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Effects of stress on healing wounds: I. Intermittent noncyclical tension
JOURNAL
OF SURGICAL
RESEARCH
Effects
20, 93-102 (1976)
of Stress
I. Intermittent
on Healing Noncyclical
Wounds: Tension’
ARNOLD. J. AREM, M.D. AND JOHN W. MADDEN,
M.D.
Department of Surgery, University of Arizona, College of Medicine, Tucson, Arizona 85724 Submitted August 1, 1975
Since the time of Hippocrates, physicians have speculated about the influence of tension on healing wounds. In some situations, stress seems to produce thin, elongated scars; and in other situations hypertrophic scar forms. The paradox that mechanical forces might generate different end products out of the same amorphous matrix remains a clinically troublesome enigma. A century ago, Wilhelm His proposed that connective tissues can alter in response to stress by stating: At every place where connective tissue is exposed to a constant and oft repeated pulling action, there is formed a fibrous band, e.g. a tendon, the direction of whose fibers is parallel to the direction of the pull.
has yielded some of its secrets to the pressure of intensive scrutiny affirming (both figuratively and literally) Wolff’s law-bone adapts to functional forces acting upon it. If this principle applies to soft tissue, proper application of stress could modify scar morphology selectively. Unfortunately, the lack of a suitable model has prevented the rigorous study of how physical forces affect scar remodeling. Accurate interpretation of data mandates the simultaneous control of many variables in a dynamic, living organism. A number of essential design criteria must be met to create an adequate model. 1. The scar must be consistent and reproducible. 2. Environmental variables must be minimized. 3. Scar measurements must be free of sampling errors. 4. The model must permit measurement of scar physical strength; quantitative and kinetic biochemistry; and histology. 5. Tension must be applied in both a controllable and measureable way. 6. Individual variability must be controlled.
This selective fiber orientation in the direction of stress has been observed repeatedly. In 1926, Bunting and Eades [5] found that mechanical tension could orient fibroblasts in fresh wounds in the direction of tension. Even mitotic spindles were aligned parallel to the applied stress, and daughter cells separated along the lines of tension. In the ensuing years may observers have noted that in wounds healing under tension, newly deposited collagen becomes oriented in the direction of stress [ 1, 2, 3,4, 10, 111.Unfortunately, none of these studies has clarified the mechanism by which such morphologic changes occur, or the functional role of stress induced changes in soft tissue. In contrast, the bone remodeling process
The purpose of this paper is to describe a model created to meet the design requirements, and the results of a preliminary experiment testing the effect of prolonged intermittent stress on wounds of varying ages.
‘This work was supported by Grant 14047, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014.
THE MODEL To create a reproducible internal scar we designed two small rectangular blocks of 93
Copyright 01976 by Academic Press, Inc. All rights of reproduction in any form reserved.
94
JOURNAL
OF SURGICAL
RESEARCH,
VOL. 20, NO. 2, FEBRUARY
1976
FIG. 1. Design of magnet assembly; the entire assembly is 7.5 cm long.
Proplast for subcutaneous implantation in rats. Proplast is a carbon-impregnated Teflon sponge material with carefully controlled pore size allowing rapid fibrous tissue ingrowth. A 3-O nylon pullout suture was used to hold each sponge pair tightly approximated for any designated period post implantation. A single 3-O nylon mattress suture, passed circumvertebrally, served to anchor the proximal sponge securely in place throughout the experiment. Any force applied to the distal sponge, once the pullout suture is removed, distracts the scar tissue forming between the fixed proximal and the mobile distal sponge. In devising a means for supplying a distracting force, we abandoned on theoretical grounds any appliance involving direct attachment to the sponges. Grappling devices tend to “cut through” with time and introduce uncontrollable artifacts. In addition, since scar tissue takes time to form, the tensile force must be withheld until scar of the desired age has developed, and tension then applied without reopening or violating the wound. Magnetism, because there are “no strings attached” and because the magnetic field can be turned on or off and manipulated at will, was adopted as the motive force. To the distal sponge of each pair we bonded a 2.5 cm length of JP-6 (6 mm) dacron reinforced Hunter Silicone Tendon
prosthesis3 providing an inelastic bridge and a longitudinal orientation to each assembly. We bonded a silicone coated $$ain. Alnico 5 bar magnet or a nonmagnetic silicone coated metal bar of similar size to the tendon rod. The completed assembly was 7.5 cm long (Fig. 1). Under Innovar anesthesia, backs of rats were shaved and prepped with alcohol and Betadine. Implants were inserted into bilateral subcutaneous pockets through dorsal midline incisions, a magnet-bearing assembly on one side and a nonmagnet-bearing assembly on the other (Fig. 2). The electromagnets used to supply the distracting force were constructed of 5 x 20 cm cylindrical soft iron cores wound with 2000 turns of #16 copper wire, cooled both through the core and around the windings. These magnets are capable of carrying a sustained current of 7 A, producing a magnetic flux of 1400 Ga at 1 cm from the poleface. Using a gaussmeter we plotted magnetic flux as a function of distance from the poleface at varying currents and at points from the central axis to the periphery of the core. Three families of curves, corresponding to the three current settings, were observed (Fig. 3). In each case, as expected, magnetic flux was highest along the central longitudinal axis of the core. The gaussmeter output was read from an oscilloscope, and in
2Generously supplied by Smith-Kline cialties.
