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A Novel Immune Competent Murine Hypertrophic Scar Contracture Model: A Tool to Elucidate Disease Mechanism and Develop New Therapies

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Hypertrophic scar contraction (HSc) following burn injury causes contractures. Contractures are painful and disfiguring. Current HSc therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune-competent murine HSc model. A third-degree burn was created on the dorsum of C57BL/6 mice. Three days post-burn, tissue was excised and grafted with ear skin. Graft contraction was analyzed by computer planimetry and tissue harvested on different time points. Outcomes were compared to human condition to validate the model. To confirm graft survival, green fluorescent protein mice (GFP) were used and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus (PC) in contraction was analyzed. Cellularity was assessed with DAPI. Collagen maturation was assessed with Picro-sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by qRT-PCR. Elastic moduli of human and murine skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ∼45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild type mice and vice-versa and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed the PC does not contribute to contraction. Microscopic analyses demonstrated that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT-PCR demonstrated upregulation of TGF-β, ASMA, NMMII, and ROCK2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.
A) Representative DAPI-stained section. All time points showed more cellularity than the normal skin. Day 7 showed the highest cellularity, whereas day 168 showed the least. (B) Representative H&E-stained sections. Granulation tissue surface area is graphically represented. Day 14 showed the greatest surface area of granulation tissue, then it decreased gradually up to day 168. (C) Representative Masson's trichrome-stained sections demonstrating collagen content of the skin grafts. Quantitative analysis of collagen index was graphically represented vs. normal skin. (D) Sirius red polarization microscopy of collagen fibers revealed an increase in collagen maturation over time. There were more immature (green, loosely packed) collagen fibers on day 7 than day 21 and day 168, respectively. Whereas, there was more mature collagen fibers (yellow, densely packed) on day 168 than on day 21 and day 7, respectively. (E) Representative CD31-stained sections demonstrating the degree of vascularity. All time points show more vascularity compared with normal skin. Day 14 showed the highest vascularity, whereas day 7 showed the least. (F) Representative F4/80-stained section demonstrating the density of macrophages. All time points show more macrophages compared with normal skin. Day 14 showed the highest number of macrophages, whereas day 168 showed the least. (G) Representative Toluidine blue-stained sections demonstrating the density of mast cells. All time points showed more mast cells compared with normal skin. Day 9 showed the highest number of mast cells, whereas day 3 showed the least. (H) mRNA expression of ASMA, NMMIIA, ROCK2, and TGF-β was assessed by qRT-PCR. Gene expression is related to ribosomal protein S9 using the ΔΔCt method and then normalized to normal skin. Data are represented as mean ± SEM. ASMA, NMMIIA, and TGF-β show an increase in expression in the graft tissue peaking at day 168. ROCK2 has the greatest increases in expression at days 28 and 168. ASMA, alpha smooth muscle actin; NMMIIA, nonmuscle myosin II A; qRT-PCR, quantitative real-time polymerase chain reaction; ROCK2, rho-associated protein kinase; TGF-β, transforming growth factor beta. *, statistical significance.
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Journal name: Wound repair and regeneration : official publication of the Wound Healing
Society [and] the European Tissue Repair Society
NIHMS ID:
NIHMS636198
Manuscript Title:
A Novel Immune Competent Murine Hypertrophic Scar Contracture Model:
A Tool to Elucidate Disease Mechanism and Develop New Therapies
Principal
Investigator:
Submitter: John Wiley And Sons Publishing (wbnih@sps.co.in, vchnih@wiley.com)
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A Novel Immune Competent Murine Hypertrophic Scar Contracture Model: A Tool to
Elucidate Disease Mechanism and Develop New Therapies
Mohamed M. Ibrahim, MD1, Jennifer Bond, PhD1, Andrew Bergeron, BA1, Kyle J. Miller, BA,1
Tosan Ehanire, BA1, Carlos Quiles, MD1, Elizabeth R. Lorden, MS3,Manuel A. Medina, MD1,
Mark Fisher, MD1, Bruce Klitzman, PhD1,3, M. Angelica Selim, MD2, Kam W. Leong, PhD3,
Howard Levinson, MD1,2
1From the Division of Plastic and Reconstructive Surgery, Department of Surgery, Duke
University School of Medicine, 2Department of Pathology, Duke University School of Medicine,
and 3the Department of Biomedical Engineering, Duke University, Durham,NC
Running head: A Novel Murine Hypertrophic Scar Contracture Model
Key words: Hypertrophic Scar; burn; woundhealing; contracture; fibrosis
Corresponding author:
Mohamed M. Ibrahim, MD
Division of Plastic and Reconstructive Surgery
Department of Surgery
Duke University School of Medicine
Durham, North Carolina
Email: mohamed.ibrahim@duke.edu
Manuscript received: 18 October 2013
Accepted in final form: 4 September 2014
ABSTRACT
Hypertrophic scar contraction (HSc) following burn injury causes contractures. Contractures are
painful and disfiguring. Current HSc therapies are marginally effective. To study pathogenesis
and develop new therapies, a murine model is needed. We have created a validated immune-
competent murine HSc model. A third-degree burn was created on the dorsum of C57BL/6 mice.
Three days post-burn, tissue was excised and grafted with ear skin. Graft contraction was
analyzed by computer planimetry and tissue harvested on different time points. Outcomes were
compared to human condition to validate the model. To confirm graft survival, green fluorescent
protein mice (GFP) were used and histologic analysis was performed to differentiate between ear
and back skin. Role of panniculus carnosus (PC) in contraction was analyzed. Cellularity was
assessed with DAPI. Collagen maturation was assessed with Picro-sirius red. Mast cells were
stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was
assessed with CD31 immune. RNA for contractile proteins was detected by qRT-PCR. Elastic
moduli of human and murine skin and scar tissue were analyzed using a microstrain analyzer.
