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. 2019 Aug 19;16(6):1354–1364. doi: 10.1111/iwj.13198

Benefits of cryopreserved human amniotic membranes in association with conventional treatments in the management of full‐thickness burns

Anne‐Sophie Hatzfeld 1,†, Louise Pasquesoone 2, Nicolas Germain 1,3, Pierre‐Marie Danzé 1, Anne‐Sophie Drucbert 5,†, Meryem Tardivel 4, Antonino Bongiovanni 4, Véronique Duquennoy‐Martinot 2, Pierre Guerreschi 2,†,✉, Philippe Marchetti 1,3
PMCID: PMC7949321  PMID: 31429202

Abstract

The use of split‐thickness skin autografts (STSA) with dermal substitutes is the gold standard treatment for third‐degree burn patients. In this article, we tested whether cryopreserved amniotic membranes could be beneficial to the current treatments for full‐thickness burns. Swines were subjected to standardised full‐thickness burn injuries, and then were randomly assigned to treatments: (a) STSA alone; (b) STSA associated with the dermal substitute, Matriderm; (c) STSA plus human amniotic membrane (HAM); and (d) STSA associated with Matriderm plus HAM. Clinical and histological assessments were performed over time. We also reported the clinical use of HAM in one patient. The addition of HAM to classic treatments reduced scar contraction. In the presence of HAM, skin wound healing displayed high elasticity and histological examination showed a dense network of long elastic fibres. The presence of HAM increased dermal neovascularization, but no effect was observed on the recruitment of inflammatory cells to the wound. Moreover, the use of HAM with classical treatments in one human patient revealed a clear benefit in terms of elasticity. These results give initial evidence to consider the clinical application of HAM to avoid post‐burn contractures and therefore facilitate functional recovery after deep burn injury.

Keywords: dermal substitute, full thickness burn, scar contracture, split‐thickness skin autograft, wound healing

Abbreviations

bFGF

basic fibroblast growth factor

ECM

extracellular matrix

EGF

epidermal growth factor

HAMs

human amniotic membranes

HE

haematoxylin and eosin

HGF

hepatocyte growth factor

KGF

keratinocyte growth factor

MMP

matrix metalloproteinase

PAI

plasminogen activator inhibitor

STSA

split‐thickness skin autografts

TGF‐β

transforming growth factor beta

TIMP‐1

tissue inhibitor metalloproteinase 1

TSP‐1

thrombospondin‐1

1. INTRODUCTION

The classic procedure for coverage of full‐thickness burns is autologous skin grafting.1 However, poor skin quality and scar contraction frequently occur in split‐skin grafted areas and lead to functional disorders and poor cosmetic outcomes. To optimise the quality of the grafted area, the use of dermal reconstruction represents a better alternative, which minimises scar contraction and improves functional recovery.2, 3 The first artificial dermis described in the late 1980s was Integra, a porous matrix of cross‐linked bovine tendon collagen and glycosaminoglycan. More recently, a new substitute, named Matriderm (Medical Z, San Antonio), was described consisting of a dimensional dermal matrix composed of bovine dermal collagen (I, III, and V) and bovine nuchal ligament elastin. One of the advantages of Matriderm is that it is suitable for a single‐stage grafting procedure with a higher skin graft take rate.4, 5, 6 It has been shown that even in complex wounds such as extensive high‐voltage injuries, the use of Matriderm in association with split‐thickness skin graft resulted in excellent take rates with a significant enhancement of skin quality.7 The dermal matrix, Matriderm, also noticeably reduces the re‐epithelialization time associated with a fast improvement of scar quality.8 In some parts of the body, such as the hands, necks, and wrists, the application of Matriderm improved the quality of scars as validated by their elasticity, pliability, and stability that are essential to recover the functionality of the region.6, 9, 10 Unfortunately, despite its positive effects, the use of Matriderm did not always lead to better long‐term clinical results in terms of elasticity, scar contraction, and patient's impression than split‐thickness skin graft alone.11 Alternatively, the therapeutic potential of human amniotic membranes (HAMs) to heal wounds like ulcers or burns has been established.12, 13 Since 1940, HAMs are routinely used in ophthalmology to restore normal ocular surface after ulcers and chemical burns.14, 15 The lack of HLA‐A, ‐B, ‐C, and ‐DR molecules confers a low immunogenicity avoiding rejection post‐transplant. Amniotic membrane promotes epithelial healing,16 diminishes inflammation by inducing the apoptosis of inflammatory cells,17 as well as switching the macrophage phenotype from a pro‐inflammatory M1 phenotype to an M2 anti‐inflammatory/regulatory phenotype.18 It was evidenced that HAM graft decreases pain, accelerates the healing process,19, 20, 21, 22 and attenuates scar formation.23, 24 HAMs also bear potent antimicrobial activities in vitro and in vivo.20, 25, 26, 27, 28, 29

