T he process of dressing selection is determined primarily by the nature of the wound, which includes the exudative, odorous, and microbial characteristics of the wound. Today, many sophisticated dressings made from a wide range of materials are available to the wound care physician. Depending upon their structure and composition, some dressings may simply absorb exudate, while others may inhibit the colonization of microorganisms, but the search for a material that eliminates both odor and exudate still continues. Wound odor is commonly produced by chronic leg or decubitus ulcers and fungating tumors. The odor may be distressing not only for patients but for their relatives and caregivers. In some cases, malodor may cause psychological problems, and a badly affected patient may refuse social contact or even appropriate care.1 The odor is generated from degradation of tissue proteins and lipids by several microorganisms. The molecules that cause malodor are short chain organic acids with a mixture of amine and diamines, such as cadaverine (1,5-pentanediamine) and putrescine (1,4-diaminobutane).2,3 The most effective odor control should focus on treating the source, but in some inoperable tumor cases or during wound treatment, the existing odor must be eliminated. A variety of odor control products, such as wound cleansers, deodorizers, charcoal briquettes, and air fresheners, have been used, but none have successfully eliminated the odor for any meaningful length of time.4 Various reports about activated charcoal dressings proved them to adsorb and eliminate the odor effectively;5,6 however, they are expensive and are not available in many countries. α-sepiolite is a natural clay mineral. Research conducted by Rodeheaver et al. showed that foreign matter containing clay applied to full-thickness wounds potentiated infection.7 Although clay may contain some a-sepiolite, a-sepiolite is not clay itself but a clay mineral. In later studies, α-sepiolite was combined with collagen and studied as an implantation material in rat calvarial defects. The study showed that the collagen-sepiolite complex did not produce any toxic effect or necrosis, and their resorption was followed by osseous regeneration.8 Additionally, several studies using human skin fibroblasts have shown that α-sepiolite does not exhibit any cytotoxic effects, since it allows normal cell adhesion and spreading on its surface, suggesting its potential use as a biomaterial.9 α-sepiolite has been used in many settings including medicine, but its odor adsorbing capacity has not been evaluated in medicine. The aim of the present study is to document the adsorptive characteristics of α-sepiolite in comparison to a well known odor adsorber: activated charcoal dressings. Materials and Methods Test materials. Group 1, gauze sponge (control group). Four layers of 10cm x 10cm gauze sponge were sterilized under 1 atmosphere of pressure at 121oC for 45 minutes. Group 2, activated charcoal with silver. A charcoal cloth consisting of 95–98% carbon is produced by carbonizing and activating a knitted viscose rayon fabric. The rayon fabric is enclosed in a sleeve of nonwoven nylon. The dressing is designed to adsorb odor, toxins, and other undesirable materials. The dressing’s primary use is to kill pathogenic organisms in a wound. It is 10.5cm x 10.5cm and sterilized with gamma irradiation. Group 3, activated charcoal with fibrous cellulose. The activated charcoal with fibrous cellulose dressing is a multilayered dressing that has both fluid absorbing and odor adsorbing properties. The inner layer of knitted viscose is backed with an absorbent layer of fibrous cellulose to which is bonded a layer of activated charcoal cloth sandwiched between 2 layers of polyethylene net. It may be placed directly on the surface of the wound. It is 10cm x 10cm and sterilized with gamma irradiation. Group 4, α-sepiolite, 5g. Particles between 297–720 microns weighing a total of 5g are enclosed in a 10cm x 10cm synthetic cellulosic fabric to avoid release of the particles into the chamber (ie, the powder is not deposited directly on the wound in clinical applications). The dressing is sterilized under 1 atmosphere of pressure at 121oC for 45 minutes (Figure 1). Group 5, α-sepiolite, 2g. Particles between 297–720 microns weighing a total of 2g are enclosed in a 10cm x 10cm synthetic cellulosic fabric to avoid release of the particles into the chamber. The dressing is sterilized under 1 atmosphere of pressure at 121oC for 45 minutes (Figure 1). Odor Adsorbing Study The odor adsorbing tests were conducted by the Medical and Aromatic Plants and Medicine Research Center of Anadolu University (TBAM). The test solution used in the study was determined to have similar molecular properties with malodorous wound fluids.2 This solution consists of sodium/calcium chloride (142mmol/L sodium ions; 2.5mmol/L calcium ions) to which is added diethylamine and 10% newborn bovine serum (B/N 0030342, Flow Laboratories, Sigma, St Louis, Mo). The test solution is first exposed to a gas chromatograph/mass spectrometer device (GC/MS, Shimadzu GCMS-QP 5050, Kyoto, Japan) for identification in later tests. The odor test apparatus consists of a hard plastic plate with a 5cm diameter and 3mm deep hollow center and plate lid. The hollow center is perforated and attached to an airtight septum that blocks the connection of inner and outer space. A 