Grafts contracted to ~45% of their original size by day 14 and maintained their size. Grafting of
GFP mouse skin onto wild type mice and vice-versa and analysis of dermal thickness and hair
follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting
and regenerated in ear skin configuration by day 30. Radiological analysis revealed the PC does
not contribute to contraction. Microscopic analyses demonstrated that grafts show increase in
cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in
collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and
mast cells were increased. qRT-PCR demonstrated upregulation of TGF-β, ASMA, NMMII, and
ROCK2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse
skin and scar tissues.
ABBREVIATIONS:
ASMA: alpha smooth muscle actin
DAPI: 4',6-diamidino-2-phenylindole
ECM: Extracellular Matrix
GFP: Green Fluorescent Protein
HPF: High Power Field
IACUC: Institutional Animal Care and Use Committee
IGF-1: Insulin-Like Growth Factor 1
MLCK: Myosin-Light-Chain Kinase
MSA: Microstrain Analyzer
NMMII: Nonmuscle Myosin II
PC: Panniculus Carnosus
qRT-PCR: Quantitative real-time polymerase chain reaction
ROCK2: Rho-associated protein kinase
STSG: Split Thickness Skin Graft
TGF-β:Transforming Growth Factor Beta
WT: Wild Type
INTRODUCTION
Dermal scarring affects more than 100 million people worldwide annually.1Over 2.4 million
Americans suffer from burns each year, and two million people are injured in motor vehicle
accidents.2Burn wounds cost billions of dollars per year worldwide.3Burnwounds often heal by
forming hypertrophic scars (HSc). HSc are firm, raised, red, itchy scars that develop over 6
months to 2 years. They are disfiguring and can have a severe impact on quality of life.4,5 HSc
contract, and when contraction occurs across a joint it restricts range-of-motion, resulting in a
scar contracture. HSc contractures are estimated to occur in up to 40% of major burn patients in
the United States, and in patients who develop contractures at least fourcorrective surgeries are
required on average.6There are presently no effective therapies to prevent HSc contractures.4
Third degree burns extend completely through the dermis and are managed by excisionof
burned tissue and subsequent skin grafting, usually three days after excision. Skin graft survival
consists of several stages. During the first 48 hours after placement, the skin graft is ischemic
and depends upon diffusion of nutrients and dissolved oxygen from the underlying
wound/granulation bed, a process called plasmatic imbibition.7Subsequently, blood vessels from
the granulation bed invade the skin graft todeliver nourishment and remove wastes via a process
called inosculation.8Inflammatory cells, including macrophages, invade the granulation bed as
the graft continues to mature. Macrophages have a large influence on scarring by removing
debris and pathogens and secreting pro-healing cytokines and growth factors.9By the third post-
operative day, blood flow is established through these anastomotic connections and fibroblasts
have begun to migrate in from the surrounding tissue, first into the granulation tissue of the
wound bed and subsequently into the skin graft.10During this process, fibroblasts begin to
differentiate into myofibroblasts in the wound bed. This differentiation is driven by the
transmission of mechanical stress, along with immune mediated release of soluble factors such as
transforming growth factor beta (TGF-β). Myofibroblasts lay down ECM components which
replace the provisional matrix and also exhibit contractile properties due to the expression of
alpha smooth muscle actin(ASMA) in actin stress fibers.11ASMA enhances the cell’s contractile
abilities,and plays a significant role in wound contraction and granulation tissue
maturation.12Once wound contraction and healing are complete, myofibroblasts should resolve
by apoptosis. A lack of myofibroblast apoptosis is thought to promote HSc.12
Despite the significance of HSc contractures, pre-clinical investigations into the
pathogenesis of HSc contraction and development of new therapies to prevent HSc contraction
are lacking. One of the major hurdles to developing an effective HSc therapy is the lack of an
immune competent murine model.5Human HSc has typically been studied in immune
compromised mice, in which multiple models have been developed. In 1989, human HSc were
transplanted into subcutaneous pockets of athymic mice.13 The partially revascularized,
ischemic, transplanted scars were considered to represent human scar tissue,but in fact they only
comprised the terminal stages of scarring and did not contain the initiating factors that led to
development of the disease. In 2004, a mouse model with genetically modified skin-humanized
mice was introduced.14 Cultured human keratinocytes were transfected with an enhanced GFP
retroviral vector and transplanted on the back of nude mice.14 However, this model was designed
to mimic excisional wounds in healthy human volunteers, not larger-size wounds requiring skin
grafts and/or dermal substitutes. In 1987, anHSc murine model was introduced, whereby full
thickness human skin grafts were transplanted onto the backs of nude mice and subsequently
burned.15 This study was repeated in 2007 by another group that observed similar HSc formation
without the burning step. These mice developed raised, red, itchy, firm HSc that histologically
resembled human HSc. The HSc was prominent after one to three months and subsequently
diminished after six months.16 Although the progressive development of improved “humanized”
mouse models has resulted in a greater understanding of human HSc tissue in vivo, none of these
models possess an intact immune system. The established importance of immunity in the process
of HSc formation reveals a major weakness in the ability of these models to adequately model
the disease conditions.17 In 2007, Gurtner et al. developed a mechanical model of HSc in
immune competent mice.18 The ensuing HSc demonstrated many histopathological similarities to
human HSc.18 While this model provides a strategy to circumvent utilizing immune
compromised animals, it does not recapitulate the present conditions in most burn scar injuries.
Therefore, the purpose of this study was to develop a novel immune competent murine HSc
contraction modelthat possesses all of the advantages of murine modelswith an abundance of
genetic variants and applicable tools, low purchase and housing costs. The goal of this work is
that this model will serve to advance the field of wound healing and scarring.