3Kindly furnished cialties, Inc.
Surgical Spe-
by Extracorporeal
Medical
Spe-
AREM AND MADDEN:
HEALING
WOUNDS
95
Measured force on bar magnet as a function of current through electromagnet and distance from poleface
160
-g In
120
;
100 2
FIG. 2. Position of implants. The circumvertehral suture anchors proximal sponges in place. A pullout suture holds each sponge pair together for several weeks. Insert demonstrates peripheral position of implants in cross-section.
no instance did we observe any magnetic ripple, eliminating any possibility of a vibrational effect induced by the ripple of the ac current. Using a sensitive transducer coupled to this magnet system, we plotted forcedistance curves to allow accurate measurement of force applied to the bar magnet (transmitted through the assembly to the developing scar tissue) at any distance from the electromagnet. Because the rat body is roughly cylindrical, with the subcutaneous implants lying peripherally in cross section (Fig. 2 insert), transducer measurements were made along the line of the core periphery. To maintain relative immobilization, we placed rats in cylindrical plastic restraining tubes (Fig. 4). The rats uniformly accli-
I?
80 60 40
Distance (cm)
FIG. 3. Force-distance curves. The force on the implanted magnet (measured along core periphery) drops off sharply with distance at any current setting.
matized to these tubes within 48 hours and slept thereafter in the tubes throughout the experiments. Rats were weighed weekly and no animal lost weight despite intermittent immobilization. EXPERIMENTAL DESIGN For the initial experiment we varied the age of the scar and maintained constant the parameters involved in the application of tension. We used female 250 gm Sprague-Dawley
FIG. 4. Rat restrained in magnetic field. Rats acclimate rapidly and sleep while in restraint tubes. Arrows indicate flow of water coolant.
96
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
rats throughout the experiments, ten rats in each group. Figure 5 summarizes the experimental design. Beginning three or 14 weeks after implantation, groups of rats were restrained at the pole ends of the electromagnets for 6 hr each day for four weeks. One day prior to sacrifice we weighed the rats and injected [3H] Proline (50 &i/ 100 g body weight) parenterally. On the day of the sacrifice, sponge assemblies were exposed, examined grossly and photographed. All implants could be dissected from the underlying tissues in a visible and reproducible plane. Representative sponge pairs were fixed in formalin, embedded in paraffin, sectioned longitudinally and stained with H & E or trichrome. We placed the remaining sponges in an Instron Tensiometer and determined the burst strength of the scar bridge between the sponges using a crosshead speed of 2 in/min. The two halves of each sponge pair were then pooled. Saline extractable collagen was assayed and net rate of collagen synthesis and deposition determined by measuring the specific activity of Hypro [7,8]. In the initial study on 3 week old scar, to determine the effect of the magnetic field it-
1976
self, we implanted magnet and nonmagnet sponge assemblies in an additional group of five rats. Beginning three weeks after implantation animals were restrained for 6 hr daily for four weeks but far removed from the electromagnets. Sponges from this “nonfield” cant rol group underwent bursting, histological and biochemical examination. Because no differences were found between these sponges and sponges from the other control groups, we deleted this control from the 14 week experiment. We were able to calculate the force transmitted to the implant at any point in time by measuring the distance from the bar magnet to poleface radiographically and plotting force on the force-distance curve for that field intensity. We used a power setting of 5A throughout the experiment. The intermagnet distance varied from 3 cm to Ya cm with a corresponding tensile force ranging from 30 to 120 gm. Several animals extruded their implants and were discarded from the study. With the exception of one animal which became paraplegic after two weeks and was discarded, there were no complications related to the circumvertebral suture.
weeks FIG. 5. Experimental design. Rats were started in magnetic field three or 14 weeks after implantation and subjected to four weeks of intermittent nonpulsatile stress.
AREM AND MADDEN:
HEALING
WOUNDS
97
FIG. 6. Appearance of sponge pairs following 4 weeks of tension on three week old scar. Lower sponge pair is from magnet side (stress), upper sponge pair from control side (no stress) of same animal.
