Congenital epidermolysis bullosa (CEB) is a phenotypically and genetically heterogeneous group of genodermatoses. Its main clinical manifestation is bullas that appear on the skin and mucous membranes after minimal mechanical impact due to hereditary defects in structural proteins that provide dermal-epidermal connections [1, 2].
Currently, there are 4 main types of CEB (simple, borderline, dystrophic — dominant and recessive forms, Kindler's syndrome) and more than 30 clinical subtypes [3—5]. For each of them the genes’ mutations that responsible for every CEB phenotype manifestation have been identified. At least 18 genes of skin structural proteins and more than 1000 mutations that associated with certain clinical manifestations of each CEB type are known [2, 6].
One of the most severe variants is autosomal recessive dystrophic epidermolysis bullosa (ARDEB) that connected with homozygous or heterozygous mutations in the COL7A1 gene encoding the VII type collagen synthesis (C7) [7, 8]; it manifests either due to complete absence of C7 (the most severe clinical variants) or its low production (milder variants of the disease) . C7 is produced by two types of cells — keratinocytes and fibroblasts and represents the main component of anchor fibers required for the stable dermal-epidermal connection (DEC) [9, 10]. Defect of C7 leads to DEC disruption and causes the bullas and erosions on the skin and mucous membranes [3, 11, 12].
ARDEB develops from the birth or from the first hours of life. It’s characterized by multiple bullas often with hemorrhagic contents that located on any part of the skin. The surface of the bulla easily ruptures; there also occur the extensive and slow healing erosions with atrophic cicatricial changes afterwards . Patients experience constant pain that affects significantly the psycho-emotional status. Exacerbations are frequent; in severe forms the multi-organ lesions and extensive scarring may develop. It leads to patients’ disability and a decreases the life expectancy [3, 4, 13—15].
Etiology-pathogenesis treatment of CEB does not exist yet. All therapeutic measures are palliative and primarily aimed at relieving the disease symptoms, preventing injuries and bullas, as well as protecting against skin infections [12, 16].
Currently, innovative approaches to the treatment of patients with CEB are being actively developed. The key therapeutic task is to restore the level and functions of structural proteins that ensure DEC. According to the materials of the DEBRA (Dystrophic Epidermolysis Bullosa Research Association) International (2012) conference, one of the most promising approaches is the biotechnology, particularly the cell therapy [17—19] that is aimed at correcting, restoring or compensating the lost skin proteins’ functions in CEB patients, such as C7 in ARDEB.
Keratinocytes and fibroblasts are mainly used for cell therapy in patients with ARDEB since these cells produce C7 in vivo [13, 20, 21]. Fibroblasts are the main cells of skin connective tissue producing collagen, elastin, growth factors / cytokines and other components of the extracellular dermis matrix providing homeostasis and morphological and functional organization of the skin [22, 23]. In addition, fibroblasts are easier to cultivate in comparison with keratinocytes; they have low immunogenicity with potential to long-term cultivation in vitro (preserving the diploid karyotype). They also do not show oncogenic properties; after transplantation into the skin their biosynthetic activity is preserved [13, 24, 25]. So, the fibroblasts namely are the cells of choice for cell therapy in patients with ARDEB [24, 26, 27].
Studies carried out on C7 hypomorphic mice (a model of ARDEB) have shown that intradermal administration of human skin fibroblasts increases the content of C7 and anchor fibrils in DEC and heals the skin wounds, accordingly .
Limited clinical trials by T. Wong et al. , N. Nagy et al. , as well as the results of a randomized, placebo-controlled, double-blind clinical study conducted by G. Petrof et al.  demonstrated an increase in COL7A1 expression (for at least 3 months) and wound healing in most ARDEB patients after single injection of allogeneic skin fibroblasts along the wound edges. In most patients the epithelialization was observed within first 28 days and obtained result lasted for at least 6 months. No side effects or adverse events have been reported. At the same time, the severity of erythema significantly decreased and patients’ life quality improved. According to the researchers, the duration and severity of clinical effect is associated with the level of COL7A1 expression.
Taking into account the results of preclinical and clinical studies mentioned above, it was decided to use the allogeneic skin fibroblasts (ASF) for wound healing in ARDEB patient (clinical case).
