Standardized qualitative evaluation of scar tissueproperties in an animal wound healing model

DIRK A. HOLLANDER, MDa; HANS J. ERLI, MD a; ALF THEISEN, DVM b; STEPHAN FALK, MD c;THOMAS KRECK, MD d; STEFAN M €UULLER, MD d

There is a great need to establish reproducible methods for evaluative studies of wound treatment and woundhealing. Validation of the healing process through optical techniques, as well as histologic and immunohistochem-ical methodologies, have been improved and to some extent have become well-established assays. Data relatingto biomechanical properties, e.g., evaluation of the tensile strength of scar tissue that forms in experimental woundtreatment strategies, are less widely available. We chose the domestic pig as an animal model in which to examineepidermal wound healing. We implanted specially made chambers that served to isolate the wounds and preventepidermal migration from the edges. We performed histologic and immunohistochemical analyses as well asevaluation of biomechanical qualities of scar tissue using laser tensiometry. Pig skin is well suited for wound healingstudies, and wound creation, implantation of the chambers, and the regular changing of dressings could all becarried out in the operating theater. In addition to established macroscopic evaluation and microscopicdocumentation, the need for objective biomechanical assessment of scar tissue by measuring tensile strength hasbeen met using laser tensiometry. By optimizing methods for measuring tensile strength, it is possible to evaluate thebiomechanical quality of scar tissue formed following different courses of wound treatment, as well as histologicassessment. (WOUND REP REG 2003;11:150–157)

There are few medical procedures, either established or

experimental, that are as poorly standardized as those

involving wound healing. The possibility to obtain com-

prehensive and reproducible evaluations of the results of

wound healing or of scar formation are equally deficient.

Only in the area of histologic and immunohistochemical

examination techniques for the appraisal and statistical

analysis of defined treatment concepts—which have

become standard procedure in studies of wound heal-

ing—has considerable progress been made in recent years.

It has also been possible to improve validation of healing

processes through noninvasive, high-quality, optical-visual

techniques, ultrasound, and computer imaging.

Despite these areas of development, the results of

wound healing, whether clinical or in animal experiments,

are still characterized largely on the basis of the empirical

experience of the experimenter and expressed in terms

such as ‘‘stable scar conditions,’’ ‘‘normo-elastic proper-

ties,’’ or ‘‘biomechanically normal.’’

The aim of this study was to standardize, in a

comprehensive manner and using a reproducible animal

model, the course of wound healing and scar formation

resulting from a defined method of treatment, in histologic

PTFE Polytetrafluroethylene

From the Department of Trauma Surgerya, UniversityHospital RWTH Aachen, Aachen; Central Facilityof Researchb, Johann Wolfgang Goethe-Uni-versity, Frankfurt; Pathology Associates Frank-furtc, Frankfurt; and Department of Trauma,Hand, and Reconstructive Surgeryd, JohannWolfgang Goethe University, Frankfurt, Ger-many.

Reprint requests: Dirk A. Hollander, MD, Departmentof Trauma Surgery, University Hospital RWTHAachen, Pauwelsstr. 30, 52074 Aachen,Germany.Fax:+49-241-80-82415;Email:dhollander@ukaachen.de.

Copyright � 2003 by the Wound Healing Society.ISSN: 1067-1927 $15.00 + 0

150

and immunohistochemical, optical-visual and, above all, in

biomechanical terms.

