Development of Acellular Dermis From Porcine Skin Using Periodic Pressurized Technique

Development of Acellular Dermis From Porcine Skin UsingPeriodic 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

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

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

Received 26 February 2007; revised 23 May 2007; accepted 25 June 2007Published 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 perio-

dic 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 enzyme-

substrate 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: 210–219, 2008

Keywords: extracellular matrix; wound healing; in vivo; tissue engineering; biocompat-

ibility/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.1–4 Because of limited supply of cadaver skin, porcine

skin is widely used to produce ADM for biomedical appli-

cations. 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.5–7

Biological scaffolds derived from decellularized tissues

and organs have been successfully used in both preclinical

animal studies and human clinical applications.8 The effi-

ciency of cell removal from tissues is dependent on the ori-

gin of tissues and specific 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 insufficient to achieve complete decellulariza-

tion.11 Decellularization technique by enzyme such as tryp-

sin 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 (2–28 days at

Correspondence to: S. Damrongsakkul (e-mail: [email protected])Contract grant sponsor: National Research Council of ThailandContract grant sponsor: Affair of Commission for Higher Education-CU Gradu-

ate Thesis Grant

' 2007 Wiley Periodicals, Inc.

210

room temperature) resulting in a disruption of collagen

structure. Walter et al.13 reported that dispase-Triton treat-

ment was more efficient 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 eo-

sin (H&E) staining. However, their method was complex

and a lengthy incubation of both enzymes was required.

In this study, a modified protocol for decellularization of

porcine skin was developed. The enzyme decellularized

process was employed incorporated with a periodic pressur-

ized technique to enhance the cell removal efficiency and

reduce incubation time. The effects of enzyme type (trypsin

and dispase II) and pressurized period on the efficiency of

cell removal quantitatively, and characteristics of ADM

were investigated.

MATERIALS AND METHODS

Materials

Fresh porcine skin from 2 to 3 months old pig was

obtained from local slaughterhouse. Dispase II from Bacil-

lus 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 soak-

ing in distilled water for 24 h.

Fat Removal Process. De-epidermis skin was freeze

thawed followed by soaking in chloroform/methanol mix-

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 influence of pressurized periods,

enzyme refreshment (change of enzyme solution), and type

of enzyme solution (trypsin and dispase II) on the effi-

ciency of cell removal. De-fat skin was divided into six

groups (CR1–CR6). The treatment for each group was sum-

marized in Table I. CR1–CR5 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. CR3–CR6 groups

were treated in a periodic pressurized process as schemati-

cally 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 25–30 s, and held for a desired

period before a burst release to 1 bar within 25–30 s. This

was defined as one pressurized period. The periodic pres-

surized 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 decellu-

larized process. For CR2–CR6 groups, enzyme solution

was also refreshed as indicated in Table I. In addition,

0.02% (w/v) sodium azide was added in every treatment

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

Group

Enzyme

Type

Enzyme

Refreshment

Number of

Pressurized Periods

Total Treatment

Time (h)

CR1 Trypsin – – 24

CR2 Trypsin Every 6 h – 24

CR3 Trypsin Every 6 h 24 12

CR4 Trypsin Every 1.5 h 36 3

CR5 Trypsin Every 45 min 36 3

CR6 Dispase II Every 1.5 h 36 3

Figure 1. Schematic diagram of periodic pressurized process.

211DEVELOPMENT OF ACELLULAR DERMIS

Journal of Biomedical Materials Research Part B: Applied Biomaterials

group to prevent bacterial growth. After the treatment,

ADM was then extensively washed and stored at 2208C.

ADM Characterization

DNA Assay. Percentage of cell removal was determined

by measuring the amount of DNA using Hoechst 33258 flu-

orescence 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 saline–sodium citrate

(SSC) was added to lyses cells. The mixture was homoge-

nized and incubated at 558C for 6 h with occasional mixing

to digest the attached cells. After the incubation, centrifu-

gation was performed at 2236g for 10 min. The mixed so-

lution was transferred to black 96-well plates and

fluorescence intensity was determined with a fluorescence

spectrophotometer (VICTOR3 Perkin–elmer 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 fibroblast cells. The percentage of cell re-

moval was calculated as follows.

Cell removal ð%Þ ¼ CR � CS

CR

� �3 100 ð1Þ

where CR represents the number of cells in the reference

and CS represents number of cells in ADM samples.

Morphology. Morphology of ADM samples and fresh

porcine skin were observed using scanning electron micro-

scope (SEM, Joel JSM 5400).

