Biomechanical behavior of scar tissue and uninjured skin in a porcine model

Biomechanical behavior of scar tissue and uninjured skin in aporcine model

David T. Corr, PhD1; Corrie L. Gallant-Behm, PhD2,3; Nigel G. Shrive4,5; David A. Hart, PhD3,5,6

1. Biomedical Engineering Department, Rensselaer Polytechnic Institute, Troy, New York,

2. Division of Plastic Surgery, Northwestern University, Evanston, Illinois,

3. Department of Microbiology & Infectious Disease,

4. Department of Civil Engineering,

5. Department of Surgery, and

6. Department of Medicine, University of Calgary, Calgary, Alberta, Canada

Reprint requests:David T. Corr, PhD, Biomedical Engineering

Department, 7042 Jonsson Engineering

Center, Rensselaer Polytechnic Institute,

110 8th Street, Troy, NY.

Tel: 11 518 276 3276;

Fax: 11 518 276 3035;

Email: [email protected]

Manuscript received: April 2, 2008

Accepted in final form: December 1, 2008

DOI:10.1111/j.1524-475X.2009.00463.x

ABSTRACT

A new method to test axial and transverse tensile properties of skin was devel-oped to improve our understanding of skin mechanical behavior, and how itchanges following injury and formation of a scar. Skin tissue was evaluated at 70days following full-thickness wounding in juvenile female pigs (N514). Sampleswere taken in the axial (cranial–caudal) and transverse (dorsal–ventral) direc-tions, for both scar tissue and uninjured skin, and were evaluated mechanically invitro using a protocol of stress relaxation followed by tensile failure. Uninjuredskin was more compliant, with a larger toe-in region, and faster load relaxation,in the axial direction than the transverse. Such directional differences were notpresent in high-load responses, such as linear stiffness or failure properties. Whencompared with uninjured skin, scars displayed a similar linear stiffness, with con-siderably reduced failure properties, and reduced low-load compliance. Scarsshowed no directional differences in low-load behavior, viscous response, or fail-ure properties. These findings suggest morphological changes that may occurwith injury that are consistent with the viscoelastic and directional changes ob-served experimentally. This improved understanding of how injury affects skinbiomechanical function provides valuable information necessary for the design ofsuccessful grafting procedures and tissue-engineered skin replacements.

Skin is a composite material consisting of collagen-rich fi-brous network embedded in a matrix of ground substance.The matrix of skin consists of a proteoglycan (PG)-richground substance, which provides the tissue its viscous na-ture at low loads.1 The main fibrous constituents, collagenand elastin, account for approximately 80 and 4% ofskin’s dry weight, respectively, and provide the structuralstiffness and elasticity to skin.2,3 This network shows apredominantly planar arrangement of fibers with a princi-pal direction of orientation. Skin exhibits anisotropic, vis-coelastic material behavior, in which the direction ofminimal extensibility correlates with this principal direc-tion of the fibrous network,4 parallel to the clinically ob-served Langer’s lines. This directionality varies with thelocation on the body, and has been observed in both hu-man4–6 and animal (e.g.,7,8) skin.

Much of our current understanding of skin behavior hascome from biomechanical evaluation, specifically tensiletesting. Tensile testing, in which the tissue is stretched andthe tensile force that develops is measured, has been usedin both human and animal skin to study the effect ofaging,9 injury,10–13 and treatments14–16 on skin function.Typically, tensile testing of skin wounds has focused onfailure properties. Tensile failure provides a measure of thebreaking strength, and thus provides of means to assess thewound quality, as well as the strength of surgical repairsand suture, based on burst strength. However, in normal

daily living, skin functions under loads much lower thanthat approaching tissue failure.

In his pioneering work, Karl Langer identified that theload deformation of skin consisted of a highly extensible ini-tial phase followed by a linear response.5,17 In the initialphase, the skin stiffens as it is stretched. This strain-stiffeningbehavior, known as the ‘‘toe-in’’ region of the load-elonga-tion curve, occurs at low loads and is attributed to progres-sive recruitment of collagen fibers: due to the straightening ofslack fibers and the rotation of collagen fibers to align withthe direction of the elongation.1 Langer also observed thetime-dependent mechanical response of skin, indicating itsviscoelastic behavior. When strained, skin will dissipate elas-tic energy stored in the fibrous network via viscous means.This viscous behavior is believed to be a result of the watercontained in the tissue (� 60–72% water18), is influenced bythe presence of PGs, and is manifest in the form of creep andstress relaxation. While burst strength and failure propertieshave typically dominated prior analyses, we are particularlyinterested in the low-load viscoelastic behavior of skin due toits relevance to the mechanical behavior of skin in its normalphysiological operating range. The importance of this low-load behavior,12 and its recovery following injury, has beenrecognized to some degree in other biological soft tissuessuch as tendon,19 ligament,20,21 and skeletal muscle.22

An important step in understanding the response of skinto injury is to better understand the effect of wounding on

Wound Rep Reg (2009) 17 250–259 c� 2009 by the Wound Healing Society250

Wound Repair and Regeneration

i:/BWUS/WRR/463/[email protected]

the viscoelastic behavior and failure properties of skin. Anappreciation of these properties, and how they change withinjury, provides insights into the ability of skin to resistelongation, bear load, and dissipate energy via viscousmeans. When studied in mutually orthogonal directions inthe plane of the skin, these measures give insight into thedirectionality of the healthy tissue, how the directionalproperties change with injury, and whether the injured tis-sue (scar) approaches ‘‘normal’’ behavior over time. Amore complete understanding of the effect of wounding onthe behavior of skin is critical to the design of more effec-tive treatments, scaffolds, or tissue engineered replacementmaterials for the repair of compromised skin.

