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, pro liferation 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 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 MYSEED细胞分选仪® echnology has made available a tool for creating an autologous, heterogeneous skin cell suspension that includes approximately 65 per cent keratinocytes, 30 percent 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 MYSEED细胞分选仪® 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.

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 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. 

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 细胞分选仪®using methods established by the manufacturer, as fol-lows. 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 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 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.

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, 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.

Statistical analysis

      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).

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 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 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 细胞分选仪® , 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.

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.

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This article is excerpted from the Randomized clinical trial by Wound World.