Development of Acellular Dermis From Porcine Skin Using

Periodic Pressurized Technique

Isarawut Prasertsung,1 Sorada Kanokpanont,1 Tanom Bunaprasert,2 Voranuch Thanakit,3
Siriporn Damrongsakkul1
1

Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand

Department of Otolaryngology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

Received 26 February 2007; revised 23 May 2007; accepted 25 June 2007

Published online 12 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30938

Abstract: In this work, a new method for producing acellular dermis (ADM), a natural
scaffold used for dermal replacement, from porcine skin was developed. Fresh porcine skin
from local slaughterhouse was dehaired by sodium sulphide following by epidermis removal
using glycerol. After fat removal by chloroform/methanol (2/1 v/v) solvent, cellular
components were removed using enzymatic treatment incorporated with a periodic
pressurized technique. The effects of enzyme type (trypsin and dispase II) and periodic pressurized conditions on the efficiency of cell removal were investigated. When periodic
pressure was applied, enzymatic treatment time could be shorten since the enzyme solution
was able to penetrate into tight dermis. As a result, cells could be easily removed from porcine
skin as noticed quantitatively by DNA assay and qualitatively by H&E staining. When enzyme
refreshment was introduced into the decellularized process, the percentage of cell removal was
further enhanced. This ensured that no inhibitions effect from the removed cells on enzymesubstrate interaction. Moreover, short-time enzymatic treatment with periodic pressurized
technique could prevent the disruption of dermal structure, as observed by SEM. Dispase II
can be used to remove cell better than trypsin in the periodic pressurized technique. However,
in vivo study indicated that numerous fibroblast from the host tissue infiltrated into ADM
prepared using both enzymes. Neo-collagen and neo-capillaries were produced in both
implanted ADMs. The result elucidated that the use of periodic pressurized technique with
enzymatic treatment has a high potential to be a new method to produce ADM for skin tissue
engineering. ' 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 85B: 210219, 2008
Keywords: extracellular matrix; wound healing; in vivo; tissue engineering; biocompatibility/soft tissue

INTRODUCTION
Acellular dermis is a natural scaffold, which has been
widely used as dermal replacement. It is produced from
cadaver or animal skin such as porcine, rat, and bovine
skin.14 Because of limited supply of cadaver skin, porcine
skin is widely used to produce ADM for biomedical applications. The similarity to human skin, the mature collagen
bundles, and the porous nature of porcine dermis are all
favorable features for a potential dermal substitute.57

Correspondence to: S. Damrongsakkul (e-mail: siriporn.d@chula.ac.th)

Contract grant sponsor: National Research Council of Thailand
Contract grant sponsor: Affair of Commission for Higher Education-CU Graduate Thesis Grant
' 2007 Wiley Periodicals, Inc.

210

Biological scaffolds derived from decellularized tissues

and organs have been successfully used in both preclinical
animal studies and human clinical applications.8 The efciency of cell removal from tissues is dependent on the origin of tissues and specic methods used. Each treatment
affects the biochemical composition, tissue ultra structure,
and mechanical behavior of remaining extracellular matrix
(ECM) scaffold.9,10 Various methods employed to remove
the immunogenic cellular constituents have been reported.
The physical treatments such as freeze-thawing method can
be used to remove cellular component, but these treatments
are generally insufcient to achieve complete decellularization.11 Decellularization technique by enzyme such as trypsin is one of the most commonly used. Oliver et al.12
reported that trypsin-treated porcine dermis was not
rejected upon transplantation. Their method, however,
required a lengthy incubation with trypsin (228 days at

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DEVELOPMENT OF ACELLULAR DERMIS

TABLE I. Cell Removal Treatment of Procine Skin (CR1-CR6 Groups)

