Abstract: A mixture of hyaluronan and iodine complex KI3 (Hyiodine®) has been developed to support wound healing. In this study, the effect of hyaluronan, KI3, and their combination on progression of wound healing in excision skin wounds in rats was determined. Methods. To evaluate the possible toxic effects of iodine after Hyiodine application for wound healing, iodine systemic absorption from the Hyiodine-treated wounds and its distribution profile were quantitatively described and compared after intravenous iodide administration using 131I as a radiolabel. Results. Treatment of rats with Hyiodine resulted in an enhancement of wound healing, proved by greater degrees of wound contraction and reduction in mean wound healing time compared to other treated rats on hours 1, 2, 24, and 216. Even if wound therapy with Hyiodine resulted in systemic iodine uptake, the estimated iodine absorbed dose in human therapy is tolerable, and the theoretical risk of its systemic toxic effects is minimal. Conclusion. A hyaluronan-iodine hydrogel has a great potential for effective treatment of wounds.
Animal models of wound repair can provide reliable and reproducible information on the behavior and response of wounds to experimental therapy. However, animal models cannot replace the ultimate verification of agents and actions in human wounds because of the substantial differences in tissue architecture and immune response. Therefore, models should strive for reproducibility, clinical relevance, humane treatment, and quantitative interpretation.1 Wound healing is a dynamic, interactive process involving many precisely interrelated phases that overlap in time and lead to the restoration of tissue integrity. The healing process reflects the complex and coordinated body response to tissue injury resulting from the interaction of different cell types and extracellular matrix (ECM) components. Hyaluronan (HYA) plays a key role in each phase of wound healing by stimulating cell migration, differentiation, and proliferation, as well as regulating ECM organization and metabolism.2 Hyaluronan is a straight chain, high molecular weight polymer of D-glucuronic acid and N-acetyl-glukosamine in the ECM. It affects cellular behavior by maintaining tissue integrity, and plays a number of roles in the healing of damaged tissues, as well as in facilitating adhesion and differentiation of cells during inflammation and embryonic development.3-6 Hyaluronan is a versatile, biocompatible, and non-immunogenic natural product, which, when applied topically to skin wounds, accelerates wound contraction and increases blood flow in the wound. The molecular weight of HYA in its native form is typically in excess of 106 Da, however, at sites of wound healing, it can de-polymerize into lower molecular weight breakdown forms. The biological effects of these fragments may be different from those of the parent HYA.3 Whereas native HYA appears to have supporting effects for tissue integrity and promotes cell quiescence, HYA fragments represent a signal that injury has occurred, and initiate an inflammatory response.3 Today, HYA hydrogels are used clinically as a wound dressing to prevent adhesions and improve wound recovery. Iodine and its compounds have been broadly used for the prevention of infection in the treatment of wounds.7 Antiseptics, such as low concentrations of iodide, are preferred to antibiotics in the treatment of open, infected wounds because of the risk of development of bacterial resistance.8 Iodine has been used for this indication for many years to reduce complications arising from infection. An advantage of using iodine-containing preparations as topical antiseptics in chronic wound care is a reduction of bacterial load. Resistance to iodine is uncharacterized, and its use reduces the need for systemic antibiotics. In addition, iodine-containing antiseptics are relatively inexpensive.9 The spectrum of antimicrobial activity of antiseptics containing iodine is wide, and includes action against resistant strains in clinical infections.9 It has also been demonstrated that topical cadexomer iodine treatment stimulates the epithelization of burns, and accelerates wound healing.8 The clinical use of antiseptics was profoundly influenced by 2 studies in which animal models were used to evaluate the toxicity of antiseptics.10,11 Irrigation of animal wounds with iodine antiseptics was found to be effective in preventing wound infection.12 A mixture of HYA and iodine complex KI3 (IC) was reported to support wound healing in animal models by stimulating wound contraction and epidermal proliferation, and also by keeping the wound moist through increased exudation.13 Hyaluronan + IC also significantly improved the healing process.14 Two possible mechanisms of iodine effects, namely its ability to inhibit apoptosis or proteinase activity, may account for its protective effect in wound healing.15 Additionally, clinical studies with HYA + IC in chronic non-healing wounds in diabetic patients have shown improvement in most of them (complete healing was evident in 14 of 18 patients within 6 weeks to 20 weeks of treatment).16 Systemic availability and toxicity of topically administered iodine should be investigated, as iodine-containing ointment treatment significantly raised iodine plasma concentrations,8 and excess iodine intake presents the theoretical risk of thyroid dysfunction. This study aimed to evaluate additional effects of KI3 on HYA hydrogel-treated wound contraction, and to analyze the biological effects of iodine in the HYA-KI3 complex employed for wound healing in excision skin wounds in rats.
