Effects of Four Preparations of 0.05% Chlorhexidine Diacetate on Wound Healing in Dogs

Veterinary Surgery, 21, 2, 107-1 12, 1992

Effects of Four Preparations of 0.05% Chlorhexidine Diacetate on Wound Healing in Dogs

Four chlorhexidine diacetate (CHD) antiseptic wound lavage preparations were evaluated in vivo to determine their effects on second intention wound healing in the dog and in vifro to determine their relative antibacterial activity against Sfaphylococcus intermedius. Chlorhexidine was diluted to 0.05% in sterile water, 0.9% sodium chloride, lactated Ringers solution (LRS), and LRS that was allowed to form a precipitate with CHD. Control solutions included sterile water and LRS. There were no significant differences in wound contraction or epithelialization. All 0.05% CHD preparations provided 100% bacterial kill.

S A WOUND IRRIGANT, 0.05% chlorhexidine (CHD) A has been shown to be effective in reducing bacterial numbers and speeding second intention healing of infected wounds.',2 Chlorhexidine is most effective in an alkaline en~ironment.~ It is toxic to tissues at commercially avail- able concentrations and must be diluted for use as a wound lavage agent,4 yet no diluent standard has been established. Chlorhexidine diacetate has been diluted with 0.9% sodium chloride, lactated Ringer's solution, and water.2.5 In electrolyte solutions, chlorhexidine forms a heavy precipitate in 1.5 hours at room temperature,* and this precipitated solution is also often used as a wound irrigant. These solutions vary in pH, osmolality,6 and particulate content.

Tissues exposed to water or electrolyte solutions suffer varying degrees of histologic damage.7 The healing rate of wounds subjected to daily lavage with antiseptic solutions may be affected by tissue damage and dysfunction the solutions cause. The antiseptic efficacy of CHD may also be affected by differences in pH or the precipitated CHD. The purpose of this study is to determine whether the type of diluent or the presence of precipitate in 0.05% CHD solutions affects the rate of wound healing or the antiseptic efficacy of CHD.

* Lozier, SM. Preliminary observations, January, 1988.

Materials and Methods

Wound Healing

Six conditioned, adult male mixed-breed dogs of approximately the same age and weight were handled and cared for in accordance with Federal standards.8 On day 0, oxymorphone (0.2 mg/kg) and acetylpromazine (0.04 mg/kg) were administered subcutaneously. Anesthesia was induced with thiopental (approximately 12 to 14 mg/kg) intravenously until tracheal intubation was possible. Anesthesia was maintained with isoflurane in oxygen.

Hair was removed with electric clippers over the dor- sum, from the caudal borders of the scapulae to the wings of the ilia and half way to ventral midline. Each dog was positioned in sternal recumbency in a V-shaped trough. Three surgical scrubs with povidone-iodine were followed by a 70% alcohol wipe. The surgical site was isolated with four quadrant drapes and a waterproof bamer drape. Six wounds were created by excising 2 X 2 cm full-thickness sections of skin with a scalpel. Three wounds were made on each side, 5 cm from the dorsal midline and 4 cm apart. The middle wound on each side was centered between the caudal border of the scapula and the wing of the ilium.

From the Department of Veterinary Medicine and Surgery, University of Missouri-Columbia, Columbia, Missouri. Funds for this project were acquired through a competitive grant (C2-00351) from the Committee on Research at the University of Missouri-

Reprint requests: Scott Lozier, DVM, Department of Veterinary Clinical Medicine and Surgery, Washington State University, Pullman, WA Columbia.

991 64-661 0.

107

108 WOUND HEALING

original wound margin

contracted wound margin

advancing epithelial margin

1 1 epithelialized area

= unhealed area

Fig. 1. Healing wound on day *‘x”. To = wound area on day 0 that lies within the original wound margin. T, = wound area on day “x” that lies within the contracted wound margin. U, = unhealed wound area on day “x” that lies within advancing epithelial margin.

