Novel Xyloglucan Sheet for the Treatment of Deep Wounds: Preparation, Physicochemical Characteristics, and in Vivo Healing Effects

Abstract

In the present study, a novel wound dressing made of xyloglucan (Xyl)–sucrose (Suc) hydrogel was developed for the treatment of deep wounds including pressure ulcers. The dressing was prepared by casing an aqueous solution of Xyl and sugar and then warming, and a hydrogel sheet was obtained. The in vitro characteristics of these sheets, such as their strength, extensibility, water content, adhesion potential, and water absorption, were examined. The strength, Young’s modulus, and adhesion strength of the sheets were greater when they had a lower water content. Furthermore, adhesion and gradual water absorbability were similar to those of commercial dressings. These in vitro features suggest that Xyl sheets possess the physicochemical properties required for wound dressings. In the in vivo experiment, a Xyl sheet made from a mixture of 3.0% (w/v) Xyl solution and 33.3% (w/w) Suc, which displayed moderate strength and water content, was selected and compared with gauze, commercial polyurethane film, and Xyl/Suc (1 : 2) hydrogel using a rat deep wound model caused by serious frostbite. Wound healing rates based on reductions in wound areas were the best in the order of the sheet > hydrogel > commercial film > gauze. The sheet resulted in better wound surface states than the other preparations by improving the conditions. Thus, the potential applicability of Xyl sheets to the treatment of deep wounds was demonstrated.

INTRODUCTION

The number of elderly individuals with pressure ulcers is expected to increase in Japan. Therefore, it is important to develop appropriate methods to treat such deep wounds.¹⁾ Recently, moist healing, including occlusive dressing therapy, is considered the most acceptable. This method can promote wound healing by sealing wounds and keeping wound healing factors.^2–5) Also, patient-friendly wound dressings are required for the enhancement of QOL.^6–8)

Ointments and creams are formulations are frequently used because they can be treated and adjusted in the wound moist environment.^9,10) However, the exchange of these medicines is sometimes accompanied by difficulty in removal and induction of pain.¹¹⁾ A spray type of medicine containing basic fibroblast growth factor (bFGF) sometimes cause side effect of exuberant granulation.¹²⁾

Considering such problems, new types of wound dressings were produced in this study. Namely, xyloglucan (Xyl) hydrogel sheets (Xyl sheets) were developed.^13,14) Xyl is a water-soluble polysaccharide, composed of a main chain of 1,4-β-glucans and side chains of xylose and galactose.^15,16) Xyl is highly safe and stable against heat and pH changes,¹⁷⁾ and can form gels in the presence of sugar or molecules with poly-alcohol.^17–19) Although a lot of gel formulations related to Xyl have been reported before, there are not so many studies for the application of Xyl sheets to wound healing.^{13,14,20–31)} For Xyl-sugar hydrogel sheets, promotion of wound healing by sugar and maintenance of appropriate moisture are expected. In the present study, their preparation, physicochemical characteristics and in vivo efficacy were investigated.

MATERIALS AND METHODS

Materials

Xyl was supplied by DSP Gokyo Food & Chemical Co., Ltd. (Osaka, Japan). Sucrose (Suc) was provided by Kozakai Pharmaceutical Co., Ltd. (Tokyo, Japan). All other chemicals were of reagent grade. Tegaderm™ hydrocolloid (3M Health Care, MN, U.S.A.) and Viewgel^® (Taiho Pharmaceutical Co., Ltd., Tokyo, Japan) were used as commercial products.

Animals

Male Wistar rats (7 weeks old, 200–210 g) were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan), and used in animal studies within one week. Rats were fed using the breeding diet MF (Oriental Yeast, Tokyo, Japan) with water ad libitum at 23 ± 1°C and a relative humidity of 60 ± 5%. The animal experiment protocol was approved by the Committee on Animal Research of Hoshi University (Tokyo, Japan). Animal experiments were executed in compliance with the Guiding Principles for the Care and Use of Laboratory Animals of Hoshi University.