In experimental settings, HAMs were found to be very versatile, resulting in the development of clinical applications for skin burns. HAM is used as a temporary biological wound dressing28 in the treatment of partial‐thickness skin burns.13, 14, 22, 30 More recently, HAMs were also successfully tested in two patients presenting with full‐thickness thermal occupational burns. Thus, adding HAMs to the care management of burn injuries can prevent amputation of the limb and reduce the formation of finger contractures.31

The healing effects of HAM have been attributed to its characteristic composition including collagen matrix with high levels of growth factors and cytokines. Amniotic epithelial cells are known to promote wound healing through high levels of expression of EGF, PDGF‐B, and eNOS.32 In addition, the amniotic membrane downregulates the expression of TGF‐β promoting re‐epithelialization and reducing the risk of fibrosis development in chronic wounds.33 Natural inhibitors of matrix metalloproteinase (MMP) such as TIMP‐1 were also detected in HAM leading to a better arrangement of the extracellular matrix.34, 35Therefore, HAMs modulate the healing process by promoting skin reconstruction rather than scar tissue formation.

In this study, we addressed the following question: What are the additional effects of HAM in addition to two of the current treatments for full‐thickness burns such as split‐thickness skin autograft (STSA) alone or its association with Matriderm?

Thus, we tested whether HAM could improve the current treatments for full‐thickness burn injuries. We first investigated (a) the effects of HAM on wound healing and on the quality of scars in a standardised full‐thickness burn porcine model; (b) then we reported the clinical use of HAM in one patient with severe burn injuries of the hand.

2. MATERIALS AND METHODS

2.1. Procurement of placenta and amniotic membrane preparation

Cryopreserved HAMs were processed from the French competent authority (ANSM) approved Tissue Bank of the Lille University Hospital. Placentas were obtained from selected donors after informed consent and medical history procurement. The placenta samples came only from caesarean section to avoid any infection risk. The donor was screened for potential risk factors such as cancer, infectious diseases, drug abuse, and sexual behaviour. On the day of the donation, a blood sample was taken in order to screen for hepatitis B and C, syphilis, HIV‐1 and ‐2, and HTLV. Immediately after delivery, the placenta was washed in a 0.9% NaCl solution (Fresenius Kabi, France) and transferred to the tissue bank at +4°C. The amniotic membrane was separated and washed in a 0.9% NaCl solution. After removing all blood clots adhering to the amniotic membrane, it was put in an antibiotic solution (amikacin 500 mg/L [Mylan, France]; amoxicillin 1 g/L [GlaxoSmithKline, France]; vancomycin 500 mg/L [Mylan, France]; and amphotericin B 50 mg/L [Brystol Myers Squibb, France]) for 2 hours at room temperature. Then, after washing in a 0.9% NaCl solution, the amniotic membrane was cut into 9 cm2 pieces. Each fragment was put on a nitrocellulose membrane (Pall Corporation, France) (epithelial side up) and transferred into a Petri dish (Becton Dickinson, France) containing Roswell Park Memorial Institute medium (RPMI) 1640 (Gibco, France) with 17% glycerol (SALF, Italy). The Petri dish was put in a sterile bag (Macopharma, France), which was then sealed and transferred at −80°C until use. One hour before application, the cryopreserved amniotic membrane was thawed at room temperature. After rinsing with balanced salt solution (BSS) (Alcon, France), the amniotic membrane was ready to use.

