2023 Volume 46 Issue 5 Pages 707-712
Wound-healing deficits of the skin, one of the most common complications in patients with diabetes, delay wound healing, significantly reducing the patient’s QOL. Therefore, the topical treatment of wound areas with drug-containing ointments and dressings is important. In this study, we investigated the effect of various ointment bases on skin wound healing in normal and streptozotocin-induced diabetic rats (STZ rats). Three ointment bases were used: white ointment (oil-based), absorbent cream (emulsion-based, w/o), and macrogol ointment (water-based). Skin wound healing in STZ rats was delayed compared with that in normal rats. Each of the three ointment bases was applied to the skin wound area in normal rats, and there was no difference in the therapeutic effect. The therapeutic effect of both white ointment and absorbent cream was higher in the STZ rats group than that in the non-treated group, and delayed wound healing was observed in STZ rats treated with macrogol ointment. In conclusion, skin wound healing in STZ rats is affected by the properties of the ointment base, and it is important to use an ointment base that controls the drying of the wound area in STZ rats. These findings provide information for the selection of ointment bases useful for application to skin wounds in patients with diabetes.
Diabetes mellitus is a metabolic disease characterized by hyperglycemia and hypoinsulinemia.1,2) The chronic hyperglycemia of diabetes mellitus is followed by damage and dysfunction of the eyes, kidneys, nerves, and blood vessels.2) Wound-healing deficits of the skin, one of the most common complications of diabetes, have a long-term impact on the patient’s QOL.3,4) Complex multiple factors, including a reduction in the level of substance P,5) decreased endothelial nitric oxide synthase activity,6) increased expression of caspase,7) decreased vasculogenesis,8) reduction in the chemotactic and phagocytic activities of neutrophils,9) and prolongation and interruption of the collagen synthesis reaction,10) cause an inflammatory response and cellular proliferation during the skin wound healing process. As a result, delayed re-epithelialization of wounds and impaired migration and proliferation of keratinocytes and fibroblasts result in the formation of chronic wounds.11,12)
Research on developing topical therapies for the treatment of wound-healing deficits using ointments and dressings containing effective drugs is ongoing to improve the prolongation and interruption of these processes. However, not only the drug but also the ointment base plays an important role in the treatment with ointments and other topical agents, and the choice of ointment base is important for effective topical therapy of skin wounds. For example, Higuchi and Kondo et al. reported that the use of volatile/nonvolatile solvent systems incorporating polymer additives significantly improves drug permeability in vitro and in vivo.13,14) Ohtani et al. reported that the skin permeability to the drug in a topical preparation is affected by the ointment base properties.15,16) In addition, the guidelines for the treatment of decubitus state that it is necessary to choose an ointment base that takes into account not only the skin permeability of the drug but also the skin condition, such as the amount of exudate and the dryness of the wound area.17) Several studies have evaluated the therapeutic effects of drug mixtures in ointment bases applied to the wound area of diabetic skin wound rats models18–20); however, little information is available on the effect of ointment base properties on diabetic skin wound healing. Therefore, an investigation of the influence of the ointment base on skin wound healing in a diabetic skin wound model would be useful in choosing an effective ointment base for skin wound healing in patients with diabetes.
The selection of an appropriate diabetic animal model is very important for evaluating the effect of ointment bases on diabetic skin wounds. In streptozotocin-induced diabetic rats (STZ rats), exposure to streptozotocin damages the pancreatic β-cells, resulting in defective insulin secretion, which leads to hyperglycemia.21) This rat model is one of the most widely studied diabetic models used to study diabetic complications, such as retinopathy,22–25) keratopathy,26) cataract,27) nephropathy,21) and skin wounds.4,18–20) In diabetic skin wound modeling STZ rats, cleaved caspase-3 activity, which acts as a major executive caspase of apoptosis, is elevated. Diabetic wounds induce apoptosis and inhibit angiogenesis, delaying wound healing.7,28) In addition, decreased expression of α-smooth muscle actin in fibroblasts impairs wound contraction and decreases collagen production, both of which are necessary for wound healing; thus, wound healing is delayed.29) The results of these studies show that the diabetic skin wound of the STZ rat model behaves similarly to the skin wounds of patients with diabetes; therefore, it is a suitable model for this study. In the present study, we used a white ointment (oil-based), an absorbent cream (emulsion-based, w/o), and a macrogol ointment (water-based) to encompass the three categories of ointment bases available30) and evaluated the effect of these ointment bases on skin wound healing in normal and STZ rats.
