Epidermal Stem Cells Cultured on Collagen-Modified Chitin Membrane Induce In Situ Tissue Regeneration of Full-Thickness Skin Defects in Mice
et al. (2014) Epidermal Stem Cells Cultured on Collagen-Modified Chitin Membrane Induce In Situ Tissue
Regeneration of Full-Thickness Skin Defects in Mice. PLoS ONE 9(2): e87557. doi:10.1371/journal.pone.0087557
Epidermal Stem Cells Cultured on Collagen-Modified Chitin Membrane Induce In Situ Tissue Regeneration of Full-Thickness Skin Defects in Mice
Yan Shen 0
Libing Dai 0
Xiaojian Li 0
Rong Liang 0
Guangxiong Guan 0
Zhi Zhang 0
Wenjuan Cao 0
Zhihe Liu 0
Shirley Mei 0
Weiguo Liang 0
Shennan Qin 0
Jiake Xu 0
Honghui Chen 0
Vipul Bansal, RMIT University, Australia
0 1 Guangzhou Institute of Traumatic Surgery, Guangzhou Red Cross Hospital Medical College, Jinan University , Guangzhou , People's Republic of China, 2 Department of Medical Laboratory, Second Affiliated Hospital of Guangzhou Medical College , Guangzhou , People's Republic of China, 3 School of Pathology and Laboratory Medicine, The University of Western Australia , Nedlands, Western Australia , Australia , 4 Student of Sophie Davis School of Biomedical Education, Mack Lipkin Fellowship , New York, New York , United States of America
A Large scale of full-thickness skin defects is lack of auto-grafts and which requires the engineered skin substitutes for repair and regeneration. One major obstacle in skin tissue engineering is to expand epidermal stem cells (ESCs) and develop functional substitutes. The other one is the scaffold of the ESCs. Here, we applied type I collagen-modified chitin membrane to form collagen-chitin biomimetic membrane (C-CBM), which has been proved to have a great biocompatibility and degraded totally when it was subcutaneously transplanted into rat skin. ESCs were cultured, and the resulting biofilm was used to cover full-thickness skin defects in nude mice. The transplantation of ESCs- collagen- chitn biomimetic membrane (ESCs-C-CBM) has achieved in situ skin regeneration. In nude mice, compared to controls with collagen-chitin biomimetic membrane (C-CBM) only, the ESCs-C-CBM group had significantly more dermatoglyphs on the skin wound 10 w after surgery, and the new skin was relatively thick, red and elastic. In vivo experiments showed obvious hair follicle cell proliferation in the full-thickness skin defect. Stem cell markers examination showed active ESCs in repair and regeneration of skin. The results indicate that the collagen-modified chitin membrane carry with ESCs has successfully regenerated the whole skin with all the skin appendages and function.
Funding: The sources of funding from: Guangdong Science and Technology Project: 2011B031300026: RMB: 30 thousand (amount to USA $:Five thousand), PI:
YS, 2012B031800338: RMB:10 thousand (amount to USA $: One thousand and Six hundred), PI:LD; Guangzhou Applied Basic Research Project 2013J4100100: RMB:
80 thousand (amount to USA $:Thirteen thousand ), PI: YS, 2008J1-C121: RMB: 100 thousand (amount to USA $:16 thousand ), PI: YS; Guangdong Natural Science
Foundation Project 9152800001000009 RMB: 30 thousand (amount to USA $:Five thousand ), PI: RL; National Natural Science Foundation Project 30973118 RMB:
30 thousand (amount to USA $:Five thousand ), PI: ZZ; Guangdong Health Department Project 2012A011046 RMB: 1 thousand (amount to USA $:One hundred
and sixty), PI: LD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Extensive dermal and full-thickness injuries are usually related
to acute incisional and excisional wound, trauma and burn [1,2,3]
The complications of these injuries may be severe, and especially,
the regeneration of a full-thickness skin is not sufficient and
pertains to dermal regeneration [4,5,6]. Autologous split skin grafts
are the gold standard for the treatment of full-thickness injuries
such as extensive burns . However, the limited donor grafts and
tedious surgeries urge the development of proper skin substitutes
through tissue engineering. Current artificial skin materials include
epidermal substitute, dermal substitute and full skin substitute
[8,9,10]. However, cells in these substitutes have a relatively weak
ability to proliferate and self-renew, which affects the outcome of
repair, with the results of blisters, scar hyperplasia and severe
contraction. For example, epidermal substitute is made by adding
epidermal cells to a scaffold to form a composite skin with some
biological activity. However, the seed cells of the dermal substitute
are mainly fibroblasts. Although fibroblasts are easily to obtain and
grow relatively quickly, their function is simple and they do not
encourage the development of skin appendages (i.e., sweat glands,
hair and hair follicles) [11,12,13,14]. This problem might be
resolved effectively if the dermal substitute is used to cover the
wound with a population of epidermal stem cells (ESC).
Stem cells demonstrate the two defining features, namely
selfrenewal and multipotency, and are instrumental for renewal,
regeneration and repair . In skin tissue engineering, ESCs
have the advantages of definite orientation, great plasticity and the
ability to induce and form skin appendages . Therefore, they
have become the first choice of seed cells in skin tissue engineering.
Recent findings suggest that the hair follicle is a major repository
of epidermal stem cells which give rise to several cell types of the
hair follicle as well as upper follicular cells . The use of ESCs
with hair follicle bulge may potentially resolve many current
problems in skin tissue engineering, including lack of skin
appendages and immune rejection. Besides the seed cells, selection
of a proper carrier for ESCs is the key to successful skin tissue
Chitin is a naturally occurring biopolymer having diversified
applications not only in the pharmaceutics for drug delivery, but
also in the biomedicine as dress material for wound healing
applications and as potential biomaterial for tissue engineering
[18,19]. The structure and function of chitin are similar to those of
mucopolysaccharides in the skin. Chitin contains an active
hydroxyl (2OH) group adjacent to the amino (2NH2) group
and is a hydrophilic cation polymer. The molecule is composed of
acetylglucosamine and glucosamine units, and it is also a
constituent of human hyaluromic acid. Chitin has excellent
biocompatibility and can be degraded into chitin oligosaccharide
by lysozyme, xylanase at a rate of absorption and utilization of
100% . It is also a relatively good adhesive, which may
enhance the proliferation and differentiation of cells on the
material, promote re-epithelization in animal skin wounds, and
accelerate wound healing . Previous studies suggest that chitin
enhances the cell proliferation and differentiation, promotes
epithelization but significantly inhibits type I collagen and
accelerates type III collagen secretion, and therefore accelerates
skin wound healing . More importantly, chitin has no
immunogenicity without any risk of immune rejection after
implantation . These properties make it a very promising
scaffold for skin tissue engineering. In our previous study . a
skin substitute with chitin membrane seeding with rat epidermal
stem cells (ESCs) has been evaluated for its promising application
in skin engineering. Chitin membrane used as the ESCs culture
vehicle has a good biocompatibility. However, shortcomings
remain. The pore size of the chitinous biomembrane material is
relatively large (500800 mm), the grid is uneven and the
compactness poor. Therefore, it can only be used as a temporary
wound dress, and is not suitable for cell adhesion and
threedimensional cell growth. Type I collagen is a good culture
substrate for many cell types. It is possible to be integrated into
chitin membrane to improve cell adhesion and proliferation,
reduce pore size.
In the current study, the chitin membrane was modified by cross
linked with type I collagen isolated from the rat tail. This
collagenchitin membrane had a great biocompatibility and degraded
totally when it was subcutaneously transplanted into rat skin. The
modified collagen-chitin biomimetic membrane (C-CBM) had a
relative small pore size (210 mm), and the adhesion of hair follicle
ESCs on the composited membrane was improved. Further, the
isolated hair follicle ESCs were seeded to the C-CBM and
transplanted into nude mouse full-thickness skin defects. The
defects were fully repaired, and the regenerated skin was proved to
contain all the skin appendages. Therefore, these data suggest that
hair follicle ESCs carried in the modified collagen-chitin scaffold is
sufficient to achieve morphological, structural and functional
reconstruction of the full-thickness skin defects.