2.2. Human case report

All procedures performed in this study were in accordance with the Helsinki declaration and ethical standards of our human research review committee. According to the European Directorate for the quality of medicine and healthcare, HAMs have been used in a specific patient on the basis of a risk/benefit analysis, taking into consideration the alternative therapeutic options. The use of HAMs in this patient has been registered by the French Agency for the Safety of Health products (ANSM). The patient was informed of the therapeutic potential and possible risks of treatment and had signed a written document of informed consent before treatment.

2.3. Animal models and anaesthesia

Three large white female LANDRAS pigs without any skin disease were used in this study and kept under standard conditions in the animal care unit of the University of Lille 2, France. Animals received human care according to the ethical standards. The protocol was approved by the experimental ethics committee.

The pigs were sedated by intramuscular injection with xylazine HCl (3 mg/kg; Ceva Sante animale, Libourne, France) and ketamine HCl (0.1 mg/kg; Virbac, Carros, France). Then pigs were intubated and placed under mechanical ventilation. Anaesthesia was pursued with a isoflurane/100% oxygen (AErrane, Baxter, Maurepas, France) mixture. Monitoring with continuous pulse oximetry was performed along all the surgery. Morphine analgesic patches (Durogesic) were used in postoperative and until the end of the experiment.

2.4. Burn model and the wound healing protocol

A detailed timeline of the evaluations is shown in Figure 1A. Four identical cutaneous deep burns were performed over paravertebral muscle in the back by applying a standardised plate heated at 82°C for 10 seconds to create full‐thickness burns. Burn areas were 33 ± 3 cm2. Afterwards, wounds were covered by a mixture of Jelonet (Smith & Nephew, London) and Betadine ointment (Meda, France). After 7 days, the dead tissue was removed under sterile conditions and the full‐thickness skin wounds were randomly assigned, one to each of the four treatment groups. All groups received STSA, but the difference resided in the presence or absence of Matriderm and/or HAM. Thus, there were four treatment groups described as follows: groups treated with a simple skin autograft alone (group STSA) or in the presence of AM (group STSA + HAM) and groups treated with the commercially available dermal substitute, Matriderm covered by skin autograft alone (group STSA + Matriderm), or with the presence of added HAM (group STSA + Matriderm + HAM). More precisely, after rinsing in saline solution, the dermal substitute Matriderm (Matriderm, Medical Z, San Antonio), was applied. Then, thin‐thickness skin fragments were manually taken by a dermatome in the posterior back and applied on the four wounds. Afterwards, an amniotic membrane was put on one wound with the Matriderm and one with the thin‐thickness skin graft alone. The surgical wounds were draped with Jelonet, and compressed and fixed with Elastoplast. Intra‐muscular prophylactic antibiotics were administered for 5 days (Ceftriaxone 1 g). At days 12, 17, and 38, surgical biopsies were performed and the dressings were changed. The animals were euthanized at the end of the experimentation using T61 (MSD Santé animale, Beaucouze, France). The graft take rates in the four groups were assessed at days 12, 17, and 38 post‐injuries.

Figure 1.

Figure 1

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A, Experimental design: wound healing and scar evaluations as described in the Materials and methods section; B) histological evaluation of wound healing after indicated treatments at day 38 post‐injury after HE staining; C, macroscopic evaluation of wound healing after indicated treatments at day 0 (the day of burn injury), at day 7 post‐injury (right after removing dead tissue), and at day 38 post‐injury (final evaluation). Photographs were taken on one representative pig out of three with similar results. White dashed lines represent the burnt area at day 7; D, above, the kinetic of the wound area evaluated after indicated treatments; below, histogram shows the scar contraction for each condition. Results are the mean ± SD of three pigs; **, P < .01

2.5. Macroscopic evaluation of wound healing

Wound contraction was analyses systematically measuring the wound size. The wound contraction was calculated in percentage by comparing the wound surface initially (at day 7 post‐injury) and at the end of the experimentation (at day 38 post‐injury). In addition, at day 38, a pinch test was performed to assess the elasticity of the scar.

