Use of an in Vitro Model of Tissue-Engineered Skin to Investigate the Mechanism of Skin Graft...

Use of an in Vitro Model of Tissue-Engineered Skin to

Investigate the Mechanism of Skin Graft Contraction

CAROLINE A. HARRISON, Ph.D.,1,4 FATMA GOSSIEL, B.Sc.,2 CHRISTOPHER M.LAYTON, Ph.D.,3 ANTHONY J. BULLOCK, Ph.D.,1 TIMOTHY JOHNSON, Ph.D.,5

AUBREY BLUMSOHN, Ph.D.,2 and SHEILA MACNEIL, Ph.D.1

ABSTRACT

Skin graft contraction leading to loss of joint mobility and cosmetic deformity remains a major clinicalproblem. In this study we used a tissue-engineered model of human skin, based on sterilized human adultdermis seeded with keratinocytes and fibroblasts, which contracts by up to 60% over 28 days in vitro, as amodel to investigate the mechanism of skin contraction. Pharmacologic agents modifying collagensynthesis, degradation, and cross-linking were examined for their effect on contraction. Collagen synthesisand degradation were determined using immunoassay techniques. The results show that skin contractionwas not dependent on inhibition of collagen synthesis or stimulation of collagen degradation, but wasrelated to collagen remodelling. Thus, reducing dermal pliability with glutaraldehyde inhibited the abilityof cells to contract the dermis. So did inhibition of matrix metalloproteinases and inhibition of lysyloxidase-mediated collagen cross-linking, but not transglutaminase-mediated cross-linking. In summary,this in vitro model of human skin has allowed us to identify specific cross-linking pathways as possiblepharmacologic targets for prevention of graft contracture in vivo.

INTRODUCTION

CONTRACTION IS A NORMAL PHYSIOLOGIC PHENOMENON that

reduces the area of a full-thickness wound. In loose-

skinned animals, the panniculus carnosus muscular layer

enables the skin to glide smoothly over the underlying tis-

sues.1 However, in man, in whom the skin is more firmly

attached, contraction leads to distortion of the surrounding

tissues with associated cosmetic deformity and limitation of

joint mobility. Application of a split-thickness skin graft to a

wound reduces wound contraction and hypertrophic scar-

ring.2 However, skin grafts also contract, leading to limita-

tion of joint mobility and causing cosmetic deformity.3

Treatment and prevention of graft contracture comprises

wearing pressure garments4 and splints5 for months after

grafting. These approaches have changed little in the last 50

years.

The majority of the published literature on contraction has

centered on the use of the fibroblast-populated collagen

lattice model and myofibroblast differentiation.6–8 Both fi-

broblasts and keratinocytes are known to contract collagen

gels.9,10 However, to date, this research has had no sig-

nificant clinical impact on the prevention of contracture. To

investigate the mechanism of contraction further, physiolo-

gically relevant models, which more closely resemble ma-

ture human skin, are needed.

We used an in vitro tissue-engineered model of human

skin, comprising sterilized human dermis seeded with

1Department of Tissue Engineering, Kroto Institute, University of Sheffield, UK.2Bone Metabolism Laboratory, Centre for Human Nutrition, Division of Clinical Sciences (North), University of Sheffield, UK.3Department of Histopathology, Northern General Hospital, Sheffield, UK.4Department of Plastic and Reconstructive Surgery, Sheffield University Hospitals NHS Foundation Trust, Sheffield, UK.5Sheffield Kidney Research Institute, University of Sheffield, UK.

TISSUE ENGINEERINGVolume 12, Number 11, 2006# Mary Ann Liebert, Inc.

3119

keratinocytes and fibroblasts and cultured at an air–liquid

interface, which contracts by 25–40% during 10 days culture

in vitro.11,12 These tissue-engineered composites have been

grafted onto patients in the release of burn scar contractures,

and contraction occurs in a similar manner to the contraction

of split-thickness skin grafts.13 In addition to the need to

decrease contraction of our tissue-engineered skin and hence

improve its clinical performance, the finding that tissue-

engineered skin contracts in vitro and in vivo offers an im-

portant opportunity to use it as an experimental model to

investigate the mechanism of contraction of normal mature

human skin. We suggest that the reconstructed skin model

consisting of mature cross-linked collagen, a basement mem-

brane, dermal fibroblasts, and well-attached keratinocytes is a

more physiologically relevant in vitro model than the fibro-

blast-populated collagen lattice in which to study skin graft

contracture. The information gained in vitro can subsequently

be applied to develop new approaches to prevent or reduce

contracture in vivo.

