144 ALVAREZ ET AL

7. Milne JA; The metabolism of androgens by sebaceous glands. Br J Dermatol 81 (suppl 2):23-28, 1969

8. Hay JB, Hodgins MB: Metabolism of androgens in vitro by human facial and axillary skin. J Endocrinol59:475-486, 1973

9. Baillie AH, Caiman KC, Milne JA: Histochemical distribution of hydroxysteroid dehydrogenase in human sebaceous glands. Br J Dermatol 77:610-616, 1965

10. Baillie AH, Thomson J, Milne JA: The distribution of hydroxyste­roid dehydrogenase in human sebaceous glands. Br J Dermatol 78:451-457, 1966

11. Caiman KC, Muir A V, Milne JA, Young H: Survey of the distri­bution of steroid dehydrogenases in sebaceous gland of human skin. Br J Dermatol 82:567- 571, 1970

12. Muir AV: Studies on the t..5-3,8-hydroxysteroid dehydrogenases in the sebaceous glands of human skin. PhD Thesis, University of Glasgow, 1970

13. Chakraborty J, Thomson J, MacSween MP, Muir A V, Caiman KC, Grant JK, Milne JA: The in vitro metabolism of [7a-3H]dehy­droepiandrosterone by human skin. Br J Dermatol 83:477-482, 1970 .

14. Burton JL, Cunliffe WJ, Stafford L, Shuster S: The prevalence of acne vulgaris in adolescence. Br J Dermatol85:119-126, 1971

15. Cunliffe WJ, Kearney JN, Simpson NB: A modified photometric technique for measuring sebum excretion rate. J Invest Dermatol 75:394-398, 1980

16. Shirley IM, Cooke BA: Metabolism of dehydroepiandrosterone by the separated zones of the human foetal and newborn adrenal cortex. J Endocrinol 44:411-419, 1969

17. Hay JB, Hodgins MB: Metabolism of androgens by human skin in acne. Br J Dermatol 94:123-133, 1974

18. Sharp F, Hay JB, Hodgins MB: Metabolism of androgens by human

0022-202X/ 83/8102·0144$02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 81:144-148, 1983 Copyright © 1983 by The Williams & Wilkins Co.

Vol. 81, No.2

foetal skin. J Endocrinol 70:491-499, 1976 19. Sharp F: The evolution and distribution of hydroxysteroid dehy­

drogenase activity in human foetal skin throughout gestatwn. Histochem J 10:517-528, 1978

20. Hay JB, Hodgins MB: Distribution of androgen metabolising: en­zymes in isolated tissues of human forehead and axillary skin. J Endocrinol 79:29-39, 1978

21. Fazekas AG, Lanthier A: Metabolism of androgens by isolated human beard hair follicles. Steroids 18:367-379, 1971

22. Fazekas AG, Sandor T: The metabolism of dehydroepiandrosterone by human scalp hair follicles. J Clin Endocrinol Metab 36:582-586, 1973

23. Archibald A: A study of factors influencing sebaceous gland activity in the rat. PhD Thesis, University of Newcastle Upon Tyne, 1973

24. Thomas JP, Oake RJ: Androgen metabolism in the skin of hirsute women. J Clin Endocrinol Metab 38:19- 22, 1974

25. Kuttenn F, Mowszowicz I, Schaison G, Mauvais-Jarvis P: Androgen production and skin metabolism in hirsutism. J Endocrinol 75:83-91, 1977

26. Sansone G, Reisner RM: Differential rates of conversion of testos­terone to dihydrotestosterone in acne and in normal human skin- a possible pathogenic factor in acne. J Invest Dermatol 56:366-372, 1971

27. Takayasu S, Wakirnoto H, Harni S, Sano S: Activity of testosterone 5a-reductase in various tissues of human skin. J Invest Dermatol 74:187-191, 1980

28. Plewig G: Acne vulgaris: proliferative cells in sebaceous glands. Br J Dermatol 90:623-630, 1974

29. Burton JL, Johnson G, Libman L, Shuster S: Skin virilism in women with hirsutism. J Endocrinol 53:349-354, 1972

Vol. 81, No.2 Printed in U.S.A.