RESULTS In sponge pairs subjected to tension three weeks post implantation, the scar bridge between the sponges was markedly elongated assuming a characteristic hourglass shape. In contrast, control sponges were tightly approximated and did not separate (Fig. 6). Histologically, the collagen fibers in stressed scars were oriented in the direction of tension, while the collagen between the nonstressed control sponges was randomly oriented (Figs. 7 and 8). Sponge pairs subjected to the same duration, timing, and magnitude of tension beginning 14 weeks after implantation did not separate when compared to non-stressed controls (Fig. 9). The collagen between the stressed sponges showed a slight tendency toward longitudinal orientation but was otherwise similar to controls, and very different from the histological appearance of the elongated younger scars (Figs. 10 and 11). The bursting strength data are shown in Table 1. By seven weeks after implantation the scar tissue between the sponges has attained over a pound of strength, and this approximately doubles by the eighteenth week. Although bursting strength of the stressed and nonstressed 18 week old scars were identical, the younger scars subjected to stress appeared to have less strength than
unstressed scars of similar age. This apparent difference is probably artifactual. As specimens are pulled apart in the tensiometer, there is a tendency for the material being distracted to undergo lateral contraction, that is, to deform and become narrower. With the elongated scars in the first experimental group, the large amount of free edge present allows the tensiometer to register bursting strength due to simple elongation and breakage. However, in control sponges, the shortness of the scar bridge constrains lateral contraction, and the deforming force registers in the tensiometer as shear in addition to elongation, resulting in a higher observed bursting strength. In the case of the older scars, the sponge pairs in both experimental and control groups were morphologically similar, and this measurement artifact occurred equally in both groups. The values for saline extractible collagen appear in Table 2, and for rate of collagen synthesis in Table 3. Under the conditions of this experiment, tension had no effect on either of these biochemical parameters of scar formation, even when the scars showed drastic morphological alterations. DISCUSSION The sponge implant model avoids the shortcomings inherent in studying the effects
98
JOURNAL
OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
1976
FIG. 7a. Histologic appearance of elongated scar tissue from stressed sponge pair shown in Fig. 6. Longitudinal section demonstrates thin, attenuated scar bridge (trichrome, 8 x). FIG. 7b. Closeup of bracketed area in Fig. 7a. Note parallel bundles of collagen oriented longitudinally in direction of stress (21 x).
of tension on dermal wounds. Animals tend to scratch and abrade surface wounds, producing additional scarring. When skin sutures are used, differences in material, placement, tightness and timing of removal become unmeasured variables. Some investigators have excised large areas of skin, relying on residual elasticity of marginal skin to produce tension, but have failed to measure the forces involved [2, 3, 5, 10, 111.
Sporadic contraction of the panniculus carnosus also affects tension and is impossible to quantitate. Interpretation of data on skin wound healing is further complicated by the existence of “skin tension lines” whose orientation with respect to the wound may influence the orientation of newly deposited collagen. Our model eliminates these sources of error, but introduces others which must be
AREM AND MADDEN:
HEALING
WOUNDS
99
FIG. 8. Histologic appearance of scar bridge from unstressed control sponge pair in Fig. 6. The narrow gap between the spongesis filled with randomly oriented collagen (trichrome 14.7x).
FIG. 9. Appearance of sponge pairs following 4 weeks of tension on 14 week old scar. Lower sponge pair is from magnet side (stress), upper sponge pair from control side (no stress) of same animal. Contrast lack of elongation of stressed older scar with marked elongation of stressed younger scar seenin Fig. 6.
considered. Any implanted sponge is a foreign body and scar tissue forms in response to the foreign substance. Although Proplast produces a reliable and reproducible artifact, scar tissue formed may not be analogous to dermal scar. Out data cannot be used to interpret skin wound healing. We can, however, draw some conclusions regarding the effects of tension on an isolated internal scar system. Our results indicate that young scars can be altered morphologically by conditions of stress that do not affect older scars. These remarkable changes in form and appearance occur without alterations in rate of synthesis, in collagen crosslinking or in bursting strength of scar collagen. However, subtle alterations in the physical properties of scar may have escaped detection. Bursting strength is a fairly crude test of the biomechanical properties of a non-homogeneous system. Perhaps an analysis of the stress-strain characteristics of the scar material between the sponges, using subbreakage forces would be more meaningful. Similarly, the biochemical data are based on measurements of the total sponge
100
JOURNAL
OF SURGICAL
RESEARCH,
VOL. 20, NO. 2, FEBRUARY
1976
F:IG. 10. Histologic appearance of stressed sponge pair shown in Fig. 9. Longitudinal section shows less str iking co11agen orientation than in stressed younger scars, but more than controls (trichrome, 15.75 x).
co1lagen. Metabolic alterations, if they ocGUI :red only in scar tissue subjected to the w :atest degree of stress, may have been mabskedby measuring the whole sponge. SePaIrate biochemical analysis of the scar
tissue between the spongesmay substani:iate this. In our primary experiment we cllose arbitrary parameters of stress and va.ried scar age. The striking morphologic dif-
FIG. I I. Histologic appearance of scar from unstressed sponge pair in Fig. 9. Longitudinal randomly oriented collagen (trichrome, 15.75 x).
