Randomized clinical trial
Randomized clinical trial of autologous skin cell suspension combined with skin grafting for chronic wounds Z.-C. Hu1 , D. Chen2 , D. Guo3 , Y.-Y. Liang1 , J. Zhang1 , J.-Y. Zhu1 and B. Tang1 Departments of 1 Burn Surgery and 2 Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, and 3 Department of Plastic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Correspondence to: Dr B. Tang, Department of Burn Surgery, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China (e-mail: [email protected])
Background: Treatment of chronic wounds using traditional surgical procedures is challenging because
of the low graft take rates. This study investigated the combination approach of split-thickness autografts with harvested skin cell suspension for chronic wound treatment. Methods: This randomized clinical trial enrolled patients with chronic wounds between March 2012 and December 2013. Patients who were assigned randomly to the active treatment received a split-thickness autograft combined with harvested skin cell suspension. Control patients received the split-thickness autograft alone. The primary outcome was the rate of complete wound closure by postoperative day 28. Analysis was by intention to treat. Patients who achieved wound closure were followed up for a minimum of 6 months to evaluate the quality of healing. Results: A total of 88 patients were included, 44 in each group. More patients achieved complete wound closure in the skin cell group than in the control group (41 versus 34 patients; P = 0⋅035). Complete wound closure was observed at a median of 14 (95 per cent c.i. 12⋅0 to 16⋅0) days in the skin cell group and 20 (15⋅7 to 24⋅3) days in the control group (P = 0⋅001). The skin cell group had significantly fewer complications (4 versus 11 patients; P = 0⋅047). The autografted sites displayed better physical attributes and a reduced tendency for wound recurrence in the skin cell group. Conclusion: Complementary split-thickness autologous skin grafting with autologous skin cells harvested using ReCell® (Avita Medical, Cambridge, UK) technology improved the healing rate of chronic wounds. Registration number: UMIN000011966 (http://www.umin.ac.jp/ctr). Click here to watch video footage recorded by the author about the contents of this paper. Paper accepted 29 September 2014 Published online in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.9688
Introduction
Treatment of chronic wounds resulting from trauma, diabetes, infections, blood vessel disorders and sustained external compression remains challenging1 – 4 . Despite varied and complex aetiologies, the factors contributing to the chronic nature of non-healing wounds are similar, including increased activity of proteases, ischaemia and hypoxia, and bacterial burdens5 , which impair cell migration, proliferation and remodelling. With increasing incidence and growing prevalence of lifestyle-related disorders, chronic wounds have become a serious global health issue, causing an enormous financial burden on healthcare systems worldwide6,7 . The conventional treatment of chronic wounds is based on the underlying aetiology8 – 10 . Unfortunately, current © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
treatment modalities act slowly and have limited success. There is strong evidence to support the use of split-thickness skin grafts in treating acute (burns) and chronic wounds11,12 . These grafts provide integrated wound coverage and a conducive healing environment by excluding pathogenic bacteria and providing extracellular matrix, keratinocytes, fibroblasts and bioactive molecules (cytokines, chemokines and growth factors) that facilitate wound repair11,12 . However, split-thickness skin grafts have some limitations, including a high rate of loss of graft skin, resulting in incomplete healing of some wounds. The low endurance of split-thickness skin grafts against pressure and friction may result in graft loss. The innovation of ReCell® (Avita Medical, Cambridge, UK) technology has made available a tool for creating BJS 2015; 102: e117–e123
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an autologous, heterogeneous skin cell suspension that includes approximately 65 per cent keratinocytes, 30 per cent fibroblasts and 3⋅5 per cent melanocytes13 . It is reported that re-epithelialization is dependent on the ability of keratinocyte to form holo-clones14 . In addition, melanocytes and fibroblasts are believed to contribute to the repigmentation process15 . These viable cells, extracted from the dermal basal layer, are capable of adhering to a wound surface and proliferating to promote healing13 . The procedure requires only a small donor population of autologous basal layer cells, and allows immediate application of the cell suspension without the need for isolation and expansion. Therefore, patients can be treated without any delays for culture. Autologous skin cell suspension has provided alternative treatment options for burn injuries, especially when the affected area is too large, or there is a lack of sufficient donor skin16,17 . It has also been used widely on cutaneous injury and skin lesions, including congenital melanocytic naevus18 and vitiligo lesions19 , and on donor sites20 . Recently, De Angelis and colleagues21 reported the use of skin cells obtained by the ReCell® system to treat chronic ulcers without autografting, which resulted in 70 per cent of patients attaining re-epithelialization between postoperative days 40 and 60. The present study was conducted to investigate whether a combination of split-thickness autologous skin grafting and autologous skin cell suspension for chronic wound treatment could improve the graft take rate and wound healing, and achieve better aesthetic outcomes. A randomized clinical trial was performed to compare the clinical outcomes of conventional split-thickness skin grafts with, and without autologous skin cells for chronic wounds.
diagnosed with other medical conditions that could hinder wound healing, such as autoimmune diseases, concomitant use of corticosteroids, immunosuppressive medications or chemotherapy, and hypersensitivity to trypsin or Hartmann’s solution, were excluded from the study. Patients considered to be at anaesthetic risk were also excluded. Suitable patients were informed about the potential benefits and risks of the procedure, and written informed consent was obtained from all study participants. Eligible patients were assigned randomly to an experimental group in a 1:1 ratio between March 2012 and December 2013. Randomization was done using opaque, consecutively numbered, sealed envelopes. Randomization was achieved by generating random numbers through a computerized randomization system (without stratification). Procedures imperative to chronic wound treatment, such as wound bed preparation, were standard for all patients included in the study. Specifically, first-stage debridement surgery was performed within 3 days of randomization. All necrotic and infected tissue was removed, followed by repeated changes of a silver-coated wound dressing until the wound bed was healthy with fresh granulation tissue. The wound bed was then tested for the absence of bacterial infection. Systemic antibiotics were used, if necessary, and in accordance with the microorganisms identified in the wound.
