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Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns
JBUR-4546; No. of Pages 11 burns xxx (2015) xxx–xxx
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Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns Xueyong Li 1, Xianghai Meng 1, Xiaolin Wang 1, Yuejun Li, Wangzhou Li, Xiaoxing Lv, Xiaoli Xu, Zhanjun Lei, Jinqing Li * Department of Burn and Plastic Surgery, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Road, Xi’an 710038, China
article info
abstract
Article history:
The potential of acellular dermal matrix (ADM) to improve cosmetic and functional out-
Accepted 5 December 2014
comes has been demonstrated; however, there have been few clinical comparative studies assessing the long-term morphological, histological and functional changes after ADM
Keywords:
placement. This study was designed to retrospectively evaluate the long-term outcomes
Acellular dermal matrix
of the cograft acellular dermal matrix with autologous thin split-thickness skin for the
Wound repair
coverage of wounds in extensively burned patients. Thirty burn patients treated with a
Long-term outcomes of ADM
composite graft of ADM with autologous split-thickness skin from January 2007 to December 2009 were enrolled in this study. Another group of thirty patients who received only an autogenous split-thickness skin implant served as the control. Our study revealed that the collagen in the dermis treated with ADM were ordered, and the proportion of collagen III/I was much higher in the control group than in the ADM group. The basement membrane was prominent and continuous. Meanwhile, the VBSS (Vancouver Burn Skin Score) was used to evaluate skin quality, which shows a significant differences between the two group (P < 0.001). Then the functional level was evaluated by the BI (Barthel Index), and the ADM group was much better than the control group (P = 0.005). Based on these results, we concluded that the composite graft of ADM with autologous thin split-thickness skin was suitable for repairing the defects in functional areas after a burn. This technique might facilitate wound management with acceptable esthetic outcomes, good functional recovery and less scar hyperplasia at the donor site. # 2014 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Burn wound coverage techniques have been greatly improved; patients with more than 90% total body surface
area (TBSA) burns can now expect a fighting chance for survival [1–3]. Nevertheless, the lack of donor sites in major burn patients and secondary hypertrophic scar formation remain two major clinical challenges. The problem of the lack of donor sites is that the need for donor sites sometimes
* Corresponding author. Tel.: +86 29 84777440; fax: +86 29 84777440. E-mail address: [email protected] (J. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.burns.2014.12.007 0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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exceeds the available unburned skin; to approach this problem, autologous thin-thickness skin grafts were developed. The intermediate meshed split skin graft for recipient site wound closure after excision of the burned area remains the gold standard for treatment. However, split-skin harvest and transplantation are accompanied by frustrating consequences, such as wound contraction, donor site morbidity, and hypertrophy [4–6]. The lack of a dermal component in burn wounds after treatment with split-thickness autografts resulting in the poor quality of healing in recipient sites created the need for alternative graft sources [7]. The advent of allo-dermal replacement has revolutionized the therapeutic potential for the success rate and quality of healing in split-skin transplantation. In 1981, a composite of bovine collagen and chondroitin-6-sulfate from shark cartilage with an outer silicone covering was first engineered as an organotypic dermia for skin grafting [8]. In the same year, Sarber et al. developed a type of skin equivalent that is composed of natural collagen extraction and fibroblasts according to a specific mixing ratio [9]. Composite skin grafting was used extensively thereafter. In 1985, Heck reported using allogeneic dermis for wound closure [10]. He found that allogeneic dermis grafts caused immunological rejection mainly due to cellular immunity [11]. Subsequently, all of the cellular components of the dermis and epidermis were processed for removal, and the extracellular matrix and basement membrane (BM) were preserved. Such ‘‘decellularized’’ allografts had been effectively used alone or in combination with cultured autologous keratinocytes for the closure of burns and chronic wounds in the next year and were considered to have the potential to be a permanent dermal alternative [11]. Until 1995, Wainwright used composite transplantation to repair burn wounds with acellular and allogeneic dermis and reticular autologous thin-split grafts; this method avoided immune rejection [12]. Subsequently, upon histological observation, researchers found that the epithelial cells were well covered and that there was less scar proliferation with good effect. Since then, composite skin grafts have been widely used in both wound healing and scar plastic surgery [13–16]. However, there is a lack of literature regarding the longterm follow-up data of randomized, controlled human studies, especially in the aspects of histological changes, contour and function. In the present study, we used the Vancouver Burn Scar Scale (VBSS) and Barthel Index (BI) Scale to conduct a long-term evaluation of contour and functional outcomes following co-transplantation of a split-thickness skin graft (STSG) with human acellular dermal matrix.
2.
Materials and methods
2.1.
Clinical data
abdominal trauma), elderly patients with co-morbid diseases such as chronic cardiovascular disease, diabetes mellitus or hypertension, and those who had significant inhalation injuries or needed intensive care were excluded from the study. Eligible patients were randomized at admission to either ADM or STSG group. Randomization was performed following a predetermined random order produced in groups of 10. This preordered format was maintained by the primary investigator. Patients who were excluded were assigned randomization but not included in the subsequent analysis. The 2 groups are as follows: the acellular dermal matrix (ADM) group (n = 30), in which the wounds were successfully covered with ADM and autologous epidermis, and the control group (n = 30), in which the wounds were covered with autologous split-thickness skin. The general patient characteristics are listed in Table 1; the types of grafts used for burn treatments are listed in Table 2. Of the 30 patients in the experimental group, 25 were male and the remaining 5 patients were female; their ages ranged from 3 to 52 years, with an average age of 30.53 10.73 years. Thirty sites from 20 patients underwent surgical burn wound excision and were grafted with acellular dermal substitutes and covered with thin split-thickness skin that was not expanded. A similar procedure was conducted on five sites of four patients with mature granulation tissue in later burn stages and on seven sites of six patients with scar plasticity. The skin graft sites included the neck, hand, elbow, shoulder, popliteal site, knee and foot back, among others. The largest area of composite skin graft was 600 cm2; the minimum area was 40 cm2. Of the control group, the mean age of the 30 patients was 25.30 15.88 years with a range of 2–57 years; 24 patients were males, and 6 were females. This study was reviewed and approved by the institutional ethics committee. Patients with electrical burns, fourthdegree burns, burns as components of multiple traumas (patients with fractures or central nervous system, thoracic and abdominal trauma), elderly patients with co-morbid diseases such as chronic cardiovascular disease, diabetes mellitus or hypertension, and those who had significant inhalation injuries or needed intensive care were excluded from the study.
2.2.
Materials
This investigation involved commercial ADM products from Jie-Ya Life Tissue Engineering (Beijing, China) that were approved by the Chinese Food and Drug Administration for transplantation. ADM is a dermal collagen matrix extracted from human skin by removing the majority of the cellular
Table 1 – General patient characteristics.
This study was reviewed and approved by the institutional ethics committee. A total of 60 patients who sustained burns provided consent to participate in this study over 3 years (2007–2009), and patients with electrical burns, fourth-degree burns, burns as components of multiple traumas (patients with fractures or central nervous system, thoracic and
Type
Average age Male/female Scar plasticity Granulation tissue Burn
Number ADM group
Control group
30.53 10.73 25/5 6 (20%) 4 (13.33%) 20 (66.67%)
25.30 15.33 24/6 2 (6.67%) 11 (36.67%) 17 (56.67%)
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Table 2 – Types of grafts for burn treatment. Type
Classification
2.3.
Number ADM group
Control group
Wound area
<10% 10–30% 31–50% >50%
8 14 4 4
15 9 3 3
Surgical site
Lower limbs Upper limbs Ankles and feet Hands Trunk
13 4 4 4 5
15 8 0 3 4
Donor site
Head Back Thigh
9 15 6
3 21 6
Area of skin grafting
<100 cm 2 100–200 cm 2 >200 cm 2
7 15 8
3 15 12
Survival rate
100% 80–99% <80%
16 12 2
19 11 0
components. Briefly, a 4-mm-thick slice is cut from cadaveric skin using a dermatome; the sample is treated with 2.5 unit/ml Dispase II and 0.2 mM CaCl2 in phosphate-buffered saline (PBS) at 4 8C for 24 h. The dermal matrix is then incubated in 0.5% Triton X-100 for an additional 24 h at room temperature and stored in a PBS solution containing sodium azide (0.02% w/v) at 4 8C. The specimen is maintained at 2–8 8C, washed with 0.9% normal saline several times before surgery, and submerged in normal saline containing 1000–2000 U/ml gentamicin for 10 min.
