Numerous substances have been advocated by others (see reviews by Whipple, 1940, and by Arey, 1936) for the stimulation of wound healing. Tissue culture tests on a number of these materials are given in Table XII. It will be seen that less than half of these alleged stimu-lating agents showed any stimulation of adult tissues in vitro. Some were definitely toxic. Of those that stimulated none showed greater ac-tion than adult serum and none gave promise of practical usefulness in aiding the healing of wounds.
The substances in Table XII (as well as those in subsequent tables) were tested on experimental wounds by Dr. Edward L. Howes in the Department of Surgery. His findings were, in general, consistent with our results in tissue cultures—except for the fact that sterility is more of a problem in wounds than in tissue culture. An agent (such as serum
C/3 ST
TABLE X (Continued)
Substance Concentration
Media Stimulatin g Iner
t Inhibitory,
toxic, o r
lethal
3
a
I
s
QX
Strontium chloride
Zinc chlorid e
Metaphosphate i n additio n t
o
8.5 m M orthophosphat e
Metaphosphate withou
t orthophosphate
"Calgon" (Sodiu m metaphos
- o ortho n t n additio phate, i
- phosphate )
0.013 % ( 0.
5 mM ) X
6 mM) UF/1 ( 2 0.05 %
0 ) UF/1 5 mM ( 0. 0.13 %
0
0.003 % ( 0.
2 mM ) UF/1 0
0.08 % (10 mM
) Pl/
3
0.05 % ( 8.
5 mM ) Pl/
3 ) UF/ 5 mM ( 8. 0.05 %
3
0.01 % Pl/4
0.01 % X6
151 E 12
7
E 9
2 F >10
0 0 A >10
A 2 8
A 3 5
E 0 E 3 8
A 4 5
174 HENRY S. SIMMS AND MARY S. PARSHLEY TABLE XI
SUMMARY OF SUBSTANCES FOUND TO BE STIMULATING TO ADULT TISSUE GROWTH«
Substance Antibacterial substances
Sulfanilamide 0.005 and 0.05%
Sulfadiazine 0.05 and 0.085%
Sulfadiazine 0.05% (in PI plus Z8)*>
Sulfamethazine 0.05%
Sulfathiazole 0.005 to 0.085%
Vitamins
Ascorbic acid 0.01% (in serum or U F / 3 )
Enzymes (preliminary digestion of tissues)
Papain 0.001 to 0.1%
Trypsin 0.05 to 0.1%
Hormones
Anterior pituitary 0.015 per ml. (in S/4)
Thyroid (fresh, chicken) 4.0%
Thyroid (crude, dried, pig)
< 0 . 3 %
Other tissue extracts
Egg white (fresh or dried) 5% and 0.12%
Embryo extract (chicken) 1/3 in U F / 3
Embryo extract (chicken) 1/3 in S/3
Heart extract 10%
Leucocyte extract 5% (in Ty) Platelet extract 0.5%
Red cell extract (laked) 0.1%
Red cell extract (laked) 5.0%
Chromatin 1.0%
Effect Aorta or skin
fibroblasts
++ + + +
+ ++ +
++ ++
+ ++
+
Inert
++++* ++
+ +
(inconsistent)
++
on the growth of Skin epithelium
++ ++
+
Toxic
Inhibitory Toxic
++
+ ++
Thyroid epithelium
++
Inhibitory
a Degree of growth stimulation is indicated as follows:
+ = up to 2 times that of the control medium + + = between 2 and 3 times that of the control medium + + + = between 3 and 4 times that of the control medium + + + + = between 4 and 5 times that of the control medium
—f—I—|—|—I— = more than 5 times that of the control medium
Except where otherwise indicated the control medium was U F / 3 or Pl/3
6 However, sulfadiazine 0.05% in Pi plus X6 was inhibitory.
c Activity compared with Ty or X6 solution control.