We chose the domestic pig as a well-known, classic

model for epidermal wound healing.1–4 Both in the anatomy

of their skin and in the physiology of wound healing, pigs

are very similar to humans.2–5 In pigs and humans dermis

and epidermis have a similar relative thickness. In addition

to the dermal papillary layers, both have epidermal rete-

ridges, sebaceous glands, apocrine sweat glands, as well as

subdermal fat and an almost identical density of hair

folliciles. However, unlike human skin, pig skin contains an

elastic membrane found in the hypodermis. The pig dermis

is also somewhat less richly vascularized than human skin

and contains no eccrine sweat glands.1,5,6

Because of the aforementioned hypodermal mem-

brane, reliable studies of a wound measuring 4 · 4 cm are

limited to about 28 days; within this time span the wound

is completely closed, mainly by contraction of the

surrounding tissue.7,8

To prevent this contraction, we implanted custom-

made wound chambers consisting of polytetrafluoroethyl-

ene (PTFE), which served to isolate the wounds that were

created and prevent epidermal migration from the wound

edges. Similar implants have already been described by a

number of other workers.7,9–11

The basis of documenting macroscopic study results

should be regularly recorded data obtained using either an

established optical-photographic, sonographic or plani-

metric method, or 3-D computer imaging.

In order to provide functional data reflecting the

quality of wound/scar tissue, it is necessary to include

biomechanical evaluation as an essential part of any test of

scar quality.

For this purpose, we decided to subject the excised

scar tissue to tensiometric testing in a laser extensiometer,

thus providing data on its tensile strength and breaking

point, as well on the degree of stretching when maximum

tension is applied to the tissue.

First descriptions of tensiometric experiments are

found in the literature of the 19th century. Paget, in 1853,12

and Chlumsky, in 1899,13 used such experiments to test the

strength of sutures applied to the skin and enteroanasto-

moses. However, it was not until 1929 that Howes et al.

rediscovered the method to investigate the strength of

healed wounds.14 Measurements of tensile strength were

again applied in the 1960s in studies investigating the

mechanical strength of wounds, although, mainly because

of technical difficulties and insufficient reproducibility, no

workable standards were established.15–20 In more recent

studies, e.g., Mansour et al. (1993),21 technical improve-

ments of the clamps were possible. The most recent

medical publication on testing tensile strength is by

B€uucheler (1999),22 who shows that using a DIN-based test

procedure it is possible to conduct comparative mechan-

ical tests of the strength of surgically stitched wounds.

In this article, the established animal model with

special wound chambers, the creation of the wounds, and

the various studies that contribute to a meaningful analysis

of the wound healing process are described in detail. As a

point of reference, the investigations were all conducted

on normal porcine skin.

MATERIALS AND METHODSThe domestic pigs, of the so-called German Landrasse,

were all castrated males weighing about 30 kg each. They

were kept in separate boxes at variable room temperature

and humidity in accordance with Guideline 86/609/EEC of

the European Commission and the Guidelines of GV-Solas.

Wound chambersThe subcutaneous wound chambers, to be implanted and

anchored below the edges of the inflicted wound, were

made in the technical department of our medical laborat-

ory to a precisely defined size from PTFE blocks (Figure 1).

The inside diameter of the chambers corresponds with the

dimensions of the wound (4 · 8 cm).

Wounding procedureAfter obtaining permission to conduct experiments with

vertebrate animals in compliance with German animal

protection laws, we made three wounds running parallel to

both sides of the animals’ backbone, each with an area of

8 · 4 cm. The exact size and shape of the wounds were

measured using the standardized PTFE wound chamber

(Figure 2). By removing epidermal, dermal, and subcuta-

neous tissues, it was possible to characterize the depth of

FIGURE 1. Mechanical PTFE wound chambers used to isolate

wounds in the pig.

WOUND REPAIR AND REGENERATIONVOL. 11, NO. 2 HOLLANDER ET AL. 151

the wounds down to the fascia of the musculus thoracicus

longus. After surgical creation of the wounds, six PTFE

chambers were anchored in them subcutaneously using

nonresorbent stitches so that isolated wound areas could

be independently treated and studied.

Following intramuscular premedication with keta-

mine/xylazine/midazolam (20 mg/2 mg/0.5 mg/kg body

weight), both flanks of the pigs’ backs were completely

shaved, first dry, then wet, and subsequently disinfected

repeatedly with 70% ethyl alcohol.