The Hea~ing of Superficial Skin Wounds Is Stimulated by External Electrical Current

OSCAR M. ALVAREZ, PH.D., PATRICIA M . MERTZ, B.A., RICHARD V. SMERBECK, B.S., AND

WILLIAM H. EAGLSTEIN, M.D.

Department of Dermatology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A.

We studied the effects of direct electric current sup­plied by an energized silver-coated electrode on dermal and epidermal wound healing. Keratome-induced wounds (0.3 mm deep) on the skin of young domestic pigs were treated with either an energized (50- 300 p.A) electrode (DC), an unenergized electrode (placebo), or left untreated. Wounds were excised on days 1-7 after wounding and the epidermis was separated from the dermis. The epidermal sheet was evaluated for reepithe­lialization and the dermis was assayed for collagen bio­synthetic capacity. Dermal collagen production among treatments did not differ markedly on days 1-4 after

Manuscript receiv"ed September 21, 1982; accepted for publication February 2, 1983. ·

This work was supported by a grant from the Sybron Corporation and Pittsburgh Skin and Cancer Foundation.

Reprint requests to: Patricia M. Mertz, Department of Dermatology, University of Pittsburgh School of Medicine, Scaife Hall RC 513, Pittsburgh, Pennsylvania 15261.

Abbreviations: CFU: colony-forming units DC: direct electric current HTso: healing time for 50% of wounds TCA: trichloroacetic acid

wounding. However, a highly significant increase (p < 0.001) in the collagen synthetic capacity was observed on days 5, 6, and 7 in wounds treated with DC. There was no significant difference in collagen synthesis among treatments when collagen production was corrected for DNA content. The rate of wound epithelialization was also significantly accelerated (p < 0.05) in DC-treated wounds. These results suggest that the proliferative and/ or migratory capacity of epithelial and connective tissue cells involved in repair and regeneration can be affected by an electrical field.

Recently the effect of electrical stimuli on tissues has been the subject of considerable experimental attention. Cells have complex electrical systems which are sensitive to electrical field changes. Varied effects, both specific and general, have been reported following electrical field alterations. Metabolic, behav­ioral, physiologic, and immunologic changes, in a wide variety of systems including bacteria, insects, plants, and mammalian cells in culture have been observed after exposure to electric fields [ 1-7].

Electrically stimulated bone healing and regeneration has been reported by many investigators [8]. Direct current has

Aug.1983

also been shown to affect nerve tissue [4,7], tumor growth [9], and skin wound healing [10]. In this study we report that direct electric current (50-300 p.A) supplied by an external silver­coated electrode promoted epidermal resurfacing and dermal collagen biosynthetic capacity in partial-thickness wounds.

MATERIALS AND METHODS Wounding Techniques and Treatments

Eleven young Yorkshire pigs weighing 5.5-8.0 kg (Delissio Stock Farms, Plumville, Pennsylvania) were fed a basal diet ad libitum and housed individually in our animal facilities with controlled temperature (19- 20°C) and light (12 h light/12 h dark) . Each animal was clipped with standard animal clippers. The skin on both sides of the animal was prepared for wounding by washing with sterile saline. The animals were anesthetized (Nembutal sodium 60 mg/2.3 kg i.p.) (Abbott Labo­ratories), and approximately 150 rectangular wounds measuring 7 X 10 mm and 0.3 mm deep were made in the paravertebral and thoracic area with an Electrokeratome (Storz Instruments), fitted with a razor blade modified so that the cutting edge was reduced to 7 mm. The wounds were separated from one another by at least 15 nun.

The wounds on each animal were divided into 3 groups and treated as follows: No treatment (control); 50-300 pA direct electric current delivered by an energized silver-coated electrode (DC); and an unener­gized silver-coated electrode (placebo). At least 5 em separated each of the treatment groups. Immediately after wounding, t he DC wounds were dressed with a saline-moistened silver-impregnated nylon sheet (contact electrode) cut to fit the treatment area. After assuring that contact was made between the electrode and the wound bed, saline­soaked gauze (Kling bandage) was layered on top of the electrode in order to maintain th e electrode moist. The treated area was then wrapped with 14 x 200 em Ace bandage and the electrode connected to the generator. The contact electrode, the return electrode, the battery for the generator, and the dressings were replaced daily during the course of the experiment. The current intensity decreased linearly from 300 p.A upon initial connection to 50 p.A at the end of a 24-h treatment period.