The aims of the study described here were to identify thelow-load viscoelastic behavior and failure properties innormal uninjured dorsal skin, and in healing wounds fol-lowing acute full-thickness excision in a porcine model.Test specimens obtained in both the axial (cranial–caudal)and transverse (dorsal–ventral) directions were analyzed invitro to provide insight to the material properties and di-rectionality of skin, and how these change with injury andsubsequent healing. These insights will allow the effects ofinjury to be identified with respect to normal skin biome-chanical function. Additionally, the improved understand-ing of uninjured skin behavior will provide a morecomplete and accurate biomechanical ‘‘target’’ to evaluatethe functionality of potential tissue replacements or clini-cal treatments. With such understanding of skin biome-chanics, it will be possible to correlate the findings with thecomposition and organization of scar tissue, as well aswith cell behavior within scars. Integration of these differ-ent aspects of tissue repair is important as new in vivo andin vitro approaches to optimize repair are developed.

METHODS

Experimental methods

Skin wounding

Approval of the Animal Care Committee of the Universityof Calgary was obtained before all experiments were con-ducted. Female [Yorkshire�Red Duroc] F1�Yorkshirebackcross pigs (N514) were purpose-bred for wound heal-ing studies, as described previously.23,24 Pigs (genetic back-ground53/4 Yorkshire11/4 Red Duroc) were enrolled inthe wound healing study at approximately 2 months ofage, when they had reached a body weight of 20–25 kg.Animals were anesthetized and subjected to surgicalwounding as described previously.24,25 Briefly, each ani-mal was fasted for 12 hours before surgery, premedicatedwith an intramuscular (IM) injection of ketamine (15mg/kg) and acepromazine (0.4mg/kg), and induced into gen-eral anesthesia using 1–2% isofluorane delivered by mask.While anesthetized, the animal was positioned and main-tained in ventral recumbency, the surgical field on the backwas shaved using animal clippers, and the dorsal skin waswashed using a full betadine scrub followed by an ethanolscrub. Arrays of 20 full-thickness wounds were surgicallycreated on each animal by excising square sections (2 cm�2 cm) of the skin, including the subcutaneous fat layer,with a scalpel. The wounds were arranged in a row of five

wounds parallel to the spine, with two parallel rows ofwounds on each side of the spine. A minimum distance of2.5 cm was maintained between all wounds. Only the twowound sites in the most cranial row of the wound matrix,distal to the left shoulder, were used for biomechanical eval-uation from each pig. The remaining sites on each animalwere reserved for longitudinal genetic analysis, as part of alarger study in the genetic aspects of wound healing.

Following wounding, hemostasis was induced, and thewounds were sprayed with a wash containing gentamycinand then bandaged. Pain management was provided by anIM injection of butorphanol (0.1mg/kg) for short-termbreakthrough pain (time < 8 hours), and a fentanyl trans-dermal patch (75mg/hour) for longer term pain control(72 hours). Bandaging continued for the first 3 days ofhealing, after which all coverings were removed, and thewounds were left open to heal.

Specimen preparation

Experimental test specimens were obtained at 70 days afterwounding, representative of the late remodeling phase ofhealing. This time point postinjury was chosen specificallybecause the scar tissue has become more normocellular, theextracellular matrix is well established, and further contin-ued remodeling occurs at a slow rate. Upon sacrifice, a sec-tion of skin (8 cm�8 cm), containing both injury sites anduninjured adjacent skin, was taken from the torso of eachanimal (60–65kg), just distal to the left shoulder. Once allsubcutaneous tissue was carefully removed via dissection,dumbbell-shaped test specimens were obtained from theskin samples using a custom designed stainless-steel punch(Figure 1). The punch was designed to provide consistentspecimen geometry (standard deviations of 1.5, 0.4, and0.6% in gauge length, width, and thickness, respectively;2.6% in cross-sectional area), and to avoid stress concentra-tions that can arise from specimen gripping. Furthermore,the aspect ratio of the test specimen ensured that local effectsdue to gripping would be far removed from the gaugelength, and that the applied load on the specimen would beuniformly distributed across the cross-sectional area withinits gauge length. Material test specimens were obtained inthe axial (cranial–caudal) and transverse (dorsal–ventral) di-rections, in both normal skin and healing wounds (Figure 1).Once punched from the surrounding skin, test specimenswere wrapped in saline-soaked gauze, stored in Petri dishesat room temperature (20 1C) until all four specimens wereobtained from the animal, and immediately evaluated me-chanically. This method of fresh specimen preparation elim-inates the possible alterations in material properties that canarise from specimen freezing.

Mechanical testing

Mounting: Before mechanical testing, specimen geometry(length, width, and thickness) was measured within thegauge length using Vernier digital calipers (� 0.01mm). Im-mediately following measurement, the specimen was securedin an INSTRON universal test machine (Model 1122, IN-STRON Corporation, Norwood, MA). The load cell waszeroed with the specimen secured in the upper grip to re-move the weight of the specimen from force measurements.

Wound Rep Reg (2009) 17 250–259 c� 2009 by the Wound Healing Society 251

Biomechanical behavior of scar tissue and skinCorr et al.

Specimen axial alignment was confirmed using an alignmentjig to center the specimen in both the thickness (fore/aft) andwidth (side-to-side) directions. Once the specimen wasaligned to ensure proper axial loading, both the upper andlower serrated soft tissue grips were fully tightened. Eachspecimen was initially mounted in a slightly slackened lengthto avoid axial loading before testing.