section demonstrates
AREM AND MADDEN:
HEALING
101
WOUNDS
TABLE 1 ference between young and old scars implies Strength Measurements that scar maturation may be reflected in an Burst strength age-related ability to resist tensile stress. Algrams, mean + SE though we were able to accurately determine the force applied to the implanted magnet, Scar age # Animals Magnet Paired control we cannot assume that this force was trans- I weeks 4 456 jl 158 538 f 48 mitted entirely to the scar bridge between 18 weeks 4 1113 f 239 1029 f 109 the sponges. The filmy attachments of the implant’s capsule to overlying skin or underlying fascia may have dampened the excursion of the implant and distributed the physical forces affect the remodeling scar applied force to a greater volume of the scar. can be of enormous practical benefit. Fibrous attachments, like the sponge Collagen is a nonliving material and any collagen, mature and strengthen, producing remodeling that occurs must ultimately a proportionately greater dampening of the depend on the ability of living cells to sense force with time. Differential force distribution may account for differences ob- and transduce mechanical force into biochemical action. Possible mechanisms served between 7 and 18 week old scars. Finally, this study fails to define ac- for this transduction have been summarized curately where the tensile forces exert their in an excellent review by C. A. L. Bassett [5]. main effect. Sponge separation may involve If tissue deformation is the mechanical triga progressive elongation of the capsule sur- ger for biological activity, then the degree of rounding the sponges as well as the scar be- force, the rate, frequency and duration of its tween them. We should be able to answer the application, and loading velocity may all be question by marking the capsule and se- important in producing connective tissue quentially studying the marker movements alterations. Our magnet system allows manipulation of these parameters singly or as the spongesseparate. in combination. In addition, the sponge The results of this study suggest that, model readily lends itself to the application under appropriately defined conditions, scar of other modalities such as ultrasound, heat, tissue morphology can be modified by stress direct current stimulation (through imand that modification may depend on a planted electrodes) across the scar bridge, or balance between age and the parameters of lathyritic agents, simultaneously with apstress. Apart from any implications relative plication of tensile force to study the to skin wound healing, data derived from studying the sponge implant model may combined action of those modalities with have practical implications for a variety of stress on scar tissue. clinical situations. If the sponge model If, as Paul Weiss [12] has said, static form proves to be analogous, for example, to is the outcome of formative dynamics, our wounds of tendon or ligament, then model will be useful in studying some of the centuries old questions about the influence of knowledge of precisely how the magnitude, rate of application, duration and timing of dynamic forces on healing wounds. TABLE II Collagen Cross Linking % Neutral salt soluble collagen mean + SE Scar age
# Samples
Magnet
Paired control
Nonfield control
I weeks 18 weeks
8 4
4.11 f .34 2.51 f .39
4.62 f .50 2.56 * .16
4.0 f .22
102
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
1976
TABLE III Rate of Collagen Synthesis Specific activity of hydroxyproline (dpm Hyplti Hyp), Mean + SE Scar age
# Samples
Magnet
Paired control
Nonfield control
I weeks 18 weeks
4 4
522 f 144 496 f 29
630 f 137 450 f 50
632 f 95
ACKNOWLEDGMENTS The authors are grateful to Ken Keown, Michael Bosnos, Zack Staples and Dr. John McCullen for their invaluable assistance and expertise; to Ellen Barker, Narcisco Tellez, Linda Tillema and Muriel Lothrop for their technical help; and to Glen Stahl and Smith-Kline Medical Specialties, Dr. James Hunter and Extracorporeal Medical Specialties, and Jim Cox and HeyerSchulte Corporation for their generous cooperation. This work was supported by U.S. P.H.S. Grant No. 14047.
REFERENCES 1. Bassett, C. A., The effect of force on skeletal tissues. In Downey and Darling (Eds.), Physiological Basis of Rehabilitation Medicine, p. 283316, New York: W. B. Saunders, 1971. 2. Berard, C., Woodward, S., Hermann, J. and Pulaski, E. J., Healing of incisional wounds in rats: The relationship of tensile strength and morphology to the normal skin wrinkle lines. Ann. Surg. 159:260-270,1964. 3. Brunius, U. and Ahren, C., Healing of skin incisions during reduced tension of the wound area. Ac?a Chir. &and. 135:383-390,1969. 4. Buck, R. C., Regeneration of tendon. J. Path. Bact. 66:1-18, 1953. 5. Bunting, C. H. and Eades, C., Effects of mechanical
tension on the polarity of growing fibroblasts. J. Exp. Med. 44:147-149.1926. 6. Forrester, J. C., Zederfeldt, B. H., Hayes, T. L. and Hunt, T. K., Tape closed and sutured wounds: A comparison by tensiometry and scanning electron microscopy. Brif. J. Surg. 51~729-737, 1970. 7. Madden, J. W. and Peacock, E. E., Studies on the biology of collagen during wound healing I. Rate of collagen synthesis and deposition in cutaneous wounds of the rat. Surgery64:288-294, 1968. 8. Madden, J. W. and Peacock, E. E., Jr., Studies on the biology of collagen during wound healing: III. Dynamic metabolism of scar collagen and remodeling of dermal wounds. Ann. Surg. 174:5I l520, 1971. 9. Peacock, E. E., Madden, J. W. and Trier, W. C., Biologic basis for the treatment of keloids and hypertrophic scars. Southern Med. J. 63:755-760, 1970. 10. Sussman, M., Effect of increased tissue traction upon tensile strength of cutaneous incisions in rats. Proc. Sot. Exp. Biol. Med. 123:38-41, 1966. 11. Thomgate, S. and Fergusen, D. J., Effect of tension on healing aponeurotic wounds. Surgery 44:619624,1958. 12. Weiss, P., From cell dynamics to tissue architecture. In Advanced Study Institute on Structure and Function of Connective and Skeletal Tissue, St. Andrews, Scotland, 1964. p. 256-263. London: Butterworths, 1965.