Electrode Components

The generator component was a self-contained, battery-operated source of constant current. The generator was equipped with a voltage limiter which kept the output voltage below 0.9 V and a liquid crystal digital display of output current (p.A). The batteries used were standard 9 V batteries (Duracell). A cable with a 5-pin connector on one end and electrode connectors on the other connected the electrode and the generator. The contact electrode (anode) was fabricated of high-purity nylon coated with silver; the return electrode (cathode) was pregelled surgical grounding pad.

The generator was mounted on a harness, designed so that the generator sat at the nape of the neck. The harness was constructed from cotton cloth and pliable elastic. The return electrode was placed on an isolated (untreated, unwounded) area (Fig 1) .

Epidermal Wound Assessment

Each day after wounding (day 0), for a period of 7 days, several wounds and surrounding normal skin from each treatment group were excised at 0.5 mm using an Electrokeratome equipped with a 22-mm blade. The excised skin containing the wound site was incubated in 0.25% trypsin (GIBCO) for 12 h at 4°C allowing a separation of the dermis from the epidermis. The epidermal sheet was examined mac­roscopically for epidermal migration as described previously [11].

Assessment of Dermal Collagen Biosynthetic Capacity

After trypsinization, the dermal portions of the dermatome-excised wounds from each treatment group from 2 animals were evaluated for collagen biosynthesis. Approximately 100 mg of the underlying dermal specimens was placed in 20-ml flasks containing 3 mi of Krebs-Ringer medium modified according to Uitto [12]. Tissues were finely minced and 5 p.Ci of .[ 14C]proline (Amershain) was added. The flasks were incubated in a shaking water bath at 37°C for 3 h. The entire incubation mixture was cooled at 0--4°C, after adding the following protease inhibitors: 1 mM phenylmethylsulphonylfluoride, 5 rnM N-ethylmaleim­ide, and 10 rnM EDT A. After rapid freezing and thawing the mixture was homogenized (Tekmar Tissuemizer) and treated with protease-free ribonuclease (20 mg/ ml) for 5 min at 37°C to cleave radioactive prolyl transfer RNA. The homogenate was then chilled to 4°C and trichloro-

WOUND HEALING 145

EN ERA TOR

FIG 1. Diagrammatic representation of experimental animal wear­ing the generator and the return electrode.

acetic acid (TCA) was added to give a final concentration of 5%. Precipitated protein was separated by centrifugation at 10,000 g and unincorporated radioactive proline in the supernatant was discarded. The precipitate was then resuspended in 5 ml cold 5% TCA and recentrifuged. The TCA from the protein pellet was extracted by 2 ethanol/ether (3:1 V / V) washes and the protein was desiccated to a powder. The dried protein was dissolved by homogenization in 0.2 N NaOH at 37°C (15 mg/ ml) and the collagen was digested by bacterial collagenase and separated from noncollagen protein. The procedure used was essentially as described by Peterkofsky and Diegelmann [13] with the fo llowing modifications: An aliquot (0.25 ml) of the dissolved substrate was transferred to each of 3 15-ml conical centrifuge tubes and partially neutralized with the addition of0.15 mi ofO.l N HCI. The incubation mixture was buffered by the addition of 0.2 mi N-2-hydrox­yethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (100 uM, pH 7.2). CaCl (0.25p.M) was added to stabilize th e collagenase. N -ethylamleimide (1.25 p.M) and phenylmethylsulfonylfluoride (0.3 mM) were added to inhibit any possible trace amount of neutral and sulfhydryl-containing proteases. The reaction mixture was adjusted to pH 7.2 and duplicate tubes received 0.01 ml of purified bacterial collagenase (20 p.g) (Ad­vanced Biofactures) in 0.05 M Tris-HCI buffer (pH 7.6) containing 5 rnM CaCI. The third tube served as an enzyme blank and received only the Tris-CaCl buffer. Collagenase digestion was canied out for 90 min at 37°C and then stopped with 0.5 ml 10% TCA-0.5% tannic acid. Collagen digestion products in the supernatant fluid were analyzed by liquid scintillation as described previously [13]. An aliquot of each collagen digest and residual noncollagen protein was analyzed for radioactive hydroxyproline according to the procedure of Peterkofsky and Prockop [14].

