ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

�Correspondfax: +353 91 49

E-mail addr

brendan.wilkins

(R.J. Collighan

abhay.pandit@

Biomaterials 29 (2008) 857–868

www.elsevier.com/locate/biomaterials

Towards development of a dermal rudiment for enhanced woundhealing response

Yolanda Garciaa,�, Brendan Wilkinsb, Russell J. Collighanc, Martin Griffinc, Abhay Panditd

aNational Centre for Biomedical Engineering Science, National University of Ireland, Galway, IrelandbAnatomy Department, National University of Ireland, Galway, Ireland

cSchool of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UKdDepartment of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland

Received 3 August 2007; accepted 29 October 2007

Available online 26 November 2007

Abstract

Enhancement of collagen’s physical characteristics has been traditionally approached using various physico-chemical methods

frequently compromising cell viability. Microbial transglutaminase (mTGase), a transamidating enzyme obtained from Streptomyces

mobaraensis, was used in the cross-linking of collagen-based scaffolds. The introduction of these covalent bonds has previously indicated

increased proteolytic and mechanical stability and the promotion of cell colonisation. The hypothesis behind this research is that an

enzymatically stabilised collagen scaffold will provide a dermal precursor with enhanced wound healing properties. Freeze-dried

scaffolds, with and without the loading of a site-directed mammalian transglutaminase inhibitor to modulate matrix deposition, were

applied to full thickness wounds surgically performed on rats’ dorsum and explanted at three different time points (3, 7 and 21 days).

Wound healing parameters such as wound closure, epithelialisation, angiogenesis, inflammatory and fibroblastic cellular infiltration and

scarring were analysed and quantified using stereological methods. The introduction of this enzymatic cross-linking agent stimulated

neovascularisation and epithelialisation resisting wound contraction. Hence, these characteristics make this scaffold a potential candidate

to be considered as a dermal precursor.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Cross-linking collagen; Microbial transglutaminase; Wound healing; Angiogenesis; Stereology

1. Introduction

Tissue repair and reconstruction occurs when animplanted scaffold has the appropriate structural andfunctional properties of the host tissue [1–3]. Collagen,being the most abundant structural protein in the dermalextracellular matrix (ECM) [4–6] maintains a highlyconserved amino acid sequence throughout the course ofevolution [7]. As a result, most of the commerciallyavailable skin substitutes are collagen based. Rangingfrom the a-cellular dressings like BioBrane (Smith

e front matter r 2007 Elsevier Ltd. All rights reserved.

omaterials.2007.10.053

ing author. Tel.: +353 91 495181x5039;

4596.

esses: yolanda.garcia@nuigalway.ie (Y. Garcia),

@nuigalway.ie (B. Wilkins), r.collighan@aston.ac.uk

), m.griffin@aston.ac.uk (M. Griffin),

nuigalway.ie (A. Pandit).

& Nephew) [8] or Integras (IntegraTM) [8,9] to theautologous keratinocyte-loaded counterparts Apligraf(Novartis) [10,11] or OrCell (Ortec International Inc.)[11]. However, all suffer from relative rapid biodegradationin vivo, frequently with a non-synchronised dermalregeneration rate [12]. Maintenance of structural andfunctional properties of the scaffold whilst maintainingcell compatibility until the healing has been completed is anecessary and challenging task to be addressed.Poor vascularisation of implants, microbial contamina-

tion, lack of drainage, lack of mechanical strength,blistering, poor healing times and inadequate cosmeticeffects are additional limitations in the current dermalsubstitutes [13]. Chemical collagen cross-linking agentshave negatively affected cell viability and physical methodsof cross-linking have proved insufficient [14,15] to maintainstructural integrity in vivo [15]. Enzymatic cross-linkers, in

ARTICLE IN PRESSY. Garcia et al. / Biomaterials 29 (2008) 857–868858

particular microbial transglutaminases (mTGase), haverecently been used to introduce covalent bonds in proteinstructures and have proven to be highly cytocompatible[16]. Other transglutaminases have also been investigatedin several other clinical applications such as biologicalglues and cell adhesion proteins for coating of medicalimplants [17]. However, mTGase shows high trans-amidating activity with the minimal peptidyl-glutaminehydrolase activity [18], therefore this enzyme’s activity isprimarily focused on cross-linking.

In this study, it is hypothesised that porous collagenscaffolds cross-linked with mTGase could address ongoingrestrictions on dermal substitutes such as vascularisation,epithelialisation and structural integrity. The e-(g-gluta-myl)lysine cross-links, introduced by mTGase, are stable toproteolytical and mechanical damage, increasing thestability and resistance to degradation of the scaffolds[18–21]. With regards to vascularisation, the enhancementin blood vessel length density found when tissue transglu-taminase (tTG) was applied topically [19] suggests thattransglutaminase activity can promote angiogenesis inwound repair. In addition, the porous nature of thescaffold will allow elimination of infective material andaccess to topically applied therapeutic products.

2. Materials and methods

2.1. Scaffold fabrication

Bovine collagen type I from calfskin (BD Biosciences, Unitech, Dublin,

Cat. no. 354231), with a concentration of 2.9mg/ml and more than 95%

purity, was used in fabricating the scaffolds. Four parts of this collagen

solution were gently and thoroughly mixed with one part 5� phosphate-

buffered saline (PBS, Sigma-Aldrich, Tallaght, Dublin, Cat. no: P-4417).

