Cultured allogeneic fibroblast injection versus fibroblasts cultured on amniotic membrane scaffold for dystrophic epidermolysis bullosa treatment Moravvej H, Abdollahimajd F, Naseh M.H, Piravar Z, Abolhasani E, Mozafari N, Niknejad H1 Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 1 School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran British Journal of Dermatology. DOI:10.1111/bjd.16338

Introduction What’s already known? Epidermolysis bullosa (EB) is a blistering disorder caused by at least 18 variable gene mutations. Recessive dystrophic EB (RDEB) is a distinct subtype of EB that is caused by different mutations in the type-VII collagen gene (COL7A1). There is no effective therapy or cure for RDEB. Treatments include multi-disciplinary healthcare and social welfare support A key therapeutic goal is to try to restore C7 function in the skin In the absence of a definitive treatment, different modalities including: Gene Therapy; Protein Replacement Therapy; Cell therapies

Introduction Cell-based treatments are promising methods; Fibroblasts are one of the most commonly studied cell types; - Intravenous injection of molecularly-engineered RDEB fibroblasts that overexpress human Col 7 can develop at lesions in murine skin and promote wound healing by producing Col 7 and forming anchoring fibrils at the DEJ. - Intradermal injection of normal human fibroblasts into mouse skin also results in wound healing. - No studies have examined the use of fibroblasts seeded on the amniotic membrane scaffold (FAMS), in the treatment of EB wounds.

Methods Patients with RDEB between10 and 40 years of age who had failed to respond to previous therapies (wound that persists more than 3 months with no response to standard treatments) Diagnosis: based on clinical features and markedly reduced Col VII staining in immunofluorescent mapping (IFM) studies. The wounds of patients with reduced (1+ but not negative) collagen VII expression were selected. Exclusion criteria: Previous treatment with allogeneic fibroblasts or amniotic membranes; Diabetes mellitus, cardiovascular disease, a history of hypersensitivity, immunosuppression; Negative collagen VII for prevention of probable production of antibodies against type VII collagen.

Methods Three wounds were treated with fibroblasts injection Three wounds were treated with FAMS One was just dressed with Vaseline gauze (control) Seven patients (4 females, 3 males) were recruited and seven wounds were assessed in each patient: 1- The lesions assigned for FAMS: Were covered gently with FAMS. 2- The lesions assigned to fibroblast injection: A suspension of fibroblasts (1 ×107cells/mL in PBS) was prepared and injected subcutaneously into the lesion. Each square centimeter of the wound (2.5 × 106 cells/cm2) was injected with 0.25 mL of the solution. The injection was carried out once, and then the lesion was dressed with Vaseline gauze. The dressing was not changed for the next 2 weeks. 3- For control wounds: Only the SWC with Vaseline gauze was applied, and patients changed the superficial dressing daily.

Results Both wound size and QWS significantly decreased in wounds treated with fibroblast injection and FAMS (P<0.0001); In contrast, no changes were observed in the controls (SWC) (P=0.29). There were no differences in wound size between FAMS and fibroblast injection groups at the baseline (P=0.83); however, after 2 and 12 weeks of treatment, wounds were significantly smaller in the fibroblast injection group (P<0.0001). In addition, the change and percent decreases in wound size were larger in the fibroblast injection group than in the FAMS group (P<0.0001). Notably, the results in both the fibroblast injection group and FAMS group were significantly better than that in the control wounds (P<0.0001).

Results Those treated with fibroblast injection: 14 out of 21 wounds (66.6%) showed complete wound closure (100% healing), with the least response in patient no. 6 (< 47% decrease in all three wounds). Those treated with FAMS: six out of 21 wounds (28%) decreased in size by more than 70%, with only one complete healing (4.7%). The percent decrease in size was <50% for 70% of the wounds Again, patient no. 6 had the least improvement, with the three FAMS-treated wounds showing <18% decreases in size. In both groups: those with complete wound closure remained closed from week 2 to week 12 of therapy (Fig. 2, 3)

Results Biopsies from the treated lesions were also assessed by IFM. The COL VII layer was continuous in lesions that were treated with fibroblast injection but was not continuous or complete in lesions treated with FAMS (Fig. 4). The COL VII layer was not present in controls. In fact, the presence of a continuous COL VII layer was the main determinant of complete wound healing.

Discussion What does this study add? In this study, we assessed two methods of cell therapy in patients with RDEB. Although FAMS resulted in good outcomes, direct injection of fibroblasts into wound was superior in terms of complete wound closure, is simpler and not as expensive to perform. Previous studies have shown that fibroblast injection has the same efficacy as injecting the vehicle containing albumin and growth factors; however, in this study, we achieved better results with lower concentrations of fibroblasts per surface area unit, supporting the need for additional studies on fibroblast injection with higher cell concentrations. Although culturing fibroblasts is an expensive and arduous task, the injection is a simple procedure that can be performed in the outpatient setting, providing improved healing of chronic wounds. Hence, this method is also recommended for use in other refractory wounds, such as burns and traumatic skin loss.

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