RU2446811C2 - Method of treating neurotpophic ulcers of extremities - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine. A method involves in the fact that autologous cell cultures of patient's fibroblasts enriched with multipotent mesenchymal stem cells (MMSCs) are once transplanted on a wound surface. A portion of the culture MMSCs makes 5 to 15%. The cells are applied on the wound dropwise and fixed on the tissue defect with the use of a fibrine carrier and protected with a bandage made of platelet-rich plasma.

EFFECT: method provides reduced length of treatment; it is safe and high-effective.

2 dwg, 1 tbl, 1 ex

Description

The invention relates to medicine, in particular to cellular technologies, and can be used to treat necrotic tissue defects, in particular, neurotrophic ulcers of the lower extremities with complications of diabetes mellitus - type II (DM).

A known method of treating neurotrophic ulcers using allogeneic transplantation, in which cryopreserved allogeneic human fibroblasts obtained from the skin of the foreskin of newborns grown on a biosorbing mesh scaffold from polyglactin are used to apply to an ulcer. The announced effectiveness of this technology is 71% [Dermograft 55, 56, 57 ... Batool Kazmi, PhD; Christopher J. Inglefield, FRCS (Plast); Mark P. Lewis, PhD Autologus Cell Therapy: Current Therapy: Current Treatments and Future Prospects // Wounds. 2009 VOLUME: 21 PUBLICATION DATE: Sep 15 2009 pp 234-242].

There is also known a method of treating ulcers by applying (applying) a two-layer artificial skin equivalent, consisting of dermal (allogeneic fibroblasts on a collagen gel matrix) and epidermal (keratinocytes grown on a preformed dermal layer) layers with a declared efficiency of 56%. [Batool Kazmi, PhD; Christopher J. Inglefield, FRCS (Plast); Mark P. Lewis, PhD Autologus Cell Therapy: Current Therapy: Current Treatments and Future Prospects // Wounds. 2009 VOLUME: 21 PUBLICATION DATE: Sep 15 2009 pp 234-242] / [Waymack P., Duff RG, Sabolinski M. The effect of a tissue engineered bilayered living skin analog, over meshed split - thickness autografts on the healing of excised burn wounds. The Apligraf Burn Study Group. Burns. 2000; 26 (7): 609-19. Kirsner R.S. The use of Apligraf in acute wounds. J. Dermatol. 1998; 25 (12): 805-11. Trent J.F., Krisner R.S. The engineered skin: Apligraf, a bi - layered living skin equivalent. J. Clin. Pract. 1998; 52 (6): 408-13].

A method for treating neurotrophic ulcers by applying autologous fibroblasts is also known, in which a three-dimensional substrate of a biocompatible (polyvinyl, etc.) inorganic substrate is used for their cultivation, and fibroblasts are pre-transfected with VEGF (vascular endothelial growth factor) containing a vector. In this case, the bioconstruction is applied to the surface of the ulcer for 8 weeks with a daily change of dressing [Naughton G.K., Mansbridge J.N., Pinney R.E., Zeltinger J. Methods for using a three - dimensional stromal tissue to promote angiogenesis. US 2003/0007954 Al. Jan. 9, 2003].

The objective of the invention is to accelerate and increase the effectiveness of the treatment of long non-healing neurotrophic ulcers of the extremities in type II diabetes using autoplasty.

The problem is solved in that in the treatment of long-term non-healing neurotrophic ulcers of the extremities in type II diabetes, cultured autologous fibroblasts are used for transplantation. At the same time, isolated and grown fibroblast cultures enriched with multipotent mesenchymal stromal cells (MMSCs) are introduced onto the ulcer surface once by droplet, while the proportion of MMSCs in the culture reaches 5-15%, and the cells are fixed on the wound surface by applying a fibrin substrate from autologous platelet-rich plasma from the patient’s blood and a wet healing dressing that is changed every three days, and before transplantation, the patient is in a state of subcompensation for the underlying disease until glycated hemoglobin reaches 5.7 to 9.0% and the presence of microbial bodies in the ulcer area is not more than 150 per square centimeter.

The novelty of the invention is that the use of autologous materials with the addition of MMSC at all stages of treatment ensures the absence of a rejection reaction and reduces the treatment time to 3-5 weeks (3-2.5 times) in comparison with the closest analogues and prototype.