3
Surgical procedure
The operation was performed within the first week of admittance if the patient’s condition was appropriate. After irrigation and hemostasis of the excised wound, grafts were applied to the wound bed for the treatment using one-step [17] placement, namely synchronous application of acellular dermis and autologous thin-thickness skin (Fig. 1). The category of the burn wounds consisted of different types of wounds with respect to the clinical appearance of depth and debridement. Full-thickness burn wounds were debrided by epifascial excision. Tangential excision was performed on paired wounds of partialthickness burn wounds. The appropriate size of acellular dermal matrix would be placed on the prepared wound and moderately stretched to maintain a certain tension. Thin split-thickness autologous skin (0.2–0.3 mm) was harvested from an uninvolved area using a Zimmer dermatome (Zimmer, Indiana, USA) and then trimmed and placed on the allogeneic dermis; this graft was fixed with skin staples. Vaseline gauze was used for dressing the graft, the area was bandaged based on the involved body parts, and the functional parts were fixed safely. After 3 days, the dressings were changed for the first time, and the staples were removed after 5–7 days. Sheet grafts were predominantly used over joints or in limited burn areas when sufficient donor areas were available.
2.4. Masson’s trichrome staining and picrosirius red staining for histological analysis 2.4.1.
Masson staining
Slide sections, 4 mm in thickness, were prepared from the paraffin-embedded skin tissue of skin grafts from both groups.
Fig. 1 – The surgical procedure of ADM and STSG. The wound surfaces before skin grafting of both ADM (panel A) and STSG (panel C), and the intra-operative photographs after grafting of the ADM (panel B) and STSG (panel D). Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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All of the sections were stained with Masson’s trichrome stain on the mounted serial 4-mm-thick sections for observation of the collagen framework. Standard procedures for this staining were performed as recommended by the manufacturers. A single pathologist, who was blinded to the treatment regimen, viewed the stained sections.
2.4.2.
Picrosirius polarization
The specimens, including full-thickness skin layers (epidermis, dermis, and hypodermis), were fixed in 4% buffered paraformaldehyde and were processed according to routine light microscope tissue processing methods, and the processed tissues were then embedded in paraffin. Tissue sections (8 mm) were selected for picrosirius red staining. Finally, picrosirius staining followed by polarized light microscopy was used to visualize the collagen fibers with digital capture and analysis [18].
2.4.3.
Image capture and analysis
Image Pro PlusTM image analysis software was used to digitally capture the images using a firewire camera. The images were stored on a personal computer. Image Pro PlusTM was used to analyze the slides in detail. Using the computer software, we were able to differentiate the red and the green areas that were selected, and the percentages of type I and type III collagen per slide were calculated. This process was repeated for each of the five randomly selected areas of the ADM and control groups. The data were then transferred directly to the spreadsheet program ExcelTM (Microsoft Corporation, Redmond, WA) for storage and further analysis.
2.5.
Immunohistochemistry
Immunohistochemistry experiments were performed on specimens from the two groups and the thin split-thickness autologous skin. Rabbit antibodies against laminin were acquired from Zhongshan Goldenbridge Biotechnology Co. LTD. Cultured tissues were fixed with cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Small pieces of each tissue were processed into 4-mm-thick transverse sections. The sections were heated at 65 8C for 30 min to soften the wax, dewaxed by incubation in xylene (2 5 min), rehydrated through a series of graded ethanol steps (100%, 2 5 min; 90%, 5 min; 80%, 5 min; 75%, 5 min), and then rinsed in tap water. After treatment with 0.3% H2O2 for 20 min, antigen retrieval was performed using 0.4% pepsin applied for 20 min, and the samples were then washed with PBS (pH 7.4). The tissue sections were blocked with 5% bovine serum albumin (BSA) for 20 min at room temperature followed by incubation with the primary antibody diluted in antibody dilution solution with background-reducing components for 18 h at 4 8C. Then, the sections were incubated with a biotinylated secondary antibody for 20 min at 37 8C. Streptavidin–biotin peroxidase was applied for 20 min at room temperature, which was followed by washing. Development was performed using diaminobenzidine followed by nuclear staining with Mayer’s hematoxylin (1 min). Finally, the specimens were observed by microscopy after balata enveloping.
2.6.
Wound site evaluation
The acceptance rate of the split-thickness autografts (sheet and mesh grafts) was defined as the percentage of the entire graft that was considered to be vital and that showed good adherence to the wound bed 5–7 days after surgery. The acceptance was assessed for both treatment groups at the same time point to allow for paired statistical analyses. In mesh graft transplantation, epithelialization of the interstices of the graft is required to achieve complete wound closure. The wound was considered closed when more than 95% of the grafted surface area was epithelialized. The necessity of regrafting was registered, which is regarded as indicative of the success of the initial transplantation.
2.7. Scar elasticity parameters: The Vancouver Burn Skin Score (VBSS) The VBSS was used to the evaluate skin quality after a minimum of 3 months [19]. This score consists of various parameters: elasticity, pliability, pigmentation and vascularization. Each parameter has a score of 0–3, and the total score is added together. The VBSS is an established, reliable and valid score in the evaluation of burn scars.
2.8.
Physical function assessment: The Barthel Index (BI)
The BI is an assessment of a patient’s functional level of independence in Activities of Daily Living (ADL) and is scored in increments of 5 points (highest possible total score = 100) [20]. The values assigned to each item are weighed according to the amount of physical assistance required if the patient could not perform the activity independently. The 10 ADL items assessed in the BI are feeding, bathing, personal hygiene, toilet use, bladder control, bowel control, dressing, wheelchair transfer to and from bed, walking, and ascending and descending stairs.
2.9.
Statistical analysis
Continuous variables with skewed distributions are presented as medians (min, max) and were analyzed using the Mann– Whitney test. P < 0.05 was considered statistically significant. All of the statistical analyses were performed using SPSS v16.0.
3.
Results
3.1.
Clinical observations
Composite skin transplantation was performed for the treatment of 42 wounds on 30 patients after burns and plastic surgery; 39 wound composite skin grafts survived, and there was partial survival in 3 grafts. The skin graft survival standard of a composite skin graft is as follows: at two weeks after the surgery, survival of more than 90% of the epidermis and dermis is regarded as complete survival; non-survival of more than 40% of the epidermis and/or dermis is regarded as no survival; survival of regions between 40 and 90% is considered partial survival. Skin flushing was apparent after
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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composite skin graft survival. After three months, the color of the composite skin was similar to the color of to the normal skin, and there was good elasticity; no composite skin graft contracture or scars were observed.
there were no blisters (Fig. 3B). Follow-up at 33 months revealed that the appearance of the composite skin was similar to the normal skin; there was good elasticity, no scar, and good joint function (Fig. 3C and D).
Typical Case 1
Typical Case 3
Following a vapor explosion, a 38-year-old woman suffered burns affecting 30% TBSA of both lower extremities. Following hospitalization, the burn pattern was identified as deep II degree. The other areas of the body were undamaged. On the 16th day after admission, escharectomy of the posterior portion of the right leg corresponding to 15% TBSA was performed, followed by coverage with composite skin (Fig. 2A); all of the grafts survived after 12 days (Fig. 2B). Follow-up after 30 months revealed that the color of the replacement appeared almost the same as the normal skin; there was good elasticity, and no scar was observed (Fig. 2C and D).