Methods
The study was approved by the Institutional Review Board of The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. A chronic wound was defined as one that failed to heal for more than 4 weeks. Patients with a chronic wound unresponsive to conventional therapies were recruited into the study. They were assessed to assure adequate perfusion, and declared eligible for skin grafting. In patients with diabetes, adequate blood supply was assessed by a dorsum transcutaneous oxygen level greater than 30 mmHg, ankle : brachial pressure index above 0⋅9, or Doppler arterial waveforms that were triphasic or biphasic at the ankle of the affected leg. Chronic venous ulcers were classified according to clinical signs of venous insufficiency, and confirmed by colour venous Duplex imaging. Pregnant women and people aged less than 18 years were not included in the trial. Patients © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
Surgical procedure All patients in the treatment group received unmeshed split-thickness skin autografts in combination with autologous skin cell suspension, whereas all patients in the control group received standard unmeshed split-thickness skin autografts. At operation, the wounds were again debrided thoroughly. All necrotic and infected tissue was removed until the healthy tissue became visible (Fig. 1a,b); nominal amounts of bleeding appropriate for the wound type, and according to the surgeon’s experience, were allowed. Haemostasis was achieved using bipolar electrocautery. Concurrently, skin cells were harvested with the ReCell® kit using methods established by the manufacturer, as follows. To achieve a cellular spread rate of 1:80, a biopsy area of 1 cm2 is required to treat a recipient area of 80 cm2 . The harvested skin biopsy was subjected to proteolytic dissociation by incubating in trypsin for 15–20 min at 37∘ C, followed by rinsing of the tissue in a sodium lactate solution (to remove residual trypsin and terminate the digestion). The dermis and epidermis were then separated mechanically, and cells (keratinocytes, melanocytes, fibroblasts and Langerhans cells) at the epidermal–dermal junction were scraped off using a sterile scalpel to generate a cell www.bjs.co.uk
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a
Ulcer on dorsum of left foot
d
Grafting of split-thickness autologous skin sheet
b
Wound debridement
e
14 days after surgery
c
Application of autologous cell suspension
f
28 weeks after surgery
A 57-year-old man with a diabetic foot ulcer for 156 weeks. a A large (approximately 180 cm2 ) ulcer on the dorsum of the left foot; b wound debridement; c application of an autologous cell suspension to the prepared wound bed by the dripping method; d grafting of split-thickness autologous skin sheet; e appearance on postoperative day 14; f appearance at week 28
Fig. 1
suspension, which was then filtered through a 100-μm pore filter for subsequent use. After curettage, the cell suspension was either dripped or sprayed over the prepared wound bed (Fig. 1c). Split-thickness skin was harvested from the unaffected thigh using a dermatome, and applied unmeshed to cover the wound area. The split-thickness skin autografts formed
a closed and continuous junction, and were secured using sutures or fibrin glue (PW®; Guangzhou Baiyun Medical Adhesive Company, Guangzhou, China) (Fig. 1d). A non-adherent hydrocolloid dressing, Urgotul® (Laboratoire Urgo, Chenôve, France) was used as the first layer to cover the wound22 . The wound was covered further using conventional dressing. Additional external fixation with
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plaster was used for chronic wounds that included a joint extremity. In the control group, all procedures were identical, but the cell suspension was not employed.
texture, elasticity, and presence of scars and blebs, according to the Manchester Scar Scale (MSS)19 , which uses a range of scores from 5 (best) to 18 (worst). In addition, these autografted sites were examined for signs of ulcer recurrence.
Postoperative care The first postoperative dressing change was done after 5 days, followed by two or three changes per week. Dressings were also changed at each follow-up visit, with Urgotul® as the primary dressing followed by conventional bandages. Compression therapy, where required, was started after the patients were discharged from hospital. Patients achieving complete wound closure were followed up for 6–9 months. At each follow-up visit, the skin autografts were evaluated for quality of healing and physical appearance.
Evaluation of wound healing The primary efficacy endpoint was the rate of complete wound healing at 28 days (full re-epithelialization, without any requirement for dressing)23 . Secondary endpoints included time to achieve complete wound closure and treatment-related complications. Independent assessments were made by two experienced and blinded surgeons. Serial wound photographs, taken with a digital camera, were reviewed separately by a blinded expert in wound care. During long-term follow-up visits, the autografts were evaluated with regard to their colour, aesthetic appearance,
The sample size calculation was based on the primary outcome (incidence of complete wound closure by postoperative day 28). Detection of a 25 per cent difference between the autologous skin cell suspension and control groups required a sample size of 43 per group with 80 per cent power and a two-tailed type I error of 0⋅05. An intention-to-treat (ITT) approach was used to analyse data that included all randomly assigned patients and protocol deviations. Continuous demographic variables are presented as mean(s.d.). Statistical significance between groups was determined by two-sample t test. The incidence of complete wound closure and the rate of postoperative complications between groups were analysed by χ2 or Fisher’s exact test, as appropriate. Median time to wound closure was determined on the basis of the number of days until complete wound closure, using Kaplan–Meier analysis of cumulative wound healing, followed by a log rank test. MSS scores are presented as median (i.q.r.) values and analysed by means of Kruskal–Wallis analysis. P < 0⋅050 (two-sided) was considered significant. All data were analysed using SPSS® version 16.0 software (IBM, Armonk, New York, USA). Assessed for eligibility n = 88
Enrolment Fig. 2
Statistical analysis
Allocation
Allocated to autologous skin cells + STSG n = 44 Received autologous skin cells + STSG n = 43 Did not receive autologous skin cells + STSG n = 1 Withdrawal of consent n = 1
Allocated to STSG n = 44 Received STSG n = 43 Did not receive STSG n = 1 Withdrawal of consent n = 1
Follow-up
Lost to follow-up n = 0
Lost to follow-up n = 1 Patient withdrew n = 1
Analysis
Randomized n = 88
Analysed n = 44
