JBUR 5321 No.

of Pages 9

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Biological function evaluation and effects of laser

micro-pore burn-denatured acellular dermal matrix

Youlai Zhang a,b , Yuanlin Zeng a,1 , Guohua Xin a, Lijin Zou a ,
Yuewei Ding a, Duyin Jiang c, *
a
Burn Center, The First Affiliate Hospital of Nanchang University, Nanchang, Jiangxi 330006, China
b
Shandong University Graduate School Jinan, Shandong 250100, China
c
Department of Emergency and Department of Burns and Plastic Surgery, The Second Hospital of Shandong University,
Jinan, Shandong 250033, China

article info abstract

Article history: Objective: In the field of burns repairs, many problems exist in the shortage of donor skin, the
Accepted 11 July 2017 expense of allograft or xenograft skin, temporary substitution and unsatisfactory extremity
Available online xxx function after wound healing. Previous studies showed that burn-denatured skin could
return to normal dermis formation and function. This study investigates the application of
laser micro-pore burn-denatured acellular dermis matrix (DADM) from an escharotomy in
Keywords:
the repair of burn wounds and evaluates the biological properties and wound repair effects of
Burn
DADM in implantation experiments in Kunming mice.
Wound healing
Methods: Specific-pathogen-free (SPF) Kunming mice were used in this study. A deep II burn
Laser micro-pore technique
wound was created on the dorsum of the mice by an electric heated water bath. The full-
Denatured dermis
thickness wound tissue was harvested. The necrotic tissue and subcutaneous tissue were
Acellular dermal matrix
removed. The denatured dermis was preserved and treated with 0.25% trypsin, 0.5% Triton X-
100. The DADM was drilled by laser micro-pore. The biological properties and grafting effects
of laser micro-pore burn-DADM were evaluated by morphology, cytokine expression levels
and subcutaneous implantation experiments in Kunming mice.
Results: We found statistical significance (P< 0.05) of the elastic modulus (MPa), maximum
load force (N) and contraction measurement (CM) of the laser micro-pore burn-DADM
(experimental group) compared to the control group (no laser micro-pore burn-DADM).
Cytokine expression level was different in the dermal matrixes harvested at various time
points after burn (24h, 48h, 72 h and infected wound group). Comparing the dermal matrix
from 24h burn tissue to infected wound tissue, the expression level of IL-6, MMP-24, VE-
cadherin and VEGF were decreased. We found no inflammatory cells infiltration in the
dermal matrix were observed in both experimental and control groups (24h burn group),
while the obviously vascular infiltration and fiber fusion were observed in the experimental
group after subcutaneous implantation experiments.

Abbreviations: DADM, denatured acellular dermal matrix; SPF, specific-pathogen-free; VEGF, vascular endothelial growth factor; TCK-1,
thymus chemokine-1; MT5-of MMP, membrane-type matrix metalloproteinase-5; MMP-24, matrix metalloproteinase-24; DDM, denatured
dermal matrix.
* Corresponding author.
E-mail address: jdybs2@vip.163.com (J. Duyin).
1
Co-first author.
http://dx.doi.org/10.1016/j.burns.2017.07.009
0305-4179/ © 2017 ISBI. Published by Elsevier Ltd. All rights reserved.

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acellular dermal matrix, Burns (2017), http://dx.doi.org/10.1016/j.burns.2017.07.009
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2 burns xxx (2017) xxx –xxx

Conclusion: There was better bio-performance, low immunogenicity and better dermal
incorporation after treated by laser micro-pore drilling and decellularized deep II burn-
DADM, which may be considered as a better substitute for dermal matrix. Furthermore, the
earlier harvested DADM after burn (24h) shows the better transplantation effect.
© 2017 ISBI. Published by Elsevier Ltd. All rights reserved.