Assessment of Wound Microbiology

On days 2, 4, and 6 after wounding, specimens from each treatment group (control, DC, and placebo) in 2 pigs were evaluated for quanti­tative and qualitative changes in aerobic resident microbial flora. Nor­mal skin and wound centers were swabbed with 70% alcohol and biopsied with a 4-mm cutaneous punch. The specimens were weighed and finely minced. The tissues were then homogenized in a 5 ml broth (0.1% peptone) with a ground-glass tissue grinder. The homogenates were then plated using an automatic spiral plater (Spiral Systems Inc.) on selective and nonselective media for isolation and quantification of the following groups of bacteria: streptococci, lipophylic diptheroids, Staphylococcus aureus, Gram negatives, P seudomonas, yeast, and fungi.

All plates were incubated at 3rC for 24-48 h or 32°C for 48-72 h. For quantification, colonies grown on nonselective media (Trypticase soy agar with/without difibrinated sheep's blood) were counted with a colony viewer equipped with a spiral grid (Spiral Systems Inc.).

Silver Analysis

The analyses of silver in tissue samples, serum, and feces were done using atomic absorption spectrophotometry according to Greenberg et al [15].

Other Assays

Aliquots of 0.2 mi were taken from the TCA-precipitated protein prior to collagenase digestion and assayed for dermal protein according

146 ALVAREZ ET AL

to Hartree [16]. Electrical field intensities and resistance were measured with a 179 TRMS Digital Multimeter (Keithley Corporation).

RESULTS

The effects of DC supplied by electrically energized silver­coated electrode on epidermal wound healing are presented in Table I. The rate of wound reepithelialization was significantly greater in the DC-treated wounds when compared to both untreated and placebo on days 2, 3, and 4 after wounding. The unenergized electrode (placebo) slightly enhanced the rate heal­ing. From the data in Table I, the time needed for 50% of the wounds in each experimental group to heal (HT50) was esti­mated by pro bit analysis [17]. The HT 50's for each experimental group and control are compared in Table II.

The effects of DC on the synthesis of hydroxyproline and collagenase digestable protein are presented in Fig 2A,B. Der­mal collagen biosynthetic capacity among treatment groups did not differ markedly on days 1-4 after wounding. However, a significant increase in collagen biosynthesis was observed on days 5, 6, and 7 in wounds treated with electric current.

The effect of the silver-generating electric current on the quantitative microflora of wounds is summerized in Fig 3. Normal unwounded pig skin has approximately 104 colony­forming units (CFU per g of tissue). Untreated wounds had 106

,

107, and 105 CFU on days 2, 4, and 6, respectively. Both De­

treated wounds and placebo-treated wounds demonstrated a marked decrease in CFU on day 2 but did not differ from untreated wounds on days 4 and 6 after wounding.

There were no significant differences in the type ofrnicroflora as a result of these treatments (data not included). Staphylo­coccus epidermidis, Micrococcus species and diphtheroids were found in both unwounded skin (day 0) and healing wounds (days 2, 4, and 6 after wounding). Transient Gram-negative (Acinetobacter species, Echerichia coli, Enterobacter cloacae, Klebsiella pneumonia, and Pseudomonas species) were occa­sionally detected but could not be associated with any specific treatment regimen.

The amount of silver detected in wounded and unwounded pig skin after treatment with DC and placebo is presented in

TABLE I. Effect of direct electric current (DC) on epidermal wound healing"

Treatments Day 1 Day 2 D ay3 Day4 Day5 Day 6

DC 0/26 3/31 21/45 35/41 39/39 (0) (9.6) b. c (46.6) b. d (85.3) b. d (100)

Placebo 0/26 0/39 11/46 18/37 23/26 19/20 (0) .(0) (23.9) c (48.6) (88.4) (95)

Untreated 0/21 0/38 2/38 9/36 27/35 17/19 (0) (0) (5.2) (25) (77.1) (89.4)

" Values are expressed as number of specimens healed/total number of specimens tested. Numbers in parentheses are percent healed.

b p < 0.05 compared with placebo. c p < 0.05 compared with untreated. "p < 0.01 compared with untreated.