Growth ratings 100-200 200-300 300-400 400-500
> 5 0 0
PROTEINS AND ADULT TISSUE GROWTH 175 TABLE XI (Continued)
Substance
Plasma (and lymph) constituents Whole plasma, lymph, or serum Serum ultrafiltrate (A Factor) Serum albumin 0.25%
Fibrinogen 1/3 Serum euglobulin 1/10 Serum gamma globulin 0.25 to
0.5%
Thrombin 0.25 to 0.5%
Amino acids and digested proteins Casein (digested by pepsin)
J-Aspartic acid 7.5 to 15 mM. 1%
Z-Asparagine 15 mM.
Z-Proline 7.5 to 15 mM.
Sulfur compounds Thiouracil 0.003%
Thiourea 0.003%
Other organic compounds Aluminum monostearate
<0.03%
Aluminum tristearate
<0.03%
Inorganic compounds Aluminum chloride 0.003%
Barium chloride 0.002%
Ferrous chloride 0.01%
Ferric chloride 0.01%
Effect on the growth of Aorta or skin
fibroblasts
+ + + ++
c++++*
Inert+ + ++ +
++++
+ + +
+ + + + + +
Skin epithelium
++++*
+ + ++
Inert
+
++ ++
++ +
+
+
Thyroid epithelium
Inhibitory
+
ultrafiltrate or aspartic acid) which aids tissue cell growth can also aid bacterial proliferation.
A. WATER SOLUBLE OINTMENT BASES ( TABLE XIII)
Of the water soluble bases, no inhibitory effect on the growth of adult fibroblasts was produced by the gels, agar, irish moss, methyl cellulose, and gum tragacanth, tested in a concentration of 1% or less.
Irish moss, inert for fibroblasts, inhibited the growth of skin epithelial cells. The water soluble carbowaxes also had no inhibitory effect on the growth of fibroblasts when tested in a concentration of 1%, but were less favorable for epithelial growth.
On the other hand, pectin, frequently recommended in the literature
176 HENRY S. SIMMS AND MARY S. PARSHLEY TABLE XII
TISSUE CULTURE TESTS ON SUBSTANCES REPORTED BY OTHERS TO BE STIMULATING TO WOUNDS«
Substance Pectin
Glycerine Triethanolamine Cod liver oil fractions Ascorbic acid Yeast extract
Trypsin (preliminary digestion) Adult serum (compared with
X6 solution control) Embryo extract Adult tissue extracts
Adrenal (whole) Heart
Thyroid»
Serum albumin Casein digest Glycine (impure) Allantoin
Urea
Sodium oxalate
Effect on Growth of Adult Aorta or skin
fibroblasts Toxic Toxic Toxic Toxic Stim. +
Inert Stim. + +
Stim. + + + + + Stim. -f
Inert Stim. + + Stim. + +
Inert Stim. + + + +
Inhibitory Inert in 0.01%
Toxic in 0.12%
Inert in 0.05%
Inhibitory in 0.09%
Inhibitory
Skin epithelium
Toxic Toxic Stim. +
Toxic Toxic
Stim. +
Stim. + Inhibitory Stim. + Stim. + + Inhibitory
a The degree of growth stimulation is indicated as follows:
Growth ratings 100-200 200-300 300-400
>400 + = up to 2 times that of the control medium
- f + = between 2 and 3 times that of the control medium + - f + = between 3 and 4 times that of the control medium
—i—|—|—]— == more than 4 times that of the control medium
The control medium was UF/3 or Pl/3 in each case, except when serum and glutathione were tested.
b When tested on thyroid epithehal cells, thyroid extract was inhibitory, thiouracil
was stimulating ( + ), and thiourea was inhibitory.
PROTEINS AND ADULT TISSUE GROWTH 177 TABLE XII (Continued)
Effect on Growth of Adult Substance
Sulfhydril compounds p-Thiocresol Cysteine
Glutathion© (not tested in
Na2S04
Thiouracil6
Thiourea0
Castillian malva Chlorophyll
UF)
Aorta or skin fibroblasts
Toxic Inhibitory (Stim.++inX6)
Stim. +
Toxic Inert to Toxic
Skin epithelium
Toxic
Inhibitory
Stim. +
Toxic for the treatment of wounds (Thompson, 1938; Tompkins et al, 1941), was completely toxic in vitro.