Materials and Methods
All experimental procedures were performed according to the
Guide for the Care and Use of Laboratory Animals and were in
compliance with the guidelines specified by the Chinese Heart
Association policy on research animal use and the Public Health
Service policy on the use of laboratory animals. The animal use
protocol has been reviewed and approved by the Animal Ethical
and Welfare Committee (AEWC) of Guangzhou Red Cross
Hospital. SD rats and BALB/c nude mice were selected from the
Experimental Animal Center of Guangdong Province. Licence :
SCXK(GD)2008-0002, SCXK(GD)2011-0029, SYXK(GD) 2007
Materials and Equipment
The following major materials were used: chitin natural
biomembrane (Kisumi, Hunan Yinghua Biomedical Co.,Changsa
Hunan, China); defined keratinocyte serum-free medium
(DKSFM, 10785, Gibco, Carlsbad, CA); rat type I collagen (3-D
Culture MatrixTM, Trevigen, Inc., Gaithersburg, MD); type IV
collagen (Sigma-Aldrich Biotechnology, L.P., St. Louis, MO);
mouse anti-rat b1 integrin (CD29) monoclonal antibody, mouse
anti-rat cytokeratin antibodies, anti-CK5, CK10, CK14, CK15
and CK19 antibodies, mouse anti-rat p63, vascular endothelial
growth factor (VEGF) and Dsg antibodies (all Santa Cruz
Biotechnology, Santa Cruz, CA); anti-CD34 and CD200
antibodies (Abcam, PLC, Cambridge, UK); diaminobenzidine
chromogenic reagent (DAB, Boster Biological Technology, Ltd.,
Fremont, CA); quantitative polymerase chain reaction (PCR)
enzyme SYBR Green PCR Master Mix (Toyobo Co., Ltd., Osaka,
Japan); RQ1 RNase-free DNase (Promega M6101) Corp.,
Madison, WI); and MaxVisionTM HRP-Polymer anti-Mouse/
Rabbit Immunohistochemistry (IHC) Kit (Shenzhen Maixin
Biotech Co., Shenzhen, China).
The following major equipment was used: real-time PCR
instrument (RT-PCR, ABI PRISMH 7300 Sequence Detection
System, Applied Biosystems, Foster City, CA); confocal laser
scanning microscope (ZEISS LSM 510 META, Oberkochen,
Germany); and scanning electron microscope (SEM, XL-30-based
Environmental Scanning Electron Microscope, Philips,
Hilversum, the Netherlands).
C-CBM Preparation and Measurement
The procedures described below are carried out on ice. Sterile
106PBS, sterile distilled water, sterile fresh 1 M NaOH solution
and type I collagen were mixed to prepare 5 mg/ml type I
collagen according to a volume ratio of 5 mg/ml type I collagen:
106PBS: 1 M NaOH = 10:5: 1. Sterile distilled water up to total
volume and adjust the pH to 6.9 with 1 N HCL. After mixture,
the final concentration of type I collagen was 0.5 mg/ml. A soft
mixture was achieved using a suction pipette. Centrifugation
(300 g610 min) was carried out to remove any air bubbles. The
collagen mixture was left to rest for 15 minutes and then the chitin
membrane was modified with type I collagen. The chitin
membrane was trimmed to 5 cm65 cm. Double-layer cross-type
placement of the chitin membrane was carried out according to
the material texture on a mold of the same area. 5 ml type I
collagen mixture was added into each mold. The mold was placed
horizontally on ice for one hour and then placed in a 37uC
incubator for another hour to promote colloid formation.
Observation of C-CBM characteristics were done with a confocal
microscope. Pre-cooling was carried out at 280uC for 20 hours or
overnight. Then, freeze drying was done at 240uC for 25 hours.
Co60 irradiation at 5 kGy was done four times for a total of
20 kGy to promote cross-linking and sterilization. Then, the
membrane was observed with a confocal laser scanning
microscope. Collagen-modified chitin membrane of 210 mm thick was
obtained and the C-CBM membrane was observed under a SEM.
Repeated cross-linking using Co60 and sterilization of C-CBM was
carried out. The specimen was fixed with 2.5% glutaraldehyde
overnight, and then rinsed with PBS three times. After gradient
alcohol dehydration, lyophilization, gold spraying and vacuum
processing, the membrane was observed with a SEM again.
Preparation of Chitin Membrane Leaching Solution
After calculation of the surface area of the chitin membrane,
10 ml DMEM culture medium was added per square centimeter,
the sample was held at 37uC for 24 h and the supernatant was
obtained. FBS was added to produce a leaching solution of 10%
FBS, which was the starting concentration. Gradient tests were
carried out according to double dilution.
Effect of Chitin Membrane Leaching Solution on ESCs
Three ESCs were chosen, and the cell concentration was
adjusted to 26104/ml after trypsin digestion. The solution was
added to 96-well plates, 100 ml in each well and incubated in 37uC
for 24 hour. The culture medium and non-adherent cells were
discarded. Double-diluted leaching solution was added to make six
groups: 1/1 (original leaching solution), 1/2, 1/4, 1/8, 1/16, 1/
32, each group filling four wells. The control group only had
culture medium added, no ESCs. After 96 hour incubation,
Alamar BlueH assay was carried out, and the results were
Isolation of ESCs, Seeding and Culture on C-CBM
Twenty clean degree 13 day old Sprague Dawley (SD) infant
rats weighing 1825 g were provided by the Experimental Animal
Center of Sun Yat-sen University. Animal experiments were
carried out in strict accordance with the established institutional
guidelines for animal care and by the authors affiliated
institutions. Full-thickness skin of the SD infant rats was harvested.
ESCs were obtained by using collagen IV adherent method with
Dispase II and trypsin. After removal of the subcutaneous tissue,
the skin was trimmed to 261 cm, then digested with 0.5% dispase
II overnight. Next, the skin was re-warmed for 15 minutes, and the
epidermal layer was separated from the dermal layer. The
epidermis was digested by 2.5% trypsin at 37uC for 1015 min
to prepare single-cell suspension. Digestion was terminated by
adding culture medium containing 10% FBS. After filtration with
a 200 mesh filter and centrifugation at 1000 rpm/min for 10 min,
the supernatant was discarded, and the pellets re-suspended in
Defined Keratinocyte-SFM (DK-SFM). Then, the solution was
placed in a culture bottle pre-coated with rat type IV collagen and
left at 37uC for 1015 min. The keratinocytes within the
supernatant were discarded. An appropriate amount of fresh
SFM was added to the adhered cells (ESCs), which were cultured
at 37uC in a 5% CO2 humidified incubator. The medium was
changed every other day. After the cells became confluent, 0.25%
trypsin and 0.02% EDTA were applied for digestion at 37uC for
1015 min to collect cells, which were used for passage or
Alamar BlueH Applied to Measure the Growth Curve of
Cells were placed in a 96-well plate. Before the end of culture,
20 ml Alamar BlueH dye was added. Colorimetric analysis was
carried out at 4 and 7 h. The absorption values were measured
directly with a microplate reader. The maximum absorption of
oxidized Alamar BlueH was at 600 nm; the maximum absorption
of deoxidized Alamar BlueH was at 570 nm, so that the value of
deoxidized Alamar BlueH dye in living cells was obtained by
subtracting ABS 570 from ABS 600. The color depth was
proportional to the number of living cells.
Measurement of Proliferation and Differentiation Markers
Surface markers of ESCs, including CD29, CD71, CD49d and
CD34, were examined by flow cytometry. ESCs were harvested,
the cell concentration adjusted to 36105/ml, then the mixture
reacted with a primary antibody at room temperature for 30 min.
