ORIGINAL RESEARCH
Umbilical Cord Mesenchymal Stem
Wounds 2015;27(5):134-140
From the 1Department of Life
Sciences and Technology, Xinxiang
Medical University, Henan, China;
2
Sanquan Medical College, Xinxiang
Medical University, Henan, China
Address correspondence to:
Zhiqing Yuan, PhD
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Department of Life Sciences and
Technology
Xinxiang Medical University
No. 601 Jinsui Road Hongqi District
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Xinxiang 453003
Henan, China
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wenbinnancn@126.com
Disclosure: The authors disclose
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no financial or other conflicts of
interest. This study was supported
by the Natural Science Research
Program of the Education
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Department in Henan Province
(grant no. 13B180214), the
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Scientific Research Fund of
Xinxiang Medical University
(grant no. 2014QN137), and
Natural Science Foundation of
China (grant no. U1304819).
134 WOUNDS® www.woundsresearch.com
Cells Combined With a Collagen-
fibrin Double-layered Membrane
Accelerates Wound Healing
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Wenbin Nan, PhD1; Rui Liu, MS2; Hongli Chen, PhD1; Zhihao
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Xu, PhD1; Jiannan Chen, MS1; Manman Wang, MS1; Zhiqing
Yuan, PhD1
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Abstract: The aim of this study was to examine the effects of human
umbilical cord mesenchymal stem cells (hUCMSCs) in combination
with a collagen-fibrin double-layered membrane on wound healing
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in mice. A collagen-fibrin double-layered membrane was prepared,
and the surface properties of the support material were investigated
using a scanning electron microscope. Twenty-four mice were pre-
pared for use as full-thickness skin wound models and randomly
divided into 3 groups: group A, a control group in which the wounds
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were bound using a conventional method; group B, a group treated
with hUCMSCs combined with a collagen membrane; and group
C, a group treated with hUCMSCs combined with a collagen-fibrin
double-layered membrane. The postoperative concrescence of the
wounds was observed daily to evaluate the effects of the different
treatments. Scanning electron microscope observation showed the
collagen-fibrin scaffolds exhibited a highly porous and interconnect-
ed structure, and wound healing in the double-layered membrane
group was better than in groups A or B. Treatment with hUCMSCs
combined with a collagen-fibrin double-layered membrane acceler-
ated wound healing.
Key words: collagen-fibrin membrane, human umbilical cord mesen-
chymal stem cells, wound healing
T
issue engineering is the use of a combination of cells, engineering,
materials, methods, and suitable biochemical and physicochemi-
cal factors to improve or replace biological functions.1 Over the
past few decades, significant progress has been made in the development
of skin tissue engineering techniques.2 Several skin substitutes for use
in skin replacement therapies are commercially available, but these sub-
stitutes are still not fully able to replace real skin tissue with its cellular
components and skin appendages.
For optimal seeding of cells for skin tissue engineering, one type of
stem cells—human umbilical cord mesenchymal stem cells (hUCMSCs)