TABLE II. Comparison of the time for 50% of the wounds in each treatment group to heal

Treatments HT,, (days) Relativ~ rate of healing (%)"

DC" Placebo Untreated

2.9 36.9 '" 29.2" 4.1 10.8c 4.6 -12.0"

, Relative Rate of Healing= HTw (control) - HTw (treatment) x (%) HTw (control)

100. b DC = Direct current supplied by an electrically energized silver­

coated electrode. c Compared to untreated. "Compared to placebo.

w"f c o ·- ..-ox ..... ~ C.c >-·­X (J) o~ ..... 0 "0 ..... >-a.. IOl ~E o -....

"<T ::2 ~ a.. ~0

5

4

3

2

._.DC o--o Unt reated ...... Placebo

2 3 4 5 Days after Wounding

Vol. 81, No.2

A

B

6 7

FIG 2. The effect of DC on synthesis of hydroxyproline and colla­genase digestable protein. Each point represents the mean ± SD of 5 values from 2 experimental animals. a = p < 0.001 compared with both placebo and untreated. • =Values for normal unwounded skin.

P------.-:l- .. ,' .·· -"""'""" / . _.rf

2 3 4 5 6 DAYS AFTER WOUND ING

FIG 3. The effect of silver-generating electric cunent on the quan­tity of normal resident microflora of healing wounds. Each point represents the mean ± SD of 3 values. a = p < 0.05 compared with untreated; • = prewounding values; 0 = untreated; • = placebo; e = DC.

Table III. More silver was recovered in the papillary dermis of wounded tissue than in the papillary dermis of normal skin. In the reticular dermis of wounds, only very small amounts of silver were detected. Silver was also recovered from placebo­treated wounds and normal skin. There were no differences in the amounts of silver detected in both serum and feces before and after treatment with DC (data not included).

Aug.1983

TABLE III . Detection of silver in wounded and unwounded pig skin

After treatment with DC After treatment with an supplied by an electri- unenergized electrode cally energized silver- (placebo)

coated electrode

Epidermis Papillary dermis Reticular dermis

Wound "

1.9 ± 0.2 0.45 ± 0.1

Normal •

2.1 ± 0.4 1.1 ± 0.2

ND

Wound" Normal •

0.9 ± 0.1 0.4 ± 0.1 ND

ND ND

Silver was not detectable in pretreatment tissue. Values expressed as p.g/ g tissue (mean ± SD) of 3 specimens. Wounds and adjacent unwounded skin were treated for 24 h. ND = not detected.

a Wounds were treated immediately after wounding. • Normal unwounded skin was excised from the area between

wounds.

DISCUSSION

There has been considerable interest in the possibility of electrical control of cell function. Activity in this area has recently increased because of the demonstration that recalci­trant bone fractures can be healed using both direct [18] and pulsating [19] currents. In vitro experiments using tissue culture dishes in contact with high-voltage external metal plates (ca­pacitive coupling) have demonstrated considerable effect on cell function including differentiation and motility [20,21).

We observed that direct electrical current significantly accel­erated epidermal resurfacing in partial-thickness wounds when compared to both placebo (unenergized) and untreated control wounds. The mechanism of action, however, remains unex­plained. We do not know whether the effects of this system are due to the direct electrical current, the silver ions, or an elec­trochemical silver complex. It is unlikely that the silver alone would enhance healing, since topical applications containing silver sulfadiazine and silver nitrate do not significantly speed up reepithelialization when compared to their vehicles [22,23). The placebo-treated wounds healed somewhat .more rapidly than did untreated wounds. This increase in the healing rate could be the result of the occlusive environment created by the dressings.

We know that the differences in wound reepithelialization observed were not due to the area of wounding since studies have demonstrated that the rate of healing in domestic pigs is independent of the area where the wound is made [11,22,24).

