New protocol treatment of pseudo-syndactyly in the Epidermolysis Bullosa hand using the dermal regeneration template glove

BACKGROUND ː The Epidermolysis Bullosa (EB) is a rare congenital 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 field in hand surgery practice. METHODS ː The Authors present their experience in the microsurgical treatment of pseudosyndactylies comparing the classic dressing with vaseline gauze with our innovative “glove protocol” using a 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 ː A total of 34 procedures were performed on 24 EB patients with hand deformities according to the proposed treatment strategy. We observed reduction of: hospital stay time, complete healing times, discomfort after dressings and average time to free recurrence. CONCLUSIONS ː The microsurgical approach followed by our new protocol described in the study has been beneficial in providing consistent and successful long-term results for these patients.


Introduction
Epidermolysis Bullosa (EB) is understood as a group of rare skin diseases that affect about 20 people out of 1 million born in the US 1 . This disease has a devastating impact on the quality of life of patients and requires multi-disciplinary team support 2 . In addition to blisters and the manifestations of skin and mucous membranes, the disease causes several functional deficiencies. In the hands and feet, for example, this disease leads to pseudo-syndactyly with partial and / or complete loss of the interdigital spaces, flexion contractures and adduction contracture of the thumb [3][4] . 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 5-6-7 . The aim of this treatment is a temporary release, as the hands of these patients have a high tendency to relapse. Furthermore the fragility of these patients limits 3 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 8 . 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. This paper aims to present our innovative Dermal regeneration template-gloves protocol for treating pseudosyndactyly of the hands in patients suffering from this disorder. Our experimental hypothesis was to demonstrate that DRTG-protocol is a safe procedure that improves clinical outcomes and QoL. The mechanism of action underlying our proposed approach was threefold, given that the use of microsurgical technique and the use of bilayer dermal regeneration template make it possible to: 1) Avoid the vascular-nervous peduncles damage; 2) Avoid skin grafting and therefore iatrogenic damages; and 3) Improve QoL by reducing pain, the length of hospital stay and recurrences.

Materials And Methods
From January 2001 to January 2016, 24 patients affected by dystrophic EB were treated (13M, 11F) at the Plastic and Reconstructive Surgery Department in Ospedali Riuniti OORR, Foggia, Italy. In all patients, the indication for surgical treatment was the functional impairment of the hands. The data of all patients treated were collected and analysed retrospectively. The range of patient age was 6-15 years old (average age 8.7).
Eleven patients, with a total of 15 mitten hands, underwent surgical release of the digital rays and dressing with vaseline gauze (Group A). Thirteen patients, with a total of 19 mitten hands, underwent surgical release of the web spaces, finger and wrist contractions, followed by reconstruction with gloves of INTEGRA® dermal regeneration template (Group B) ( Table I). This group of patients had a minimum follow up of 18 months (range 1.5-9 years). 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 (Table II).
In 2001, we began to treat mitten hand in Epidermolysis bullosa patients. Our therapeutic protocol consisted of debridement of the pseudo-syndactyly and, in severe cases, 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. Healing with complete reepithelization of the hands occurred in about 75 days (Fig 2), during which patients were medicated every 48-72 hours and, in selected cases, given intravenous sedation. Dressings were painful for the patients, in particular when, to completely release their fingers, the vascular-nerves pedicels were exposed. Patients were also often children, so the need for a sedation or nerve block was greater. In order to carry out these procedures, it was necessary to have an average hospital stay of about 30 days with great difficulties for the patient and the family members. Since 2006, we have started an innovative protocol the we called IGP as "INTEGRA®-gloves protocol". 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), we perform surgical debridement of the pseudo-syndactyly. In fact, the use of microsurgical technique allows us to perform more precisely and safely the release of scar contractions with greater preservation of vascular-nerve axes which especially in paediatric patients, are very thin and easily vulnerable. Due to this attentiveness, we do not have partial or total cutaneous necrosis in patients treated with the current protocol. We apply K-wires in all hands to fix the metacarpo-phalangeal joints in extension, taking care not to induce ischemia in the digital rays.
After release, we perform coverage through the use of a double-layer dermal substitute (INTEGRA® dermal regeneration template) (Fig 3). In fact, surgical procedures involve a degloving of the hands of these patients. Through "tailoring" we make double-layer, custom-made gloves with INTEGRA® and apply them to the hands of patients with absorbable sutures. We use bioengineered tissue on all the surface of the hands, for the known INTEGRA® anti-inflammatory properties. The rationale for this choice being that a reduction in tissue inflammation is very effective in reducing pain during postoperative dressings. After finishing the coverage, we use vaseline-gauze dressings. All patients undergo perioperative short term prophylaxis with penicillin (according to their weight) and paracetamol is used for pain control. The average hospital stay time is approximately 72 hours. The first dressing change is performed on the eighth postoperative day, without the need for analgesic sedation or nerve block. Indeed, the presence of the non-adhering silicone layer of the dermal substitute results in a renewal of painless dressings. Around the 30th day, the silicone layer is removed and almost the entire surface of the hands is completely healed (spontaneous reepithelialization) thanks to the INTEGRA® cover. The few residual areas are medicated with Vaseline gauze with complete healing time in about 40 days (Fig 4). We use silicone night splints for 60 days ( Fig 5).
The study protocol was approved by the Local Medical Ethics Committee in accordance with the Helsinki Declaration of 1975, as amended in 2008. Informed consent was obtained from all patients for inclusion in the study.
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 significant.