Contraction, by definition, involves a decrease in surface

area.14 The aim of this study was to use the tissue-engineered

model to determine whether this decrease is predominantly

due to changes in the rate of collagen synthesis, degradation,

or simply to rearrangement of existing collagen fibrils. We

used 2 different approaches to investigate the mechanism of

cell-mediated dermal contraction. First, we aimed to de-

crease the pliability of the dermis using glutaraldehyde15 and

investigate the influence of this on the ability of the cells

to cause contraction. Second, we selected pharmacologic

agents predicted to modify collagen turnover or cross-linking

and investigated their effect on contraction. Agents predicted

to promote collagen synthesis, such as insulin-like growth

factor-1 (IGF-1),16 estrone, and 17b-estradiol,17 were added

to the culture medium. Agents predicted to inhibit collagen

synthesis, specifically corticosteroids,18 basic fibroblast

growth factor (bFGF),19 tumor necrosis factor-a (TNF-a),20

and prostaglandin-E2 (PGE2),21 were also studied. During

extracellular matrix remodelling, both collagen synthesis and

degradation take place in parallel. We have previously de-

monstrated matrix metalloproteinase-2 (MMP-2) and MMP-

9 activity in tissue-engineered skin22 and have shown that

galardin, a broad-spectrum MMP inhibitor, inhibits contrac-

tion of tissue-engineered composites but at the expense of

creating hyperkeratosis in the keratinocyte layer.12 In this

study, we used catechin23 and a2-macroglobulin (a2mg)24 to

inhibit MMP activity.

If the keratinocytes use tractional forces to contract the

composites, it is conceivable that the collagen fibrils slide

over one another in the underlying dermis. To maintain this

contraction, the collagen fibrils may become covalently cross-

linked to one another, thus stabilizing their new conformation.

To investigate this hypothesis, the role of the cross-linking

enzymes lysyl oxidase25 and transglutaminase26 were studied

using the lysyl oxidase inhibitor b-aminopropionitrile

(b-APN)27 and the transglutaminase inhibitor putrescine.28

Also, NTU283 and NTU285, novel thioimidazolium trans-

glutaminase inhibitors synthesized by Professor M. Griffin at

Nottingham Trent University,29,30 were investigated. The

effects of pharmacologic agents on collagen synthesis, de-

gradation, and soluble collagen content were related to the

ability of the skin cells to cause contraction.

MATERIALS AND METHODS

Skin cell culture

Skin was obtained from patients undergoing breast reduc-

tions and abdominoplasties who gave informed consent for

use of their skin for research purposes under a protocol ap-

proved by Sheffield University Hospitals NHS Trust Ethics

Committee.

Fibroblasts and keratinocytes were isolated from skin

according to the methods described by Ghosh et al.31 Pas-

sage 1 keratinocytes and passage 3–7 fibroblasts were used.

Preparation of acellular de-epidermized dermis

De-epidermized dermis (DED) was prepared from split-

thickness human skin using the method described by

Chakrabarty et al.32 For any 1 experiment, DED from a

single source was used, wherever possible cut from the

same sheet. The DED was cut into squares approximately

15�15mm and the papillary surface was orientated up-

permost in 6-well plates (Corning, Corning, NY).

Production of tissue-engineered skin composites

Composites were produced using the method of Chakra-

barty et al.32 and were cultured for 28 days. Media changes

were performed every 7 days, or more frequently if the media

was visibly depleted, indicated by a color change from red-

purple to orange-yellow. Each time, a 1.5-mL aliquot of

conditioned medium from each culture well was stored at

�208C for later analysis.

Pretreatment of dermis with glutaraldehyde

Glutaraldehyde (Sigma Chemical Co, Dorset, UK) con-

centrations of 0.0125–0.1% were prepared by serial dilution

in phosphate-buffered saline (PBS). Squares of DED mea-

suring 2�2 cm were immersed in the glutaraldehyde at 48Cfor 20 h. The DED was then washed twice with PBS and

incubated in an excess of 10% Greens medium at 378C for 4

weeks prior to use. This ensured that any residual glutar-

aldehyde was washed out of the dermis prior to the addition

of cells. Composites were then prepared and media ex-

change performed as above.

Supplementation of culture medium

with pharmacologic agents

b-APN, putrescine, estrone, 17b-estradiol (water so-

luble), ascorbic acid-2-phosphate, PGE2, (þ)-catechin,

3120 HARRISON ET AL.

verapamil, and dexamethasone were purchased from Sigma

Chemical Company. TNF-a and a2-mg were purchased from

Roche Diagnostics Ltd. (Lewes, UK). IGF-1 and bFGF were

obtained from R&D Systems Ltd. Transglutaminase in-

hibitors R283 and R285were synthesized by ProfessorMartin

Griffin (Nottingham Trent University, U.K.). Known con-

centrations of each agent in ascorbic acid-supplemented

Greens medium were prepared by serial dilution for experi-

mental use.

Assessment of composite contraction by digital

photography and image analysis

The 6-well plate was placed alongside a scale bar and

digitally photographed using a camera mounted vertically

above the plate. The images were imported into ImageJ

software (National Institutes of Health, Bethesda, MD) and

the scale bar was used to calibrate the image. The margin of

the composite was traced freehand on screen and the soft-

ware automatically calculated the area of this plot relative to

the calibration derived from the scale bar. The area of each

composite on the day of being raised to an air–liquid inter-

face was designated 100% and all changes in area were

expressed relative to this initial measurement.

Assays of collagen synthesis and degradation

Collagen synthesis was measured indirectly using the

concentration of the amino terminal propeptide of type I

collagen (P1NP). P1NP is cleaved by endopeptidases during

the synthesis of type I collagen and is used as a biochemical

marker of increased collagen turnover in serum and urine in

pathologic conditions such as Paget disease of bone.33 A new

electrochemiluminescence immunoassay (ECLIA) has re-

cently been developed for the measurement of P1NP in

serum (Roche Diagnostics GmbH, Mannheim, Germany).