Analysed n = 44
CONSORT diagram for the trial. STSG, split-thickness skin graft
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Results
Eighty-eight patients were enrolled between March 2012 and December 2013; two patients did not receive treatment but were included in the ITT analysis (Fig. 2). The protocol deviations were uncommon and distributed equally. Although the ITT analysis is reported here, the results of the per-protocol analysis were similar. The median size of the chronic wounds was 48 (range 20–300) cm2 . The two patient groups had similar demographics and wound properties (Table 1). Thirty-six patients in the autologous skin cell group and 38 patients in the control group received additional immobilizing devices. Complete wound closure (Fig. 1e) was achieved within 28 days in significantly more patients in the treatment group than in the control group (41 versus 34; P = 0⋅035). In addition, time to healing was significantly shorter in patients who had the skin cell application: the Kaplan–Meier median estimate for complete wound closure was 14 (95 per cent c.i. 12⋅0 to 16⋅0) days versus 20 (15⋅7 to 24⋅3) days in controls (P = 0⋅001) (Fig. 3). Postoperative complications, including haematoma, infection, liquefaction and necrosis, are shown in Table 2. Significantly fewer complications occurred after additional application of autologous skin cells than in controls (4 versus 11; P = 0⋅047). However, no significant differences were observed for any of the complications. Patients were followed up for a mean(s.d.) of 7⋅2(1⋅2) months. During follow-up visits, the skin grafts with additional skin cell grafting appeared to possess good elasticity and texture, were similar in colour to Table 1
Patient demographics and wound characteristics Autologous skin cells (n = 44)
Control (n = 44)
48(20) 30:14
44(18) 28:16
14 8 9 6 1 3 3 36(44)
11 9 10 8 2 1 3 34(42)
17 13 8 3 3 65⋅0(48⋅3)
16 15 8 3 2 58⋅8(47⋅2)
Age (years)* Sex ratio (M:F) Cause of wound Diabetes Trauma Residual burn wounds Vascular Pressure Scar Other Duration of wound (weeks)* Location of wound Foot Leg Arm Trunk Head Area of wound (cm2 )* 2
*Values are mean(s.d.). †χ test, except ‡two-sample t test.
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P† 0⋅389‡ 0⋅653 0⋅933
0⋅785‡ 0⋅990
0⋅553‡
Cumulative wound healing
100
80
60
40
Autologous skin cells
20
Control 7
0
14
21
28
Time to complete closure (days) No. at risk Skin cells 43 Control 43
43 43
20 29
9 20
2 9
Kaplan–Meier estimates of time taken to achieve complete wound closure in autologous skin cell and control groups. P = 0⋅001 (log rank test)
Fig. 3
Table 2
Complications
Haematoma Liquefaction Necrosis Infection
Autologous skin cells* (n = 44)
Control† (n = 44)
χ2
d.f.
P‡
0 4 1 1
1 7 4 3
– 0⋅935 1⋅908 1⋅048
– 1 1 1
1⋅000§ 0⋅334 0⋅065 0⋅125
*Two patients had two complications and two had one complication; †four patients had two complications and seven had one complication. ‡χ2 test, except §Fisher’s exact test.
surrounding skin, and displayed less scar tissue growth at the border between normal and grafted skin (Fig. 1f ). Median (i.q.r.) MSS scores were lower in the autologous skin cell group than in the control group (9 (7⋅25–10) versus 10 (9–12) respectively; P = 0⋅005). No recurrent ulcers were recorded in the autologous skin cell group, whereas three patients in the control group developed new wounds, which included one diabetic wound, one pressure wound and one vascular wound. These patients underwent secondary surgical intervention. Discussion
The present study examined the clinical outcomes of using a combination of split-thickness autologous skin grafts and autologous skin cell suspension to treat chronic wounds. The results suggest this combination therapy is more effective and safer than split-thickness skin grafts alone. More patients who had the combination therapy achieved complete wound closure within 28 days, and their www.bjs.co.uk
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healing rate was significantly quicker. The autologous skin cell group also had significantly fewer complications. Long-term follow-up data on the aesthetic outcomes further confirmed the efficacy of split-thickness skin grafts plus autologous skin cells. The recipient sites displayed good elasticity and texture, similar pigmentation to the surrounding skin, reduced scarring at the wound edges, and no recurrent ulceration. In general, the healing rate of refractory wounds is low owing to inherent problems associated with wound aetiology. Under normal physiological conditions, wound healing is a complex process that requires adequate circulation, nutrition and immune competence to induce the migration, proliferation and differentiation of multiple cell types including keratinocytes, fibroblasts, endothelial cells, macrophages and platelets. These cells fight infection at the site of injury, form new tissue and ultimately lead to wound closure. In contrast, chronic wounds are characterized by interrupted wound healing, bacterial colonization, diminished capillary perfusion, local tissue hypoxia, and altered cellular and systemic stress responses. Here, the wound bed preparation, including clearing of bacteria and debridement of necrotic tissue, was critical to the improved wound healing rate in the autologous skin cell group. The use of autologous skin was necessary to provide integrated wound coverage. Creation of a favourable healing environment allowed the harvested autologous cell suspension, consisting of keratinocytes, melanocytes, fibroblasts and Langerhans cells, to function effectively. The migration, infiltration, proliferation and differentiation of these healthy basal layer cells culminated in the formation of new tissue and speedy re-epithelialization. It is known that collagenase plays an important role in initiating the wound healing process by dissolving necrotic tissue and digesting redundant collagen that can impair healing. It is possible that the autologous skin cell suspension might release collagenase into the wound site; however, this needs to be corroborated with further experimental evidence. The autologous cell suspension consists predominantly of keratinocytes and fibroblasts, along with some melanocytes and Langerhans cells13 . It has been shown that cellular interactions at wound sites promote fibroblast attachment and produce new connective tissue matrix. The viable cells extracted from the basal lamina in the harvest procedure contain actively proliferating cells, which remain viable, migrate through the microenvironment of the wound site and self-organize to form a differentiated epidermis. When sprayed on to a recently autografted wound bed, cells may differentiate into vascular endothelial cells, fibroblasts or melanocytes, resulting in quicker re-epithelialization of the wound.