Results
In both groups treated, no patient reported major complications such as infection and total necrosis of the fingers. 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). 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®. 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. The average time to free recurrence was 19.1 ± 5.3 months for group A and 31.1 ± 4.7 months for B.
Comparison of the two treatment groups was performed using a Mann-Whitney U test with a value of p less than 0.05 considered statistically significant.
There were a statistically significant difference in hospital stay, in healing time, in complete healing times, in discomfort after dressings and, finally, in time to free recurrence. 6

Discussion
Recessive dystrophic forms of EB are burdened with a high psychological impact for patients and family members. A multidisciplinary approach to manage the disease is crucial. In addition, "mitten hands" compromise the functional development of the hands in children with EB. So, an early surgical approach is of paramount importance. In our experience, but also in literature, pseudosindatiles in patients with this disease are characterized by high rates of recurrence [6][7] . The scar tissue of the hands can alter the anatomical structures, including the vascular-nerve pedicels. Conventional surgical techniques for "mitten hand" are limited to the release of pseudosyndactylies and wound coverage. During surgical procedures, care should be taken not to damage the vascular peduncles, in order to avoid necrotic skin phenomena with additional iatrogenic damage. Split-thickness skin grafts provide coverage of wide areas, however, availability of healthy skin for autologous grafts of dermis and difficulty healing at the donor site can be major problems in EB patients. The use of skin substitutes in patients with bullous epidermolysis has been described in the literature; often in combination with skin grafts [9][10] . The surgical approach is limited to a temporary resolution [7][8][9][10] . Of paramount importance are the function preservation and the increase of free recurrence time.
Through a careful analysis of our technique, it was possible to improve our results thanks to the use of microsurgical technique and the use of INTEGRA® dermal regeneration template.
In fact, we believe that magnifying loupes have allowed us to preserve the vascular-nervous peduncles 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.
Artificial 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, a flexible 7 adherent covering for the wound surface, and heat and moisture modulation of the wound, prevent formation of granulation tissue on the matrix surface and adds increased tear strength to the device.
Whereas the inner layer, composed of bovine tendon type I collagen cross-linked to chondroitin-6sulfate, is a biologic-based acellular dermal scaffold and promotes cellular ingrowth (it's histoconductive/ histoinductive and allow for fibroblast 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 first stage. It acts as a template for the ingrowth of host fibroblasts and endothelial cells and is gradually replaced by host (endogenous) collagen, forming a new dermal layer (neodermis). At approximately 3 weeks, when well-vascularized neodermis is usually formed, the silicone layer may be peeled off and replaced with cultured epithelial cells or autologous thin split-thickness skin graft (applied directly to the surface of the vascularized neodermis).
The major disadvantage of the artificial dermis use is the need for a second operative procedure to provide epidermal coverage; moreover, 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 artificial 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-inflammatory 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 peduncles, 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 slight 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 8 the patient at their initial submission are also considerable factors, which will be studied in subsequent 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 infiltration, or deep sedation with ketamine and fentanyl plus local anaesthetic infiltration. 11 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 justified. 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 qualitative rather than standardized and based on the Surgeon's experience. In spite of these limitations, the significant differences between both groups cannot be ignored, suggesting the clear repercussions of this new protocol on long lasting healing and on quality of life 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 findings in this study may be useful for other surgery Units; however, it would be desirable to have a multi-center study with other Hand Surgery departments that take care of these deformities. The multidisciplinary approach is fundamental in the treatment of the "mitten hand" deformity in patients affected by EB.
Moreover, the potential improvement in QoL for patients with EB should be rated at its absolute inestimable value.  Follow-up range (years, range) 3.7 -8 1.5-9

Tables
Major complications 0 0 Figures 12 Figure 1 13 appearance of mitten-hand with imprisonment of the fingers, thumb adduction and extensory deficit.  silicone splints are necessary for 60 days at least.