We have previously employed this ECLIA to investi-

gate the influence of a range of putative collagen modu-

lators on collagen synthesis by fibroblasts alone and in the

presence of keratinocytes in a 2D culture model.34 Cumula-

tive P1NP production was calculated for the 28-day culture

period.

Collagen degradation was assessed using an ECLIA for

bL-CTX (b-CrossLaps; Roche Diagnostics GmbH). The

assay uses murine monoclonal antibodies to a synthetic

peptide based on part of the C-terminal telopeptide of type I

collagen.35,36 Total CTX was calculated for the aliquots

collected between days 14 and 21 of the incubation.

Composite soluble collagen assay

The keratinized layer of the composites was peeled off

using forceps and the wet dermal weight measured and

recorded. Acid- and pepsin-soluble collagen extraction was

performed using the method of Miller and Rhodes37 to

allow examination of the acute turnover of collagen during

composite contraction. The collagen was quantified using

the Sircol soluble collagen assay (Biocolor, Newtownabbey,

Northern Ireland), which is based on the binding of Sirius

red to the basic amino acids in collagen, which then pre-

cipitate out of solution and can be measured using a multi-

well plate reader (l¼ 492 nm). Total extracted collagen was

calculated and expressed as a percentage of the wet weight

of the skin composite (w/w).

Histology

Composites were fixed with 10% phosphate-buffered for-

maldehyde. Collagen IV immunohistochemistry was per-

formed using murine anti-human collagen IV monoclonal

antibody (COL94; Sigma Chemical Co.) and goat anti-mouse

IgG-AP conjugate (Dako, Carpenteria, CA; 1:50 in 0.05M

TRIS-HCl buffer). Hydrogen peroxide avidin–biotin complex

and diaminobenzidine substrates were used to give a brown

endpoint. Pancytokeratin immunohistochemistry was per-

formed using an AE1/AE3 monoclonal antibody (Dako). For

details of method see Sun et al.38

Statistical analysis

Statistical analysis was performed using the SPSS soft-

ware (Version 11.0). Statistical advice was obtained from

Dr. Edward Casson, Statistical Services Unit, University of

Sheffield. Replicate data within each experimental condition

was presented as mean values� standard error of the mean

(SEM). A 2-way ANOVA, with Bonferroni correction for

multiple comparisons, was used to compare the effect of

different concentrations of the test drug with the untreated

composite. A p-value of< 0.05 was considered to indicate a

significant difference.

RESULTS

Glutaraldehyde pretreatment significantly reduces

contraction of tissue-engineered skin in vitro

Pretreatment of DED with increasing glutaraldehyde

concentrations (0.0125–0.1%) reduced composite contrac-

tion (Fig. 1A). Immunoassay for type I procollagen (P1NP)

showed that pretreatment of DED with increasing con-

centrations of glutaraldehyde appeared to decrease P1NP

concentration in the conditioned medium, although this did

not achieve statistical significance (Fig. 1B). Cell viability

was unaffected by glutaraldehyde pretreatment, with the

keratinocytes establishing an organized, well-differentiated

epidermis (Figs. 1C, D). The keratinized layer in glutar-

aldehyde pretreated composites was slightly thinner than

the control composites. The dermo–epidermal junction was

more convoluted, giving an appearance similar to rete

ridges. Dermal morphology and fibroblast penetration were

not affected.

MECHANISM OF SKIN GRAFT CONTRACTION 3121

Estradiol and estrone significantly increase

composite contraction

Composite contraction was increased by 0.1–1.0 nM es-

trone and 1 nM estradiol (Fig. 2A). There was no significant

change in P1NP concentration in conditioned medium

from these composites and no significant change in CTX

concentration or soluble collagen extraction (data not pre-

sented). However, on histologic examination a dense cel-

lular infiltrate was seen in the dermis of composites treated

with estrone (Fig 2B), but not with estradiol (not shown).

Pancytokeratin immunohistochemistry revealed that these

cells were keratinocytes (Fig. 2C). This infiltration was

associated with loss of immunostaining for type IV col-

lagen in composites treated with estrone (Fig. 2D). (Estrone

had no effect on collagen IV in cell-free DED.)

Dexamethasone increases collagen degradation

but has no effect on composite contraction

There was no effect on the rate or extent of composite

contraction on supplementing the culture medium with

10 nM to 1 mM dexamethasone (Fig. 3A). A trend toward

increased P1NP concentration was seen, although this did

not achieve statistical significance (data not presented).

However, CTX concentrations increased up to 30-fold on

supplementing culture medium with dexamethasone (Fig.

3B), and a significant decrease in acid- and pepsin-soluble

collagen extraction was seen (Fig. 3C). These changes were

reflected in the composite histology where reduced dermal

density was seen (Figs. 4A, B).