2.2. Establishment of the animal model

1. Introduction
There are some problems in the field of burn repairs which
includes shortage of donor skin, the expense of allograft or
xenograft skin, temporary substitutions and unsatisfactory
extremity function after wound healing [1]. In our previous
studies, we found there is a special layer of dermal tissue
which was not burned to necrosis in the deep II and mixed
degree burned skin, in which the cells are disorders and
dysfunction. The morphology of this special layer of dermal
tissue is abnormal, but with improvement of local environ-
ment, the special layer of dermal tissue could recover to
normal morphology and function [2]. Research showed
systemic application of P188 in the deep II and the burn-
denatured skin could return to normal dermis formation and
function in the early burn stage [3]. Our previous study showed
that the deep II burn-denatured dermal matrix treated with
0.25% trypsin and 0.5% Triton X-100 has lower immune
rejection and better tissue compatibility after implantation
in animal [2,4]. However, after the thermal injury, the burn
toxins are released and can induce adverse effects in patients
[2]. Furthermore, denatured dermal matrix has the following
issues that need to be resolved, including tissue fragility and
“toxin” residue. Vascular infiltration and cells growth slowly
were also observed in implantation studies [4]. Study shows
that laser micro-pore PADM grafting in combination with split-
thickness autografting can improve wound healing [5]. We
applied a laser micro-pore technique on deep II burn-
denatured dermis, after that the laser micro-pore burn-DADM
was treated with trypsin and Triton-100. The biological
properties and grafting effects of laser micro-pore burn-DADM
were evaluated by examining the properties and cytokine level
and performing an implantation experiment in Kunmimg
mice.
2. Materials and methods
2.1. Laboratory animal selection and preparation of DADM
A total of 60 health specific-pathogen-free (SPF) Kunming
mice, aged 12–14 weeks and weighing 403g, were housed in
the standard animal environment. The mice were allowed to
adapt to the environment of the lab for 5–7days and were
randomly selected to receive a deep II burn wound. All
animals were purchased from Experimental Animal Center of
Shandong University. The certificate number was SCXK (Lu)
20150002. These mice were raised in accordance with the
Guidelines for the Care and Use of Laboratory Animals
published by National Institutes of Health of the USA (1996
Revision).
First, 10% chloral hydrate (30mg/kg) was used to anesthetize
the animals. After successful anesthesia, the experimental
animal was secured in a stationary position. Freshly prepared
depilatory were evenly applied on the dorsum to completely
wet the hair. After 3s, the hairs were removed, sterile saline
was used to wipe off the depilatory, and this skin area was
dried.
An electric heated water bath was used to control the
water temperature. The temperature to make a burn injury
was set at 90  C. The surgeon wore thick waterproof gloves.
The animal was secured in a stationary position, and its
dorsum was immersed in the hot bath for 12 s [2]. The
animal was removed from the hot bath immediately after
the timer went off. A dry towel was used to dry the dorsum,
and then iodophor was applied to the wound site. The
injured animal received Ringer’s solution (40 ml/kg) via
intraperitoneal injection for fluid resuscitation. The animal
was housed alone after it awoke from the anesthesia.
Povidone-iodine was applied to the burn wound twice daily.
Adequate care was taken to prevent the animals from biting
each other.
2.3. Preparation and grouping of DADM
The animals were sacrificed by cervical dislocation at 24,
48h, 72 h after the burn. After povidone-iodine disinfection,
the skin at the burn wound was harvested and repeatedly
washed in sterile saline. Using aseptic technique, the
subcutaneous fascia, fat and muscles were trimmed and
removed. The deep II burn skin tissues were selected for this
study by histological examination (H&E) for a small piece of
skin, and the remaining skin tissues were stored at 4  C for
future use.
The deep II burn skin tissues were soaked in a 0.1%
benzalkonium bromide solution for 30 min and then cut into
32-cm2 DADM sheets, which were placed in a 250ml jar
with 0.25% trypsin and 0.5% Triton X-100 for 15min. The
solution was titrated to 100ml with PBS. The jar was put on a
37  C thermostat shaker at 100 rpm vortex for 2h. After
vortex, the DADM sheets were washed in PBS for 12h. The
PBS was replaced with fresh solution every 6h until the PBS
in the jar was completely clear. The DADM sheets were
randomly divided into two groups: the experimental group
and the control group. The specific laser template drilling
technique was then used to produce a total of 300 micro-
holes in a 32-cm2 portion of the dermal matrix sheet under
laser template guidance. The diameter of the micro-holes is
135 mm, and the distance between the micro-holes is 1mm
[6].