Patient N., 26 years old, a man, has been observed in the dermatological department of the Moscow center of dermatovenerology and cosmetology for the last 10 years. The medical history: diagnosis Q81.2. Recessive dystrophic epidermolysis bullosa (diagnosed from the birth). Dermatological status: on the body skin there are multiple erosions of various diameters characterized by slow healing — from 4 days to several months. By patient aging there is a progressive deterioration in wounds epithelialization that requires increased attention and care. Pathological data: subepidermal blisters . After wound healing the cicatricial skin atrophy is noted; it accompanied by the scars restricting the movement. Pseudosyndactyly and fingers contractures are recorded from the first months of life; these changes have progressive character. There are also lesions of oral mucosa, anomalies in teeth location with its partial loss, disorders in the musculoskeletal system accompanied by myalgia, arthralgia, osteoporosis, growth retardation and decreased mobility.
The patient carries out daily the local therapy of skin wounds with antiseptic solutions and atraumatic non-adhesive dressings with antibacterial, analgesic and regenerating components. Despite the therapy, there is a slow healing of wounds / erosions and the constant appearance of new erosive foci and bullas that significantly reduces the patient's life quality.
After prolonged symptomatic therapy with no clear clinical effect a specialists’ council was held at the Institute of Plastic Surgery and Cosmetology aimed to use the innovative methods of regenerative medicine; it was concluded to carry out a limited clinical study on cultured ASF application in order to restore and maintain the patient’s skin integrity and his life quality.
Objective: to investigate the ASF in order to restore and maintain the skin integrity in recessive dystrophic congenital epidermolysis bullosa.
1. To evaluate the effectiveness of ASF in ARDEB.
2. To study the duration of carryover clinical effect.
A pilot clinical study was carried out in 1 patient (male, 26 years old) with Q81.2. Recessive dystrophic epidermolysis bullosa (diagnosed at birth).
Material: ROS suspension from a healthy donor (immunophenotype of cells: CD34-, CD45-, cytokeratins CD34-, CD45-, collagen I, III, vimentin) in 1 ml of saline; a study of cellular material for biosafety was carried out in order to exclude contamination with bacterial and fungal agents.
Cultured fibroblasts do not express the antigens of main complex histocompatibility class II (MHC II), therefore allogeneic fibroblasts after transplantation do not induce immune reactions [24, 31] but in 2 weeks MHC II begin appear on the cells surface and they are eliminated from the body .
Methods: the cell suspension was injected along the erosion edges in a linear-retrograde style using 32G,12 mm needles. Before procedure the zones of cell suspension introduction were treated with anesthetic cream Acriol Pro. After procedure the patient continued with standard wound care. The observation trial was 6 months.
Results: while preparation to ASF injection the results of general physical examination and blood laboratory tests were at reference values. After the patient signed an informed consent, the cell suspension was injected into the edges of 8 erosions using linear technique; its sizes ranged from 2 to 10 cm2 in the back region. (it should be noted that patient felt pain during the cell suspension injection despite local anesthesia).
Wound healing was assessed monthly for 6 months. Complete epithelization of small erosions (2-4 cm) was observed in one month; complete epithelization of large wounds (8, 10 cm2) was recorded in 4 months. The achieved effect lasted for 6 months. Allergic reactions and other side effects, as well as undesirable effects when using ASF were not identified. A significant reduction of skin erythema was recorded. As patient mentioned, a significant life quality improvement was noted including daily time shortening for routine wound care.
Based on the results, it can be concluded that in ARDEB with a single use of ASF a complete epithelization of small and large wounds is possible (in the conditions of COL7A1 expression) . Skin fibroblasts do not express antigens of the major histocompatibility complex class II  and these cells introduction does not lead to any pathological reaction in patient.
It should also be noted, if erosion recurred the procedure can be repeated within a short time since ASF processing is not difficult. The only problem is adequate skin anesthesia before the procedure because the fibroblasts’ suspension introduction is quite painful.
The study result concludes that ASF is a possible alternative treatment for patients with ARDEB that requires further research.
The concept and design of the study: M.V. Rubanenko, N.E. Manturova, N.N. Potekaev, A.Yu. Ustiugov, V.L. Zorin, A.I. Zorina
Collecting and interpreting the data: M.V. Rubanenko, A.Yu. Ustiugov, O.V. Porshina, V.V. Petunina
Drafting the manuscript: M.V. Rubanenko
Revising the manuscript: N.E. Manturova, N.N. Potekaev, A.Yu. Ustiugov, V.L. Zorin, A.I. Zorina
The authors declare no conflict of interest.