Next, it was washed with PBS twice, and then reacted with FITC
or PE-conjugated secondary antibody for 30 min in the dark. The
cells were re-suspended with PBS on ice, and then measured by
Measurement of CK15, CK19 and p63 Expression by IHC
using Strept Avidin-Biotin Complex (SABC)
ESCs were placed in a mixed solution of 30% H2O2 and
methanol (1:10, v/v), soaked at room temperature for 10 min to
inactivate endogenous enzymes, and washed three times with PBS
for 5 min each. After heat-induced antigen retrieval and
membrane rupture, 50 ml normal goat serum blocking solution
was added together with anti-CK15, CK19 and p63 monoclonal
antibodies. The mixture was then placed in a wet box at 4uC
overnight. Next, it was placed in a 37uC box for 20 min for
rewarming, and then washed three times with PBS for 3 min each
time. Biotinylated secondary antibody was added and the reaction
carried out at 37uC for 20 min, and then the mixture was washed
with PBS three times for 3 min each time. SABC was added, the
reaction carried out at 37uC for 20 min, then the mixture was
washed with PBS three times for 3 min each time. DAB was added
and the mixture incubated for 30 s to 5 min,and subsequently
washed with distilled water. Hematoxylin counterstaining,
dehydration, vitrification and mounting were then done.
Seeding of ESCs on CBM and C-CBM In vitro
The modified chitin membrane was trimmed to a
10 mm610 mm square, sterilized with ultraviolet irradiation,
balanced by culture medium and preserved. Cultured ESCs were
digested and rendered into suspension, and the cell concentration
was adjusted to 26106/ml. Surface implantation was carried out
with 0.5 ml cell suspension, which was dropped onto the surface of
the modified chitin membrane and put aside for 1 h. DK-SFM
containing 20% FBS was added and the membrane was placed in
a 37uC, 5% CO2 incubator. Routine culture was applied for 1
2 w, with the medium changed every other day. When a
biomimetic skin covering of ESCs-modified chitin membrane
formed, it was used for measurement or animal experiment.
Characterization of Cellular Behavior on Chitin
The states of cell growth on the material before and after
modification were compared. ESCs were planted on non-modified
chitin membrane and chitin membrane modified with type I rat
tail collagen. Colony-formation was observed after 12 w in both
groups. The relationship between the cells and the material and
the cell spreading status was observed with a laser scanning
confocal microscope. The membrane was treated described as
above, and then observed with SEM.
Establishment of Rat Full-thickness Skin Defect Animal
Model and in vivo Degradation Test of Chitin Membrane
Twenty-four healthy SD rats were selected from the Laboratory
Animal Center of Guangdong Province and used for both the
material group and the sham-surgery (control) group. Each rat
received two back wounds: one on the upper left side (the material
group) and one on the lower right side (the sham-surgery group).
No material was implanted in the sham-surgery group. Rats were
anesthetized with 50 mg/kg 0.6% sodium pentobarbital and small
incisions (about 2 cm long) were made at bilateral thick-fleshed
sites of the spine about 3 cm apart. Incisions of about 1 cm61 cm
were made 1.5 cm from the spine. The incisions were deep into
the subcutaneous layer to form a mechanically injured animal
model. For the material group, 1 cm2 of chitin membrane blank
material was implanted in each wound. Compressive dressing was
applied. Each rat was put in a separate cage. Four specimens were
harvested at 1, 2, 4, 6, 8 and 10 w each, embedded with paraffin
and stained with hematoxylin and eosin (H&E) stain.
In situ Induction of Epidermal Regeneration by
ESCsmodified Chitin Membrane in Nude Mice Full-thickness
Skin Defect Model
Sixty-three BALB/c nude mice were randomly divided three
groups which were the type I collagen, experimental group
(ESCsC-CBM) and the control group (C-CBM without cells).each group
divide into seven timeslot. Nude mice were anesthetized with
50 mg/kg 0.6% sodium pentobarbital and incisions made 1.5 cm
from the spine to produce a full-thickness skin defect model as
described above, with the upper left injury belonging to the
experimental group, and the lower right injury to the control
group. The type I collagen group was independent as biomaterial
control. For each injury, 1 cm2 ESCs-C-CBM was implanted into
the experimental injury and 1 cm2 C-CBM implanted into the
control injury including the type I collagen group and C-CBM
without cells group. All injuries were sutured at the four angles and
compressive dressing applied. Each mouse was put in a separate
cage. Three specimens were harvested at d 1 and 3, and 1, 2, 4, 6
and 10 w. H&E staining was performed and histological changes
in the wound observed.
Histological and Immunohistochemical Examinations of
H&E staining: Paraffin-embedded sections were placed in a
55uC oven for 20 min, and xylene dewaxing carried out for 5
10 min. Then, each section was placed into a mixture of xylene
and pure alcohol (1:1) for about 5 min. Gradient alcohol hydration
and hematoxylin counterstaining were performed. Color
separation was carried out with 0.51% hydrochloric-alcohol solution.
Eosin staining was performed for 15 min. Gradient alcohol
dehydration, xylene vitrification and mounting were carried out.
IHC Measurement of in vivo ESCs Markers CD34 and
Each specimen was fixed with 10% neutral formalin and
embedded with paraffin. A conventional tissue section was
prepared and the specimen was dewaxed with xylene. Gradient
alcohol hydration was performed and the specimen was placed in
distilled water for later application. High-temperature and
highpressure retrieval was carried out using pH 6.0 citrate buffer at
120uC for 2 min. The specimen was washed with PBS three times
for 3 min each time, incubated together with peroxidase blocking
agent at room temperature for 10 min, and then washed with PBS
three times for 3 min each time. Fifty ml primary anti-rat antibody
CD34 or CD200 (1:100) was added and the specimen placed at
4uC overnight, then washed with PBS three times for 5 min each
time. Fifty ml HRP-Polymer anti-rat antibody was added and the
specimen was incubated at room temperature for 20 min, then
washed with PBS three times for 3 min each time. Coloration was
carried out by adding 2 drops or 100 ml newly-prepared DAB
reagent for 35 min. After the specimen was profusely washed
with water, hematoxylin counterstaining, dehydration, vitrification
and mounting were carried out.
Western Blot Analysis of Early-stage Protein Expression in
ESCs and Protein Expression of Transient Amplifying Cells
(TAC) during ESCs-C-CBM Induced Skin Repair
The tissue and protein were obtained using the Trizol method.
Protein sample loading buffer was added by ratio. Boiling was
done for 5 min to obtain sample loading protein solution.
SDSPAGE electrophoresis was conducted as follows. Electrophoresis
was done at 80 V for 4050 min, and then electrophoresis was
applied at 120 V. Low temperature membrane transfer was
performed at 200 mA for 1 h. The specimen was then washed
with TBST three times for 5 min each time. Five percent skim
milk powder was added and the specimen was held closed at room
temperature for 1 h. It was washed with TBST three times for
5 min each time, then primary antibody (1:1000) added and the
sample held at 4uC overnight. It was again washed with TBST
three times for 5 min each time. Mouse-HRP secondary antibody
working solution was added and the specimen was incubated at
37uC for 1 h. It was again washed with TBST three times for
5 min each time. Chamber exposure was carried out and the
results were scanned and preserved. Early markers of ESCs like
CD29, p63 and VEGF-A, as well as transient amplifying cell
(TAC) markers CK5, CK10, CK14 and CK15 were measured by
Quantitative RT-PCR Analysis and miR-203
Quantitative RT-PCR was used to analyze the mRNA
expression levels of CD29, CK15, p63 and VEGF-A throughout
the ESCs-modified chitin membrane skin repair process and to
track changes in the regulatory factor microRNA-203 (miR-203).
To evaluate the genetic changes of ESCs-modified chitin
membrane at the early stage, the nude mice full-thickness skin
defect model was examined through day 42. Primers of the target
fragments were designed and synthesized according to Table 1.