Materials and Methods
Chemicals. Hyaluronan (HYA; M.W. 1.2 MDa) was obtained from Streptococcus sp. (Contipro C Ltd, Dolni Dobrouc, Czech Republic). The iodine complex (IC) was prepared by dissolving iodine in a solution of potassium iodide (Riedel de Haen, Seelze, Germany). Radioiodide-131I-sodium iodide carrier-free (LACOMED, Rez, Czech Republic), activity 925 MBq/ml, was also obtained for the study. Test formulation (HYA + IC) consisted of 1.5 g sodium HYA, 0.15 g KI, 0.1 g I2, and 100 ml water for injection. The mixture is commercially available as Hyiodine (Contipro, Dolni Dobrouc, Czech Republic). Hyaluronan + IC labeled with 131I: 0.1 ml 131I-sodium iodide in 0.01 mol/l NaOH (activity of 92.5 MBq) was added to 20 ml of HYA + IC. The test formulation was stirred, sealed, and shaken for 1 hour. The formulation then stood for 24 hours to equilibrate the mixture. Animals. Animal studies were carried out using both male outbred Wistar, and inbred non-diabetic ZDF, rats weighing 260 g to 300 g. The animals were fasted overnight before the experiment to empty the bowels, but had free access to water. The rats were maintained during the experiment in standard animal facilities that comply with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. The animals were fed pelleted food and had free access to food (after the first night of fasting) and water. The State Veterinary and Food Administration of the Slovak Republic and the Ethics Committee for Control of Animals Experimentation on the National Institute of Rheumatic Diseases approved the experimental protocol and all procedures.
Induction of wounds. The ZDF rats were anesthetized by intramuscular administration of 100 mg/kg ketamine plus 16 mg/kg xylazine (Spofa a.s., Prague, Czech Republic). After shaving the dorsal hair and disinfecting the exposed skin with 70% ethanol, full-thickness excision quadratic wounds (20 mm x 20 mm) were made on the back of the rats by the modified method described by DiPietro and Burns.17 Each wounded animal was housed in a separate sterile polypropylene cage during the study period. At the end of experiments, the animals were sacrificed after euthanasia by injecting with a fatal dose of barbiturate (sodium thiopental) after administration of anesthetics (ketamine and xylazine). Treatment. The tested substances were administered in corresponding doses from hour 0 of the study. Solutions of HYA, IC, and their combinations were prepared in distilled water to reach the required concentration, and were applied directly on the wounds at a dose of (0.2 ml/cm2) at 0, 1, 2, 24, 72, and 144 hours. A fresh solution of the tested substances was prepared for each administration. The untreated groups received the vehicle (sterile saline) in the same manner. The wounds were covered with sterile gauze after administration of test substances. Rat groups. The animals were divided into the following 4 groups of 6: Group 1, untreated controls; Group 2, rats treated with HYA; Group 3, rats receiving IC; and Group 4, rats administered the combination of HYA + IC.
Quantitative data are presented as the mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for statistical analysis of the results, and P < 0.05 was taken as the significance limit for all comparisons.
Determination of wound contraction. The wound size was measured using a digital caliper. The process of healing was calculated using the following equation (expressed as a percentage): Degree of wound contraction (%) = ([Ahour0 - AhourX] / Ahour0) x 100 where X = 1, 2, 24, 72, 144, or 216 hours after wound creation, A = wound surface area.18 Progression of the healing of excision wounds in the inflammatory period was assessed by periodic measurement of wound contraction. The area of each wound was expressed as the percentage of its original size at 1, 2, 24, 72, 144, and 216 hours (Figure 1). The maximum wound contraction was observed at 216 hours post-surgery in then HYA+IC group. In control rats, there were increased values of wound size in the first 2 hours. The degree of contraction was similar in both HYA and IC treated groups. Hyaluronan, IC, and their combination treatment resulted in a significant enhancement of wound contraction at all time intervals under study. Neither HYA nor IC alone exhibited significant difference in effect on wound healing at 216 hours post-surgery. The macroscopic morphology of wound contraction after treatment with hyaluronan (HYA), iodine complex (IC), combination HYA+IC, and control are shown in Figure 2.