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2.4. The test parameters and methods
2.4.1. Observation of the gross specimens
Gross specimens observation included the color, texture,
durability and ductility of the acellular dermal matrix.
2.4.2. H&E
H&E staining for skin tissues from the experimental and
control groups. The tissue morphology images were captured
using a ordinary optics microscope.
2.4.3. Physical property test
Mechanical property testing: The prepared DADM was cut into a
2.01.0-cm2 dumbbell-shaped sheet. Each group included five
DADM sheets. Both ends of the DADM sheet were clipped on a
universal testing machine. The DADM sheet was stretched
along the fiber-running direction at a rate of 1mm/min until the
maximum load force was 3MPa. Then, the stretched DADM
sheet was relaxed at the same rate until the maximum load
force was 0MPa. This stretch-relax process was repeated three
times to obtain a stable stress-strain curve. When this process
was repeated for the fourth time, the stress value was recorded
when the maximum load force reached 3MPa. Another DADM
sheet was stretched at a rate of 1mm/min until the DADM sheet
ruptured. The load force and the stretching length were
recorded at the time of rupture. All data were recorded and
computed by the JBK measurement control system. The
measurements of the DADM graft were performed at 25  C
and 75% humidity to prevent dehydration of the DADM sheet.
2.5. Cytokine expression of acellular dermal matrix
2.5.1. Sample preparation for protein arrays
All procedures were performed on ice. Three pieces of a 3.02.0-
cm2 DADM were harvested from experimental groups at 24h,
48h, 72h after burn or burn wound with infection respectively.
Samples were weighed and quickly stored in liquid nitrogen.
2.5.2. Sample dialysis
The tissue samples were lysed, and the sample lysates were
dialyzed using dialysis tubing (Item A) before they were labeled
with biotin. 200 ml of each sample lysate was added to the
dialysis tubing. The tubing was dialyzed in 4000ml of 1 PBS (pH
8) at 4  C with stirring. The dialysis buffer was changed every 3h.
2.5.3. Determination of protein concentration
After dialysis, the concentration of the purified protein was
determined (BCA assay, Pierce, lot number: 23227). A standard
curve was prepared by plotting the absorbance of the BSA
standards. The standard curve and dilution factor of the samples
was used to determine the protein concentration of each
unknown sample. The measurement results are shown below:
Sample Con. of protein (mg/ml)
24 h 12,252.53
48 h 11,596.67
72 h 8296.667
Infected 7678.559
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3
2.5.4. Biotinylation of the samples
During biotinylation of the samples, all reagents should not be
contaminated with amines or sodium azide (for example, Tris,
glycine). To make the solution, the vial with biotin powder was
centrifuged briefly, add 100 ml of 1 PBS to the powder, then the
solution was mixed well by pipetting up and down to prepare
1 biotin solution. An appropriate amount of biotin solution
was added into centrifugation tubes containing the samples
and mixed immediately. The centrifugation tubes were put on
a shaker and incubated at room temperature for 30min. The
centrifugation tubes were flicked every 5min to mix the
reagents. At the end of this step, 3 ml of stopping buffer was
added to stop the reaction, and unconjugated biotin was
removed using the dialysis tubing. For this, 150 ml of the
sample solution was added to the dialysis tubing. The tubing
was dialyzed in 4000ml of 1 PBS (pH 8) at 4  C with stirring.
The dialysis buffer was changed every 3h.
2.5.5. Completely dry the chip slides
The bag of the chip slides was opened after 30min warm up at
room temperature. The seals were removed, and then, the chip
slides were dried in a vacuum dryer at room temperature for
2h.
2.5.6. Blocking and incubation
400 ml of 1 blocking buffer was applied in each well of the chip
slide, avoiding air bubbles. The slides were incubated on a
shaker for 1h at room temperature. After removing the
blocking buffer, 400 ml of sample was transferred to each well.
Note that one sample was used for each array. Next, the chip
slide was incubated at 4  C on a shaker overnight. Biotin-
labeled samples were diluted in blocking buffer (60-fold
dilution). After the samples were removed, the slides were
rinsed with 1ml of 1 wash buffer I for 45min at RT. After the
1 wash buffer I was removed, the slides were rinsed with 1ml
of 1 wash buffer II for 35min at room RT. After 1 wash
buffer II was removed, 400 ml of Cy3-streptavidin-biotin (1:1500
dilution in blocking buffer) was applied to each well, and the
plate was incubated in the dark on a shaker at RT for 2h.
2.6. Fluorescence detection
An InnoScan 300 Microarray Scanner (Innopsys, Parc d’Acti-
vités Activestre, 31 390 Carbonne, France) was used to scan the
plate at the Cy3 or the green channel (excitation frequen-
cy=532nm). The scanning parameters were as follows:
wavelength: 532nm; resolution: 10 mm. The manufacturer’s
software was used for data acquisition, and AAM-BLG-1
software was used for the data analysis.
2.7. Subcutaneous implantation experiment (Fig. 1)
The two groups of DADM were implanted subcutaneously to
compare tissue compatibility and degradation in vivo. A total
of 40 SPF Kunming mice, 40–45g weight, were anesthetized
with 10% chloral hydrate (0.3ml/kg) via intraperitoneal
injection. The surgical field on the dorsum of the mice was
prepared. The two groups of DADM were cut into 1010-mm2
DADM grafts. A longitudinal middle incision was made on the
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Fig. 1 – The process of laser micro-pore DADM embedding.