The solutions tested were chlorhexidine diacetatet diluted to 0.05% in sterile water$ (CHD/H20), CHD diluted to 0.05% in sterile 0.9% sodium chloride solutions (CHD/ NaCI), CHD freshly diluted to 0.05% in sterile lactated Ringer’s solution(( that had been titrated to pH 7.4 with sodium bicarbonate7 (CHD/LRS), and CHD diluted to 0.05% in sterile lactated Ringer’s solution that had been titrated to pH 7.4 with sodium bicarbonate and allowed to stand long enough to form a heavy white precipitate (CHD/PPT). The control solutions were sterile water and LRS that was titrated to pH 7.4 with sodium bicarbonate. The pH and osmolality of the test and control solutions and 2% chlorhexidine diacetate were determined with a pH meter and a freezing point osmometer.** This was done before the addition of sodium bicarbonate to solutions containing LRS. The pH meter was also used throughout the study to determine the correct volume of sodium bicarbonate to add to the LRS solutions.

From days 0 to 24, each wound was lavaged once a day with one of the six solutions. All six solutions were used on each dog, and each solution was assigned to a different wound position on each dog. The wounds were lavaged

t Nolvasan Solution, Fort Dodge Laboratories, Inc., Fort Dodge, Iowa t Sterile water for irrigation USP, Kendall McGaw Laboratories, Inc.,

0 0.9% Sodium Chloride for irrigation USP, Kendall McGaw Labo-

11 Lactated Ringer’s injection USP, Baxter Healthcare Corporation,

ll LyphoMed, Sodium Bicarbonate injection USP, LyphoMed, Inc.,

Accumed electrometer model 630, Fisher Scientific Co., Pittsburg,

** Micro-osmette model 5004, Precision Systems, Inc., Natick, Mas-

Irvine, California.

ratories, Inc., Irvine, California.

Deerfield, Illinois.

Rosemont, Illinois.

Pennsylvania.

sachusettes.

with 10 mL of the assigned agent under low pressure by using a 12-mL syringe and an 18-gauge needle. Contact with other wounds was prevented by placing a plastic shield around the wound while it was imgated. Each wound was covered with a sterile nonadherent pad?? and two sterile 2 X 2 cm gauze squares. Strips of 2-inch white tape were placed from side to side and from cranial to caudal over the gauze. A layer of conforming gauze wrapped around the dog’s body was held in place by 3- inch elastic tape at the cranial and caudal edges of the gauze that overlapped onto the hair. A cotton bandage covered with fiberglass cast material was placed around each dog’s neck to prevent self-inflicted trauma and dis- ruption of the wounds. The dogs and bandages were examined 2 to 3 times each day. Analgesics were administered as necessary.

On days 0,3, 7, 10, 14, I7,21, and 24, with the animal anesthetized and positioned as before, the dimensions of each wound were recorded by tracing the wound and epithelial margins on sterile transparent polyethylene sheets.$$ Crusts and regrowing hair were removed with sterile scalpel blades when they impaired accurate tracing. On days 7 and 14, all wounds were cultured for aerobic bacteria with sterile swabs.§§ The swabs were plated on blood agar medium. On day 7, surface bacteria quanti- tation was attempted by using a velvet pad rinse technique.’JO

Tracings from the wounds were transferred onto clear acetate sheets.)) 1) The surface areas within the day 0 wound margin (To), within the contracted wound margin (Tx), and within the advancing epithelial margin (U,) were determined with a computerized planimetric analysis sys- temll at each measurement (day X) (Fig. 1). All measurements were repeated 3 times and averaged. Three wound healing parameters were calculated from the surface area measurements.