2.6. Histological evaluation

Full‐thickness skin biopsies, including the underlying muscle and fascia, were performed on each wound according to the timeline indicated in Figure 1A. Each biopsy was fixed in 4% buffered formalin and embedded in paraffin and sectioned (4–5 μm). The quality of the dermis was evaluated via histochemistry staining. (a) Neovascularization and collagen deposition: Masson's trichrome staining was performed in order to either detect neovessels and to quantify the neovascularization in each condition, or to measure collagen deposition. Neovessels were counted in five independent 0.05 mm2 areas; collagen fibres were quantified by the collagen index based on a method using the ImageJ software.36, 37 (b) Dermis colonisation by fibroblasts: Haematoxylin and eosin (HE) staining was performed and the number of fibroblasts was evaluated counting them in five independent 0.05 mm2 areas. The cells consisting of an elongated cytoplasm in the dermis were considered as fibroblasts. (c) Inflammation: HE staining was performed and the number of inflammatory cells in the dermis was evaluated counting them in five independent 0.05 mm2 areas. Round small cells with a small cytoplasm were considered as inflammatory cells. (d) Elastin staining: Orcein staining was performed in order to evaluate elastic fibres in the dermis. Briefly, after removing the paraffin, sections were put in an orcein solution for 30 to 60 minutes. After washing with distilled water, orcein excess was removed with 95° alcohol solution, and staining was ended with an absolute alcohol bath in order to obtain black elastic fibres.

2.7. Image acquisition and analysis

Acquisition was performed using a slide scanner microscope Axio Scan, Z1 (Zeiss, Jena, Germany) with a ×20 dry lens (NA 0,8). Images were processed with ZEN software (Zeiss Efficient Navigation).

To quantify the surface and the length of elastic fibres, images analysis was performed using the ImageJ software (https://imagej.nih.gov/ij/).

RGB images were unmixed using the Colour Deconvolution plugin (Ruifrok, A.C. & Johnston, D.A. 2001) to obtain three channels corresponding to the background, collagen fibres, and elastic fibres. This last channel was converted to binary by thresholding, where a foreground pixel is assigned the maximum value (255) and background pixels are assigned the minimum possible value (0), and measured as an individual particle. With ImageJ (National Institutes of Health, MA) in conjunction with the Elastic Tools plugin we developed (available at https://github.com/antoninolillefacility/CampusHU/blob/master/Elastic%20fibers%20analysis.ijm), automated calculations involving elastic fibres were used.

2.8. Statistical analysis

Data are represented as mean ± SD and are subjected to one‐way analyses of variance (one‐way ANOVA) (Prism 8, GraphPad, San Diego). Significance levels were set at P < .05.

3. RESULTS

3.1. Wound healing and scar contraction

We first compared the four treatments in an experimental model for deep burns (cf. the Materials and Methods section) to evaluate the effects of HAM on wound healing following the procedures indicated in Figure 1A. All pigs recovered quickly from the burn injuries and the subsequent treatment procedures that occurred 7 days after the burns. No haematoma or sign of infection were detected in all graft conditions. In all cases, the epidermis and dermis were reconstructed with a papillary basal membrane (Figure 1B). No difference in wound healing speed was evidenced between all groups (data not shown and Figure 1B). At a later timepoint (day 38 post‐burn injury), wound healing was achieved regardless of treatment conditions. There were no macroscopic differences in skin graft take rate and surface roughness between all the different conditions (data not shown).

However, macroscopic differences were observed in terms of scar contraction. Figure 1C shows that scar surfaces were smaller at day 38 than at day 0 or day 7 for STSA alone or associated with Matriderm. Interestingly, the scar surface remained larger with HAM. Indeed, it appeared that in the presence of HAM, the wound healing exhibits a low retraction rate while, without HAM, retraction rates were higher regardless of the treatment conditions (Figure 1D). Altogether these results indicate that HAM reduces scar contraction when associated with split‐thickness autograft alone or with Matriderm.