OF SURGICAL
RESEARCH
Effects
20, 93-102 (1976)
of Stress
I. Intermittent
on Healing Noncyclical
Wounds: Tension’
ARNOLD. J. AREM, M.D. AND JOHN W. MADDEN,
M.D.
Department of Surgery, University of Arizona, College of Medicine, Tucson, Arizona 85724 Submitted August 1, 1975
Since the time of Hippocrates, physicians have speculated about the influence of tension on healing wounds. In some situations, stress seems to produce thin, elongated scars; and in other situations hypertrophic scar forms. The paradox that mechanical forces might generate different end products out of the same amorphous matrix remains a clinically troublesome enigma. A century ago, Wilhelm His proposed that connective tissues can alter in response to stress by stating: At every place where connective tissue is exposed to a constant and oft repeated pulling action, there is formed a fibrous band, e.g. a tendon, the direction of whose fibers is parallel to the direction of the pull.
has yielded some of its secrets to the pressure of intensive scrutiny affirming (both figuratively and literally) Wolff’s law-bone adapts to functional forces acting upon it. If this principle applies to soft tissue, proper application of stress could modify scar morphology selectively. Unfortunately, the lack of a suitable model has prevented the rigorous study of how physical forces affect scar remodeling. Accurate interpretation of data mandates the simultaneous control of many variables in a dynamic, living organism. A number of essential design criteria must be met to create an adequate model. 1. The scar must be consistent and reproducible. 2. Environmental variables must be minimized. 3. Scar measurements must be free of sampling errors. 4. The model must permit measurement of scar physical strength; quantitative and kinetic biochemistry; and histology. 5. Tension must be applied in both a controllable and measureable way. 6. Individual variability must be controlled.
This selective fiber orientation in the direction of stress has been observed repeatedly. In 1926, Bunting and Eades [5] found that mechanical tension could orient fibroblasts in fresh wounds in the direction of tension. Even mitotic spindles were aligned parallel to the applied stress, and daughter cells separated along the lines of tension. In the ensuing years may observers have noted that in wounds healing under tension, newly deposited collagen becomes oriented in the direction of stress [ 1, 2, 3,4, 10, 111.Unfortunately, none of these studies has clarified the mechanism by which such morphologic changes occur, or the functional role of stress induced changes in soft tissue. In contrast, the bone remodeling process
The purpose of this paper is to describe a model created to meet the design requirements, and the results of a preliminary experiment testing the effect of prolonged intermittent stress on wounds of varying ages.
‘This work was supported by Grant 14047, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014.
THE MODEL To create a reproducible internal scar we designed two small rectangular blocks of 93
Copyright 01976 by Academic Press, Inc. All rights of reproduction in any form reserved.
94
JOURNAL
OF SURGICAL
RESEARCH,
VOL. 20, NO. 2, FEBRUARY
1976
FIG. 1. Design of magnet assembly; the entire assembly is 7.5 cm long.
Proplast for subcutaneous implantation in rats. Proplast is a carbon-impregnated Teflon sponge material with carefully controlled pore size allowing rapid fibrous tissue ingrowth. A 3-O nylon pullout suture was used to hold each sponge pair tightly approximated for any designated period post implantation. A single 3-O nylon mattress suture, passed circumvertebrally, served to anchor the proximal sponge securely in place throughout the experiment. Any force applied to the distal sponge, once the pullout suture is removed, distracts the scar tissue forming between the fixed proximal and the mobile distal sponge. In devising a means for supplying a distracting force, we abandoned on theoretical grounds any appliance involving direct attachment to the sponges. Grappling devices tend to “cut through” with time and introduce uncontrollable artifacts. In addition, since scar tissue takes time to form, the tensile force must be withheld until scar of the desired age has developed, and tension then applied without reopening or violating the wound. Magnetism, because there are “no strings attached” and because the magnetic field can be turned on or off and manipulated at will, was adopted as the motive force. To the distal sponge of each pair we bonded a 2.5 cm length of JP-6 (6 mm) dacron reinforced Hunter Silicone Tendon
prosthesis3 providing an inelastic bridge and a longitudinal orientation to each assembly. We bonded a silicone coated $$ain. Alnico 5 bar magnet or a nonmagnetic silicone coated metal bar of similar size to the tendon rod. The completed assembly was 7.5 cm long (Fig. 1). Under Innovar anesthesia, backs of rats were shaved and prepped with alcohol and Betadine. Implants were inserted into bilateral subcutaneous pockets through dorsal midline incisions, a magnet-bearing assembly on one side and a nonmagnet-bearing assembly on the other (Fig. 2). The electromagnets used to supply the distracting force were constructed of 5 x 20 cm cylindrical soft iron cores wound with 2000 turns of #16 copper wire, cooled both through the core and around the windings. These magnets are capable of carrying a sustained current of 7 A, producing a magnetic flux of 1400 Ga at 1 cm from the poleface. Using a gaussmeter we plotted magnetic flux as a function of distance from the poleface at varying currents and at points from the central axis to the periphery of the core. Three families of curves, corresponding to the three current settings, were observed (Fig. 3). In each case, as expected, magnetic flux was highest along the central longitudinal axis of the core. The gaussmeter output was read from an oscilloscope, and in
2Generously supplied by Smith-Kline cialties.