MATERIALS AND METHODS
Mice
Female C57BL/6 mice, 10-12 weeks-old, weighing 18 to 23 g (Jackson Laboratories, Bar
Harbor, ME), were used as wild type (WT) mice throughout the study. Female hemizygous
C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ carriers (green fuorescent protein;GFP) and non-
carrier control littermates, 10-12 weeks-old, weighing 18 to 23 g (Jackson Laboratories, Bar
Harbor, ME) were used for WT-GFP and GFP-WT studies. All mice were monitored for signs of
toxicity including changes in: weight, grooming, irritability, and respiratory rate.The mice were
housed under protocols approved by the Institutional Animal Care and Use Committee (IACUC)
of Duke University.
Producing a third-degree thermal injury
All procedures were performed in accordance with a protocol approved by the Duke University
IACUC. Mice were anesthetized using gas anesthesia (oxygen, 2L/min, isoflurane, 2%). The
recipient mouse was anesthetized and the back of the mouse was shaved with metallic clippers.
The back was then sterilized using alcohol. The mouse was placed in a restraining tube. A metal
brass rod 8mm in diameter, weighing 65g, was placed in boiling water for 15min, the brass rod
was confirmed to be 100°C.The surface of the brass rod was wiped dry and then placed on the
back of the mouse for 1s to produce a third-degree burn. The burn was left for 3 daysand a 14mm
diameter circle over the burn site was excised for recipient skin grafting.
Skin transplantation
The donor mouse was anesthetized. Its ears were cleansed with alcohol and cut at the base with
curved surgical scissors. The dorsal skin surface of the ear was carefully separated from the
cartilaginous ventral surface using sharp fine tipped scissors, and the cartilaginous surface was
discarded. Three fenestrations were created in the dorsal ear skin using a #10 scalpel, 2mm
apart.Two donor skin ears were laid over an excised 14mm diameter burn wound. The edges of
the grafts and the edges of the skin were approximated and sutured with interrupted stitches (5
stitches for each ear and 2 stitches between the 2 ears) using 6-0 silk suture. The skin grafts were
then secured with a padded bolster. The bolster was removed on post-operative day 3.
Tagging the panniculus carnosus
The panniculus carnosus muscle layer around the skin graft were identified, excised and the
margins were tagged with 6 surgical titanium micro clips (Microclip TM,Teleflex Medical,
Research Triangle Park, NC) in a circular arrangement. X-ray images were captured using an X-
ray device (Faxitron Bioptics, Tucson, AZ). The mice were placed with the skin graft in the
central gridline of the device. The X-ray images were captured on days 3, 10, 17,and 24 post-
surgery. The surface area surrounded by the titanium clips was measured using ImageJ software
(National Institutes of Health, Bethesda, MD).
Tissue collection
The mice were euthanized and tissue was collected on post-operative days 3, 7, 9, 11, 14, 28, 70,
and 168. The collected tissues were cut into equal halves. One half was preserved in 10%
formalin and paraffin embeded for histological analyses, while the other half was immediately
frozen in liquid nitrogen for additional analyses. Prior to staining,tissue sections were de-
paraffinized by warming at 65°C overnight, immersing in xylene for 15 min, rehydrating with
decreasing concentrations of ethanolin distilled water.
Histology
Routine H&E, DAPI, Toluidine blue,and picrosirius red staining to enhance polarization of
collagen fibers was performed on 5-μm-thick paraffin-embedded sections. For collagen index,
the color information from each Masson trichrome high power field (HPF) image was quantified
using colorimetric analysis. The collagen index value was calculated as collagen index = (B + G)
/ (2R + B + G) for each pixel within the image (where R, G, and B represent the red, blue, and
green pixel values, respectively). The value of the collagen index ranged from0 for extremely
red objects to 1 for completely blue-green objects. The average collagen index of 3 (HPF)
images for each time point was graphed.
Immunohistological Staining
Tissue sections were immersed in 3% hydrogen peroxidefor 10min to inhibit endogenous
peroxidase. Slides were placed into citrate pH 6 antigen retrieval solution (Target Retrieval
Solution, Dako North America Inc. Carpinteria, CA, USA) and brought to >85°C in a water bath
for 20min, followed by 30min cool down. After rinsing sections with deionized water and 1X
tris-buffered saline (TBS, TBS Automation Washing Buffer, Biocare Medical, Concord, CA,
USA), sections were treated with 10% goat serum (Normal Goat Serum S-1000, Vector
Laboratories, Burlingame, CA, USA) for 1h at room temperature to block non-specific antibody
binding.
Macrophage F4/80 staining
F4/80 (14-4801-83, 1:1500 dilution, eBioscience, San Diego, CA) was incubated for 1h at room
temperature. After washing with TBS, the slides were incubated with biotinylated rabbit anti rat
(BA-4000, 1:200 dilution, Vector Laboratories, Burlingame, CA, USA) for 30min at room
temperature.
Staining of endothelial cells for CD31
Anti-CD31 antibody (AB28364, 1:50 dilution, abcam, Cambridge, MA) was incubated for 1h at
room temperature. After washing with TBS, the slides were incubated with biotinylated goat anti
rabbit (BA-1000, 1:50 dilution, Vector Laboratories, Burlingame, CA, USA) for 30min at room
temperature
Following incubatio n in secondary antibodies, the sections were then incubated with
avidin-biotin complex reaction (PK-7100, Vector Laboratories, Burlingame, CA, USA) for
30min. The sections were incubated with DAB substrate solution(DBC859L10, Biocare
Medical, CA, USA) for 3min after rinsing with 1X TBS. The slides were quickly dipped in
hematoxylin solution for counter stain solution, and then were rinsed in running tap water for
20min. After dehydration, labeled cells were visualized by use of a Nikon eclipse E600
microscope and images were captured with a Nikon DXM 1200 digital camera under the same
settings. Five HPF images were analyzed from each tissue section and the average number of
cells was assayed
Quantitative real-time polymerase chain reaction
Total RNA from was isolated using RNeasy Plus Universal Mini Kit (73404,Qiagen, Valencia,
CA, USA) including DNase treatment. Diluted by 10mM Tris buffer, RNA concentration and
purity of the samples were measured by use of spectrophotometer (Beckman Coulter, Brea, CA,
USA). Afterwards, following the standard one-step RT-PCR protocol for use with the
QuantiTect SYBR Green RT-PCR Kit (204243, Qiagen, Valencia, CA, USA) and Mx3005P
QPCR System (Agilent Technologies Inc, CA, USA), template RNA (10ng/reaction) was added
to prepare reaction mix. The one-step PCR cycling conditions were: 50for 30min, 95for
15min, 40 cycles of 94for 15s, 55for 30s and 72for 30s. S9 was employed as a
housekeeping gene. Data were collected and analyzed by MxPor QPCR software (Agilent
Technologies Inc, CA, USA), relative gene expression is calculated as a ratio of Ct of the
interested gene to that of S9. All qRT-PCR primers were purchased from Qiagen.