Group
CR1
CR2
CR3
CR4
CR5
CR6

Enzyme
Type
Trypsin
Trypsin
Trypsin
Trypsin
Trypsin
Dispase II

Enzyme
Refreshment
Every
Every
Every
Every
Every

6h
6h
1.5 h
45 min
1.5 h

room temperature) resulting in a disruption of collagen

structure. Walter et al.13 reported that dispase-Triton treatment was more efcient to remove cell than NaCl-SDS
treatment. However, ADM components are generally more
abundant in NaCl-SDS ADM than dispase-Triton ADM.
The former may more readily support cell attachment and
proliferation. A cell removal process on xenogeneic such as
porcine skin was reported by Chen et al.14 Trypsin, dispase
II, and SDS solution were utilized in the decellularized
method. They claimed that the cells could be completely
removed, qualitatively observed from hematoxylin and eosin (H&E) staining. However, their method was complex
and a lengthy incubation of both enzymes was required.
In this study, a modied protocol for decellularization of
porcine skin was developed. The enzyme decellularized
process was employed incorporated with a periodic pressurized technique to enhance the cell removal efciency and
reduce incubation time. The effects of enzyme type (trypsin
and dispase II) and pressurized period on the efciency of
cell removal quantitatively, and characteristics of ADM
were investigated.
MATERIALS AND METHODS
Materials

Number of
Pressurized Periods

Total Treatment
Time (h)

24
36
36
36

24
24
12
3
3
3

ture (2/1 v/v) at room temperature for 2 h. The dermis was

then extensively washed with PBS buffer for 6 h.
Cell Removal Process.
This experiment was
designed to study the inuence of pressurized periods,
enzyme refreshment (change of enzyme solution), and type
of enzyme solution (trypsin and dispase II) on the efciency of cell removal. De-fat skin was divided into six
groups (CR1CR6). The treatment for each group was summarized in Table I. CR1CR5 groups were treated in 1%
(w/v) trypsin solution while 0.24% (w/v) dispase II solution
was used in CR6 treatment. CR1 and CR2 treatments were
performed under continuous stirring. CR3CR6 groups
were treated in a periodic pressurized process as schematically shown in Figure 1. In brief, the pressure of the system
containing de-fat skin in enzyme solution was suddenly
increased to 8 bar within 2530 s, and held for a desired
period before a burst release to 1 bar within 2530 s. This
was dened as one pressurized period. The periodic pressurized conditions, that is number of pressurized periods,
and period time, were varied as shown in Figure 2. This
therefore results in different total treatment time of decellularized process. For CR2CR6 groups, enzyme solution
was also refreshed as indicated in Table I. In addition,
0.02% (w/v) sodium azide was added in every treatment

Fresh porcine skin from 2 to 3 months old pig was

obtained from local slaughterhouse. Dispase II from Bacillus polymyxa (0.5 U/mg) was purchased from Roche
Applied Science (German). Trypsin from hog pancreas (95
U/mg) and Hoechst dye 33258 were obtained from Fluka
(Germany).
ADM Preparation
Dehair and De-epidermis Process. Fresh porcine skin
was cut in pieces and washed thoroughly with water. After
the complete cleaning, subcutaneous fat was excised off.
To remove hairs, the skin was treated in 20% (w/v) sodium
sulphide. To remove epidermis, dehaired skin was soaked
in 1M of NaCl solution at room temperature for 24 h and
85% (w/v) glycerol solution for 14 days, followed by soaking in distilled water for 24 h.
Fat Removal Process.
De-epidermis skin was freeze
thawed followed by soaking in chloroform/methanol mixJournal of Biomedical Materials Research Part B: Applied Biomaterials

Figure 1. Schematic diagram of periodic pressurized process.

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PRASERTSUNG ET AL.

Morphology.
Morphology of ADM samples and fresh
porcine skin were observed using scanning electron microscope (SEM, Joel JSM 5400).
Histological Examinations.
ADM samples were rst
dehydrated with an increasing concentration series of alcohol and then embedded in parafn. Parafn-embedded
ADMs were sectioned at a thickness of 5 lm. After removing the parafn, samples were stained with hematoxylin
and eosin, then the samples were examined using light microscopy.
In Vivo Study

Figure 2. Treatment pattern of periodic pressurization for CR3CR6

treatments.

group to prevent bacterial growth. After the treatment,

ADM was then extensively washed and stored at 2208C.