A 28-year-old individual presented to our burn unit following a sulfuric acid spill at work; the spill resulted in 16% TSBA deep partial-thickness burns and 5% TSBA full-thickness burns, mainly on both lower limbs. The surgical management involved the debridement of all of the non-viable burned tissue. An acellular allogeneic human dermis matrix was used to cover the wound with no meshed split-thickness skin graft (Supplemental Fig. 1). Good acceptance of the graft was accomplished at the first dressing change 12 days post-surgery (Supplemental Fig. 1). After a follow-up period of 34 months, the appearance of the transplanted composite skin was close to the appearance of normal skin, with good elasticity, and no scar was observed (Supplemental Fig. 1).
Typical Case 2 A previously healthy 28-year-old male sustained a flame injury to 46% TBSA. The injury consisted of deep partialthickness burns and full-thickness burns to both lower extremities. The patient received fluid resuscitation, and enteral nutritional support was initiated via a nasogastric tube. The wounds were dressed with topical silver sulfadiazine. On postburn day three, the patient underwent surgery for escharectomy and wound closure with a composite skin graft on the bilateral knees and a micro skin graft on other parts of the legs. On the twelfth post-operative day, the dressing change revealed 100% survival of the skin grafts (Fig. 3A). The elasticity of the composite skin was good after 31 d, and
Comparative study The two patients in the control group with autogenous split-thickness skin obtained non-satisfactory outcomes (Fig. 4B, C, E, F, H and I). The appearance, elasticity, and function of the grafts in the ADM group (Fig. 4A, D and G) were much better compared to the split-thickness group.
3.2. Masson’s trichrome staining and picrosirius red staining for histological analysis Masson’s trichrome staining was performed on both allogeneic dermal tissue and tissue with no implanted
Fig. 2 – Photographs of Case 1. The intra-operative site after coverage with a one-stage sheet autograft (panel A). A photograph taken 12 days after surgery shows the preliminary results (panel B). A close-up of the front portion of the lower extremity revealing skin without scar lines (panel C). The functional outcome 30 months after epidermal skin replacement (panel D). Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Fig. 3 – Photographs of Case 2. The post-operative view at 12 days after surgery (panel A). A photograph taken 31 days after surgery shows the post-operative view (panel B). After 33 months of follow-up, there were satisfactory effects with a good appearance and good function (panel C, panel D).
allogeneic dermis. The different layers of the epidermis were well identified in the histological images (including the stratum corneum disjunctum, stratum corneum compactum, stratum granulosum, stratum spinosum, and stratum basale), and the collagen fibers in the dermis layer were very clear (Fig. 5A); the cells in the epidermis and the vessels in the dermis stained red, collagen stained blue, and nuclei stained black. The collagen of the acellular dermal tissue appeared stretched and aligned in the same plane as the epidermis; there were few blood vessels, and there was good continuity of the basal layer (Fig. 5B). The collagen in the tissue with the autogenous split-thickness skin implant appeared relaxed and was arranged in a random array; there was poor continuity of the basal layer, there were a large number of blood vessels, and scars appeared to form easily (Fig. 5C). Collagen deposition increases with scar tissue, and the fibers became thicker, changing from yellow/red (type I collagen) to green (type III collagen). The proportion of collagen III/I was much higher in the hypertrophic scars than in the normal skin [19,20]. We determined that the ratio of collagen III/I was higher in the tissue with autogenous split-thickness skin implants and that the ratio was much lower in the ADM tissue compared to normal skin (Fig. 6).
3.3.
Immunohistochemistry of the basement membrane
Tissues were stained with laminin antibodies against the major BM constituents. The staining demonstrated that the autologous thin-thickness skin, which covered the ADM, excluded the BM (Fig. 7A). Intense and continuous lines of
laminin deposits were observed (Fig. 7B). Compared with the samples of the ADM skin, the BM of the control group was not clearly observed (Fig. 7C).
3.4.
The Vancouver Burn Skin Score outcome
The outcomes of the scar evaluation parameters by VBSS are shown in Table 3. The recovery time was regarded as an important factor of the effectiveness of wound repair. However, the use of ADM was another significant means for obtaining acceptable esthetic outcomes. The differences in the total score at 6 months were statistically significant (Z = 6.302, P < 0.001) (Table 4). The median of the ADM group was 9, and the median of the control group was 13. The differences in the total score at 30 months were also statistically significant (Z = 6.680, P < 0.001). The median of the ADM group was 5, and the median of the control group was 9. The medians of the ADM group were lower compared to those of the control group.
3.5.
The Barthel Index outcome
The statistical results of the physical functional assessment by the BI are shown in Table 5. The ADL index of the ADM group was higher compared to the ADL index of the control group. When the total scores of the ADM and control groups (Table 6) were compared, the differences were statistically significant (Z = 2.814, P = 0.005). The median of the ADM group was 100, which was higher than the median of the control group (99.50).
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Fig. 4 – Photographs of the appearance, elasticity, and function of the patients’ grafts. The case with the ADM allograft shows a good appearance (panel A). The views of the control group with autogenous split-thickness skin (panel B, panel C). The suitable elasticity of the case with the ADM implant (panel D). The views of the control group with autogenous split-thickness skin were not much better than the views of the ADM group (panel E, panel F). The good function of the case with suitable elasticity with the ADM implant; the patient was able to squat easily (panel G). The functional abilities of the control group were not as prominent (panel H, panel I).
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Fig. 5 – Histopathological features of the skin. The tissue with the ADM allograft shows the entire structure of the skin (panel A, T400). The collagen was arranged in an orderly manner in the skin with the ADM (panel B, T100). The control group with autogenous split-thickness skin showed irregular collagen arrangements (panel C, T100). The skin with autogenous splitthickness skin grafting revealed a high content of collagen III (panel D, T200). However, the content of collagen III was much lower in the group with the ADM allograft (panel E, T200).
4.
Discussion
In the treatment of major burns, it is important to focus on ensuring that the ‘‘quality of life is worth the pain of survival’’ [21]. Normal skin is repeatedly harvested from donor sites in the treatment of extensive burns, which limits the transfer of autologous skin, especially after harvesting the medium split-thickness skin. In the past 30 years, there have been many attempts toward improving skin replacement, such as the use of meshed skin, meek grafts, micro-skin transplantations and others [22–24]. In these successful techniques, cultured epithelium autografts appear to be a potential
Fig. 6 – Graph showing the type III/I collagen ratios in the ADM and control groups.
alternative. In 1984, Gallico showed that full-thickness skin burn patients could be saved using cultured epithelium autografts (CEAs). Unfortunately, because of the wide patient-to-patient variation and the lack of controlled clinical research, it is difficult to determine if there is a place for cultured epithelial grafts in the treatment of major burns [25]. Nevertheless, studies have shown the dermal component deficiency and the increased possibility of contractive scar formation at the recipient sites [26–28]. This situation could be changed by the combined use of the STSG with an allogeneic acellular dermal component [29], which preserves the natural mixture of structural and functional proteins that constitute the extracellular matrix, such as collagen, fibronectin, laminin and vimentin [30–32]; the cell components, which possess strong immune cell composition in allogeneic acellular dermal matrices, are excluded during this process. The allo-dermal matrix is the carrier of cell transplantation and the ‘‘soil’’ of cell proliferation; additionally, the three-dimensional structure of dermal tissue could support fibroblast infiltration, neovascularization, and keratinocyte migration from overlying STSG [29] and could improve the state of mechanical force in a wound, promoting tissue remodeling, reducing hypertrophic scar formation, and improving the healing quality of the wound. The present study notes that ADM shows good affinity and excellent appearance and is easy to slide (Fig. 4). The histological staining revealed that after the composite skin is transferred, the structure of the composite graft survival skin is similar to the normal skin area in the region receiving the composite skin transplantation (Fig. 7). Because the split-thickness skin is transplanted on the dermal matrix, hyperplasia is decreased, which could prevent the occurrence of scar contractures.