The solution was neutralised by the addition of 3M sodium hydroxide

(Sigma-Aldrich, Tallaght, Dublin, Cat. no. 30620) until a final pH of 7–7.5

was reached. The solution was kept in an ice bath to delay gel formation

until the corresponding reagent was added. Three different types of

scaffolds were fabricated: collagen alone, collagen cross-linked with

mTGase and collagen cross-linked with mTGase but loaded with the site-

directed mammalian transglutaminase inhibitor R283 (1,3-dimethyl-

2[(oxopropyl)thio]-imidazolium) [22]. This inhibitor, which is highly

specific for transglutaminase, was originally designed for the inhibition

of Factor XIII but is also highly potent against TG2 (tTG) [23] and

displays minimal inhibition of mTGase (data not shown). mTGase

(ActivaTM WM, Ajinomoto Corporation Inc. Japan) was purified as

previously stated, and R283 (1,3-dimethyl-2[(oxopropyl)thio]-imidazolium

was synthesised according to published procedures [23]. MTGase and

R283 were added at concentrations of 0.1mg/ml and 1mM, respectively,

and the solution was subsequently placed in a humidified-atmosphere

incubator at 5% CO2 and 37 1C overnight. All scaffolds were freeze-dried

under the same conditions using a freeze-dryer (VirTis AdvantageTM,

Wizard 20, SP Industries, New York, USA). The processing parameters

were freeze temperature �25 1C, condenser set-point �45 1C and vacuum

set point: 100–200mTorr.

The treatment groups

Treatment 1: collagen scaffold.

Treatment 2: collagen scaffold cross-linked with mTGase.

Treatment 3: collagen scaffold cross-linked with mTGase and inhibitor

R283.

Treatment 4: no treatment identified in the graphs as ‘‘control’’.

2.2. In vivo model

All procedures performed were conducted under an animal licence no.

B100/3316 and were approved by the Animals Ethics Committee of the

National University of Ireland, Galway. In addition, animal care and

management followed the Standard Operating Procedures of the Animal

Facility at the National Centre for Biomedical Engineering Science.

Eighteen Sprague Dawley male rats, ranging between 250 and 300 g of

body weight, were used in the study. The experimental design included

three time periods (3, 7 and 21 days) with six animals per time period with

four treatments per animal. The agents used to anesthetise the animals

were intra-peritoneal xylazine and ketamine (Xylapan and Narketan,

Vetoquinol Ltd., UK) at a dose rate of 100 and 10mg/kg, respectively.

The animals’ skin was clipped, the positions (wounds at each side of the

dorsal midline) and dimensions of the wounds were marked with a

permanent ink marker, and the surgical field was disinfected prior to

surgery. Four full thickness 1 cm2 wounds were placed at least 1 cm apart.

All the procedures were performed under standard general practice

principles of asepsis and no further antimicrobial therapy was provided.

The positioning of the four treatments was randomised and recorded. An

outline of all the wounds was traced on sheets of pre-labelled sterile

tracing paper and additional photographic records were taken. The

wounds were covered with transparent polyurethane dressing (Opsites

Smith & Nephew). Colour-coded and numbered jackets were used on all

the animals with the intention of preventing wound disturbance and

facilitating the identification of experimental groups. Extra measures were

taken to minimise wound disruption by housing all the animals

individually for the duration of the study.

2.3. Harvesting and processing of tissue

The animals were sacrificed with an overdose of sodium pentobarbitone

at different post-implantation time periods (3, 7 and 21 days). Jackets and

dressings were carefully removed and the wounds were traced and

photographed prior to excision. All samples were identified by animal

number (1–6), time period (red jacket: 3 days, yellow; 7 days and white: 21

days) and position in the animal (AL: anterior left; AR: anterior right;

ML: medial left; MR: medial right; PL: posterior left; PR: posterior right)

to avoid bias during analysis. Samples were fixed in 10% neutral buffered

formalin for 24 h to be subsequently processed (Leica ASP300), blocked

(LeicaEG1150H and EG1150C) and sectioned (Leica RM 2235) perpen-

dicularly to the wound surface in 5mm consecutive sections. The samples

were stained with haematoxylin–eosin (H&E), Mason’s Trichrome (MT),

Picrosirius Red and Collagen type III immunohistochemical stain for

subsequent stereological analysis.

2.4. Explant analysis

The efficacy of the treatments on wound healing was evaluated by the

analysis of parameters such as wound closure, epithelialisation, angiogen-

esis, inflammatory reaction and scarring.

2.4.1. Wound closure

Wound closure was assessed by comparing the area reduction of

different treatments in the sheets of tracing paper at different time periods

with respect to day 0. The sheets of tracing paper were scanned to a 1:1

scale; the area was measured using image analysis software (IMAGE-

PROs PLUS, Media Cybernetics, Silver Spring, MD, USA), and the

reduction in these areas was expressed as a percentage. In addition to the

tracings, wound areas in transversal sections were measured in � 40

histological samples at different time periods using the same image

analysis technique. The measurements were expressed in square microns.

2.4.2. Epithelialisation

Epithelialisation was evaluated as a measure of percentage of

epithelialisation (%E), epithelialisation rate (ER) and percentage of

ARTICLE IN PRESSY. Garcia et al. / Biomaterials 29 (2008) 857–868 859

contraction (%C). Images of sections were captured on � 40 images by

drawing two digital lines limiting the wound at each lateral margin, and

measuring the distance between them in microns to capture wound length

(W). The %E was determined by the total epithelial layer over the wound

at both ends (E1+E2) divided by the length of the original wound (Wo)

(Eq. (2.1)). The original wound size (Wo) was 1 cm and was kept constant

during surgery. The contraction rate (%C) was determined by the

reduction in length of the wound divided by the original wound size

(Eq. (2.2)). The ER was calculated as per Pandit et al. [24], taking into

consideration the effects of contraction

%E ¼ðE1 þ E2Þ

Wo� 100, (2.1)

%C ¼ðWo �W Þ

Wo� 100, (2.2)

ER ¼W �%E

2

� ��

%C �%E�Wo

2

� �. (2.3)

The thickness of the epithelium was also measured in the 21-days time-

period samples, by using stereological methods [25,26]. Orthogonal

lines where drawn from the base of the epithelial layer to the skin surface

in � 100 magnified images. Four fields of view were examined per

slide and a minimum of 10 measurements was taken in each field. The

mean of these length values was considered the mean thickness of the

epithelial layer.