Specimens were harvested regularly after surgery and total RNA
extraction carried out. RNase-free DNase was applied and the
reaction solution was prepared, digested at 37uC for 30 min, then
inactivated at 65uC for 10 min. The purity and integrity of total
RNA were checked. Quantitative PCR was performed after
reverse transcription. The internal control segment used was 18S
rRNA-112 bp and the primer of the target fragment designed and
synthesized according to Table 1. The reaction system used was
5.0 ml cDNA (1:25) in 10 mM solution, the forward primer was
0.5 ml in 10 mM solution and the reverse primer was 0.5 ml of 2x
SYBR with 10 ml Green PCR Master Mix and 4.0 ml dH2O, for a
total volume of 20 ml. Reaction conditions were obtained by
predenaturation at 95uC for 10 min, denaturation at 95uC for 15 s,
annealing at 60uC for 15 s and extension at 72uC for 30 s. Plates
were read after 40 cycles. To analyze the melting curve, the
temperature ranged 6095uC and was read every minute.
MiR-203 changes were measured simultaneously by the method
described as above. The target segment was designed according to
the details in Table 2.
One way ANOVA was performed using SPSS 13.0 (SPSS Inc.,
Chicago, IL) to analyze experimental data. The values were
expressed as mean 6 standard deviation (SD). A significance level
of 0.05 was adopted.
Biological Characteristics and Verification of Rat
Epidermal Stem Cells
Primary rat epidermal stem cells (ESCs) were isolated from
Sprague Dawley rats by using dispase II and trypsin and cultured
on type IV collagen. Identification of these ESCs was determined
by the unique biological characteristics and marker expression
(Figure 1). (Figure 1A) ESC colony formation was observed after
three to six days of culture, and ESCs can be identified by their
round or polygonal morphological shape (Figure 1A, a). After one
to two weeks of culture, ESCs formed a large flakiness appearance
(Figure 1A, b).
ESCs were also confirmed by immunocytochemical staining of
their unique markers. High levels of Cytokeratin 15 (CK15),
Cytokeratin 19 (CK19) and p63 protein were detected in the
isolated ESCs (Figure 1A, d,f,h). CK15+ and CK19+ brown
particles are expressed in the cytoplasm (Figure1 A, d and f,
respectively), and p63+ brown particles are expressed in the
nucleus (Figure 1A, h). This is in contrast to the negative control
cells which show no brown particles (Figure 1A, c,e,g).
Growth rate of ESCs was examined by Alamar Blue colorimetry
(Figure 1B). The growth curve shows that the doubling growth
time of ESCs is 48 hours. Furthermore, flow cytometry analysis of
the ESCs cell cycle showed that 98% of cells were in the G0G1
phases (Figure 1C).
ESCs were identified based on their phenotypic expression of
b1-Integrin (CD29)+, CD45d+, CD712 and CD342 by flow
cytometry (FCM; Figure 1D). The percentage of b1-Integrin,
CD45d, CD71 and CD34 in M1 phases is 97.765%, 85.0362%,
8.6763% and 0.9465%, respectively. Rat ESCs express CD29
and CD49d, but not CD71 and CD34, indicating that these ESCs
were of high proliferation, but low differentiation potential. These
results show that ESCs isolated from rat skin could successfully
proliferate with ESCs marker.
Biocompatibility of C-CBM Material and ESCs, and
Microcharacteristics of their Co-culture
Though CBM has been shown to be a successful carrier
medium for ESCs, there are still some flaws, such as the uneven
spread of ESCs and the weak adherence of ESCs to the covering.
The addition of type-I collagen to the membrane carrier (modified
CBM) allowed the ESCs to better adhere to the membrane and for
greater proliferation. Type I collagenchitin biomimetic (C-CBM)
material was prepared with different concentrations of type I
collagen (0.0, 0.5 and 1.0 mg/ml). (Figure 2). Analysis of the
different concentrations of type I collagen was done in order to see
which preparation was most suitable for growth of ESCs. This was
determined by factors such as the abundant spread of ESCs and
the production of the smallest pore size in the membrane.
Non-modified CBM (0.0 mg/ml collagen) are electrospun
fabric obtained commercially. (Figure 2A, CBM). However, the
pore diameter was relatively large (200500 mm). In contrast,
1 mg/ml modified CBM prevented grid formation because the
collagen was too thickly bonded together, and the ESCs also fell
off easily after lyophilization (Figure 2A, CBM+1 mg/ml type I
collagen). However, if the concentration is too low, the grid
formation becomes uneven and the pore diameters are not
uniform. In the current study, 0.255 mg/ml filtration showed
that 0.5 mg/ml type I collagen was the most conducive
concentration to grid formation. 0.5 mg/mL modified CBM
showed a web-like grid with relatively small pores (210 mm in
diameter). Thus, the spread of ESCs on this scaffold was
distributed fully and evenly (Figure 2A, CBM +0.5 mg/ml type I
collagen). Thus, this concentration of CBM showed to be most
suitable for ESCs proliferation and differentiation.
The effect of the degradation process of C-CBM in the rat
fullthickness skin defect models was also examined to ensure
compatibility of chitin with the natural microenvironment of the
skin (Figure 2B). In the experimental group, C-CBM was
embedded beneath the full-thickness skin post-injury
(postsurgery). Sham operations were performed on rats in the control
group and contained neither C-CBM nor cells. An inflammatory
response ensued two to four weeks after the operation in the group
of rats dressed with C-CBM. At six to eight weeks after the initial
injury, C-CBM fibers were hydrolyzed, and had a rod-like
appearance encircled by connective tissues. At 10 weeks, these
fibers were largely degraded and newly repaired tissue took their
place. However, granulations appeared in the repaired tissue,
though the amount was not significant, especially when compared
to the control group. Also, this granulated mass softened over time.
Non-modified CBM was also compared with modified C-CBM
(0.5 mg/ml) to evaluate the spread of ESCs on the material. ESCs
and either CBM or C-CBM were co-cultured in-vitro to further
examine the morphology of the ESC culture (Figure 2C). When
CBM and ESCs were co-cultured and observed under a laser
scanning confocal microscope at two to four weeks, the ESCs were
visible on the grid pattern of the dressing (Figure 2C, b). The ESCs
were also labeled with DiI. When observed under a fluorescence
microscope, ESCs were shown to have adhered to the fiber
junctions only. Though there was an affluent amount of colonies,
each colony only contained a few cells. Furthermore, the
distribution of these cells were uneven over a round or oval area.
Also, the adhesion of the ESCs to the material was tenuous and the
cells easily fell off. In the middle layer, the original CBM fibers as
observed with yellow reflected light showed excellent growth of
Figure 1. Biological characteristics and verification of rat ESCs. (A) The unique morphology of rat ESCs and cell colonies: (a) ESC colonies
formed at d3 under an inverted microscope (1006); rat ESCs have a round or polygonal morphology; (b) ESCs morphology under the phase contrast
microscope at d5 (2006), a large flakiness appearance can be seen, characteristic of ESCs; (c) Immunocytochemistry of CK15+ negative control shows
no brown granules after staining (1006); (d) Positive ESCs visualized by brown granules: CK15+ expression in the cytoplasm (1006); (e)
Immunocytochemistry of CK19+ negative control; (f) Positive ESCs visualized by brown granules: CK19+ expression in the cytoplasm (1006); (g)
Immunocytochemistry of p63+ negative control; (h) Positive ESCs visualized by brown granules: p63+ expression in the nucleus (1006); (B) ESCs
growth curve; (C) Analysis of cell cycle of rat ESCs showed 95% were in stages G0G1; (D) ESCs phenotypes identify CD29+, CD45d+, CD712 and
CD342 by flow cytometry.