In the present study, patients in the autologous skin cell group had significantly fewer complications. This may have been due to the expedited wound healing in this group. Other advantages include the easy use of the ReCell® kit, and the quick and immediate application of the autologous cell suspension. Although only a small site is required for harvesting donor cells, the resulting cell suspension is sufficient to cover a wound 80 times larger than the donor site. The present study suggests that treatment of chronic wounds using the combination of split-thickness skin autograft and autologous skin cell suspension has several advantages, including improved wound healing, long-term aesthetic appearance and fewer clinical complications. Larger studies are necessary for further confirmation of the efficacy of this combination approach for treating different types of wound.
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Acknowledgements
Z.C.H., D.C. and D.G. contributed equally to this study. The authors thank F. Zhou for her support in randomizing patients and statistical analyses. This study was supported by grants from the National Natural Science Foundation of China (nos 81272096 and 30973128), the Research Fund for the Doctoral Programme of Higher Education of China (PhD supervisor; no. 20110171110064) and the Sun Yat-sen University Clinical Research 5010 Programme (no. 2013001). Disclosure: The authors declare no conflict of interest. References 1 Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999; 341: 738–746. 2 Jiang Y, Huang S, Fu X, Liu H, Ran X, Lu S et al. Epidemiology of chronic cutaneous wounds in China. Wound Repair Regen 2011; 19: 181–188. 3 Mekkes JR, Loots MA, Van Der Wal AC, Bos JD. Causes, investigation and treatment of leg ulceration. Br J Dermatol 2003; 148: 388–401. 4 Angel MF, Ramasastry SS, Swartz WM, Basford RE, Futrell JW. The causes of skin ulcerations associated with venous insufficiency: a unifying hypothesis. Plast Reconstr Surg 1987; 79: 289–297. 5 Mustoe TA, O’Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg 2006; 17(Suppl): 35S–41S. 6 Wicke C, Bachinger A, Coerper S, Beckert S, Witte MB, Königsrainer A. Aging influences wound healing in patients with chronic lower extremity wounds treated in a specialized wound care center. Wound Repair Regen 2009; 17: 25–33. 7 Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK et al. Human skin wounds: a major and snowballing
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threat to public health and the economy. Wound Repair Regen 2009; 17: 763–771. Schultz GS, Sibbald RG, Falanga V, Ayello EA, Dowsett C, Harding K et al. Wound bed preparation: a systematic approach to wound management. Wound Repair Regen 2003; 11(Suppl): 1S–28S. Fan K, Tang J, Escandon J, Kirsner RS. State of the art in topical wound-healing products. Plast Reconstr Surg 2011; 127(Suppl): 44S–59S. Attinger CE, Janis JE, Steinberg J, Schwartz J, Al-Attar A, Couch K. Clinical approach to wounds: débridement and wound bed preparation including the use of dressings and wound-healing adjuvants. Plast Reconstr Surg 2006; 117(Suppl): 72S–109S. Tang B, Zhu B, Liang YY, Bi LK, Chen B, Hu ZC et al. Early escharectomy and concurrent composite skin grafting over human acellular dermal matrix scaffold for covering deep facial burns. Plast Reconstr Surg 2011; 127: 1533–1538. Mahmoud SM, Mohamed AA, Mahdi SE, Ahmed ME. Split-skin graft in the management of diabetic foot ulcers. J Wound Care 2008; 17: 303–306. Wood FM, Giles N, Stevenson A, Rea S, Fear M. Characterisation of the cell suspension harvested from the dermal epidermal junction using a ReCell® kit. Burns 2012; 38: 44–51. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A 1987; 84: 2302–2306. Yamaguchi Y, Morita A, Maeda A, Hearing VJ. Regulation of skin pigmentation and thickness by Dickkopf 1 (DKK1). J Investig Dermatol Symp Proc 2009; 14: 73–75. Wood F, Martin L, Lewis D, Rawlins J, McWilliams T, Burrows S et al. A prospective randomised clinical pilot
study to compare the effectiveness of Biobrane® synthetic wound dressing, with or without autologous cell suspension, to the local standard treatment regimen in paediatric scald injuries. Burns 2012; 38: 830–839. Gravante G, Di Fede MC, Araco A, Grimaldi M, De Angelis B, Arpino A et al. A randomized trial comparing ReCell system of epidermal cells delivery versus classic skin grafts for the treatment of deep partial thickness burns. Burns 2007; 33: 966–972. O’Neill TB, Rawlins J, Rea S, Wood F. Treatment of a large congenital melanocytic nevus with dermabrasion and autologous cell suspension (ReCELL®): a case report. J Plast Reconstr Aesthet Surg 2011; 64: 1672–1676. Mulekar SV, Ghwish B, Al Issa A, Al Eisa A. Treatment of vitiligo lesions by ReCell vs. conventional melanocyte– keratinocyte transplantation: a pilot study. Br J Dermatol 2008; 158: 45–49. Campanella SD, Rapley P, Ramelet AS. A randomised controlled pilot study comparing Mepitel® and SurfaSoft® on paediatric donor sites treated with ReCell®. Burns 2011; 37: 1334–1342. De Angelis B, Migner A, Lucarini L, Agovino A, Cervelli V. The use of a non cultured autologous cell suspension to repair chronic ulcers. Int Wound J 2013; 28. doi: 10.1111/ iwj.12047. Meaume S, Senet P, Dumas R, Carsin H, Pannier M, Bohbot S. Urgotul: a novel non-adherent lipidocolloid dressing. Br J Nurs 2002; 11(Suppl): 42S–43S, 46S–50S. US Food and Drug Administration. Guidance for Industry: Chronic Cutaneous Ulcer and Burn Wounds – Developing Products for Treatment; 2006. http://www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm071324.pdf [accessed 27 May 2012].