20-gauge intravenous (IV) cannula (Mediflon™, Eastern Medikit Limited, Delhi, India) is introduced in the center of the septum. The dressing material to be tested is placed over the hollow center. The undersurface of the material is sealed and covered with impermeable adhesive tape. The test solution is introduced into the dressing through the IV cannula at a rate of 30mL/hour by a Lifecare 5000 pump (Abbott Laboratories, Abbott Park, Ill). Another airtight septum is placed at the center of the lid to observe the diethylamine concentration released by the dressing to the space created by the plate and lid. The concentration of diethylamine in the space is measured throughout the procedure at the delivery times of the solution in amounts of 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 22, 25, 30, 40, and 50mL by a solid-phase microextraction (SPME) microinjector in the lid septum (Figure 2). After addition of each of the aforementioned amounts, a 3-minute interval is given for the microinjector to adsorb the diethylamine concentration of the space. The microinjector is injected into the gas chromatograph immediately. Each injection analysis lasted 10 minutes. These tests were repeated 5 times for each group. pH measurement Each dressing material is placed in glass beakers with 30mL of deionized water. The beakers are held at 37oC for 24 hours. After 24 hours, the pH of the resultant solution is measured by Coleman 28 C Perlin-Elmer metrion IV pH meter. Fluid Absorption As water-holding capacity would not reflect the ability of a dressing to absorb wound exudate, a test solution was used to simulate the density of exudate. The test solution is composed of sodium/calcium chloride containing 142mmol/L of sodium ions and 2.5mmol/L calcium ions—values typical of those found in serum and wound fluid.10 Each dressing material is placed in a glass beaker with 50mL of test solution and held at 37oC for 24 hours. At 2, 4, 24, and 48 hours, the dressing materials are removed from the solution. Excess fluid is allowed to passively drain for 15 minutes, then the dressing material is weighed with sensitive electronic balance (Mark, BEL Engineering electronic balance, Monza, Italy). After each measurement, the material is placed in the same beaker for the other measurements. At each time point, the amount of solution per gram of dressing material is recorded. Statistical Analysis The results of odor adsorption were evaluated by using 2-way analysis of variance (ANOVA). The results of fluid absorption tests were analyzed by 1-way ANOVA. The results of pH measurements were evaluated by Student’s t-test. Results Odor adsorption. The data obtained from the gas chromatograph are shown in Figure 3. There were no differences between groups at 50mL of solution (p>0.05). More diethylamine was released into the closed space by gauze (p0.05). Differences among the other odor-adsorbing dressings were less significant, with the main results being that the 5g α-sepiolite dressing suppressed odor detection at all flow rates up to 40mL compared to all dressings except the 2g α-sepiolite dressing, which was comparable in odor suppression below 13mL flow rates. pH measurement. All dressings had neutral pH except for the 2 activated charcoal dressings, with silver (pH 5) and with fibrous cellulose (pH 8) (Figure 4). Fluid absorption test. Fluid absorption did not change over time for any dressing (p>0.05) (Table 1), but statistically significant differences existed among groups (Figure 5). At all time points, the weight of fluid handled by α-sepiolite was greater than other groups. The fluid absorbed per 1g of α-sepiolite did not differ in Groups 4 and 5 (p>0.05). Group 2 had the least ability to absorb the test solution and was different from all other groups at all time points. Group 3 absorbed the test solution more than Groups 1 and 2 but less than Groups 4 and 5 (Figure 5). Discussion Sepiolite is a natural clay mineral with a formula of magnesium hydrosilicate (Si12)Mg8O30(OH)6(OH2)4.8H2O. Its unique fibrous structure with interior channels (3.6 x 10.6Ao) allows penetration of organic and inorganic ions into the structure of sepiolite and assigns sepiolite an excellent industrial importance in adsorptive, rheologic, and catalytic applications.11 α-sepiolite is a natural product and has been widely used in many areas. In medicine, sepiolite combined with collagen is used as an osseous implantation material and has been found to be biocompatible.11,12 Sepiolite has been used in antacid and antidiarrheal drugs for several years because of its adsorptive properties. Ohta et al.12 reported satisfactory antibacterial affectivity of sepiolite combined with povidone-iodine. The authors did not observe any irritative reactions to a sepiolite preparation containing povidone-iodine when it was applied to a rabbit auricle, ophthalmic mucosa, and human skin.12 Adsorptive properties of α-sepiolite imply the high ability to adsorb the odor and absorb the fluid. Although it is widely used as an odor adsorber in pet litter, α-sepiolite has not been used in medicine to eliminate wound odor. An objective of the present study is to measure the odor adsorbing capacity of α-sepiolite by a quantitative assay procedure. In many reports, odor assessments were made on patients by using subjective criteria, such as the Baker and Haig scales.3,4 These scales classify the odor presence and severity