- To - T, wound healed by contraction). TO

- T, - ux Percent epithelialization (percent of -~ day 0 wound healed by epithelialization). TO

To - ux day 0 wound healed by

-~ Percent contraction (percent of day 0

Percent healed wound (percent of - --

epithelialization and contraction). T,

tt Telfa non-adherent dressings, The Kendall Company, Hospital

$$ Saran Wrap, The Dow Chemical Co., Indianapolis Indiana. $5 Culturette, Marion Scientific Culturette Collection and Transfer

System, Did. Marion Laboratories, Inc., Kansas City, Missouri. 11 11 Transparencies, Xerox Corporation, Rochester, New York. lIll Sigma Scan, Jandel Scientific, Corte Madera, California.

Products, Boston, Massachusetts.

109 LOZIER, POPE, AND BERG

Each parameter was grouped by solution type for every day that measurements were taken and differences were determined by ANOVA. Differences were considered significant when p 5 .05.

On day 24, the dogs were euthanatized, and the wounds were excised and fixed in 10% buffered formalin. Trans- verse sections from the cranial, middle, and caudal thirds of each specimen were embedded in paraffin, cut into lop sections, and stained with hematoxylin and eosin. The slides were examined by the senior author and a veterinary pathologist.

Antibacterial Activity

In vitro, Mcfarland standards of S. intermedius were diluted I : 10,000 in phosphate buffered saline and then diluted 1 : 10 in each of the test and control solutions and phosphate buffered saline for 60 seconds. Each bacterial suspension (0.1 mL) was plated on blood agar and in- cubated at 37°C for 24 hours. Bacterial colonies on each plate were counted. Results from five trials were averaged. Bacterial kill of the test and control solutions was ex- pressed as a percentage of the colonies on the matched phosphate buffered saline plates. Differences in antibacterial activity were determined by the Quades test. Dif- ferences were considered significant when p s .05.

Results

Two percent CHD was slightly alkaline (pH 7.5) and had a low osmolality (45). Sterile water had a neutral pH (7.4) and an osmolality of zero. Stock LRS was acidic (pH 7.1) and had an osmolality of 325. The addition of 2% CHD to water or LRS and precipitation of CHD/LRS had little effect on the pH and osmolality. Chlorhexidine diacetate diluted to 0.05% in 0.9% sodium chloride was the most acidic (pH 6.8) of the solutions tested and had an osmolality of 270 (Table 1).

Wound Healing

No postoperative analgesia was necessary. The dogs tolerated the bandages well. Some bandages occasionally slipped and were replaced. The neck bandages were effective in preventing self-inflicted trauma while allowing good neck motion.

There were no significant differences in the wound healing parameters between any of the lavage agents at any time during the wound healing process (Fig. 2). All wounds except those lavaged with CHD/PPT expanded initially. Granulation tissue first appeared in all wounds

Prepared from 20X stock solution of 170g NaCI, 13.6 g KH2P04, 3.0 g NaOH, q.s. to 1000 mL with distilled HzO.

TABLE 1. Osmolality and pH of Extracellular Fluid, Stock Chlor- hexidine, Physiologic Saline, Control, and Test Solutions

Osmolality

295-31 0

Solution PH

ECF' 7.4 7.5 45 2% CHD

NaCI' 5.4 31 0

Control Solutions

H20 LRS

7.4 7.1

Test Solutions

0 325

CHD/H20 7.4 9 CHD/NaCI 6.8 270 CHD/LRS 7.1 31 6 CHD/PPT 7.1 31 3

ECF = extracellular fluid; 2% CHD = stock chlorhexidine; NaCl = physiologic saline; H20 = sterile water; LRS = lactated Ringer's solution; CHD = chlorhexidine diluted to 0.05%; PPT = lactated Ringer's solution with precipitate.

Values reported by others6

between days 3 and 5. Wound contraction began between days 3 and 7 and accounted for a mean of 78% (range, 76% to 80%) of total wound healing by day 24 (Fig. 2A). Epithelialization was detectable grossly by day 7 in most wounds and accounted for a mean of 20% (range, 18% to 22%) of total wound healing by day 24 (Fig. 2B). The wounds were healed an average of 98% (range, 97% to 98%) by day 24 (Fig. 2C). No gross purulence was evident at any time during the healing process.