Anesthesia during the operation was by intravenous

administration of ketamine/midazolam (33 mg/1.0 mg/kg/h)

using a perfusor (perfusor VI or perfusor fm; Braun-

Melsungen). During the operation, 0.01 mg/kg buprenor-

phine was administered intravenously. The same dose was

administered again 12 hours later. The wounds were made

in the aforementioned manner, while animals were under

full anesthesia and after disinfecting the skin once again.

After creating the wounds, the standardized PTFE

wound chambers were implanted. Before starting a

particular treatment protocol, each wound was washed

with sterile normal saline solution and checked for

bleeding. The defined wound treatment was performed

as follows: The undamaged skin surrounding the wound

chambers was cleaned and the wound chamber itself,

depending on the defined treatment, covered with a

secondary dressing, a plaster dressing, and subsequently,

in order to protect the wound chambers, a close-fitting

hose dressing around the whole animal was applied.

Dressings were changed under sedation as described

above.

After terminating the experiment, the animals were

euthanized by intracardial injection of T61 (1 ml/5 kg body

weight).

Photographic and optical-visual documentationAt defined points in time, depending on the particular plan

of treatment, standardized photographs were taken with a

Canon EOS 500 camera (28–80 mm zoom lens), with a

distance of 30 cm and right-angled to the wound surface

(Figure 3).

Histology and immunohistochemistry proceduresMicroscopic documentation of the course of wound

morphology was performed at regular intervals by taking

biopsies reaching deep into the dermal tissue using a

standardized skin punch (4 mm diameter). After fixation

for 24 hours in 4% neutral buffered formaldehyde solution,

the samples are embedded in paraffin, 4-lm-thick sections

were made perpendicular to the skin surface from the

resulting paraffin blocks, and they were stained. For

histology, well-established and standard laboratory proce-

dures for hematoxylin and eosin and Goldner’s trichrome

stain were used (Figure 4).

A B

C D

FIGURE 3. Macroscopic visualization of

wound healing. (A) Individual area of skin

defect down to muscle fascia, isolated by

the wound chamber. (B–D) Healing pro-

gression at weeks 1, 2, and 3, respectively.

A BFIGURE 2. Surgical wounding procedure.

(A) Experimental animal under full

anesthesia with internal diameters of

chambers outlined. (B) Experimental ani-

mal with implanted wound chambers.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2003152 HOLLANDER ET AL.

All immunohistochemical investigations were per-

formed using a standard immunoalkaline phosphatase

technique. Briefly, sections were deparaffinized, rehy-

drated, and incubated overnight with antibodies at optimal

dilution. Antibody binding was then visualized by incuba-

tion with an alkaline-phosphatase-coupled secondary

antibody directed against the primary mouse antibody

with subsequent addition of a chromogen, Neufuchsin.

Binding of the primary antibody, i.e., detection of the

epitope in question, resulted in a red reaction product

easily identifiable in sections counterstained by hematox-

ylin. The antibodies listed in Table 1 were used to identify

a variety of antigens (Figure 5). Antibody reactivity

was assessed semiquantitatively by two independent

observers.

Laser-tensiometryAs a standardized approach to measuring biomechanical

stability of scar tissue, tensile strength was determined in

dM[N/cm2 ], i.e., the force needed to break a piece of tissue

in relationship to its cross section. Width of the sample was

standardized by the punch used to obtain it, and its depth

was measured to an accuracy of about 1 mm using a pair of

calipers. Extension of the tissue was documented under

conditions of maximum tension (tensile strength ¼ eM[%]). The rate of tension is 10 mm/min with a 2 bar plug

pressure of the test clamps at the beginning of the test,

running up to a maximum of 7 bar. The tensiometer used

was a general testing device made by Hounsfield (model

H10K).

Excision of developing scar tissue for tensiometry was

carried out at defined intervals. After removal of the PTFE

chambers, the corresponding area of newly formed scar

tissue was carefully dissected down to the muscle fascia

and removed, post mortem, without disrupting its conti-

nuity. The excised tissue was transported and stored in

200 ml of Ringer solution at 4�C. During periods of

experimental intervals, the samples were kept at room

temperature and 50% relative humidity.