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Table 4 – Vancouver Score analysis. Grouping ADM group Control group Z P
6 months
30 months
9.0 (8.0, 11.0) 13.0 (10.0, 14.0) 6.302 <0.001
5.0 (4.0, 5.0) 9.0 (5.0, 11.0) 6.680 <0.001
Table 5 – Barthel Index Scale Scores. Item
Scores (mean SD)
1—Feeding 2—Bathing 3—Personal hygiene 4—Toilet use 5—Bladder control 6—Bowel control 7—Dressing 8—Wheelchair transfer to and from bed 9—Walking 10—Ascending and descending stairs Total
Fig. 7 – Immunohistochemistry of the BM. The autologous thin-thickness skin that covered the allogeneic dermis contains no BM (panel A, T400). The outcome of the ADM skin showed a continuous BM. The black arrow indicates the areas of the BM, which are strongly stained for laminin (panel B, T400). The BM of the control group was not clearly observed (panel C, T400).
The role of the BM is important for maintaining the integrity and structure of an epithelial layer, as it acts as a mechanical support and forms the physical interface between epithelial cells and the surrounding connective tissue [34,35]. What would the epidermal–dermal composites after a cograft be like? As described in the information sheets, the J-1 acellular dermis reserved the BM. In our study, the autologous split-thickness skin harvested from the patient’s scalp or thigh were a thickness of 0.2 mm and excluded the BM, as shown in Fig. 7A. After the cograft, the epidermal–dermal composites survived well in principle. Immunohistological staining revealed that the autogenous epithelial sheets consisted of
ADM group
Control group
9.80 0.61 4.73 0.94 4.90 0.31 10.00 0.00 10.00 0.00 10.00 0.00 9.93 0.37 14.90 0.55
9.87 0.51 4.80 0.41 4.70 0.47 9.27 0.98 10.00 0.00 10.00 0.00 9.53 0.86 14.40 1.22
15.00 0.00 9.93 0.37
14.83 0.91 9.73 0.69
99.20 2.34
97.13 4.57
more layers of contiguous cells that are anchored to the integrated BM, which separates the epithelium from the underlying connective tissues (Fig. 7). A solid combination of autologous epidermal and allogeneic dermis components plays an important role in maintaining the skin’s durability, abrasion resistance, pressure resistance and resistance to shear stress to allow wound healing and to avoid contractures. If the skin is not able to endure the wear and tear of normal existence without this structure, then blisters and other phenomena between the epidermis and dermis would appear [36]. The BM between the epidermis and dermis of the skin could effectively tolerate the horizontal stress. Although there have been many reports in the literature regarding the use of ADM, our results are preliminary and subject to confirmation in a randomized and controlled trial with longer follow-up periods. Remarkably, the patients who received ADM coverage achieved apparently better results than the control group. The VBSS was used to gain an impression of the outcome of the healing quality. The data suggests an anticontraction effect of the ADM, and the esthetic outcomes in the cograft group were obviously better than those in the control group. The patients who received ADM achieved better appearance, elasticity, and pliability than the
Table 3 – Vancouver Burn Skin Score. Item
Pigmentation Thickness Vascularization Pliability Total score
6 months after surgery (mean SD)
30 months after surgery (mean SD)
Control group
ADM group
Control group
ADM group
2.87 0.35 3.47 0.57 2.80 0.48 3.67 0.48 12.8 1.47
2.27 0.45 2.33 0.48 2.17 0.38 2.40 0.50 9.17 0.95
1.9 0.31 2.4 0.56 1.87 0.43 2.43 0.57 8.60 1.48
1.20 0.41 1.00 0.37 1.10 0.31 1.23 0.43 4.53 0.51
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Table 6 – Barthel Index analysis. Grouping
N
M (min, max)
ADM group Control group
30 30
100.0 (89.0, 100.0) 99.5 (84.0, 100.0)
Z
P 2.814
0.005
control group. The results also indicate that dermal matrices could increase the flexibility of the grafted skin, could decrease wound contracture and scar formation, and are crucial factors in wound-healing quality. In terms of function, we chose the BI as the standard by which to measure the functional recovery after surgery for patients with deep burn wounds who did and did not receive ADM. The BI is a weighted and summed index designed to reflect a patient’s dependency in ADL (such as transfer, toilet use, fecal continence, and dressing). Initially, the BI was developed for the assessment of patients with neuromuscular and musculoskeletal disorders, but soon after its inception, it was widely used to assess functional change in the rehabilitation stroke patients [33]. The BI has subsequently been used to measure ADL in a wide range of disabling conditions. The scores for all items is added, and the higher the total score, the lower the restrictions in ADL. The BI is administered by doctors, therapists, and nurses. In the control group, our data indicate that the knee joint could perform only a 908 squat; thus, it could hardly meet the ADL. In contrast, the patients in the cograft group could achieve nearly normal function. Although the evenness of the composite skin at sites involving joints was slightly worse than that in nonjoint sites, as indicated by shrinkage or mild contractures at the former sites, this unevenness did not affect the anatomic function of the joint. Besides, because the ages of patients we chose were in average of 25 (control group)–30 (ADM group), so there was no significant differences between the two groups. And from the outcomes of our research, the sex has no significant impact on the final results. Through follow-up observations, we determined that the appearance, elasticity, and abrasion resistance were good after transplantation with ADM composite skin; recovery was rapid and no scar formation was observed.
5.
Conclusions
The immediate application of thin autografts onto an excised burn bed covered with ADM could become an optimal surgical procedure, with rapid donor site healing and excellent longterm results. However, the limited availability of cadaver skin, the expense of purchasing human ADM, and the risk of disease transmission limit the use of human ADM. Further research in molecular biology, wound healing, and immunology would likely yield better skin substitutes with which to treat patients in the future. Eventually, a synthetic bilayer membrane of quality equal to that of skin might be available for application in a method as simple as a dressing change.
Conflict of interest statement The authors have no conflicts of interest to declare.
Acknowledgments We thank all of the patients who took part in this study. The authors received no financial support for conducting this study.
Supplemental Fig. 1 Supplemental Fig. 1 associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. burns.2014.12.007.
references
[1] Feng X, Tan J, Pan Y, Wu Q, Ruan S, Shen R, et al. Control of hypertrophic scar from inception by using xenogenic (porcine) acellular dermal matrix (ADM) to cover deep second degree burn. Burns 2006;32:293–8. [2] Grisolia G, Pelli P, Pinzauti E, Panozzo G, Stuto A, Danti D, et al. Skin substitutes in the treatment of deep partial skin thickness burns in children: clinical experience and longterm results. Burns 1991;17:52–5. [3] Atherton DD, Tang R, Jones I, Jawad M. Early excision and application of matriderm with simultaneous autologous skin grafting in facial burns. Plast Reconstr Surg 2010;125:60e–1e. [4] Hinshaw JR, Miller ER. Histology of healing split-thickness, full-thickness autogenous skin grafts and donor sites. Arch Surg 1965;91:658–70. [5] Herd A, Hall P, Widdowson P, Tanner N. Mesh grafts—an 18 month follow-up. Burns 1987;13:57–61. [6] Matsuzaki K, Sugishita K, Fujii N, Miyajima K. Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry 1995;34:3423–9. [7] Valencia IC, Falabella AF, Eaglstein WH. Skin grafting. Dermatol Clin 2000;18:521–32. [8] Burke JF, Yannas IV, Quinby Jr WC, Bondoc CC, Jung WK. Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Ann Surg 1981;194:413–28. [9] Sarber R, Hull B, Merrill C, Soranno T, Bell E. Regulation of proliferation of fibroblasts of low and high population doubling levels grown in collagen lattices. Mech Ageing Dev 1981;17:107–17. [10] Heck EL, Bergstresser PR, Baxter CR. Composite skin graft: frozen dermal allografts support the engraftment and expansion of autologous epidermis. J Trauma 1985;25: 106–12. [11] Cuonoa C, Langdonb R, McGuire J. Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1986;327:1123–4. [12] Wainwright D. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns 1995;21:243–8. [13] Lee JT, Bhuta S, Calcaterra TC, Blackwell KE. Acellular dermal matrix grafts for prevention of microarterial thrombophlebitis. Arch Otolaryngol Head Neck Surg 2007;133:42–5. [14] Levin F, Turbin RE, Langer PD. Acellular human dermal matrix as a skin substitute for reconstruction of large periocular cutaneous defects. Ophthal Plast Reconstr Surg 2011;27:44.