ESCs (Figure 2C, d). On the other hand, ESCs co-cultured with
C-CBM showed more favorable outcomes. ESCs labeled with
GFP formed dense colonies on the C-CBM material (Figure 2C,
e), and spread widely and evenly. Layered scanning showed that
the material surface was covered by cells. Spindle-shaped cells
were evenly distributed across the grid, unlike in CBM (Figure 2C,
f). Extracting solution of the chitin membrane had no effect on the
cells. Comparison between the P3 ESCs group and the control
group showed F = 0.781, P.0.05, indicating no significant
difference (Figure 2C g). The secretion of the extracellular matrix
was also not significant.
ESCs were observed under a Scanning Electron Microscope
(SEM) to see whether or not the ESCs spread sufficiently in their
respective carrier mediums (Figure 2D). Pure type I collagen is a
good culture substrate for ESCs (Figure 2D, a,d,g and i) and many
cell types. But it can dissolve in cell culture media only be
maintained for a maximum of three days. This is relatively short
compared to type I collagen modified CBM, which showed large
amounts of proliferating ESCs and incessancy for at least 4 weeks
(Figure 2D, c,f,i and l). Furthermore, C-CBM material showed
densely porous wire-mesh structures, evenly distributed, with pore
diameters of the mesh aperture ranging 210 mm (Figure 2D, i),
compared to the unmodified chitin which produced pores ranging
in size from 200500 mm (Figure 2D b). Also, it was observed that
the extracellular matrix secreted by cells was woven into the
natural mesh grid together with the collagen-modified grid
(CCBM). Cells were evenly spread over the grid and actively growing
with vigorous secretion of the extracellular matrix and increased
proliferation over controls and clear evidence of mitosis
These results indicated that the modified chitin membrane was
fundamentally a better surface structure for the culturing of ESCs.
Fresh rat tail type I collagen mixture was prepared to a final
concentration of 0.5 mg/ml type I collagen and a pH 6.9. It will
be assumed from this point on that C-CBM refers to a
concentration of 0.5 mg/ml of type I collagen.
Re-epithelialization and Re-growth of Skin Appendages
in the Wound Area
In vivo experiments were carried out to compare the effects of
CCBM and ESCs-C-CBM in nude mice with full-thickness skin
defects. Factors that were evaluated include the presence of early
biomarker expression and wound healing capability by
immunohistochemical (IHC) assay.
The nude mice were divided into two groups: (1) C-CBM group
and (2) ESCs-C-CBM group, and observed for up to ten weeks
post-injury. Nude mice wounds dressed with ESCs-C-CBM
produced new skin that was relatively thicker, redder and more
elastic (Figure 3A). The thickness of the skin in the repaired wound
could be observed by the naked eye. The morphology of the
repaired skin in this group was similar to that found in unwounded
rat full-thickness skin defect models at week 4, 6 and 10. Paraffin and H&E staining methods were utilized. Embedded chitin material (blue arrows)
were degraded by week 10 (100X). (C) Biocompatibility of the co-culture of C-CBM or CBM with ESCs. (a) Observation of CBM material with the naked
eye; (b) Observation of CBM material with ESCs cell culture with the naked eye; grid-like colonies formed for 24 weeks; (c) Pore diameter of
nonmodified CBM (100X); (d) Pore diameters in CBM with ESCs stained with DiI (100X); ESCs adhered only to junction; (e) Pore diameters in C-CBM
material (100X); (f) ESCs-C-CBM stained with GFP (100X); (g) Effect of culture ESCs in media from leaching solution of CBM material. (D)
Characterization of cellular behavior on CBM by SEM. Scale bars 100 mm and Scale bars 20 mm. Group 3: Type I collagen, Chitin biomiemtic
membrand (CBM) and 0.5 mg/ml type I collagen-chitin biomimetic membrane (C-CBM). (a,g) Type I collagen; (d,j) ESCs grown on the Type I collagen;
b,h: Chitin biomiemtic membrand (CBM), (e,k) ESCs grown on the CBM; (c,i) C-CBM, (f,l) ESCs grown on the C-CBM.
or peripheral tissue, which was soft and red. On the other hand,
the control group (C-CBM), The reconstruction skin only formed
a thin layer of epidermis, the repaired wound was thin, light purple
and had a tendency to bleed easily. When only type I collagen was
applied, the wounds took a much longer time to heal and had a
dark purplish appearance and wound shrinking.
Furthermore, in vivo experiments showed more obvious hair
follicle cell proliferation in the full-thickness skin defect nude mice
dressed with ESCs-C-CBM compared to those dressed with
CCBM and type I collagen alone (Figure 3B). Haematoxylin Eosin
(H&E) staining showed no significant inflammatory reaction in the
dermal connective tissue under microscopic examination in the
both groups. Also, in ESCs-C-CBM, formation of round or oval
areas of net-like epidermis (blue arrows) increased regularly from
the second week, was most intense at four to six weeks, and began
to reduce after eight to 10 weeks post-injury. Moreover, the
netlike epidermis was detected by CD200 and CD34 in wound repair
by IHC (Figure 4AD). The round or oval areas of net-like
epidermis were actually different cross-sections of hair follicle cells.
Reassembly and analysis of those various sections provides a
complete diagram of the hair follicle structure. These events were
absent in the control group (C-CBM).
CD34 and CD200 are early-stage markers of hair follicle stem
cells. The group in ESCs-C-CBM had more CD34 and CD200
positive cells than the group in C-CBM. Figure 4A shows that
CD34 was mainly expressed in the hair follicle bulge cell group in
ESCs-C-CBM. This expression occurred mainly at day three after
surgery and was maintained from day 5 to week 4, then decreased
after six weeks. However, CD34 was expressed up to week 4 in the
group C-CBM. These results indicate that ESCs were highly
proliferative before week 4 without differentiation and maintained
their ESCs characteristics.
Figure 4B shows that CD200 was located at the hair follicle in
the ESCs-C-CBM group. It was expressed at day 3 after surgery
and maintained until the sixth week, which is relatively a long
duration. In the group C-CBM, CD200 was expressed at day 7
and the expression started to decrease after the fourth week, which
suggests that the cells already started to differentiate, and the
phenotypic expression of CD200 is decreased. CD34 was relatively
strongly expressed at the hair bulb at day 5, while CD200 was
significantly expressed at the outer hair root sheath at day 3.
(Figure 4C, D).
To further examine the early protein expression and mRNA in
the wound samples, Western blots and Quantitative RT-PCR
were performed. Analysis of these tests showed early protein
expression markers were significantly increased; the levels of
CD29, p63, VEGFA and Dsg3 at day 3 of ESCs-C-CBM were
much higher than those found in the C-CBM group (Figure 5A).
Results showed that the ESCs were viable in vivo and became
active. On the other hand, there were no significant differences
between the two groups in the expression levels of transit
amplifying cells (TAC) markers in CK5, CK10, CK14 and CK15.
The hair follicle mRNA markers: CD29 (Figure 5B, a),
transcription factor p63 (Figure 5B, b) and VEGF-A (Figure 5B,
c) and CK15 (Figure 5B, d) were highly expressed in group
ESCsC-CBM at day 3 compared to the group C-CBM without ESCs.
Of these, the sustained expression of high levels of CK15 was most
noticeable; expression increased three-fold at day 3, four-fold at
day 5, eight-fold at day 28, and was continued to increase by a
2.5fold up to day 42. Moreover, p63 mRNA group increased five-fold
over ESCs-C-CBM than group C-CBM, which was maintained
until day 28. In addition, CD29 mRNA expression, suggesting
early-stage transcription, increased eight-fold at day 3. VEGF-A
was highly expressed from day three to 28, and decreased to
normal by day 42. CD19 was highly expressed at day 28.
Regulatory factor miR-203 did not show a significant difference
between the two groups before day 7, but its expression increased
in the group C-CBM at day 28 and the two groups significant
difference (P,0.01) (Figure 5B, f).