in a patient’s room.4 However, the varying wound dimensions and characteristics of different patients may limit the objective findings of in-vivo studies. To compare the objective findings, the authors preferred to use the standard analytic technique of gas liquid chromatography. By this technique, it is possible to measure the amount of odor released by a particular dressing material to a closed space and compare the results. In the present study, measured values for odor adsorbing capacity of α-sepiolite is found to be higher than simple gauze and activated charcoal cloths, and this capacity is increased with the volume of the material. Diethylamine is the odor molecule present in malodorous wounds that has been used by other scientists;2 however, questions may remain as to the variety of odorous molecules present in the wound fluid from chronic or infected wounds that may interact differently with the tested materials. The mechanism of odor adsorbance of α-sepiolite depends on the silisium-hydroxite groups, which are present on the α-sepiolite fibers. These groups have the ability to create covalent bonding with certain organic reactives. In addition, the amine molecules are reported to cling both in micropores and at the outer surface of α-sepiolite. When heated to 300oC, α-sepiolite loses some of the odor adsorption capacity because of the demolished porous structure.13 The charcoal dressings also have pores that adsorb odor. The charcoal cloth is activated by heating to 800–900oC in the presence of CO2 or steam, which breaks down the carbon surface to form large numbers of pores.14 Both α-sepiolite and activated charcoal trap smaller odor molecules in their micropores and hold the larger molecules on their surfaces, possibly by electrical forces. As previously stated, while heating activates charcoal, heating over 300oC inactivates α-sepiolite. In conventional sterilizers where the heat is approximately 134–135oC, α-sepiolite can be sterilized without any functional loss. Removal of excess exudate is a general performance parameter of an optimal dressing.15 Exotoxins or cell debris in exudate may extend the inflammatory phase and retard healing. Thomas identified that exudate production from leg ulcers averages within a range of 4–12g/10cm2/24 hours in his study.16 These values are similar to those reported by Lamke, who reported the evaporative water loss from burns.17 The balance between humidity and absorption is critical. The activated charcoal dressing has limited or no fluid absorption ability.2 In a study by Mulligan et al., activated charcoal was reported to reduce the amount of exudate; however, this study depended on the investigators’ weekly assessments of wounds of 97 people.6 The fluid absorption capacity of charcoal dressings is improved by adding a polyethylene-coated contact layer.18 Certain varieties of α-sepiolite are known to be able to hold water up to 200–250 times their original weights. In this study, the sepiolite type used absorbed 5 times its weight in test solution. The hydrophilic character depends on the silanol groups of the sepiolite surface, microporous structure, and zeolitic channels.13 The authors’ data support that α-sepiolite absorbed the test solution at a greater rate than the simple gauze sponge and activated charcoal dressings with or without an absorbent layer. Since α-sepiolite forms a gelinatous structure after absorbing liquids, it cannot easily leave the fabric and has no tendency to drop particles. If it soils the wound, it does not show toxic or detrimental effects either systemically or locally and can be removed from the wound easily by washing. It has been shown that α-sepiolite does not have any carcinogenic properties and has been used in medicine, such as antacid drugs and antidiarrheal drugs, for several years.13 Wound exudate and cell debris cause an acidic medium that increases capillary permeability and edema. The acidic pH during the early phases of wound healing shift through neutral to alkaline conditions as wound healing progresses.19 Creating a constant acidic or alkaline pH environment with dressing materials may interfere with the healing process. Additionally, the constant acidic environment decreases or inhibits the activity of some antibiotics, especially systemic or local aminoglycosides. For example, gentamicin20 is 90 times more effective at pH 7.8 than at pH 5.5. Among the tested materials, activated charcoal dressings showed acidic pH, while simple gauze sponge and α-sepiolite maintained neutral pH. Conclusion α-sepiolite is particularly effective for adsorbing odor caused by diethylamine and absorbing exudate under simulated conditions of use. Maintaining a neutral pH, α-sepiolite should contribute favorably to the healing process. A limitation of the present study is that its clinical relevance is questionable, because it is an in-vitro study and may not accurately represent clinical wound fluid or the cellular or biological interactions between α-sepiolite and a real wound. The effects of a-sepiolite on odor adsorption, exudate absorption, or pH in acute or chronic wounds where these effects may be altered by more complex physiologic or biochemical interactions remain to be investigated. Future studies may prove that α-sepiolite incorporated into a dressing material kept in an envelope away from the wound surface can be suitable in malodorous and exudative wounds.