Distribution and elimination studies in rats
Distribution studies. Radioiodide-131I (10-4 mol/l and 5x10-2 mol/l) was administered to Wistar rats intravenously in a volume of 0.2 ml. At 5 minutes, and again at 1, 2, 24 and 48 hours after dosing, the carotid artery was exposed under ether anesthesia, and a blood sample was collected in glass tubes containing dry heparin. After exsanguination under ether anesthesia, the tissues and organs of interest were removed and weighed, and their radioactivity was measured in a gamma counter. HYA + IC labeled with 131I. The treatment application on the wound surface was described above. At 1, 2, 24, and 72 hours after dosing, the same procedures were performed as described in the previous paragraph. Elimination studies. Radioiodide-131I or HYA + IC labeled with 131I were administered to rats as described above. Following administration, the animals were individually placed into separate glass metabolic cages, the construction of which allows reliable separation of the urine and solid excrements. The animals had free access to standard diet and water. Two hours after administration the rats were forced to empty their urinary bladders by handling (immobilization), and urine and feces were collected. The procedure was repeated at 24 hours and 48 hours (for HYA + IC labeled with 131I, the procedure was also repeated at 72 hours) after administration. All the pharmacokinetic animal experiments were approved by the Ethics Committee of the Faculty of Pharmacy, Charles University. The distribution of 131I-radioactivity (% administered dose per organ) after intravenous administration of 131I-iodide at a dose of 5x10-8 mol and 10-5 mol is summarized in Tables 1 and 2. In other organs not shown in Table 1 and 2, namely the adrenals, heart, spleen, testes, and brain, less than 1% of administered 131I activity was found. The later dose approximately corresponds to the total iodine amount in the HYA + IC used for application to the wound in this study; however, in patients, substantially lower HYA + IC amount should be expected, calculated based on the body mass index: 0.8 ml of the test formulation for a rat in the present study and usually 1.5-8 ml of the test formulation for patients (oral communication, Lubos Sobotka, MD, PhD, Head of Department of Gerontology and Metabolic Care, Faculty of Medicine, Charles University, Hradec Kralove, Czech Republic, March 2012). Distribution of 131I-radioactivity after 131I - (HYA + IC) treatment of experimental wounds is shown in Table 3. Under these experimental conditions, a portion of the HYA + IC was applied on the wound and covered with sterile wound dressing. The activities in the samples of marginal wound, in the polyethylene foil (covering the wound dressing to prevent loss of the test formulation), and wound dressing were thus determined. Cumulative urinary/fecal excretion of radioactivity after intravenous 131I-iodide dosing and 131I-(HYA + IC) wound treatment is summarized in Tables 4 and 5.
Wound dressings are generally used to promote healing, prevent infection, and provide mechanical protection of the wound. The goal of (HYA + IC) - containing dressings is both to increase healing rates and to reduce bacterial bioburden. Wound contraction can be defined as the centripetal movement of the edges of a full-thickness wound to facilitate closure of the defect.18,19 This process in rats can be readily measured by noninvasive, morphometric techniques.1 Inflammatory phase of the wound healing process begins immediately after removal of skin tissue. The wound size in the negative control of rats demonstrated minimal change or became slightly enlarged in the first hours after wound induction, compared to treated groups. The authors assumed the tested substances have anti-inflammatory activity. The second possibility is that the hydrophilic HA molecule increased wound contraction in the early stages of wound healing. These factors probably had important influence on wound-induced contraction in the first hours of wound healing following wound induction. In this study, application of HYA significantly enhanced wound contraction at all time points of the study, compared to either the untreated control groups or the group treated with IC alone. Significant differences were obtained between groups of rats treated with a combination of HYA + IC and treated rats with HYA monotherapy at 1, 2, 24, and 216 hours. Wound contraction progressed with time, and a steady contraction of the excision wound was noted in all treated groups. The findings of this study demonstrated that the administration of HYA + IC in this wound model significantly improved the excisional wound healing process. In agreement with our studies, Slavkovsky et al13 reported contraction was significantly accelerated in the first days by the mixture of HYA and KI3 treatment. To characterize the fate of iodine in the body after HYA + IC application to the wounds, the authors assessed the biodistribution of radioactivity after intravenous injection of 131I-iodide in the first step. Two different iodide concentrations were utilized in the experiments: an equivalent amount of iodine in the HYA + IC used in these experiments, and an estimated dose for human use, corrected for body weight. Blood radioactivity-time courses were mutually