A 10% concentration of glycerine, reported as beneficial to wound healing by Kulkarni (1939) was found to be toxic for the growth of fibroblasts, as was "glycerite" of starch. The addition of glycerine to inert tragacanth containing a noninhibitory concentration of sulfathia-zole resulted in complete toxicity. Brush and Lam (1942) also report no consistent healing of guinea pig wounds with either pectin or gly-cerine, although Robson and Wallace (1941) described a sulphonamide glycerine base as harmless in the treatment of burns.
B. WATER INSOLUBLE (GREASY) BASES (TABLE XIV)
The only greasy bases tested which were well tolerated in vitro were pure preparations of mineral oil and vaseline, and a mixture of vaseline and lanolin ("Aquaphor"). These were tested by floating on the surface of a protective medium containing the tissue. Repeated heating of vase-line rendered it toxic. The importance of the quality of the preparation is evident from Table XIV, which shows the variability of results ob-tained with different preparations.
Of a large number of bases, antiseptics, and detergents tested on rat wounds by Baker (1944) petrolatum, motor oil, and boric acid ointment were the only substances found not to cause necrosis. Hawking (1942) commented on the undesirable tissue reaction to oily preparations of the sulfa drugs used in treatment of wounds and recommended the use of sahne solutions.
TABLE hydroxide ge
l
TABLE XIII (Continued)
Substance Concentration
Media (%)
Stimulating Inert
Inhibitory, toxic, o
r lethal
3
o>
Ö
Tragacanth-glycerine bas
e sußathiazole) (0.5%
Carbowax 150 0
Carbowax 400 0
Carbowax 600 0
Navy SP-E P ointmen
t x ) (containing beeswa and petrolatum
S S
S
10 1.0 1.0 1.0
5.0 2.0
1.0
UF/10 UF/3 UF/3 UF/3 UF/3 Pl/3 Pl/3
F A 71 76
F >10
0 E 8
3
A 0 E 3 7
E 1 0
A 3 E 66
E 7 3
8 3
TABLE mineral oi l 1/
2
(Fresh vaseline 1/
2 monooleate 10
% \
PROTEINS AND ADULT TISSUE GROWTH 181
C. STEARATES AND GREASY BASES CONTAINING STEARATES (TABLE XV)
Traces of salts of the alkaline earth metals and stearic acid, used as emulsifiers in wound-healing preparations, were almost inert. In fact, two preparations of aluminum stearate stimulated the growth of fibro-blasts in vitro. However, greasy bases containing these stearates were not all inert. This variability may be due to differences in purity of the greases used in these preparations.
D. DETERGENTS, EMULSIFIERS, PENETRANTS, ETC. (TABLE XVI)
Although the glycols appeared to have no inhibitory effect on growth in vitro when tested in low concentrations, esters of the glycols and stearic acid showed an increased tendency to inhibit growth with in-crease in the number of carbon atoms. A preparation of dodecaethylene glycol monolaurate was completely toxic. Of a large number of other organic compounds tested for use as emulsifiers, penetrants, and de-tergents, only propylene and polypropylene glycol and sorbitol in low concentrations were found to be inert. Most of these substances com-pletely inhibited growth in vitro. Triethanolamine, recommended as a vehicle for treating wounds with sulfadiazine (Pickrell, 1941; Adams and Crawford, 1942) was completely toxic. Noninhibitory iodochlorhydroxy-quinoline ("Vioform") dissolved in noninhibitory 0.1% propylene glycol formed a toxic preparation.
E. ANTIBACTERIAL SUBSTANCES
1. Sulfa Drugs (Table XVII)
The sulfa drugs have been recommended for the treatment of wounds on the basis of their lack of toxicity. There is some disagreement as to whether they inhibit growth (Whipple, 1940; Holmes, 1942). Undoubt-edly, the concentration and the method of application affect the results.
In the treatment of wounds Zintel et al. (1942) reported that sulfanila-mide and sulfathiazole administered orally to maintain therapeutic blood levels did not prolong the time of healing, and Smelzer (1944) found that the sulfonamides applied to abraded or burned rat corneas as oint-ments, powders, or solutions had no deleterious effect on healing. On the other hand, the local application of solid sulfonamides to wounds has been reported to retard blood clotting, and to depress phagocytic activity and fibroblastic proliferation, although not seriously interfering with healing (Glynn, 1941); Key and Frankel, 1941; Harbison and Key, 1942). The undissolved portion acts as a foreign body.
