New treatment protocol for pseudosyndactyly in epidermolysis bullosa hand using a dermal regeneration template glove.

BACKGROUND (cid:0) The Epidermolysis Bullosa (EB) is a rare genetic disease associated with blistering skin caused by minimal trauma. Because of its exposed position and its continued use in daily activities, the hand is constantly at risk of microtrauma and is therefore one of the organs most affected by the disease with highly disabling deformities that represents a challenging eld in hand surgery practice. METHODS (cid:0) The Authors present their experience in the microsurgical treatment of pseudosyndactylies comparing the classic dressing with vaseline gauze with our innovative “glove protocol” using Integra® dermal regeneration template. The endpoints analyzed were: healing times, hospital stay time, discomfort for the patient, free-recurrence interval, follow-up range and major complications. RESULTS (cid:0) A total of 34 procedures were performed on 24 EB patients with hand deformities. Compared with the dressing with vaseline gauze, microsurgery followed by application of dermal regeneration template gloves allowed a signicant reduction of hospital stay, healing time, and dressing pain as well as an increased recurrence-free interval. CONCLUSIONS (cid:0) The microsurgical approach followed by our new protocol described in the study has been benecial in providing consistent and successful long-term results for these patients.


Background
Epidermolysis Bullosa (EB) is a group of rare genetic muco-cutaneous diseases characterized by fragile skin and mucosal tissues that blister following minimal trauma (Nikolsky sign). 1 EB may be caused by changes (deleterious mutations) in at least 18 genes that, encoding keratin laments, play various roles in the structure, integrity, and repair of the skin. 2 It affects about 20 people out of 1 million born in the US and prevalence is 8.2 cases /1000000 live births. 2 The clinical diagnosis is con rmed by histology and genetic testing. 3 Although 30 subtypes are described, the main clinical types of EB are: simplex (if the blisters are within the basal keratinocytes), junctional (within the lamina lucida), dystrophic (in the super cial papillary dermis), and Kindler syndrome (a mixed type). 4 Dystrophic EB may be transmitted as an autosomal dominant or recessive subtype.
The recessive dystrophic EB (RDEB) is the most frequent form with a prevalence of 2 in 100000 5 and it is related to the loss of collagen VII expression that causes generalized skin blisters at birth, involvement of mucous tissue (gastrointestinal tract), chronic anemia, poor nutrition, recurrent infection, severe renal complication and squamous cell carcinomas. 6 This disease has a devastating impact on the quality of life of patients and requires multi-disciplinary team support 7 . In addition to blisters and the manifestations of skin and mucous membranes, the disease causes several functional de ciencies. In the hands, for example, the repeated minimal trauma and subsequent healing cause dermal brosis that leads to pseudosyndactyly with partial and / or complete loss of the interdigital spaces, exion contractures of the joints and adduction contracture of the thumb [8][9] . The severe form of contractures is known as "mitten hand" or "cocoon hand" deformity.
Although various gene and protein therapies have been investigated, nowadays there is not cure for EB.
The treatment consists of skin care, prevention of skin trauma, adequate nutrition, and medical and surgical treatment if indicated. 10 Surgical treatment of these patients is a challenge for the hand surgeon; obtaining successful results and avoiding recurrences are still major problems. In the literature, surgical approaches have been described to correct hand malformations in EB [11][12][13] . The aim of this treatment is a temporary release, as the pseudosyndactyly of these patients have a high tendency to relapse. Furthermore, the fragility of these patients limits their resilience to anesthesiological procedures. Post-operative treatment is also burdened with very painful dressings and, especially in children, intravenous sedation or a nerve block are often required 14 . In our Department, we have 15 years of experience treating "mitten hands" (Fig. 1) in patients with EB; over the years our surgical and anesthesiological protocol has changed, becoming less invasive and more tolerated by patients. In this manuscript, we describe our protocol combining microsurgery and the use of bilayer dermal regeneration template-gloves for treatment of hand pseudosyndactyly in EB patients. Outcomes in a series of EB patients treated with the new protocol were compared with results of a previous protocol based on surgery followed by dressing with vaseline gauze. The novel protocol proved superior in terms of healing time, hospital stay time, dressing pain, and recurrence-free interval.

