The Nature of Wound Healing: *
Experimental Tensile Strength Studies with Deca Durabolin and S35
GEORGE T. WATTS, CH.M., F.R.C.S., MICHAEL R. BADDELEY, M.B., B.SC., F.R.C.S.,
ROBERT WELLINGS, JANICE EVANS
From the Division of Wound Healing Research, Department of Surgery,
University of Birmingham, Birmingham, England
UNION of the two sides of a wound is be-
lieved to be made by the deposition of
collagen fibers in a matrix of granulation
tissue. Studies of the granulation layer,
however, suggested that the changes origi-
nated in cells in the surrounding tissue.5
This concept is supported by the work of
Griubo2 who showed that new fibroblasts
develop in the tissue around wounds. The
object of the present experiments was to
compare changes in the wound cavity con-
tents with morphologic change expressed
by the tensile strength.
Methods
For the biochemical studies, square skin
wounds in guinea pigs were used as de-
scribed previously.4 The incised wounds for
measurement of tensile strength were stand-
ardized by using a rubber stamp. This
marked the site of the wound, the position
of stitches and the points at which dis-
traction was applied to measure tensile
strength. The wounds divided skin and
panniculus carnosus. To measure the tensile
strength small metal plates were cemented
to the skin edges with an impact adhesive.
Threads through these were attached to the
testing machine. A full description of this
is being presented elsewhere.
Experiment 1. One group of animals was
used for controls and a second was treated
with an anabolic agent; 2.5 mg. of deca
* Submitted for publication June
7, 1964.
Supported in part by the United States National
Institute of Health Grant H 5772, the Medical Re-
search Council and the Endowment Fund of the
United Birmingham Hospitals.
109
durabolin was given by intramuscular in-
jection 4 days before wounding. The ana-
bolic effect was shown by increase in kid-
ney weight (expressed as a per cent of the
original weight of the animal) and by gain
in total body weight (Fig. 1).
There was no significant difference be-
tween the treated and control animals with
respect to total tissue, total desoxyribo-
nucleic acid (DNA), hydroxyproline (OHP)
and concentration of DNA and hydroxy-
proline in these wounds (Fig. 2-6). Com-
paring the rate of contraction of the wound
area, treated wounds contract more rapidly
(Fig. 7). There was also a difference in
tensile strength, most easily demonstrated
by regression lines (Fig. 8). (These are
mathematical representations and do not
show actual values in wounds.) Adminis-
tration of deca durabolin advances tensile
1-3
OsI*2 j %\.
2,
oC 12
1 I
/ \
0~
3 4 5 6 7 8 9 10
DAYS AFTER WOUNDING
FIG. 1. To show anabolic effect produced by
deca durabolin expressed as a % of original weight
of animal (p = 0.05 - 0.02). 2.5 mg. deca dura-
bolin were given in 0.1 ml. of ethyl oleate. Con-
trol animals were given a similar volume of ethyl
oleate ( In all diagrams controls are shown by
solid lines; treated animals are shown by broken
lines).
110 WATTS, BADDELEY, WELLINGS AND EVANS Annals of Surgery
July 1965
80 strength levels between 1 and 2 days in the
earlier stages of healing. Later (by the 10th
0 day) there is no significant difference.
z 60
0
If we regard DNA as an index of granu-
lation tissue cells and hydroxyproline of
I-
40 collagen, these results indicate that tensile
0
strength may increase without change in
-J
0- -o the cell or collagen levels in the wound
a- 20
0
cavity. These, therefore, cannot be the only
I.- mediators of union. While improved con-
traction would facilitate healing, it does not
3 4 5 6 7 a 9 10
alter the need for an agent to unite the sur-
DAYS AFTER WOUNDING faces. The alternative to collagen is the
FIG. 2. Total weight of granulation tissue in con-
cement which joins the cells, fibers and
trol and deca durabolin treated animals. other components of healthy, unwounded
tissue, the principle component of which
is mucopolysaccharide.
2001 Experiment 2. We measured the uptake
of S35 labelled sodium sulphate in two
groups of animals, one used as controls and
o 150 the other treated with deca durabolin as
z
0 before. The granulation tissue was har-
100
vested on the appropriate days and the S35
z
uptake measured by the method of Kodicek
0
-J 0
and Loewi.3 The tissue is fused with sodium
0
- s50 0 sulphate, dissolved in hydrochloric acid and
then precipitated as barium sulphate by
addition of barium chloride. A Geiger Miul-
o
3 4 5 6 7 a 9 10
ler tube is used for counting. Administra-
DAYS AFTER WOUNDING tion of radioactive sulphate alters the rate
FIG. 3. Comparison of desoxyribonucleic acid totals of healing, delaying the time of maximum
in treated and control animals. formation of granulation tissue about 3
days, and affects the animal's total weight.
/-
0
z 1.0 z) 40