ADM obtained from CR5 (trypsin model) and CR6 treatments (dispase II model) were freeze-dried and steriled
using ethylene oxide treatment prior to subcutaneous implantation onto the back of 4-week-old female Wistar rats
(National Laboratory Animal Center, Nakornpathom, Thailand). All animal experiments were performed in accordance to the Home ofce guidelines on the scientic use of
animals (Scientic procedures, Act 1986). The implanted
samples were removed at 1-week, 2-week, and 4-week
postoperatively (n 5 3). The retrieved sample were xed in
10% (v/v) formalin for at least 3 days prior to the histological and scanning electron microscopic (SEM) examinations.
RESULTS

ADM Characterization

Cell Removal

DNA Assay. Percentage of cell removal was determined

by measuring the amount of DNA using Hoechst 33258 uorescence dye.15 Each group of samples were grounded
with mortar in liquid nitrogen. One hundred milligrams of
ground sample were transferred to a micro centrifugal tube
and 1 mL of 0.02% (w/v) SDS in salinesodium citrate
(SSC) was added to lyses cells. The mixture was homogenized and incubated at 558C for 6 h with occasional mixing
to digest the attached cells. After the incubation, centrifugation was performed at 2236g for 10 min. The mixed solution was transferred to black 96-well plates and
uorescence intensity was determined with a uorescence
spectrophotometer (VICTOR3 Perkinelmer USA) at the
excitation and emission wavelengths of 355 and 460 nm,
respectively. Three measurements were repeated for each
sample. The untreated sample (no cell removal) was used
as a reference. The calibration curves were performed using
L929 mouse broblast cells. The percentage of cell removal was calculated as follows.

The percentage of cell removal from each treatment, determined by the DNA assay, is shown in Figure 3. It was
noticed that, the number of cell in ADM obtained from
trypsin treatment under stirring (CR1) was poorly removed
(33% cell removal). After the addition of enzyme refreshment (CR2), however, percentage of cell removal was



CR
CS
Cell removal %
3 100
CR

where CR represents the number of cells in the reference

and CS represents number of cells in ADM samples.

Figure 3. Percentage of cell removal from porcine skin determined

by DNA assay. * Represented a signicant difference at p \ 0.01,
** represented a signicant difference at p \ 0.05.
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DEVELOPMENT OF ACELLULAR DERMIS

213

Figure 4. SEM micrographs of freeze-dried porcine skin and ADM samples from different treatments: (a) fresh porcine skin, (b) CR1, (c) CR2, (d) CR3, (e) CR4, (f) CR5, and (g) CR6.

slightly increased (38% cell removal). Percentage of cell

removal in a short-time treatment under periodic pressurization (12 h, CR3) was much higher than those with long
treatment time with continuous stirring (24 h, CR2). This
Journal of Biomedical Materials Research Part B: Applied Biomaterials

indicated that the periodic pressurized process was able to

reduce time of cell removal process and progressively
improve the efciency of cell removal by 34%. When the
incubation time of enzyme was decreased from 12 to 3 h

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Figure 5. Histological photographs of fresh porcine skin and ADM samples stained with hematoxylin and eosin (H & E) (3400 magnication): (a) fresh porcine skin, (b) CR4, (c) CR5, (d) CR6 (arrows
indicated broblast cells). [Color gure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]

and pressurized period was increased from 24 to 36 periods

as compared CR3 with CR4 treatment, percentage of cell
removal was increased from 72 to 77%. Results from group
CR4 and CR5 revealed the inuence of enzyme refreshing
frequency on the efciency of cell removal. The percentage
of cell removal signicantly increased when enzyme solution was refreshed more frequently. Comparing the type of
enzymes; trypsin and dispase II used in the CR4 and CR6
treatments, respectively, the cell removal using dispase II
was more effective than using trypsin (77 and 92% cell removal for trypsin and dispase II, respectively).
Morphology and Histological Examination
of ADM Samples