The achieved non-obvious therapeutic effect consists in closing the necrotic skin defect for a shorter period compared to the described analogs of the time (3-5 weeks) and more fully filling the tissue deficiency in the area of the defect itself, and when using a culture of enriched MMSC fibroblasts, all layers are most fully restored dermis and underlying tissues, in particular subcutaneous fat and fascia. The proliferative potential of MMSC provides the formation of a new microcircular vascular bed.

The method of treatment is carried out in stages as follows: first, with medical methods, a state of subcompensation for diabetes is achieved using sugar-lowering therapy until glycated hemoglobin reaches 5.7 to 9%. At the same time, neurotropic, angiotropic therapy is carried out, if necessary, the limb is immobilized using a discharge bandage.

During the treatment, regardless of the stage of the wound healing process, tissue material is taken from the gluteal region or the anterior abdominal wall (lower horizontal fold).

MMSCs (multipotent mesenchymal stromal cells) are extracted from subcutaneous fat by an enzymatic method and cultured on a nutrient medium that limits differentiation. In parallel, fibroblasts are cultured for 30-38 days. During the cultivation of cells, the wound is sanitized using antibiotics, antimycotics and sanitizing discharge dressings.

The fibroblast culture is enriched with MMSC up to 5-15% of the total volume and is applied dropwise to the wound surface in the form of a mixed culture in the amount of at least 200 thousand cells per square meter. see. The applied cells are fixed on the surface of the wound using a fibrin substrate for the dressing obtained from platelet-rich plasma made from the patient’s blood immediately before applying the mixed cell culture to the wound. The presence of a fibrin substrate from the patient’s blood guarantees the retention of cellular elements in the wound and the provision of cells with growth factors containing a wide spectrum of activity contained in platelets. The latter stimulate the proliferative activity of fibroblastic differential cell and MMSC. Due to this, cells of a mixed autologous culture repair tissue defects faster, despite the lack of trophic supply of the affected skin areas.

The main indicators and limiting parameters of the proposed method, such as: the proportion of MMSC content in the introduced culture from 5 to 15%; the use of a fibrin substrate made before applying the cells from platelet-rich plasma obtained from the blood of a patient; the number of cells of mixed culture introduced to the surface of the ulcer, not less than 200 thousand per square. see wounds and requirements for the patient as subcompensated - identified and substantiated in practice as a result of numerous laboratory experiments, taking into account the theoretical study of analogues, prototypes and the experience of previous studies and clinical trials.

After a detailed testing in laboratory conditions of cultivation methods, preparation and cultivation of fibroblasts, clinical trials of the proposed method were carried out in the conditions of the regional clinical hospital No. 40 (Yekaterinburg) in 10 patients with severe type II diabetes. The average age of the patients was 65.16 years (from 50 to 79 years). Experience of diabetes - 8.16 years (2-25 years). Examination of all patients revealed complications of diabetes - diabetic microangiopathy of the lower extremities and severe polyneuropathy.

Patients were selected with the voluntary informed consent signed by patients on the basis of anamnestic data, the results of a medical examination, and laboratory tests for contamination with infectious agents (HIV, SMB, hepatitis, myco- and toxoplasma (Axsym, Abbott laboratories).

The criterion for inclusion in the test was the presence of a peptic ulcer with an area of at least 35x10 mm (on the foot) and 65x50 mm (on the lower leg) for 6 months or more and the failure of previously performed conservative treatment.

The explant was taken regardless of the stage of the wound process under conduction anesthesia. Manufacturers of the skin and underlying subcutaneous fat were manufacturers with the gluteal region 7-8 cm below the ilium wing, the explant was placed in a PBS solution containing two concentrations of cultured antibiotics, stored at a temperature of 4 ° C for no more than 12 hours.

Cells were cultured at 37 ° C, CO ₂ concentrations _of 5% and 95% humidity in DMEM / Ham F-12 medium (Sigma) supplemented with 10% fetal bovine serum (Well Clone). In addition to culturing skin cells from an insignificant amount of subcutaneous adipose tissue (VT), multipotent mesenchymal stromal cells (MMSCs) were extracted in an enzymatic manner in order to obtain a reproducible cell line. The limited volume of VT made it possible to obtain three such lines: patients No. 2; 4 and 5. The affiliation of MMSCs to the stem pool was confirmed by differentiation of cells in fibrogenic, chondrogenic and osteogenic directions.