0 tn
I-

an I--

0.75) 3 35
13
I

w
If
z 0
2 30
0 0.50 I-

4(
I z
0

25
(i
4 z
I~- 0_ _Io 0
0 u
1-

o 3 4 5 6 7 a 9 10
3 4 5 6 7 a 9 10 DAYS AFTER WOUNDING
DAYS AFTER WOUNDING
FIG. 5. To show absence of any difference in
FIG. 4. Comparison of hydroxyproline totals in concentration of desoxyribonucleic acid between
treated and control animals. control and treated wounds.
Volume 162
Number 1
THE NATURE OF WOUND HEALING ill
U,,S
,, 12001.
In
w 2o I
Iooo0
3 2-0 E
a

-T
0 I Z//
o
z
600
oo w

0 1.0
3
(" 400
1.0
Zi
z
Z 200
-I hi

09
IL
3 4 5 6 7 S
DAYS AFTER WOUNDING
FIG. 6. To show absence of any difference in
concentration of hydroxyproline between control
and treated wounds.
These effects make day-to-day comparison
of treated and untreated animals impos-
sible. We therefore compared the total S35
uptake of each group of animals (Fig. 9)
and found a significant difference. That
the increased uptake of S35 after treatment
with deca durabolin results from increased
mucopolysaccharide formation is shown by
electrophoretic analysis-the S35 on the
autoradiograph of an electrophoretic strip
is at the same position as the mucopolysac-
charide on a stained strip of tissue or con-
trol solutions (Fig. 10). Since the sulphate
will be taken up in the chondroitin sul-
phate, deca durabolin presumably increases
the deposition of chondroitin sulphate.
/
0lS
0 1 2 3 4 5 6 7 8 9 10
DAYS
FiG. 8. Extrapolation of regression lines for cal-
9 0 culated best fit show a significant difference in in-
tercept (p < 0.001) indicating that development of
tensile strength in treated animals is advanced as
compared with controls.
Discussion
Administration of an anabolic agent can
increase tensile strength in a wound by an
increase of chondroitin sulphate without in-
crease of collagen. If the effect was pro-
duced through integration in collagen fibers,
the total collagen laid down should in-
crease, but this does not happen. It there-
fore appears that mucopolysaccharides
alone can be the agents for producing ten-
sile strength. Elsewhere in the body they
act as tissue cement and where the tissue
is predominantly cellular, the strength of
the tissue depends mainly on this action.
Where epithelial healing occurs collagen