White ointment was purchased from Maruishi Pharmaceutical Co., Ltd. (Osaka, Japan). Absorptive cream was obtained from NIKKO Pharmaceutical Co., Ltd. (Gifu, Japan), and macrogol ointment was provided by Yoshida Pharmaceutical Co., Ltd. (Tokyo, Japan). Isoflurane was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All other chemicals used were of the highest commercially available purity.
AnimalsSeven-week-old Wistar rats (male, normal rats) were obtained from Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). The rats were injected with streptozotocin for two days (100 mg/kg/d, intraperitoneally (i.p.)), and housed for one week (STZ rats). All animal experiments were conducted in accordance with the Kindai University Experimental Animal Regulations (Approval Code: KAPS-31-009; April 1, 2019), and handled according to the guideline of Kindai University and the Japanese Pharmacological Society. All surgery was performed under isoflurane inhalation anesthesia, and all efforts were made to minimize suffering. The rats were housed in a temperature-controlled room at 25 °C under a 12-h light/dark cycle (2–3 rats/cage). All rats had free access to commercial diet (CE-2, Clea Japan Inc., Tokyo, Japan) and water, and rat’s health and behavior were observed daily.
Measurement of Blood Glucose and Insulin LevelsFasting blood glucose and insulin levels were measured one week after streptozotocin administration. Blood was collected from the rat tail vein using an anticoagulant-filled capillary tube (Terumo). Immediately after blood collection, centrifugation (9100 × g, 5 min) was performed using an MX-200 (Tomy Seiko Co., Ltd., Tokyo, Japan) to harvest serum. The plasma glucose concentration was calculated using a Glucose Assay Kit (Bio Vision), and the blood insulin concentration was calculated using an ultrasensitive rat insulin measurement kit (Morinaga Co., Ltd., Tokyo, Japan).
Image Analysis of Skin Wound Healing in Rats Treated with Ointment BaseOn the day before the start of the experiment, the hair on the ventromedial area of both the normal and STZ rats was carefully removed using an electric clipper and razor. The following day, the rats were anesthetized by inhalation with 2.0–2.5% isoflurane using BS-400T (Brain Science Idea Co., Ltd., Osaka, Japan), and four wounds in the ventromedial skin of each rat were made by debriding the epidermis and dermis. The epidermis and dermis were debrided by using a biopsy punch (6 mm in diameter), and the area which is more concave than normal skin were recognized as the wound. The skin-debrided rats were allowed to rest for 48 h for hemostasis and stabilization and then used in the following experiments. Each ointment base (white ointment, absorptive cream, and macrogol ointment) was fixed to the ventromedial skin with adhesive and immediately sealed with adhesive tape. The amount of ointment base was set at 0.3 g, which is sufficient to cover the wound according to previous studies.31) Each ointment base was applied to the skin of the rats once daily for 14 d and washed with saline before application. In addition, the wounds were photographed daily, and the wound areas were measured by selecting an area using image J.
Statistical AnalysisStatistical analyses were performed using JMP ver. 5.1 (SAS Institute, NC, U.S.A.). All data are expressed as mean ± standard error (S.E.) and the significance level was set at p < 0.05. For comparison of body weight and plasma glucose and insulin levels between normal and STZ rats was analyzed using Student’s and Aspin–Welch’s t-test. Time-related changes in wound area in normal and STZ rats were compared by Student’s t-test. Time-related changes in wound area in normal and STZ rats with each ointment base were compared by one-way ANOVA followed by Dunnett’s multiple comparisons and two-way (repeated measured) ANOVA.
Figure 1 shows the changes in body weight (A), plasma glucose (B), and insulin levels (C) in normal and STZ rats. There was no significant difference in body weight between normal and STZ rats; however, a decreasing trend was observed in STZ rats. The plasma glucose levels in STZ rats were significantly higher than those in normal rats. Furthermore, the plasma insulin level was significantly lower in STZ rats than that in normal rats. These results confirm that the STZ rats developed diabetes mellitus with hyperglycemia and hypoinsulinemia.
Normal, normal rat. STZ, STZ rat. Means ± S.E., n = 6. * p < 0.05, vs. normal rat for each category (Student’s and Aspin–Welch’s t-test). Hypoinsulinemia and hyperglycemia were observed in STZ rats, and the body weights of STZ rats were lower than that of normal rats.