Study population
In a retrospective study the Authors collected the data of 24 consecutive patients affected by dystrophic EB who underwent surgical treatment at the Plastic and Reconstructive Surgery Department in Ospedali Riuniti OORR, Foggia, Italy, between January 2001 to January 2016. All patients suffered by severe pseudosyndactylies or mitten hands, with the functional impairment. The aim of this study was to compare our new protocol in the treatment of mitten hand in EB patients with our previous surgical treatment. We have evaluated the following variables: healing time, hospital stay time, discomfort for the patient (through VAS-scale) during dressings, free-recurrence interval.
First protocol for pseudosyndactyly surgical treatment From 2001 to 2005, treatment was based on debridement of the pseudosyndactyly and, in mitten hand, temporary arthrodesis in extension of the digital rays with application of K-wires. Coverage of the bloody areas was provided by dressings with vaseline gauze.
The patients were medicated every 48-72 hours and, in selected cases, given intravenous sedation, until complete epithelialisation. Usually 3 weeks after hand release, the hand therapist fashioned a custommade splint in perforated thermoplastic material (Fig.2), and started with functional exercises.
Innovative protocol for pseudosyndactyly surgical treatment The surgical procedure is performed in nerve block anesthesia (using lidocaine 0.5-1% with naropine 1%) and intravenous sedation. Through the use of magnifying loupes (3.5X), the surgeons performed surgical debridement of the pseudosyndactylies more precisely and safely. In fact, the use of microsurgical technique allows us to perform the release of scar contractions with greater preservation of vascularnerve pedicles which, especially in paediatric patients, are very thin and easily vulnerable.
In all hands K-wires were applied to x the metacarpo-phalangeal joints in extension, taking care not to induce ischemia in the digital rays. After release, the coverage was performed through a "tailoring" of a double-layer dermal substitute (INTEGRA® dermal regeneration template). These custom-made gloves ( Fig. 3) were applied to the hands of patients with absorbable sutures.
The rst dressing change was performed on the eighth postoperative day, without the need for analgesic sedation or nerve block. Successive dressing changes were performed every 7 days in outpatient department. Around the 30th day, the silicone layer was removed and the few residual areas denuded were medicated with vaseline gauze until complete healing. The use silicone night splints were recommended for 60 days.
A speci c physiotherapy was carried out by experienced hand therapists, followed by home therapy.

Statistical analyses
We present summary statistics as means with standard deviation (Std). The continuous data were assessed for normality of distribution using a Kolmogorov-Smirnov test that revealed a normal Gaussian distribution. Comparison of the two treatment groups was performed using a Mann-Whitney U test for continuous consequent variables. An expert bio-statistician performed the statistical analysis using Statistical Package for Social Sciences (SPSS version 16.0). A value of p less than 0.05 was considered statistically signi cant.