2.4.3. Fibroblast and inflammatory cell infiltrations

Both parameters, fibroblast and inflammatory cell infiltrations, were

measured in relative terms of nuclear volumes (Eq. (2.4)) [26,27]. In

practical terms, this means that a grid mask (5� 5mm2) was applied to

each image with a magnification of � 1000 (five fields of view per slide, six

slides per treatment, four treatments per animal) and the numbers of grid

intersections that intersect the nucleus of fibroblasts or inflammatory cells

(neutrophils, eosinophils, macrophages and/or unidentified mononuclear

cells, lymphocytes) were tagged. All the images were taken in the wound

area immediately under the epithelium, when present, or in the most

centralised areas of the wound. The cell types were identified following the

criteria shown in Table 1 [28,29]. The volume fraction (Vv) of each

parameter (Eq. (2.4)) [27] was expressed as the fraction of tagged

intersections (Pv) in relation to the total number of intersections (PT) in

the grid:

Vv ¼Pv

PT. (2.4)

2.4.4. Angiogenesis

Neoangiogenesis of the wound was measured in terms of surface area

(SA) and total length (LT). Preliminary calculations of surface density (SV)

and length density (LV) were needed and performed in the capillary bed

under the epithelium, when present. These two stereological parameters

were estimated using a cycloid grid of known length of total cycloid lines

(L) and counting of the number of intersections (I) of those lines with

Table 1

Criteria for the morphological identification of fibroblasts and inflammatory c

Cell type Description

Fibroblasts Large oval nucleus, large nucleolus, tapering spindle-shaped m

Neutrophils Multilobed nucleus joined by fine strands of nuclear material

Eosinophils Bilobed nucleus with strongly eosinophilic granules in the cyto

Lymphocytes Cells with a large round nucleus that occupies 90% of the cell

Macrophages Varied morphologic appearance, normally round with large n

pseudopodia. Small darkly stained nuclei normally eccentrical

blood vessels. Surface density was estimated using the horizontal

positioning of the grid in relation to the skin surface (Eq. (2.5))

[27,30,31] and length density was estimated using the vertical positioning

(Eq. (2.6)) [27,30], TS being the thickness of the section. Wound volumes

were calculated by multiplying the section thickness (5mm) by the

corresponding wound areas. Angiogenesis, in terms of SA ((Eq. (2.8))

[26,27] and LT (Eq. (2.9)) [26], was assessed by multiplying the values

(Sv and Lv) by the wound volume. Radial diffusion, Rdiff ((Eq. (2.7)

[27,32], was an additional parameter calculated from length density

estimations that gave an indication of the efficiency of the capillary

network by measuring the diffusion zone around the vessels:

SV ¼ 2�I

L, (2.5)

Lv ¼ð2� ðI=LÞÞ

TS, (2.6)

Rdiff ¼1ffiffiffiffiffiffiffiffiffiffiffiffiffiffi

p� Lv

p , (2.7)

SA ¼ SV � V , (2.8)

LT ¼ LV � V . (2.9)

2.4.5. Collagen fibre orientation

Quantitative polarised microscopy was performed on paraffin em-

bedded [33], 5 mm thick, sections stained with Sirius red dye (Sircol

Collagen Assay, Lot no: B44, Bicolor Ltd., N. Ireland), a birefringence

enhancer of collagens. The polarigraphs were captured using a birefrin-

gence imaging transmission microscope (Metripol Oxford Cryosystems,

Ltd.) equipped with a rotating polariser. Collagen, as a birefringent

material [34], alters the orientation of polarised light and separates the

light in two rays. The delay of one ray in relation to the other is

represented in pseudo-colours, which can subsequently be analysed with

image analysis software (ImagePros Plus, Media Cybernetics). This

software correlates colours with angles and quantifies the percentage area

of each colour, allowing the estimation of the degree of organisation and

orientation of collagen fibre in the wound bed. Images of the most

superficial areas of the wound edge and centre were captured at � 40 [35],

six slides per treatment at two different time periods (7 and 21 days) were

analysed and the values were plotted in terms of percentage of wound area

per angle range (140–1651, 108–1401, 85–1081, 64–851, 35–641, 10–351 and

165–1801 or 1–101). Additionally, the slides stained with Sirius red dye

were used to differentiate between the two types of collagens, I and III.

Collagen type I-like fibres were identified as yellow and red and collagen

type III-like fibres in green [36]. In the visualisation of the images a simple

polarising attachment BX-POL (Olympus) was used in an Olympus bright

field microscope. This device consists of a polariser (U-POT) inserted in

the illumination path and an analyser (U-ANT) oriented orthogonal to the

polarised beam.

ells

orphology with basophilic cytoplasm

and pale stained cytoplasm

plasm

ular space with a thin basophilic rim of cytoplasm surrounding it

umber of lysosomes. Cytoplasm can display slight granularity and

ly positioned

ARTICLE IN PRESSY. Garcia et al. / Biomaterials 29 (2008) 857–868860

2.4.6. Collagen type III deposition

Collagen type III production by the infiltrated fibroblasts in the wound

area was elucidated using immune-staining and quantified using stereo-

logical methods in terms of volume fraction. The antibodies used in the

identification were mouse monoclonal to collagen III (ab6310, Abcam plc,

Cambridge, UK) as a primary (dilution: 1/500) and goat polyclonal to

mouse IgG and IgM (ab2891, Abcam plc, Cambridge, UK) as a secondary

antibody (ready-to-use format). The chromogen was developed with

amino-ethyl carbazol (AEC) (Sigma-Aldrich, Tallaght, Dublin Cat. no.