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BJS 2015; 102: e117–e123
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Randomized clinical trial of autologous skin cell suspension combined with skin grafting for chronic wounds Z.-C. Hu1 , D. Chen2 , D. Guo3 , Y.-Y. Liang1 , J. Zhang1 , J.-Y. Zhu1 and B. Tang1 Departments of 1 Burn Surgery and 2 Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, and 3 Department of Plastic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Correspondence to: Dr B. Tang, Department of Burn Surgery, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China (e-mail: [email protected])
Background: Treatment of chronic wounds using traditional surgical procedures is challenging because
of the low graft take rates. This study investigated the combination approach of split-thickness autografts with harvested skin cell suspension for chronic wound treatment. Methods: This randomized clinical trial enrolled patients with chronic wounds between March 2012 and December 2013. Patients who were assigned randomly to the active treatment received a split-thickness autograft combined with harvested skin cell suspension. Control patients received the split-thickness autograft alone. The primary outcome was the rate of complete wound closure by postoperative day 28. Analysis was by intention to treat. Patients who achieved wound closure were followed up for a minimum of 6 months to evaluate the quality of healing. Results: A total of 88 patients were included, 44 in each group. More patients achieved complete wound closure in the skin cell group than in the control group (41 versus 34 patients; P = 0⋅035). Complete wound closure was observed at a median of 14 (95 per cent c.i. 12⋅0 to 16⋅0) days in the skin cell group and 20 (15⋅7 to 24⋅3) days in the control group (P = 0⋅001). The skin cell group had significantly fewer complications (4 versus 11 patients; P = 0⋅047). The autografted sites displayed better physical attributes and a reduced tendency for wound recurrence in the skin cell group. Conclusion: Complementary split-thickness autologous skin grafting with autologous skin cells harvested using ReCell® (Avita Medical, Cambridge, UK) technology improved the healing rate of chronic wounds. Registration number: UMIN000011966 (http://www.umin.ac.jp/ctr). Click here to watch video footage recorded by the author about the contents of this paper. Paper accepted 29 September 2014 Published online in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.9688
Introduction
Treatment of chronic wounds resulting from trauma, diabetes, infections, blood vessel disorders and sustained external compression remains challenging1 – 4 . Despite varied and complex aetiologies, the factors contributing to the chronic nature of non-healing wounds are similar, including increased activity of proteases, ischaemia and hypoxia, and bacterial burdens5 , which impair cell migration, proliferation and remodelling. With increasing incidence and growing prevalence of lifestyle-related disorders, chronic wounds have become a serious global health issue, causing an enormous financial burden on healthcare systems worldwide6,7 . The conventional treatment of chronic wounds is based on the underlying aetiology8 – 10 . Unfortunately, current © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
treatment modalities act slowly and have limited success. There is strong evidence to support the use of split-thickness skin grafts in treating acute (burns) and chronic wounds11,12 . These grafts provide integrated wound coverage and a conducive healing environment by excluding pathogenic bacteria and providing extracellular matrix, keratinocytes, fibroblasts and bioactive molecules (cytokines, chemokines and growth factors) that facilitate wound repair11,12 . However, split-thickness skin grafts have some limitations, including a high rate of loss of graft skin, resulting in incomplete healing of some wounds. The low endurance of split-thickness skin grafts against pressure and friction may result in graft loss. The innovation of ReCell® (Avita Medical, Cambridge, UK) technology has made available a tool for creating BJS 2015; 102: e117–e123
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Z.-C. Hu, D. Chen, D. Guo, Y.-Y. Liang, J. Zhang, J.-Y. Zhu and B. Tang
an autologous, heterogeneous skin cell suspension that includes approximately 65 per cent keratinocytes, 30 per cent fibroblasts and 3⋅5 per cent melanocytes13 . It is reported that re-epithelialization is dependent on the ability of keratinocyte to form holo-clones14 . In addition, melanocytes and fibroblasts are believed to contribute to the repigmentation process15 . These viable cells, extracted from the dermal basal layer, are capable of adhering to a wound surface and proliferating to promote healing13 . The procedure requires only a small donor population of autologous basal layer cells, and allows immediate application of the cell suspension without the need for isolation and expansion. Therefore, patients can be treated without any delays for culture. Autologous skin cell suspension has provided alternative treatment options for burn injuries, especially when the affected area is too large, or there is a lack of sufficient donor skin16,17 . It has also been used widely on cutaneous injury and skin lesions, including congenital melanocytic naevus18 and vitiligo lesions19 , and on donor sites20 . Recently, De Angelis and colleagues21 reported the use of skin cells obtained by the ReCell® system to treat chronic ulcers without autografting, which resulted in 70 per cent of patients attaining re-epithelialization between postoperative days 40 and 60. The present study was conducted to investigate whether a combination of split-thickness autologous skin grafting and autologous skin cell suspension for chronic wound treatment could improve the graft take rate and wound healing, and achieve better aesthetic outcomes. A randomized clinical trial was performed to compare the clinical outcomes of conventional split-thickness skin grafts with, and without autologous skin cells for chronic wounds.
diagnosed with other medical conditions that could hinder wound healing, such as autoimmune diseases, concomitant use of corticosteroids, immunosuppressive medications or chemotherapy, and hypersensitivity to trypsin or Hartmann’s solution, were excluded from the study. Patients considered to be at anaesthetic risk were also excluded. Suitable patients were informed about the potential benefits and risks of the procedure, and written informed consent was obtained from all study participants. Eligible patients were assigned randomly to an experimental group in a 1:1 ratio between March 2012 and December 2013. Randomization was done using opaque, consecutively numbered, sealed envelopes. Randomization was achieved by generating random numbers through a computerized randomization system (without stratification). Procedures imperative to chronic wound treatment, such as wound bed preparation, were standard for all patients included in the study. Specifically, first-stage debridement surgery was performed within 3 days of randomization. All necrotic and infected tissue was removed, followed by repeated changes of a silver-coated wound dressing until the wound bed was healthy with fresh granulation tissue. The wound bed was then tested for the absence of bacterial infection. Systemic antibiotics were used, if necessary, and in accordance with the microorganisms identified in the wound.