The histologic appearance of all wounds was similar. Fibrous tissue was progressively more mature from the base to the surface of each wound. Hair particles engulfed by phagocytic cells were found at the base of all wounds. Exposed granulation tissue composed of inflammatory cells, fibrin, vascular buds, and fibroblasts was evident in wounds that had not completely epithelialized.

Aerobic bacteria were cultured from two of 36 wounds on day 7, and four of 36 wounds on day 14. One wound yielded a positive culture on both sample dates. One wound with positive cultures had been irrigated with a solution containing CHD. Four wounds with a positive culture had been irrigated with LRS. Beta hemolytic Streptococcus sp. was isolated from the cultures that were positive on day 14. Attempts at quantitating wound bacteria with the velvet pad adhesion technique yielded no bacterial growth on any wounds.

Antibacterial Activity No significant differences in bacterial kill were detected

between any of the CHD solutions. One hundred percent

110

20

WOUND HEALING

-

2 0

Yo Epithelialization

-

O.\Q

40

20

0

J 0 3 7 10 14 17 21 24

days after surgery

- 2 0 '

- H20 LRS CHDIHZO

CHDlNaCl 0 CHDIPPT - CHDILRS

A.

YO Healed Wound % healed

120

100

60

40

bacterial kill was observed with all solutions containing CHD. Antibacterial activity of all CHD solutions differed significantly from the controls (p < .01). There was no significant bacterial kill with water or LRS.

Discussion

At the University of Missouri-Columbia Veterinary Teaching Hospital, the dog is the animal most frequently presented with wounds that require lavage. Comparisons of the effects of topical medicaments on different species emphasize that all species do not necessarily react simi-

In vitro fibroblast toxicity studies have not been reliable predictors of the in vivo wound response.' For these reasons, live dogs were selected for use in this in- vestigation.

Chlorhexidine has rapidly gained popularity as an antiseptic agent for wound lavage. Its advantages over povidone-iodine include greater residual antibacterial activity, better antibacterial activity in the presence of blood, pus and other organic debris, less systemic absorption and

"I 10

5 t

0- 3 7 14 17 21 24 10

days after surgery

LRS CHDIHZO

CHDlNaCl 0 CHDIPPT

- - HZO - CHDILRS

B.

Fig. 2. Differences in wound healing parameters after lavage of wounds with test and control solutions. CHD/ = chlorhexidine diacetate to 0.05% in;, H20 = sterile water, LRS = lactated Ringer's solution, NaCl = physiologic saline, PPT = lactated Ringer's solution with precipitate, To = area within day 0 wound margins. (A) Percent day 0 wound area healed by contraction. (B) Percent of the day 0 wound area healed by epithelialization. (C) Percent day 0 wound healed by epithelialization and contraction. There are no statistically significant differences between any of the solutions at any time for any of the parameters shown.

toxicity, and more rapid wound ' - I 3 Re sults of previous studies have helped establish a concentration range for CHD that is effective yet not deleterious to wound healing.4,14 For Staphylococcus aureus, this range is approximately 0.05 - 0.1 %.4*14

Water, LRS, and 0.9% NaCl differ in electrolyte composition, osmolality, and P H . ~ Of these fluids, LRS most closely approximates extracellular fluid composition; it is followed by NaCl and H20.6 Stock CHD (2%) has low osmolality and is slightly alkaline. It has little effect on diluent pH and osmolality when diluted to 0.05% (Table 1). Use of a wound imgant that is similar to extracellular fluid should result in less cytologic disturbance and less delay in wound healing. Because CHD is reported to be more effective in an alkaline en~ironment,~ the lower pH of NaCl and LRS compared with extracellular fluid make them poorer choices for CHD diluents. In this study, the pH of all LRS solutions was adjusted to 7.4 to achieve a more physiologic solution that might be less deleterious to the healing wound and possess greater antibacterial activity than NaCI.