—have the ability for self-renewal and omnidirectional
transformation into specific cell populations, and are
highly effective for repairing skin wounds when com-
bined with tissue engineering materials.3,4 Among the
many tissue engineering materials available, collagen,
a type of natural protein present in animal skin, bone,
tendon, ligament, and cornea tissue, is extensively ac-
cepted in tissue engineering as a drug carrier and a
raw material for building active 3-dimensional (3D)
scaffolds.5 This fibrous protein is also often used to
fabricate 3D scaffolds in tissue engineering.6 However,
collagenous fiber only dissolves in an acidic environ-
ment, and it is difficult to uniformly disperse water-
soluble proteins, cytokines, and other nutrient content
in a neutral environment.7 Although fibrous protein has
good hemostatic function, its mechanical strength is
low,8 restricting its potential clinical applications.Thus,
the shortcomings of these 2 materials could be com-
pensated for to a certain extent by combining collagen
and fibrin into a composite scaffold.9 The aim of the
present study was to employ a collagen-fibrous protein
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composite material as a scaffold in combination with
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hUCMSCs to repair full-thickness skin wounds, and to
evaluate its therapeutic effect to provide a reference
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for clinical practice in the application of tissue-engi-
neered skin.
Materials and Methods
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Isolation and incubation of human umbilical
cord mesenchymal stem cells. The human umbilical
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cord used in this experiment was collected at the
Third Affiliated Hospital of Xinxiang Medical College
(Xinxiang, China). The mother who donated the mate-
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rial was considered healthy— all indexes of routine
blood examination were normal; HbsAg, anti-HCV, anti-
HIV, and syphilis antibody checks were all negative ex-
pression; and the baby was delivered by cesarean sec-
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tion at full-term. The mother and her family provided
written, informed consent. The experimental plan was
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approved by the Xinxiang Medical College Medical
Ethics Committee.
Human umbilical cord mesenchymal stem cells were
cultured using the tissue explants adherent method.10
A 3 cm section of the umbilical cord was collected in
aseptic conditions, digested using a collagen enzyme,
then filtrated and centrifuged before the cells were col-
lected for culture. When the cells reached 80% conflu-
ence, they were digested and centrifuged, and added
to a new culture apparatus. Cells were maintained in
Nan et al
Keypoints
• 
Human umbilical cord mesenchymal stem cells
(hUCMSCs) are highly effective for repairing skin
wounds when combined with tissue-engineering
materials.3,4
• 
Collagen is accepted in tissue engineering as a
drug carrier and raw material for building active
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3-dimensional scaffolds.5
• 
The study employed a collagen-fibrous protein
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composite material as a scaffold in combina-
tion with hUCMSCs to repair full-thickness skin
wounds.
a saturated humidity incubator at 37°C in a 5% CO2
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atmosphere to observe their growth.
Preparation of collagen. Based on past studies, a
collagen sponge scaffold was created by freeze-drying
a collagen dispersion after it had been centrifuged,
stirred, and poured into a flat, stainless steel mold.5 The
lyophilized collagen sponge scaffold was soaked in a
0.3% glutaraldehyde solution with a pH of 8.4 for 2
hours, making the bracket for collagen cross-linking,
and washed in deionized water 10 times for adequate
removal of the residual glutaraldehyde from the brack-
et. Samples were then precooled in a -80°C ultra-low
temperature freezer, and returned to the freeze-drying
apparatus for a second round of freeze-drying, to pro-
duce the collagen scaffold.5
Preparation of collagen-fibrin glue composite ma-
terials. Fibrinogen was evenly mixed by magnetic stir-
ring, thrombin was added, and the resulting mixture
was added to the prepared collagen scaffold immedi-
ately, along with fibrin glue. A collagen mass ratio of 1:1
to 1:3 was used to prepare the collagen-fibrin double
composite scaffold materials. The prepared scaffold
Keypoints
• 
Human umbilical cord mesenchymal stem cells
(hUCMSCs) were cultured using the tissue ex-
plants adherent method10 after obtaining consent
from the donating mother.
• A collagen mass ratio of 1:1 to 1:3 was used to
prepare the collagen-fibrin double composite scaf-
fold materials.
• 
Third-generation hUCMSCs were prepared and
placed on the sterile collagen-fibrin double mem-
brane and incubated for 24 hours. The resulting
composite hUCMSCs collagen-fibrin composite
material was used for this study.
Vol. 27, No. 5 May 2015 135
 Nan et al

materials were cut into circles with a diameter of

14 mm, placed into individual polyethylene film bags,
and sealed after gamma radiation sterilization (60 Co,
2 × 106 rad).
Preparation of full-thickness skin wounds in mice
models. Healthy, clean male mice (n = 24, weighing
25 g - 30 g) were provided by the Xinxiang Medical

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College Laboratory Animal Center. The dorsi of the
mice were shaved, and the mice were anesthetized via

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intramuscular injection of ketamine, followed by regu-
lar disinfection. Circular full-thickness sections of skin,
1 cm in diameter, were excised from both sides of the
back, and wounds were stanched with sterile gauze.
Figure 1. Morphological character of human umbilical cord
Human umbilical cord mesenchymal stem cell sur-

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mesenchymal stem cells.
face antigen identification. Flow cytometry was used
to detect mesenchymal stem cell phenotypes, CD90,
Keypoints CD105, CD73, CD34, CD45, HLA-DR, CD79a, and CD14.
• Circular full-thickness sections of skin, 1 cm in di-
ameter, were excised from both sides of the back of
24 healthy male mice weighing 25 g - 30 g.
• Mice were randomly divided into group A, a con-
PL Preparation of human umbilical cord mesenchy-
mal stem cell double-composite materials. Third-gen-
eration hUCMSCs were incubated in suspension at a
density of 2 × 107 cells/mL for 30 minutes, and washed
trol group treated with collagen-fibrin double- with phosphate buffered saline (PBS). Cells were re-
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layered membrane without human umbilical cord suspended in α-Dulbecco’s modified eagle medium
mesenchymal stem cells (hUCMSCs); group B, (α-DMEM) culture medium with 10% fetal bovine se-
treated with hUCMSCs combined with a collagen
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rum (FBS). Cells were placed on the prepared sterile