similar for both iodide concentrations. When biodistribution profiles in other organs and tissues were compared, differences in radioactivity uptake in the thyroid gland were found. There are several reports regarding inhibition of the thyroid iodine uptake due to rapid increases in the plasma iodide concentration. Iodine is an essential element of thyroid hormones, and plays a key role in the regulation of thyroid gland function. The active transport of iodide from the blood stream into the thyroid follicular cells is mediated by sodium iodide symporter, which is localized in the basolateral membrane of thyrocytes.20 Iodide is incorporated into thyroglobulins in the follicular compartment of the thyroid. Higher plasma iodide concentrations reversibly inhibit the iodide transport mechanisms, and incorporation of iodine into organic molecules, due to the inhibition of several steps of thyrocyte activity (the Wolff-Chaikoff effect).20 An excess of iodide thus influences numerous processes involved in thyroid function, including hormone secretion. This intrinsic regulatory system protects the thyroid from dangerously high intracellular concentrations of iodide.19 For these reasons, radioactivity accumulation in the thyroid gland was several-fold lower after a higher dose of iodide in the longer time intervals after injection. With regard to elimination, the majority of the radioactivity was excreted in the urine, and no significant differences in elimination pathways between the 2 iodide doses were identified. When the 131I radioactivity-time profile in the body after topic administration of HYA + IC on wounds and intravenous iodide injection were compared, both methods of iodide administration yielded surprisingly similar distribution profiles. Although approximately 1/3 of the radioactivity after HYA + IC treatment remained on the wound dressing, distribution characteristics in the blood and other organs were comparable for both topical HYA + IC administration and intravenous iodide injection, beginning 1 hour after dosing. These results indicate the available iodine in HYA + IC was rapidly absorbed from the experimental wound to the central distribution compartment. Significant plasma iodide concentration increases have also been reported after the use of cadexomer-iodine ointment in wound healing by Lamme et al.8 Rapid absorption of iodine from the HYA + IC-treated wounds into the systemic circulation indicated its anti-bacterial effect was not only topical, but also works at deeper regions of the wound. This study quantitatively assesses the fate of iodine after topical HYA + IC application on a model for excisional wounds. The authors’ finding that iodine is gradually absorbed to the systemic circulation raises the concern of potential systemic toxic effects of iodine after HYA + IC administration for wound healing. It is known that chronic excess iodide may result in growth anomalies,20 hypothyroidism and goiters.21 A review of iodine toxicity reports concluded that iodine intakes of up to 1,000 mg per day are probably safe for the majority of the population, but may cause adverse effects in some subjects.22 An extensive study on the toxicity of high single doses of iodide is described in an epidemiological study related to the Chernobyl accident,23 where 90 million doses of 100 mg of KI, of which 18 million doses were administered to children, were used to reduce the thyroid radioiodine burden. According to the results, there were no significant differences in serum thyroid-stimulating hormone levels between the protected and unprotected groups, no serious adverse reactions, and only a few side effects—mainly mild gastrointestinal disorders and skin rashes—occurred in both children and adults. No medical assistance was required, and it is still unclear if these problems were caused by iodide toxicity, or by psychological factors. In patients, an estimated volume of HYA + IC solution applied to the wound is between 5 ml and 25 ml. Hyaluronan + IC is administered by sterile gauze soaked in HYA + IC, or is directly applied to deeper wounds.16 This volume of HYA + IC corresponds to iodine doses between 10 mg and 50 mg. Even if wound therapy with HYA + IC results in systemic iodine uptake, the iodine dose should be tolerable, and the theoretical risk of its systemic toxic effects would be minimal, if any. However, caution should be advised in subjects with autonomous thyroid disease or with iodine-sensitivity; infants and children; and pregnant or lactating women.
The findings of this study suggest administration of combination HYA + IC significantly improved wound-healing activity. Therefore, HYA + IC therapy may be helpful in acute wound repair, as iodine supports the positive effects of HYA on the excisional wound healing process. The results in animals demonstrated that most of the iodine on the wound was gradually absorbed into the bloodstream, but the theoretical risks of iodine toxic effects are minimal, if any.
The authors thank technicians Jarmila Hoderova, Eva Teichmanova, Marcela Vasinova, and Katarina Vandakova for their useful technical assistance. This study was supported by the CPN Ltd, Dolni Dobrouc, Czech Republic. Authors Vladimir Velebny, PhD, and Karol Svik, PhD, are partly employed in the firm producing the tested formulation.