The isolation and culture of ESCs in vitro and creation of a
three-dimensional (3D) tissue skin structure in vitro for their clinical
utilization has been a challenge for clinical application in its advent
to improve the process of wound healing . Kojma et al
suggested that chitin was able to stimulate endogenous collagen
production . Currently, chitin membrane has been used as
wound coverings in clinical practices. However, the addition of
ESCs to this membrane, as well as modifying the membrane with
type I collagen, has not been done before. The objective of this
study was to see whether chitin with ESCs culture and exogenous
collagen-modification would be a more effective wound covering
than just chitin itself, and to observe whether such wound covering
would be in accordance with the natural microenvironment
required for proper wound healing.
First, we described a procedure for isolation and the culture of
ESCs (with its markers) from a rats skins backside (Figure 1).
Then, we compared two different cell scaffolds: (1) CBM and (2)
C-CBM. CBM is chitin membrane without type I collagen added.
It is purchased from commerce and has been pre-electrospun. The
other type, C-CBM, is coated chitin that has been modified with
type I collagen. In a previous experiment, Lee coated type I
collagen to the surface of chitin scaffold and used sodium chloride,
and showed that it produced large pores that were 260330 mm in
diameter . Results showed that 0.5 mg/ml collagen
concentrations produced a patterned web-like grid (Figure 2A) with pores
as small as 210 mm in diameter. Thus, cells were able to spread
fully and evenly throughout the matrix. This is in contrast to
CBM, which contained pores that were 200500 mm in diameter
(Figure 2C c). The implanted fibroblasts showed relatively good
affinity and proliferative ability after 14 days. If the collagen
concentration is too high and the bonded powder too thick, cells
easily fall off. If the collagen concentration is too low to form a grid
or the mesh diameter is too large, the cells cannot adhere well.
Second, we compared type I collagen coated on the surface of
chitin scaffold (C-CBM) with and without ESCs in vivo on nude
mice. Patterned surface modification affects the relationship of
cells with each other, the extracellular matrix, and soluble factors
to change the differentiation and modification of stem cells.
Figure 3. Observation of nude mice full-thickness defect model. (A) Observation of nude mice full-thickness defect model dressed with type I
collagen only, C-CBM, or ESCs-C-CBM at 1 d, 1 w, 4 w, and 10 w. Type I collagen group shows that wounds are much slower to heal. Group C-CMB
shows the repaired wound skin in the control group was relatively thin and heliotrope with a tendency to bleed; Group ESCs-C-CMB shows the
repaired wound in the experimental group was relatively thick and red with re-epithelialization. Scale bar = 1cm. (B) Formation of epidermal nests on
the wound surface repaired by epidermal stem cells- collagen-chitin biomimetic (ESCs-C-CBM) membrane compared with C-CBM. Paraffin and stained
with hematoxylin and eosin (H&E) stain. Yellow arrows point to chitin and blue arrows point to epidermal nests increased in the ESC-C-CBM group at
210 w (100X).
Improving the biological induction material requires increasing
the contact between the cells and the surface or the interface of the
material, forming a bio-mimetic surface with a
microenvironment that optimizes the distribution and reconstruction of such
active materials as the extracellular matrix and cytokines for
Furthermore, to explore the use of C-CBM with implanted
ESCs for wound repair we examined the effects of cell
differentiation. Previous experiments showed that the chitin
group C-CBM, which shows CD34 was expressed 4 w after surgery; (B) CD200 was located at the hair follicle group ESCs-C-CMB. It was expressed at
d3 after surgery and maintained until 6w compare the control group C-CMB which CD200 was expressed at d 7 and the expression decreased from
4w. (C) Measurement of early-stage in vivo CD34/CD200 markers of hair follicle stem cells in the epidermal nests. Hair follicle longitudinal sections and
hair follicle transverse sections are both shown. (D) CD34 and CD200 present on ESCs during early stage in vivo. CD34 was strongly expressed in the
hair bulb at d5. CD200 was highly expressed at the outer hair root sheath at d3.
membrane was a suitable carrier medium . The C-CBM
produced in vitro use of ESCs for a wound covering in vivo and
helps ESCs survive on the wound surface. C-CBM is degraded
automatically 23 weeks after application and becomes detached.
Figure 3B shows numerous round and oval epidermal nests
formed under the skin of in situ repair in the animal model of
fullthickness skin defects. These nests may be related to replication
and migration, during which ESCs differentiate into epidermal
cells. A single application of ESCs without C-CBM support will
not result in the cells entering the wound surface because the
leaked tissue fluid quickly removes them. As an important
medium, collagen can be combined with integrins in ESCs to
produce dense connections between cells. C-CBM was applied in
the current study to significantly increase the rate of proliferation
of ESCs, and was interwoven into a network with the extracellular
matrix secreted by the cells. With regard to early-stage protein
expression, we found increased m-RNA transcription in CD29,
p63, VEGF-A, sustained expression of high levels of CK15 and
low levels of the regulatory factor miR-203, suggesting that the
chitin membrane promotes early-stage protein synthesis at the
wound surface and enhances the healing speed together with ESCs
which is play an important role in this process, perhaps because
the signaling molecules secreted by these cells may induce ESCs
differentiation, resulting in self-repair and regeneration of tissues
Clinically, the formation of normal re-epithelialization and
follicular orifices is very important for skin wound repair. The
follicular orifice is used for sweat gland secretion, and also speeds
up wound healing and reduces scar formation that raise the quality
of wound healing. Additionally, hair follicular stem cells (FSCs)
play a major role in this process. It is well-known that cytokeratin
15 (CK15), CK19, CD29, CD200 and CD34 are highly expressed
surface markers of hair FSCs, which correlated with our data.
These results indicated that the surface markers of FSCs were
present. The others include a6-integrin (CD29), CD71, CK19,
CK15, CD34 [29,30,31]. Because CD29 is highly expressed on
the surface of and transiently proliferating cells, but not in
postmitotic and terminal cells, it can be used to differentiate ESCs and
transiently proliferating cells. CD71 is a transferrin receptor on the
surface of ESCs. Some epidermal cells with low levels of CD71
have the characteristics of ESCs. Relatively strong expression of
CK15 was previously observed in mouse ESCs . CK15 and
Kl9 are keratins expressed at the early stage of cell differentiation.
During FSC differentiation, CK15 expression is reduced earlier
than that of CK19, suggesting that decreased CK15 expression
may be the earliest sign of cellular differentiation of transiently
proliferating cells. Cells with negative CK15 and positive CK19
may be early-stage transiently proliferating cells. CK15 may
therefore be a more meaningful marker of FSC differentiation
than CK19. In the current study, cytokeratin 15 is a specific
marker of stem cells of the hair-follicle bulge expression of CK15
continued at day 28, implying that the characteristics and function
of ESCs may be maintained for 28 days in this microenvironment
and the transiently proliferating cells appear relatively late. CKl9 is
scattered at the basal layer of the epidermis and deep in the
reticular spine in accordance with the location of label-retaining
cells (LRCs). CK19 positive cells within the bulge are LRCs and
slow cycling stem cells . Advances now indicate there are a
number of stem cell repositories within the epidermis, two of
which, the interfollicular epidermis and the bulge region of the
hair follicle, may supply each other when damaged .
CD34 is a specific marker of hematopoietic stem cells, as well as
a phenotype marker of vascular endothelial cells. Trempus
demonstrated that CD34 is a special marker of skin bulge cells,
showing it to be highly expressed in the bulge . CD34 and
K15 positive cells are located at the same place in the rat hair
follicle, but K15 expression is decreased or absent in the bulge of
human hair follicles . Therefore, CD34 is also a marker of rat
ESCs and vascular endothelial cells.
