermal matrix (DM), combined with autologous flap [WANG Jing,SUN Shu-guang, WANG Liang-xi, WU Jing, MAO Xue-fei "Repair of scar deformities by composite grafting of acellular dermal matrix with razor thin autoskin" Journal of Southeast China National Defence Medical Science 2006-04]. The method is used to eliminate post-burn scar deformities. Produce the excision of the scar to the subcutaneous fat. At the donor site harvest autologous split skin graft in proportion to the defect. The wound sequentially stacked DM with autologous skin graft of the patient. The main disadvantage of this method is the increase in wound surface twice in the harvesting of autologous flap of skin commensurate esecanna scar. When this method is used only in the treatment of the wound, generated by the elimination of post-burn scar deformities. In addition, to eliminate scar requires an additional operation with adequate anesthesia Handbook.

Thus, for the treatment of patients with extensive traumatic soft tissue defects requires the development of a new method, applicable when the wounds of different etiology, at different stages of treatment, allowing to combine DM with stem cells, including the CE ALLO - and auto-MSC and resident dining cells.

Achievable technical result of the proposed method is to optimize the wound healing of skin and soft tissues due to the stimulation of regeneration by stem cells, including ALLO - and auto-MSC and resident dining cells. The result is achieved through the use of DM as the optimal biocompatible carrier for cells and to create the necessary microenvironment at the wound, stimulation of regeneration processes by combination with homogeneous autologous skin or by injection of allogeneic MSC in the wound area. The proposed method can be applied at different stages of treatment: during PHO wounds and delayed after preparation of the bottom of the wound.

Transplanted allogeneic cells in the wounds remain viable for 2-3 days. When the destruction of the highlights of biologically active substances, promoting angiogenesis and cell migration in the affected area, thereby reducing phase of the wound healing process and thereby accelerate the process of regeneration.

Autologous crushed leather, made in the perforation of the dermal matrix, is the source of native resident of table cells. While autologous fragments of skin can settle down on the bottom of the wound, forming Islands of epithelialization and as a source of growth factors, especially in the tenderly with extensive skin defects, when healing by second intention can take a long time.

Experimental rationale for the use of waste tissues as a source of autologous cells

To assess the possibility of using waste tissue as a source of autologous cells carried out the isolation and cultivation of cells from the flap of skin removed during primary surgical treatment ripped here-contused wound of the shoulder. After excision of the damaged tissue was placed in a medium for culturing cells DMEM with the addition of gentamicin (10:1) and kept to trypsinization 11.5 hours at a temperature of +4°C.

In a sterile box fragment of the flap was removed from the transport medium was placed in a Petri dish with a diameter of 90 mm and fragmentarily scissors into pieces of 1-3 mm3. The crushed material is washed three times with saline, and then poured the wash liquid was added 10 ml of 0.25% trypsin solution, warmed to 37°C. the Petri dish with pieces of fabric and trypsin were placed in a thermostat with a temperature of 37°C for 30 minutes.

After incubation, the mixture was pietravalle to separate pieces of fabric of individual cells and small conglomerates, the action of trypsin inhibited by the addition of fetal serum company "Gibco".

The resulting mixture was transferred into centrifuge tubes and centrifuged for 10 minutes n and a speed of 1000 Rev/min

The supernatant was removed, and the residue resuspendable in a nutrient medium for cell cultures, DMEM with glutamine, with the addition of 10% fetal serum, gentamicin (40 µg/ml) and insulin (5 μg/ml). Within 1 minute and allowed to settle large pieces of fabric and then the supernatant was divided into 2 Petri dishes with a diameter of 40 mm, one of which was a bandage on the basis of type I collagen person.

When viewing drops of cell suspension, resulting trypsinization and stained with the vital dye, was found clusters of different morphology of living cells.

After 8 days after planting, the primary cell suspension many rounded cells of different sizes attached to the bottom of the Petri dishes, and some of them - are in suspension of collagen fibers. Celebrated cells located in metabolically active state. Of small rounded cells started to form a single fibroblast - elongated cells fusiform shape. Watched mitotically dividing cells. After 9 days after planting, the primary cell suspension was formed a loose monolayer of cells. Fibroblasts form a "cell" network, which houses the group keratinocytes. After 11 days after planting primary cell suspensions from large conglomerates of cells adherent to the bottom of the Petri dishes, about what was azoulas monoclona mixed composition of culture, consisting of fibroblasts and keratinocytes. The cells were produced collagen.

Thus, from scrap fabrics, esecanna during the primary surgical treatment of wounds, it is possible to obtain viable cells that make up the cultivation of organ-specific colony. This confirms the possibility of using waste tissue as a source of autologous progenitor cells of the skin.

A method of combining autologous skin with DM in the wound of the patient.

The indication for the use of this method are extensive traumatic wounds with soft tissue defect. Thus a prerequisite for the implementation of this method is the availability of sufficient quantity of waste skin flaps.