dorsum of the mouse, and two subcutaneous pouches were
created by blunt dissection in the left and right side of the
incision. The depth of the pouch was 15mm. The DADM graft of
the experimental group was placed in the left pouch, and the
graft of the control group was placed in the right pouch. The
incisions of the skin and subcutaneous tissue were closed. The
two DADM grafts were not in contact with each other. The
mouse was housed alone in a single cage, and 1% povidone-
iodine solution was applied daily on the skin incision. The
observation parameters included (1) when the mice were
sacrificed at the 1, 2, 3, and 4 weeks after implantation, the
appearance, texture, elasticity, fiber formation and surface
capillary growth in the two DADM graft groups were
documented; (2) ten mice were sacrificed at the 1, 2, 3, and 4
weeks after implantation. The DADM graft with its surround-
ing tissue was harvested and fixed with 10% formaldehyde for
and dehydrated with sequential concentration of alcohol and
finally with xylene, embedded in paraffin, and sections were
made at 7-mm thickness. To examine tissue morphology,
standard H&E staining was used. The images of collagen
arrangement, cell growth, and angiogenesis were captured by
ordinary optics microscope.
2.8. Statistical methods
homogeneity of variance are expressed as xs. The differ-
ences among the groups were compared using Student’s t-test
(two groups) or analysis of variance. P <0.05 was considered
statistically significant.
3. Results
3.1. Gross specimen and histological observation
The freshly prepared DADM appeared porcelain white and was
soft, elastic and durable. A light microscope examination
showed the absence of epithelial cell residue and structure in
the DADM but the presence of dermal collagen hyaline
degeneration (Fig. 2a) and partial breakage of the collagen
fibers after the laser micro-pore drilling (Fig. 2a, bottom).
3.2. Biomechanical properties
In the denatured dermal matrix with laser micro-pore drilling,
the elastic modulus and the maximum load capacity all
increased, but the stretching length decreased. The differ-
ences were statistically significant (P<0.05) between the two
groups (Table 1).

The SPSS 19.0 software package was used for the statistical 3.3. Implantation of the DADM graft in animals
analysis. The normality and homogeneity of variance of all the
experimental data were tested using the Shapiro–Wilk test and After implantation, the DADM graft did not show shrinkage,
the Levene method. All data with normal distribution and folds, apparent exudation, redness or swelling. A fiber

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Fig. 2 – H&E staining (100). (a) Showing absence of epithelial cell residue and structure in the DADM but the presence of dermal
collagen hyaline degeneration and partial breakage of the collagen fibers after laser template micro-pore drilling. (b) Showing
that nuclear ruptures were predominant in the experimental group (top panel). The “foam-like” gaps were observed among the
fibers after cell lysis. Irregular arrangements of the elastic fibers were present (1 week after implantation). (c) The bottom panel
showed more obvious vascular infiltration than the top panel. There was more infiltration in the experimental group (2 weeks).
(d) Showing some cellular component growth near the surface of the implanted DADM graft was present in the experimental
group (bottom panel). No inflammatory cell infiltration was shown in either group (3 weeks). (e) Showing that the fiber fusion
phenomenon was more pronounced. Some cellular component growth near the surface of the implanted DADM graft was
present. Vascular infiltration and fiber fusion were present predominately in the experimental group. No inflammatory cell
infiltration was shown in either group (4 weeks).