Stress relaxation: For stress relaxation testing, the IN-STRON force-scaling factor was set to 1.0 (full-scaleload510N). Specimen zero length was established byelongating the specimen until the measured force increasedto 10.01N. Upon establishing zero length, the specimenwas slowly stretched (0.04mm/second) until the desired0.1N preload was reached, then elongated 1.0mm (ap-proximately 6.0% of gauge length) at a constant rate(2.4mm/second), and held isometrically for 240 seconds.

During the isometric period the force was allowed to relaxwhile the specimen was held at the prescribed 1.0-mm dis-placement (Figure 2). After 240 seconds of relaxation, thespecimen was returned to zero length, then extended tofailure in tension (Figure 3).

Tensile failure: For tensile failure testing, the INSTRONforce-scaling factor was set to 50.0, increasing the full-scaleload to 500N. The specimen was then elongated from zerolength to failure at the same constant rate (2.4mm/second)(Figure 3). All data were recorded using a PC-based dataacquisition system, at a rate of 20Hz. During both relax-ation and failure testing, the tissue was kept hydrated dur-ing testing with a spray of phosphate-buffered saline,applied over the entire gauge length.

Data interpretation

Stress relaxation

Two measurements were analyzed from the force–time dataof the stress relaxation experiments: (a) peak force duringrelaxation, and (b) the rate of relaxation. In the stress relax-ation experiments, the peak force occurred at the end of theelongation phase, at the instant the specimen reached max-imum length. This value serves as an indirect measure of tis-sue stiffness: a higher peak force indicates that more force isdeveloped for the given elongation, and thus the tissue isstiffer over the initial 6% of elongation. Because the 6%elongation is within the low-load region of the load–dis-placement curve, the maximum force generated during re-laxation is indicative of the tissue’s low-load stiffness.

To analyze the rate of relaxation, the time was adjustedso that t50 corresponded to the time of peak load devel-opment (end of elongation). The 240-second period, inwhich the load was allowed to relax while the tissue washeld at 6% elongation, was analyzed. Raw, unfiltered,force–time relaxation data were fit using a power law for-mulation: starting at an initial time point 10 times largerthan the rise time of the 6% elongation, and ending at 240seconds. This starting time point was chosen to avoid tran-sient effects of a displacement that is not a pure step func-tion.26,27 The power law formulation models the transientforce decay as,

FðtÞ ¼ At�n ð1Þwhere A is the magnitude of force and n is the ‘‘rate’’ ofrelaxation. This method describes the rate of relaxation,indicative of the tissue’s viscous energy dissipation, using asingle variable, and has been used previously in ligamentstudies, showing excellent agreement with the mechanicalstress relaxation data.28

Tensile failure

Raw, unfiltered data were analyzed. Time was adjusted sothat t50 corresponded to the onset of displacement. Sixmeasures were obtained from the tensile failure load–dis-placement data: failure load, failure displacement, tough-ness (energy to failure), linear stiffness, limit displacement,and toe-in duration. The tissue’s failure properties weredescribed by the displacement and load at the point of fail-ure, as well as toughness, or the energy absorbed before

Figure 1. (A) Stainless-steel specimen punch dimensions. (B)

Representative test specimens of healing wound (left) and unin-

jured skin (right) taken in the axial direction, from the same animal.


Biomechanical behavior of scar tissue and skin Corr et al.

failure. To obtain toughness, the area under the load vs.displacement curve, from the onset of load to the point offailure, was determined using trapezoidal integration (Fig-ure 3B). Toughness indicates a material’s general resis-

tance to failure, whereas the failure load and displacementidentify the ultimate strength andmaximum extensibility ofthe material, respectively. The linear stiffness was deter-mined as the slope of the load–displacement curve over therange from 50–80% of the failure displacement (Figure3A). This region of the data was highly linear (R2 > 0.997),and describes the stiffness for elongations that extend be-yond the toe-in region (21,29 in ligament). Thus, the linearstiffness is a measure of the high-load stiffness, attributedto the amount of collagen in the cross-sectional area.30 Thelow-load behavior of the skin was characterized using twodifferent methods: a limit displacement approach and

Figure 3. Representative force–displacement data obtained

from tensile failure testing. (A) All specimens exhibited a similar

general behavior with increasing elongation: a nonlinear, strain-

stiffening behavior at low loads, followed by a linear force–dis-

placement response (dark gray area of line), and a softening be-

havior before specimen failure. The extent of the strain-stiffening

region (toe-in) was directly calculated, and approximated using a

measure of the limit displacement (arrow). (B) Force and dis-

placement values corresponding to the point of failure (arrow)

were recorded, and the energy to failure was determined as the

area under the force–displacement curve, calculated from the

onset of load to the point of failure (shaded area). Data shown are

for uninjured skin tested in the axial direction.Figure 2. Raw, unfiltered force–elongation–time data from a

representative stress relaxation experiment in skin. (A) The

measured force peaks when maximum elongation is first

reached (t51 second), and relaxes over the 240-second iso-

metric period, thereby illustrating the tissue’s ability to release

stored elastic energy via viscous dissipation. (B) The first 10

seconds of the test show that peak load is reached at the end of

elongation, and indicates the tissue’s stiffness at small dis-

placements (6%).



direct quantification of the toe-in region. The limit dis-placement was defined as the intercept of the linear stiffnessslope on the displacement axis, similar to limit strain mea-surements previously used to evaluate the low-load behav-ior in human skin.6 The limit displacement represents anapproximate measure of the toe-in region of the load–dis-placement curve, characteristic of the strain-stiffening be-havior of skin at low loads (Figure 3A). The toe-in regionwas also quantified as the displacement at which the in-creasing slope of the low-load region first equaled the linearstiffness. To begin, the first half of the load–displacementcurve (0–50% of failure displacement) was fit with a qua-dratic function,

FðxÞ ¼ c1x2 þ c2xþ c3 ð2Þ

where x is displacement and c1, c2, and c3, are arbitraryconstants. This function was then differentiated with re-spect to displacement to get an expression of the slope as afunction of displacement,

slopeðxÞ ¼ dFðxÞdx

¼ 2c1xþ c2: ð3Þ

The size of the toe-in region (mm) was then determinedby solving (Eq. 3) for the displacement at which the slopefirst equaled the linear stiffness. This method determinesthe point at which the increasing slope of the low-load re-gion equals the linear slope, and thus the point at whichthe strain-stiffening mechanical behavior that character-izes the toe-in region ceases to be nonlinear.