Histological Examinations. ADM samples were first

dehydrated with an increasing concentration series of alco-

hol and then embedded in paraffin. Paraffin-embedded

ADMs were sectioned at a thickness of 5 lm. After remov-

ing the paraffin, samples were stained with hematoxylin

and eosin, then the samples were examined using light mi-

croscopy.

In Vivo Study

ADM obtained from CR5 (trypsin model) and CR6 treat-

ments (dispase II model) were freeze-dried and steriled

using ethylene oxide treatment prior to subcutaneous im-

plantation onto the back of 4-week-old female Wistar rats

(National Laboratory Animal Center, Nakornpathom, Thai-

land). All animal experiments were performed in accord-

ance to the Home office guidelines on the scientific use ofanimals (Scientific procedures, Act 1986). The implanted

samples were removed at 1-week, 2-week, and 4-week

postoperatively (n 5 3). The retrieved sample were fixed in

10% (v/v) formalin for at least 3 days prior to the histolog-

ical and scanning electron microscopic (SEM) examina-

tions.

RESULTS

Cell Removal

The percentage of cell removal from each treatment, deter-

mined 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 refresh-

ment (CR2), however, percentage of cell removal was

Figure 2. Treatment pattern of periodic pressurization for CR3–CR6

treatments.

Figure 3. Percentage of cell removal from porcine skin determined

by DNA assay. * Represented a significant difference at p \ 0.01,** represented a significant difference at p\ 0.05.

212 PRASERTSUNG ET AL.


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

removal in a short-time treatment under periodic pressur-

ization (12 h, CR3) was much higher than those with long

treatment time with continuous stirring (24 h, CR2). This

indicated that the periodic pressurized process was able to

reduce time of cell removal process and progressively

improve the efficiency of cell removal by 34%. When the

incubation time of enzyme was decreased from 12 to 3 h

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



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 influence of enzyme refreshing

frequency on the efficiency of cell removal. The percentage

of cell removal significantly increased when enzyme solu-

tion 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 re-

moval for trypsin and dispase II, respectively).

Morphology and Histological Examinationof 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(d–g). Apparently, the structural

pattern of collagen fiber still remained in these ADM sam-

ples. In comparison of Figure 4(e–g) with Figure 4(a), the

collagen structure of ADM samples looked to be more fi-

brous than that of fresh porcine skin.

To confirm the cell removal, the ADM from CR4, CR5,

and CR6 treatments were examined using histological ex-

amination (Figure 5). Histological photograph of porcine

skin without any treatments served as the control [Figure

5(a)] exhibited a dense structure of collagen fiber (pink

color) with a lot of fibroblast cells-bound (bluish purple

color indicated by arrows). For the samples of CR4–CR6

groups shown in Figure 5(b–d), fewer amount of fibroblast

cells were observed. There were much fewer fibroblast

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 fibroblast 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 implanta-

tion, neo-capillaries were observed in both models. It was

noted that no significant change in the appearance of

implanted ADM was noticed at 2-week postoperatively. Af-

ter 4-week of implantation, some parts at the edge of

implanted ADM disappeared markedly in both models. In

Figure 5. Histological photographs of fresh porcine skin and ADM samples stained with hematoxy-

lin and eosin (H & E) (3400 magnification): (a) fresh porcine skin, (b) CR4, (c) CR5, (d) CR6 (arrows

indicated fibroblast cells). [Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]



addition, the ADM samples from both models were filled

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 defined by C1, C2, and C3 as shown in Figure

7. The C1 section represented region of tissue at the sam-

ple edge, C2 at the 0.25 cm from the edge, and C3 at the

center of implanted samples. Figure 8 shows the SEM pho-

tographs of the implanted ADM from CR5 treatment. The

results showed that cells adhered at the edge of ADM dur-

ing the first week. After that, cells could further infiltrate

into ADM. After 4-week implantation, a number of cells

could be found at the center of the ADM samples. Similar

phenomena of cell infiltration could be observed in the

case of CR6 samples, as illustrated in Figure 9. These

results indicated that a number of cells could infiltrate 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-

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 af-

ter 2-week, (c) CR5 after 4-week, (d) CR6 after 1-week, (e) CR6 after 2- week, and (f) CR6 after 4-week. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.

com.]



and 4-week postoperatively. After 2-week implantation

[Figure 10(a,b)], inflammatory and fibroblast cells were

able to infiltrate into the open space of the samples. The

inflammatory cells are mainly lymphocyte. No evidence of

polymorphonuclear leukocytes for other acute inflammatory

was noticed. The depth of cell infiltration into both ADM

samples was similar. In addition, neo-collagen fibril 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 filled with cells.