Preparation of Xyl Sheets

Xyl was dissolved in water with a magnetic stirrer for 24 h, leading to a polymer solution of 3, 4.5, and 6% (w/v). Xyl solution (4 g) mixed with Suc (1, 2, or 4 g) was poured into a square plastic box (4 × 4 × 2 cm (in depth)), and the box was placed horizontally on a hot plate warmed at 37°C. The time at which the fluidity of the mixture was lost was evaluated as the sheet formation time.

Measurement of Physical Characteristics

Tensile strength and extensibility were measured using the rheometer FUDOH RHEO METER RTC-2005D·D (Rheotech Co., Ltd., Tokyo, Japan). The sheet was cut into 3 × 4 cm, and the upper side (1.5 cm) and bottom side (1.5 cm) were fixed using clamps. The sheet was pulled at 6 cm/min. Strength immediately before the sheet broke was measured as tensile strength. Extensibility (%) was calculated from the breaking time. Young’s modulus was calculated using the following equation.

  

Adhesion characteristics to the plastic plate were investigated by referring to the 12th edition of the Japanese Pharmacopoeia³²⁾ and Frandol^® tape interview form.³³⁾ The sheet (4 × 4 cm) was fixed to the bottom plate of the rheometer. A disk-shaped bakelite plate (2 cm) fixed to the upper plate of the rheometer was attached to the above sheet with a force of 10 N for 1 min. The lower plate was then pulled down at 2 cm/min. The time at which the sheet separated from the bakelite plate was defined as the adhesion time, and strength immediately before separation was obtained as adhesion strength. Tegaderm™ hydrocolloid and Viewgel^® were tested in the same manner.

Examination of Water Content

Sheets were placed and warmed on a hot plate at 37°C. The water content of the sheets was calculated from change in sheet weight.

Measurement of Water Absorption

The water absorption test was performed according to the method by Nakamura et al.³⁴⁾ A filter membrane (pore size, 0.8 µm) was fixed on the glass filter surface, which was in contact with water. A sheet sample (1 × 1 cm) was placed on the membrane surface. The amount of water absorbed by the sheet was monitored for 5 h. As references, 1.5 g of Tegaderm™ hydrocolloid or Viewgel^® was placed on the membrane surface, and water absorption was measured similarly.

Efficacy Studies in Rat Wound Models

The deep wound model was created according to the method reported by Machida et al.³⁵⁾ Rats were anesthetized by an intraperitoneal (i.p.) injection of pentobarbital at 25 mg/kg (4 mL/kg in saline), and their back hair was shaved. A 1.0-cm circular flap of skin located 1.0 cm from the midline to the right was removed. A brass cylindrical tube with a circular basement (diameter: 1.0 cm) and wall thickness of 0.02 cm was charged dry ice–acetone mixture. This tube was placed on the wound, which was covered with a plastic film, for 3 min using tube weight only. After the frostbite had been produced, the wound was covered with 6 sheets of gauze and protected by surgical adhesive tape. Each injured rat was bred with one animal per cage.

The treatment was performed 24 h after the production of frostbite. A Xyl sheet (3.0% Xyl/33.3% (w/w) Suc) was used in the in vivo test. Previously reported Xyl/Suc hydrogel,³⁵⁾ made by mixing 3.0% Xyl solution (4.0 g) and Suc (4.0 g), was used as Xyl hydrogel. A CATHEREEP^® FS ROLL was used as a polyurethane film. After these preparations were applied to wound surfaces, they were covered with 3 sheets of gauze. Gauze alone (3 sheets) was used as the control. Finally, each was fixed using surgical adhesive tape. Each preparation was changed to new one once every day, when wound surface state and size were observed. Every time changing the preparation, the wound surface was washed with saline and wiped gently with a non-woven wiper. The wound area was determined as follows, and its ratio to the area immediately before treatment was calculated.