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[15] Patton JH. Use of human acellular dermal matrix in complex and contaminated abdominal wall reconstructions. Am J Surg 2007;193:360–3. [16] Zhang X, Deng Z, Wang H, Yang Z, Guo W, Li Y, et al. Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration. Biomaterials 2009;30:2666–74. [17] Soejima K, Chen X, Nozaki M, Hori K, Sakurai H, Takeuchi M. Novel application method of artificial dermis: one-step grafting procedure of artificial dermis and skin, rat experimental study. Burns 2006;32:312–8. [18] Cuttle L, Nataatmadja M, Fraser JF, Kempf M, Kimble RM, Hayes MT. Collagen in the scarless fetal skin wound: detection with picrosirius-polarization. Wound Repair Regen 2005;13:198–204. [19] Sullivan T, Smith J, Kermode J, McIver E, Courtemanche DJ. Rating the burn scar. J Burn Care Rehabil 1990;11:256–60. [20] Mahoney FI, Barthel DW. Functional evaluation: the Barthel Index. Md State Med J 1965;14:61–5. [21] Wood FM. Quality assurance in burn patient care: the James Laing Memorial Essay, 1994. Burns 1995;21:563–8. [22] Alexander JW, MacMillan BG, Law E, Kittur DS. Treatment of severe burns with widely meshed skin autograft and meshed skin allograft overlay. J Trauma 1981;21:433–8. [23] Kopp J, Magnus Noah E, Rubben A, Merk HF, Pallua N. Radical resection of giant congenital melanocytic nevus and reconstruction with meek-graft covered integra dermal template. Dermatol Surg 2003;29:653–7. [24] Wardhana A. Clinical applications of micro-skin grafting for skin defect coverage. J Plast Rekonstr 2012;1:98–101. [25] Rea SM, Goodwin-Walters A, Wood FM. Surgeons and scars: differences between patients and surgeons in the perceived requirement for reconstructive surgery following burn injury. Burns 2006;32:276–83. [26] Hansbrough JF, Dore C, Hansbrough WB. Clinical trials of a living dermal tissue replacement placed beneath meshed,
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns Xueyong Li 1, Xianghai Meng 1, Xiaolin Wang 1, Yuejun Li, Wangzhou Li, Xiaoxing Lv, Xiaoli Xu, Zhanjun Lei, Jinqing Li * Department of Burn and Plastic Surgery, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Road, Xi’an 710038, China
article info
abstract
Article history:
The potential of acellular dermal matrix (ADM) to improve cosmetic and functional out-
Accepted 5 December 2014
comes has been demonstrated; however, there have been few clinical comparative studies assessing the long-term morphological, histological and functional changes after ADM
Keywords:
placement. This study was designed to retrospectively evaluate the long-term outcomes
Acellular dermal matrix
of the cograft acellular dermal matrix with autologous thin split-thickness skin for the
Wound repair
coverage of wounds in extensively burned patients. Thirty burn patients treated with a
Long-term outcomes of ADM
composite graft of ADM with autologous split-thickness skin from January 2007 to December 2009 were enrolled in this study. Another group of thirty patients who received only an autogenous split-thickness skin implant served as the control. Our study revealed that the collagen in the dermis treated with ADM were ordered, and the proportion of collagen III/I was much higher in the control group than in the ADM group. The basement membrane was prominent and continuous. Meanwhile, the VBSS (Vancouver Burn Skin Score) was used to evaluate skin quality, which shows a significant differences between the two group (P < 0.001). Then the functional level was evaluated by the BI (Barthel Index), and the ADM group was much better than the control group (P = 0.005). Based on these results, we concluded that the composite graft of ADM with autologous thin split-thickness skin was suitable for repairing the defects in functional areas after a burn. This technique might facilitate wound management with acceptable esthetic outcomes, good functional recovery and less scar hyperplasia at the donor site. # 2014 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Burn wound coverage techniques have been greatly improved; patients with more than 90% total body surface
area (TBSA) burns can now expect a fighting chance for survival [1–3]. Nevertheless, the lack of donor sites in major burn patients and secondary hypertrophic scar formation remain two major clinical challenges. The problem of the lack of donor sites is that the need for donor sites sometimes
* Corresponding author. Tel.: +86 29 84777440; fax: +86 29 84777440. E-mail address: [email protected] (J. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.burns.2014.12.007 0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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exceeds the available unburned skin; to approach this problem, autologous thin-thickness skin grafts were developed. The intermediate meshed split skin graft for recipient site wound closure after excision of the burned area remains the gold standard for treatment. However, split-skin harvest and transplantation are accompanied by frustrating consequences, such as wound contraction, donor site morbidity, and hypertrophy [4–6]. The lack of a dermal component in burn wounds after treatment with split-thickness autografts resulting in the poor quality of healing in recipient sites created the need for alternative graft sources [7]. The advent of allo-dermal replacement has revolutionized the therapeutic potential for the success rate and quality of healing in split-skin transplantation. In 1981, a composite of bovine collagen and chondroitin-6-sulfate from shark cartilage with an outer silicone covering was first engineered as an organotypic dermia for skin grafting [8]. In the same year, Sarber et al. developed a type of skin equivalent that is composed of natural collagen extraction and fibroblasts according to a specific mixing ratio [9]. Composite skin grafting was used extensively thereafter. In 1985, Heck reported using allogeneic dermis for wound closure [10]. He found that allogeneic dermis grafts caused immunological rejection mainly due to cellular immunity [11]. Subsequently, all of the cellular components of the dermis and epidermis were processed for removal, and the extracellular matrix and basement membrane (BM) were preserved. Such ‘‘decellularized’’ allografts had been effectively used alone or in combination with cultured autologous keratinocytes for the closure of burns and chronic wounds in the next year and were considered to have the potential to be a permanent dermal alternative [11]. Until 1995, Wainwright used composite transplantation to repair burn wounds with acellular and allogeneic dermis and reticular autologous thin-split grafts; this method avoided immune rejection [12]. Subsequently, upon histological observation, researchers found that the epithelial cells were well covered and that there was less scar proliferation with good effect. Since then, composite skin grafts have been widely used in both wound healing and scar plastic surgery [13–16]. However, there is a lack of literature regarding the longterm follow-up data of randomized, controlled human studies, especially in the aspects of histological changes, contour and function. In the present study, we used the Vancouver Burn Scar Scale (VBSS) and Barthel Index (BI) Scale to conduct a long-term evaluation of contour and functional outcomes following co-transplantation of a split-thickness skin graft (STSG) with human acellular dermal matrix.
2.
Materials and methods
2.1.
Clinical data
abdominal trauma), elderly patients with co-morbid diseases such as chronic cardiovascular disease, diabetes mellitus or hypertension, and those who had significant inhalation injuries or needed intensive care were excluded from the study. Eligible patients were randomized at admission to either ADM or STSG group. Randomization was performed following a predetermined random order produced in groups of 10. This preordered format was maintained by the primary investigator. Patients who were excluded were assigned randomization but not included in the subsequent analysis. The 2 groups are as follows: the acellular dermal matrix (ADM) group (n = 30), in which the wounds were successfully covered with ADM and autologous epidermis, and the control group (n = 30), in which the wounds were covered with autologous split-thickness skin. The general patient characteristics are listed in Table 1; the types of grafts used for burn treatments are listed in Table 2. Of the 30 patients in the experimental group, 25 were male and the remaining 5 patients were female; their ages ranged from 3 to 52 years, with an average age of 30.53 10.73 years. Thirty sites from 20 patients underwent surgical burn wound excision and were grafted with acellular dermal substitutes and covered with thin split-thickness skin that was not expanded. A similar procedure was conducted on five sites of four patients with mature granulation tissue in later burn stages and on seven sites of six patients with scar plasticity. The skin graft sites included the neck, hand, elbow, shoulder, popliteal site, knee and foot back, among others. The largest area of composite skin graft was 600 cm2; the minimum area was 40 cm2. Of the control group, the mean age of the 30 patients was 25.30 15.88 years with a range of 2–57 years; 24 patients were males, and 6 were females. This study was reviewed and approved by the institutional ethics committee. Patients with electrical burns, fourthdegree burns, burns as components of multiple traumas (patients with fractures or central nervous system, thoracic and abdominal trauma), elderly patients with co-morbid diseases such as chronic cardiovascular disease, diabetes mellitus or hypertension, and those who had significant inhalation injuries or needed intensive care were excluded from the study.