During the cultivation and growth of the cell mass (on average 37.6 days), the wound was sanitized using standard dressings and atraumatic dressings. In one case, mycoplasma DNA was detected in the cell culture of the test subject, in connection with which the culture was discarded, and the recaptured explant was cultured with the addition of a 20-fold (from the culture) concentration of the antiparasitic antibiotic tylosin (Sigma), which led to decontamination culture to the third passage.

Prior to cell transplantation, all patients underwent correction of sugar-lowering therapy to achieve a subcompensated state of carbohydrate metabolism. Indicators of glycated hemoglobin (HbAlc) ranged from 5.7 to 9.0% (an average of 7.73%). A necessary condition for cell transplantation was the presence of a “clean” ulcer defect (the number of microbial bodies was not more than 150 per cm ² ) and active granulations. Patients with localization of an ulcer on the foot were immobilized with a limb using an unloading dressing.

To determine the area of the wound defect, the formula for calculating the area of the circle was used: S = πR ² . With uneven edges of the wound, the average value obtained by adding the largest and smallest defect radii with the subsequent division of the obtained value into two was taken as the radius.

As part of the implementation of the safety program, before the cell therapy, a three-stage control of the obtained cell lines was performed on the probability of mutations of the p53, k-ras, b-raf genes. At the same time, among the cells collected from the culture surfaces, the proportion of Stro-1 positive cells (MMSCs) was determined by the immunofluorescence method.

In order to ensure the possibility of repeated therapeutic use of the obtained cell lines, some of the cells were cryopreserved according to standard methods.

During the tests, enrichment of dermal fibroblast cultures with MMSC lines in an autogenous version was undertaken. Moreover, the proportion of Stro-1-positive cells in the cultures of patients No. 2; four; 5 increased tenfold - to 0.6%. The transplantation of enriched cultures in the Clinical Hospital № 40 was performed using the "blind" method with the mandatory passage of all stages of pre-transplant control.

For a single application to the wound surface, a cell suspension containing at least 8x106 living cells was used. The number of applied cells varied depending on the size of the ulcer defect.

To fix the cells on the surface of the ulcer, an autologous fibrin substrate enriched with platelets and a special dressing providing “wet” wound healing were used. The manufacture of an autologous fibrin substrate was carried out immediately before transplantation, almost at the patient’s bedside by centrifuging the patient’s blood, followed by heating of the clot to form a film.

Ulcer healing was monitored over the course of 26 weeks.

Table 1 presents the results of observations at the clinical hospital No 40 and the dynamics of the reduction of ulcerative defects in patients.

Table 1 Patient No. Before treatment,% Treatment Results - Weeks 1 week 3 weeks 5-6 weeks 8-9 weeks 11 weeks 21 weeks one 0 54 73 90 one hundred 2 (MMSK) 0 75 89 98 one hundred 3 0 10 80 one hundred 4 (MMSK) 0 fifty one hundred 5 (MMSK) 0 85 one hundred 6 0 four 40 75 7 0 36 77 98 one hundred 8 0 46 69 98 one hundred 9 0 60 96 one hundred 10 0 35 58 80 one hundred

From table 1 it follows that after the first week after cell transplantation, a decrease in the ulcer surface by more than 50% was observed in half of the patients (the arithmetic mean reduction in the ulcer defect was 45.5%). Moreover, in patients with transplantation of cultures enriched in MMSCs, after seven days, a decrease in ulcer area was recorded by 75% (patient No. 2), 50% (No. 4) and 85% (No. 5). By the end of the third week, patient No. 4 had complete ulcer healing, and in eight patients, the ulcer surface was reduced by more than 60%. Six weeks later, test No. 5 showed complete wound epithelization, No. 2 — 98% closure of the ulcer area, and 58–80% in the remaining patients. By the end of the second month, patient No. 2 had complete healing. By the eleventh week, 7 out of 10 patients had complete closure of wound defects, another 2 were at the final stage of healing. Final epithelization in 9 patients was confirmed by 21 weeks of observation. One patient had 75% healing at this time. Complete epithelization of the ulcer defect in this subject was not achieved due to a violation of the recommended methodology (use of an absorbent gauze dressing to close the wound surface).

In one case, 6 days after application of the cells, the patient had an increase in temperature to febrile numbers. When conducting crops of the wound, the growth of Staphylococcus aureus was revealed, which required the appointment of broad-spectrum antibiotics. No other complications were detected in the subjects. There were no allergic reactions during the study.

The average duration of treatment (from the time of autofibroblast transplantation to wound epithelialization) with localization of an ulcer on the foot was 9.5 weeks, with localization on the lower leg - 19.3 weeks. In this case, the average healing time when using enriched MMSC cultures in the autogenous form was 4.5 ± 1.5 weeks.