  

Statistical Analysis

In the in vivo studies, each dressing was compared with the control (gauze alone) using ANOVA followed by the Dunnett’s post hoc test. The criterion for significant difference was set at p < 0.01.

RESULTS AND DISCUSSION

Formation of Xyl Sheets

Xyl forms a gel under the coexistence of sugar of 40–65% (w/w) in the solution.¹⁷⁾ Therefore, Xyl and Suc were mixed in water, and water was evaporated gradually on the hot plate at 37°C to form the wet sheet. In every formulation, sheets were formed within 4 h (Fig. 1). Therefore, Xyl sheets were produced by warming at 37°C for 6 h in all formulations.

Fig. 1. Film Formation Time after the Addition of Suc

The results are expressed as the mean ± standard deviation (S.D.) (n = 3).

Physical Characteristics of Xyl Sheets

Physical characteristics of Xyl sheets were examined at several concentrations of Xyl and Suc (Table 1). The thickness of the sheet was slightly larger when the concentrations of Suc were higher. At 6 and 24 h, tensile strength was greater when the concentration of Suc was lower. The gelation time became shorter as the concentration of Suc increased. Tensile strength was higher at 24 h than at 6 h in every formulation, suggesting the sheet strength should be greater with the decrease in water content (see next section). Extensibility decreased with time in every formulation, which was due to the loss of water with prolonged warming (24 h). Young’s modulus increased after prolonged warming (24 h) in all formulations.

Table 1. Physical Parameters of Sheets Made of the Different Xyl and Suc Contents 6 and 24 h after Warming Xyl Solution at 37°C

Xyl (%)	Suc (%)	Physical parameter a)
		Tensile strength (MPa)		Extensibility (%)		Young modulus (MPa)		Adhesion strength (Mpa)	Adhesion time (s)
		6 h	24 h	6 h	24 h	6 h	24 h	6 h	6 h
3	20	1.36 ± 0.03	2.51 ± 0.12	300.57 ± 16.61	125.10 ± 3.65	0.45 ± 0.01	2.01 ± 0.04	0.04 ± 0.00	5.25 ± 0.38
	33.3	0.29 ± 0.01	0.55 ± 0.12	140.67 ± 3.06	108.20 ± 7.53	0.21 ± 0.01	0.51 ± 0.08	0.03 ± 0.00	5.60 ± 0.17
	50	0.06 ± 0.01	0.13 ± 0.02	218.07 ± 15.35	125.93 ± 6.56	0.03 ± 0.00	0.10 ± 0.01	0.00 ± 0.00	4.83 ± 0.55
4.5	20	1.33 ± 0.04	3.01 ± 0.27	196.00 ± 50.78	88.07 ± 11.42	0.63 ± 0.13	3.44 ± 0.35	0.02 ± 0.00	4.53 ± 0.24
	33.3	0.39 ± 0.10	1.03 ± 0.21	165.40 ± 0.30	136.57 ± 0.21	0.24 ± 0.06	0.75 ± 0.04	0.01 ± 0.00	4.29 ± 0.16
	50	0.06 ± 0.03	0.20 ± 0.02	209.03 ± 40.34	168.33 ± 10.90	0.03 ± 0.01	0.12 ± 0.01	0.00 ± 0.00	6.35 ± 0.68
6	20	1.22 ± 0.10	3.04 ± 0.45	136.70 ± 27.01	75.78 ± 12.68	0.90 ± 0.01	4.06 ± 0.73	0.01 ± 0.00	3.15 ± 0.57
	33.3	0.60 ± 0.13	1.49 ± 0.10	226.67 ± 41.63	165.50 ± 3.82	0.27 ± 0.02	0.90 ± 0.08	0.01 ± 0.00	3.61 ± 0.79
	50	0.06 ± 0.02	0.16 ± 0.02	195.00 ± 19.50	170.87 ± 34.78	0.03 ± 0.01	0.10 ± 0.01	0.00 ± 0.00	6.41 ± 2.90

a) The results are expressed as the mean ± S.D. (n = 3).