AEC101) and the sample was counterstained with Mayer’s haematoxylin

(Sigma-Aldrich, Tallaght, Dublin, Cat. no. 51275). Three images were

taken per slide and six slides per treatment. The location of analysis was

medial and the magnification used was � 400. Volume fractions of

collagen type III were calculated (Eq. (2.4)) using a grid size of 2.5 mm2.

2.4.7. Statistical analysis

One way analysis of variance (ANOVA) was used to evaluate the

data with post-hoc differences between groups using Tukey’s Method

(MINITABTM version 13.32, Minitab, Inc.). p-Values of less than 0.05

were considered to be statistically significant within a given time period.

The outliers were previously calculated using Grubb’s test and eliminated

from the results.

3. Results

3.1. Wound closure

Wound closure, from tracing paper measurements, wasanalyzed for the three time-periods as a percentage of areareduction. No statistical difference in wound closure wasdetected between the treatments (data not shown). How-ever, the wound area calculated in the histological samplesrevealed that cross-linked collagen scaffold-treated woundsmaintain the wound size for longer (Fig. 1), by showingstatistically significant larger wound areas. Additionalimages supporting the integrity of the implanted scaffoldafter 3 days can be seen in Fig. 1(b–d). In subsequent timeperiods this structural integrity is difficult to assess due tothe intense fibroblastic infiltration and tissue integration,making it impossible to differentiate between host tissueand implanted scaffold.

3.2. Epithelialisation

Epithelialisation, when measured as a percentage(Fig. 2(a)), showed no statistical difference betweentreatments at 3 and 21 day time-periods. However, at the7-day time-period, the percentage of epithelialisation inenzymatically cross-linked collagen treated wounds andcontrol wounds was significantly smaller than in the othertreatments; coinciding, as seen in Fig. 2(b), with a decreasein the percentage of contraction. When the effect of woundcontraction is eliminated (Fig. 2(c)), the results show astatistically significant increase in the ER in cross-linkedscaffolds after 7 days of treatment. Structurally stablescaffolds (cross-linked scaffolds) markedly prevented con-traction. This fact is also corroborated by the increasedwound size of cross-linked scaffold treated wounds (Fig. 1).The effect of the cross-linked scaffolds on the final qualityof the epithelium was also evaluated in terms of average

thickness. All measurements were performed at 21 days,when all the wounds had the epithelial cover fully restored,and no statistically significant difference between treat-ments (Fig. 2(d)) was found.

3.3. Fibroblast and inflammatory cell infiltrations

No significant difference in fibroblast infiltration wasobserved between treatments at 3 and 7 days post-implantation (Fig. 3(a)) with the exception of controlwounds with no scaffold that showed significantly lowervolume fractions of fibroblasts in the wound area at 7 days.After 21 days post-injury all wounds showed statisticallyequal volume fractions of fibroblasts.After 3 days, inflammatory cell infiltration does not vary

in collagen cross-linked scaffolds (Fig. 3(b)) when com-pared to the positive control (collagen alone). At 7 days,the number of inflammatory cells in collagen cross-linkedscaffolds appears to be higher than collagen alone andcontrol. However, at 21 days there is no difference ininflammatory reaction between treatments.

3.4. Angiogenesis

Angiogenic parameters were expressed in terms ofabsolute values of surface area and total vascular lengthof the capillary network in wounded areas. In normalwound healing, the surface area of new vasculaturebecomes more significant after 7 days in the proliferativephase of wound healing. At this point collagen cross-linkedtreated wounds with or without the loading of mammaliantransglutaminase inhibitor showed a significantly increasedsurface area of capillary networks (Fig. 4(a)) as well astotal blood vessel length (Fig. 4(b)). When assessing theseparameters in control groups it has to be taken intoconsideration that the measurements might have comefrom the wound bed itself and not strictly from newvascularised areas. For at least the first 3 days, the scaffoldsconstitute an additional physical barrier for cell migration,delaying endothelial tubular development in the area. Theradial diffusion of the new vasculature did not varybetween treatments, the capillaries being equally effectivein perfusing blood in all the groups.

3.5. Scarring

3.5.1. Collagen fiber orientation

The arrangement of the collagen fibers after 7 and21 days maintained normal architecture, with equallyarranged collagen fibers independently to the treatmentapplied. The majority of the collagen fibers were parallel tothe skin surface (data not shown) [37] and presumably inthe direction of the wound’s tensile forces. With polarisedlight microscopy (Fig. 5) the thickness of the fibers in themost central parts of the wound were seen to be a lotthinner than normal skin collagen fibers. The thickness ofthe fibers increased with the proximity to the wound edge.

ARTICLE IN PRESS

0

2000000

4000000

6000000

8000000

10000000

12000000

3 days 7 days 21 days

Time Periods

Wo

un

d A

rea

s (

mic

ron

s2)

Collagen Collagen +TG Control Collagen-TG+R283

***

100 µm

100 µm

100 µm

Fig. 1. Wound areas at 3, 7 and 21 days: (a) shows wounds areas at different time-points (n ¼ 6, pp0.05). (*) After 3 and 7 days cross-linked collagen

scaffolds resist wound contraction, maintaining wound size when compared to collagen alone and control wounds. The arrow in the following images

shows the presence of the applied scaffold. (b) Image of � 40 Mason’s Trichrome stained 3 days wound treated with collagen alone. (c) Image of � 40

Mason’s Trichrome stained 3 days wound treated with cross-linked collagen. (d) Image of � 40 Mason’s Trichrome stained 3 days not treated wound.