Figure 1 and figure 2 presents photographs of the stages of the application of the method of treatment of neurotrophic ulcers of the extremities in patients during clinical trials in OKB No. 40 (Yekaterinburg).

Figure 1 - patient No. 2, 50 years old. Type 2 diabetes mellitus, identified in 1997, severe, subcompensation.

VTS. Neuro-ischemic type, (predominance of neuropathy) (amputation).

Exarticulation of the third to fifth toes of the left foot (02/15/2008 regarding gangrene). Trophic ulcer of the right foot for more than 1 month.

Taking a skin biopsy on 03.21.08. The culture is enriched by MMSK.

04.24.08 implantation of a mixed culture of autologous fibroplasts and MMSCs on the skin defect area was performed.

A - before treatment; B - in a week; B - after 3 weeks.

Figure 2 - patient No. 4, 69 years old. Type 2 diabetes mellitus, first detected, severe.

Trophic ulcer of the right lower leg since October 2007, more than 8 months.

08/10/08 on the lower third of the right lower leg there is an ulcerative defect of vesiculate form 4.5 cm wide, 7.7 cm wide including the spur, 7.5 cm long.

Autologous fibroplasts enriched in MMSCs were transplanted to the area of the ulcer defect.

A - before treatment; B - in a week; B - after 5 weeks.

After ulcer epithelialization, a monthly scar was monitored for 6 months. Relapse of the disease during the observation period was not observed.

The results of clinical trials of the proposed method for the treatment of neurotrophic ulcers in patients with severe type II diabetes indicate that the enrichment of MMSCs of cultured fibroblasts and their use in the autogenous version, compared with the use of fibroblastic differon cells, is accompanied by a twofold acceleration of the wound healing process. This confirms the assumption that MMSCs participate in the design of the dermal and epithelial layers of the skin, as well as the vascular bed, preventing re-ulceration.

The proposed method of treatment, including a single application to the surface of ulcers in the diabetic foot of cultured dermal fibroblasts enriched in MMSCs in an autogenous form when covered with a fibrin backing under a bandage, is accompanied by a significant increase in wound epithelialization compared to using unenriched cultures. This suggests that MMSC-mediated activation of the engrafment of the transplanted cells and differentiation in the lesion area, their proliferation with accelerated replacement of the tissue destruction zone.

Clinical trials of the proposed method for the treatment of lower limb ulcers in type II diabetes convincingly indicate the technical feasibility of the method and the creation of a practical basis for the development of a safe and effective technology for cell therapy of neurotrophic ulcers, which can significantly reduce treatment time with its high effectiveness, expanding the possibilities of using cell autoplasty cultures in the treatment of neurotrophic ulcers of the extremities.

Claims (4)

1. A method of treating neurotrophic ulcers of the extremities, including preparing the patient with medication, applying cell cultures to the surface of the ulcer, fixing the cell cultures on the surface, closing the dressing and monitoring the course of treatment, characterized in that autologous cell cultures of the patient’s fibroblasts are transplanted once, enriched with multipotent mesenchymal stem cells (MMSCs), the percentage of MMSCs contained in the culture is from 5 to 15%, and the cells inflicting droplets on the wound and fixed to the tissue defect site with fibrin substrate via a bandage made of a platelet-rich plasma. 2. A method for the treatment of neurotrophic ulcers of the extremities according to claim 1, characterized in that MMSCs are extracted from the patient’s subcutaneous fat tissue by an enzymatic method and cultured on a nutrient medium that limits differentiation, and a mixed culture of autologous fibroblasts and autologous MMSCs is used on the wound surface in an amount of not less than 200 thousand cells per square cm of wound. 3. A method of treating neurotrophic ulcers of the extremities according to claims 1 and 2, characterized in that the platelet-rich plasma for the manufacture of the fibrin substrate is obtained from the blood of the patient himself, and the wound defect with the cells fixed to it by the fibrin substrate is covered with a dressing for wet healing, and the bandage is changed every three days. 4. A method for the treatment of neutrotrophic ulcers of the extremities according to claims 1 and 2, characterized in that before the transplantation of cell cultures, the patient achieves a state of subcompensation for the underlying disease until the glycated hemoglobin from 5.7 to 9.0% and the presence of microbial bodies in the area ulcers no more than 150 per square. cm.

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