Clin. North America. 58:1233-1248; 1978. 7. Thornton, J.W.; Tavis, M.J.; Harney, J.H. Graft adherence to wound surfaces: Collagen fibrin interactions. Burns 3:23 ...
Annals of Biomedical Eng&eering, Vol. 21, pp. 51-55, 1993
0090-6964/93 $6.00 + .00 Copyright 9 1993 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
Development of a Device for Measuring Adherence of Skin Grafts to the Wound Surface C. Do~rc,* E. M E A D , * R. SKALAK,* Y.C. F U N G , * J.C. D~aES,* R.L. ZAPATA-SIRVENT,t C. ANDREE,t G. GREENLEAF,t M. COOPER,t and J.F. HANSBROUGHt *Department of Applied Mechanics and Engineering Sciences, tDepartment of Surgery, University of California, San Diego, La Jolla, CA
Abstract-Adherence of a biological graft to the wound surface is the most important factor influencing the ultimate success of graft viability. A machine has been developed to test the adherence of biological graft materials to a suhstrate such as a wound surface. The peeling mode, which yields reproducible quantitative measurements of adherence, is a standard method for testing adhesives. The device is designed to continuously measure the force required to peel the graft from the substrate at a constant rate. This force is a function of the energy of adhesion per unit area of adhered surface. This device has been used to measure the peeling force of (2 x 2 cm) skin grafts which are applied to full-thickness wounds on mice. Results of tests on adherence of autografts on mice show that the peeling force increases significantly with time over the first 9 days of healing. Thus, this device is useful in quantitative comparison of various skin grafting techniques and artificial grafts.
plied perpendicular to the skin surface required to dislodge a tiny (6 m m diameter) skin graft on animals (5). The m a x i m u m force required to dislodge the graft was then recorded on a strip chart. We have designed a device for measuring adherence of much larger (2 x 2 cm) grafts which are applied to full-thickness wounds on mice. The equipment allows for the reproducible, quantitative measurement of early adherence forces, and should be useful for the study of adherence properties o f various biological and prosthetic grafts. The present device operates in a peeling mode at constant velocity, and the data is reduced to an average value. The peeling mode is a standard method o f testing adhesives and is expected to be a more reproducible method than perpendicular force testing of a small area.
Keywords-Skin grafts, Adherence, Tensiometer.
MATERIALS A N D METHODS
INTRODUCTION
Application of Skin Grafts to Wounds
The most important property of a biological or artificial graft applied to a wound is adherence to the wound surface (4,6). Without rapid adherence the biological graft will not be vascularized by the underlying tissues and the graft material will rapidly develop subgraft fluid collections and will quickly dislodge from the wound surface. Adherence of grafts to wound surfaces has generally been divided into two periods. "Early" adherence occurs within the first 72 hours o f graft placement, and is thought to be dependent upon fibrin interactions between the wound surface and the graft (7). "Late" adherence begins after 72 hours and involves fibrovascular ingrowth into the graft material, and is more a measure of wound healing than of interface adherence. Adherence of graft materials to the wound bed is difficult to quantitate in a reproducible fashion. An earlier report described a tensiometer that measured forces ap-
All animal experiments were performed with approval o f the U.C.S.D. Animal Research Committee and in accordance with guidelines of the National Institutes of Health (Guide for the Care and Use o f Laboratory Animals, N I H publication No. 85-23, revised 1985). Female CF-1 outbred mice (Charles Rivers Laboratories) were used for all experiments. The dorsal skin was shaved. Surgery was then performed in laminar-flow hoods under anesthesia using 10 mg intraperitoneal (i.p.) Avertin in tert-butyl alcohol (Aldrich Chemical Co., Milwaukee, WI) in 0.4 ml normal saline. The dorso-lateral surface of the mouse was washed with povidone-iodine and 70% isopropanol. A 2 x 2 cm full thickness skin section was excised, sparing the panniculus carnosus. The excised skin was then rotated 180 ~ (the caudal edge was turned cephalic) and immediately sutured onto the wound defect using 6.0 silk. Grafts were then covered with X e r o f o r m " (Sherwood Medical, St. Louis, MO) and cotton gauze, and secured with a light bandage circumferentially around the b o d y of the animal. Surgical mortality was less than 3%.