An inflammatory response (0–3 d after injury) and cellular proliferation (3–12 d after injury) are related to the skin wound healing process, and any prolongation of or interruption of these phases results in a delayed skin wound healing process.10) Figures 2A and B show the images of the skin wounds and the wound areas in normal and STZ rats at 0–12 d post skin debridement. No differences in skin condition, determined by the presence of exudate, were observed in either normal or STZ rats. The skin wounds of the normal rats tended to heal more quickly than those of the STZ rats (Fig. 2A). In addition, in normal rats, the wound area decreased to less than 50% at two days post skin debridement and almost disappeared by six days post skin debridement. On the other hand, STZ rats require four days for the wound area to be less than 50%, and the wound was still present after eight days (Fig. 2B). STZ rats showed delayed skin wound healing compared with normal rats.
Normal, normal rat. STZ, STZ rat. Means ± S.E., n = 6. * p < 0.05, vs. normal rat for each category (Student’s t-test). The skin wound healing levels in STZ rats were lower than that in normal rats.
Figures 3 and 4 show the images (Fig. 3) and wound areas (Fig. 4) during the skin wound healing process in normal rats treated with three types of ointment bases (white ointment, absorptive cream, and macrogol ointment). The skin wound healing levels were similar among the non-treated rats and the rats treated with each ointment based, with wound repair occurring six days after skin debridement. These results demonstrate that the treatment of skin wounds in normal rats was not affected by the type of ointment base.
The skin wound healing levels were similar among the rats treated with white ointment, absorptive cream, and macrogol ointment.
Means ± S.E., n = 6. * p < 0.05, vs. normal for each category (one-way ANOVA followed by Dunnett’s multiple comparisons). No significant difference was observed between the rats treated with white ointment, absorptive cream, or macrogol ointment, with all wounds being repaired six days after the abrasion.
Figures 5 and 6 show the images (Fig. 5) and wound areas (Fig. 6) during the skin wound healing process in STZ rats treated with three types of ointment bases (white ointment, absorptive cream, and macrogol ointment) compared to the same rats without treatment (Control). The rate of skin wound healing in the STZ rats was increased by treatment with white ointment and absorptive cream when compared with the control group. In contrast, the skin wound healing rate in STZ rats treated with macrogol ointment was delayed compared to that of the non-treatment group. In this study, we also analyzed data sets of time-series of wound area when each ointment base is used on normal and STZ rats by using a two-way (repeated measured) ANOVA. As a results, there was a significant difference in wound area behavior between normal and STZ rats when white ointment, absorptive cream, and macrogol ointment were applied, respectively. These p-value in white ointment, absorptive cream, and macrogol ointment were 2.41 × 10−9, 1.49 × 10−18, and 4.35 × 10−22, respectively. The high p-values between normal and STZ rats when white ointment and absorptive cream were applied, showed that the wound healing effect is higher than that of the macrogol ointment.
Skin wound healing of the STZ rats increased with the treatment of white ointment or absorptive cream. In contrast, the skin wound healing in the STZ rats treated with macrogol ointment was delayed in comparison with the non-treatment group.
Means ± S.E., n = 6. * p < 0.05, vs. normal for each category (one-way ANOVA followed by Dunnett’s multiple comparisons). The skin wounds of the STZ rats treated with white ointment and absorptive cream healed after six and eight days, respectively. On the other hand, the skin wound in macrogol ointment-treated STZ rats healed 12 d after the abrasion.
Diabetes mellitus is characterized by hyperglycemia and hypoinsulinemia, which induce the formation of reactive oxygen species, pro-inflammatory cytokines and recruitment of inflammatory cells, in turn degrading components of the extracellular matrix and the growth factors necessary for wound healing.32) As a result, the inflammatory process of healing is prolonged, granulation tissues fail to develop, and eventually chronic wounds form.33) Therefore, the development of topical therapies to improve the delayed wound healing of patients with diabetes is important and ongoing. Several studies have evaluated the therapeutic effects of drug mixtures in ointment bases applied to the wound areas of diabetic skin wound rat models.18–20) However, little information is available on the effects of ointment base properties on diabetic skin wound healing. Therefore, we investigated the influence of the ointment base on wound healing in a rat diabetic skin wound model.