Mechanical testing of tissue samples
Scar and uninjured skin samples were gathered from humanand murine donors. Human skin
samples were donated from discarded human tissue from Duke Hospital operating rooms under
exemption by Duke IRB. Uninjured skin was gathered from breast resection, while scar tissue
was taken from excised keloid, radiated forearm, and rejected skin graft. Tissue from three
human donors was used with 3-5 biological replicates per donor. Uninjured murine tissue
samples were collected from the dorsum of 10-12 week-old C57BL/6 mice. Murine scar tissue
was taken from contracted day 28 skin grafted mice. Tissue from 5 murine donors was used,
with three biological replicates per donor. All human and murine tissues were kept moist on
damp gauze between collection and mechanical testing, and analyzed within 1-5h of collection.
Prior to testing, underlying tissue was removed and samples were cut into uniform strips. Elastic
moduli of human and murine skin samples were analyzed using a microstrain analyzer (MSA)
(TA Instruments RSA III). Samples were cut into strips 3mm wide and 4cm long, and loaded
with a 5mm gap between the grips. The samples were strained at a rate of 0.1mm/s at room
temperature (23°C) until failure. Many of the human skin samples overloaded the maximum
force of the MSA (35N) and had to be stopped prior to failure. Stress-strain curves were graphed
in Excel, and the elastic modulus of the material (E) calculated as stress/strain (σ/ε) in the initial
linear portion of the graph, prior to necking of the material.
Statistical analysis
Data were analyzed using Microsoft® Excel software. The statistical significance of values
among groups was evaluated by the analysis of variance (ANOVA), followed by least significant
difference t-test. All values used in figures and text are expressed as mean +/-standard error of
the mean (SEM). The difference was considered significant when the p -value was 0.05 or less.
RESULTS
Establishing a third degree burn murine model
A third-degree burn injures the full thickness of the skin: the epidermis and the
dermis.19Histological analysis revealed that a 1s burn contact time with a 65g brass rod heated to
100°C, held in place by gravity, resulted in a reproducible full-thickness burn (Figure 1A). As is
noted in the figure, there was necrosis throughout the epidermis and dermis, but not into the deep
muscular layer.
Skin graft survival
Both donor and recipient mice were immune competent. This was intentionally done so that one
could study the immune system as it relates to HSc and skin graft survival. The burned tissue of
the recipient mouse was excised after 3 days and skin grafted to mimic clinical practice. Mouse
ear skin was used as a donor for the skin graft (Figure 1B) and was transplanted onto the
granulation tissue bed. During optimization of the model, we tested alternative skin donor sites
including murine back skin, tail skin, and abdominal skin but had poor success due to the
thickness of the skin graft. We attempted split-thickness skin graft harvesting utilizing a
dermatome, but found the harvested layers to be inconsistent in quality and thickness. A bolster
as a dressing for the split thickness skin graft (STSG)was necessary for STSG survival as an
adhesive bandage was not reliable.20
To confirm the survival of the skin graft, we used GFP mice in two experiments. We used
donor ear skin from a WT mouse and transplanted it to a GFP recipient mouse. The recipient
GFP mouse appeared green while the transplanted WT skin graft appeared grey indicating skin
graft survival. We also used donor ear skin from a GFP mouse and transplanted it onto a WT
recipient mouse. The recipient WT mouse appeared grey while the transplanted GFP skin graft
appeared green, confirming skin graft survival (Figure 1C). Analysis was done on Day 30.
Mature skin grafts had the same dermal thickness and number of hair follicles as ear skin
To further confirm that we were observing skin graft survival and not traditional wound healing,
we performed H&E analysis over a period of 6 months. Our analysis of dermal thickness
demonstrated that mature skin graft had the same dermal thickness as normal ear skin, ~90μm.
We also found that the number and frequency of hair follicles (Figure 2A) in mature skin graft
correspond to normal ear skin. This confirmed that the skin graft survived as donor ear skin and
was not replaced by fibrous tissue or back skin (Figure 2A).
Interestingly, our analysis also revealed that the hair follicles disappeared in the early
time point of skin graft survival, only to reappear again later on day 30 (Figure 2B).
Skin grafts contracted but did not disappear;PC did not contribute to skin graft
contraction
Our macroscopic observation and measurement of skin graft size showed that the grafts
contracted to ~45% of the original wound size by day 14, after whichno further contraction was
observed. In comparison, a non-grafted woundsfully contracted by day 14 (Figure 3A). Grafts
were initially red and flat, in contrast to the raised nature of early human HSc. However, human
HSc often flatten as they mature (Figure 3B).
Rodents possess a subcutaneous muscle layer called the PC. The PC has been shown to
promote wound contraction and thus expedite scar contraction. 21Our radiolo gical analysis on
post-operative days 3, 10, 17,and 24 showed no significant change in the surface area of PC
underneath the skin graft, indicating that the PC did not contribute to skin graft contraction
(Figure 3C).