3.2. Scar elasticity

The pinch test was performed to test the elasticity of the scar at day 38 post‐burn injury. Irrespective of the treatments (STSA, or STSA and Matriderm), the presence of HAM skin wound healing increased elasticity (Figure 2A). Since skin elasticity was better under conditions with amniotic membranes, we further examined the presence of elastic fibres in dermis by orcein staining (Figure 2B). With STSA alone or STSA and Matriderm, elastic fibres were mainly short and small. Upon comparison, at day 38 post‐burn injury, when HAM was added, the elastic fibre network was more dense and well organised in the presence of long elastic fibres (Figure 2C, right). No increase in the total quantity of elastic fibres was seen with HAM. Thus, HAM promotes the formation of a dense network with long elastic fibres to maintain scar elasticity.

Figure 2.

Figure 2

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Comparison of the effects of indicated treatments on skin elasticity assessed at 38 days after burn injury (A) by pinch test and (B) histological staining of elastin fibres in the dermis performed by orcein (magnification ×63). White arrows display elastin fibres in wounded skin and black arrows display elastin fibres in healthy skin. This result displays one representative experiment out of three with similar results. C, left panel, quantification of the surface of elastic fibres in the dermis over time after indicated treatments; right panel, repartition of elastic fibres in three indicated groups according to their length. Results are mean ± SD of three independent experiments

3.3. Fibroblast colonisation of dermis and collagen evaluation

Fibroblasts are the major cellular agents of wound repair since they produce collagen for skin regeneration. In fact, the migration of fibroblasts into the wound is a crucial step of the healing process. At day 12 post‐burn, a very low number of fibroblasts were detected after STSA. In contrast, when STSA was associated with Matriderm, an increase in fibroblasts was evidenced. Interestingly, the addition of HAM significantly increased the recruitment of fibroblasts in the dermis, regardless of the initial treatment (Figure 3A,B)). The recruitment effect of HAM was validated later at day 17 post‐burn (Figure 3B). Finally, regardless of the experimental conditions, fibroblasts in the dermis reached normal density at day 38 post‐burn (Figure 3B). Thus, our results indicate that the presence of HAM does accelerate the recruitment of dermal fibroblasts to the wound site. However, we did not observe significant accumulation of collagen in the dermis under HAM conditions (Figure 4B).

Figure 3.

Figure 3

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Histological evaluation of the dermis upon treatments with HE staining. A, Representative photographs of dermis at day 38 post‐burn. Quantitative evaluation of fibroblasts (B) or inflammatory cells (C) in the dermis of pigs at the indicated days after burn injury. Diagram displays the mean ± SD of five independent counting performed after HE staining on areas of 0.05 mm2 for each condition. **, P < .01; *, P < .5

Figure 4.

Figure 4

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Assessment of dermis structure after indicated treatments with Masson's staining. A, Representative photographs of dermis for each condition at day 38 post‐burn after Masson's trichrome colour staining. White arrows show typical vessels. B, Quantification of collagen fibres at day 38 post‐burn. Histogram represents the mean ± SD of three pigs. C, Revascularization at day 38 post‐burn assessed by the percentage of revascularization of superficial dermal related to healthy skin. Results display the mean ± SD of five independent counting of neovessels

3.4. Inflammatory cell recruitment

The process of wound repair is associated with an early inflammatory phase characterised by the recruitment of inflammatory cells in the wound. Figure 3A,C shows that in comparison to healthy skin, there was a transient increase (day 12‐17 post‐burn injury) in the number of inflammatory cells in the dermis, regardless of the treatments. The presence of HAM did not change the recruitment of inflammatory cells in the wound site.

3.5. Neovascularization

Angiogenesis is a critical process that allows the production of granulation tissue in the wound. The addition of Matriderm did not favour neovascularization (Figure 4A,C). In contrast, the addition of HAM increased neovessels in the dermis when burns were treated with STSA alone or associated with Matriderm (Figure 4A,C). Thus, the presence of HAM increased angiogenesis.