Methods
The study was approved by the Institutional Review Board of The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. A chronic wound was defined as one that failed to heal for more than 4 weeks. Patients with a chronic wound unresponsive to conventional therapies were recruited into the study. They were assessed to assure adequate perfusion, and declared eligible for skin grafting. In patients with diabetes, adequate blood supply was assessed by a dorsum transcutaneous oxygen level greater than 30 mmHg, ankle : brachial pressure index above 0⋅9, or Doppler arterial waveforms that were triphasic or biphasic at the ankle of the affected leg. Chronic venous ulcers were classified according to clinical signs of venous insufficiency, and confirmed by colour venous Duplex imaging. Pregnant women and people aged less than 18 years were not included in the trial. Patients © 2015 BJS Society Ltd Published by John Wiley & Sons Ltd
Surgical procedure All patients in the treatment group received unmeshed split-thickness skin autografts in combination with autologous skin cell suspension, whereas all patients in the control group received standard unmeshed split-thickness skin autografts. At operation, the wounds were again debrided thoroughly. All necrotic and infected tissue was removed until the healthy tissue became visible (Fig. 1a,b); nominal amounts of bleeding appropriate for the wound type, and according to the surgeon’s experience, were allowed. Haemostasis was achieved using bipolar electrocautery. Concurrently, skin cells were harvested with the ReCell® kit using methods established by the manufacturer, as follows. To achieve a cellular spread rate of 1:80, a biopsy area of 1 cm2 is required to treat a recipient area of 80 cm2 . The harvested skin biopsy was subjected to proteolytic dissociation by incubating in trypsin for 15–20 min at 37∘ C, followed by rinsing of the tissue in a sodium lactate solution (to remove residual trypsin and terminate the digestion). The dermis and epidermis were then separated mechanically, and cells (keratinocytes, melanocytes, fibroblasts and Langerhans cells) at the epidermal–dermal junction were scraped off using a sterile scalpel to generate a cell www.bjs.co.uk
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a
Ulcer on dorsum of left foot
d
Grafting of split-thickness autologous skin sheet
b
Wound debridement
e
14 days after surgery
c
Application of autologous cell suspension
f
28 weeks after surgery
A 57-year-old man with a diabetic foot ulcer for 156 weeks. a A large (approximately 180 cm2 ) ulcer on the dorsum of the left foot; b wound debridement; c application of an autologous cell suspension to the prepared wound bed by the dripping method; d grafting of split-thickness autologous skin sheet; e appearance on postoperative day 14; f appearance at week 28
Fig. 1
suspension, which was then filtered through a 100-μm pore filter for subsequent use. After curettage, the cell suspension was either dripped or sprayed over the prepared wound bed (Fig. 1c). Split-thickness skin was harvested from the unaffected thigh using a dermatome, and applied unmeshed to cover the wound area. The split-thickness skin autografts formed
a closed and continuous junction, and were secured using sutures or fibrin glue (PW®; Guangzhou Baiyun Medical Adhesive Company, Guangzhou, China) (Fig. 1d). A non-adherent hydrocolloid dressing, Urgotul® (Laboratoire Urgo, Chenôve, France) was used as the first layer to cover the wound22 . The wound was covered further using conventional dressing. Additional external fixation with
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plaster was used for chronic wounds that included a joint extremity. In the control group, all procedures were identical, but the cell suspension was not employed.
texture, elasticity, and presence of scars and blebs, according to the Manchester Scar Scale (MSS)19 , which uses a range of scores from 5 (best) to 18 (worst). In addition, these autografted sites were examined for signs of ulcer recurrence.
Postoperative care The first postoperative dressing change was done after 5 days, followed by two or three changes per week. Dressings were also changed at each follow-up visit, with Urgotul® as the primary dressing followed by conventional bandages. Compression therapy, where required, was started after the patients were discharged from hospital. Patients achieving complete wound closure were followed up for 6–9 months. At each follow-up visit, the skin autografts were evaluated for quality of healing and physical appearance.
Evaluation of wound healing The primary efficacy endpoint was the rate of complete wound healing at 28 days (full re-epithelialization, without any requirement for dressing)23 . Secondary endpoints included time to achieve complete wound closure and treatment-related complications. Independent assessments were made by two experienced and blinded surgeons. Serial wound photographs, taken with a digital camera, were reviewed separately by a blinded expert in wound care. During long-term follow-up visits, the autografts were evaluated with regard to their colour, aesthetic appearance,
The sample size calculation was based on the primary outcome (incidence of complete wound closure by postoperative day 28). Detection of a 25 per cent difference between the autologous skin cell suspension and control groups required a sample size of 43 per group with 80 per cent power and a two-tailed type I error of 0⋅05. An intention-to-treat (ITT) approach was used to analyse data that included all randomly assigned patients and protocol deviations. Continuous demographic variables are presented as mean(s.d.). Statistical significance between groups was determined by two-sample t test. The incidence of complete wound closure and the rate of postoperative complications between groups were analysed by χ2 or Fisher’s exact test, as appropriate. Median time to wound closure was determined on the basis of the number of days until complete wound closure, using Kaplan–Meier analysis of cumulative wound healing, followed by a log rank test. MSS scores are presented as median (i.q.r.) values and analysed by means of Kruskal–Wallis analysis. P < 0⋅050 (two-sided) was considered significant. All data were analysed using SPSS® version 16.0 software (IBM, Armonk, New York, USA). Assessed for eligibility n = 88
Enrolment Fig. 2
Statistical analysis
Allocation
Allocated to autologous skin cells + STSG n = 44 Received autologous skin cells + STSG n = 43 Did not receive autologous skin cells + STSG n = 1 Withdrawal of consent n = 1
Allocated to STSG n = 44 Received STSG n = 43 Did not receive STSG n = 1 Withdrawal of consent n = 1
Follow-up
Lost to follow-up n = 0
Lost to follow-up n = 1 Patient withdrew n = 1
Analysis
Randomized n = 88
Analysed n = 44