Table 1 – Biomechanical changes of the DADM after laser micro-pore drilling (X  s).

Dermal matrix Elastic modulus (MPa) Maximum load capacity (N) Stretching length (cm)
Control group 35.802.17 67.40 3.51 2.66  0.05
Experimental group
Group 24h 71.602.30 118.00 3.54 2.48  0.23
Group 48h 70.202.85 107.30 6.15 2.52  0.18
Group 72h 71.802.40 123.55 4.75 2.33  0.31
Group infected 67.403.53 101.00 3.22 2.10  0.27
P value 0.000 0.000 <0.05

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6 burns xxx (2017) xxx –xxx
Fig. 3 – Images of AAM-BLG-1 expression.
membrane layer of micro-vascular network was present on
the surface of the embedded dermis matrix. The histological
analysis showed the differences of cell infiltration and
angiogenesis in the experimental group. One week of after
implantation, a large amount of nuclear condensation and
rupture presents in the central portion of the implanted DADM
graft in both groups, but there is more predominant in the
experimental group compare to the control group. The “foam-
like” gaps were observed among the fibers due to cell lysis.
Irregular arrangement of the elastic fibers was present (Fig. 2b).
Two weeks of after implantation, partial lysed cells near the
surface of the implanted DADM graft were absorbed; fiber
hyaline degeneration and nuclear condensation appeared;
and local vascular infiltration was observed and was more
obvious in the experimental group (Fig. 2c). Three weeks of
after implantation, all lysed cells near the surface of the
implanted DADM graft were absorbed and disappeared; local
vascular infiltration was present; many regions of fiber hyaline
degeneration were still observed; and the fiber fusion was
more obvious. Some cellular component growth near the
surface of the implanted DADM graft was present in the
experimental group. No inflammatory cell infiltration was
shown in either group (Fig. 2d). Four weeks of after implanta-
tion, all lysed cells near the surface of the implanted DADM
graft were absorbed and disappeared in both groups; local
vascular infiltration was present; some fiber hyaline degener-
ation was still present; and the fiber fusion phenomenon was
more pronounced. Some cellular component growth near the
surface of the implanted DADM graft was present. Vascular
infiltration and fiber fusion were more obvious in the
experimental group, and no inflammatory cell infiltration
was shown in either group (Fig. 2e).
3.4. Protein expression of the dermal matrix at various
wound
The protein analysis showed that cytokine expression was
different in the dermal matrixes harvested at various time
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acellular dermal matrix, Burns (2017), http://dx.doi.org/10.1016/j.burns.2017.07.009
points (24h, 48h, 72h and the infected wound group) (Fig. 3).
Comparing the dermal matrix from 24h, 48h, 72h to infected
wound group, the expression of MMP-24/MT5-of MMP, VE-
cadherin, TCK-1 and VEGF decreased, but the most remaining
tested cytokine levels increased (Fig. 4).
4. Discussion
Large, deep burn wounds are prone to infection, and this is a
continual problem as long as the burn wounds remain
unhealed [7]. The preserved denatured dermis can become
necrotic because they are vulnerable to wound swelling,
infection, and severe fever after escharotomy in patients
with large deep II burns [3]. Hence, the denatured dermis is
usually removed along with completely necrotic tissue
during escharotomy. This method later necessitates autol-
ogous thin-skin grafting in the wounds, especially in
wounds involving a functional part of the skin. After wound
healing, however, scarring can affect the joint function [8].
The recycling of the denatured dermis, i.e., “Turning waste
to treasure”, has been a research direction focus in our
group.
Our previous study showed that the deep II burn-
denatured dermal matrix had advantages, including lower
immune rejection and better tissue compatibility after they
were treated with 0.25% trypsin and 0.5% Triton X-100 for
decellularization [2]. However, it is known that denatured
dermal matrix (DDM), a dermal substitute, has the following
issues that need to be resolved, including tissue fragility, a