SEM examination of ADM samples from each cell removal

treatment is shown in Figure 4. The SEM micrographs of
fresh porcine skin [Figure 4(a)] as a reference, showed a
dense porous structure. After treatment with trypsin for 24
h with continuous stirring, the dermal structures as shown
in Figure 4(b,c) were altered. They looked less porous than
the original porcine skin. This indicated that a lengthy
treatment time with trypsin-induced collagen degradation in
dermal structure. The effects of decreasing incubation time
and increasing pressurized periods on the structure of ADM
were observed in Figure 4(dg). Apparently, the structural
pattern of collagen ber still remained in these ADM sam-

ples. In comparison of Figure 4(eg) with Figure 4(a), the

collagen structure of ADM samples looked to be more brous than that of fresh porcine skin.
To conrm the cell removal, the ADM from CR4, CR5,
and CR6 treatments were examined using histological examination (Figure 5). Histological photograph of porcine
skin without any treatments served as the control [Figure
5(a)] exhibited a dense structure of collagen ber (pink
color) with a lot of broblast cells-bound (bluish purple
color indicated by arrows). For the samples of CR4CR6
groups shown in Figure 5(bd), fewer amount of broblast
cells were observed. There were much fewer broblast
cells found in the CR5 sample comparing to CR4 sample.
Moreover, complete removal of cells can be observed in
the samples of CR6 treatment. No broblast cells were
noticed among the dermal matrix.
In Vivo Study

Figure 6 shows a photograph of the samples retrieved at 1-,

2-, and 4-week postoperatively of CR5 (trypsin model) and
CR6 (dispase II model) groups. After 2-week of implantation, neo-capillaries were observed in both models. It was
noted that no signicant change in the appearance of
implanted ADM was noticed at 2-week postoperatively. After 4-week of implantation, some parts at the edge of
implanted ADM disappeared markedly in both models. In
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DEVELOPMENT OF ACELLULAR DERMIS

215

Figure 6. Photographs of retrieved ADM samples produced from CR5 (trypsin model) and CR6
(dispase II model) treatment at 1-, 2-, and 4-week postoperatively: (a) CR5 after 1-week, (b) CR5 after 2-week, (c) CR5 after 4-week, (d) CR6 after 1-week, (e) CR6 after 2- week, and (f) CR6 after 4week. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.
com.]

addition, the ADM samples from both models were lled

with numerous numbers of capillaries from the host tissue.
Scanning electron microscopy was employed to visualize
cell migration into the implanted ADM. The section of
samples was dened by C1, C2, and C3 as shown in Figure
7. The C1 section represented region of tissue at the sample edge, C2 at the 0.25 cm from the edge, and C3 at the
center of implanted samples. Figure 8 shows the SEM photographs of the implanted ADM from CR5 treatment. The
results showed that cells adhered at the edge of ADM durJournal of Biomedical Materials Research Part B: Applied Biomaterials

ing the rst week. After that, cells could further inltrate
into ADM. After 4-week implantation, a number of cells
could be found at the center of the ADM samples. Similar
phenomena of cell inltration could be observed in the
case of CR6 samples, as illustrated in Figure 9. These
results indicated that a number of cells could inltrate into
ADM samples prepared from both trypsin and dispase II
models.
Figure 10 showed the histological stained samples of
CR5 (trypsin model) and CR6 (dispase model) groups at 2-

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PRASERTSUNG ET AL.

lular tissue. Instead, broblast (migrating from the host tissue), neo-collagen bril, neo-capillaries, and red blood cell
were observed. The depth of cell inltration into ADM was
signicantly greater than that observed at 2-week implantation. In addition, the number of cells inltrated into the
samples of CR6 was much greater than that of CR5.
DISCUSSION
Figure 7. Schematic diagram of sectional direction on implanted
ADM samples prior to cell inltrated observation of cell inltration.