Statistical analysis

All statistical analyses were conducted using parametricanalyses in SPSS. Directional differences were analyzedwhile keeping the material constant (both within skin andwithin scar, not between skin and scar). Similarly, thedifferences between uninjured skin and scar were observedwithin the same material direction (in axial direction andin transverse direction, but not between axial and trans-verse directions. Because no outliers were present, com-parisons were made on the complete data sets usingStudent’s t test parametric analysis (SPSS Statistical soft-ware, 12.0, SPSS Inc., Chicago, IL), with a level ofp < 0.05 to establish statistical significance.

RESULTS

Uninjured skin

Comparing the behavior of normal, uninjured skin in theaxial and transverse directions identified interesting visco-elastic differences. Uninjured skin displayed similar high-load behavior in both directions. The axial and transversedirections exhibited a similar resistance to failure, withsimilar loads, displacements, and energy at failure (Table1), as well as no directional difference in linear stiffness(Figure 4). At low loads, skin was more compliant in theaxial direction than the transverse: as evidenced by signifi-cantly larger limit displacements (Figure 5A) and toe-inregions (Figure 5B). Furthermore, the axial direction dis-played significantly lower peak relaxation forces (Figure6), and thus a more compliant (less stiff) low-load behav-ior. Relaxation tests further identified that normal, unin-jured skin relaxed much faster in the axial direction thanthe transverse (Figure 7).

Effect of scarring

Healing wounds were much less resistant to failure thanuninjured skin, in both the axial and transverse directions.Scars were weaker, less extensible, and less tough thannormal skin (Table 1). However, despite the large reduc-tion in failure properties with wounding, there was no sig-nificant change in linear stiffness with scarring (Figure 4).While linear stiffness was relatively unaffected by scarring,the low-load behavior showed a more dramatic response.The toe-in region was significantly shorter in the scar tissuethan in uninjured skin, in both the transverse and axial di-rections (Figure 5B). This reduction in low-load compli-ance with scarring was also reflected in the limitdisplacement (Figure 5A). The peak relaxation force (de-veloped at 6% elongation) was elevated with scarring inthe axial direction, indicating a significant reduction inlow-load compliance, and appeared to decrease with scar-ring in the transverse direction, although the observed re-ductions were not statistically significant (p50.09) (Figure6). Wounding did not affect the rate of relaxation in theaxial direction, but caused a significant increase in thetransverse relaxation rate (Figure 7).

Although there was no axial/transverse difference in lin-ear stiffness of the uninjured skin, wounding producedscars with higher axial linear stiffness than transverse

Table 1. Failure properties for uninjured skin and scar

Uninjured skinn Scarn

Axial Transverse Axial Transverse

Failure load (N) 203.9� 44.4 178.9� 49.2 82.2� 46.8w 61.5� 26.6w

Failure displacement (mm) 13.90� 1.87 11.96� 2.54 4.75� 1.62w 5.39� 1.28w

Energy to failure (mJ) 1,394� 325 1,222� 373 225� 160w 230� 144w

Data are shown as mean� standard deviation.nNo directional differences observed between axial and transverse specimens within either uninjured skin or scar.wScar specimens displayed a significant reduction in all failure properties (load, displacement, and energy to failure) with respect to

uninjured skin, in both axial and transverse directions (p < 0.01).



(Figure 4). Aside from linear stiffness, healing wounds dis-played strikingly similar viscoelastic behavior in the axialand transverse directions: exhibiting no difference in fail-ure properties (Table 1), low-load behavior (Figures 5 and6), or relaxation rate (Figure 7).

DISCUSSION

Scar tissue and uninjured skin exhibited significant direc-tional differences in biomechanical behavior, as observedin the low-load viscoelastic response of the tissue. Whilethe more traditional measurements of failure properties(load, displacement, and energy) clearly identified differ-ences between scars and uninjured skin, they were unableto appreciate the axial–transverse directional differenceswithin each tissue. This insensitivity highlights the need forviscoelastic analyses within the low-load region. Such an-alyses measure the biomechanical behavior of the tissue ina physiologically relevant deformation range, and provideinsight into the structure of uninjured tissue and the mor-phological changes that may occur during healing after anacute injury.

In its native, uninjured state, skin displayed a materialdirectionality where axial specimens relaxed more quickly,and were more compliant at low loads, than those in thetransverse direction. It is possible to explain these ob-served behaviors by a single orientation of fibers within the

tissue structure. If the principal direction of collagen fiberswere biased toward the transverse direction in uninjuredskin, collagen fibers would begin to bear load at a lowertissue strain in the transverse specimens than the axial. As

Figure 5. Extent of low-load, strain-stiffening behavior, as mea-

sured by (A) a limit displacement approximation and (B) direct

calculation of toe-in region. Uninjured skin exhibits a significantly

larger strain-stiffening region in the axial direction than the trans-

verse (#p < 0.01). Wounding appeared to reduce this region,

with significant strain-stiffening reductions observed in scars in

both directions (np < 0.001, zp < 0.005). The strong directional-

ity originally present in skin was lost with wounding, as evi-

denced by the virtually identical low-load behavior of scar in the

two directions (p > 0.7). The limit displacement approximation

(A) yielded toe-in region estimates that were lower in magnitude,

but with identical trends as those found by direct calculation (B).