The protease inhibitors catechin

and a2-macroglobulin reduce

composite contraction

Figures 5A and B demonstrate the reduction in compo-

site contraction seen in response to 1–100 mg/mL catechin

and 0.1–1.0U/mL a2mg. P1NP was markedly reduced in

response to a2mg (Fig. 5C), and collagen degradation was

markedly reduced in response to catechin (Fig. 5D). So-

luble collagen extraction was significantly increased

in response to catechin (Fig. 5E) and a2mg (data not

FIG. 1. Effect of glutaraldehyde pretreatment on tissue-engineered skin. (A) A dose-dependent reduction in contraction is seen with

increasing concentrations of glutaraldehyde (0.0125–0.1%; n¼ 3 replicates). Data are presented as mean values� SEM. *Significant

difference in surface area from untreated composite on day 28 (2-way ANOVAþBonferroni correction). *p< 0.05; **p< 0.01;

***p< 0.001. (B) Glutaraldehyde pretreatment has no significant effect on cumulative P1NP in conditioned medium ( p> 0.05, 2-way

ANOVAþBonferroni correction) when compared with untreated composites (n¼ 3 replicates). (C) Histology of composite cultured in

ascorbic acid-supplemented 10% Greens medium without glutaraldehyde pretreatment. H&E stain. Scale bar¼ 150 mm. (D) Histology

of composite after pretreatment of dermis with 1% glutaraldehyde. H&E stain. Scale bar¼ 150 mm. Good keratinocyte attachment,

proliferation, and differentiation are seen, despite glutaraldehyde pretreatment of the dermis.


presented). On histologic examination, dermal density was

visibly increased in response to both catechin (data not

presented) and a2mg (Figs. 4C, D).

Transglutaminase inhibitors have no

effect on composite contraction

No effect on composite contraction was seen on supple-

mentation of the culture medium with putrescine (0.5–

2.0mM; Fig. 6A), NTU283 (Fig. 6B), or NTU285 (Fig. 6C).

A marked reduction in P1NP was seen in response to pu-

trescine (Fig. 6D). P1NP was not measured in conditioned

medium from composites incubated with NTU283 or

NTU285 owing to resource constraints. CTX was not sig-

nificantly affected by culture with putrescine, NTU283, or

NTU285 (data not presented). Soluble collagen was un-

affected by culture with putrescine (data not presented), but

increased in response to NTU283 (Fig. 6E) and NTU285

(Fig. 6F). Histologic examination revealed keratinocyte

hyperproliferation and parakeratosis, which is the subject of

further investigation.

The lysyl oxidase inhibitor b-aminopropionitrilereduces composite contraction

Figure 7A demonstrates the reduction in composite

contraction seen in response to b-APN (50–200 mg/mL).

P1NP concentration was reduced (Fig. 7B) and CTX con-

centration was increased (Fig. 7C). Soluble collagen ex-

traction was unchanged (data not presented). Histologic

examination revealed good keratinocyte attachment, pro-

liferation, and differentiation, accompanied by an alteration

in the appearance of the dermal matrix (Figs. 4E, F).

Composite contraction was unaffected by

IGF-1, bFGF, TNF-�, and PGE2

IGF-1 (5–20 ng/mL) and bFGF (5–20 ng/mL) had no

effect on composite contraction (data not presented). There

was no significant effect on P1NP, CTX, or soluble

collagen extraction. Histologic examination was unremark-

able. Similarly, TNF-a (100–500U/mL) and PGE2 (10 nM–

1 mM) had no effect on composite contraction, P1NP, CTX,

FIG. 2. Effect of estrone and estradiol on composite contraction and histology. (A) Estrone and estradiol increase composite

contraction (mean� SEM; n¼ 3 experiments, 3 replicates per experiment). *Significant difference in surface area from untreated

composite on day 28 (2-way ANOVAþBonferroni correction). *p< 0.05; **p< 0.01; ***p< 0.001. (B) A dense dermal infiltrate is

seen on culture with estrone (1 nM). H&E stain. (C) This dermal infiltrate stains positive for pancytokeratin. (D) Collagen IV staining

reveals preservation of the basement membrane in control composites, and (E) loss of basement membrane during culture with 1 nM

estrone. Scale bar¼ 100 mm.


or soluble collagen extraction (data not presented). How-

ever, hyperproliferation and abnormal keratinocyte differ-

entiation were seen in response to TNF-a (Figs. 4G, H). This

is the subject of further investigation. The effect of all of the

pharmacologic agents tested on contraction, P1NP con-

centration, CTX concentration, and soluble collagen ex-

traction is summarized in Table 1.

DISCUSSION

Wound contraction contributes to the closure of wounds,

but also results in significant tissue distortion with loss of

joint mobility and cosmetic disfigurement. A major practical

problem for any research in this area has been the lack of

appropriate in vitro or in vivo models of graft contraction;

wound healing in loose-skinned animal models differs sig-

nificantly from human skin. Studies of collagen gels being

contracted by skin cells lack the complexity of mature cross-

linked human dermal collagen.