tendency to crack, weak stretchiness, and “toxin” residue.
Vascular infiltration and cells growth slowly were observed in
implantation studies. We attempted to solve the above
mentioned issues by making micro-pores in the acellular
dermal matrix via a laser micro-pore drilling technique, which
enhances the elastic modulus and maximum load force of the
acellular dermal matrix and can significantly enhance their
biological properties and convenience for application.
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Fig. 4 – Protein expression of dermal matrix form 24-h, 48-h, 72-h to infected burn wounds.
However, this technique can result in a shorter stretching
length, which we believe would not affect its application.
After dermal matrix grafting, the graft may lack or have
insufficient blood supply if angiogenesis is slow. This may
cause graft fusion with animal slowly and necrosis of the
dermal matrix graft. Moreover, the denatured dermal matrix is
not fully nourished in itself and in the ecological state [6]. It is
key for successful dermal matrix grafting to how to improve
the acellular denatured dermal matrix permeability to obtain
adequate nutrition and to enhance its angiogenesis rate. Study
shows that cross-linking can result in a better tissue structure
to reduce the extent of tissue degradation and tissue
inflammation [9]. However, the residual toxicity of medication
for cross-linking can easily lead to cytotoxicity, which will
affect the angiogenesis and cellularization of the dermal
matrix. Laser punch is a practical laser processing technique
and has been widely used in processing material, laser
punching is a physical heating process at the interfere between
laser and materials. When the high energy laser beam is
focused on the surface of a material to be processed, the
material obtains the heat by absorbing laser energy. Micro-
pores are formed as the solid material gasifies from which the
local temperature rises, the advantages make it the best choice
for processing a large number of minute pores in high density.
We use this technique to process uniformly distributed micro-
pores on DADM, which could facilitate the passage of tissue
fluids and penetration of new formed micro-vessels [5]. In this
study, a micro-pore drilling technique (micro-pore diameter:
135 mm, distance between micro-pores: 1mm) can cause a little
damage on the dermal matrix [6]. The micro-pores were
considered as extra room for angiogenesis and can be helpful
in early blood plasma supply of the DADM graft to promote
angiogenesis. This study demonstrated the coverage of a
vascular membrane on the DADM graft and partial vascular
infiltration at the 2 weeks after grafting. Compared with the
previous results [4], the angiogenesis speed was increased by
1/3. At the 3 weeks after grafting, some cellular growth near the
surface of the implanted DADM graft was present in the
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acellular dermal matrix, Burns (2017), http://dx.doi.org/10.1016/j.burns.2017.07.009
7
experimental group. The cellular component presence was
observed 1 week earlier than that in the control group.
This study using micro-pore burn-DADM after 48h burn
wound to implant the animal. This is parallel to the clinic that
patients will be performed the escharectomy after shock
period. H&E staining results show that there is no evidence of
significant inflammation in the animal after 1, 2, 3, and 4 weeks
after implantation.
Transplant rejection is an important factor to determine
whether the allografting is successful [10]. There are a lot of
cytokines involved in the burn wound healing. MT5-of MMP is
a new member of the MT-MMPS family and was cloned from
human brain tissue. It is expressed in normal tissue and tumor
tissue and is known to participate in embryogenesis, angio-
genesis and wound healing in physiological conditions and be
involved in tissue remodeling in pathological conditions [11].
VE-cadherin is a key factor of vascular endothelial cell
connection, the main ingredient of vascular endothelial cell
adhesion, is regulated by the SRC kinase. VE-cadherin plays an
important role in regulating cell differentiation and prolifera-