Figure 4. Linear stiffness of skin and scar (mean� standard devi-

ation), measured over the linear range at high loads (50–80% of

failure load). Similar linear stiffnesses were observed in both axial

and transverse directions of uninjured skin, as well as between

skin and scars in each direction. However, one directional differ-

ence was observed in the scars: significantly higher linear stiffness

in the axial direction than the transverse (nnp < 0.01). Despite this

slight difference, the linear stiffness appeared relatively insensitive

to specimen orientation or the effect of wounding.



a result, transverse toe-in and limit strain would besmaller, and the low-load stiffness would be higher, as ob-served (Figures 5 and 6). This argument can also be ex-tended to explain the viscous behavior. A decrease in therate of relaxation has been previously observed with in-creasing tissue strains in ligament.28 The authors attrib-uted this effect to ‘‘wringing out’’ of the tissue, wherestraightening of collagen fibers would cause fluid to exudefrom the tissue, and thus decrease the viscous response. Inskin, the collagen would strain more, causing a greater‘‘wringing out’’ of the tissue, for a strain applied along theLanger’s line. Therefore, if the fiber orientation were bi-ased toward the transverse direction, transverse specimenswould experience a greater viscous reduction, resulting inslower rates of relaxation than axial specimens, as evi-denced in uninjured skin experiments (Figure 7). Thus,these biomechanical results suggest a tissue morphologywith collagen fibers oriented with a bias toward the trans-verse (dorsal–ventral) direction.

Following acute injury, scars displayed distinctly differ-ent biomechanical behavior to that of uninjured skin. Thesignificant reduction in failure properties (load, displace-ment, and energy) observed in scars, indicated their com-promised bursting strength, extensibility, and toughness,with respect to normal uninjured skin; similar to previousobservations in the guinea pig.11 Additionally, the low-

load behaviors showed that scars relax at a faster rate, in-dicating greater viscous content. Scars also displayed re-duced toe-in durations and stiffer responses at low loads,as reflected in the increased loads developed at small (6%)elongations. The increase in viscous behavior can resultfrom elevated water content due to higher concentrationsof PGs and other water-binding molecules, or a reductionin the water loss with stretching, i.e., less ‘‘wringing out’’ ofthe tissue. Because the scars exhibited significantly stifferresponses at low loads, a greater strain would be expectedin the collagen fibers for the given applied elongation(6%), and thus a greater ‘‘wringing out’’ of the tissue. Thefact that increased viscous behavior was observed in a tis-sue that should exude more water suggests that scars muststart with significantly higher water content, most likelydue to increased PGs. In the future, water content assess-ments of normal and scar tissue should likely be under-taken to address this point, as well as studies to determinethe quality and quantity of the PG present. Interestingly, asimilar elevated viscous behavior has been observedin healing ligaments when compared with uninjured con-trols; however, the specific cause(s) of this increase was notidentified.31

Direct quantification of the toe-in region gives a moreaccurate measure of the strain-stiffening duration than thelimit displacement approach that has been previouslyused.6 Furthermore, the limit displacement is significantlyaffected by linear slope value: a larger slope will predict alarger limit strain. Direct quantification simply relies onthe point at which the curve becomes linear, and thus is

Figure 6. Peak load measured during stress relaxation

(mean� standard deviation). Peak load always occurred at the

end of the 6% stretch, and thus the start of the relaxation por-

tion of the protocol. The force developed from the 6% applied

stretch indicates the resistance within the tissue, and thus an

indirect measure of the tissue stiffness at low loads. These re-

sults show skin developed significantly higher loads in the

transverse direction when compared with axial (#p < 0.01),

and a significant increase in axial peak load with wounding

(np < 0.01). Similar to the results of the toe-in region (Figure 5),

the significant directionality originally present in the skin was

lost with wounding, as evidenced by scars with no directional

difference (p50.5).

Figure 7. Load relaxation rate measured in skin and scar

(mean� standard deviation), indicating the viscous response

of the tissues. The significant directional difference originally

present in the skin, in which load relaxed much faster in the ax-

ial direction (#p < 0.01), was lost with wounding. Axial and

transverse scars relaxed at the same rate (p50.4), and the rates

in scars were elevated compared with skin. A large, significant

increase in relaxation rate was observed with wounding in the

transverse direction (np < 0.01), and scars appeared to relax

faster than skin in the axial direction as well, although not sig-

nificantly (p50.08).



completely unaffected by the value of the linear slope. Be-cause the linear stiffnesses observed in this study were rel-atively unaffected by injury, we expect the limitdisplacement and direct quantification methods to yieldsimilar trends, as evidenced in our data (Figure 5).

Toe-in region duration is affected by the contributionsof elastin, the amounts of collagen cross-linking and PGspresent, as well as the structural orientation of the collagenfibers.2,32 Uninjured skin had a significantly larger toe-in(and limit displacement) in the axial direction, suggesting aprincipal fiber direction biased toward the transverse di-rection. This material directionality was lost with scarring,resulting in similar toe-in regions in both axial and trans-verse scar directions. Thus, fiber organization in the scarsmust have lost its directional preference, either as a resultof random fiber orientations or fiber alignment in a direc-tion that has no bias toward either the axial or transversedirections. Because these tissues were analyzed in the late-remodeling phase of healing, we expected the scar to showsome reorganization of the fibrous components, ratherthan a purely random network. In a pilot histologicalanalysis, uninjured skin displayed a highly organizedcollagen fiber network with a principal direction biasedtoward the transverse direction, as expected from our bio-mechanical analyses (Figure 8). Scars also showed a highdegree of organization, but with the fibers aligned to anangle with virtually identical components in the axial andtransverse directions (Figure 8). While these results arecurrently far from a definitive study of skin morphology,the findings do corroborate our predictions based on theobserved biomechanical changes that occurred with in-jury. However, if the biomechanical changes were solelydue to a change in the orientation of the fibrous network,the measured values (toe-in, limit strain, and peak relax-ation load) would: (a) be equal in both directions, and (b)have values between the axial and transverse values in un-injured skin. While the measured values were equal in bothdirections of the scar, only the peak relaxation load dis-played values within the bounds of the axial and transversevalues in uninjured skin. Thus, it appears that other struc-tural changes, such as higher elastin concentrations andincreased collagen cross-linking, would be necessary tomore fully explain the reduction of toe-in beyond thetransverse values observed in normal skin.