2.2.
Materials
This investigation involved commercial ADM products from Jie-Ya Life Tissue Engineering (Beijing, China) that were approved by the Chinese Food and Drug Administration for transplantation. ADM is a dermal collagen matrix extracted from human skin by removing the majority of the cellular
Table 1 – General patient characteristics.
This study was reviewed and approved by the institutional ethics committee. A total of 60 patients who sustained burns provided consent to participate in this study over 3 years (2007–2009), and patients with electrical burns, fourth-degree burns, burns as components of multiple traumas (patients with fractures or central nervous system, thoracic and
Type
Average age Male/female Scar plasticity Granulation tissue Burn
Number ADM group
Control group
30.53 10.73 25/5 6 (20%) 4 (13.33%) 20 (66.67%)
25.30 15.33 24/6 2 (6.67%) 11 (36.67%) 17 (56.67%)
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Table 2 – Types of grafts for burn treatment. Type
Classification
2.3.
Number ADM group
Control group
Wound area
<10% 10–30% 31–50% >50%
8 14 4 4
15 9 3 3
Surgical site
Lower limbs Upper limbs Ankles and feet Hands Trunk
13 4 4 4 5
15 8 0 3 4
Donor site
Head Back Thigh
9 15 6
3 21 6
Area of skin grafting
<100 cm 2 100–200 cm 2 >200 cm 2
7 15 8
3 15 12
Survival rate
100% 80–99% <80%
16 12 2
19 11 0
components. Briefly, a 4-mm-thick slice is cut from cadaveric skin using a dermatome; the sample is treated with 2.5 unit/ml Dispase II and 0.2 mM CaCl2 in phosphate-buffered saline (PBS) at 4 8C for 24 h. The dermal matrix is then incubated in 0.5% Triton X-100 for an additional 24 h at room temperature and stored in a PBS solution containing sodium azide (0.02% w/v) at 4 8C. The specimen is maintained at 2–8 8C, washed with 0.9% normal saline several times before surgery, and submerged in normal saline containing 1000–2000 U/ml gentamicin for 10 min.
3
Surgical procedure
The operation was performed within the first week of admittance if the patient’s condition was appropriate. After irrigation and hemostasis of the excised wound, grafts were applied to the wound bed for the treatment using one-step [17] placement, namely synchronous application of acellular dermis and autologous thin-thickness skin (Fig. 1). The category of the burn wounds consisted of different types of wounds with respect to the clinical appearance of depth and debridement. Full-thickness burn wounds were debrided by epifascial excision. Tangential excision was performed on paired wounds of partialthickness burn wounds. The appropriate size of acellular dermal matrix would be placed on the prepared wound and moderately stretched to maintain a certain tension. Thin split-thickness autologous skin (0.2–0.3 mm) was harvested from an uninvolved area using a Zimmer dermatome (Zimmer, Indiana, USA) and then trimmed and placed on the allogeneic dermis; this graft was fixed with skin staples. Vaseline gauze was used for dressing the graft, the area was bandaged based on the involved body parts, and the functional parts were fixed safely. After 3 days, the dressings were changed for the first time, and the staples were removed after 5–7 days. Sheet grafts were predominantly used over joints or in limited burn areas when sufficient donor areas were available.
2.4. Masson’s trichrome staining and picrosirius red staining for histological analysis 2.4.1.
Masson staining
Slide sections, 4 mm in thickness, were prepared from the paraffin-embedded skin tissue of skin grafts from both groups.
Fig. 1 – The surgical procedure of ADM and STSG. The wound surfaces before skin grafting of both ADM (panel A) and STSG (panel C), and the intra-operative photographs after grafting of the ADM (panel B) and STSG (panel D). Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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All of the sections were stained with Masson’s trichrome stain on the mounted serial 4-mm-thick sections for observation of the collagen framework. Standard procedures for this staining were performed as recommended by the manufacturers. A single pathologist, who was blinded to the treatment regimen, viewed the stained sections.
2.4.2.
Picrosirius polarization
The specimens, including full-thickness skin layers (epidermis, dermis, and hypodermis), were fixed in 4% buffered paraformaldehyde and were processed according to routine light microscope tissue processing methods, and the processed tissues were then embedded in paraffin. Tissue sections (8 mm) were selected for picrosirius red staining. Finally, picrosirius staining followed by polarized light microscopy was used to visualize the collagen fibers with digital capture and analysis [18].
2.4.3.
Image capture and analysis
Image Pro PlusTM image analysis software was used to digitally capture the images using a firewire camera. The images were stored on a personal computer. Image Pro PlusTM was used to analyze the slides in detail. Using the computer software, we were able to differentiate the red and the green areas that were selected, and the percentages of type I and type III collagen per slide were calculated. This process was repeated for each of the five randomly selected areas of the ADM and control groups. The data were then transferred directly to the spreadsheet program ExcelTM (Microsoft Corporation, Redmond, WA) for storage and further analysis.
2.5.
Immunohistochemistry
Immunohistochemistry experiments were performed on specimens from the two groups and the thin split-thickness autologous skin. Rabbit antibodies against laminin were acquired from Zhongshan Goldenbridge Biotechnology Co. LTD. Cultured tissues were fixed with cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Small pieces of each tissue were processed into 4-mm-thick transverse sections. The sections were heated at 65 8C for 30 min to soften the wax, dewaxed by incubation in xylene (2 5 min), rehydrated through a series of graded ethanol steps (100%, 2 5 min; 90%, 5 min; 80%, 5 min; 75%, 5 min), and then rinsed in tap water. After treatment with 0.3% H2O2 for 20 min, antigen retrieval was performed using 0.4% pepsin applied for 20 min, and the samples were then washed with PBS (pH 7.4). The tissue sections were blocked with 5% bovine serum albumin (BSA) for 20 min at room temperature followed by incubation with the primary antibody diluted in antibody dilution solution with background-reducing components for 18 h at 4 8C. Then, the sections were incubated with a biotinylated secondary antibody for 20 min at 37 8C. Streptavidin–biotin peroxidase was applied for 20 min at room temperature, which was followed by washing. Development was performed using diaminobenzidine followed by nuclear staining with Mayer’s hematoxylin (1 min). Finally, the specimens were observed by microscopy after balata enveloping.
2.6.
Wound site evaluation
The acceptance rate of the split-thickness autografts (sheet and mesh grafts) was defined as the percentage of the entire graft that was considered to be vital and that showed good adherence to the wound bed 5–7 days after surgery. The acceptance was assessed for both treatment groups at the same time point to allow for paired statistical analyses. In mesh graft transplantation, epithelialization of the interstices of the graft is required to achieve complete wound closure. The wound was considered closed when more than 95% of the grafted surface area was epithelialized. The necessity of regrafting was registered, which is regarded as indicative of the success of the initial transplantation.
2.7. Scar elasticity parameters: The Vancouver Burn Skin Score (VBSS) The VBSS was used to the evaluate skin quality after a minimum of 3 months [19]. This score consists of various parameters: elasticity, pliability, pigmentation and vascularization. Each parameter has a score of 0–3, and the total score is added together. The VBSS is an established, reliable and valid score in the evaluation of burn scars.
2.8.
Physical function assessment: The Barthel Index (BI)
The BI is an assessment of a patient’s functional level of independence in Activities of Daily Living (ADL) and is scored in increments of 5 points (highest possible total score = 100) [20]. The values assigned to each item are weighed according to the amount of physical assistance required if the patient could not perform the activity independently. The 10 ADL items assessed in the BI are feeding, bathing, personal hygiene, toilet use, bladder control, bowel control, dressing, wheelchair transfer to and from bed, walking, and ascending and descending stairs.