Ointment bases are mainly classified as oil, emulsion, and water-based, which have a variety of properties.30) Oil-based ointment bases have a coating, protective, and softening effect on the skin surface and are used to treat dry skin wounds. Typical oil-based ointment bases include white petrolatum, liquid paraffin, and white ointment.30) The emulsion-based ointment bases consist of emulsions of oil- and water-based components with surfactants and may be further grouped into oil-in-water (o/w) and water-in-oil (w/o) types. These are good for the treatment of dry skin wounds; however, they cannot be used for wet skin wounds because of their emulsifying effect and secretory reabsorption at the wound area. Typical emulsion-based ointment bases include hydrophilic cream, hydrophilic petrolatum, and absorptive cream.30) Water-based ointment bases are recommended for moist skin wounds because they can absorb and remove secretions from the wound area. A typical water-based ointment base is macrogol ointment.30) The following three ointment bases were used in this study; white ointment (oil-based), absorbent cream (emulsion-based, w/o), and macrogol ointment (water-based), to investigate the effects of the ointment bases on the skin wound-healing ratio in the normal and diabetic rat models.
In this study, we established STZ rats by intraperitoneal administration of streptozotocin and then compared the wound-healing process between the normal and STZ rats. First, we investigated the therapeutic effect of the ointment base on skin wound healing in normal rats. The skin wound healing levels were similar between the non-treated rats and the rats treated with each ointment based (Figs. 3, 4). Thus, our findings suggest that the properties of the ointment base do not affect skin wound healing in healthy rats. Next, we investigated the therapeutic effects of the ointment base on skin wound healing in STZ rats. The STZ rats developed diabetes mellitus with hyperglycemia and hypoinsulinemia (Fig. 1), and skin wound healing in STZ rats was delayed in comparison with normal rats (Fig. 2). The present findings in the STZ rats are consistent with previous literature.31) In contrast to the results in normal rats, the skin wound healing level in STZ rats was increased by treatment with white ointment and absorptive cream. In contrast, the skin wound healing level in STZ rats treated with macrogol ointment was delayed compared to that in the non-treatment group (Figs. 5, 6). The skin wound healing process consists of four sequential phases: (1) hemostasis, (2) inflammatory response, (3) cellular proliferation, and (4) remodeling. There is any prolongation or interruption that occur during these phases result in an delayed of the skin wound healing process.10,34–36) In addition, it is known that skin wound healing is delayed in patients with diabetes by the inhibition of cell proliferation, angiogenesis, and collagen production due to hyperglycemia.37,38) Taken together, it was suggested that the skin wound healing process was maintained normally and healed early in normal rats, resulting in no difference regardless of which ointment base was applied. On the other hand, the STZ rats showed delayed skin wound healing process and healing took longer, so the effect of the ointment base may have been significant.
It is important to discuss the mechanism of skin wound healing by the ointment bases. White ointment (oil-based) and absorbent cream (emulsion-based) are recommended for the treatment of dry skin wounds, while macrogol ointment (water-based) is recommended for treat moisture skin wounds.30) The pathology of patients with diabetes is associated with dry skin, such as xeroderma and skin infections, in addition to retinopathy, keratopathy, cataract, and nephropathy.39) Moreover, Sekijima et al. reported that hyperglycemia activates inflammatory cytokines, such as tumor necrosis factor (TNF)-α, and decreases hyaluronic acid production, resulting in reduced skin hydration and a dry skin condition in type 2 diabetic model rats.40) We showed that there was no difference in the presence of exudate in the skin wounds of normal and STZ rats in this study (Fig. 2). The improved wound healing efficiency that occurs due to protecting and moisturizing the wound area is a consequence of maintaining an appropriately moist environment at the wound area, which promotes fibroblast and endothelial cell proliferation and angiogenesis.41,42) This finding suggests that it is important to use an ointment base that controls the drying of the wound area of patients with diabetes; thus, the white ointment and absorbent cream, which are more effective in skin protection and moistening, may be more effective for diabetic skin wound healing than macrogol ointment, which is more effective in absorbing exudate. Further histological studies are needed to clarify the behavior of fibroblasts, endothelial cells, and angiogenesis during ointment base application in diabetic skin wounds.
In conclusion, we demonstrated that skin wound healing in STZ rats was delayed compared to normal rats, and it was affected by the properties of the ointment base.30) Delayed skin wound healing in STZ rats was observed when a water-based ointment base was used for treatment, demonstrating the importance of maintaining an appropriately moist environment in the wound area for diabetic skin wounds. These findings will provide information for the choice of ointment bases for skin wounds in patients with diabetes mellitus.
The authors declare no conflict of interest.