Molecular interpretations

These biomechanical observations suggest a number ofpotential mechanisms, in both the elastic fibrous networkand viscous PG-rich ground substance matrix, which mayoccur in the healing response to full-thickness skin injury.The reduced toe-in region of the load–elongation curvesobserved in scars could be attributed to increases in colla-gen cross-linking, increased amounts of elastin, or a com-bination of the two. Cross-links act to stiffen the collagenfibrils and the entire collagen network.33 Thus, an increasein collagen cross-linking, beyond the baseline levels of un-injured skin, would provide a mechanism for the decreasedlow-load compliance. However, we anticipate that at 70days postinjury the collagen cross-links are only just be-ginning to form in our porcine model.34 Alternately, due toits dominant mechanical role at low strains, an increase inelastin could explain the increased low-load stiffness, and

thus the observed decrease in toe-in region duration. Be-cause of its dominant mechanical role at low strains, in-creased elastin would reduce the toe-in region without asignificant increase in linear stiffness (high load), as ob-served in the data presented.While elastin is not critical forwound repair,35 and its fibers may take years to reform inhumans (if at all), elastin fibers have been shown to reap-pear within the first 6 weeks of healing in porcinewounds.36 This makes elastin a particularly attractivemechanism to explain the observed biomechanicalchanges. However, these suggestions are purely specula-tive, and future studies that measure the amounts collagencross-linking and elastin, as well as their structural organi-zation, are needed to truly identify the specific changes tothe elastic fiber network.

Some of the observed changes in low-load behaviorcould also be attributed to changes in the surrounding ma-trix material, particularly PGs. Heparan sulfate PGsare structural molecules involved with protein–proteininteractions that have shown biomechanical effects.Increased breaking strength was observed in woundstreated with heparan sulfate PG gel.37 A similar increasein breaking strength was observed in incisional woundsthat overexpressed the PG fibromodulin.38 Fibromodulinmodifies the activity of the growth factor TGF-b, which inturn affects collagen synthesis. While both of these PGshave shown the ability to increase bursting strength, theireffects on low-load properties of healing wounds remainunknown. One of the largest PGs in skin, versican, hasseveral glycosaminoglycan (GAG) chains that draw waterinto the skin, and thus increase its viscous content. It istypically present in small amounts in normal skin, slightlyhigher amounts in mature normal scar, and in largeramounts in hypertrophic scar.39 The major large PG ofcartilage, aggrecan, is likely similar to versican in activityand expression; however, much less is known about its role

Figure 8. Polarized light microscopy image of sections in the

plane of the skin (left) and scar (right), showing collagen net-

work organization (�100) with respect to the axial and trans-

verse axes. There are obvious morphological differences

between the skin and scar, many of which correlate with the

observed viscoelastic behaviors. Uninjured skin exhibits orga-

nization of the collagen fibers in a principal direction biased to-

ward the transverse direction. Scars healed with considerable

fiber organization, in a direction with similar components in

both directions. Fiber bundles appear to be much thinner in

scars than uninjured skin.



in skin. In addition to their viscous contributions, thesePGs also strengthen the matrix material, and thus provideadditional stiffness at low strain. The increased relaxationrate and decreased toe-in duration displayed by scars inour study, would both be consistent with an increase in theamount of versican (or aggrecan) in the healing wounds.As with the elastic network, changes in many of these PGscould explain many of the observed biomechanicalchanges; however, their precise study and quantificationis necessary to test if these proposed mechanisms truly ac-count for the observed altered viscoelastic behavior. On-going studies are designed to validate these possiblecorrelations between mechanical properties and biome-chanical composition and organization.

Relevance to human skin

The present experiments were carried out in a porcinemodel of wound healing that has been described previ-ously,23–25 and one shown to exhibit a similar healing re-sponse to normal human cutaneous healing.23 Porcine skinis notably similar in structure and function to human skin.Pigs are tight-skinned animals (as are humans) and healthrough a combination of reepithelialization and woundcontraction. The wound contractions associated with heal-ing in the animals used in the present studies (� 50%)23

are similar to those observed in normal human woundhealing (25–50%).24 In contrast, loose-skinned animals(e.g., rodents, lagomorphs) heal predominantly via con-traction, with wound contractions of up to 90%.40 Thescar tissue assessed in the present studies also exhibitedfunctional deficits similar to those previously observed inhuman skin injury. In addition to reduced failure proper-ties, these healing porcine wounds displayed a reduced toe-in region compared with uninjured skin. This is similar tothe pronounced reduction in toe-in (limit displacement)observed in human hypertrophic scarring.41 The similari-ties observed in structure, function, and healing responsehighlight why the pig is an attractive model for studyinghuman wound healing.