111 LOZIER, POPE, AND BERG

Chlorhexidine diluted in LRS forms a visible precipitate within 3 to 4 hours at room temperature.* In NaC1, it precipitates within 1.5 hours.* Although the exact nature of the precipitate is not known, some have assumed the solution's antibacterial activity to be negated.*** Still, precipitated CHD solutions are often used clinically. The precipitated particles within the wound may act as foreign material, harbor bacteria, and inhibit host defense mech- anisms and potentiate wound infe~tion.'~ Delayed wound healing may result.

In this study, none of the 0.05% CHD solutions significantly affected the rate of wound healing relative to the other CHD test solutions or relative to the sterile water and LRS controls. This finding differs from a similar study in which wounds lavaged with CHD had more healed wound area on days 7 and 14 and had more wound contraction than saline controls.* In that study, many of the wounds were contaminated with bacteria, and the wounds lavaged with CHD had a significantly lower contamination rate than wounds lavaged with saline.* Reduction in the number of bacteria may explain the differences in beneficial effect of CHD in that study. Fewer wounds lavaged with CHD in this study became contaminated than wounds lavaged with control solutions. However, the low contamination rate of all wounds minimized the bacterial influence on wound healing.

The pH and osmolality of the solutions apparently were not different enough from normal extracellular fluid to cause deleterious effects on granulation, contraction, or epithelialization. Even the precipitated solution did not delay wound healing. In fact, although it was not statistically significant, wounds lavaged with CHD/PPT did not retract initially as did all other wounds. Retraction of wound margins is not an active process but is thought to result from preexisting skin tension.I6 If the difference in wound retraction was real, an explanation was not readily apparent. Aside from hair, no foreign particulate matter or unexplained granulomas characteristic of foreign body reactions were found in any of the tissue sections. It is possible that the precipitate dissolves in tissue fluids, thus negating its action as a foreign material.

Water has been advocated as an initial lavage agent for wounds that are heavily contaminated. The effects of water on wound healing have not been compared in vitro to more physiologic electrolyte solutions. Prolonged exposure of tissues to sterile water, distilled water, and tap water causes marked histologic alterations.' Sodium chloride is less injurious to tissues than water and causes only slight edema.7 Tyrode's solution, a physiologic mul- tiple electrolyte solution similar to LRS, causes no tissue damage.7 Our results indicate that sterile water used as a

*** Personal Communication, February 1990, Fred C. Zimmermann, DVM, Fort Dodge Laboratories, Fort Dodge, Iowa.

daily irrigant does not impede wound healing. No differ- ential tissue damage was detected by light microscopy after repeated exposure of the wounds to any of the lavage agents used.

To minimize bacterial influence on wound healing and prevent masking of the effects of the lavage agents, the wounds were not intentionally contaminated. They were cultured during the healing period to document a low contamination rate. The velvet pad technique was initially developed to estimate the degree of surface contamination during We used the technique to compare the relative in vivo antibacterial efficacies. Successful trials with the technique using grossly infected wounds indicated that this technique would be useful, but low numbers of bacteria and poor bacterial transfer probably led to failure in this study. Collection of tissue samples may be a more reliable method of determining the presence and degree of infection. l7 Tissue samples were not collected because they would have interfered with wound healing and tracings of the epithelial and original wound margins.

Results of the in vitro portion of this study indicated that the antibacterial activity of all the 0.05% CHD solutions against S. intermedius was similar. This bacterium was selected for testing because it is a common skin in- habitant in dogs and is a potential wound pathogen. 18,19

All solutions containing CHD exhibited 100% bacterial kill. Use of a larger number of trials with a CHD concentration in which only partial bacterial kill is documented may differentiate antibacterial activity of the solutions tested.

The experimental model used in the in vivo study ap- pears to be adequate for assessing the effects of wound imgation. In all controlled studies, a positive and negative control are ideal. This study lacked a negative wound healing control such as a more concentrated CHD solution that caused a detectable delay in wound healing.4 The sensitivity of this study may therefore be questioned, but it does not detract from the clinical information gained.

