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Abstract
Cellular toxicity of dermal wound cleansers may be an important consideration in wound healing. To study the effects of dermal wound cleansers on wound closure, human fibroblasts were cultured and survival rates were established. Cellular integrity of both red and white blood cells was established by quantifying the number of intact cells using a hemocytometer. In addition, the concentration of hemoglobin in plasma was determined. All tests were conducted against saline controls. Cellular viability and function in each experiment were used to rank solutions according to the cytotoxic effects demonstrated. Significant differences were found in the toxicity profile of available cleansers.
Cellular toxicity of dermal wound cleansers is an important consideration in wound healing. Initial steps in wound management should not undermine subsequent phases in wound healing.
Introduction
Awareness of the cytotoxicity of disinfectant irrigation solutions has spurred the development of nontoxic dermal wound cleansers. Cytotoxic effects can damage viable tissue, possibly slowing wound closure and increasing the wound's susceptibility to infection.1,2
Dermal wound cleansers are agents used to remove bacteria and other contaminants from wounds. Wound cleansers may or may not contain antimicrobials, and are not considered antiseptic agents. Unlike disinfectants, which have been shown to cause tissue damage and interfere with tissue function, wound cleansers ideally would not interfere with healing processes.3 Fibroblast cells are considered the key cells for the laying down of collagenous dermal tissue and are the initiators of wound contraction. Human fibroblasts in vitro have been used to study the cytotoxicity of a variety of topical agents.4 In the series of Lineweaver's experiments, the antiseptic agent was exposed to fibroblasts for 25 minutes before incubation. Although the assay offers a quantitative measure of cytotoxicity of fibroblast cells, the time of exposure to the antiseptic agent is an experimental variable. In 1968, Bettley suggested that the effect of cleansers on blood could be used as a comparative measurement of the cellular toxicity.5
In this study, a number of commercially available dermal wound cleanser formulations were tested to evaluate their potential cytotoxicity on in vitro human fibroblasts. Dermal wound cleansers tested included: Cara-Klenz™ (CK), Carrington Laboratories, Inc., Irving, TX; Constant-Clens™ (CC), Sherwood Medical Company, St. Louis, MO; Saf-Clens™ (SaC), Calgon Vestal Laboratories, St. Louis, MO; Ultra-Klenz™ (UK), Carrington Laboratories, Inc., Irving, TX; and Shur-Clens® (ShC), Calgon Vestal Laboratories, St. Louis, MO. All of these wound cleansers were mixed with culture medium at a 20 percent (v/v) ratio and remained in contact with the fibroblast cell throughout the entire culture period. Dermal wound cleansers were also tested to evaluate their potential cytotoxicity to red and white blood cells in vitro. pH of the dermal wound cleansers was measured as a function of their suitability to promote cell viability.
Materials and methods
Dermal wound cleansers tested were obtained from commercial sources, had identification removed, and were blindly coded for the experiments.
pH measurement
pH of wound cleanser formulations was measured at 20°C on a Corning 610 A pH meter. pH was measured on dermal wound cleansers directly and also on wound cleanser formulations mixed with DMEM culture medium at a concentration of 20 percent (v/v).
Human fibroblast cultures
The cell line used for this study (ATTC CLLS 11D Detroit 551 Normal Diploid Female) was derived from the skin of a Caucasian female embryo by Drs. Simpson, Peterson and Stulbert in 1965. Cells are described as a typical diploid human fibroblast-like line which was established in the same manner as were lines with abnormal chromosomal configuration developed for the American Type Culture Collection. The Detroit 551 cells may be considered normal and may serve as controls for the study of cells having mutant chromosomes. The cell line has a finite life span characteristic of this fibroblast type of cell.
Five culture flasks for each formulation were prepared with Detroit 551 cells and allowed to establish (48 hours) in medium without wound cleanser compound. Following 48 hours, DMEM medium containing 0 and 20 percent (v/v) wound cleanser was added to four flasks with one flask serving as a control culture. DMEM was replaced every 24 hours for a period of six days and growth was observed and measured with a hemocytometer. After six days, cultures were washed with PBS (Dulbecco's), stained with 0.1 percent Methylene Blue, and live versus dead cells were counted under an inverted scope at 200X using an ocular micrometer.
Cellular integrity of red and white blood cells following exposure to dermal wound cleansers
Human blood samples from three male volunteers were drawn into tubes containing ethylenediamine tetra—acetate and used immediately for these experiments. Blood samples were pooled and combined with an equal volume of a dermal wound cleanser to be tested. Blood samples with dermal wound cleansers were maintained at 37°C for 10, 30, and 60 minutes and each sample was replicated three times.