Sulfadiazine in vitro (8 and 80 mg. per 100 ml.) had no effect on phagocytosis by leucocytes but supersaturated solutions were inhibitory
TABLE stearate greas
e (vaseline) Calcium stearate greas
e ) (mineral oil Calcium
ί
stearate greas e 1/
2 j 2 C l 1/ l oi Almay minera Aluminum
monostearate (Bristol-Meyer)
Aluminum
tristearate (Bristol-Meyer)
Aluminum
tristearate (commercial)
Barium 12-hydroxystearate
Glyceryl monohydroxystearat
TABLE XV (continued)
3
o> t g Iner Media Stimulatin Concentration Substance
Inhibitory, toxic, o
r lethal
Diethylene glyco l monostearat e S
Nonaethylene glyco l monostearat e
Propylene glyco l monostearat e P
Related compoun d Dodecaethylene glyco
l monolaurat e
1 .0 0 .2
<
0.1
<
0 .2 1 .0
%
%
%
%
%
(25 ( 5 ( 5 (29
mM) mM) mM) mM)
Pl/3 UF/3 UF/3 UF/3 Pl/3
0.1 % UF/3
E 13 0
30
E F E E F
28 38 53 1 4 2 5
EF 0
s
TABLE XV
I , PENETRANTS DETERGENTS, EMULSIFIERS
, SOLVENTS
" " "Tergitol 7 Triethanolamine Triethanolamine wit h 0.09
PROTEINS AND ADULT TISSUE GROWTH 185 (Reed and Orr, 1942). Several hundred milligrams per 100 ml. of so-dium sulfathiazole was required to inhibit the migration of leucocytes in vitro (Herrell and Heilman, 1943), and concentrations of 5 and 50 mg. per 100 ml. did not inhibit the growth of young guinea pig fibro-blasts while concentrations of 100 mg. per 100 ml. inhibited growth but did not kill the cells (Reed et al. 1942).
We have found (Table XVII) that concentrations of sulfanilamide and sulfathiazole as low as 5 mg. per 100 ml., will stimulate the growth of adult chicken aorta fibroblasts in tissue culture, and that a concen-tration of 50 mg. per 100 ml. of sulfanilamide, sulfadiazine, sulfametha-zine, or sulfathiazole, usually had a stimulating eflEect on the growth of both adult fibroblasts and skin epithelial cells. Marfanil and sulfamera-zine in the same concentration inhibited these cells. Sulfamethasulfamera-zine
(50 mg. per 100 ml.) was found to be highly stimulating to the growth of adult chicken thyroid epithelium. The reaction of the tissues to dif-ferent concentrations of sulfadiazine and sulfathiazole (5 to 85 mg. per 100 ml.) varied with the type of medium. A reduction in bicarbonate favored the stimulation of the growth of fibroblasts by the higher con-centrations of sulfadiazine and sulfathiazole, while a reduction in calcium and an increase in phosphate in the medium, resulted in stimu-lation of skin epithelial cells by sulfadiazine. However, sulfadiazine tended to inhibit these cells when applied in X6 solution, which favors the growth of fibroblasts.
2. Miscellaneous Antibacterial Substances (Table XVIII)
In our experience the following commonly used antiseptics com-pletely inhibited the growth of adult cells in tissue culture: boric acid, 0.1%; hydrogen peroxide, 0.01 to 1.0%; iodine, <0.05%; phenol, 0.5%;
"Zephiran," 0.02%; zinc peroxide, <0.01%. Baker (1944) found boric acid ointment to be harmless in the treatment of rat wounds, but stated that hydrogen peroxide caused necrosis of the muscle. Zinc peroxide has been recommended as nonirritating (Holmes, 1942) and growth-stimulating (Connell, 1940) in the treatment of wounds. According to Herrell and Heilman (1943), "Zephiran" is hemolytic in low con-centrations and, although inferior to penicillin and sodium sulfathiazole, compares favorably with gramicidin in regard to toxicity for leucocytes in tissue cultures. We have found gramicidin to be relatively inert in tissue cultures of adult fibroblasts and skin epithelial cells.