2.9.
Statistical analysis
Continuous variables with skewed distributions are presented as medians (min, max) and were analyzed using the Mann– Whitney test. P < 0.05 was considered statistically significant. All of the statistical analyses were performed using SPSS v16.0.
3.
Results
3.1.
Clinical observations
Composite skin transplantation was performed for the treatment of 42 wounds on 30 patients after burns and plastic surgery; 39 wound composite skin grafts survived, and there was partial survival in 3 grafts. The skin graft survival standard of a composite skin graft is as follows: at two weeks after the surgery, survival of more than 90% of the epidermis and dermis is regarded as complete survival; non-survival of more than 40% of the epidermis and/or dermis is regarded as no survival; survival of regions between 40 and 90% is considered partial survival. Skin flushing was apparent after
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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composite skin graft survival. After three months, the color of the composite skin was similar to the color of to the normal skin, and there was good elasticity; no composite skin graft contracture or scars were observed.
there were no blisters (Fig. 3B). Follow-up at 33 months revealed that the appearance of the composite skin was similar to the normal skin; there was good elasticity, no scar, and good joint function (Fig. 3C and D).
Typical Case 1
Typical Case 3
Following a vapor explosion, a 38-year-old woman suffered burns affecting 30% TBSA of both lower extremities. Following hospitalization, the burn pattern was identified as deep II degree. The other areas of the body were undamaged. On the 16th day after admission, escharectomy of the posterior portion of the right leg corresponding to 15% TBSA was performed, followed by coverage with composite skin (Fig. 2A); all of the grafts survived after 12 days (Fig. 2B). Follow-up after 30 months revealed that the color of the replacement appeared almost the same as the normal skin; there was good elasticity, and no scar was observed (Fig. 2C and D).
A 28-year-old individual presented to our burn unit following a sulfuric acid spill at work; the spill resulted in 16% TSBA deep partial-thickness burns and 5% TSBA full-thickness burns, mainly on both lower limbs. The surgical management involved the debridement of all of the non-viable burned tissue. An acellular allogeneic human dermis matrix was used to cover the wound with no meshed split-thickness skin graft (Supplemental Fig. 1). Good acceptance of the graft was accomplished at the first dressing change 12 days post-surgery (Supplemental Fig. 1). After a follow-up period of 34 months, the appearance of the transplanted composite skin was close to the appearance of normal skin, with good elasticity, and no scar was observed (Supplemental Fig. 1).
Typical Case 2 A previously healthy 28-year-old male sustained a flame injury to 46% TBSA. The injury consisted of deep partialthickness burns and full-thickness burns to both lower extremities. The patient received fluid resuscitation, and enteral nutritional support was initiated via a nasogastric tube. The wounds were dressed with topical silver sulfadiazine. On postburn day three, the patient underwent surgery for escharectomy and wound closure with a composite skin graft on the bilateral knees and a micro skin graft on other parts of the legs. On the twelfth post-operative day, the dressing change revealed 100% survival of the skin grafts (Fig. 3A). The elasticity of the composite skin was good after 31 d, and
Comparative study The two patients in the control group with autogenous split-thickness skin obtained non-satisfactory outcomes (Fig. 4B, C, E, F, H and I). The appearance, elasticity, and function of the grafts in the ADM group (Fig. 4A, D and G) were much better compared to the split-thickness group.
3.2. Masson’s trichrome staining and picrosirius red staining for histological analysis Masson’s trichrome staining was performed on both allogeneic dermal tissue and tissue with no implanted
Fig. 2 – Photographs of Case 1. The intra-operative site after coverage with a one-stage sheet autograft (panel A). A photograph taken 12 days after surgery shows the preliminary results (panel B). A close-up of the front portion of the lower extremity revealing skin without scar lines (panel C). The functional outcome 30 months after epidermal skin replacement (panel D). Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Fig. 3 – Photographs of Case 2. The post-operative view at 12 days after surgery (panel A). A photograph taken 31 days after surgery shows the post-operative view (panel B). After 33 months of follow-up, there were satisfactory effects with a good appearance and good function (panel C, panel D).
allogeneic dermis. The different layers of the epidermis were well identified in the histological images (including the stratum corneum disjunctum, stratum corneum compactum, stratum granulosum, stratum spinosum, and stratum basale), and the collagen fibers in the dermis layer were very clear (Fig. 5A); the cells in the epidermis and the vessels in the dermis stained red, collagen stained blue, and nuclei stained black. The collagen of the acellular dermal tissue appeared stretched and aligned in the same plane as the epidermis; there were few blood vessels, and there was good continuity of the basal layer (Fig. 5B). The collagen in the tissue with the autogenous split-thickness skin implant appeared relaxed and was arranged in a random array; there was poor continuity of the basal layer, there were a large number of blood vessels, and scars appeared to form easily (Fig. 5C). Collagen deposition increases with scar tissue, and the fibers became thicker, changing from yellow/red (type I collagen) to green (type III collagen). The proportion of collagen III/I was much higher in the hypertrophic scars than in the normal skin [19,20]. We determined that the ratio of collagen III/I was higher in the tissue with autogenous split-thickness skin implants and that the ratio was much lower in the ADM tissue compared to normal skin (Fig. 6).
3.3.
Immunohistochemistry of the basement membrane
Tissues were stained with laminin antibodies against the major BM constituents. The staining demonstrated that the autologous thin-thickness skin, which covered the ADM, excluded the BM (Fig. 7A). Intense and continuous lines of
laminin deposits were observed (Fig. 7B). Compared with the samples of the ADM skin, the BM of the control group was not clearly observed (Fig. 7C).
3.4.
The Vancouver Burn Skin Score outcome
The outcomes of the scar evaluation parameters by VBSS are shown in Table 3. The recovery time was regarded as an important factor of the effectiveness of wound repair. However, the use of ADM was another significant means for obtaining acceptable esthetic outcomes. The differences in the total score at 6 months were statistically significant (Z = 6.302, P < 0.001) (Table 4). The median of the ADM group was 9, and the median of the control group was 13. The differences in the total score at 30 months were also statistically significant (Z = 6.680, P < 0.001). The median of the ADM group was 5, and the median of the control group was 9. The medians of the ADM group were lower compared to those of the control group.
3.5.
The Barthel Index outcome
The statistical results of the physical functional assessment by the BI are shown in Table 5. The ADL index of the ADM group was higher compared to the ADL index of the control group. When the total scores of the ADM and control groups (Table 6) were compared, the differences were statistically significant (Z = 2.814, P = 0.005). The median of the ADM group was 100, which was higher than the median of the control group (99.50).
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Fig. 4 – Photographs of the appearance, elasticity, and function of the patients’ grafts. The case with the ADM allograft shows a good appearance (panel A). The views of the control group with autogenous split-thickness skin (panel B, panel C). The suitable elasticity of the case with the ADM implant (panel D). The views of the control group with autogenous split-thickness skin were not much better than the views of the ADM group (panel E, panel F). The good function of the case with suitable elasticity with the ADM implant; the patient was able to squat easily (panel G). The functional abilities of the control group were not as prominent (panel H, panel I).
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Fig. 5 – Histopathological features of the skin. The tissue with the ADM allograft shows the entire structure of the skin (panel A, T400). The collagen was arranged in an orderly manner in the skin with the ADM (panel B, T100). The control group with autogenous split-thickness skin showed irregular collagen arrangements (panel C, T100). The skin with autogenous splitthickness skin grafting revealed a high content of collagen III (panel D, T200). However, the content of collagen III was much lower in the group with the ADM allograft (panel E, T200).
4.