There were still some inflammatory cells presented in acel-

lular tissue. Instead, fibroblast (migrating from the host tis-

sue), neo-collagen fibril, neo-capillaries, and red blood cell

were observed. The depth of cell infiltration into ADM was

significantly greater than that observed at 2-week implanta-

tion. In addition, the number of cells infiltrated into the

samples of CR6 was much greater than that of CR5.

DISCUSSION

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 com-

bined enzymatic method to mechanical method using peri-

odic pressurization to enhance the efficiency of cell

removal and reduce enzyme incubation time.

The use of mechanical agitation with enzymatic treatment

was insufficient to achieve a complete cell removal as shown

in the CR1 treatment. Since porcine skin has a tightly struc-

ture, the access of enzyme solution into the dermis is diffi-

cult. 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 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.

Figure 7. Schematic diagram of sectional direction on implanted

ADM samples prior to cell infiltrated observation of cell infiltration.



number of protease inhibitor can be released from disrupted

cells, resulting in the inhibition of enzyme-substrate interac-

tion.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 re-

moval. 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 enzyme–substrate interaction,

though the activity of enzyme after 12 h of incubation signif-

icantly decreased as mentioned previously. These results

indicated that the efficiency of cell removal using periodic

pressurization for a short time was much greater than the

continuous stirring for a long time. When the pressurized pe-

riod was increased from 24 to 36 periods and the enzyme

incubation time was shorten from 12 to 3 h (CR4), the per-

centage of cell removal was further increased. Moreover, in

CR5 treatment, the higher percentage of cell removal was

achieved with more frequent enzyme refreshment. This sug-

gests that more pressurized periods within a short time could

improve the efficiency 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 fibroblast 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 con-

sistent with the highest percentage of cell removal deter-

mined 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 fibro-

blast 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 confirmed that after trypsin treatment for 24 h, collagen

degradation was observed in dermis structure [Figure 4(b,c)].

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 cmfrom the edge, and C3 is at the center of implanted samples.



When the enzyme incubation time was shorten, the struc-

tural pattern of collagen fiber less degraded. This proved

that the treatment with periodic pressurized method was

beneficial 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 mod-

els were not significantly changed. After 4-week implanta-

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

the ADM samples and the degradation of porcine ADM

were observed (Figure 6). It was noted that, infiltration of

cells was accompanied with degradation of acellular tis-

sue.16 The implants provoked cellular responses which led

to invasion by various cells such as macrophages, and

fibroblasts.17,19 As appeared in the SEM micrographs (Fig-

ures 8 and 9), after 1-week implantation, there was poor

cell adhesion on the surface of ADM, possibly due to los-

ing of some cell-adhesion ligands during the preparation

Figure 10. Histological photographs of CR5 (trypsin model) and CR6 (dispase II model) implanted

ADM at 2- and 4-week postoperatively (3100 magnification): (a) CR5 at 2-week, (b) CR6 at 2-week, (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 fibroblasts with newly formed capillaries. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]



process. Cell adhesion improved after 2 weeks of implanta-

tion. The absence of cell at the center of tissues probably

results from tightly compacted network of collagen bun-

dles, blocking the migration of cells.5 After 4-week implan-

tation, a lot of cells infiltrated into the center of ADM

prepared from both models (Figures 8 and 9, C3). SEM

results corresponded to histological examinations, from

which inflammatory cells and fibroblast cells were pre-

sented in both ADM samples (Figure 10). Liang et al.18

reported that, once inflammatory cells infiltrated into ADM

scaffolds, proteolytic enzymes such as collagenase secreted

by macrophages started to degrade the original porcine tis-

sue fibrils. This could be one possible mechanism of degra-

dation occurred allowing fibroblasts from the host tissue

(rat) to migrate into the ADM. Consequently, more neo-

collagen fibrils were produced. Moreover, cell can pene-

trate 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

infiltration.

CONCLUSION

In our study, the protocol of decellularized porcine dermis

was developed. Periodic pressurized technique can be effec-

tively utilized in decellularized process, providing advan-

tages 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 fibroblast 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 fibro-

blasts from the host tissue migrated into the ADM prepared

using either trypsin or dispase II and neo-collagen fibrils as

well as neo-capillaries were produced.

The animal experiments have been approved by the EthicsCommittee of the Faculty of Medicine, Chulalongkorn University,Bangkok, Thailand. The authors are grateful to Prof. YasuhikoTabata, Kyoto University, and Mr. Preecha Sangtherapitikul fortheir generous advice.

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Development of Acellular Dermis From Porcine Skin Using Periodic Pressurized Technique