Effects of Cortisone and Vitamin A on Wound Healing
H. PAUL EHRLICH, M.A., THOMAS K. HUNT, M.D.
From the Departments of Surgery, University of California School of Medicine,
and San Francisco General Hospital, San Francisco
ANTI-INFILAMMATORY steroids such as
cortisone decrease the rate of healing of
surgical wounds.7 Many attempts have
been made to interpret this phenomenon,
but no satisfactory explanation has been
found.
The outstanding characteristic of the in-
hibitory effect of cortisone on wound heal-
ing is that it occurs only when the drug
is given in moderate or large amounts
within the first 2 or 3 days after injury.12
Cortisone given in moderate or large doses
after the third day following injury does
not inhibit wound strength and apparently
does not irnhibit collagen synthesis.
Cortisone delays the appearance of al-
most all elements of healing, including in-
-flammatory cells, ground substance, fibro-
blasts, collagen, regenerating capillaries,
and epithelial migration.
All anti-inflammatory steroids have an
inhibitory effect on wound healing, and
inflammation dominates the phase of heal-
ing in which the effect of cortisone ap-
pears. The most commonly accepted expla-
nation for the effect of cortisone on healing
is that it prevents the inflammatory phase
which is somehow, essential to the healing
process. However, this explanation is little
more than a description of the events. It
would be much more helpful to know spe-
Submitted for publication September 15, 1967.
Please address correspondence to: Thomas K.
Hunt, M.D., U. C. Surgical Service, San Francisco
General Hospital, 3rd Floor Solarium, 22nd and
Potrero, San Francisco, California.
This study was supported, in part, by a grant
from the Committee on Medical Research, Uni-
versity of California School of Medicine.
324
cifically which aspect of inflammation is
involved.
One possible explanation for the effect
of cortisone lies in the area of lysosome
physiology. Cortisone and other anti-
inflammatory steroids increase the integrity
of the lysosome, the subcellular particle
which contains an assortment of acid hy-
drolases. Lysosomal enzymes are known
to take a prominent part in the inflamma-
tory process.3 If the effect of cortisone on
wound healing is mediated through the
lysosomal membrane, we would expect
that administration of Vitamin A, a lyso-
somal labilizer, would return the rate of
healing toward normal. The rationale be-
hind this hypothesis is as follows: In vitro
studies show that acid hydrolytic enzymes
are released into the supernatant of liver
homogenates when Vitamin A is added.4
Vitamin A in vivo causes the release of
hydrolytic enzymes which results in patho-
logic destruction of connective tissue.5
Vitamin A and cortisone have antagonistic
effects upon the lysosomal membrane.17
To test the hypothesis that the inhibition
of wound healing by cortisone may be me-
diated through the lysosome, a system has
been devised in which the effects of Vita-
min A and cortisone on wound healing can
be determined.
Method
Male Sprague-Dawley rats, 4 to 6 months
old and weighing 350 to 450 Gm., were
placed in individual cages and were given
water and Purina Chow ad libitum. No
dietary supplements were given.
Volume 167 EFFECTS OF CORTISONE AND VITAMIN A ON WOUND HEALING
Number 3
The animals were anesthetized with
ether and their backs were shaved and
cleaned with tincture of iodine. One stand-
ard wound 6 cm. long was made on the
back of each animal by the Sandberg meth-
ods.12 The wound was placed 1 cm. lateral
to the midline. In half the wounds were
placed on the right side and in the other
half on the left. The wounds were closed
with running sutures of 4-0 silk. Immedi-
ately after operation, the rats were placed
in collars to prevent damage to the wounds.
The animals were divided into four main
groups.
Group I. Eighteen rats served as control
animals and received no injections.
Group II. Fifteen animals received Vita-
min A alone. They were given 1,500 inter-
national units of Vitamin A in peanut oil
intraperitoneally on Days 1, 3, and 5.*
Group III. Twenty-five animals received
cortisone only. Each rat was given an injec-
tion of 10 mg. of cortisone acetate into the
muscle of the right thigh starting on Day
0 and daily thereafter through Day 7. Each
animal also received 0.2 cc. of sterile
peanut oil intraperitoneally on Days 1, 3
and 5.