Following exposure over time, aliquots were removed from each sample and the number of intact red and white blood cells were quantitated using a hemocytometer. Immediately following aliquot removal, the remainder of the samples was centrifuged to form a cellular pellet overlaid with plasma. The concentration of hemoglobin in the plasma was determined using a modification of the benzidine method as described by Crosby and Furth in 1956.6
Blood exposed to 0.9 percent of NaCl for 10, 30, and 60 minutes at 37°C served as a control for each treatment, respectively. Data were analyzed by pair—wise comparisons using Scheffe's test.7
pH
The pH of undiluted and diluted wound cleanser formulations with culture media 20 percent (v/v) is shown in Table 1. CK and ShC both exhibited a pH considered outside a physiological range in undiluted samples. Dilution with culture media elevated the pH considerably. However, CK and ShC both remained below a pH of 7.00.
Human fibroblast assay
Wound cleanser formulations differed in cytotoxicity to human fibroblast cells when used at a level of 20 percent (v/v) with culture medium. (Figure 1)
Blood cell assay
Exposure of white blood cells to CC and SaC for 10, 30, and 60 minutes did not damage cells compared to controls. UK, CK and ShC significantly damaged white blood cells (P £ 0.05) compared to controls at all times tested. (Table 2) ShC destroyed red blood cells at almost a two—fold logarithmic decrease at all times tested compared to all other compounds and controls. (Table 3)
ShC demonstrated a significant rise in hemoglobin in the first ten minutes of exposure. A lesser but still significant increase in hemoglobin was observed for UK and SaC at 30 and 60 minutes, and with CK at only 60 minutes, compared to controls. Hemoglobin levels were non—detectable for CC at all time periods tested. (Table 4)
Discussion
A wide range in pH was found among the wound cleanser products. In general, wound cleanser products which had the lowest pH were also the most cytotoxic to fibroblast cultures and blood cells.
CC and SaC were significantly (P < 0.05) less cytotoxic to fibroblast cells compared to all other wound cleanser products tested. CK was found to be slightly cytotoxic to fibroblast cells compared against saline exposed controls, but significantly less cytotoxic (P < 0.05) compared to UK. ShC was the most cytotoxic product tested with only 34.5 percent of the fibroblast cells surviving the culture.
CC was superior to all other agents tested for being nondestructive to white blood cells for all time periods tested. UK, CK and SaC were found to destroy white blood cells at a higher level compared to CC. White blood cell destruction occurred during the initial ten minutes of exposure to agents, with only CK having continued destruction at the 30—minute exposure time.
Destruction of red blood cells exposed to dermal wound cleansers was less compared to white blood cells. CC and UK showed no statistical difference from controls for destruction of red blood cells. CC, as well as controls, was the only compound to demonstrate non—detectable hemoglobin levels at all time periods tested. ShC was approximately 100 fold more toxic to red blood cells than the other dermal wound cleansers tested. CK and SaC, although not toxic following a ten minute exposure time, did show some toxicity at 30 and 60 minutes compared to controls. Since most of the destruction occurred during the initial ten minute exposure period and little more at 30 and 60 minutes, that suggests a population difference among blood cells exists. Previous work measured blood cell destruction at only a 30 minute exposure time,8 and this observation merits further investigation.
Conclusion
Results of this study showed differences in the ability of some commercially available dermal wound cleansers in cellular toxicity to both red blood cells and white blood cells in vitro. Clearly static conditions employed in vitro are not comparable to the ever changing physiology of a wound. However, in vitro methods do allow the study of a single effect, i.e. toxicity of blood cells in a controlled environment. Of course, this aspect is extremely valuable in the understanding of the role of dermal wound cleansers in wound management. The premise that all dermal wound cleansers are created equal is flawed. The superiority of one dermal wound cleanser over another must ultimately be tested on the healing of human wounds with enough patients included to obtain meaningful results.
References
1. Faddis D, Daniel D, Boyer J: Tissue toxicity of antiseptic solutions. A study of rabbit articular and periarticular tissues. Journal of Trauma 1977;17:895—897.
2. Rodeheaver GT, William B, Kody M, Spatafora G, Fitton L, Leyden K, Edlich R: Bacterial activity and toxicity of iodine—containing solutions in wounds. Archives of Surgery 1982;117:181—186.
3. Leaper D, Simpson R: The effects of antiseptics and topical antimicrobials on wound healing. Journal of Antimicrobial Chemotherapy1986;17:135—137.
4. Lineweaver W, Howard R, Soucy D, McMorris 5, Freeman J, Crain C, Robertson J, Rumley T: Topical antimicrobial toxicity. Archives of Surgery 1985;120:267—270.
5. Bettley FR: The toxicity of soaps and detergents. British Journal of Dermatology 1968;80:635-642.
6. Crosby WH, Furth FW: A modification of the benzidine method for measurement of hemoglobin in plasma and urine. Blood 1956;380—383.
7. Scheffe H: The Analysis of Variance. Wiley, NY, 1959.
8. Rodeheaver GT, Klutz L, Kricher BJ, Edlich RF: Pluronic F—68: A promising new skin wound cleanser. Annals of Emergency Medicine 1980;9:572-576.
Raymond W. Wright, Jr. PhD.
Dr. Wright is a Research Professor and Fellow in the Health Science Center at Washington State University in Spokane, WA.
Robert Orr
Mr. Orr is a Wound Care Associate at Sherwood Medical Company in St. Louis, MO.