Skin grafts showed an increase in cellularity and granulation tissue formation
Histological analysis of DAPI-stained sections revealed significant increases in cellularity at all
time points compared to normal mouse skin (Figure 4A). There was also a significant increase in
granulation tissue surface area at all time points compared to normal mouse skin (Figure 4B).
Collagen maturation has increased over time
Picrosirius red analysis demonstrated increase in collagen maturation over time;there were more
immature (green, loosely packed) collagen fibers on day 7 than day 21 and day 168. More
mature collagen fibers (yellow, densely packed) were present on day 168 than on day 21 and day
7(Figure 4D).
Skin grafts showed an increase in the vascular density
Immunohistochemistry showed increased number of endothelial cells, vascular density, at all
time points of the skin grafts compared to the normal skin(Figure 4E).
Skin grafts showed an increase in the numbers of macrophages and mast cells
Immunohistochemistry using F4/80 as a marker, demonstrated wide areas of positive
macrophage staining in the skin grafts, and quantification showed an increase in the number of
macrophages at all the time points of the skin grafts compared to the normal skin (Figure 4F).
Histological analysis of Toluidine blue-stained sections showed an increase in the number of
mast cells at all time points in the skin grafts compared to normal skin (Figure 4G).
Up-regulated mRNA expression of TGF-β and cytoskeletal proteins in skin grafts
TGF-β, ASMA, NMMIIA and ROCK2 have all been reported to coordinately regulate the
development of fibrotic conditions.22qRT-PCR analysis showed increase mRNA expression of
TGF-β, ASMA, NMMIIA, and ROCK2 at all time points compared to normal skin. Surprisingly,
day 168 skin graft showed the highest mRNA expression (Figure 4H).
Skin grafts showed the same elasticity as HSc when compared to normal skin
Prior microstrain analysis studies have been conducted on human and mouse tissue.18,23 Most of
these tests utilize a strain rate of several mm/min or above. The measured elastic moduli are
highly dependent upon the strain rate since skin is a viscoelastic tissue.23 We chose a smaller
strain rate of 0.1 mm/second in order to better approximate the slow speed of contraction exerted
by cellular forces on wounds. A comparison of elastic modulus between mouse scar, mouse
normal skin, human scar and human normal skin illustrated that mouse scar is stiff, akin to
human scar, whereas unwounded mouse skin is more elastic like unwounded human skin (Figure
5).
Comparison ofnovel immune competent murine hypertrophic scar contracture model as a
hypertrophic scar model
Animal model validity is discussed in terms of similarities between the model and the human
condition.24 In humans, a third-degree burn injury is managed through excision of the dead tissue
and delayed skin grafting.25 The skin graft heals and contracts over 6 months. Our murine HSc
contracture model shares many similarities with human HSc (Table 1).26
DISCUSSION
This study introduces a validated immune competent murine HSc contraction model. First, a
third-degree burn was created on the dorsum of a C57Bl/6 mouse. The burned tissue was
subsequently excised and a skin graft fashioned from donor murine ear skin was sutured over the
wound. We initially secured the skin grafts with adhesive bandages, as described in transplant
models, but this approach led to skin graft death. Murine ear skin, as opposed to skin from other
donor sites such as murine flank, tail, or abdomen, was chosen due to the relative thinness of ear
skin. Thin skin grafts were more likely to survive than thick skin grafts and thin split thickness
skin grafts are the preferred method of treatment in human burn wounds. Transplanted skin
grafts demonstrated viability at two weeks post-operation. Skin grafts contracted to ~45% of
their original size in two weeks, as compared to excisional wounds which were contracted closed
at two weeks post-wounding. The rate of murine skin graft contraction was much faster than
what is reported inhuman split thickness skin grafts of the scalp.27Over time the murine skin
graftsbecame flat and pale, as is observed in some but not all human HSc.28,29 The process of
murine skin graft contraction demonstrated many gross morphological and microscopic
characteristics similar to human HSc (Table 1).29
The healing process following deep dermal injury is a stepwise sequence of overlapping
events which often results in scar. Shortly after wounding hemostasis is achieved changes in
blood vessel walls allow inflammatory cells to migrate into the wound. While inflammatory cells
such as macrophages may appear 48 hours after wounding, prolonged inflammation is associated
with skin fibrosis and HSc.30 Macrophages, in particular, are a major source of cytokines and
growth factors.31TGF-βis one such protein released by macrophages that promotes HSc
contraction and has been shown to be a potent chemotaxin for inflammatory cells, including
macrophages and mast cells.32Dysfunction of normal macrophage response can result in
numerous pathologic conditions, including ulcers, chronic wounds, keloids, and HSc.32
Mast cells are another type of inflammatory cell that participatesin wound healing. As
granulation tissue develops interstitial collagen, mast cells appear. It has been previously
reported that HSc contain significantly greater numbers of mast cells than normal skin, and mast
cells may be a stimulus for HSc contraction.33 HSc contraction is caused by mast cells release of
histamine and arachidonic acid metabolites, as well as angiotensin II.34 Our group has previously
demonstrated that increased levels of angiotensin II activate the contractile protein NMMIIAand
promote dermal fibroblast contractility.22
As previously mentioned, increased vascular density has been observed in HSc. While
the reason for increased vascularity is unclear, prior studies have suggested that increased
vascularity could account for the persistent high inflammatory cell density observed in HSc.26
Conversely, persistent inflammation could itself contribute to increased vascularity through
positive feedback loops.35 Our present study showed an increase in the vascularity of skin grafts
compared to unwounded skin. Along with increased vascular density, we observed high
concentrations of fibroblasts. Blood vessels and fibroblasts are the major constituents of a
critically important transitional connective tissue called granulation tissue.36 Granulation tissue
may persist for up to 6.5 months post-injury, due to diminished cell apoptosis. This persistenceof
increased cell numbers contributes to HSc contraction because granulation tissue fibroblasts
become aligned along lines of mechanical stress.3712
Aligned fibroblasts differentiate into contractile and secretory myo fibroblasts.