A characteristic increase in the collagen biosynthetic capacity after wounding was observed in the dermis of excised wounds from all treatment groups. This initial increase in collagen biosynthetic capacity is a normal response to healing and has been observed in both superficial [24) and full-thickness wounds [25-27]. It is known that during dermal wound repair, mesen­chymal, fibroblasts, and endothelial cells adjacent to the site of injury increase their migratory, synthetic, and proliferative activities. The excised dermis from wounds subjected to 50-300 p.A electric current demonstrated not only a 28% increase in protein content (data not included) but also a 109% increase in labeled collagen (Fig 2B) between days 4 and 5 after wounding. This rise in collagen biosynthesis observed in DC-treated wounds could be due to either a local increase in the number of collagen-producing cells present (either by migration or mitosis) or to an increased ability to synthesize collagen by the cells already there. To answer this question, we expressed collagen production per DNA content. We found that when collagen synthesis is corrected for cell number (DNA) there were no differences among the treatment groups (data not included). Therefore, the increase in the collagen synthetic capacity in the dermis of DC-treated wounds was due to an augmentation of collagen-producing cells. Increased number of cells present at the wound site could be the result of proliferation and/or chemoattraction. Bassett and Herman [20] observed that cul­tured fibroblasts (3T-6) subjected to an external electrical field not only proliferated more rapidly (20% increase in DNA) but

WOUND HEALING 147

also increased their collagen biosynthetic activity (100%) when compared to unenergized controls. Increased proliferation (DNA synthesis) as a result of electric fields has also been reported in other cells [28).

The antimicrobial potential of this system during wound healing was also evaluated. We found that on the first day (day 2 after wounding) both the placebo and the silver-generating electric current initially inhibited the growth of normal resident microflora compared to untreated control wounds. However, on the subsequent days of evaluation (days 4 and 6) the 3 treatment groups had the same numbers of microorganisms. It may be that during the early days after wounding before the epidermal barrier is restored, there was a greater penetration of the current/silver into the wound tissue. As the wound heals there is both a restoration of the epidermal barrier and an increased electrical resistance which could feasibly reduce the antimicro­bial effect. Alternatively, microorganisms resistant to the re­leased silver could reproduce to equal numbers by day 4. It is important to realize that occlusion enhances the growth of microorganisms [29). Considering the stimulatory effect of oc­clusion on microbial growth, the reduction in the number of microorganisms produced by the silver-generating electric cur­rent is especially impressive. The finding that the unenergized electrode (placebo) also inhibited the quantity of microflora indicated that silver was being released from the electrode. This finding was subsequently confirmed when traces of silver were found in dermis and epidermis of wounded and unwounded skin treated with the unenergized electrode (Table III). In addition, in vitro experiments revealed that the unenergized electrode produced a zone of inhibition in agar seeded with S. aureus (data not included) . Silver, unlike many other trace metals, is known to have bactericidal and antifungal properties in vitro both as topical agent [30) and in ionic form [31,32]. The in vivo studies show that silver ion liberated by DC did not significantly inhibit the growth of microorganisms when com­pared to its unenergized counterpart.

More silver was found in the papillary and reticular dermis of wounded skin than was found in the papillary and reticular dermis of unwounded skin. The fact that the epidermis is absent from 24-h-old wounds probably accounts for these differences. The failure to detect differences in silver from serum or feces before and after treatment with DC suggests that silver released from the contact electrode was sequestered by the skin.

Since skin temperatures under both unenergized and ener­gized electrodes were similar, we felt it unlikely that heat generated from the electrical field accounted for our findings.

We found that the low-intensity direct current generated from the energized electrode linearly decreased over time. Twenty-four hours after initial connection the current recorded was approximately 27% of the current at time zero (initial connection). A constant current at different intensities may prove to be either more or less beneficial.

Wu et al [33) studied the effect of DC on the tensile strength of full-thickness incision wounds in rabbits. These authors reported that neither the polarity nor the current intensity (40-400 p.A) had any apparent effect on wound dehiscence. The differences in our findings and those of Wu et al may be the result of different electrodes and/or different methods of eval­uating healing. Wu et al used platinum and stainless steel electrodes, whereas we used silver, a more conductive material. Wu et al used tensile strength measurements as an evaluation of healing. Wound tensile strength is a physical measurement which reflects the degree of intermolecular collagen cross-link­ing, not collagen biosynthesis. We measured collagen synthesis and epidermal resurfacing. Electrical current may affect colla­gen production but not collagen maturation or remodeling.

We have demonstrated that an external DC delivered by a silver-impregnated nylon electrode enhances both dermal and epidermal wound repair in superficial wounds. This effect seems to be unrelated to the antimicrobial action of the liberated silver.

148 ALVAREZ ET AL

We would like to thank Dr. Elaine Jeveli and Robert Behl for their assistance throughout the project and Mr. John R. Taylor for his excellent technical assistance.

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