Results
From January 2001 to January 2016, 24 patients affected by dystrophic EB were treated (13M, 11F). The range of patient age was 6-15 years old (mean age 8.7). Eleven patients (7M-4F), with a total of 15 mitten hands, underwent surgical release of the digital rays and dressing with vaseline gauze (Group A) (Fig.4). Thirteen patients (6M-7F), with a total of 19 mitten hands, underwent surgical release of the web spaces, nger and wrist contractions, followed by reconstruction with gloves of INTEGRA® dermal regeneration template (Group B) ( Fig.5 and 6). The mean age in Group A was 10.1 ± 0.7, and 7.3 ± 2.5 in group B. (See Table I).
In both groups treated, no patient reported major complications such as infection and total necrosis of the ngers. In Group A, the average hospital stay time was 29.6 ± 1.6 days (range 16 to 45 days), while in Group B it was 3.6 ± 0.8 days (range 2-5 days), with a p-value=0.021 Complete healing times (with reepithelialization of more than 90% of the hand) were 46.4 ± 3.4 days for Group A and 24.6 ± 2.9 days for the group treated with INTEGRA®, with a p-value=0.008.
In group A, patients had discomfort after dressings, with an average value of 7.6 on the VAS scale, whereas B group scored an average of 0.6, with a p-value=0.034.
Dressings were painful for the patients of Group A, in particular when, to completely release their ngers, the vascular-nerves pedicles were exposed. Patients also need for a sedation or nerve block. In order to carry out these procedures, it was necessary to have an average hospital stay of about 30 days with great di culties for the patient and the family members.
In patients of Group B we use bioengineered tissue on all the surface of the hands, for the known INTEGRA® anti-in ammatory properties. The rationale for this choice being that a reduction in tissue in ammation is very effective in reducing pain during post-operative dressings. Indeed, the presence of the non-adhering silicone layer of the dermal substitute results in a painless renewal of dressings.
The average time to free recurrence was 19.1 ± 5.3 months for group A and 31.1 ± 4.7 months for B, with a p-value=0.045.
There was a statistically signi cant difference in hospital stay, in healing time, in complete healing times, in discomfort after dressings and, nally, in time to free recurrence.

Discussion
Recessive dystrophic forms of EB are burdened with a high psychological impact for patients and their families. A multidisciplinary approach to manage the disease is fundamental and surgery plays a critical role. "Mitten hands" compromise the functional development of the hands in children with EB. So, an early surgical approach is of paramount importance. Fine et al. showed that about 61% of recessive dystrophic EB patients require hand surgery, typically needing 5 or more operations. 5 In our experience, but also in literature, pseudosyndactylies in patients with this disease are characterized by high rates of recurrence 1,7,9,[11][12][13]15 .
Moreover, other challenges include impaired wound healing and risk of iatrogenic trauma. In fact, the scar tissue of the hands can alter the anatomical structures, including the vascular-nerve pedicles; so minimal inadvertence during surgical procedures can cause local ischemia and subsequent partial or total skin necrosis.
However, surgery is crucial to correct severe hand deformities, improving patient's quality of life. In a study of 1995, Ciccarelli at al. 15 proposed these indications for surgery: palmar contracture, contracture of the proximal IP joint >30°, severe SF deformity, pseudosyndactyly extending to the proximal IP joint, severe impairment of daily activities.
Conventional surgical techniques for "mitten hand" are limited to the release of pseudosyndactylies and wound coverage, achieving optimal epithelialisation, beginning early mobilization, and providing longterm stability with minimal recurrence. 7 Various methods are described, but no consensus exists.
Moreover, split-thickness skin graft usually provides unstable coverage with high risk of early recurrences. 1,2,7,11 Full-thickness skin graft is useful in the rst web space and delays recurrence of contracture as compared with STSG, but the donor sites are very limited. 1 The use of skin substitutes in patients with EB has been described in the literature; often in combination with skin grafts.
Recent studies focused on allogenic skin substitutes derived from human keratinocytes and broblasts.
In particular, the studies of Fivenson 19 20,25 , however it is demonstrated that the new dermal support created by the acellular dermal matrix can prevent recurrence of the pseudosyndactylies in the long term. 26 Hand function preservation and the time interval to relapse are the most important parameters in evaluating surgery e cacy.
Following a careful analysis of our surgical procedures, we were able to improve outcomes in our patients by combining the use of microsurgery with the application of Integra ® dermal regeneration template".
In fact, we believe that magnifying loupes have allowed us to preserve the vascular-nervous pedicles in these patients, especially if in paediatric age. The higher skills and expertise of the Surgeons using the microscopic lenses in the release of pseudosyndactylies also helped to gain better results avoiding surgical damages.
We believe that through our protocol using a bilayer dermal regeneration template it is possible to avoid skin grafting and therefore iatrogenic damage and further surgery.
Arti cial dermis, as Integra®, is an acellular purely bilaminate synthetic construct consisting of an outer silicone (polysiloxane) semipermeable membrane and an inner porous matrix of collagenglycosaminoglycan.
The outer layer serves as an epidermal substitute and provides mechanical protection, and heat and moisture modulation of the wound, prevents formation of wound granulation tissue and increases tear strength of the custom-made gloves.
On the other hand, the inner layer, composed of bovine tendon type I collagen cross-linked to chondroitin-6-sulfate, is a biologic-based acellular dermal scaffold and promotes cellular ingrowth (it is histioconductive/histioinductive and allows for broblast proliferation and migration into the dermal scaffold and capillary growth). In particular glycosaminoglycan provides elasticity to the matrix, controls the biodegradation rate, and maintains a more open pore structure that allows cell migration into the matrix.
The porous layer of the matrix is strictly applied to the wound bed at the rst stage. It acts as a template for the ingrowth of host broblasts and endothelial cells and is gradually replaced by host (endogenous) collagen, forming a new dermal layer (neodermis).
Integra® dermal regeneration template has been originally designed for treatment of full thickness skin lesions in a two-step procedure, where the second step consists of peeling off the silicone layer and applying an autologous split-thickness skin graft on the neodermis. 27,28 Importantly, in our protocol for EB hands we use the Integra®-glove as an advanced wound dressing avoiding the second phase of skin graft and obtaining an almost complete and spontaneous re-epithelialization.
On the other hand, the use of arti cial dermis offers many advantages, such as: immediate availability, possibility to cover large defects, no donor-site morbidity, good scarring, and early recovery.
In addition, the anti-in ammatory properties and physical characteristics of INTEGRA ® allowed us to perform pain-free dressing and achieve more stable coverage over time. The coverage of noble structures, such as vascular and nerve pedicles, has been found to perform well. We believe that coverage through our technique has allowed a lower rate of early recurrence. In fact, inter-digital spaces have had a reduced tendency toward fusion during the healing phase, probably thanks to the INTEGRA® layer. The severity of the primary disease, the degree of hand deformity and the age of the patients at their initial referral are also relevant factors, which need to be addressed in future studies.
Finally, these surgical procedures are light and can be carried on in local anaesthesia and sedation; in particular, we performed: regional anaesthesia (brachial plexus), or deep sedation with fentanyl and propofol plus local anaesthetic in ltration, or deep sedation with ketamine and fentanyl plus local anaesthetic in ltration. 29 We avoid to perform general anaesthesia because of high risk of iatrogenic damage of intubation in this kind of patients. With our new protocol the medications are painless, so we don't use anaesthesia.