Myofibroblasts lay down ECM and exhibit increasedexpression of ASMA in actin stress
fibers.11Expression of ASMA in stress fibers confers at least a twofold stronger contractile
activity to these specialized cells.38Myofibroblast cell contractility is regulated by myosin light-
chain (MLC) phosphorylation in a manner similar to regulation of smooth muscle cell
contraction.39 Two kinase systems seem to regulate MLC phosphorylation: calcium (Ca2+)-
dependent myosin-light-chain kinase (MLCK) and Rho-kinase.40Myofibroblast contraction tends
to be rapid and short-lived, as increasesin intracellular Ca2+are transient and active myosin
phosphatase terminates the reaction by removing the phosphate group from MLC. In the Rho
kinase pathway, RhoA (a small Rho family GTPase) activatesROCK2.41 Activated ROCK2
increasesMLC phosphorylation by two mechanisms: first, by direct phosphorylation of MLC
and second by inactivating myosin phosphatase through phosphorylation of the myosin-binding
subunit.42,4344 ActivatedMLC binds to the neck domain of NMMII, which leads to NMMII
sliding actin in a slip-ratchet fashion. Actomyosin contractility causes cell migration, adhesion,
and contractility.45NMMII levels are increased under increased levels of matrix stiffness from
collagenorganization(such as scar tissue).46
Collagen organization plays a key role in the strength and elasticit y of healthy skin and
scar tissue.47 In unwounded adult animals, collagen bundles are thick, arranged in a basketweave
formation, and have a low index of organization. In HSc, collagen fibers are thin andarranged in
parallel.48 In our model we observed an increase in immature collagen lo osely packed in parallel
fashionwith blood vessels perpendicular to the surface.These immature fibers gradually gave
way to an abundance of mature, densely packed collagen fibers by day 168 (Figure 4D).
In HSc, the epidermal surface is devoid of appendages, such as hair follicles, and lacks
the ingrowth of rete pegs.49Interestingly, in our model we noted the disappearance of hair
follicles early after skin graft application, with later regeneration of hair follicles around day 30.
The reappearance of hair follicles is unexpected, and suggests the possibility of cellular
reprogramming or stem cell migration into the graft tissue.
Scar tissue exhibits different mechanical properties than unwounded tissue. HSc is less
extensible, requires more energy to be stretched in the physiologic range,and stores strain energy
less efficiently than unwounded skin.23,50Clark et al. demonstrated an up to seven times increase
in stiffness in hypertrophic scars versus normal skin tissue. Our results demonstrated that scar
tissue from our mouse model graft site was stiffer (79.46 +/-16.62 kPa) than unwounded mouse
skin (36.41 +/-3.68 kPa).This relationship is similar to previously published findings and
consistent with our findings in human tissue, with hypertrophic scars possessing an elastic
modulus nearly three times as large as unwounded human skin (Figure 5).18,23
The lack of an ideal HSc contraction small animal model has greatly hindered both the
understanding of this disease,and thedevelopment of effective therapies for its prevention.5,16,51
The mouse remains the most commonly used animal for biologic research, owing in large part to
its ease of care, economic advantage, and wealth of mouse-specific reagents and genetic tools.52
As previously mentioned, present murine models of HSc either lack an immune system or fail to
effectively model the pathologic condition.14-16,18,53In addition to murine models, scientists have
utilized other animals to study HSc. An athymic rat model of human scar transplantation was
developed,but fibroblast proliferation was diminished when compared to human samples.54The
rabbit ear excisional HSc modelwas developed in 1997 and has been used frequently since then
to elucidate molecular pathways in HSc and testing of potential therapies.55Chemically-induced
hypertrophic scars have also been demonstrated to occur in incisional wounds in guinea pigs.56
In the guinea pig HSc model, glucose-6-phosphate dehydrogenase levels and histologic analysis
were similar to human HSc, but HSc development was inconsistent and unpredictable between
animals.
While our murine HSc model shares many characteristics with the human condition
(Table 1), there are differences between mice and humans.This model does not utilize a truesplit
thickness skin graft because harvesting of mouse skin with a dermatome is unreliable.
Additio nally,thescars we observed in our model were initially flat and red, eventually becoming
pale. HSc in humans are classically described as red, raised, and itchy.28This may be due to a
diminished level of mechanical tension in the murine skin graft bed.28,29
HSc development is a common debilitating condition with a high morbidity and a high
cost to patients both financially and in terms of quality of life.1-5 Several animal models have
been developed in order to meet a growing need for modeling the pathogenesis of HSc. No
model has yet to meet the criteria of a reproducible, low-cost model with adequate scientific
tools that closely resembles the human pathology.5,51 To this end, we have developed a novel,
validated, immune competent, murine HSc contraction model which has been closely modeled
after present-day burn scar conditions and management. This model possesses all of the
advantages of murine models, including an abundance of genetic variants and applicable tools,
low cost, and practical husbandry techniques, all of which will aid in the research and
development of novel therapeutic approaches for the treatment of HSc contraction.
ACKNOWLEDGMENTS
The authors would like to thank Eugenia H. Chofor her technical assistance and Gloria Adcock
for her assistance with tissue processing.
Funding: This work was supported by a grant from the National Institutes of Health,K08 GM
085562-05.
Conflict of Interest: The authors declare that there are no conflicts of interest.