Limitations

In this study, wound healing was evaluated at a single timepoint following injury in juvenile pigs. We understand thatthis limits the interpretation of the results to the late-re-modeling phase of healing. While direct extrapolation ofthe present results to other models of wound healing (e.g.,differences in animal, location of the body, time postinju-ry, sex and age of animal, etc.) may not yet be possible dueto the single time point chosen, some parallels have beennoted between these results and other findings in ligamentrepair (unpublished data). Further studies conducted atdifferent stages of development (e.g., juvenile, sexuallymature, and skeletally mature) would be necessary to iden-tify and quantify any sex- and age-related differences inthe wound-healing response, and longitudinal studieswould be necessary to address temporal factors, whichwould strengthen potential conclusions regarding the mo-lecular and biomechanical aspects of healing. Further-more, our study was limited to dorsal full-thickness skinexcisions. Because skin cells,’42 structure, and mechanicaldemands likely vary with location, future investigations

using tissue from both dorsal and ventral wounds could de-termine if location of the injury plays a significant role in thehealing response. Similarly, future studies with skin frompurebred Yorkshire and Red Duroc animals may lead newinsights into how genetics may influence outcomes.

In conclusion, uninjured porcine skin exhibits a mechan-ical directionality, most likely attributed to structural ori-entation of collagen in the tissue. In this study, the precisemeasurement and analysis of the low-load behavior un-iquely identified changes in biomechanical behavior thatwere not appreciated through the traditional analysis ofhigh-load linear stiffness and failure properties. In additionto considerably reduced failure properties, scars showed al-tered behavior at low-loads, and a loss of the directionalbias that was present in the uninjured tissue. These biome-chanical findings suggest tissue morphological changes inskin scars following acute full-thickness injury contributeto the observed differences. This considerable reduction inbiomechanical function coupled with the decreased failureresistance of scars highlights the need for clinical treat-ments of wounds and tissue-engineered constructs to re-store the low-load viscoelastic behavior of the native,uninjured surrounding skin to the wound site. This wouldincrease load transfer and strain compatibility between theskin and scar, thereby potentially leading to improvedfunctionality and esthetics of the healed wound.

ACKNOWLEDGMENTS

The authors acknowledge the financial support of The Al-berta Ingenuity Fund (DTC), Ernst & Young Fellowshipin Joint Injury and Arthritis Research (DTC), The Arthri-tis Society, CIHR, and NSERC. DAH is the CalgaryFoundation-Grace Glaum Professor and NGS is a KillamProfessor at the University of Calgary.

REFERENCES

1. Lanir Y. Skin mechanics. In: Skalak R, Chien S, editors.Handbook of bioengineering. Dallas, TX: McGraw-Hill,1987: 11.1–.25.

2. Lanir Y. The fibrous structure of the skin and its relation tomechanical behaviour. In: Marks R, Payne PA, editors. Bioen-gineering and the skin. Hingham, MA: MTP Press, 1981: 93–6.

3. Smith LT, Holbrook KA, Byers PH. Structure of the dermalmatrix during development and in the adult. J Invest Derma-tol 1982; 79 (Suppl. 1): 93s–104s.

4. Stark HL. Directional variations in the extensibility of hu-man skin. Br J Plast Surg 1977; 30: 105–14.

5. Langer K. Zur Anatomie und Physiologie der Haut. I. Uber die

Spaltbarkeit der Cutis. Sitzungsber Akad Wien 1891; 44: 19–46.6. Gibson T, Stark H, Evans JH. Directional variation in ex-

tensibility of human skin in vivo. J Biomech 1969; 2: 201–4.7. HusseinMA. The orientation of connective tissue fibres in rat

skin. Acta Anat (Basel) 1972; 82: 549–64.8. Vogel HG. Directional variations of mechanical parameters

in rat skin depending on maturation and age. J InvestDermatol 1981; 76: 493–7.



9. Vogel HG. Tensile strength, relaxation and mechanical re-covery in rat skin as influenced by maturation and age. JMed1976; 7: 177–88.

10. Howes EL, Sooy JW, Harvey SC. The healing of wounds asdetermined by their tensile strength. J AMA 1929; 92: 42–5.

11. White WL, Brody GS, Glaser AA,Marangoni RD, BeckwithTG, Must JS, Lehman JA Jr. Tensiometric studies of un-wounded and wounded skin: results using a standardizedtesting method. Ann Surg 1971; 173: 19–25.

12. Mansour JM, Davis BR, Srour M, Theberge R. A methodfor obtaining repeatable measurements of the tensile proper-ties of skin at low strain. J Biomech 1993; 26: 211–6.

13. Hollander DA, Erli HJ, Theisen A, Falk S, Kreck T, MullerS. Standardized qualitative evaluation of scar tissue proper-ties in an animal wound healing model. Wound Repair Regen2003; 11: 150–7.

14. Oxlund H, Fogdestam I, Viidik A. The influence of cortisolon wound healing of the skin and distant connective tissueresponse. Surg Gynecol Obstet 1979; 148: 876–80.

15. Kim LR, Pomeranz B. The sympathomimetic agent, 6-hydroxydopamine, accelerates cutaneous wound healing.Eur J Pharmacol 1999; 376: 257–64.

16. Fung LC, Mingin GC, Massicotte M, Felsen D, Poppas DP.Effects of temperature on tissue thermal injury and woundstrength after photothermal wound closure. Lasers Surg Med1999; 25: 285–90.

17. Gibson T. Karl Langer (1819–1887) and his lines. Br J PlastSurg 1978; 31: 1–2.

18. Vitellaro-Zuccarello L, Cappelletti S, Dal Pozzo Rossi V,Sari-Gorla M. Stereological analysis of collagen and elasticfibers in the normal human dermis: variability with age, sex,and body region. Anat Rec 1994; 238: 153–62.