Our aim was to use an in vitro model of skin contraction

based on mature human dermis for investigation of how skin

cells contract mature collagen. This mature dermis is popu-

lated with keratinocytes and fibroblasts and has a stable

basement membrane. Although the density of the fibroblasts

within the dermis is lower than that seen in normal mature

human skin, we feel that this model is much more physiolo-

gically relevant than those models composed of reconstituted

collagen lattices, which form the majority of the published

literature in this subject area. We have also used sensitive

assays of collagen synthesis and degradation, previously used

to study turnover of bone,36 but not previously applied to

organotypic skin models. We have, however, recently pub-

lished successful use of the ECLIA for P1NP in 2D culture of

keratinocytes and fibroblasts.34

Previous studies have examined the effect of collagen

deposition on wound contraction. Stephens et al.39 found a

lack of correlation between wound contraction and collagen

synthesis. Similarly, Grillo and Gross demonstrated that

contraction occurs in the presence of marked vitamin C

FIG. 3. Effect of dexamethasone (Dex) on (A) composite contraction, (B) CTX concentration, (C) soluble collagen extraction, and

(D) composite histology. (A) Dexamethasone (10 nM–1 mM) has no effect on composite contraction (mean� SEM; n¼ 3 experiments, 3

replicates per experiment). p > 0.05 (2-way ANOVAþBonferroni correction). (B) Culture of composites with dexamethasone results in

a marked increase in CTX concentration in conditioned medium (mean� SEM; n¼ 3 replicates). (C) Acid- and pepsin-soluble collagen

extraction is significantly decreased after culture of composites with 100 nM dexamethasone (mean� SEM; n¼ 3 replicates). *p< 0.05;

**p< 0.01; ***p< 0.001 (2-way ANOVAþBonferroni correction).


deficiency and hence is unaffected by reduction in collagen

synthesis.40 Woodley et al.41 identified an increase in lysyl

oxidase-mediated collagen cross-linking that parallels con-

traction of fibroblast-populated collagen lattices in vitro.

Although there has been extensive research into the role of

transglutaminase in wound healing,42–45 its importance in

contraction has not previously been determined.

Here, increased glutaraldehyde concentrations resulted in

reduced composite contraction, decreased P1NP production

(indirect marker of collagen synthesis), and decreased CTX

production (collagen degradation). These results are likely to

indicate reduced collagen turnover owing to reduced cellular

matrix remodelling. Keratinocytes cultured on glutaraldehyde

pretreated DED showed good cellular attachment and pro-

liferation. Dermal morphology was normal with normal fi-

broblast penetration throughout the dermis. The lack of

toxicity seen in vitro after thorough washing of the glutar-

aldehyde–cross-linked dermal matrix, and the established use

of glutaraldehyde as a cross-linking agent in the manufacture

of other dermal substitutes such as Permacol and the collagen/

chondroitan-6-sulphate dermal substitute Integra, indicate

that the use of glutaraldehyde may be a simple approach for

decreasing contraction and enhancing the in vivo application

of the tissue-engineered composite. However, because of its

cellular cytotoxicity, it is unsuitable for decreasing contrac-

tion of normal human skin autografts.

A marked increase in composite contraction was seen in

response to the addition of estrone and estradiol to the culture

medium. Topical application of estrogens to healing wounds

has been demonstrated to result in increased collagen con-

tent and increased tensile strength.46 Estrogen treatment has

been linked to an increase in transforming growth factor-b1(TGF-b1) expression

47 and TGF-b1 stimulates deposition of

collagen,48 inhibits metalloproteinase activity,49,50 and upre-

gulates the synthesis of proteinase inhibitors.51 A marked

increase in dermal density was seen, consistent with the ob-

servations of Ashcroft et al.,46,47 that estrogens increase col-

lagen deposition.

FIG. 4. Effect of supplementation of culture medium with pharmacologic agents on histology of composites cultured at air–liquid

interface for 28 days. (A) Control composite and (B) composite cultured with dexamethasone (1mM). Decreased dermal density is seen

on culture with dexamethasone. (C) Control composite and (D) composite cultured with a2mg 1U/mL. Dermal density is increased

during culture with a2mg. (E) Control composite and (F) composite cultured with b-APN 200 mg/mL. Keratinocyte proliferation and

differentiation are good in the presence of b-APN. Dermal morphology is altered by culture with b-APN. (G) Control composite and

(H) composite cultured with TNFa 100U/mL. Keratinocyte hyperproliferation and disordered epidermal morphology are seen on

culture with TNF-a. H&E stain. Scale bar¼ 100 mm.


FIG. 5. (A) Effect of catechin (1–100 mg/mL) on composite contraction (mean� SEM; n¼ 3 experiments, 3 replicates per experi-

ment). One hundred mg/mL catechin inhibits composite contraction. (B) Effect of a2mg (0.1–1U/mL) on composite contraction (mean�SEM; n¼ 3 experiments, 3 replicates per experiment). a2mg 1U/mL significantly inhibits composite contraction. (C) Cumulative P1NP

concentration in conditioned medium is reduced by culture with a2mg (0.1–1.0U/mL; mean� SEM; n¼ 3 experiments, 3 replicates per

experiment). (D) CTX concentration is reduced during culture with catechin 1–100 mg/mL (mean� SEM; n¼ 3 replicates). (E) Acid-

and pepsin-soluble collagen extraction is increased during culture with 100 mg/mL catechin (mean� SEM; n¼ 3 replicates). *p< 0.05;

**p< 0.01; ***p< 0.001 (2-way ANOVAþBonferroni correction).


A dense cellular infiltrate was seen in the dermis of

composites treated with estrone (0.1–1.0 nM). Pancytoker-

atin immunohistochemistry demonstrated that these cells

were keratinocytes that had crossed the basement mem-

brane. The mechanism of keratinocyte invasion through the

basement membrane in response to estrone is not clear.