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Table 1. Characteristics of human and murine hypertrophic scars
Parameter Human HSc Murine HSc
Initial injury Burn Third-degree burn Third-degree burn
Management Surgical Excision and skin graft Excision and skin graft
Gross
Appearance
Scar contraction Increased Increased
Matrix Collagen maturation
/type I
Increased Increased
Cellularity VascularityIncreased Increased
Macrophage densityIncreased Increased
Mast cell densityIncreased Increased
ProliferationIncreased Increased
Cytokines TGFIncreased Increased
Cytoskeletal
Changes
ASMA
NMMIIA
ROCK2
Increased
Increased
Increased
Increased
Increased
Increased
Elasticity Elastic modulus Increased compared to
uninjured human skin
Increased compared to
uninjured mouse skin
FIGURE LEGENDS
Figure 1.Third-degree burn injury model and skin graft survival. (A) Masson’s trichrome
staining of mouse skin revealed a third-degree burn (e-epidermis, d-dermis, m-muscle, black
arrow-third-degree burn). Magnified section shows intact muscular layer. (B) Representative
photographs of the dorsal ear skin graft before and after transplantation on the dorsum of the
rodent. (C) Top row: Donor ears were obtained from C57BL/6 mice and were grafted on GFP
transgenic mice. Bottom row: Donor ears were obtained from GFP transgenic mice and were
grafted onto C57BL/6 mice. Mice were photographed under normal light (left) and the same
mice were photographed under GFP excitation light demonstrating graft survival.
Figure 2. Skin graft survival. (A) H&E stained-sections showing dermal thickness and
density and number of hair follicles (h) per unit area in normal ear skin and normal back skin.
The average number of hair follicles/field is graphically represented in normal ear skin, normal
back skin and day 168 skin graft. Mature skin grafts have the same number of hair follicles and
same average dermal thickness as normal ear skin indicating the ear skin grafts persist. (B) The
hair follicles were absent in the early time points of the skin grafts, then hair follicles have
regenerated again starting by day 30.
Figure 3. Skin graft contraction.(A) Graph showing the relative skin graft size vs. the
relative open wound size indicating that rodent skin grafts behave like human skin grafts. Open
wounds were contracted and closed by day 14, whereas skin grafts stopped contraction by day 14
and remained 50% of the initial graft size. (B) The murine burn contracture model is analogous
to the human condition with faster graft contraction: human skin grafts contract ~35% by 6
weeks, the murine skin graft contracts to ~45% of the original size by 4 weeks and the murine
open non-grafted wound contracts ~95% by 14 days. Note that contracted skin grafts are
hypertrophic scars but this does not mean that contracted hypertrophic scars are elevated. (C)
Graph showing no significant change in the panniculus carnosus size at different time points,
whereas the skin graft contracted to ~45% of the original size; these data show that the
panniculus carnosus does not play a role in skin graft contraction in this model.
Figure 4. (A) Representative DAPI-stained section. All time points showed more cellularity
than the normal skin. Day 7 showed the highest cellularity whereas day 168 showed the least. (B)
Representative H&E-stained sections. Granulation tissue surface area is graphically represented.
Day 14 demonstrated the greatest surface area of granulation tissue then it decreased gradually
up to day 168. (C) Representative Masson’s trichrome-stained sections demonstrating collagen
content of the skin grafts. Quantitative analysis of collagen index was graphically represented vs.
normal skin. (D) Sirius red polarization microscopy of collagen fibers revealed an increase in
collagen maturation over time. There were more immature (green, loosely packed) collagen
fibers on day 7 than day 21 and day 168, respectively. Whereas, there was more mature collagen
fibers (yellow, densely packed) on day 168 than on day 21 and day 7, respectively. (E)
Representative CD31-stained sections demonstrating the degree of vascularity. All time point
show more vascularity compared to normal skin. Day 14 showed the highest vascularity whereas
day 7 showed the least. (F) Representative F4/80-stained section demonstrating the density of
macrophages. All time point show more macrophages compared to normal skin. Day 14 showed
the highest number of macrophages whereas day 168 showed the least. (G) Representative
toluidine blue-stained sections demonstrating the density of mast cells. All time points showed
more mast cells compared to normal skin. Day 9 showed the highest number of mast cells
whereas day 3 showed the least. (H) mRNA expression of ASMA, NMMIIA, ROCK2and TGF-
βwasassessed by qRT-PCR. Gene expression is related to ribosomal protein S9 using the ΔΔCt
method and then normalized to normal skin. Data are represented as mean ± SEM. ASMA,
NMMIIA and TGFshow an increase in expression in the graft tissue peaking at day 168.
ROCK2 has the greatest increases in expression at days 28 and 168.
Figure 5. Comparison of measured elastic modulus between uninjured (normal) skin and
scar tissue for human and mouse samples. Each marking represents a unique sample. Data is
presented in this way to show variability of scar tissue and relative stiffness of human and mouse
scars.
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
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... Ibrahim et al. 2014 developed a model to analyse scar contracture in hypertrophic tissue on immunocompetent mice (83). The thermal injury was created using a brass metal rod 8 mm in diameter heated to 100°C in boiling water for 15 min; then placing the rod on the mouse for 1 s. ...
... The authors reported that the skin grafts contracted but did not disappear. Interestingly they found that the PC did not contribute to the contraction of the skin graft (83). The scars were reported as flat and initially red but later becoming pale (83). ...
... Interestingly they found that the PC did not contribute to the contraction of the skin graft (83). The scars were reported as flat and initially red but later becoming pale (83). ...
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Introduction A reproducible, standardised model for cutaneous scar tissue to assess therapeutics is crucial to the progress of the field. A systematic review was performed to critically evaluate scarring models in both animal and human research. Method All studies in which cutaneous scars are modelling in animals or humans were included. Models that were focused on the wound healing process or those in humans with scars from an existing injury were excluded. Ovid Medline ® was searched on 25 February 2019 to perform two near identical searches; one aimed at animals and the other aimed at humans. Two reviewers independently screened the titles and abstracts for study selection. Full texts of potentially suitable studies were then obtained for analysis. Results The animal kingdom search yielded 818 results, of which 71 were included in the review. Animals utilised included rabbits, mice, pigs, dogs and primates. Methods used for creating scar tissue included sharp excision, dermatome injury, thermal injury and injection of fibrotic substances. The search for scar assessment in humans yielded 287 results, of which 9 met the inclusion criteria. In all human studies, sharp incision was used to create scar tissue. Some studies focused on patients before or after elective surgery, including bilateral breast reduction, knee replacement or midline sternotomy. Discussion The rabbit ear scar model was the most popular tool for scar research, although pigs produce scar tissue which most closely resembles that of humans. Immunodeficient mouse models allow for in vivo engraftment and study of human scar tissue, however, there are limitations relating to the systemic response to these xenografts. Factors that determine the use of animals include cost of housing requirements, genetic traceability, and ethical concerns. In humans, surgical patients are often studied for scarring responses and outcomes, but reproducibility and patient factors that impact healing can limit interpretation. Human tissue use in vitro may serve as a good basis to rapidly screen and assess treatments prior to clinical use, with the advantage of reduced cost and setup requirements.