3.6. Clinical case report

A 33‐year‐old man presented with 42% total body surface area burns, wherein 20% of the surface was a third‐degree burn. This burn was located on the entire right upper limb including the right hand (Figure 5A). Tangential excision and STSA were performed at day 5 post‐admission as recommended. (Figure 5B). By day 15 post‐graft, the first skin graft was totally lysed and a necrosis of the distal fingers had been developed. A second procedure with STSA was performed. This surgery failed with loss of the autograft and local infections. The wound evolution of the right hand was dramatic with the beginning of a contracture scar formation and finger dorsal retraction (Figure 5C). Afterwards, we decided to use a dermal substitute (Matriderm) with a cryopreserved HAM dressing to reduce the dorsal retraction. The retractile scar and remaining wound fibrin located on the dorsal area of the hand were excised with manual dermatome. The dermal substitute (Matriderm) was applied on the wound bed, and then an autologous thin‐thickness skin graft was immediately performed in a one‐stage procedure. The amniotic membrane was applied and the dressing was ended by applying oily tulle (Figure 5D). We then followed the macroscopic evolution. From day 9 postoperation, the healing was achieved (Figure 5E). In addition, the dermis was well integrated without any oedema or inflammation. No infection was detected and no early hand retraction was noticed. The patient was able to leave the hospital quickly on day 30 post‐graft. Interestingly, after 24 weeks postoperation, on the dorsal skin of the hand, area which received the amniotic membrane, no retraction was detected (Figure 6F). This was associated with good elasticity tested with the pinch test (Figure 6G). By contrast, in the palmar region that did not receive HAM, we noticed a high rigidity with a strong retraction (Figure 6H).

Figure 5.

Figure 5

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Evolution of a burnt hand of 33‐year‐old patient burned by fire. Photographs represent A, hand on the day of the burn; B, day 5 after burn; C, before the third excision; D, just after amniotic membrane + Matriderm graft; E, day 9 post‐graft; and F,G,H, week 24 post‐graft, where F, grafted dorsal side, G, pinch test on the dorsal side, and H non‐grafted palmar side

4. DISCUSSION

After a deep burn, the medical care of patients allows the optimization of wound healing quality in order to obtain the best skin quality in terms of function and cosmetic outcomes. In this context, several artificial dermal substitutes have been developed to improve the quality of the wound healing. Nevertheless, some results indicate that dermal substitutes are often not sufficient especially in areas that need a good elasticity and pliability such as the hands, neck, and wrists.

In this work, we tested the effect of the amniotic membrane alone and also the Matriderm dermal substitute in the care management of a pig model with third‐degree burn. On a clinical level, the retraction scar was reduced in the presence of an amniotic membrane, resulting in a better cosmetic outcome. This is consistent with a recent study, which evidenced that a HAM extract decreases retraction of fibroblast‐embedded lattices.38 In addition, pinch tests highlight a more pliable skin in the presence of an amniotic membrane. This could be explained by the presence of a well‐formed network of elastin fibres observed only when the amniotic membrane was applied. A recent study described that the application of an amniotic membrane on a minipig with a third‐degree burn induced a significant decrease of αSMA+ cells in the dermis compared to the Integra artificial dermis.39 In addition, it was described that the presence of elastin fibres is crucial in a regenerated dermis to decrease wound contraction and improve scar appearance and functionality.40 Matriderm does not contain any elastin fibres. Here, we show that an amniotic membrane management should stimulate the synthesis of elastin fibres and induce a well‐formed elastin fibre network completing the already described improved effects of dermal substitutes on wound‐healing quality.