Group IV. This group of 60 animals was
divided into three sub-groups. All 60 ani-
mals received cortisone intramuscularly on
the same schedule as Group III. Vitamin A
ester, 1,500 I.U. in peanut oil, was given
to 22 of the 60 rats. Vitamin A alcohol,
1,500 I.U., was dissolved with ethanol in
peanut oil, and was given to 15 of the 60
rats.** Vitamin A acid, 1,500 I.U., was dis-
solved in ethanol and peanut oil, and was
given to 23 of the 60 rats.
* The
day of wounding is referred to as Day
1, and the day prior to wounding as Day 0.
** Cortisone acetate was given in the form of
Cortone®9 Acetate, Merck and Company. Vitamin
A alcohol and Vitamin A acid were given in the
form of Retinol®, type X, Retinoic acid®, type
XX, Sigma Chemical Co. Vitamin A ester was
supplied by Eastman Kodak and Company.
325
Tensile strength of the wounds was de-
termined in all four groups on Day 7. The
rats were anesthetized with ether and the
sutures were removed. A tensiometer, de-
scribed by Sandbloom, Peterson and
Muren,13 was placed over the wound and
the hooks of the tensiometer were inserted
into the skin 1 cm. on each side of the
wound. The instrument gives a constant
rate of distraction at its point of attach-
ment to the skin. Tensile strength was mea-
sured to the nearest gram.
Each wound was tested twice, once pos-
teriorly and once anteriorly. There was no
significant variation between the two mea-
surements. In half of the animals, the ante-
rior end of the wound was tested first. In
the remaining rats the posterior end of the
wound was tested first. If any wound ap-
peared to be infected, results from this
animal were discarded.
Results
The mean tensile strength of control
wounds in the animals from Group I was
340 Gm. The mean tensile strength of
wounds in Group II animals that were
given Vitamin A alone was 361 Gm. The
difference between Group I and Group II
was not statistically significant. The mean
tensile strength of wounds in the animals
in Group III that were given cortisone
alone was 244 Gm., a decrease of 28%o
from control levels. This difference is sta-
tistically significant by the t-test at the
0.001 level. In Group IV, the mean tensile
strength of the sub-group that was given
Vitamin A ester plus cortisone was 328
Gm. In the animals that received Vita-
min A alcohol plus cortisone, the tensile
strength was 380 Gm. and in the Group
that received Vitamin A acid plus corti-
sone it was 326 Gm. All of these values
were statistically indistinguishable from
the controls but were statistically signifi-
cantly different from the group receiving
cortisone alone (Table 1).
 326 EHRLICH AND HUNT Annals of Surgery
March 1968
TABLE 1. Effect of Vitamin A and Cortisone on Wound Healing
Mean
Tensile
No. of Strength S.D.
No. Group Animals (Gm.) (Gm.) t Value
I Control 18 340 84 0.0
II Vitamin A 15 361 81 0.925
ester alone
III Cortisone 25 244 62 4.44
IV
A Cortisone 22 328 78 0.738
plus
Vitamin A
ester
B Cortisone 15 380 93 0.213
plus
vitamin A
alcohol
C Cortisone 23 326 84 0.743
plus
vitamin A
acid

The weight of the control animals and
the animals that received Vitamin A alone
did not change during the experiment. In
animals that received cortisone alone, the
mean body weight decreased by 64 11
Gm. In Group IV, animals that received
cortisone plus Vitamin A had a mean
weight loss of 67 + 8 Gm. during the ex-
periment. Weight loss in the sub-group
that was given cortisone alone was similar
to that reported by other investigators.12
Results are summarized in Table 1.
Discussion
The 28%o decrease in wound strength
produced by cortisone agrees with the re-
sults reported by Sandberg 12 in identical
experimental conditions. Vitamin A alone
did not increase wound strength over con-
trol levels. However, Vitamin A did pre-
vent cortisone from affecting the wounds.
Animals that received Vitamin A plus cor-
tisone lost the same amount of weight as
animals that were given cortisone alone.
This indicates that no direct chemical an-
tagonism exists between the two agents,
and further supports Sandberg's conten-
tion that cortisone does not inhibit healing
through weight loss. The antagonistic ef-
fect of the two agents appears to be at a
common site, a site which directly influ-
ences wound healing.
Although much research has been done
in this field, the details of metabolism in
healing wounds are not completely known.