%A
301
so z
0
i

/
2 20 ~~~~/
60 ..0
z
/\ ,~~
0
/
z
C 40 0
z A- se
0 In
-
on

-~0
20

3 4 5 6 7 a 9 10

DAYS AFTER WOUNDING

0 1 2 3 4 5 6 7 a 9 10 FIG. 9. Total uptake of S'5 labelled sodium sul-

DAYS AFTER WOUNDING phate (0.336 millicuries/100 Gm. total body weight
given intraperitoneally 48 hours before sacrifice).
FIG. 7. Contraction expressed as a % of original Increased uptake is shown in treated animals.
wound Overall significance of increased con-
area. Values of p: 3rd day 0.1-0.05; 4th day >0.1;
traction in the first 6 days (p <0.001). 7th day 5th day 0.05; 6th day <0.001; 7th day 0.01 -
onwards p = 0.15 0.20. -
0.001; 8th day >0.1; 10th day 0.01 - 0.001.
112 WATTS, BADDELEY, WELLINGS AND EVANS
A B C ates development of tensile strength in
FIG. 10. A) Autoradiograph of electrophoretic
strip of tissue hydrolysed with caustic soda to show
position of S' containing elements. B) Same prepa-
ration stained with alcian blue. C) Control solution
of mucopolysaccharide stained with alcian blue.
is unimportant-well demonstrated in the
lowest animals with a single cell epithelial
covering and in acute ulceration in higher
animals in similar tissues such as the ali-
mentary tract lining.
Although collagen does fulfill a useful
role in the healing of many wounds, it may
not be an essential agent for healing. It is
possible that in an "ideal" wound no col-
lagen, but only intercellular cement, would
be needed for union. Healing would then
restore the situation in normal tissue where
cellular and supportive elements are inte-
grated by the ground substance. It is known
that the surgical wound which has least
scar tissue is ultimately the strongest and
that wound sutures in young patients can
be removed earlier than in older ones as
wound strength develops more rapidly. It
has been shown 1 that young tissue contains
more mucopolysaccharide relative to col-
lagen. We have observed that wounds heal-
ing in young animals develop less collagen
than those in old ones.
Presumably, mucopolysaccharides are uti-
lized by tissues in healing by a process of
polymerization similar to that occurring in
some modern adhesives.* This may be re-
lated to the polymerization which occurs as
* We have attempted to use these
polymerizing
adhesives for repair of wounds but have found that
certain of their physicochemical properties result
in inferior healing.
Annals ofJulySurgery
1965
part of collagen formation. It is, however,
possible that collagen deposition is unneces-
sary for wound healing and that it is 1)
part of a nonspecific tissue reaction associ-
ated with tissue protection and immune
processes and 2) as nonspecific to wounds
as fever to infection.
Summary
Administration of deca durabolin acceler-
wounds without affecting collagen or cell
content of the wound granulation tissue.
This development is accompanied by in-
creased formation of mucopolysaccharide
when measured by uptake of S35 labelled
sulphate.
Clinical experience indicates that muco-
polysaccharide and not collagen is the im-
portant agent in the healing of wounds.
Young tissues have a higher mucopoly-
saccharide to collagen ratio and heal more
rapidly.
Collagen deposition may be a nonspe-
cific process and not a fundamental part of
healing.
Acknowledgments
We should like to express our appreciation to
Professor A. L. d'Abreu, University Department of
Surgery, Birmingham, for the facilities provided,
Dr. N. Crawford, Miss S. Parkinson and Miss P.
Cole for their assistance, and Dr. J. A. H. Water-
house for advice in our statistical analyses.
References
1. Clausen, B.: Influence of Age on Connective
Tissue: Uronic Acid and Uronic Acid-hydroxy-
proline Ratio in Human Aorta, Myocardium
and Skin. Lab. Invest., 11:1340, 1962.
2. Grillo, H. C.: Origin of Fibroblasts in Wound
Healing: An Autoradiographic Study of In-
hibition of Cellular Proliferation by Local X-
irradiation. Ann. Surg., 157:453, 1963.
3. Kodicek, E. and G. Loewi: The Uptake of S5
Sulphate by Mucopolysaccharides of Granu-
lation Tissue. Proc. Roy. Soc., 144B:100,
1955-6.
4. Watts, G. T., R. M. Baddeley, N. Crawford
and R. Wellings: Wound Healing: Biochemi-
cal Methods for Observation of the Cellular
and Intercellular Phases. Brit. J. Clin. Pract.,
16:733, 1962.
5. Watts, G. T., R. M. Baddeley and R. Wellings:
Significance of Cells in Wound Granulation
Tissue. Lancet, 2:1031, 1963.