membrane; and group C, treated with hUCMSCs
collagen-fibrin double membrane and incubated for
combined with the collagen-fibrin double-layered
membrane. 24 hours. The resulting composite hUCMSCs collagen-
fibrin composite material was used in for this study.
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Figure 2. Surface markers of human umbilical cord mesenchymal stem cells.

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Animal grafting experiment. Mice models were ran-
domly divided into 3 groups: group A, a control group
treated with collagen-fibrin double-layered membrane
without hUCMSCs; group B, treated with hUCMSCs
combined with a collagen membrane; and group C,
treated with hUCMSCs combined with the collagen-
fibrin double-layered membrane.
Histological observation.The cicatrization of the in-
jured areas was observed and histologically examined.
Results were recorded and compared between groups.
Results
Culture and identification of human umbilical
cord mesenchymal stem cells. A small piece of umbili-
cal cord Huatong glue tissue was cultivated in a nu-
trient solution. Newly generated cells could be seen
after 5-7 days. The cells grew, adhering to the wall, and
were shuttle-shaped or polygons; their distribution was
mostly non-uniform (Figure 1). Examination by flow
cytometry showed the phenotype of the hUCMSCs
conformed with the phenotypic characteristics of
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mesenchymal stem cells: CD90(+), CD105(+), CD73(+),
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CD34(-), CD45(-), HLA-DR(-), CD79a(-), CD14(-) (Fig-
ure 2). The following percentages of antigen expres-
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sion were observed: CD90, CD105, CD73 ≥ 95%; CD34,
CD45, HLA-DR, CD79a, CD14 ≤ 2%.
Scanning electron microscopy images of scaffold.
Figure 3 shows a cross-section of the double-layered
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Figure 3. Scanning electron microscopy images of scaffold. (A) Collagen layer of scaffold; (B) fibrin layer of scaffold.
Nan et al
collagen scaffold, stent microporous interlinked with
each other. The 3D structure was clear, and the aper-
ture of the scaffold material was measured to be 100
µm and 200 µm by scanning electron microscopy. Fig-
ure 3 shows the cross-section of the fibrin glue com-
posite material; the line is a branch of the 3D reticular
structure.
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Full-thickness skin wound healing. Figure 4 shows
the wound healing rates of the 3 groups. At 5 days, the
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wound healing percentages of the 3 groups were as fol-
lows: group A, 25.76% ± 2.91%; group B, 56.32% ± 3.47%;
and group C, 62.62% ± 6.27%. The healing effect in
group C was higher than in the group A. At 10 days, the
wound healing percentages of the 3 groups were as fol-
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lows: group A, 48.36% ± 3.75%; group B, 68.79% ± 4.78%;
and group C, 88.47% ± 7.14%. This result showed the
healing effect of group C was better than groups A or B.
The wound healing percentages of the 3 groups at 15
days were 62.72% ± 5.42% for group A; 81.27% ± 4.26%
for group B; and 96.14% ± 4.56% for group C. In group
C, the wounds had almost completely healed. The differ-
ences between the 3 groups were significant (P < 0.05).
Full-thickness skin wounds, histological examina-
tion. After 2 weeks, the wounds in group C had almost
completely healed. In group A, the wounds were par-
tially covered with granulation tissue and ingrowth of
fibroblasts had formed granulation tissue (Figure 5A).
In wounds treated with hUCMSCs combined with the
Vol. 27, No. 5 May 2015 137
 Nan et al

Discussion
Wound healing is an
intricate process in which
the skin (or other organ
tissue) repairs itself after
injury.11 Although dam-
aged skin tissue has a ca-

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pacity for self-repair, in
certain large areas of skin

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trauma and chronic skin
wounds this is limited,12,13
and human intervention
is necessary to promote
wound healing. In skin tis-

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sue engineering research,
wounds are often treated
with stem cells combined