Collagen IV staining revealed loss of the basement mem-

brane in composites cultured with 1 nM of estrone. Estro-

gens stimulate keratinocyte proliferation52 and reduce

apoptosis53; hence, the increase in keratinocyte proliferation

seen in the composite model is consistent with the published

literature. However, expression of MMPs is generally re-

FIG. 6. (A) Putrescine (0.5–2.0mM) has no effect on composite contraction (mean� SEM; n¼ 3 experiments, 3 replicates per experi-

ment). (B) NTU283 (25–100 mM) has no effect on composite contraction (mean� SEM; n¼ 3 experiments, 3 replicates per experiment).

(C) NTU285 (25–100 mM) has no effect on composite contraction (mean� SEM; n¼ 3 experiments, 3 replicates per experiment). (D) P1NP

concentration is reduced in conditioned medium from composites cultured with putrescine (0.5–2.0mM; mean� SEM; n¼ 2 experiments,

3 replicates per experiment). Acid- and pepsin-soluble collagen extraction is increased from composites cultured with (E) NTU283 25–100

mM and (F) NTU285 25–100 mM (n¼ 3 replicates). *p< 0.05; **p< 0.01; ***p< 0.001 (2-way ANOVAþBonferroni correction).


duced in response to 17b-estradiol54 and the production of

tissue inhibitors of metalloproteinases is upregulated.55

Also, laminin-5 synthesis is increased by 17b-estradiol,56

and one would expect basement membrane integrity to be

enhanced rather than impaired. Previous work by our group

has shown that when basement membrane antigens are re-

moved from DED prior to addition of fibroblasts and kera-

tinocytes, the keratinocytes form a poor attachment to the

FIG. 7. (A) b-APN (200 mg/mL) decreases composite contraction (mean� SEM; n¼ 3 experiments, 3 replicates per experiment).

(B) b-APN (50–100 mg/mL) decreases P1NP concentration in conditioned medium (mean� SEM; n¼ 3 experiments, 3 replicates per

experiment). (C) b-APN (200 mg/mL) increases CTX concentration in conditioned medium (mean� SEM; n¼ 3 replicates). *p < 0.05;

**p < 0.01; ***p < 0.001 (2-way ANOVAþBonferroni correction).

TABLE 1

Pharmacological agent Contraction

Collagen

synthesis

(P1NP)

Collagen

degradation

(CTX)

Soluble

collagen

Glutaraldehyde pre-treatment (0.0125–1%) ; ; ; ; ;Insulin-like Growth Factor-1 (IGF-1) (5–20 ng/ml) No effect No effect No effect

Estrone (0.1–1 nM) or Estradiol (1–10 nM) : : No effect No effect No effect

Dexamethasone (10 nM–1 mM) No effect No effect : : ;Basic fibroblast growth factor (bFGF) (5–20 ng/ml) No effect No effect No effect

Tumour necrosis factor-a (TNFa) (100–500U/ml) No effect No effect No effect No effect

Prostaglandin-E2 (PGE2) (10 nM–1 mM) No effect No effect No effect

Catechin (1–100mg/ml) ; ; ; :a2-macroglobulin (0.1–1U/ml) ; ; ; No effect :b-aminopropionitrile (b-APN) (50–200 mg/ml) ; ; : No effect

Putrescine (0.5–2mM) No effect ; ; No effect No effect

NTU283 or NTU285 (25–100 mM) No effect No effect : :


underlying dermis with reduced proliferation and a lack of

hemidesmosome formation.57 However, despite the lack of

basement membrane, we have never previously seen any

invasion of the keratinocytes into the dermis.22,57 Richard-

son et al.58 studied the effect of estrone and 17b-estradiol oninvasion of a melanoma cell line through a fibronectin ma-

trix and found dose-dependent inhibition of invasion. Si-

milar findings have also been reported by Dewhurst et al.59

However, with respect to keratinocyte-derived carcinomas,

17b-estradiol has been reported to enhance the development

of basal cell carcinoma and squamous cell carcinoma in

rodents in response to the tumor promoter 3-methylcholan-

threne.60 No mechanism was identified in these studies.

The large increase in contraction in response to the es-

trogens is also somewhat difficult to explain. No significant

effect was seen collagen synthesis, degradation, or solubility

in this model. In addition, no effect on P1NP concentra-

tion was noted in fibroblast monocultures or co-cultures of

keratinocytes and fibroblasts on tissue-culture plastic cul-

tured with estrone or estradiol.34 Hence, it is likely that these

estrogens are either exerting their effect on the non-

collagenous proteins of the dermal matrix or are in someway

manipulating collagen cross-linking. It is known that estro-

gens upregulate TGF-b1 synthesis47 and that TGF-b1 upre-

gulates lysyl oxidase expression.61,62 It is therefore possible

that increased lysyl oxidase–catalyzed cross-linking is re-

sponsible for the increased composite contraction.