3Kindly furnished cialties, Inc.
Surgical Spe-
by Extracorporeal
Medical
Spe-
AREM AND MADDEN:
HEALING
WOUNDS
95
Measured force on bar magnet as a function of current through electromagnet and distance from poleface
160
-g In
120
;
100 2
FIG. 2. Position of implants. The circumvertehral suture anchors proximal sponges in place. A pullout suture holds each sponge pair together for several weeks. Insert demonstrates peripheral position of implants in cross-section.
no instance did we observe any magnetic ripple, eliminating any possibility of a vibrational effect induced by the ripple of the ac current. Using a sensitive transducer coupled to this magnet system, we plotted forcedistance curves to allow accurate measurement of force applied to the bar magnet (transmitted through the assembly to the developing scar tissue) at any distance from the electromagnet. Because the rat body is roughly cylindrical, with the subcutaneous implants lying peripherally in cross section (Fig. 2 insert), transducer measurements were made along the line of the core periphery. To maintain relative immobilization, we placed rats in cylindrical plastic restraining tubes (Fig. 4). The rats uniformly accli-
I?
80 60 40
Distance (cm)
FIG. 3. Force-distance curves. The force on the implanted magnet (measured along core periphery) drops off sharply with distance at any current setting.
matized to these tubes within 48 hours and slept thereafter in the tubes throughout the experiments. Rats were weighed weekly and no animal lost weight despite intermittent immobilization. EXPERIMENTAL DESIGN For the initial experiment we varied the age of the scar and maintained constant the parameters involved in the application of tension. We used female 250 gm Sprague-Dawley
FIG. 4. Rat restrained in magnetic field. Rats acclimate rapidly and sleep while in restraint tubes. Arrows indicate flow of water coolant.
96
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
rats throughout the experiments, ten rats in each group. Figure 5 summarizes the experimental design. Beginning three or 14 weeks after implantation, groups of rats were restrained at the pole ends of the electromagnets for 6 hr each day for four weeks. One day prior to sacrifice we weighed the rats and injected [3H] Proline (50 &i/ 100 g body weight) parenterally. On the day of the sacrifice, sponge assemblies were exposed, examined grossly and photographed. All implants could be dissected from the underlying tissues in a visible and reproducible plane. Representative sponge pairs were fixed in formalin, embedded in paraffin, sectioned longitudinally and stained with H & E or trichrome. We placed the remaining sponges in an Instron Tensiometer and determined the burst strength of the scar bridge between the sponges using a crosshead speed of 2 in/min. The two halves of each sponge pair were then pooled. Saline extractable collagen was assayed and net rate of collagen synthesis and deposition determined by measuring the specific activity of Hypro [7,8]. In the initial study on 3 week old scar, to determine the effect of the magnetic field it-
1976
self, we implanted magnet and nonmagnet sponge assemblies in an additional group of five rats. Beginning three weeks after implantation animals were restrained for 6 hr daily for four weeks but far removed from the electromagnets. Sponges from this “nonfield” cant rol group underwent bursting, histological and biochemical examination. Because no differences were found between these sponges and sponges from the other control groups, we deleted this control from the 14 week experiment. We were able to calculate the force transmitted to the implant at any point in time by measuring the distance from the bar magnet to poleface radiographically and plotting force on the force-distance curve for that field intensity. We used a power setting of 5A throughout the experiment. The intermagnet distance varied from 3 cm to Ya cm with a corresponding tensile force ranging from 30 to 120 gm. Several animals extruded their implants and were discarded from the study. With the exception of one animal which became paraplegic after two weeks and was discarded, there were no complications related to the circumvertebral suture.
weeks FIG. 5. Experimental design. Rats were started in magnetic field three or 14 weeks after implantation and subjected to four weeks of intermittent nonpulsatile stress.
AREM AND MADDEN:
HEALING
WOUNDS
97
FIG. 6. Appearance of sponge pairs following 4 weeks of tension on three week old scar. Lower sponge pair is from magnet side (stress), upper sponge pair from control side (no stress) of same animal.