Identical tissue samples for laser tensiometry were

obtained using a standardized punch. In consideration of

scar size, we chose test bodies of type 5 (Figure 6),

BA FIGURE 4. Histological examination of

wound tissue (A) Standard H & E stain of

normal pig skin showing normal epidermal

squamous epithelium (arrow a) with scat-

tered capillaries (arrow b). (B) Trichrome

stain after Goldner; differential staining of

keratin (arrow a), epidermis (arrow b), and

collagen fibers of the dermis (arrow c).

Scale bar represents 18 lm. (Original mag-

nification · 66).

Table 1. Antibodies appropriate for wound healing studies and their targets

Target Antibody Antigen Dilution Source@

Keratinocytes Anti-CK 10 Cytokeratin 10 1 : 250 DAKO, Hamburg, GermanyBasement membrane Anti-laminin Laminin 1 : 200 DAKOProliferating cells MIB 5 Ki 67 1 : 100 DiANOVA, Hamburg, GermanyCapillaries, angiogenesis Anti-vWF Von Willebrand

Factor (F VIII-ass. Antigen)1 : 900 DAKO

Myeloid/histiocytic cells MAC 387 Calprotein 1 : 1000 DAKOFibroblasts

(other mesenchymal cells)Vim 3B4 Vimentin 1 : 220 DAKO

Myofibroblasts 1A4 a-smooth muscle actin 1 : 100 DAKO

@This is one supplier of the indicated antibody. Other sources of comparable antibodies are available from different companies.

WOUND REPAIR AND REGENERATIONVOL. 11, NO. 2 HOLLANDER ET AL. 153

corresponding to the European Standard of the Interna-

tional Organization for Standardization, EN ISO 527.

In studying the tensile strength of scar tissue, the

method of clamping it into the tensiometer is of great

importance. The sample must be held firmly enough so as

not to slip out of the clamps’ grip as it is subjected to

increasing tension, while at the same time it must suffer no

damage as a result of clamping.

After conducting a number of preliminary experi-

ments, we decided on the pneumatic clamps made by

Instron (Darmstadt, Germany) with a maximum plug

pressure of 7 bar. The clamps measured 25 · 20 mm with

a grip profile of 0.5 mm.

RESULTSAs already mentioned, on account of its similarity with

human skin, pig skin is well suited for studies of wound

healing. The animals, kept in the central animal research

facility under veterinary care, were easy to deal with.

Wound creation, implantation of the PTFE chambers, the

regular changing of dressings and other measures could be

carried out in the operating theater, with the animals under

either sedation or full anesthesia without any problems or

complications.

Wound chambersThe PTFE chambers are easily sutured into place

beneath the edges of the artificial wound. Thus,

A B

C D

E F

G

FIGURE 5. Series of immunohistochemical

stains used to follow changes in important

cellular and structural elements of healing

wounds. (A) Cytokeratin 10 staining of

epidermal squamous epithelium (arrow).

(B) Staining with KI 67 antibody highlights

epidermis with basal cell proliferation

activity (arrows). (C) Staining of basal

basement membrane with anti-laminin

antibody (arrow). (D) Dermal vessel endo-

thelium marked by factor VIII (arrows). (E)

Individual macrophages shown by CD 68

antibody staining (arrows). (F) Profusion of

mesenchymal cell elements stained with

vimentin-antibody. (G) Actin antibody

stains smooth muscle cells in dermal tissue

(arrow a). Small arterioles (arrow b) serve

as internal control. Scale bar represents 14

lm in B and D, and 28 lm in A, C, E, F, and

G. (Original magnification · 33 in A, C, E, F,

G and · 66 in B, D).

B2 B1

L1

L2

FIGURE 6. Standard test punch type 5, EN ISO 527 (B1 6 mm, B2

25 mm, L1 33 mm, L2115 mm).