Adhesion strength increased as the concentration of Suc decreased (Table 1). A lower concentration of Xyl slightly gave stronger adhesion. The adhesion time was not markedly affected by changes in the Suc concentration.

The adhesion properties of the 3% Xyl/33.3% Suc sheet were compared with those of Tegaderm™ hydrocolloid and Viewgel^® (Fig. 2). The adhesion strength of the Xyl sheet was significantly greater than those of the commercial dressings. On the other hand, Tegaderm™ hydrocolloid showed the longest adhesion time, which might be because Tegaderm™ hydrocolloid has an adhesion layer. Shorter adhesion time is considered better in removal from wound surface. In addition, the high adhesion strength may be useful to fixation to the application site. The adhesion features of the Xyl sheet were similar to those of commercial formulations, suggesting that the sheet would be acceptable for handling.^36,37)

Fig. 2. Adhesion Strength and Adhesion Time of Xyl Sheet (3% Xyl/33.3% Suc), Tegaderm™ Hydrocolloid and Viewgel^®

The results are expressed as the mean ± S.D. (n = 3).

Water Content of Xyl Sheets

The water contents of Xyl sheets are shown in Fig. 3. In every Xyl concentration, the water content increased as the concentration of Suc became higher. Even after warming for 24 h, the water content was greater at higher Suc concentrations. This might be due to the water uptake by the formation of hydrogen bond networks with Suc. The commercially available dosage forms, Reflap^® ointment (water-in-oil emulsion base) have similar water contents.³⁸⁾ Therefore, Xyl sheets may give wet condition adequately.

Fig. 3. Water Contents of Sheets Made of the Different Xyl and Suc Contents 6 and 24 h after Warming Xyl Solution at 37°C

The results are expressed as the mean ± S.D. (n = 3).

Water Absorption by Xyl Sheets

Water absorption, caused because of the compensation for water loss by warming, progressed gradually. The water absorbed at 5 h was 0.5–0.7 mL at the conditions of 20% Suc and 33.3% Suc, but at a Suc concentration of 50%, the water of 0.3–0.5 mL was absorbed at 5 h. Xyl sheets of high Suc concentration might tend to show less water absorption. The water absorption profiles were compared among the 3% Xyl/33.3% Suc sheet, Tegaderm™ hydrocolloid and Viewgel^® (Fig. 4). The Xyl sheet showed a water absorption profile with a very slight deviation. The absorption rate was similar to that of Tegaderm™ hydrocolloid, whereas Viewgel^® had a slower absorption rate. The Xyl sheet showed similar water absorbability to the commercial dressings.

Fig. 4. Water Absorption Profiles of Different Types of Wound Dressings

Data are Xyl sheet of 3% Xyl/33.3% Suc (◆), Tegaderm™ hydrocolloid (□), and Viewgel^® (▲). The results are expressed as the mean ± S.D. (n = 3).

Efficacy in the Severe Wound Model

Changes in wound areas are shown in Fig. 5. After 7 d, the decrease was significantly greater with Xyl sheet than with gauze (p < 0.01). On day 15, the wound area almost completely disappeared. For the Xyl hydrogel, the wound area decreased faster at the later period. After 9 d, the wound surface area with the Xyl hydrogel was significantly smaller than that with gauze (p < 0.01). Gauze and the polyurethane film removed exudates poorly, while the Xyl sheet and Xyl hydrogel effectively removed exudates, resulting in good healing effects. The Xyl sheet appeared to show higher absorption of exudate than the Xyl hydrogel, which would be related to water absorption characteristics of the Xyl sheet. This point might be the difference in effect between sheet and hydrogel.

Fig. 5. Changes in Wound Surface Area after Application of Different Preparations in Rats with Deep Wound

The results are expressed as the mean ± standard error (S.E.) (n = 4).