Interestingly, our human case report displays similar results, that is, improved wound compared to split‐thickness skin graft alone with better elasticity recovery. In developing country where cadaver skin banks are rare, surgeons have described amnion grafts in the context of the care management of second‐ and third‐degree burns. Ravishanker et al clearly described that the use of amniotic membrane was beneficial to patients with (a) decreased pain, (b) good adherence of the amniotic membrane on the wound, which was made for an easier application, and (c) good recovery of limb and joint mobility.24 In facial burns, a shorter healing time and a long‐term follow‐up of the scar led to a better cosmetic outcome.24 A recent study reported that a split‐thickness skin graft in association with an amniotic membrane dressing yielded better and faster take rates than skin graft only.41, 42 In leg ulcers, the benefits of amniotic membranes in the wound healing process were widely reported. In this context, the amniotic membrane induced the decrease of matrix metalloproteinases (such as MMP‐2 and MMP‐9) activities measured in wound exudates.43 This is partly because of the presence of the potent MMP inhibitors, such as tissue inhibitor of metalloproteinases‐1 (TIMP‐1), type‐1 plasminogen activator inhibitor (PAI‐1), and thrombospondin‐1 (TSP‐1) present in the amnion dressings. Fibroblasts play a crucial role in the balance between extra cellular matrix (ECM), compound secretion, and their inhibitors in order to generate a structured ECM network. This balance is essential in wound healing to obtain a good quality scar. It has been described that the amniotic membrane downregulates TGF‐β and its receptor expression by the fibroblasts and, in doing so, reduces the risk of fibrosis. Also, a HAM extract increases the in vitro secretion of extracellular matrix molecules such as glycosaminoglycans, procollagen‐I, MMP‐1, and TIMP‐1 by the fibroblasts.38 Then HAM promotes tissue reconstruction rather than scar tissue formation.44 Amniotic membrane compound should contribute to reducing the differentiation of fibroblasts into myofibroblasts and thus promote better tissue reconstruction.

In our study, the dermis colonisation by fibroblasts is enhanced by the application of amniotic membrane without increasing the number of myofibroblasts. Interestingly, it was recently described that ulcer‐issued fibroblasts transiently increase their proliferation in the presence of a HAM extract without any αSMA expression.38

In parallel, a strong increase in neovessel formation was detected in the condition STSA+Matriderm and amniotic membrane. The angiogenesis effect of amniotic membrane remains controversial. Several previous studies described anti‐angiogenesis effects mainly in a context of ocular surface reconstruction. Nevertheless, in a full‐thickness wound in a pig mode, a higher vessel proliferation with amniotic membrane and with dermal substitute was recently reported.39 The triggering of this proangiogenic effect could be due to the amniotic membrane, which is permeable to oxygen.45 Also, oxygen and nutrients could reach the wound bed more rapidly and enhance the wound healing process.

One risk in promoting neovascularization could be to increase the recruitment of inflammatory cells. Nevertheless, we did not detect any difference in the number of inflammatory cells among the different conditions. In addition, in the human case described here, a good graft take rate was observed when the amniotic membrane was applied unlike the two previous grafts without amniotic membrane that ended in a dramatic graft rejection and inflammatory reaction. This should reflect the previously described anti‐inflammatory effect of amniotic membrane. As previously reported, amniotic membrane induces apoptosis of inflammatory cells and induced TGB β secretion, and cytokine is well known to induce a tolerance immune response.

More recently, a hypothermically stored amniotic membrane (at +4°C) was proposed to be used in an environment of chronic wound.46 Also dehydrated amniotic membrane is already used in diabetic foot ulcers,47, 48 venous leg ulcers,49 and several chronic wounds. It was shown to be equivalent to cryopreserved ones in terms of (a) growth factors composition, (b) induction of fibroblast and keratinocyte proliferation, and (c) angiogenesis induction.46, 50, 51 Storage of dehydrated AM is easier to use in comparison with the cryopreserved amniotic membrane. It would be interesting to continue this study using hypothermic or dehydrated stored amniotic membrane.

In conclusion, this study reveals promising results concerning a beneficial effect of amniotic membrane in the context of deep burns treated with a split‐thickness skin graft and dermal substitute. Owing to good results obtained in elasticity recovery, all these data encourage us to enrol a larger number of patients in a clinical trial in order to demonstrate a real effect on humans, especially in areas of the body requiring good elasticity and pliability.

CONFLICT OF INTEREST

The authors declare no potential conflict of interest.

ACKNOWLEDGEMENTS

This study was supported by INSERM, CHU de Lille, and Université de Lille.

Hatzfeld A‐S, Pasquesoone L, Germain N, et al. Benefits of cryopreserved human amniotic membranes in association with conventional treatments in the management of full‐thickness burns. Int Wound J. 2019;16:1354–1364. 10.1111/iwj.13198

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