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2003154 HOLLANDER ET AL.

individual areas of skin defects are created, which can be

independently treated and evaluated. During the course

of the experiments, the chambers showed only a small

degree of deformation and remained sufficiently stable;

they did not cause any allergic reactions. Occasionally,

due to strong mechanical stress (e.g., the animal rolling

on the floor), the sutures with which the chambers were

anchored had to be renewed or strengthened. Following

post mortem removal, the chambers were thoroughly

cleaned, sterilized, and individually packed, and thus

ready for renewed use.

Photographic and optical-visual documentationStandardized photography of the wounds (Figure 3)

permits the progress of treatment and healing to be

documented over a defined period of time, thus facilitating

detailed description and macroscopic comparisons

(wound coatings, blood supply, granulation, wound secre-

tions, signs of inflammation or infection, epithelialization,

wound measurement, etc.). A typical time-to-healing curve

cannot be provided in this model, because a complete

healing process, i.e., complete epithelialization, is preven-

ted by the wound chambers.

Histologic findingsThe stains that were used are well-established and

standard laboratory procedures that can be easily carried

out by any institute of pathology. Hematoxylin and eosin

staining highlights basophilic and acidophilic structures

and is thus used as a general survey stain. Nuclei take on a

blue/black color, while components of the cytoplasm,

intercellular substances, as well as collagen fibers, are

stained red (Figure 4A). Goldner’s trichrome stain differ-

entiates connective tissue collagen fibers from the sur-

rounding tissue. Nuclei are stained black, cytoplasm red,

and collagen fibers green, thus facilitating assessment of

collagen fiber thickness and its relationship (ratio) to

fibroblasts. As expected, during the early stages of wound

healing there are many fibroblasts and relatively few

collagen fibers, while in later stages progressively more

fibers and fewer fibroblasts are found (Figure 4B).

Immunohistochemical analysisKeratin is a fibrillar protein that, together with actin

filaments and microtubules, forms an essential part of the

cytoskeleton found particular in epithelial tissue. Cytoker-

atin is a characteristic component of keratocytes. The

antibody DAKO-CK10 stains keratin 10, a 56.5-kDa,

intermediate filament protein, which is synthesized mainly

in suprabasal layers of multilayered epithelium. With the

help of this stain it is possible to study whether, and to

what extent, keratinocytes contribute to the formation of

physiological epithelium in wound or scar tissue. The

thickness of the layer of keratinocytes can be accurately

measured using an eyepiece micrometer, thus facilitating

assessment of the formation of a functional covering of

wound surface and its aesthetic implications. Keratino-

cytes in hair follicles and sweat gland ducts are also stained

by this antibody (Figure 5A).

All cells in the S-phase of their cell cycle synthesize the

nuclear, proliferation-associated antigen, Ki-67. This can

be stained using the MIB5 antibody, thus facilitating the

recognition of proliferating cells. Through quantitative

evaluation it is possible to estimate proliferation activity of

cells in wound and scar tissue and compare it with that in

normal skin. Based on the relationship between cell

proliferation and the rate of wound healing, rapid healing

progress can be expected in situations when high cell

proliferation is present (Figure 5B).

As a component of the lamina rara externa, the

polypeptide laminin forms an essential part of the basement

membrane (up to 1 lm thick). The mentioned antibody

stains laminin, thus offering a way of studying whether a

basement membrane is formed during the course of wound

healing. Because the basement membrane is a functional

component of natural skin, its formation, or lack of it, has

been selected as an important parameter in assessing the

wound healing process. The formation of a basement

membrane would indicate the formation of a physiological

dermal–epidermal junction because cells can attach to it via

coupling proteins. Laminin, a noncollagen glucoprotein

consisting of three polypeptide chains, is a ubiquitous

structural element of the basement membrane and also

plays a regulating role in cell differentiation.

The development of a basement membrane, or its

components, promotes the formation and differentiation of

multilayered skin. Studies of wounds in human skin have

shown that after 4 days the first basement membrane

structures are detectable (Figure 5C).