Discussion
In the treatment of major burns, it is important to focus on ensuring that the ‘‘quality of life is worth the pain of survival’’ [21]. Normal skin is repeatedly harvested from donor sites in the treatment of extensive burns, which limits the transfer of autologous skin, especially after harvesting the medium split-thickness skin. In the past 30 years, there have been many attempts toward improving skin replacement, such as the use of meshed skin, meek grafts, micro-skin transplantations and others [22–24]. In these successful techniques, cultured epithelium autografts appear to be a potential
Fig. 6 – Graph showing the type III/I collagen ratios in the ADM and control groups.
alternative. In 1984, Gallico showed that full-thickness skin burn patients could be saved using cultured epithelium autografts (CEAs). Unfortunately, because of the wide patient-to-patient variation and the lack of controlled clinical research, it is difficult to determine if there is a place for cultured epithelial grafts in the treatment of major burns [25]. Nevertheless, studies have shown the dermal component deficiency and the increased possibility of contractive scar formation at the recipient sites [26–28]. This situation could be changed by the combined use of the STSG with an allogeneic acellular dermal component [29], which preserves the natural mixture of structural and functional proteins that constitute the extracellular matrix, such as collagen, fibronectin, laminin and vimentin [30–32]; the cell components, which possess strong immune cell composition in allogeneic acellular dermal matrices, are excluded during this process. The allo-dermal matrix is the carrier of cell transplantation and the ‘‘soil’’ of cell proliferation; additionally, the three-dimensional structure of dermal tissue could support fibroblast infiltration, neovascularization, and keratinocyte migration from overlying STSG [29] and could improve the state of mechanical force in a wound, promoting tissue remodeling, reducing hypertrophic scar formation, and improving the healing quality of the wound. The present study notes that ADM shows good affinity and excellent appearance and is easy to slide (Fig. 4). The histological staining revealed that after the composite skin is transferred, the structure of the composite graft survival skin is similar to the normal skin area in the region receiving the composite skin transplantation (Fig. 7). Because the split-thickness skin is transplanted on the dermal matrix, hyperplasia is decreased, which could prevent the occurrence of scar contractures.
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Table 4 – Vancouver Score analysis. Grouping ADM group Control group Z P
6 months
30 months
9.0 (8.0, 11.0) 13.0 (10.0, 14.0) 6.302 <0.001
5.0 (4.0, 5.0) 9.0 (5.0, 11.0) 6.680 <0.001
Table 5 – Barthel Index Scale Scores. Item
Scores (mean SD)
1—Feeding 2—Bathing 3—Personal hygiene 4—Toilet use 5—Bladder control 6—Bowel control 7—Dressing 8—Wheelchair transfer to and from bed 9—Walking 10—Ascending and descending stairs Total
Fig. 7 – Immunohistochemistry of the BM. The autologous thin-thickness skin that covered the allogeneic dermis contains no BM (panel A, T400). The outcome of the ADM skin showed a continuous BM. The black arrow indicates the areas of the BM, which are strongly stained for laminin (panel B, T400). The BM of the control group was not clearly observed (panel C, T400).
The role of the BM is important for maintaining the integrity and structure of an epithelial layer, as it acts as a mechanical support and forms the physical interface between epithelial cells and the surrounding connective tissue [34,35]. What would the epidermal–dermal composites after a cograft be like? As described in the information sheets, the J-1 acellular dermis reserved the BM. In our study, the autologous split-thickness skin harvested from the patient’s scalp or thigh were a thickness of 0.2 mm and excluded the BM, as shown in Fig. 7A. After the cograft, the epidermal–dermal composites survived well in principle. Immunohistological staining revealed that the autogenous epithelial sheets consisted of
ADM group
Control group
9.80 0.61 4.73 0.94 4.90 0.31 10.00 0.00 10.00 0.00 10.00 0.00 9.93 0.37 14.90 0.55
9.87 0.51 4.80 0.41 4.70 0.47 9.27 0.98 10.00 0.00 10.00 0.00 9.53 0.86 14.40 1.22
15.00 0.00 9.93 0.37
14.83 0.91 9.73 0.69
99.20 2.34
97.13 4.57
more layers of contiguous cells that are anchored to the integrated BM, which separates the epithelium from the underlying connective tissues (Fig. 7). A solid combination of autologous epidermal and allogeneic dermis components plays an important role in maintaining the skin’s durability, abrasion resistance, pressure resistance and resistance to shear stress to allow wound healing and to avoid contractures. If the skin is not able to endure the wear and tear of normal existence without this structure, then blisters and other phenomena between the epidermis and dermis would appear [36]. The BM between the epidermis and dermis of the skin could effectively tolerate the horizontal stress. Although there have been many reports in the literature regarding the use of ADM, our results are preliminary and subject to confirmation in a randomized and controlled trial with longer follow-up periods. Remarkably, the patients who received ADM coverage achieved apparently better results than the control group. The VBSS was used to gain an impression of the outcome of the healing quality. The data suggests an anticontraction effect of the ADM, and the esthetic outcomes in the cograft group were obviously better than those in the control group. The patients who received ADM achieved better appearance, elasticity, and pliability than the
Table 3 – Vancouver Burn Skin Score. Item
Pigmentation Thickness Vascularization Pliability Total score
6 months after surgery (mean SD)
30 months after surgery (mean SD)
Control group
ADM group
Control group
ADM group
2.87 0.35 3.47 0.57 2.80 0.48 3.67 0.48 12.8 1.47
2.27 0.45 2.33 0.48 2.17 0.38 2.40 0.50 9.17 0.95
1.9 0.31 2.4 0.56 1.87 0.43 2.43 0.57 8.60 1.48
1.20 0.41 1.00 0.37 1.10 0.31 1.23 0.43 4.53 0.51
Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007
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Table 6 – Barthel Index analysis. Grouping
N
M (min, max)
ADM group Control group
30 30
100.0 (89.0, 100.0) 99.5 (84.0, 100.0)
Z
P 2.814
0.005
control group. The results also indicate that dermal matrices could increase the flexibility of the grafted skin, could decrease wound contracture and scar formation, and are crucial factors in wound-healing quality. In terms of function, we chose the BI as the standard by which to measure the functional recovery after surgery for patients with deep burn wounds who did and did not receive ADM. The BI is a weighted and summed index designed to reflect a patient’s dependency in ADL (such as transfer, toilet use, fecal continence, and dressing). Initially, the BI was developed for the assessment of patients with neuromuscular and musculoskeletal disorders, but soon after its inception, it was widely used to assess functional change in the rehabilitation stroke patients [33]. The BI has subsequently been used to measure ADL in a wide range of disabling conditions. The scores for all items is added, and the higher the total score, the lower the restrictions in ADL. The BI is administered by doctors, therapists, and nurses. In the control group, our data indicate that the knee joint could perform only a 908 squat; thus, it could hardly meet the ADL. In contrast, the patients in the cograft group could achieve nearly normal function. Although the evenness of the composite skin at sites involving joints was slightly worse than that in nonjoint sites, as indicated by shrinkage or mild contractures at the former sites, this unevenness did not affect the anatomic function of the joint. Besides, because the ages of patients we chose were in average of 25 (control group)–30 (ADM group), so there was no significant differences between the two groups. And from the outcomes of our research, the sex has no significant impact on the final results. Through follow-up observations, we determined that the appearance, elasticity, and abrasion resistance were good after transplantation with ADM composite skin; recovery was rapid and no scar formation was observed.
5.
Conclusions
The immediate application of thin autografts onto an excised burn bed covered with ADM could become an optimal surgical procedure, with rapid donor site healing and excellent longterm results. However, the limited availability of cadaver skin, the expense of purchasing human ADM, and the risk of disease transmission limit the use of human ADM. Further research in molecular biology, wound healing, and immunology would likely yield better skin substitutes with which to treat patients in the future. Eventually, a synthetic bilayer membrane of quality equal to that of skin might be available for application in a method as simple as a dressing change.
Conflict of interest statement The authors have no conflicts of interest to declare.
Acknowledgments We thank all of the patients who took part in this study. The authors received no financial support for conducting this study.
Supplemental Fig. 1 Supplemental Fig. 1 associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. burns.2014.12.007.
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Please cite this article in press as: Li X, et al. Human acellular dermal matrix allograft: A randomized, controlled human trial for the long-term evaluation of patients with extensive burns. Burns (2015), http://dx.doi.org/10.1016/j.burns.2014.12.007