In the composite model, dexamethasone did not affect

contraction. Corticosteroid injection is a mainstay of

treatment of hypertrophic scarring and corticosteroids re-

duce fibroblast proliferation,63 reduce collagen synthesis,64

and increase collagen degradation by a reduction in TIMP

synthesis.65 CTX concentrations were increased 30-fold by

100 nM dexamethasone, consistent with increased collagen

degradation. This was reflected in the decreased dermal

density seen on histologic examination. The lack of effect

of dexamethasone on contraction strongly suggests that

contraction is unrelated to collagen synthesis and de-

gradation, but instead is related to either the mechanical

gathering in of the underlying dermis or to modification of

the cross-linking between adjacent collagen fibrils. Simi-

larly, Guidry and Grinnell found an absence of collagen

degradation in contracting collagen gels and concluded that

rearrangement of existing fibrils was taking place during

contraction.7

The effect of inhibition of MMP activity was studied using

catechin and a2mg, both of which inhibited contraction.

a2mg is a broad spectrum MMP inhibitor66 that also binds to

many growth factors and cytokines, controlling their activ-

ity.67–70 Catechins are antioxidants with free radical–

scavenging properties.71,72 They inhibit TNF-a synthesis73

and increase keratinocyte transglutaminase (TG1) con-

centration and activity by a calcium-independent mechan-

ism74 and increase keratinocyte differentiation.75 In addition,

catechins increase the stability of collagen fibrils by pro-

moting the formation of intermolecular hydrogen bonds; they

also interfere with collagenase binding sites.76 In vitro, ca-

techins inhibit the activity of MMP-2 and MMP-9.77,78 In-

hibition of MMP-2 reduces keratinocyte migration.79

Decreased P1NP levels were seen using a2mg. As P1NP

results from the cleavage of the amino-terminal propeptide

from procollagen by proteinase activity, it is predictable

that a proteinase inhibitor would reduce P1NP concentra-

tion. The reduction in P1NP levels reflects reduction in

extracellular processing of procollagen, rather than reduc-

tion in gene transcription. Collagen fibril generation is

therefore inhibited as cleavage of the propeptides is a ne-

cessary prerequisite for collagen fibril self-assembly. The

marked reduction of CTX concentration seen with catechin

parallels MMP inhibition. Both a2mg and catechin increased

acid and pepsin-extractable collagen, probably partly due to

suppression of MMP activity, with increased levels of col-

lagen that would otherwise have been degraded by MMPs.

Also, lysyl oxidase is secreted into the extracellular space as

the proenzyme prolysyl oxidase, and requires proteolytic

cleavage of its propeptide for activation.80 MMP inhibitors

may therefore reduce lysyl oxidase activity and hence re-

duce collagen cross-linking. This in turn increases the

amount of acid and pepsin-soluble collagen.

From the results described, topical administration of

MMP inhibitors to healing wounds and grafts could be ex-

pected to markedly reduce collagen degradation and graft

contraction. However, administration of an MMP inhibitor

may inhibit processes such as angiogenesis and fibrinolysis,

essential in wound healing and in successful ‘‘take’’ of a

graft onto its recipient bed. Additional problems would be

anticipated owing to inhibition of proteolytic activation of

cytokines such as TNF-a.81,82 Therefore, we suggest that theuse of MMP inhibition as a therapeutic approach for pre-

venting contraction is unlikely to be taken forward into the

clinical arena. Nevertheless, the effectiveness of MMP in-

hibitors in reducing contraction of tissue-engineered skin

in vitro is an indicator of the importance of MMP-mediated

extracellular matrix remodelling in contraction.

The nonspecific transglutaminase inhibitor putrescine and

transglutaminase-specific inhibitors NTU283 and NTU285

were used to investigate the role of transglutaminase-induced

cross-linking on contraction. Transglutaminases are a family

of enzymes including the keratinocyte-specific isoforms ker-

atinocyte and epidermal transglutaminase, and the ubiquitous

tissue transglutaminase (TG2; reviewed in Griffin et al.83).

TG2 expression is upregulated in sites of wound healing43 and

hypertrophic scarring.84 It plays a role in the covalent stabi-

lization of collagen fibrillar formation through cross-linking of

the core fibrils85 and facilitates deposition of matrix pro-

teins.85,86 Addition of exogenous TG2 to primary human

dermal fibroblasts leads to accumulation of matrix-associated

collagen86 and a reduction in the rate of degradation, partly

owing to the resistance of cross-linked collagen to proteolytic

degradation.87 TG1 and epidermal transglutaminase (TG3)

catalyze the cross-linking of cornified envelope precursors.

Inhibition of these enzymes, by reducing intracellular calcium


or by addition of specific transglutaminase inhibitors, would

be expected to inhibit keratinocyte differentiation.