RESULTS In sponge pairs subjected to tension three weeks post implantation, the scar bridge between the sponges was markedly elongated assuming a characteristic hourglass shape. In contrast, control sponges were tightly approximated and did not separate (Fig. 6). Histologically, the collagen fibers in stressed scars were oriented in the direction of tension, while the collagen between the nonstressed control sponges was randomly oriented (Figs. 7 and 8). Sponge pairs subjected to the same duration, timing, and magnitude of tension beginning 14 weeks after implantation did not separate when compared to non-stressed controls (Fig. 9). The collagen between the stressed sponges showed a slight tendency toward longitudinal orientation but was otherwise similar to controls, and very different from the histological appearance of the elongated younger scars (Figs. 10 and 11). The bursting strength data are shown in Table 1. By seven weeks after implantation the scar tissue between the sponges has attained over a pound of strength, and this approximately doubles by the eighteenth week. Although bursting strength of the stressed and nonstressed 18 week old scars were identical, the younger scars subjected to stress appeared to have less strength than
unstressed scars of similar age. This apparent difference is probably artifactual. As specimens are pulled apart in the tensiometer, there is a tendency for the material being distracted to undergo lateral contraction, that is, to deform and become narrower. With the elongated scars in the first experimental group, the large amount of free edge present allows the tensiometer to register bursting strength due to simple elongation and breakage. However, in control sponges, the shortness of the scar bridge constrains lateral contraction, and the deforming force registers in the tensiometer as shear in addition to elongation, resulting in a higher observed bursting strength. In the case of the older scars, the sponge pairs in both experimental and control groups were morphologically similar, and this measurement artifact occurred equally in both groups. The values for saline extractible collagen appear in Table 2, and for rate of collagen synthesis in Table 3. Under the conditions of this experiment, tension had no effect on either of these biochemical parameters of scar formation, even when the scars showed drastic morphological alterations. DISCUSSION The sponge implant model avoids the shortcomings inherent in studying the effects
98
JOURNAL
OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
1976
FIG. 7a. Histologic appearance of elongated scar tissue from stressed sponge pair shown in Fig. 6. Longitudinal section demonstrates thin, attenuated scar bridge (trichrome, 8 x). FIG. 7b. Closeup of bracketed area in Fig. 7a. Note parallel bundles of collagen oriented longitudinally in direction of stress (21 x).
of tension on dermal wounds. Animals tend to scratch and abrade surface wounds, producing additional scarring. When skin sutures are used, differences in material, placement, tightness and timing of removal become unmeasured variables. Some investigators have excised large areas of skin, relying on residual elasticity of marginal skin to produce tension, but have failed to measure the forces involved [2, 3, 5, 10, 111.
Sporadic contraction of the panniculus carnosus also affects tension and is impossible to quantitate. Interpretation of data on skin wound healing is further complicated by the existence of “skin tension lines” whose orientation with respect to the wound may influence the orientation of newly deposited collagen. Our model eliminates these sources of error, but introduces others which must be
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FIG. 8. Histologic appearance of scar bridge from unstressed control sponge pair in Fig. 6. The narrow gap between the spongesis filled with randomly oriented collagen (trichrome 14.7x).
FIG. 9. Appearance of sponge pairs following 4 weeks of tension on 14 week old scar. Lower sponge pair is from magnet side (stress), upper sponge pair from control side (no stress) of same animal. Contrast lack of elongation of stressed older scar with marked elongation of stressed younger scar seenin Fig. 6.
considered. Any implanted sponge is a foreign body and scar tissue forms in response to the foreign substance. Although Proplast produces a reliable and reproducible artifact, scar tissue formed may not be analogous to dermal scar. Out data cannot be used to interpret skin wound healing. We can, however, draw some conclusions regarding the effects of tension on an isolated internal scar system. Our results indicate that young scars can be altered morphologically by conditions of stress that do not affect older scars. These remarkable changes in form and appearance occur without alterations in rate of synthesis, in collagen crosslinking or in bursting strength of scar collagen. However, subtle alterations in the physical properties of scar may have escaped detection. Bursting strength is a fairly crude test of the biomechanical properties of a non-homogeneous system. Perhaps an analysis of the stress-strain characteristics of the scar material between the sponges, using subbreakage forces would be more meaningful. Similarly, the biochemical data are based on measurements of the total sponge
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F:IG. 10. Histologic appearance of stressed sponge pair shown in Fig. 9. Longitudinal section shows less str iking co11agen orientation than in stressed younger scars, but more than controls (trichrome, 15.75 x).
co1lagen. Metabolic alterations, if they ocGUI :red only in scar tissue subjected to the w :atest degree of stress, may have been mabskedby measuring the whole sponge. SePaIrate biochemical analysis of the scar
tissue between the spongesmay substani:iate this. In our primary experiment we cllose arbitrary parameters of stress and va.ried scar age. The striking morphologic dif-
FIG. I I. Histologic appearance of scar from unstressed sponge pair in Fig. 9. Longitudinal randomly oriented collagen (trichrome, 15.75 x).