CD200 is a recently-found marker of ESCs. Most residual cells
with slow cycling markers are located at the hair follicle bulge,
CD200 positive cells showed greater ability to form clones 
and CD200 is expressed in cells on the outer root sheath of the
hair follicle . This niche microenvironment found in the hair
follicles bulge possesses intrinsic stemness features without
restricting the establishment of epithelial polarity or changes in
gene expression . Thus, bulge cells can develop into epidermal
During rodent experiments, the speed of wound healing of the
hair follicle was quicker at the anagen period than at the telogen
phage. In autologous skin graft surgery, the scalp can usually be
used as the donor site for repeated skin harvesting without scar
formation. This practice suggests that the hair follicle is closely
related to wound healing. Taylor et al. reported that FSC can not
only differentiate into various types of cells within the hair follicle,
but also differentiate into skin epidermal cells during the periodic
cycle of the hair follicle . Tumbar et al. studied histone-GFP
mice to show that GFP positive cells from the bulge differentiated
into the outer root sheath of the hair follicle, hair and inner root
sheath cells at the anagen phase . Moreover, labeled slow cells
moved out from the bulge, migrated and differentiated into the
basal membrane and epidermal cells when the skin was injured.
After labeling and implanting isolated dermal cells of the rat hair
follicle in wounds in the rat ear and back, researchers observed
that the labeled cells took part in the repair of dermal tissue, acting
just like fibroblasts taking part in wound healing . Amoh et al.
also found in rat experiments that some parts of new capillaries
originated from labeled cells in the anagen phase of the hair follicle
during skin wound repair [41,42]. These results suggest that FSC
may take part in dermal vascularization during wound healing.
FSCs can differentiate into sebaceous glands or epithelial cells in
the sweat gland . As a transcription factor, p63 is related to the
genotypic change of stem cells or transiently proliferating cells. A
homolog of p53, it is localized in the nucleus and is involved in
epidermal development. It plays a decisive role in maintaining the
biological properties and proliferation and differentiation of ESCs
MiR-203 is a regulation gene of ESCs differentiation. If
expressed too early in epidermal cells in the basal layer,
miR203 may result in premature differentiation and defects of
proliferative potential. MiR-203 expression is obvious during
epidermis differentiation and development. Moreover, it evolves
into conservative miRNA. MiR-203 targets Np63 mRNA, and
acts as a switch in the proliferation and differentiation of
Figure 5. Early-stage markers of protein level and m-RNA level of epidermal stem cells (ESCs). (A) Western blot analysis to detect marked
protein. (B) Quantitative real time polymerase chain reaction (RT-PCR) analysis of m-RNA levels: (a) CD-29 increased at d3; (b) p63 was maintained at a
relatively high level; (c) VEGF increased at d35; (d) CK15 was maintained at a relatively high level; (e) CK-19 was still highly expressed at d28; (f)
miR203 was significantly low at d28 compared to the control group, *P,0.01.
keratinocytes in the adult epidermis. Therefore, miR-203 is a key
molecule controlling the differentiation of keratinocytes from the
basal state to the basal layer. MiR-203 regulates p63 by
suppressing translation, and strongly inhibits p63 expression. After
the defects of miR-203, p63 translation in the basal epidermis will
increase. Over-expressed miR-203 reduces Np63 mRNA . It
has been reported that p63 expression is strongly inhibited during
low-calcium culture of primary mouse keratinocytes transfected
with wild-type miR-203, which indicates that miR-203 regulates
p63 by translational suppression . Although VEGF-A is an
important regulatory factor of carcinogenesis, it is also expressed in
certain normal tissues. Angiogenesis during embryonic
development depends on normal VEGF-A expression. Neovascularization
is needed during wound repair and wound healing may be affected
by abnormal VEGF expression . In the present study,
VEGFA disappeared after d28, suggesting that increased VEGF-A
production is important during early phase wound healing, then
turns off subsequently.
The present study showed that ESCs produced in vitro with the
related scaffold can form a temporary biomimetic covering. ESCs
can differentiate into epidermal cells to enhance wound healing,
inducing in situ regeneration of nude mice full-thickness skin
defects. This shows that biological material containing seed cells
can promote wound healing. However, the final turnover of the
epidermal nests should be further identified by labeling.
Simulation experiments of the structure and function of the ESCs
microenvironment showed production of biological induction
materials imitating the natural extracellular matrix with signaling
molecules effectively regulating the differentiation of ESCs to
stimulate and induce self-repair and regeneration. The result was
the morphological and structural regeneration and functional
reconstruction of damaged tissue.
Our findings that 0.5 mg/ml rat tail type I collagen can be used
for surface modification of the chitin membrane, creating a
relatively good biocompatible material Cells secrete various
bioactive substances, which closely cooperate and coordinate with
each other and its surrounding microenvironment. ESCs grow
well on C-CBM and are important in wound repair. The results of
both in vivo and in vitro experiments showed ESCs were highly
spread over the C-CBM surface and the proliferation rate
increased significantly. ESCs-C-CBM plays a role in induction
of hair follicular stem cells (FSCs) and are the main ESCs growing
on the modified chitin membrane. Reconstruction of the
epidermal and the dermal layer could be observed in full-thickness
skin defects in an in vivo experiment, with more hair follicle stem
cells, and the skin on the repaired wound was relatively thick and
red with clear signs of re-epithelialization.
Hair FSCs can differentiate into epidermal cells and skin
appendages to achieve initial repair of the epidermis and dermis
without having to engineer both skin tissues separately. It can be
applied to both superficial and full-thickness wound repair in nude
mice. ESCs-C-CBM produced by tissue engineering using this
matrix grid can reconstruct tissues with structures and metabolic
activities similar to the natural skin in a relatively short period, and
is an important candidate for clinical wound repair for such
injuries as burns, skin wounds and diabetic foot ulcers.
Conceived and designed the experiments: YS XL HC. Performed the
experiments: YS LD RL SQ. Analyzed the data: YS GG WC. Contributed
reagents/materials/analysis tools: LD ZZ ZL WL. Wrote the paper: YS JX
1. Coruh A , Yontar Y ( 2012 ) Application of split-thickness dermal grafts in deep partial- and full-thickness burns: a new source of auto-skin grafting . J Burn Care Res 33 : e94 - e100 .
2. Figus A , Leon-Villapalos J , Philp B , Dziewulski P ( 2007 ) Severe multiple extensive postburn contractures: a simultaneous approach with total scar tissue excision and resurfacing with dermal regeneration template . J Burn Care Res 28 : 913 - 917 .
3. Rennekampff HO ( 2009 ) Skin graft procedures in burn surgery . Unfallchirurg 112 : 543 - 549 .
4. Chou TD , Chen SL , Lee TW , Chen SG , Cheng TY , et al. ( 2001 ) Reconstruction of burn scar of the upper extremities with artificial skin . Plast Reconstr Surg 108 : 378 - 384 : discussion 385.
5. Faulhaber J , Felcht M , Teerling G , Klemke CD , Wagner C , et al. ( 2010 ) Longterm results after reconstruction of full thickness scalp defects with a dermal regeneration template . J Eur Acad Dermatol Venereol 24 : 572 - 577 .
6. Haik J , Weissman O , Hundeshagen G , Farber N , Harats M , et al. ( 2012 ) Reconstruction of full-thickness defects with bovine-derived collagen/elastin matrix: a series of challenging cases and the first reported post-burn facial reconstruction . J Drugs Dermatol 11 : 866 - 868 .
7. Haslik W , Kamolz LP , Manna F , Hladik M , Rath T , et al. ( 2010 ) Management of full-thickness skin defects in the hand and wrist region: first long-term experiences with the dermal matrix Matriderm . J Plast Reconstr Aesthet Surg 63 : 360 - 364 .
8. Liu X , Ma L , Liang J , Zhang B , Teng J , et al. ( 2013 ) RNAi functionalized collagen-chitosan/silicone membrane bilayer dermal equivalent for full-thickness skin regeneration with inhibited scarring . Biomaterials 34 : 2038 - 2048 .