1. Davidson JM. Experimental Animal Wound Models. WOUNDS. 2001;13:9-23. 2. Chen WY, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair Regen. 1999;7:79-89. 3. Noble PW. Hyaluronan and its catabolic products in tissue injury and repair. Matrix Biol. 2002;21:25-29. 4. Vazquez JR, Short B, Findlow AH, et al. Outcomes of hyaluronan therapy in diabetic foot wounds. Diabetes Res Clin Pract. 2003;59:123-127. 5. Stern R, Asari AA, Sugahara KN. Hyaluronan fragments: an information-rich system. Eur J Cell Biol. 2006;85:699-715. 6. Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol. 2007;23:435-461. 7. Fleischer W, Reimer K. Povidone-iodine in antisepsis - state of art. Dermatology. 1997;195 (Suppl 2):3-9. 8. Lamme EN, Gustafsson TO, Middelkoop E. Cadexomer-iodine ointment shows stimulation of epidermal regeneration in experimental full-thickness wounds. Arch Dermatol Res. 1998;290:18-24. 9. Leaper DJ, Durani P. Topical antimicrobial therapy of chronic wounds healing by secondary intention using iodine products. Int Wound J. 2008;5:361-368. 10. Lineaweaver W, Howard R, Soucy D, et al. Topical antimicrobial toxicity. Arch Surg. 1985;12:267-70. 11. Brennan SS, Leaper DJ. The effect of antiseptics on the healing wound: a study using the rabbit ear chamber. Br J Surg. 1985;72:780-782. 12. Edlich RF, Custer J, Madden J, et al. Studies in management of the contaminated wound. Am J Surg. 1969;118:21-30. 13. Slavkovsky R, Kohlerova R, Jiroutova A, et al. Effects of hyaluronan and iodine on wound contraction and granulation tissue formation in rat skin wounds. Clin Exp Dermatol. 2010;35:373-379. 14. Frankova J, Kubala L, Velebny V, Ciz M, Lojek A. The effect of hyaluronan combined with KI3 complex (Hyiodine wound dressing) on keratinocytes and immune cells. J Mater Sci Mater Meds. 2006;17:891-898. 15. Wormser U, Sintov A, Brodsky B, Amitai Y, Nyska A. Protective effect of topical iodine preparations upon heat-induced and hydrofluoric acid-induced skin lesions. Toxicol Pathol. 2002;30:552-558. 16. Sobotka L, Smahelova A, Pastorova J, Kusalova M. A case report of the treatment of diabetic foot ulcers using a sodium hyaluronate and iodine complex. Int J Low Extrem Wounds. 2007;6:143-147. 17. DiPietro LA, Burns AL. Wound healing methods and protocols. Humana Press, 2003. 18. Agren MS, Mertz PM, Franzen L. A comparative study of three occlusive dressings in the treatment of full-thickness wounds in pigs. J Am Acad Dermatol. 1997;36:53-58 19. Kumar P, Jagetia GC. Modulation of wound healing in mice. In: Peacock EE, ed. Wound Repair, Third Edition. Philadelphia, PA: WB Saunders, 1984:39-55. 20. Dayem M, Navarro V, Marsault R, et al. From the molecular characterization of iodide transporters to the prevention of radioactive iodide exposure. Biochimie. 2006;88:1793-1806. 21. Baker DH. Iodine toxicity and its amelioration. Exp Biol Med. 2004;229:473-478. 22. Pennington JA. A review of iodine toxicity reports. J Am Diet Assoc. 1990;90:1571-1581. 23. Nauman J, Wolff J. Iodide prophylaxis in Poland after the Chernobil reactor accident: benefits and risks. Am J Med. 1993;94:524-532. Milan Laznicek, PhD; and Alice Laznickova, PhD are from the Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic. Vladimir Velebny, PhD is from the Laboratory of Wound Healing, CPN, Dolni Dobrouc, Czech Republic. Karol Svik, PhD is from the Laboratory of Wound Healing, CPN, Dolni Dobrouc, Czech Republic, and the National Institute of Rheumatic Diseases, Piestany, Slovak Republic. Address correspondence to: Milan Laznicek, PhD Faculty of Pharmacy Charles University Heyrovskeho 1203, CZ-50005 Hradec Kralove, Czech Republic email@example.com