Factor VIII is synthesized by endothelial cells, megak-

aryocytes, blood platelets, and other cells of endothelial

origin. It is also formed in the tunica interna vasorum and

can serve as a marker for the smallest, newly created

capillaries, the numbers of which per unit area can be thus

determined, providing information on the degree of

vascularization in wound or scar tissue (Figure 5D).

The cells detected with the CD68 antibody are a

parameter for measuring infiltration of a wound with

macrophages, and thus of chronic wound inflammation

and the wound tissue’s capacity for resorption. Evaluation

is quantitative, measured in positive cells per surface area

(Figure 5E).

Vimentin is a 57-kDa intermediate filament protein that

occurs in cells of mesenchymal origin, thus facilitating the

WOUND REPAIR AND REGENERATIONVOL. 11, NO. 2 HOLLANDER ET AL. 155

identification of lymphoid and endothelial cells as well as

smooth muscle cells and fibroblasts. It is the latter that are

of interest here, i.e., their infiltration into the wound tissue

(Figure 5F).

Actin is a contractile element that occurs in fibroblasts

that have undergone differentiation to myofibroblasts.

These elements can be counted and their number placed in

relation to the number of vimentin-positive and actin-

negative fibroblasts, thus providing an indication of the

degree of differentiation. This in turn allows an estimation

of the amount of expected scar contraction and the

resulting loss of elasticity (Figure 5G).

Laser-tensiometryA piece of scar tissue clamped in the tensiometer is shown

in Figure 7B. The tissue was formed 4 weeks after wound

creation in the course of a defined wound healing

experiment. It shows breakage at 24.54% extension under

a force of 90.0 N and at the same rate of extension of

10 mm/min (Figure 7, A–C).

In a typical control study with healthy pig skin, the

punched sample broke when extended by 25.66% under a

force of 275.5 N at a rate of extension of 10 mm/min.

(Figure 8).

DISCUSSIONNumerous in vitro and in vivo wound healing models have

been described in the medical literature. Based on the well-

known similarities in anatomy and physiology of the

wound healing processes between porcine and human

skin, the choice of the pig as the most suitable animal

model for investigating questions related to wound healing

seems clear.

We recommend hematoxylin and eosin staining, which

highlights basophilic and acidophilic structures and thus

should be used as a general survey stain. Goldner’s

trichrome stain differentiates connective tissue collagen

fibers from the surrounding tissue. Elastica stain differen-

tiates the elastic fibers from collagen fibers.

Using cytokeratin 10-antibody it is possible to

characterize squamous epithelium as one measure of

keratinocyte function. The MiB 5 antibody stains the

epidermis with a predominantly basally located prolifer-

ation marker. A clear visualization of the basement

membrane in normal skin is obtained through the use of

the laminin antibody. Factor VIII marks the endothelium

of dermal blood vessels. In normal pig skin, individual

macrophages can be shown using a CD 68 antibody.

Staining with a vimentin antibody shows that the normal

A B C

FIGURE 7. Tissue undergoing biomechanical testing. (A) Excised scar tissue at the end of a wound healing study and prepared for testing.

(B) The beginning of a tensiometric test with the laser tensiometer. (C) The test sample tears in the defined test region.

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80 90 100Dehnung [%]

Kra

ft [

N]

FIGURE 8. Diagram of tensile strength in normal pig skin.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2003156 HOLLANDER ET AL.

pig skin is rich in dermal mesenchymal cells and the

actin antibody stains smooth muscle cells in the dermis

of normal skin.

In addition to the established macroscopic and optical-

visual evaluation of the course of wound healing, and

histological and immunohistochemical documentation,

there is also an important need for biomechanical evalu-

ation of scar tissue.

By optimizing methods of measuring tensile strength,

it is possible to evaluate in a systematic, reproducible, and

standardized way the biomechanical quality of scar tissue

formed following different courses of wound treatment.

We suggest that the procedures described here present

a minimum set of parameters necessary to evaluate an

animal model of wound healing.

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