Cortisone has a number of metabolic ef-
ects, both in the wound and in the host
organism. Vitamin A influences a number
of metabolic reactions of the host. Hereto-
fore, the relationship between Vitamin A
and wound healing has not been studied.
The effects of Vitamin A and cortisone are
so complex that any number of explana-
tions for the antagonistic effect demon-
strated here can be postulated. Further-
more, the exact role of lysosomes in wound
healing has been debated." However, the
antagonism of Vitamin A and cortisone sug-
gests that the effect of cortisone is, in part
at least, mediated through the lysosome.
Volume 167
Number 3
EFFECTS OF CORTISONE AND VITAMIN A ON WOUND HEALING
Lysosomes have been defined biochemi-
cally and histochemically as cytoplasmic
granules composed of a single-unit lipopro-
tein membrane surrounding a variety of
acid-hydrolytic enzymes. Fell and Thomas 6
found that cortisone increased the stability
of the surrounding membrane but Vitamin
A increased the availability of the hydro-
lytic enzymes. Cohn and Hirsch 2 demon-
strated that lysosomes are particularly
plentiful in inflammatory cells, and inflam-
matory cells are obviously particularly
prominent in the early phases of healing.
The effects of Vitamin A administration
have been related to connective tissue me-
tabolism. When Vitamin A is given in large
quantities, it stimulates lysis of connective
tissue.17 On the other hand, Vitamin A
deficiency leads to impaired collagen syn-
thesis. Furthermore, other lysosome labi-
lizers, the action of which appears to be
similar to that of Vitamin A, have been
related both to cortisone and to healing.
Singh and Udupa 15 demonstrated that
anabolic steroids inhibit the effect of cor-
tisone on bone healing. The authors attrib-
uted this inhibitory effect to the nitrogen-
sparing properties of anabolic steroids.
Nevertheless, testosterone, an anabolic ste-
roid, is known to be a lysosome labilizer
with actions somewhat similar to those of
Vitamin A.4 14
Recently, Prudden and Wolarsky 10 dem-
onstrated that cartilage powder applied to
fresh wounds before closure "reversed" the
cortisone effect in rats. Their results dif-
fered from ours in two major ways, how-
ever. Cartilage powder had only a slight
effect against the doses of cortisone that
we used, whereas "reversal" by Vitamin A
was complete. Secondly, cartilage powder
increased the rate of healing in control rats
but Vitamin A did not.
The concept of lysosomes, lysosome labi-
lization, and lysosome stabilization inevi-
tably allies with that of anabolism and
catabolism. Catabolic reactions generally
follow trauma, and it is well known that
327
the process of wound healing derives es-
sential components from catabolized tis-
sue.8'9 Catabolic reactions in the wound
site may also supply vital nutritional ele-
ments for healing. Collagen lysis is promi-
nent during the first few days after injury.'
The results of these experiments cannot
be interpreted as proof that the antagonism
between these compounds occurs within
the healing wound, but it seems likely that
the major portion of the effect is there.
Another possible explanation for the re-
sults of this experiment might be related to
mucopolysaccharide synthesis. Mucopoly-
saccharides, such as condroitin sulfate, are
rapidly synthesized in the healing wound.
Cortisone inhibits fixation of sulfate in
wounds and thus prevents synthesis of
chondroitin sulfate.9 ATP-sulfurylase acti-
vates sulfate, prior to its transfer to chon-
droitin sulfate. An activating lipid factor
associated with this enzyme appears to
contain a Vitamin A derivative.'6 There
have been no reports, however, that corti-
sone affects the ATP-sulfurylase system.
Obviously Vitamin A has a possible
clinical application in the prevention of
inhibitory effects of cortisone on healing.
However, this study has focused entirely
on the effects of cortisone and Vitamin A
on the healing wound. The effect of Vita-
min A on the other actions of cortisone is
unknown. Therefore, clinical attempts to
antagonize the effect of cortisone with
Vitamin A must be undertaken cautiously.
Further research is needed before Vitamin
A can be given to patients who are taking
cortisone.
Summary
Vitamin A has been shown to overcome
the inhibitory effects of cortisone on the
rate of gain in tensile strength in early
stages of healing, yet Vitamin A alone has
no effect on wound healing. Several pos-
sible explanations for this effect are dis-
cussed.
 EHRLICH AND HUNT Annals of Surgery
328~~~~~~~~~~~~~~~~~~~~~~~~~Mrh16
328
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