tion in addition [12–15]. Structural and functional abnormali-
ties of this protein can cause dissociation of endothelial cell
connections and result in increased permeability of the
endothelial cell layer, local tissue oedema, ischaemia, hypoxia,
and functional restriction. It’s disruption could be one of the
causes of decreased expression of IL6 and MMP-24 in the local
wound. TCK-1 is a member of the CXC chemokine family and
can bind with CXCR2 receptor to directly or indirectly promote
angiogenesis in the local micro-environment [16]. In addition,
VEGF is a specific heparin-binding growth factor of vascular
endothelial cells and can promote endothelial cell prolifera-
tion by binding to the corresponding receptors on the vascular
endothelium [17]. Meanwhile, it can increase vascular perme-
ability to promote endothelial cell migration and is considered
the most powerful angiogenic factor to date [18]. IL6 plays a
major role in enhancing the effect of other cytokines, the
expression of other inflammatory cytokines was higher in the
infected group than the uninfected group. All burn patients
JBUR 5321 No. of Pages 9
8 burns xxx (2017) xxx –xxx
showed initial elevation in IL-6 levels, and mean IL-6 levels
were found to increase with the increase in the percentage of
burn injury and in the infection burn patient [19], but study
result shows that the expression of IL-6 decreased in infected
burn wound, it remains need to be further research, but
current evidence does not support a direct effect of IL-6-174 G/
C polymorphism on the risk of sepsis [20].
Thus, it is important to determine whether the dermis can
be harvested any time after the burn to prepare the acellular
dermal matrix for grafting. To further explore the existence of
“burn toxins”, we compared the protein expression of acellular
dermal matrix harvested at 24h, 48h and 72h after the burn to
the infected burn wound. The results showed that the
expression of MMP-24/MT5-of MMP, VE-cadherin, TCK-1 and
VEGF decreased, but that the remaining cytokines significantly
increased in their expression with a longer time course.
Decreased expression of these factors may not function in
favor of dermal matrix implantation and incorporation.
Therefore, DADM harvested with different timing may have
different responses to inflammation. A short time course may
be related to less inflammation. Considering the timing of
grafting for patients with extensive burns, we suggested that
dermal matrix harvested from an uninfected wound after burn
is feasible for use, and it seemed that the better transplanta-
tion effect was discovered in the earlier harvested DADM after
burn.
5. Summary
The deep II burn-DADM from a non-infected wound can be
converted to micro-pore acellular dermal matrix by means of
laser micro-pore drilling. The advantages of this micro-pore
acellular dermal matrix include better bio-performance, low
immunogenicity, better dermal compatibility and active
cytokines, which can improve the wound healing. Therefore,
this laser micro-pore burn-DADM may be considered a suited
substitute for dermal matrix.
Funding and role of the funding source
This work was supported by grants from the National Natural
Science Foundation Project [grant numbers 30772258,
81071560, and 81372074] and the Scientific and Technological
Project of Shandong Province [grant numbers 2009GG10002078
and 2015GSF118041].
Conflict of interest
No conflict of interest exits in the submission of this
manuscript, and manuscript is approved by all authors for
publication. I would like to declare on behalf of my co-authors
that the work described was original research that has not
been published previously, and not under consideration for
publication elsewhere, in whole or in part. All the authors
listed have approved the manuscript that is enclosed.
Please cite this article in press as: Y. Zhang, et al., Biological function evaluation and effects of laser micro-pore burn-denatured
acellular dermal matrix, Burns (2017), http://dx.doi.org/10.1016/j.burns.2017.07.009
REFERENCES
[1] Philandrianos C, Andrac-Meyer L, Mordon S, Feuerstein JM,
Sabatier F, Veran J, et al. Casanova comparison of five dermal