Y. Garcia et al. / Biomaterials 29 (2008) 857–868 861

ARTICLE IN PRESS

-10

0

10

20

30

40

50

60

70

3 days 7days 21 days

Time Periods

3 days 7days 21 days

Time Periods

Pe

rce

nta

ge

of

Ep

ith

eli

ali

sa

tio

n (

%)

Collagen Collagen + TG Control Collagen+TG+R283 Collagen Collagen + TG Control Collagen+TG+R283

Collagen Collagen + TG Control Collagen+TG+R283

3 days 7days 21 days

Time Periods

0

10

20

30

40

50

60

70

80

90

100

Pe

rce

nta

ge

of

Co

ntr

ac

tio

n (

%)

-1500

-1000

-500

0

500

1000

1500

Ep

ith

eli

ali

sa

tio

n R

ate

(m

icro

ns

)

0

10

20

30

40

50

60

70

Collagen Control

Treatment Group

Ep

ith

eli

al

Th

ick

ne

ss

(m

icro

ns

)

**

**

*

**

Collagen +TG+R283

Collagen+TG

Fig. 2. Epithelialisation at 3, 7 and 21 days: (a) compares the percentage of epithelium developed in treated wounds at different time periods (n ¼ 6,

pp0.05). (*) After 7 days collagen cross-linked scaffold and control has a statistically reduced percentage of epithelialisation when compared to other

treatments. (b) Shows the percentage of contraction of the treated wounds at different time periods (n ¼ 6, pp0.05). (*) After 3 and 7 days there is

statistically obvious reduced contraction in cross-linked scaffolds, which is not seen at 21 days. (c) Epithelialisation rate of the treated wounds was

expressed in microns (n ¼ 6, pp0.05). (*) After 3 and 7 days the epithelialisation rate of cross-linked collagen scaffolds is significantly higher than any

other treated wounds. The negative values observed in the graph are due to the high contraction values when applying the formula. (d) The epithelial

thickness is not affected by any treatment after 21 days (n ¼ 6, pp0.05).

Y. Garcia et al. / Biomaterials 29 (2008) 857–868862

3.5.2. Collagen type III

The volume fractions of collagen type III found incollagen scaffold treatment alone and collagen cross-linkedtreated wounds were statistically similar after 21 days(Fig. 6). Only wounds additionally treated with mamma-lian transglutaminase inhibitor R283 and the controlwounds (without a collagen scaffold) show a statisticallysignificant increase in collagen III deposition. The distribu-tion pattern of the collagen type III fibers is more obviouson the most superficial areas of the wound, in the samemanner as seen in normal skin [36]. This distribution can beassociated with the most immature areas in the woundhealing process.

4. Discussion

The in vitro effects of mTGase cross-linked proteinscaffolds have been extensively investigated includingstudies done by our research group [16,37–40]. However,the potential use of these scaffolds in dermal woundhealing has not been analysed to date. This is a first studythat reports the in vivo dermal response to enzymaticallycross-linked scaffolds and cross-linked scaffolds loadedwith the mammalian transglutaminase inhibitor R283.In the evaluation of wound closure, the use of sterile

tracing papers at different time periods was estimated to bean objective method for measuring wound areas. In vivo

ARTICLE IN PRESS

0

0.05

0.1

0.15

0.2

0.25

3 days 7days 21days

Time Periods

3 days 7days 21days

Time Periods

Vo

lum

e F

racti

on

Collagen Col+TG Control Col-TG+R283

Collagen Col+TG Control Col-TG+R283

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

Vo

lum

e F

racti

on

*

*

*

Fig. 3. Fibroblast and inflammatory cell infiltration at days 3, 7 and 21: volume fraction of the cell types in Table 1 at different time periods (n ¼ 6,

pp0.05). (a) Shows fibroblast cell infiltration in different scaffolds. (*) at 7 days control wounds appear to have significantly reduced fibroblast density

with regard to other treatments. (b) Shows total inflammatory cell infiltration in different scaffolds. At 7 days cross-linked scaffolds show and increased

inflammatory reaction when compared to control and collagen alone that is not present at 21 days.

Y. Garcia et al. / Biomaterials 29 (2008) 857–868 863

and post-excisional wound photographs were used asvisual aids in the assessment of wound healing but theywere not considered an option for quantification purposes.Measurements cannot be performed accurately on photo-graphs of non-flat surfaces and even if the wounds weresurgically removed the obvious limitation is that it wouldbe impossible to obtain data corresponding to day 0. Oneof the major difficulties in the evaluation of wound closureby this method was to determine an objective visualboundary between wounded and healed areas after 7 and21 days. In practical terms, it was difficult to distinguish thechange in color tones with the corresponding epithelialisedareas. The assessment of epithelialisation proved, at

posteriori, that all wounds were fully healed after 21 daysand what were traced at 21 days were not wounds butdiscolored areas, invalidating the data collected fromwound closure at this time point.The lack of panniculus carnosus on the more cranial

wounds was originally thought to negatively influence thewound closure by contraction; however, no statisticaldifference (data not shown) was observed in this parameterbetween treatment locations or between treatments. Whenevaluating wound epithelialisation at the earliest stages, itbecame evident that in some wounds the precautionstaken to avoid wound disturbance were not alwayssuccessful. The scaffold did not always constitute a

ARTICLE IN PRESS

0

200

400

600

800

1000

1200

1400

1600

3 days 7 days 21 days

Time Periods

3 days 7 days 21 days

Time Periods

Su

rfa

ce

Are

a (

mic

ron

s2x

10

00

)

Collagen Col+TG Control Col-TG+R283

Collagen Col+TG Control Col-TG+R283

0

50

100

150

200

250

300

Va

sc

ula

r L

en

gth

(m

icro

ns

x1

00

0)

*

*

5 µm 5 µm

Fig. 4. Angiogenesis: (a) vascular surface area at 3, 7 and 21 days (n ¼ 6, pp0.05). (*) Collagen cross-linked scaffold with no inhibitor significantly

increases new vascularisation by increasing the surface area of capillary profiles at the wound bed. (b) Total vascular length correlates with the results of

surface area at 3, 7 and 21 days. (*) Collagen cross-linked scaffold with no inhibitor significantly increases angiogenesis by increasing the total vascular

length of capillaries at the wound bed. (c) and (d) are images taken from the same animal at 7 day time period. Their magnification is � 400 and both

images are stained with Mason’s Trichrome. (c) Granulation tissue of wound treated with non-cross-linked collagen scaffold. (d) Granulation tissue of

wound treated with mTGase cross-linked collagen scaffold where increased length and density of blood vessels is visually evident.