19. Butler DL, Juncosa-Melvin N, Boivin GP, Galloway MT,Shearn JT, Gooch C, Awad H. Functional tissue engineeringfor tendon repair: a multidisciplinary strategy using me-senchymal stem cells, bioscaffolds, and mechanical stimula-tion. J Orthop Res 2008; 26: 1–9. Review.

20. Hurschler C, Provenzano PP, Vanderby R Jr. Scanning elec-tron microscopic characterization of healing and normal ratligament microstructure under slack and loaded conditions.Connect Tissue Res 2003; 44: 59–68.

21. Thornton GM, Shrive NG, Frank CB. Ligament creep re-cruits fibres at low stresses and can lead to modulus-reducingfibre damage at higher creep stresses: a study in rabbit medialcollateral ligament model. J Orthop Res 2002; 20: 967–74.

22. Corr DT, Leverson GE, Vanderby R Jr., Best TM. Anonlinear rheological assessment of muscle recovery fromeccentric stretch injury. Med Sci Sports Exerc 2003; 35:1581–8.

23. Gallant-Behm CL, Reno C, Tsao H, Hart DA. Genetic in-volvement in skin wound healing and scarring in domesticpigs: assessment of molecular expression patterns in (York-shire�Red Duroc)�Yorkshire backcross animals. J InvestDermatol 2007; 127: 233–44.

24. Gallant-Behm CL, Hart DA. Genetic analysis of skin woundhealing and scarring in a porcine model.Wound Repair Regen2006; 14: 46–54.

25. Wang JF, Olson ME, Reno CR, Wright JB, Hart DA. Thepig as a model for excisional skin wound healing:characterization of the molecular and cellular biology, andbacteriology of the healing process. Comp Med 2001; 51:341–8.

26. Turner S. Creep in glassy polymers. In: Haward RH, editor.Thephysics of glassy polymers. London: Applied Science PublishersLtd., 1973: 250–63.

27. Lakes RS, Vanderby R. Interrelation of creep and relaxation:a modeling approach for ligaments. J Biomech Eng 1999; 121:612–5.

28. Provenzano P, Lakes R, Keenan T, Vanderby R Jr. Nonlin-ear ligament viscoelasticity. Ann Biomed Eng 2001; 29: 908–14.

29. Thornton GM, Shrive NG, Frank CB. Healing ligamentshave decreased cyclic modulus compared to normal liga-ments and immobilization further compromises healing liga-ment response to cyclic loading. J Orthop Res 2003; 21:716–22.

30. Manschot JF, Brakkee AJ. The measurement and modellingof the mechanical properties of human skin in vivo—I. Themeasurement. J Biomech 1986; 19: 511–5.

31. Thornton GM, Leask GP, Shrive NG, Frank CB. Early me-dial collateral ligament scars have inferior creep behaviour.J Orthop Res 2000; 18: 238–46.

32. Eshel H, Lanir Y. Effects of strain level and proteoglycandepletion on preconditioning and viscoelastic responses ofrat dorsal skin. Ann Biomed Eng 2001; 29: 164–72.

33. Puxkandl R, Zizak I, Paris O, Keckes J, Tesch W, BernstorffS, Purslow P, Fratzl P. Viscoelastic properties of collagen:synchrotron radiation investigations and structural model.Philos Trans R Soc Lond B Biol Sci 2002; 357: 191–7.

34. Frank C, McDonald D, Wilson J, Eyre D, Shrive N. Rabbitmedial collateral ligament scar weakness is associated withdecreased collagen pyridinoline crosslink density. J OrthopRes 1995; 13: 157–65.

35. Davidson JM, Giro M. Healing slack skin. J Invest Dermatol2006; 126: 2563–4.

36. Lamme EN, Van Leeuwen RT, Brandsma K, Van Marle J,Middelkoop E. Higher numbers of autologous fibroblasts inan artificial dermal substitute improve tissue regenerationand modulate scar tissue formation. J Pathol 2000; 190: 595–603.

37. Tong M, Zbinden MM, Hekking IJ, Vermeij M, BarritaultD, van Neck JW. RGTA OTR 4120, a heparan sulfate pro-teoglycan mimetic, increases wound breaking strength andvasodilatory capability in healing rat full-thickness excisionalwounds. Wound Repair Regen 2008; 16: 294–9.

38. Stoff A, Rivera AA, Mathis JM, Moore ST, Banerjee NS,Everts M, Espinosa-de-los-Monteros A, Novak Z, VasconezLO, Broker TR, Richter DF, Feldman D, Siegal GP, Stoff-Khalili MA, Curiel DT. Effect of adenoviral mediated over-expression of fibromodulin on human dermal fibroblasts andscar formation in full-thickness incisional wounds. J MolMed 2007; 85: 481–96.

39. Scott PG, Dodd CM, Tredget EE, Ghahary A, RahemtullaF. Chemical characterization and quantification of pro-teoglycans in human post-burn hypertrophic and maturescars. Clin Sci (London) 1996; 90: 417–25.

40. Hayward PG, Robson MC. Animal models of wound con-traction. Prog Clin Biol Res 1991; 365: 301–12.

41. Clark JA, Cheng JC, Leung KS, Leung PC.Mechanical char-acterisation of human postburn hypertrophic skin duringpressure therapy. J Biomech 1987; 20: 397–406.

42. de Hemptinne I, Gallant-Behm CL, Noack CL, Parreno J,Hart DA. Dermal fibroblasts from red Duroc and Yorkshirepigs exhibit intrinsic differences in the contraction of collagengels. Wound Repair Regen 2008; 16: 132–42.



Biomechanical behavior of scar tissue and uninjured skin in a porcine model

Documents

Transcript of Biomechanical behavior of scar tissue and uninjured skin in a porcine model