In addition to completely inhibiting growth, p-chlorophenol, iodine, phenol, proflavine, and propamidine delayed plasma clotting from 1 to 4 days. p-Chlorophenol, recommended for use with penicillin by Me-laney et ah (1946) because of its activity against gram-negative
organ-TABLE XVII SUBSTANCES, A ANTIBACTERIAL . SULF
A DRUG S
Substance Concentration
Media Stimulating Iner
t Inhibitory,
toxic, o
r lethal
Marfanil" (4-amino-2-methyl
- ) benzene sulfonamide
0.05 % 0.1 % 0.1 % ( 2.
5 mM
) mM) mM) ( 5( 5
UF/3 UF/3
Pl/3
Sulfanilamide Sulfadiazine Sulfamerazine
0.5 % 0.01 % 2.5 mM
) 0.5 mM ) Pl/3
Pl/3
0.005%
0.05 % 0.05 % 0.05 %
0.085%
0.009%
0.05 % 0.085%
0.085%
0.05 % 0.005%
0.05 % 0.05 %
0.05 % 0.05 %
( 0.3mM
) ( 2.9mM
) ) ) (29 mM ( 2.9mM
( 3.
4 mM
) ) ) ) ) ) ) ) mM) ( 0.3mM( 2( 3.4mM( 3.4mM( 2.0mM( 0.2mM( 2.0mM( 2.0mM
( 2.0mM
) ( 2.0mM
)
UF/10
UF/10 UF/10
X6 D. UF/1
0 D. UF/1
0 X6 X6 X6 UF/10 UF/10 P1/3(X6) Pl/3 (Z8
) UF/3 Pl/3
A A
A
A A A
E
70 146
250
175 150
131 260
A 10 2
A A
E F F
E E
0 10 46
0 44 24 40
A A
A E
A E
56 53
71 52
68 57
TABLE XVI I (Continued)
Substance Concentration
Media Stimulating
Inert Inhibitory,
toxic, o
r lethal
3
OC /3 Sulfamethazine
Sulfasuxidine Sulfathiazole
0.05 % 0.05 % 0.05 % 0.10 % 0.10 % 0.085%
0.05 % 0.085%
0.005%
0.005%
0.085%
( 2.
0 mM
) ( 2.0mM
) 0 mM ( 2.
)
( 2.
8 mM
) ( 2.8mM
)
( 3.3mM
) 9 mM ( 2.
) 3 mM ( 3.
) 2 mM ( 0.
) 2 mM ( 0.
) ( 3.3mM
)
UF/10 Pl/3 (X6
) ) Pl/3 (Z8
X6 UF/10
D. UF/1
0 X6 X6 UF/10 UF/10 UF/10
E A E Thy
A A A
145 155 240 280
175 174 210
A A A
A A
75 130 90
88 43
CO
W CJ
8 I
H W
TABLE XVIII SUBSTANCES, B ANTIBACTERIAL . MISCELLANEOU - methoxyamine 0.17 %
These solution a
s delaye
TABLE XVIII (Continued)
Substance penicillin
("Superoxol")
TABLE XVIII (Continued)
CD O penicillin
Sodium penicillin
Phenol
TABLE XVIII (Continued)
Substance
192 HENRY S. SIMMS AND MARY S. PARSHLEY
isms, and considered to be well tolerated by the tissues, was completely toxic to adult cells in vitro. The acridine antiseptics, proflavine and propamidine, have been recommended in dilution as fairly acceptable substitutes for penicillin in the treatment of wounds (Russell and Fal-coner, 1943). Thrower and Valentine (1943) stated that 0.1% pro-pamidine has little effect on the phagocytic activity of leucocytes, but that concentrations in excess of this, used on wounds, caused necrosis of granulation tissue.
The data on penicillin were obtained prior to 1945 and do not apply to the purer preparations available today.