Absolute contraindications are bite wounds in the acute period (first three days after injury according to Appendix No. 2 to the Ministry of health of the Russian Federation No. 297 07/10/1997).

Relative contraindication is the presence at the bottom of the wounds of the major neurovascular bundles, bones or internal organs when reservation is plastic soft tissues.

The first stage produces a PHO operating in compliance with the rules of asepsis and antisepsis by the standard procedure with excision of the edges.

Immediately after excision of the skin is thoroughly treated with antiseptic solution, dissect from the remnants of the subcutaneous is igrovoy fiber. Preserving sterility, crushed in a tray with a scalpel or scissors to the size of the fragments 1-2 mm.

Sterile DM made from the skin of the donor-cadaver, in compliance with the rules of asepsis and antisepsis perforined scalpel in the tray to allow outflow of wound, for example, with a frequency of 1 perforation length of 5 mm to 1 cm2.

Perforated DM stack on the bottom of the wound so that between the edges of the perforations was diastasis, perforations make all the prepared fragments of the waste of the patient's skin, evenly distribute, then the wound is covered with a sterile gauze bandage. The first ligation produced after 48 hours, assess the presence of signs of inflammation, the amount and nature of wound, the degree of fixation of DM and fragments of skin. Further dressings make every 48 hours.

The way the combination of DM and allogeneic MSC.

For stimulation of regeneration processes in bite wounds and, if necessary, stimulation of regeneration processes in the deferred period has been developed a method of combining DM and allogeneic MSC. Also this method can be applied at the time of admission to the hospital. The indication for the use of this method are extensive traumatic wounds with soft tissue defects of various origins.

Relative contraindication is the presence of the no wounds major neurovascular bundles, bones or internal organs when reservation is plastic soft tissues. The presence of acute inflammatory process in the wound.

A suspension of allogeneic cells is prepared in the culture unit in compliance with the rules of asepsis and antisepsis. Use MMSC third or fourth passage, derived from bone marrow donors. A solution of trypsin-versene cells separated from the culture plastic and prepare a cell suspension in 0.9% NaCl solution. To assess the quality MMSC is cytometrical study. The cellular material suitable for transplantation if confirmed his biological and infectious safety, it 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 transplantation prepare injectable form of the drug in 0.9% NaCl solution with a concentration of cells in a suspension of 0.5-2 million/ml, preferably 1 million/ml Drug MMSC suitable for use within 6 hours.

In the case of bite wounds spend PHO in the operating conditions in compliance with the rules of asepsis and antisepsis according to Appendix No. 2 to the Ministry of health of the Russian Federation No. 297 07/10/1997, which is abundant washing of the wound with running water and soap (or any detergent), processing of the wound edges 70° alcohol. For the eat is the transplantation of allogeneic MSC in suspension, containing 0.5-2 million MSC in 1 ml, preferably 1 million/ml, in the bottom of the wound and the surrounding soft tissue injection to a depth of 0.1-0.3 cm with a frequency of 10 injections on 1 cm2the volume of each injection of 0.05-0.1 ml of the bottom of the wound cover, sterile DM, modeled on the shape of the wound, with perforations to create outflow of wound, preferably with a frequency of 1 perforation length of 5 mm to 1 cm2then the wound is covered with a sterile gauze bandage.

When applying the method in the deferred period, the manipulation is carried out in the dressing room, with observance of the rules of asepsis and antisepsis. The patient removed the bandage from the wound. The wound triple handle sterile gauze swab, moistened with a 5% aqueous solution of chlorhexidine, get wet sterile dry gauze pad. To prevent contamination of wounds of the skin around the wound three-treated with 0.5%alcoholic chlorhexidine, avoiding the wound. Then in compliance with the rules of asepsis and antisepsis is the transplantation of allogeneic MSC in the bottom of the wound and the surrounding soft tissue injection of a suspension containing 0.5-2 million MMSC, preferably of 1 million/ml in 1 ml of the solution to a depth of 0.1 to 0.3 mm with a frequency of 10 injections on 1 cm2the volume of each injection of 0.05-0.1 ml Then the bottom of the wound cover, sterile DM, modeled on the shape of the wound, with perforaciones create outflow of wound, preferably with a frequency of 1 perforation length of 5 mm to 1 cm2then the wound is covered with a sterile gauze bandage. The first ligation produced after 48 hours, assess the presence of signs of inflammation, the amount and nature of wound, the degree of fixation of the dermal matrix. Further dressings make every 48 hours.

Clinical examples of the application of the proposed method

Clinical example 1

Patient K., 43 years old, he enrolled in the Institute of JV with a diagnosis of extensive ripped here-bitten patchwork wound middle third of the right tibia.

The bottom of the wound was subcutaneous fat (Agrocomplex). The base of the flap is directed distally. The flap was a layer of skin and Agrocomplex area of 20 cm2.

At admission according to Appendix No. 2 to the Ministry of health of the Russian Federation No. 297 07/10/1997 was the toilet of the wound with soap and water and antiseptic solution, the wound is covered with sterile towels soaked in 5% aqueous solution of chlorhexidine.