Analysed n = 44
CONSORT diagram for the trial. STSG, split-thickness skin graft
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Results
Eighty-eight patients were enrolled between March 2012 and December 2013; two patients did not receive treatment but were included in the ITT analysis (Fig. 2). The protocol deviations were uncommon and distributed equally. Although the ITT analysis is reported here, the results of the per-protocol analysis were similar. The median size of the chronic wounds was 48 (range 20–300) cm2 . The two patient groups had similar demographics and wound properties (Table 1). Thirty-six patients in the autologous skin cell group and 38 patients in the control group received additional immobilizing devices. Complete wound closure (Fig. 1e) was achieved within 28 days in significantly more patients in the treatment group than in the control group (41 versus 34; P = 0⋅035). In addition, time to healing was significantly shorter in patients who had the skin cell application: the Kaplan–Meier median estimate for complete wound closure was 14 (95 per cent c.i. 12⋅0 to 16⋅0) days versus 20 (15⋅7 to 24⋅3) days in controls (P = 0⋅001) (Fig. 3). Postoperative complications, including haematoma, infection, liquefaction and necrosis, are shown in Table 2. Significantly fewer complications occurred after additional application of autologous skin cells than in controls (4 versus 11; P = 0⋅047). However, no significant differences were observed for any of the complications. Patients were followed up for a mean(s.d.) of 7⋅2(1⋅2) months. During follow-up visits, the skin grafts with additional skin cell grafting appeared to possess good elasticity and texture, were similar in colour to Table 1
Patient demographics and wound characteristics Autologous skin cells (n = 44)
Control (n = 44)
48(20) 30:14
44(18) 28:16
14 8 9 6 1 3 3 36(44)
11 9 10 8 2 1 3 34(42)
17 13 8 3 3 65⋅0(48⋅3)
16 15 8 3 2 58⋅8(47⋅2)
Age (years)* Sex ratio (M:F) Cause of wound Diabetes Trauma Residual burn wounds Vascular Pressure Scar Other Duration of wound (weeks)* Location of wound Foot Leg Arm Trunk Head Area of wound (cm2 )* 2
*Values are mean(s.d.). †χ test, except ‡two-sample t test.
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P† 0⋅389‡ 0⋅653 0⋅933
0⋅785‡ 0⋅990
0⋅553‡
Cumulative wound healing
100
80
60
40
Autologous skin cells
20
Control 7
0
14
21
28
Time to complete closure (days) No. at risk Skin cells 43 Control 43
43 43
20 29
9 20
2 9
Kaplan–Meier estimates of time taken to achieve complete wound closure in autologous skin cell and control groups. P = 0⋅001 (log rank test)
Fig. 3
Table 2
Complications
Haematoma Liquefaction Necrosis Infection
Autologous skin cells* (n = 44)
Control† (n = 44)
χ2
d.f.
P‡
0 4 1 1
1 7 4 3
– 0⋅935 1⋅908 1⋅048
– 1 1 1
1⋅000§ 0⋅334 0⋅065 0⋅125
*Two patients had two complications and two had one complication; †four patients had two complications and seven had one complication. ‡χ2 test, except §Fisher’s exact test.
surrounding skin, and displayed less scar tissue growth at the border between normal and grafted skin (Fig. 1f ). Median (i.q.r.) MSS scores were lower in the autologous skin cell group than in the control group (9 (7⋅25–10) versus 10 (9–12) respectively; P = 0⋅005). No recurrent ulcers were recorded in the autologous skin cell group, whereas three patients in the control group developed new wounds, which included one diabetic wound, one pressure wound and one vascular wound. These patients underwent secondary surgical intervention. Discussion
The present study examined the clinical outcomes of using a combination of split-thickness autologous skin grafts and autologous skin cell suspension to treat chronic wounds. The results suggest this combination therapy is more effective and safer than split-thickness skin grafts alone. More patients who had the combination therapy achieved complete wound closure within 28 days, and their www.bjs.co.uk
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healing rate was significantly quicker. The autologous skin cell group also had significantly fewer complications. Long-term follow-up data on the aesthetic outcomes further confirmed the efficacy of split-thickness skin grafts plus autologous skin cells. The recipient sites displayed good elasticity and texture, similar pigmentation to the surrounding skin, reduced scarring at the wound edges, and no recurrent ulceration. In general, the healing rate of refractory wounds is low owing to inherent problems associated with wound aetiology. Under normal physiological conditions, wound healing is a complex process that requires adequate circulation, nutrition and immune competence to induce the migration, proliferation and differentiation of multiple cell types including keratinocytes, fibroblasts, endothelial cells, macrophages and platelets. These cells fight infection at the site of injury, form new tissue and ultimately lead to wound closure. In contrast, chronic wounds are characterized by interrupted wound healing, bacterial colonization, diminished capillary perfusion, local tissue hypoxia, and altered cellular and systemic stress responses. Here, the wound bed preparation, including clearing of bacteria and debridement of necrotic tissue, was critical to the improved wound healing rate in the autologous skin cell group. The use of autologous skin was necessary to provide integrated wound coverage. Creation of a favourable healing environment allowed the harvested autologous cell suspension, consisting of keratinocytes, melanocytes, fibroblasts and Langerhans cells, to function effectively. The migration, infiltration, proliferation and differentiation of these healthy basal layer cells culminated in the formation of new tissue and speedy re-epithelialization. It is known that collagenase plays an important role in initiating the wound healing process by dissolving necrotic tissue and digesting redundant collagen that can impair healing. It is possible that the autologous skin cell suspension might release collagenase into the wound site; however, this needs to be corroborated with further experimental evidence. The autologous cell suspension consists predominantly of keratinocytes and fibroblasts, along with some melanocytes and Langerhans cells13 . It has been shown that cellular interactions at wound sites promote fibroblast attachment and produce new connective tissue matrix. The viable cells extracted from the basal lamina in the harvest procedure contain actively proliferating cells, which remain viable, migrate through the microenvironment of the wound site and self-organize to form a differentiated epidermis. When sprayed on to a recently autografted wound bed, cells may differentiate into vascular endothelial cells, fibroblasts or melanocytes, resulting in quicker re-epithelialization of the wound.