Address correspondenceto Richard Skalak, Department of Applied Mechanics and Engineering Sciences, Universityof California, San Diego, La Jolla, CA 92093-0412. (Received 3/16/91; Revised 12/5/91) 51
52
C. DoNa et al. Development o f the Test Equipment
The test equipment (Fig. 1) is designed to measure the peeling force af a constant peeling speed. As shown in Fig. 1, the test specimen is clamped along one edge of the test square and is placed on a platform whose height is adjustable along the platform post. The platform post is fixed on the base of the device. The clamp is about 2 cm long and is mounted onto two clamp arms. The peeling force is applied to one edge (the peeling edge) of the test surface on the specimen via a triangular metal yoke. This metal yoke is made from stainless steel of very light gage and its shape is designed as a right isosceles triangle (Fig. 2). Side BC is glued (by Super Glue, Cyanoacrylate Adhesive, Super Glue Corporation) onto the peeling edge. The force is applied at the corner A (Fig. 2). The peeling force is generated by the motor (Holtzer-Cabot) with a constant rate rotation of 2 rpm. The total peeling resistance will be balanced by the force produced by the string winding onto the motor shaft 0.63 cm in diameter. A silk suture is used to connect the motor shaft and corner A o f the metal piece via a pulley which is placed on a rod fixed between two pulley posts. A force transducer (Gould-Statham Model No. UTC3) is installed under the balance arm to record the reaction which is proportional to the peeling force, while the motor is mounted on the other side of the arm. This balance arm is supported by frictionless hybrid flexure pivot (Free Flex Pivot | and
balanced by weight (W) as shown in Fig. I. The force transducer can be moved in either direction along the lever to achieve the best sensitivity of reading. The device is calibrated before each set of tests. Calibration is accomplished by removing the animal platform and hanging standard weights from a silk thread. The voltage generated by the force transducer is magnified by an amplifier (Validyne M1-3) and plotted by a calibrated plotter (HP 7090A Measurement Plotting System, Hewlett Packard). Determination of the Adhesive Energy of Skin Grafts to the Wound
The adhesion of skin graft to the wound surface can be evaluated in terms of the energy of adhesion 3' per unit area of adhered surface. This can be measured by the peeling experiment: 3' ---
adhesive surface energy area of contact
peeling force length of peeling line
Newtons Meter To determine 3', we need to measure the peeling force and length of the peeling line perpendicular to the applied force. The peeling force is measured by using the device described above; a data set recorded on the plotter is illus-
haft
~m t
Load cell FIGURE 1. The test equipment designed to measure the peeling force of a biological graft at constant peeling speed.
Measuring Adherence of Skin Grafts
B Glue line
53
Glue line
Specimenxx(L~\ a,(:; Clamp
Metalyoke .
Peeling force
Sub=rate
Peeling force
C
Top View (a)
Side View (b)
The peeling force is applied to one edge of the specimen via a triangular metal yoke. Side B,C is glued onto the peeling edge. The force is applied at the corner A. FIGURE 2.
trated in Fig, 3, The force measured here is the total peeling resistance of the adhesion between the skin graft and the wound surface. It has been measured in such a way (steady peeling) that the elastic effect of the skin graft can be neglected so that the total peeling force is a measure of the adhesive energy. The peeling clamp was sufficiently stiff and attached to the entire width o f the graft so that the entire width peeled uniformly. The edges of the graft were cut through the skin thickness prior to each test to eliminate edge tearing effects. Grafts were installed on one side or the other of the mid-sagital plane so the effects of body curvature were minimized. The peeling time for each test is defined as beginning at the first local m a x i m u m of the peeling force after the initial nonlinear rise (Fig. 3) and ending at the last local m a x i m u m prior to the sudden drop near the end of the recording. In Fig. 3, the beginning and ending times were 22 and 78 s, respectively. A simple averaging is used to obtain the mean value of the peeling force f r o m the actual data plot for each individual peeling test. Consider a total number of points measured to be Nm in the m th test record. The mean force if,, during the m th test is calculated by:
E
3O 2s
~ eo 0 i,
15
~
5
/
At"
,^ i/M/"
I
t~ A,
Ii 2,
o 20
40
60
80
1 100
Time (Seconds) A data set recorded on the plotter. Force due to peeling resistance of the adhesion between the skin graft and the wound surface is recorded vs. time during steady peeling. FIGURE 3.
l fT.,
- Jo Fm = -Tm
1 Nm - i=l ~aF(ti)Atm F ( t ) dt = -T,~
(1)
in which
T,.
Atm = - Nm
(2)
where Tm is the entire peeling time period in the m th test and i = 1,2,3...Arm. I f there are total M t e s t s in one group, (m = 1,2 . . . . . M ) , the standard deviation o f all the F,, in this group is expressed by:
s=
•J ~
1
M
N(,e
_F)~
(3)
m=l
in which P is a mean o f all the Fm in the group defined by: M
1 ~ , p~ .
(4)
"1~= mm=l
The Standard Error o f the mean (SEM) is given by s/~f-A$. In the experiments, reported here, five different groups o f tests on CF-1 mice were selected to study the adherence of skin grafts to the wound surface. The mice were under anesthesia (the same as used to p e r f o r m the skin graft) at the time of peeling, and they were sacrificed right after the procedure by cervical dislocation. A total of 56 female CF-1 outbred mice (Charles River) were used in these experiments. The number o f mice (M) in each o f the five groups is shown within the bars in Fig. 4. Each group of tests were identical in test specimen size and procedure of measurement of peeling force, but tested at different times after placement of grafts onto the wound. The statistical significance of differences in the results
54
C. DONG et al.
400
-
taJ
* P( 0.05 COMPAREDTO PREVIOUSDAY
ee 7 O o
LL
Z
O_
300-
x o
200-
,
100-
'
2 hours
1
day 1
1
day 3
'
day 6
I
day 9
TIME AFTER GRAFTING FIGURE 4. The mean peeling force (• SEM) of skin grafts tested
at various times after surgery ranging from 2 hours to 9 days, The numbers inside the bars indicate the number of mice (M) tested in each group. The asterisk indicates a statistically significant difference in the means between consecutive days according to a two tailed T-test with p < 0 , 0 5 .