... For common scald burn models, the drawback is that they frequently require flame-resistant molds to cover the animals before immersing exposed skin into boiling water, thus it is difficult to manipulate (Hiyama et al. 2013;Abdullahi, Amini-Nik, and Jeschke 2014). In contrast, dry burn models using heated metal blocks are simple but the previous studies rarely mention how the metal blocks are applied with the same pressures for each wound (Stevens et al. 1994;Ibrahim et al. 2014). Besides, the metal blocks after heating in boiling water are then often wiped dry, thus their temperature can be differently changed before wounding (Ibrahim et al. 2014). ...
... In contrast, dry burn models using heated metal blocks are simple but the previous studies rarely mention how the metal blocks are applied with the same pressures for each wound (Stevens et al. 1994;Ibrahim et al. 2014). Besides, the metal blocks after heating in boiling water are then often wiped dry, thus their temperature can be differently changed before wounding (Ibrahim et al. 2014). Furthermore, using boiling water is dangerous for experimenters. ...
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Fibroblast growth factor 2 (FGF-2) is a multifunctional protein that has major roles in wound healing, tissue repair, and regeneration. This therapeutic protein is widely used for burn treatment because it can stimulate cell proliferation and differentiation, angiogenesis, and extracellular matrix remodeling. In this study, we developed a simple method using a controlled heated brass rod to create a homogenous third-degree burn murine model and evaluated the treatment using recombinant human FGF-2 (rhFGF-2). The results indicated that the wound area was 0.83 ± 0.05 cm2 and wound depth was 573.42 ± 147.82 μm. Mice treated with rhFGF-2 showed higher rates of wound closure, granulation tissue formation, angiogenesis, and re-epithelialization than that of phosphate-buffered saline (PBS)-treated group. In conclusion, our lab-made rhFGF-2 could be a potentially therapeutic protein for burn treatment as well as a bioequivalent drug for other commercial applications using FGF-2.
... 4,8,9 Myofibroblasts are present in contracting scars for 6 months post-wounding. 10 After 6 months, the extracellular matrix (ECM) is crosslinked and stabilized, and cells are considered stress shielded from mechanical tension. 10 Stress shielding signals myofibroblast apoptosis, indicating completion of adult wound healing. ...
... 10 After 6 months, the extracellular matrix (ECM) is crosslinked and stabilized, and cells are considered stress shielded from mechanical tension. 10 Stress shielding signals myofibroblast apoptosis, indicating completion of adult wound healing. 11 We hypothesize the key to preventing HSc is dampening of mechanical tension throughout the wound healing process. ...
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... Upon further examination of macrophage subtypes, M2 macrophages were found to be elevated, and higher levels of pro-inflammatory cytokines were noted in HTS-like samples [31] . An increase in both mast cells and macrophages has also been reported in a mouse model of hypertrophic scar contracture [32] . ...
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... Whilst both methods may be informative for in vivo studies of hypertrophic scarring, they do not reflect the pattern of genetic predisposition in humans (Zhu et al. 2013), and the knock-down target does not correlate with known protective genetic variants (Sood et al. 2015). It is suggested that concomitantly xenografting human skin cells into the wound may improve the validity of the mouse burns model by promoting a more extensive scar phenotype (Ibrahim et al. 2014;Momtazi et al. 2013). However, this could be confounded by the immunogenic effects of xenografting skin onto an immunocompetent mouse (Racki et al. 2010). ...
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... Studies have shown that mast cells are prominent in HTS-like lesions that develop in mechanically loaded wounds with increased tension [67]. Elevated mast cell numbers have also been reported in a mouse model of HTS contracture [68] and in a model that used human skin grafting in nude mice to induce HTS [69]. ...
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Scars are generated in mature skin as a result of the normal repair process, but the replacement of normal tissue with scar tissue can lead to biomechanical and functional deficiencies in the skin as well as psychological and social issues for patients that negatively affect quality of life. Abnormal scars, such as hypertrophic scars and keloids, and cutaneous fibrosis that develops in diseases such as systemic sclerosis and graft-versus-host disease can be even more challenging for patients. There is a large body of literature suggesting that inflammation promotes the deposition of scar tissue by fibroblasts. Mast cells represent one inflammatory cell type in particular that has been implicated in skin scarring and fibrosis. Most published studies in this area support a pro-fibrotic role for mast cells in the skin, as many mast cell-derived mediators stimulate fibroblast activity and studies generally indicate higher numbers of mast cells and/or mast cell activation in scars and fibrotic skin. However, some studies in mast cell-deficient mice have suggested that these cells may not play a critical role in cutaneous scarring/fibrosis. Here, we will review the data for and against mast cells as key regulators of skin fibrosis and discuss scientific gaps in the field.
... Hypertrophic scars are raised, and highly erythematous although they remain within the confines of the original cutaneous wound site (32,34). A number of murine models of hypertrophic scars have shown prominent mast cell staining in the scar tissue, with some demonstrating an increased number of MCs present in scar tissue compared to normal skin (35)(36)(37)(38). High MC numbers have also been described in large animal models of hypertrophic scarring (39,40). ...
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