section demonstrates
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WOUNDS
TABLE 1 ference between young and old scars implies Strength Measurements that scar maturation may be reflected in an Burst strength age-related ability to resist tensile stress. Algrams, mean + SE though we were able to accurately determine the force applied to the implanted magnet, Scar age # Animals Magnet Paired control we cannot assume that this force was trans- I weeks 4 456 jl 158 538 f 48 mitted entirely to the scar bridge between 18 weeks 4 1113 f 239 1029 f 109 the sponges. The filmy attachments of the implant’s capsule to overlying skin or underlying fascia may have dampened the excursion of the implant and distributed the physical forces affect the remodeling scar applied force to a greater volume of the scar. can be of enormous practical benefit. Fibrous attachments, like the sponge Collagen is a nonliving material and any collagen, mature and strengthen, producing remodeling that occurs must ultimately a proportionately greater dampening of the depend on the ability of living cells to sense force with time. Differential force distribution may account for differences ob- and transduce mechanical force into biochemical action. Possible mechanisms served between 7 and 18 week old scars. Finally, this study fails to define ac- for this transduction have been summarized curately where the tensile forces exert their in an excellent review by C. A. L. Bassett [5]. main effect. Sponge separation may involve If tissue deformation is the mechanical triga progressive elongation of the capsule sur- ger for biological activity, then the degree of rounding the sponges as well as the scar be- force, the rate, frequency and duration of its tween them. We should be able to answer the application, and loading velocity may all be question by marking the capsule and se- important in producing connective tissue quentially studying the marker movements alterations. Our magnet system allows manipulation of these parameters singly or as the spongesseparate. in combination. In addition, the sponge The results of this study suggest that, model readily lends itself to the application under appropriately defined conditions, scar of other modalities such as ultrasound, heat, tissue morphology can be modified by stress direct current stimulation (through imand that modification may depend on a planted electrodes) across the scar bridge, or balance between age and the parameters of lathyritic agents, simultaneously with apstress. Apart from any implications relative plication of tensile force to study the to skin wound healing, data derived from studying the sponge implant model may combined action of those modalities with have practical implications for a variety of stress on scar tissue. clinical situations. If the sponge model If, as Paul Weiss [12] has said, static form proves to be analogous, for example, to is the outcome of formative dynamics, our wounds of tendon or ligament, then model will be useful in studying some of the centuries old questions about the influence of knowledge of precisely how the magnitude, rate of application, duration and timing of dynamic forces on healing wounds. TABLE II Collagen Cross Linking % Neutral salt soluble collagen mean + SE Scar age
# Samples
Magnet
Paired control
Nonfield control
I weeks 18 weeks
8 4
4.11 f .34 2.51 f .39
4.62 f .50 2.56 * .16
4.0 f .22
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TABLE III Rate of Collagen Synthesis Specific activity of hydroxyproline (dpm Hyplti Hyp), Mean + SE Scar age
# Samples
Magnet
Paired control
Nonfield control
I weeks 18 weeks
4 4
522 f 144 496 f 29
630 f 137 450 f 50
632 f 95
ACKNOWLEDGMENTS The authors are grateful to Ken Keown, Michael Bosnos, Zack Staples and Dr. John McCullen for their invaluable assistance and expertise; to Ellen Barker, Narcisco Tellez, Linda Tillema and Muriel Lothrop for their technical help; and to Glen Stahl and Smith-Kline Medical Specialties, Dr. James Hunter and Extracorporeal Medical Specialties, and Jim Cox and HeyerSchulte Corporation for their generous cooperation. This work was supported by U.S. P.H.S. Grant No. 14047.
REFERENCES 1. Bassett, C. A., The effect of force on skeletal tissues. In Downey and Darling (Eds.), Physiological Basis of Rehabilitation Medicine, p. 283316, New York: W. B. Saunders, 1971. 2. Berard, C., Woodward, S., Hermann, J. and Pulaski, E. J., Healing of incisional wounds in rats: The relationship of tensile strength and morphology to the normal skin wrinkle lines. Ann. Surg. 159:260-270,1964. 3. Brunius, U. and Ahren, C., Healing of skin incisions during reduced tension of the wound area. Ac?a Chir. &and. 135:383-390,1969. 4. Buck, R. C., Regeneration of tendon. J. Path. Bact. 66:1-18, 1953. 5. Bunting, C. H. and Eades, C., Effects of mechanical
tension on the polarity of growing fibroblasts. J. Exp. Med. 44:147-149.1926. 6. Forrester, J. C., Zederfeldt, B. H., Hayes, T. L. and Hunt, T. K., Tape closed and sutured wounds: A comparison by tensiometry and scanning electron microscopy. Brif. J. Surg. 51~729-737, 1970. 7. Madden, J. W. and Peacock, E. E., Studies on the biology of collagen during wound healing I. Rate of collagen synthesis and deposition in cutaneous wounds of the rat. Surgery64:288-294, 1968. 8. Madden, J. W. and Peacock, E. E., Jr., Studies on the biology of collagen during wound healing: III. Dynamic metabolism of scar collagen and remodeling of dermal wounds. Ann. Surg. 174:5I l520, 1971. 9. Peacock, E. E., Madden, J. W. and Trier, W. C., Biologic basis for the treatment of keloids and hypertrophic scars. Southern Med. J. 63:755-760, 1970. 10. Sussman, M., Effect of increased tissue traction upon tensile strength of cutaneous incisions in rats. Proc. Sot. Exp. Biol. Med. 123:38-41, 1966. 11. Thomgate, S. and Fergusen, D. J., Effect of tension on healing aponeurotic wounds. Surgery 44:619624,1958. 12. Weiss, P., From cell dynamics to tissue architecture. In Advanced Study Institute on Structure and Function of Connective and Skeletal Tissue, St. Andrews, Scotland, 1964. p. 256-263. London: Butterworths, 1965.