and 4-week postoperatively. After 2-week implantation

[Figure 10(a,b)], inammatory and broblast cells were
able to inltrate into the open space of the samples. The
inammatory cells are mainly lymphocyte. No evidence of
polymorphonuclear leukocytes for other acute inammatory
was noticed. The depth of cell inltration into both ADM
samples was similar. In addition, neo-collagen bril and
neo-capillaries were observed in all ADM samples. After
4-week implantation [Figure 10(c,d)], the results showed
that acellular tissues were degraded and lled with cells.
There were still some inammatory cells presented in acel-

In this study, the protocol of decellularized was developed

to produce ADM from porcine skin. Because of the fact
that porcine dermis has a tightly compacted network of
thick collagen bundles,5 the decellularized process combined enzymatic method to mechanical method using periodic pressurization to enhance the efciency of cell
removal and reduce enzyme incubation time.
The use of mechanical agitation with enzymatic treatment
was insufcient to achieve a complete cell removal as shown
in the CR1 treatment. Since porcine skin has a tightly structure, the access of enzyme solution into the dermis is difcult. In addition, at such a long incubating time, the activity
of trypsin greatly dropped by 60% after 12 h of incubation
(data not shown here). During the decellularized process, a

Figure 8. SEM micrographs of CR5 (trypsin model) implanted samples retrieved at 1-, 2-, and 4week postoperatively: C1 represents region at the sample edge, C2 is at the depth of 0.25 cm from
the edge, and C3 is at the center of implanted samples.
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DEVELOPMENT OF ACELLULAR DERMIS

217

Figure 9. SEM micrographs of CR6 (dispase II model) implanted samples retrieved at 1-, 2-, and
4-week postoperatively: C1 represents region at the sample edge, C2 is at the depth of 0.25 cm
from the edge, and C3 is at the center of implanted samples.

number of protease inhibitor can be released from disrupted

cells, resulting in the inhibition of enzyme-substrate interaction.9 From this information, the decellularized process using
trypsin was carried out by changing trypsin solution at every
6 h (CR2). The percentage of cell removal was slightly
increased, when enzyme refreshment was introduced. This
obviously implied that protease inhibitors released from the
disrupted cells was not a main reason for such a poor cell removal. While the periodic pressurized technique was
employed and the enzyme incubation time was shorten from
24 to 12 h (CR3), the percentage of cell removal was greatly
increased. Under pressurized condition, enzyme solution
could possibly penetrate into porcine skin more easily. A
greater increase in the percentage of cell removal probably
resulted from an increasing of enzymesubstrate interaction,
though the activity of enzyme after 12 h of incubation significantly decreased as mentioned previously. These results
indicated that the efciency of cell removal using periodic
pressurization for a short time was much greater than the
continuous stirring for a long time. When the pressurized period was increased from 24 to 36 periods and the enzyme
incubation time was shorten from 12 to 3 h (CR4), the percentage of cell removal was further increased. Moreover, in
CR5 treatment, the higher percentage of cell removal was
Journal of Biomedical Materials Research Part B: Applied Biomaterials

achieved with more frequent enzyme refreshment. This suggests that more pressurized periods within a short time could
improve the efciency of cell removal. A number of released
protease inhibitors were eliminated by enzyme refreshment,
ensuring high performance of enzyme reaction.
To compare the effect of trypsin and dispase II on the
decellularized process as represented in CR4 and CR6
treatment, dispase II could remove more cells than trypsin
(92 and 77% cell removal in CR6 and CR4, respectively).
Histological examinations revealed that broblast cells can
be found in the ADM prepared from trypsin model, but not
dispase II model (Figure 5). Complete removal of cells
observed in the samples of dispase II treatment was consistent with the highest percentage of cell removal determined by DNA assay. However, the quantitative cell
removal in this case did not reach 100%. The discrepancy
in DNA assay possibly due to the use of L929 mouse broblast in the calibration. Dispase II treatment of skin has
been shown to remove cellular components from the dermis
effectively.1
As reported previously, the lengthy incubation with
enzyme led to disruption of dermal structure.9 The present
study conrmed that after trypsin treatment for 24 h, collagen
degradation was observed in dermis structure [Figure 4(b,c)].