PL with other materials.14 This

method does not just pro-
mote wound healing, but
also plays a long-term re-
pair role in the healing pro-
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cess, because the cytokines
are released from tissue-en-
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gineering materials.15,16 In
Figure 4. Wound healing rate of 3 groups.
this study, collagen and fi-
hUCMSCs: human umbilical cord mesenchymal stem cells
brin were combined to pre-
pare a new collagen-fibrin
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Keypoints double-layered membrane which produced highly ef-

• In this study, collagen and fibrin were combined ficient wound healing.
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to prepare a new collagen-fibrin double-layered The collagen-fibrin double-layered membrane

membrane which produced highly efficient wound showed a good 3D conformation. A cross-section of
healing. the collagen-fibrin scaffolds exhibited a highly porous
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To detect the wound-healing rate after treatment

•  and interconnected structure with an even pore size
with the different methods, histological examina- and a clear 3D structure. A cross-section of the com-
tion for new skin tissue was performed by hematox- pact fibrin network was made up of branched fibrin
ylin-eosin staining. fibers. The presence of the fibrin layer is beneficial to
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add mechanical strength to the collagen, and thus has

collagen membrane or the collagen-fibrin double-lay- broad clinical application prospects.The current study
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ered membrane (group B and C), a good combination
between the treatment material and wound edge was
formed. The basal layer, stratum spinosum, stratum gran-
ulosum, stratum lucidum, and stratum corneum could
clearly be observed. In group C, the reepithelialization
process was rapid. Collagen was evenly distributed un-
der the neonatal keratinocytes. The basal layer was flat
and closely combined with the collagen, and formation
of epithelization was observed by histological examina-
tion (Figures 5B and 5C).
138 WOUNDS® www.woundsresearch.com
used 3 experimental groups for treatment of full-thick-
ness skin wounds in mice to investigate the effect of
wound healing using stem cells combined with the
double-layered material. Group A was a control group
treated with a collagen-fibrin double-layered material
without hUCMSCs; group B was treated with hUC-
MSCs combined with a single-layer collagen material;
and group C was treated with hUCMSCs combined
with the collagen-fibrin double-layered material. To de-
tect the wound-healing rate after treatment with the

Figure 5. Histologic findings of skin tissue. (A) Control group (group A); (B) human umbilical cord mesenchymal stem cells
(HUCMSCs) combined with collagen membrane treated group (group B); (C) HUCMSCs combined with collagen-fibrin double
layer membrane treated group (group C).
different methods, histological examination for new
skin tissue was performed by hematoxylin-eosin stain-
ing. When compared with group A 5 days after surgery,
groups B and C showed improved wound healing (dif-
ferences were significant); 10 days after surgery, group
C was better than the other 2 groups (differences
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were significant); and after 15 days, the wounds had
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almost completely healed in group C, which hastened
the reepithelialization process. Collagen was evenly
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distributed under the neonatal keratinocytes. The bas-
al layer was flat and combined closely with the col-
lagen, and epithelization was observed by histological
examination. These results indicate that the collagen-
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fibrin double-layered membrane may be used as a skin
substitute. If the material is combined with stem cells,
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such as hUCMSCs, it could play a long-term repair role
in wound healing, because the cytokines are released
from tissue-engineering materials.
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Umbilical cord mesenchymal stem cells are a popu-
lation of multipotent cells.Wound healing can be accel-
erated using hUCMSCs treatments.17 The mechanism
of wound repair involves many aspects.Early covering
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of the wound with a dressing material can help pre-
vent infection, and stem cells play an important role
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in accelerated wound healing by promoting cell dif-
ferentiation, homing, and paracrine secretion.18,19 The
current study’s results showed the presence of a fibrin
layer is beneficial for mechanical strengthening of col-
lagen, and the collagen-fibrin double-layered material
combined with hUCMSCs improved wound healing.
It appears stem cells grow well in the double-layered
material, and that the material encourages the stem
cells to release cytokines. This mechanism is worthy
of further research.
Nan et al
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Keypoints
• If a collagen-fibrin double-layered material is com-
bined with stem cells, such as human umbilical
cord mesenchymal stem cells (hUCMSCs), it could
play a long-term repair role in wound healing, be-
cause the cytokines are released from tissue-engi-
neering materials.
• The current study’s results showed the presence of
a fibrin layer is beneficial for mechanical strength-
ening of collagen, and the collagen-fibrin double-
layered material combined with hUCMSCs improved
wound healing.
Conclusion
The collagen-fibrin double-layered material de-
veloped for this study could be used as a tissue en-
gineering material for promoting wound healing. The
double-layered material adds mechanical strength and,
when combined with hUCMSCs, the material improved
wound healing. These results widened ideas of tissue-
engineering skin research.
References
1. Biedermann T, Boettcher-Haberzeth S, Reichmann E.
Tissue engineering of skin for wound coverage. Eur J
Pediatr Surg. 2013;23(5):375-382.
2. Groeber F, Holeiter M, Hampel M, Hinderer S, Schenke-
Layland K. Skin tissue engineering--in vivo and in vitro
applications. Adv Drug Deliv Rev. 2011;63(4-5):352-
366.
3. Zhang Y, Hao H, Liu J, Fu X, Han W. Repair and regen-
eration of skin injury by transplanting micropar-
ticles mixed with Wharton’s jelly and MSCs from the
human umbilical cord. Int J Low Extrem Wounds.
Vol. 27, No. 5 May 2015 139
 Nan et al