a m o n g these five groups is evaluated by the T-test on the basis of the P-value at a 5~ level of significance. RESULTS Adherence Studies
Measurement of adherence of grafts to the wound surface at 2 hours after placement revealed a low but measurable and reproducible peeling force (Fig. 4). The mean peeling force at 2 hours was 33.64 + 4.90 (SEM) N • 10 -3 . G r a f t adherence was markedly increased at 24 hours postgrafting, with a mean value of 83.94 + 11.67 (SEM) N • 10 -3. At 6 days after grafting, values were not statistically different f r o m the 72 hour values. This latter value suggests that appreciable fibrovascular ingrowth into the graft has not yet occurred by the 6th day after grafting. However, as shown in Fig. 4, there was a large increase in the peeling force between day 6 and day 9, presumably due to vascularization and the fibrous ingrowth that accompanies it. Visual inspections of the wound surfaces after peeling indicate an increase of microvascular development between day 6 and day 9. DISCUSSION The equipment described here has the advantage of a reproducible, controlled, constant rate peeling process, which gives an accurate measure of the force required to overcome the adhesion of the graft to its substrate. In tests
in which a force is applied perpendicular to the surface of the skin, there is a greater deformation of the specimen in an uncontrolled manner. In such a test, peeling also probably occurs, but it is not at a constant rate or over a controlled length o f the peeling line. In the present tests, the use o f the balance beam allows accurate measurement o f the small forces involved, while the constant speed m o t o r provides the controlled, constant rate o f peeling. It should be noted that the forces reported herein are for the full 2 cm width of the graft. There may be a slight contraction during healing and also due to the peeling force. Rather than estimating these effects, the specimens are prepared to standard dimensions and the full force of peeling is reported. This device should be helpful for developing and improving both natural and prosthetic graft materials. Modifications of current biologic grafts and membranes might benefit very early adherence properties and improve "take" of grafts containing synthetic materials (2). In addition, pharmacologic and biologic agents such as growth factors (I) and attachment peptides (3), which may promote fibrovascular ingrowth from the wound surface into the graft, m a y be shown to accelerate the onset of "late" adherence of grafts. In the results shown here (Fig. 4), there was no statistically significant increase in the adherence values f r o m the 3rd to the 6th day following graft placement, suggesting that significant fibrovascular ingrowth into the graft had not yet occurred. This was confirmed by the finding that little bleeding was seen during any of the tests when the skin was peeled from the wound bed. However, by day 9, the peeling force is dramatically increased, presumably due to neovascularization. This leaves a potentially ample opportunity for biologic effectors to improve the fibrovascular response of the wound bed to the graft, Further studies with the adherence measuring device should allow us to quantitate the effects of biological modifiers for each healing process. REFERENCES
1. Cooper, M.L.; Hansbrough, J.F.; Foreman, T.J.; Sakabus, S.; Laxer, J.A. The effects of epidermal growth factor and basic fibroblast growth factor on epithelialization of meshed skin graft interstices. In: Barbul, A., ed. Clinical and experimental approaches to dermal and epidermal repair: Normal and chronic wounds. Proceedings of the 3rd International Symposium on Tissue Repair, Miami, FL. New York: Wiley/Liss Inc.; 1991: pp. 529-442. 2. Cooper, M.L.; Hansbrough, J.E; Spielvogel, R.L.; Cohen, R.; Barrel, R.L.; Naughton, G. In vivo optimization of a living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials 12:243-248; 1991.
Measuring Adherence of Skin Grafts 3. Cooper, M.L.; Hansbrough, J.E; Foreman, T.J.; Laxer, J.A. In vitro and in vivo effects of matrix peptides on a cultured dermal-epidermal composite skin substitute. J. Surg. Res. 48:528-533; 1990. 4. Hansbrough, J.F. Biologic dressings. In: Boswick, J., ed. The art and science of burn care. Rockville, MD: Aspen Publishers; 1987. 5. Tavis, M.J.; Thornton, J.W.; Harney, J.H.; Woodroof,
55
E.A.; Bartlet, R.H. Graft adherence to de-epithelialized surfaces. Ann. Surg. 184:594-600; 1976. 6. Tavis, M.J.; Thornton, J.; Danet, R.; Bartlett, R.H. Current status of skin substitutes. Surg. Clin. North America 58:1233-1248; 1978. 7. Thornton, J.W.; Tavis, M.J.; Harney, J.H. Graft adherence to wound surfaces: Collagen fibrin interactions. Burns 3:23; 1977.