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Figure 10. Histological photographs of CR5 (trypsin model) and CR6 (dispase II model) implanted
ADM at 2- and 4-week postoperatively (3100 magnication): (a) CR5 at 2-week, (b) CR6 at 2week, (c) CR5 at 4-week, (d) CR6 at 4-week. Dash line represented an interface between the host
tissue (rat) and the implanted sample. Insight (3400) showed lymphocytes and few plasma cells
admixed with broblasts with newly formed capillaries. [Color gure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]

When the enzyme incubation time was shorten, the structural pattern of collagen ber less degraded. This proved
that the treatment with periodic pressurized method was
benecial not only effectively cell remove but also preserve
natural structure of dermal matrix in ADM.
From in vivo study of ADM derived from CR5 (trypsin
model) and CR6 (dispase II model), it was found that after
2-week implantation, capillaries were observed on both
models. However, porcine acellular tissues from both models were not signicantly changed. After 4-week implanta-

tion, the neo-capillaries from the host tissue were lled in

the ADM samples and the degradation of porcine ADM
were observed (Figure 6). It was noted that, inltration of
cells was accompanied with degradation of acellular tissue.16 The implants provoked cellular responses which led
to invasion by various cells such as macrophages, and
broblasts.17,19 As appeared in the SEM micrographs (Figures 8 and 9), after 1-week implantation, there was poor
cell adhesion on the surface of ADM, possibly due to losing of some cell-adhesion ligands during the preparation
Journal of Biomedical Materials Research Part B: Applied Biomaterials

DEVELOPMENT OF ACELLULAR DERMIS

process. Cell adhesion improved after 2 weeks of implantation. The absence of cell at the center of tissues probably
results from tightly compacted network of collagen bundles, blocking the migration of cells.5 After 4-week implantation, a lot of cells inltrated into the center of ADM
prepared from both models (Figures 8 and 9, C3). SEM
results corresponded to histological examinations, from
which inammatory cells and broblast cells were presented in both ADM samples (Figure 10). Liang et al.18
reported that, once inammatory cells inltrated into ADM
scaffolds, proteolytic enzymes such as collagenase secreted
by macrophages started to degrade the original porcine tissue brils. This could be one possible mechanism of degradation occurred allowing broblasts from the host tissue
(rat) to migrate into the ADM. Consequently, more neocollagen brils were produced. Moreover, cell can penetrate through sample derived from CR6 treatment (dispase
II model) deeper than that from CR5 treatment (trypsin
model). This might imply low antigenicity or relatively
loose structure of dispase II-derived ADM favorable to cell
inltration.
CONCLUSION
In our study, the protocol of decellularized porcine dermis
was developed. Periodic pressurized technique can be effectively utilized in decellularized process, providing advantages over typical enzymatic methods. The process uses
lesser time, removes more cells, and can preserve collagen
native structure compared with the conventional enzymatic
process. The performance of cell removal, both quantitatively
determined by DNA assay and qualitatively examined by
H&E, showed that there were very few broblast cells or
almost none left in the dermal matrix. Dispase II can be used
to remove cell better than trypsin in our pressurized cycle
technique. However, in vivo study indicated numerous broblasts from the host tissue migrated into the ADM prepared
using either trypsin or dispase II and neo-collagen brils as
well as neo-capillaries were produced.
The animal experiments have been approved by the Ethics
Committee of the Faculty of Medicine, Chulalongkorn University,
Bangkok, Thailand. The authors are grateful to Prof. Yasuhiko
Tabata, Kyoto University, and Mr. Preecha Sangtherapitikul for
their generous advice.

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