Neither putrescine nor NTU283 and NTU285 had any ef-

fect on contraction in the composite model. However, pu-

trescine showed a marked dose-dependent reduction in P1NP

concentration. This is consistent with the published literature,

suggesting a reduction in collagen synthesis after topical

putrescine treatment of healing wounds44 and hypertrophic

scars.88 The authors attributed the decrease in collagen

synthesis to decreased collagen cross-linking, resulting in an

increased pool of soluble collagen and therefore exerting a

negative feedback effect on collagen synthesis.

No effect on CTX concentration was seen with either

NTU283 or NTU285. However, a large increase in acid and

pepsin-soluble collagen was seen with both inhibitors. This

may represent an increase in collagen synthesis in response to

culture with the transglutaminase inhibitors or an increased

susceptibility to proteolytic digestion of uncrosslinked col-

lagen. An increase in collagen synthesis initially appears

contradictory to published evidence; inhibition of transglu-

taminase would be expected to increase the pool of soluble

collagen and hence decrease collagen synthesis.44,84 How-

ever, transglutaminase knockout cells synthesize significantly

increased amounts of collagen III and IV in mesangial cells

but deposit reduced amounts of mature insoluble collagen

compared with wild-type cells.89 Nardacci et al.90 studied

carbon tetrachloride-induced liver fibrosis in TG2 knockout

mice. The knockout mice displayed histologic evidence of

progressive accumulation of extracellular matrix components

compared to wild-type mice. Hence, TG2 inhibition can

paradoxically result in increased accumulation of extra-

cellular matrix components.

Finally, b-APN was used in the composite model as an

inhibitor of the lysyl oxidase cross-linking enzyme. Previous

studies by Redden and Doolin have demonstrated that in-

hibition lysyl oxidase reduces fibroblast-populated collagen

gel contraction,91 and Woodley et al.41 have demonstrated

that the number of lysyl oxidase catalyzed collagen cross-

links is proportional to the degree of collagen gel contrac-

tion. In the composite model, culture with b-APN also re-

sulted in marked inhibition of contraction, associated with a

reduction in P1NP concentration. This finding is consistent

with research by Giampuzzi et al.,92 who demonstrated in-

creased type III collagen transcription in primary human

skin fibroblasts overexpressing lysyl oxidase and showed

that this increase in type III collagen transcription could be

completely suppressed by b-APN. This establishes the linkbetween lysyl oxidase catalytic activity and collagen

synthesis, independent of common gene promoter activity. It

is possible that increased cross-linking reduces the pool of

soluble collagen and therefore provides a negative feedback

stimulus for further collagen synthesis.

b-APN increases collagen degradation in the composite

model, consistent with decreased lysyl oxidase activity lead-

ing to an increased pool of soluble collagen. The presence of

soluble collagen has previously been shown to increase col-

lagenase activity.93 Gerstenfeld et al.94 proposed that collagen

cross-linking increases irreversible retention of collagen fi-

brils in the extracellular matrix, reducing the pool of soluble

collagen and decreasing collagen susceptibility to proteolytic

degradation. Hence, by decreasing collagen cross-linking, the

collagen becomes more susceptible to degradation by col-

lagenase.95 Nelson et al.96 studied the effect of b-APN on

fibroblasts in vitro and found a dose-dependent inhibition of

migration, with no effect on proliferation, collagen synthesis,

or noncollagenous protein synthesis. In the composite model

studied herein, composite morphology and keratinocyte dif-

ferentiation appear to be unaffected by culture with b-APN.This research provides several useful pointers to the me-

chanism of contraction of tissue-engineered skin. In the re-

sults presented, no clear relationship was established between

collagen synthesis or collagen degradation and composite

contraction. It was particularly striking that dexamethasone

increased CTX concentration 30-fold in conditioned medium,

yet had no effect on composite contraction. Similarly, the

female sex steroids estrone and estradiol led to a major in-

crease in contraction, without any significant effect on col-

lagen synthesis or degradation. These data strongly suggest

that collagen synthesis and degradation do not themselves

directly affect contraction in this model and therefore would

be inappropriate targets for pharmacologic modulation of skin

contraction. Inhibition of lysyl oxidase catalyzed collagen

cross-linking, however, appears to be a promising potential

target for preventing contraction. The data presented using

this in vitro tissue-engineered model suggest that further

in vivo studies, possibly using human tissue-engineered skin

grafted onto nude mice, are merited to determine how ef-

fective topical lysyl oxidase inhibitors such as b-APNmay be

in the treatment of skin graft contracture.

ACKNOWLEDGMENTS

Caroline Harrison was the recipient of a Research Fel-

lowship of the Royal College of Surgeons of England with

support from the Freemasons and the Rosetrees Trust and also

received a Peel Medical Trust Research Award. Tim Johnson

was supported by a National Kidney Research Fund Senior

Fellowship.

We are also grateful to Professor Martin Griffin, Notting-

ham Trent University for permitting the use of transgluta-

minase inhibitors NTU283 and NTU285 in this study.

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Address reprint requests to:

Miss Caroline Harrison

Department of Plastic Surgery

Northern General Hospital

Herries Road, Sheffield, S5 7AU, UK

E-mail: [email protected]


Use of an in Vitro Model of Tissue-Engineered Skin to Investigate the Mechanism of Skin Graft...

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