9. Cronin H , Goldstein G ( 2013 ) Biologic skin substitutes and their applications in dermatology . Dermatol Surg 39 : 30 - 34 .
10. Franco RA , Min YK , Yang HM , Lee BT ( 2013 ) Fabrication and biocompatibility of novel bilayer scaffold for skin tissue engineering applications . J Biomater Appl 27 : 605 - 615 .
11. Kuroyanagi Y ( 2006 ) Regenerative medicine for skin . Nihon Ronen Igakkai Zasshi 43 : 326 - 329 .
12. Wang TW , Sun JS , Wu HC , Tsuang YH , Wang WH , et al. ( 2006 ) The effect of gelatin-chondroitin sulfate-hyaluronic acid skin substitute on wound healing in SCID mice . Biomaterials 27 : 5689 - 5697 .
13. Spiekstra SW , Breetveld M , Rustemeyer T , Scheper RJ , Gibbs S ( 2007 ) Woundhealing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes . Wound Repair Regen 15 : 708 - 717 .
14. Windsor ML , Eisenberg M , Gordon-Thomson C , Moore GP ( 2009 ) A novel model of wound healing in the SCID mouse using a cultured human skin substitute . Australas J Dermatol 50 : 29 - 35 .
15. Blanpain C , Lowry WE , Geoghegan A , Polak L , Fuchs E ( 2004 ) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche . Cell 118 : 635 - 648 .
16. Christiano AM ( 2004 ) Epithelial stem cells: stepping out of their niche . Cell 118 : 530 - 532 .
17. Taylor G , Lehrer MS , Jensen PJ , Sun T-T , Lavker RM ( 2000 ) Involvement of Follicular Stem Cells in Forming Not Only the Follicle but Also the Epidermis . Cell 102 : 451 - 461 .
18. Croisier F , Jerome C ( 2013 ) Chitosan-based biomaterials for tissue engineering . European Polymer Journal 49 : 780 - 792 .
19. Dhandayuthapani B , Krishnan UM , Sethuraman S ( 2010 ) Fabrication and characterization of chitosan-gelatin blend nanofibers for skin tissue engineering . J Biomed Mater Res B Appl Biomater 94 : 264 - 272 .
20. Aranaz I , Mengbar M , Harris R , Pan os I , Miralles B , et al. ( 2009 ) Functional Characterization of Chitin and Chitosan . Current Chemical Biology 3 : 203 - 230 .
21. Yang Y , Xia T , Zhi W , Wei L , Weng J , et al. ( 2011 ) Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor . Biomaterials 32 : 4243 - 4254 .
22. Amidi M , Hennink WE ( 2010 ) Chitosan-based formulations of drugs, imaging agents and biotherapeutics . Preface. Adv Drug Deliv Rev 62 : 1 - 2 .
23. Songjiang Z , Lixiang W ( 2009 ) Amyloid-beta associated with chitosan nanocarrier has favorable immunogenicity and permeates the BBB . AAPS PharmSciTech 10 : 900 - 905 .
24. Shen Y , Li XJ , Liang R , Li YC , Zhang Y , et al. ( 2009 ) An experimental study of using chitinous membrane as the culture scaffold for epidermal stem cells . Chin J Burns 25 : 197 - 201 .
25. Lei XH , Ning LN , Cao YJ , Liu S , Zhang SB , et al. ( 2011 ) NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure . PLoS One 6 : e26603 .
26. Kojima K , Okamoto Y , Miyatake K , Fujise H , Shigemasa Y , et al. ( 2004 ) Effects of chitin and chitosan on collagen synthesis in wound healing . J Vet Med Sci 66 : 1595 - 1598 .
27. Lee SB , Kim YH , Chong MS , Lee YM ( 2004 ) Preparation and characteristics of hybrid scaffolds composed of beta-chitin and collagen . Biomaterials 25 : 2309 - 2317 .
28. Thibault M , Astolfi M , Tran-Khanh N , Lavertu M , Darras V , et al. ( 2011 ) Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes . Biomaterials 32 : 4639 - 4646 .
29. Jensen KB , Collins CA , Nascimento E , Tan DW , Frye M , et al. ( 2009 ) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis . Cell Stem Cell 4 : 427 - 439 .
30. Horsley V , Aliprantis AO , Polak L , Glimcher LH , Fuchs E ( 2008 ) NFATc1 balances quiescence and proliferation of skin stem cells . Cell 132 : 299 - 310 .
31. Rhee H , Polak L , Fuchs E ( 2006 ) Lhx2 maintains stem cell character in hair follicles . Science 312 : 1946 - 1949 .
32. Liu Y , Lyle S , Yang Z , Cotsarelis G ( 2003 ) Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge . J Invest Dermatol 121 : 963 - 968 .
33. Michel M , L' Heureux N , Auger FA , Germain L ( 1997 ) From newborn to adult: phenotypic and functional properties of skin equivalent and human skin as a function of donor age . J Cell Physiol 171 : 179 - 189 .
34. Hodgkinson VC , ELFadl D , Agarwal V , Garimella V , Russell C , et al. ( 2012 ) Proteomic identification of predictive biomarkers of resistance to neoadjuvant chemotherapy in luminal breast cancer: a possible role for 14-3-3 theta/tau and tBID? J Proteomics 75 : 1276 - 1283 .
35. Trempus CS , Morris RJ , Bortner CD , Cotsarelis G , Faircloth RS , et al. ( 2003 ) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34 . J Invest Dermatol 120 : 501 - 511 .
36. Cotsarelis G ( 2006 ) Gene expression profiling gets to the root of human hair follicle stem cells . J Clin Invest 116 : 19 - 22 .
37. Ohyama M , Terunuma A , Tock CL , Radonovich MF , Pise-Masison CA , et al. ( 2006 ) Characterization and isolation of stem cell-enriched human hair follicle bulge cells . J Clin Invest 116 : 249 - 260 .
38. Rosenblum MD , Olasz EB , Yancey KB , Woodliff JE , Lazarova Z , et al. ( 2004 ) Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance ? J Invest Dermatol 123 : 880 - 887 .
39. Tumbar T , Guasch G , Greco V , Blanpain C , Lowry WE , et al. ( 2004 ) Defining the epithelial stem cell niche in skin . Science 303 : 359 - 363 .
40. Gharzi A , Reynolds AJ , Jahoda CA ( 2003 ) Plasticity of hair follicle dermal cells in wound healing and induction . Exp Dermatol 12 : 126 - 136 .
41. Amoh Y , Li L , Yang M , Moossa AR , Katsuoka K , et al. ( 2004 ) Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells . Proc Natl Acad Sci U S A 101 : 13291 - 13295 .
42. Amoh Y , Kanoh M , Niiyama S , Hamada Y , Kawahara K , et al. ( 2009 ) Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheralnerve injury: an advantageous alternative to ES and iPS cells . J Cell Biochem 107 : 1016 - 1020 .
43. Morris RJ , Liu Y , Marles L , Yang Z , Trempus C , et al. ( 2004 ) Capturing and profiling adult hair follicle stem cells . Nat Biotechnol 22 : 411 - 417 .
44. De Felice B , Ciarmiello LF , Mondola P , Damiano S , Seru R , et al. ( 2007 ) Differential p63 and p53 expression in human keloid fibroblasts and hypertrophic scar fibroblasts . DNA Cell Biol 26 : 541 - 547 .
45. Lena AM , Shalom-Feuerstein R , Rivetti di Val Cervo P , Aberdam D , Knight RA , et al. ( 2008 ) miR-203 represses 'stemness' by repressing DeltaNp63 . Cell Death Differ 15 : 1187 - 1195 .
46. Yi R , Poy MN , Stoffel M , Fuchs E ( 2008 ) A skin microRNA promotes differentiation by repressing 'stemness' . Nature 452 : 225 - 229 .
47. Yla-Herttuala S ( 2009 ) Gene therapy with vascular endothelial growth factors . Biochem Soc Trans 37 : 1198 - 1200 .