2012;11(4):264-270. wound healing by local application of mesenchymal

4. Biazar E, Keshel SH. The healing effect of stem cells
loaded in nanofibrous scaffolds on full thickness skin
defects. J Biomed Nanotechnol. 2013;9(9):1471-1482.
5. Chen H, Chen H, Liu L, Yuan P, Zhang Q. The study of
improved controlled release of vincristine sulfate from
collagen-chitosan complex film. Artif Cells Blood Sub-
stem cells derived from human umbilical cord blood.
Wound Repair Regen. 2010;18(5):506-513.
18. Liu L, Yu Y, Hou Y, et al. Human umbilical cord mesen-
chymal stem cells transplantation promotes cutane-
ous wound healing of severe burned rats. PloS One.
2014;9(2):e88348. doi: 10.1371/journal.pone.0088348.

E
stit Immobil Biotechnol. 2008;36(4):372-385. 19. Yang S, Huang S, Feng C, Fu X. Umbilical cord-derived
6. de la Puente P, Ludena D. Cell culture in autologous mesenchymal stem cells: strategies, challenges, and

AT
fibrin scaffolds for applications in tissue engineering. potential for cutaneous regeneration. Front Med.
Exp Cell Res. 2014;322(1):1-11. 2012;6(1):41-47.
7. Brouwer KM, van Rensch P, Harbers VE, et al. Evaluation
of methods for the construction of collagenous scaf-
folds with a radial pore structure for tissue engineer-

IC
ing. J Tissue Eng Regen Med. 2011;5(6):501-504.
8. Whelan D, Caplice NM, Clover AJ. Fibrin as a delivery
system in wound healing tissue engineering applica-

9.
tions. J Control Release. 2014;196:1-8.
PL
Lai VK, Frey CR, Kerandi AM, Lake SP, Tranquillo RT, Bar-
ocas VH. Microstructural and mechanical differences
between digested collagen-fibrin co-gels and pure col-
lagen and fibrin gels. Acta Biomater. 2012;8(11):4031-
U
4042.
10. Harris DT. Umbilical cord tissue mesenchymal stem
D

cells: characterization and clinical applications. Curr

Stem Cell Res Ther. 2013;8(5):394-399.
11. Zhong SP, Zhang YZ, Lim CT. Tissue scaffolds for skin
wound healing and dermal reconstruction. Wiley Inter-
T

discip Rev Nanomed Nanobiotechnol. 2010;2(5):510-

525.
O

12. Rea S, Giles NL, Webb S, et al. Bone marrow-derived

cells in the healing burn wound--more than just inflam-
mation. Burns. 2009;35(3):356-364.
N

13. Falanga V. Wound healing and its impairment in the dia-

betic foot. Lancet. 2005;366(8498):1736-1743.
14. Hayward CJ, Fradette J, Morissette Martin P, Guignard R,
Germain L, Auger FA. Using human umbilical cord cells
O

for tissue engineering: a comparison with skin cells.

Differentiation. 2014;87(3-4):172-181.
D

15. Li H, Fu X, Ouyang Y, Cai C, Wang J, Sun T. Adult bone-

marrow-derived mesenchymal stem cells contribute
to wound healing of skin appendages. Cell Tissue Res.
2006;326(3):725-736.
16. Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu
H. Mesenchymal stem cells are recruited into wound-
ed skin and contribute to wound repair by transdif-
ferentiation into multip skin cell type. J Immunol.
2008;180(4):2581-2587.
17. Luo G, Cheng W, He W, et al. Promotion of cutaneous

140 WOUNDS® www.woundsresearch.com