Conclusion

In dogs, dilution of CHD to 0.05% in wound imgants other than sterile water offers no detectable advantage in second intention wound healing. The precipitate that forms when CHD is diluted to 0.05% in LRS does not delay the wound healing process. Sterile water does not delay wound healing more than LRS. There is no difference in antibacterial activity against S. interrnedius between CHD diluted to 0.05% in water, NaCI, LRS, or LRS with a precipitate. Of the solutions tested, the ideal diluent for preparation of CHD as an antiseptic wound irrigant depends upon availability, cost, and personal preference.

112 WOUND HEALING

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antimicrobial efficacy and tissue reaction of four antiseptics on canine wounds. Vet Surg 1983; 12:63-68

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3. Russell AD. Chlorhexidine: Antibacterial action and bacterial resis- tance. Infection 1986: 14212-214.

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6. Webb AI. Fluid therapy in hypotensive shock. Vet Clin North Am (Small Anim Pract) 1982; 12:515-534.

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8. Department of Health and Human Services Guide for the Care and L k of Laboratory Animals. NIH Publication No. 86-23, Revised 1986.

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10. Raahave D. Velvet pad surface sampling of anaerobic and aerobic bacteria: An in vitro laboratory model. J Clin Pathol 1982;35:

ASSOC 1988;24:77-84.

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11. Amber El, Swaim SF. An update on common wound antiseptics. Aust Veterinary Pract 1984: 14:29-33.

12. Swaim SF, Lee AH. Topical wound medications: A review. J Am Vet Med Assoc 1987; 190: 1588- 1593.

13. Rosenberg A, Alatary SB, Peterson AF. Safety and efficacy of the antiseptic chlorhexidine gluconate. Surg Gynecol Obstet 1976; 143:

14. Sanchez IR, Swaim SF, Nusbaum KE, et al. Chlorhexidine diacetate and povidone-iodine cytotoxicity to canine embryonic fibroblasts and Staphylococcus aureus. Vet Surg 1988; 17:182-185.

15. Haury BB, Rodeheaver GT. Inhibition of nonspecific defenses by soil infection potentiating factors. Surg Gynecol Obstet 1977: 144: 19-24,

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789-792.

Abstract of Current Literature

ANTERIOR SPINAL FIXATION AFTER LUMBAR CORPECTOMY: A STUDY IN DOGS Zdeblick TA, Shirado 0, McAfee PC, deGroot H, Warden KE Journal of Bone and Joint Surgery 1991; 73-A:527-534

An animal model was developed to simulate an unstable burst fracture that had been treated with corpectomy. A 5th lumbar laminectomy, partial facetectomy, and corpectomy were performed in 21 mature coon hounds. In seven dogs (controls, Group I), a biodegradable polymer spacer was used to create fusion failure. Seven dogs were treated by traditional anterior arthrodesis with a keyed-in autogeneic ulnar strut-graft and without fixation (Group 11). Seven dogs were treated with the same grafting procedure and fixation with appropriately sized anterior Kaneda instrumentation (Group 111). At week 24, the results were analyzed for rate of fusion, biomechanical rigidity, neuropathologic findings, and histomorphometric data on the vertebral response. Rate of fusion and stiffness in torsion were significantly higher in Group I11 than in Groups I and 11. Axial compressive loading did not reveal a difference between the groups. There was no difference between groups in terms of the number of neuropathologic changes in the spinal cord. Histomorphometric analysis showed that device-related osteopenia was not significant in the vertebrae that were spanned by the Kaneda device. Trabecular density was increased in the vertebrae in which the instrumentation was anchored. This study confirmed the efficacy of anterior instrumentation of the lumbar spine after corpectomy and strut-grafting. Instrumentation can safely lead to a higher percentage of fusions and a more rigid spinal segment.

Documents

Effects of Four Preparations of 0.05% Chlorhexidine Diacetate on Wound Healing in Dogs