substitutes in full-thickness skin wound healing in a porcine
model. Burns 2012;38(6):820–9.
[2] Yu G, Ye L, Tan W, Zhu X, Li Y, Jiang D. A novel dermal matrix
generated from burned skin as a promising substitute for
deep-degree burns therapy. Mol Med Rep 2016;13(3):2570–82.
[3] Shi Y, Li L, Chai J, Sun T. Effect of Poloxamer 188 on deepening
of deep second-degree burn wounds in the early stage. Burns
2012;38(1):95–101.
[4] Wang XC, Li C, Shan F, Wang WT, Zhu XG, Jiang DY.
Experimental study on the recycling of denatured acellular
dermal matrix after burn. Chin J Burns 2012;28(3)201–6 [in
Chinese].
[5] Chai JK, Liang LM, Yang HM, Feng R, Yin HN, Li FY, et al.
Preparation of laser micropore porcine acellular dermal
matrix for skin graft: an experimental study. Burns 2007;33
(6):719–25.
[6] Luo X, Wan L, Li AL, Xu JJ, Xia WD, Li YL, et al. Preparation and
application of novel laser micropore porice acellular dermal
matrix. Chin J Inj Repair Wound Healing (Electronic Edition)
2012;7(3)240–4 [in Chinese].
[7] Patil NK, Luan L, Bohannon JK, Guo Y, Hermandez A,
Fensterheim B, et al. IL-15 superagonist expands mCD8+ T, NK
and NKT cells after burn injury but fails to improve outcome
during burn wound infection. PLoS One 2016;11(2):e0148452.
[8] Singer AJ, Thode Jr. HC, McClain SA. The effects of epidermal
debridement of partial-thickness burns on infection and
reepithelialization in swine. Acad Emerg Med 2000;7(2):114–9.
[9] Grover CN, Gwynne JH, Pugh N, Hamaia S, Farndale RW, Best
SM, et al. Crosslinking and composition influence the surface
properties, mechanical stiffness and cell reactivity of
collagen-based films. Acta Biomater 2012;8(8):3080–90.
[10] Leto Barone AA, Mastroianni M, Farkash EA, Mallard C,
Albritton A, Torabi R, et al. Genetically modified porcine split-
thickness skin grafts as an alternative to allograft for provision
of temporary wound coverage: preliminary characterization.
Burns 2015;41(3):565–74.
[11] Baranger K, Marchalant Y, Bonnet AE, Crouzin N, Carrete A,
Paumier JM, et al. MT5-MMP is a new pro-amyloidogenic
proteinase that promotes amyloid pathology and cognitive
decline in a transgenic mouse model of Alzheimer’s disease.
Cell Mol Life Sci 2016;73(1):217–36.
[12] Harris ES, Nelson WJ. VE-cadherin: at the front, center, and
sides of endothelial cell organization and function. Curr Opin
Cell Biol 2010;22(5):651–8.
[13] Toit AD. Mechanotransduction: VE-cadherin lets it flow. Nat
Rev Mol Cell Biol 2015;16(5):268.
[14] Ikezoe T, Yang J, Nishioka C, Umezawa K, Yokoyama A.
Thrombomodulin blocks calcineurin inhibitor-induced
vascular permeability via inhibition of Src/VE-cadherin axis.
Bone Marrow Transplant 2017;52(2):245–51.
[15] Liu R, Cao Z, Tu J, Pan Y, Shang B, Zhang G, et al. Lycorine
hydrochloride inhibits metastatic melanoma cell-dominant
vasculogenic mimicry. Pigment Cell Melanoma Res 2012;25
(5):630.
[16] Song J, Hu D, He C, Wang T, Liu X, Ma L, et al. Novel biomarkers
for early prediction of sepsis-induced disseminated
intravascular coagulation in a mouse cecal ligation and
puncture model. J Inflamm (London, England) 2013;10(1):7.
[17] Zhou J, Ni M, Liu X, Ren Z, Zheng Z. Curcumol promotes
vascular endothelial growth factor (VEGF)-mediated diabetic
wound healing in streptozotocin-induced hyperglycemic rats.
Med Sci Monit 2017;23(1):555–62.
JBUR 5321 No. of Pages 9

burns xxx (2017) xxx –xxx 9

[18] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak
HF. Tumor cells secrete a vascular permeability factor that
promotes accumulation of ascites fluid. Science 1983;219
(4587):983–5.
[19] Modi S, Rashid M, Malik A, Shahid M. Study of complement
activation, C3 and interleukin-6 levels in burn patients and
their role as prognostic markers. Indian J Med Microbiol
2014;32(2):137–42.
[20] Gao JW, Zhang AQ, Pan W, Yue CL, Zeng L, Gu W, et al.
Association between IL-6-174G/C polymorphism and the risk
of sepsis and mortality: a systematic review and meta-
analysis. PLoS One 2015;10(3):e0118843.

Please cite this article in press as: Y. Zhang, et al., Biological function evaluation and effects of laser micro-pore burn-denatured
acellular dermal matrix, Burns (2017), http://dx.doi.org/10.1016/j.burns.2017.07.009