Y. Garcia et al. / Biomaterials 29 (2008) 857–868864

uniform undisturbed layer filling the wound space intotality (Fig. 1). This finding could partially account forthe wide standard deviations in the collected data. Theevaluation of wound epithelialisation, when expressed as a

percentage, does not provide enough information to drawconclusions about any of the treatments. However, oncethe influence of contraction (Fig. 2) on wound closure iseliminated (Eq. (2.3)), the enzymatically cross-linked

ARTICLE IN PRESS

0

0.05

0.1

0.15

0.2

0.25

0.3

Collagen Col+TG Control ColTG+R283 Normal Skin

Treatment Group

Co

lla

ge

n I

II/I

Ra

tio

*

*

*

Fig. 5. Collagen fiber orientation: explant sections stained with picrosirius dye and viewed under polarised light to detect collagen fibres. Green stained

fibres demonstrate the presence of collagen type III-like fibres and yellow and red stained fibres demonstrate the presence of collagen type I-like fibres.

Time point: 21 days. (a) The ratios of collagen-like type III/I are significantly reduced in collagen, collagen cross-linked (without R283) and control-treated

wounds (n ¼ 6� 400 magnification, pp0.05). (b) Collagen scaffold-treated wound, (c) collagen cross-linked scaffold-treated wound, (d) control-treated

wound, (e) collagen cross-linked and R283-treated wound, (f) Normal skin and (g) transition zone (scale bar of 30mm).

Y. Garcia et al. / Biomaterials 29 (2008) 857–868 865

ARTICLE IN PRESS

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Collage

n

Collage

n+TG

Con

trol

Collage

n+TG

+r28

3

Nor

mal

Treatment Group

Vo

lum

e F

rac

tio

n o

f C

oll

ag

en

III

*

*

Fig. 6. Collagen type III deposition at 21 days: (a) graph comparing volume fraction of collagen type III deposition with different treatments. (b) Central

section of collagen cross-linked-treated wound immune-stained for collagen type III. (c) Central section of control-treated wound immune-stained for

collagen type III. (d) Central section of collagen cross-linked and inhibitor R283-treated wound immune-stained for collagen type III. (e) Normal

unwounded areas of rat skin were used as control to compare normal collagen type III distribution and amount.

Y. Garcia et al. / Biomaterials 29 (2008) 857–868866

scaffolds show an increase in the ER at 3 and 7 days. Themammalian transglutaminase inhibitor did not affect thisprocess. The structural appearance of this epithelium wasalso analysed after 21 days, with no effect detected on thestructure. This is quite surprising for scaffolds loaded with

R283 since this inhibitor is active across the mammaliantransglutaminase range [41].The effect that cross-linked collagen scaffolds have in

preventing wound contraction is not only shown whencalculating the percentage of contraction in the wound but

ARTICLE IN PRESSY. Garcia et al. / Biomaterials 29 (2008) 857–868 867

additionally corroborated when measuring wound areas.The area values of the cross-linked scaffold-treated groupswith or without transglutaminase inhibitor are higherfor a period of 7 days of wound repair. This effect issignificant from the clinical perspective. Wounded areasthat risk restriction of functionality due to contracted scarswill potentially benefit from scaffolds that resist contrac-tion forces and therefore skin tension until healing iscomplete [42,43].

The positive effect that enzymatically cross-linkedcollagen scaffolds without the mammalian transglutami-nase inhibitor have on new vascularisation of the woundbed is probably the most important characteristic for theapplication of this scaffold as a dermal substitute incompromised wounds. Evidence of the higher surface areaof blood vessels combined with the increased vessel lengthensures an effective and increased perfusion of the woundarea. This effect could initially be attributed to the largerwound areas in cross-linked-treated wounds more than theeffect of the scaffold in angiogenesis, however, the relativevalues of surface density and length density support ourconclusion that it is due to the effect of the scaffold(data not shown). The surface of endothelial cells is animportant source of tTG [44]; an enzyme that is believed tobe involved in cell migration [44,45] and cell adhesion[20,44] without using its transamidating activity [46].Inhibiting tTG using R283 at 1mM concentration has beenreported as having no effect on in vitro models of vasculartubular development [44]. It can be speculated that thereason why angiogenesis with R283-loaded collagencross-linked scaffolds does not match its inhibitor-freecounterpart, is that the migration of endothelial cells isaffected by the inhibition of tTG. This direct involve-ment of tTG in angiogenesis endorses the report byHaroon et al. in 1999 of increased length density of thevascular network after the topical application of recombi-nant tTG to wounded skin [46]. However, it was also foundin tumour models that addition of increased tTG couldinhibit angiogenesis suggesting that although necessary,increased amounts could be non-advantageous by promot-ing matrix accumulation by protein cross-linking [44].However, it can be hypothesised that an advantage of tTGinhibition would be down-regulation of the local expres-sion of cytokines directly involved in wound repair(transforming growth factor b1, interleukin-6 and tumournecrosis factor-a) [19] which would therefore affectangiogenesis.