On the first day after injury to stimulate regeneration, prevention of ischemic complications in the flap with the rules of asepsis and antisepsis was performed transplantation of allogeneic MSC in the form of a suspension containing 1 million MSC in 1 ml in the bottom of the wound and the surrounding soft tissue injection to a depth of 0.1 to 0.3 mm with a frequency of 10 injections of 1 cm and create OptimaLineStroy in the wound on the bottom of the wound laid sterile perforated DM. The patient received combined therapy, during which he received antibacterial therapy, HBOT, ERT.

After removal of the DM on the 5th day after transplantation the bottom of the wound covered with bright granulations, the flap of skin color tightly span with the underlying tissues.

On the 6th day after injury with a goal early closure of the wound defect in the dressing room, with observance of the rules of asepsis and antisepsis was combined plastic surgery wounds: sterile DM made from the skin of the donor-cadaver was performable with a frequency of 1 perforation length of 5 mm to 1 cm2to create ways of outflow of wound and put on the bottom of the wound so that between the edges of the perforations was diastasis, perforations, in a checkerboard pattern, made of autologous skin of the patient, the wound was covered with a sterile gauze bandage.

On the 3rd day since plastics were observed engraftment of skin fragments in the perforations DM, there is a strong edge epithelialization under DM.

At all stages of treatment produced the capture of crops from the wound. According to the results of crop growth pathological microflora was not detected.

Clinical example 2

Patient P. 70 years old, he enrolled in the Institute of JV with a diagnosis of multiple bite wounds of the extremities, including extensive patchwork wound with a skin defect on the back surface of the right forearm with distain the m base of the flap. The bottom of the wound was undamaged tendons of the extensors of fingers, the muscles of the forearm and crushed subcutaneous fat.

In the ward the patient received comprehensive treatment: antibiotic therapy, HBOT, ERT, were conducted daily dressing of wounds. On the 7th day hospital stay, despite treatment, a marked increase in ischemic skin flap in the lower third of the forearm.

To stimulate the regeneration and prevention of ischemic complications in the flap with the rules of asepsis and antisepsis was performed transplantation of allogeneic MSC in the form of a suspension containing 1 million MSC in 1 ml in the bottom of the wound and the surrounding soft tissue injection to a depth of 0.1 to 0.3 mm with a frequency of 10 injections on 1 cm2. Then the bottom of the wound was covered with sterile DM, modeled on the shape of the wound, with perforations to create outflow of wound, a frequency of 1 perforation length of 5 mm to 1 cm2then the wound was covered with a sterile gauze bandage.

On the fifth day after transplantation of cells positive dynamics: the area of ischemia decreased, the bottom of the wound is 80% covered with granulation tissue, expressed marginal epithelization. After filling the wound of forearm bright fine juicy granulation tissue when expressed epithelialization order defect closure stitches is on autodermoplasty free skin flap. At the moment plastics size of the wounds was 48 CSM First ligation produced on the 6th day after autodermoplastiki. The result of autodermoplastiki regarded as good - transplanted skin flap stuck at 90%. On the 7th day after the operation the patient was discharged to outpatient aftercare.

Long-term results of treatment were evaluated after 8 months since the injury. The skin area of the former wound movable without trophic disorders and scars. Function hands fully restored.

Thus, the proposed method allows to reduce terms of regeneration of wounds by stimulating the process of epithelialization (earlier activation of proliferation and migration of keratinocytes), reduction of the inflammatory phase and accelerate the formation and maturation of granulation tissue.

1. Method of recovery of the skin in patients with extensive traumatic wounds with soft tissue defects, including excision waste of skin at the edges of the wound, isecheno dissect the skin from the remnants of subcutaneous fat and crushed to fragments of a size of 1-2 mm2simulate dermal matrix made from the skin of the donor-cadaver, the shape of the wound, perforined to create ways of outflow of wound and lay on the bottom of the wound so that between the edges of the perforations was diastasis, punch the AI staggered contribute waste fragments of the patient's skin, then the wound is covered with a sterile gauze bandage.

2. The method according to claim 1, wherein the graft perforined over the entire surface with a frequency of one perforation 5 mm 1 cm2.

3. Method of recovery of the skin in patients with extensive traumatic wounds with soft tissue defects, including transplantation into the bottom of the wound allogeneic mesenchymal multipotent stromal cells (MSC) in suspension by injection to a depth of 0.1 to 0.3 mm with a frequency of 10 injections of 1 cm, the volume of each injection of 0.05-0.1 ml, simulate dermal matrix made from the skin of the donor-cadaver, the shape of the wound and perforined to create paths wound, lay on the bottom of the wound, the wound is covered with a sterile gauze bandage.

4. The method according to claim 3, in which the graft perforined over the entire surface with a frequency of one perforation 5 mm 1 cm2.

5. The method according to claim 3, in which the suspension MSC contains 1 million MSC in 1 ml.



 

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