Conclusions
The proposed protocol is proven safe, repeatable, and simple to execute. Patients reported less discomfort after Integra®-gloving than after the previous treatment option, and we believe that every effort to reduce patients' distress and shorten the number of hospital visits is justi ed. The high rate of recurrence after treatment of "mitten hands" necessitates a lasting approach that offers stable coverage with less discomfort for patients. The limitations in our study are: its small simple size and a method for assessing wound healing that is based on the surgeon's experience and qualitative rather than standardized. Moreover, it doesn't compare the results of different dermal substitutes; at last, there is not preoperative evaluation of pseudosyndactylies using a validated scale.
In spite of these limitations, the signi cant differences between both groups cannot be ignored, suggesting the clear repercussions of this new protocol on long lasting healing and on quality of life (QoL) of EB patients.
The results of this relatively small study (34 hands) have led to an adaptation of the EB-hands treatment at our Centre; we are consequently collecting and analyzing other data in order to expand this study in future and produce a "stronger" analysis.
The Authors are convinced that the ndings in this study may be useful for other surgery Units. We believe that the use of this dermal regeneration template in dystrophic EB hand deformities should be included in the routine surgical treatment protocol.
However, it would be desirable to have a multi-center study with other Hand Surgery departments that take care of these deformities. Finally, a future study should also measure with validated instruments the impact of pseudosyndactyly surgical treatment on quality of life of EB patients, as QoL represents a primary and essential outcome of any treatment strategy for this extremely disabling disease manifestation.