The evaluation of scarring after 21 days was prematureas the collagen fibres were still at the early stages ofremodelling (thin bundles), though, if the time periods inthe animal model were prolonged the identification ofwounded areas could have become an issue and the animalmodel should be adapted accordingly (bigger wounds orwound frames to counteract wound closure by contrac-tion). Cross-linking with mTGase does not have a directeffect on collagen fibre orientation as seen by polarisedlight microscopy of the edge and centre of the wound. The

instauration over time of normal collagen fibre structureand distribution in transition areas [Fig. 5(g)] indicates thatcollagen architecture will return to normal in most centralareas with the exception of the presence of dermal adnexa.Immunohistochemical staining showed that inhibition

of tTG, which is likely to be the prime mammaliantransglutaminase present in the dermis initially, up-regulates collagen type III expression and deposition.Although surprising given other data [19], this findingcorrelates with the inhibition of collagen type III, I and IVexpression found after exogenous administration of tTG toin vitro models of angiogenesis [42]. Moreover in ourmodel the collagen implant presented to cells is alreadyhighly cross-linked making it much more complex. It istherefore difficult at this stage to provide a mechanism forhow the inhibitor is affecting matrix deposition but clearlyit is playing some role in this process. tTG is known tostabilise and promote ECM accumulation and when thisenzyme is present in excess , for example after continuoustissue insult then fibrosis can result [19]. Therefore itbecomes paramount that for the success of any implantedscaffold not only to focus on topical therapy but also toinvestigate systemic means to preserve tissue integrity bymaintaining physiological levels of tTG and ECM duringthe remodelling process without inducing an excess andpromoting fibrosis [19].

5. Conclusions

mTGase cross-linked scaffolds provide an optimumsubstrate for cell migration, preventing wound contraction,stimulating epithelialisation and neoangiogenesis withoutinducing significant inflammatory reaction. The additionof a mammalian transglutaminase inhibitor to modulatematrix deposition does not in this model add anyadvantage. Our results suggest that this scaffold can beused as a dermal substitute with or without additionalcellular components.

Acknowledgements

This experimental work has been funded by The IrishHigher Education Authority’s Program for Research in ThirdLevel Institutions, Enterprise Ireland Research InnovationFund and EPSRC (Grant reference GR/S21755/02).The comments provided by Prof. Peter Dockery and

Jerome Henry on areas of stereology and statisticsrespectively are considered a valuable and essentialcontribution to this manuscript.

References

[1] Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive

extracellular microenvironments for morphogenesis in tissue engi-

neering. Nat Biotechnol 2005;23(1):47–55.

[2] Stock UA, Vacanti JP. Tissue engineering: current state and

prospects. Annu Rev Med 2001;52:443–51.

ARTICLE IN PRESSY. Garcia et al. / Biomaterials 29 (2008) 857–868868

[3] Ponec M. Skin constructs for replacement of skin tissues for in vitro

testing. Adv Drug Deliv Rev 2002;54(Suppl 1):19–30.

[4] Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J

Pharm 2001;221(1-2):1–22.

[5] Friess W. Collagen-biomaterial for drug delivery. Eur J Pharm

Biopharm 1998;45(2):113–36.

[6] Yannas IV, Burke JF, Gordon PL, Huang C, Rubenstein RH. Design

of artificial skin II. Control on chemical composition. J Biomed

Mater Res 1980;14:131–70.

[7] Badylak SF. The extracellular matrix as a scaffold for tissue

reconstruction. Semin Cell Dev Biol 2002;13(5):377–83.

[8] Supp DM, Boyce ST. Engineered skin substitutes: practices and

potentials. Clin Dermatol 2005;23(4):403–12.

[9] Enoch S, Grey JE, Harding KG. Recent advances and emerging

treatments. Br Med J Clin Res Ed 2006;332(7547):962–5.

[10] Balasubramani M, Kumar TR, Babu M. Skin substitutes: a review.

Burns 2001;27(5):534–44.

[11] Ehrenreich M, Ruszczak Z. Update on tissue-engineered biological

dressings. Tissue Eng 2006;12(9):2407–24.

[12] Eisenbud D, Huang NF, Luke S, Silberklang M. Skin substitutes and

wound healing: current status and challenges. Wounds 2004;16(1):

2–17.

[13] Jones I, Currie L, Martin R. A guide to biological skin substitutes. Br

J Plast Surg 2002;55(3):185–93.

[14] Angele P, Abke J, Kujat R, Faltermeier H, Schuman D, Nerlich M,

et al. Influence of different collagen species on physico-chemical

properties of crosslinked collagen matrices. Biomaterials 2004;25(14):

2831–41.

[15] Orban JM, Wilson LB, Kofroth JA, El-Kurdi MS, Maul TM, Vrop

DA. Crosslinking of collagen gels by transglutaminase. J Biomed

Mater Res 2004;68(4):756–62.

[16] Chau DY, Collighan RJ, Verderio EA, Addy VL, Griffin M. The

cellular response to transglutaminase-cross-linked collagen. Bioma-

terials 2005;26(33):6518–29.

[17] Collighan RJ, Cortez JA, Griffin M. The biotechnological applica-

tions of transglutaminases. Minerva Biotechnol 2002;14(2):143–8.

[18] Griffin M, Casadio R, Bergamini CM. Transglutaminases: nature’s

biological glues. Biochem J 2002;368(Pt 2):377–96.

[19] Verderio EA, Johnson T, Griffin M. Tissue transglutaminase in

normal and abnormal wound healing: review article. Amino Acids

2004;26(4):387–404.

[20] Heath DJ, Christian P, Griffin M. Involvement of tissue transgluta-

minase in the stabilisation of biomaterial/tissue interfaces important

in medical devices. Biomaterials 2002;23(6):1519–26.