In the present study, patients in the autologous skin cell group had significantly fewer complications. This may have been due to the expedited wound healing in this group. Other advantages include the easy use of the ReCell® kit, and the quick and immediate application of the autologous cell suspension. Although only a small site is required for harvesting donor cells, the resulting cell suspension is sufficient to cover a wound 80 times larger than the donor site. The present study suggests that treatment of chronic wounds using the combination of split-thickness skin autograft and autologous skin cell suspension has several advantages, including improved wound healing, long-term aesthetic appearance and fewer clinical complications. Larger studies are necessary for further confirmation of the efficacy of this combination approach for treating different types of wound.
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Acknowledgements
Z.C.H., D.C. and D.G. contributed equally to this study. The authors thank F. Zhou for her support in randomizing patients and statistical analyses. This study was supported by grants from the National Natural Science Foundation of China (nos 81272096 and 30973128), the Research Fund for the Doctoral Programme of Higher Education of China (PhD supervisor; no. 20110171110064) and the Sun Yat-sen University Clinical Research 5010 Programme (no. 2013001). Disclosure: The authors declare no conflict of interest. References 1 Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999; 341: 738–746. 2 Jiang Y, Huang S, Fu X, Liu H, Ran X, Lu S et al. Epidemiology of chronic cutaneous wounds in China. Wound Repair Regen 2011; 19: 181–188. 3 Mekkes JR, Loots MA, Van Der Wal AC, Bos JD. Causes, investigation and treatment of leg ulceration. Br J Dermatol 2003; 148: 388–401. 4 Angel MF, Ramasastry SS, Swartz WM, Basford RE, Futrell JW. The causes of skin ulcerations associated with venous insufficiency: a unifying hypothesis. Plast Reconstr Surg 1987; 79: 289–297. 5 Mustoe TA, O’Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg 2006; 17(Suppl): 35S–41S. 6 Wicke C, Bachinger A, Coerper S, Beckert S, Witte MB, Königsrainer A. Aging influences wound healing in patients with chronic lower extremity wounds treated in a specialized wound care center. Wound Repair Regen 2009; 17: 25–33. 7 Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK et al. Human skin wounds: a major and snowballing
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threat to public health and the economy. Wound Repair Regen 2009; 17: 763–771. Schultz GS, Sibbald RG, Falanga V, Ayello EA, Dowsett C, Harding K et al. Wound bed preparation: a systematic approach to wound management. Wound Repair Regen 2003; 11(Suppl): 1S–28S. Fan K, Tang J, Escandon J, Kirsner RS. State of the art in topical wound-healing products. Plast Reconstr Surg 2011; 127(Suppl): 44S–59S. Attinger CE, Janis JE, Steinberg J, Schwartz J, Al-Attar A, Couch K. Clinical approach to wounds: débridement and wound bed preparation including the use of dressings and wound-healing adjuvants. Plast Reconstr Surg 2006; 117(Suppl): 72S–109S. Tang B, Zhu B, Liang YY, Bi LK, Chen B, Hu ZC et al. Early escharectomy and concurrent composite skin grafting over human acellular dermal matrix scaffold for covering deep facial burns. Plast Reconstr Surg 2011; 127: 1533–1538. Mahmoud SM, Mohamed AA, Mahdi SE, Ahmed ME. Split-skin graft in the management of diabetic foot ulcers. J Wound Care 2008; 17: 303–306. Wood FM, Giles N, Stevenson A, Rea S, Fear M. Characterisation of the cell suspension harvested from the dermal epidermal junction using a ReCell® kit. Burns 2012; 38: 44–51. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A 1987; 84: 2302–2306. Yamaguchi Y, Morita A, Maeda A, Hearing VJ. Regulation of skin pigmentation and thickness by Dickkopf 1 (DKK1). J Investig Dermatol Symp Proc 2009; 14: 73–75. Wood F, Martin L, Lewis D, Rawlins J, McWilliams T, Burrows S et al. A prospective randomised clinical pilot
study to compare the effectiveness of Biobrane® synthetic wound dressing, with or without autologous cell suspension, to the local standard treatment regimen in paediatric scald injuries. Burns 2012; 38: 830–839. Gravante G, Di Fede MC, Araco A, Grimaldi M, De Angelis B, Arpino A et al. A randomized trial comparing ReCell system of epidermal cells delivery versus classic skin grafts for the treatment of deep partial thickness burns. Burns 2007; 33: 966–972. O’Neill TB, Rawlins J, Rea S, Wood F. Treatment of a large congenital melanocytic nevus with dermabrasion and autologous cell suspension (ReCELL®): a case report. J Plast Reconstr Aesthet Surg 2011; 64: 1672–1676. Mulekar SV, Ghwish B, Al Issa A, Al Eisa A. Treatment of vitiligo lesions by ReCell vs. conventional melanocyte– keratinocyte transplantation: a pilot study. Br J Dermatol 2008; 158: 45–49. Campanella SD, Rapley P, Ramelet AS. A randomised controlled pilot study comparing Mepitel® and SurfaSoft® on paediatric donor sites treated with ReCell®. Burns 2011; 37: 1334–1342. De Angelis B, Migner A, Lucarini L, Agovino A, Cervelli V. The use of a non cultured autologous cell suspension to repair chronic ulcers. Int Wound J 2013; 28. doi: 10.1111/ iwj.12047. Meaume S, Senet P, Dumas R, Carsin H, Pannier M, Bohbot S. Urgotul: a novel non-adherent lipidocolloid dressing. Br J Nurs 2002; 11(Suppl): 42S–43S, 46S–50S. US Food and Drug Administration. Guidance for Industry: Chronic Cutaneous Ulcer and Burn Wounds – Developing Products for Treatment; 2006. http://www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm071324.pdf [accessed 27 May 2012].
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