[21] Lorand L, Graham RM. Transglutaminases: crosslinking enzymes

with pleiotropic functions. Nat Rev 2003;4(2):140–56.

[22] Freund KF, Doshi KP, Gaul SL, Claremon DA, Remy DC, Baldwin

JJ, et al. Transglutaminase inhibition by 2-(2-oxopropyl)thio

imidazolium derivatives—mechanism of factor Xiiia inactivation.

Biochemistry 1994;33(33):10109–19.

[23] Balklava Z, Verderio E, Collighan R, Gross S, Adams J, Griffin M.

Analysis of tissue transglutaminase function in the migration of swiss

3T3 fibroblasts—the active-state conformation of the enzyme does

not affect cell motility but is important for its secretion. J Biol Chem

2002;277(19):16567–75.

[24] Pandit A, Ashar R, Feldman D. The effect of TGF-beta delivered

through a collagen scaffold on wound healing. J Invest Surg 1999;

12(2):89–100.

[25] Mayhew TM. The new stereological methods for interpreting

functional morphology from slices of cells and organs. Exp Physiol

1991;76(5):639–65.

[26] Garcia Y, Breen A, Burugapalli K, Dockery P, Pandit A.

Stereological methods to assess tissue response for tissue-engineered

scaffolds. Biomaterials 2007;28(2):175–86.

[27] Glaser JR, Glaser EM. Stereology, morphometry, and mapping: the

whole is greater than the sum of its parts. J Chem Neuroanatomy

2000;20(1):115–26.

[28] Amenta PS, Amenta PS. Connective tissue. In: Histology, from

normal microanatomy to pathology, 7th ed. Padova, Italy: Piccin

Nuova Libraria S.p.A.; 1997. p. 84–92.

[29] Stevens A, Lowe J. Immune system. In: Human histology. 3rd ed.

Philadelphia: Elsevier Mosby; 2005. p. 125–44.

[30] Nyengaard JR. Stereologic methods and their application in kidney

research. J Am Soc Nephrol 1999;10(5):1100–23.

[31] Nyengaard JR, Bendtsen TF, Russell B, Lokkegaard A, Tang Y,

Gundersen HJG. A stereological approach to capillary networks. In:

Sharma AK, editor. Morphometry applications to medical science.

New Delhi: Macmillan India Ltd.; 1996. p. 217–31.

[32] Madibally SV, Solomon V, Mitchell RN, Van De Water L, Yarmush

ML, Toner M. Influence of insulin therapy on burn wound healing in

rats. J Surg Res 2003;109(2):92–100.

[33] Rowe AJ, Finlay HM, Canham PB. Collagen biomechanics in

cerebral arteries and bifurcations assessed by polarizing microscopy.

J Vasc Res 2003;40(4):406–15.

[34] Brody S, Pandit A. Microarchitectural characterization of the aortic

heart valve. Adv Exp Med Biol 2004;553:167–86.

[35] Cuttle L, Nataatmadja M, Fraser JF, Kempf M, Kimble RM,

Hayes MT. Collagen in the scarless fetal skin wound: detection

with picrosirius-polarization. Wound Repair Regen 2005;13(2):

198–204.

[36] Druecke D, Lamme EN, Hermann S, Pieper J, May PS, Steinau HU,

et al. Modulation of scar tissue formation using different dermal

regeneration templates in the treatment of experimental full-thickness

wounds. Wound Repair Regen 2004;12(5):518–27.

[37] O’Halloran DM, Collighan RJ, Griffin M, Pandit AS. Characteriza-

tion of a microbial transglutaminase cross-linked type II collagen

scaffold. Tissue Eng 2006;12(6):1467–74.

[38] Chen RN, Ho HO, Sheu MT. Characterization of collagen matrices

crosslinked using microbial transglutaminase. Biomaterials 2005;

26(20):4229–35.

[39] Ito A, Mase A, Takizawa Y, Shinkai M, Honda H, Hata K, et al.

Transglutaminase-mediated gelatin matrices incorporating cell adhe-

sion factors as a biomaterial for tissue engineering. J Biosci Bioeng

2003;95(2):196–9.

[40] Harrison CA, Layton CM, Hau Z, Bullock AJ, Johnson TS, MacNeil

S. Transglutaminase inhibitors induce hyperproliferation and para-

keratosis in tissue-engineered skin. Br J Dermatol 2007;156(2):

247–57.

[41] Jones RA, Kotsakis P, Johnson TS, Chau DY, Ali S, Melino G, et al.

Matrix changes induced by transglutaminase 2 lead to inhibition of

angiogenesis and tumor growth. Cell Death Differentiat 2006;

13(9):1442–53.

[42] Brissett AE, Sherris DA. Scar contractures, hypertrophic scars and

keloids. Facial Plast Surg 2001;17:263–72.

[43] Billingham RE, Russell PS. Incomplete wound contracture and the

phenomenon of hair neogenesis in rabbits’ skin. Nature 1956;177:

791–2.

[44] Akimov SS, Belkin AM. Cell surface tissue transglutaminase is

involved in adhesion and migration of monocytic cells on fibronectin.

Blood 2001;98(5):1567–76.

[45] Verderio EA, Telci D, Okoye A, Melino G, Griffin M. A novel RGD-

independent cell adhesion pathway mediated by fibronectin-bound

tissue transglutaminase rescues cells from anoikis. J Biol Chem

2003;278(43):42604–14.

[46] Haroon ZA, Hettasch JM, Lai TS, Dewhirst MW, Greenberg CS.

Tissue transglutaminase is expressed, active, and directly involved in

rat dermal wound healing and angiogenesis. Faseb J 1999;13(13):

1787–95.