Although the wound states were only observed visually, the Xyl sheet tended to suppress wound severity as shown in Fig. 6. In this efficacy studies, the Xyl sheet was softened obviously, which might be partly due to the absorption of the exudate. Suc was considered to accelerate the granulation of the wound surface from its biological function.³⁹⁾ From these results, the Xyl sheet was proposed to be superior for wound healing.

Fig. 6. Wound States during the Treatment Using Different Preparations

One interval of the scale = 1 mm.

CONCLUSION

Xyl sheets could be produced using a simple casting method. The sheets had appropriate physical features for handling, and exhibited water absorption and retention similar to those of commercial wound dressings. The Xyl sheet exerted good healing effects in a rat severe wound model. The Xyl sheets are suggested to be useful as a novel wound dressing. However, its safety must be confirmed for practical use. Furthermore, the Xyl sheet with antibacterial agents will be needed for more suppression of wound infection. Those should be studied in more detail in the future.

Acknowledgments

This study was supported in part by a research grant from DSP Gokyo Food & Chemical Co., Ltd. The authors wish to thank DSP Gokyo Food & Chemical Co., Ltd.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Arroyo AA, Casanova PL, Soriano JV, Torra i Bou JE. Open-label clinical trial comparing the clinical and economic effectiveness of using a polyurethane film surgical dressing with gauze surgical dressings in the care of post-operative surgical wounds. Int. Wound J., 12, 285–292 (2015).
• 2) Winter GD. Effect of air exposure and occlusion on experimental human skin wounds. Nature, 200, 378–379 (1963).
• 3) Yamamoto N, Kiyosawa T. Histological effects of occlusive dressing on healing of incisional skin wounds. Int. Wound J., 11, 616–621 (2014).
• 4) Dyson M, Young SR, Hart J, Lynch JA, Lang S. Comparison of the effects of moist and dry conditions on the process of angiogenesis during dermal repair. J. Invest. Dermatol., 99, 729–733 (1992).
• 5) Wood RA, Hughes LE. Silicone foam sponge for pilonidal sinus: a new technique for dressing open granulating wounds. BMJ, 4, 131–133 (1975).
• 6) Kim JH, Jin SG, Ku SK, Nam DH, Sohn YT, Ryu DS, Do E-S, Jang SW, Son MW, Yong CS, Choi H-G, Kim JO. Wound healing evaluation of benzalkonium chloride-loaded hydrocolloid in the wound infection model. J. Pharm. Investig., 42, 327–333 (2012).
• 7) Sütterlin S, Tano E, Bergsten A, Tallberg AB, Melhus A. Effects of silver-based wound dressings on the bacterial flora in chronic leg ulcers and its susceptibility in vitro to silver. Acta Derm. Venereol., 92, 34–39 (2012).
• 8) Boonkaew B, Kempf M, Kimble R, Supaphol P, Cuttle L. Antimicrobial efficacy of a novel silver hydrogel dressing compared to two common silver burn wound dressings: Acticoat™ and PolyMem Silver^®. Burns, 40, 89–96 (2014).
• 9) The Japanese Society of Pressure Ulcers Guideline Revision Committee. JSPU guidelines for the prevention and management of pressure ulcers (4th ed.) Japanese Journal of Pressure Ulcers, 18, 455–544 (2016).
• 10) Noda Y, Watanabe K, Sanagawa A, Sobajima Y, Fujii S. Physicochemical properties of macrogol ointment and emulsion ointment blend developed for regulation of water absorption. Int. J. Pharm., 419, 131–136 (2011).
• 11) Sharp NE, Aguayo P, Marx DJ, Polak EE, Rash DE, Peter SD, Ostlie DJ, Juang D. Nursing preference of topical silver sulfadiazine versus collagenase ointment for treatment of partial thickness burns in children. J. Trauma Nurs., 21, 253–257 (2014).
• 12) Miyahara M. Effective method for the use of b-FGF spray in pressure ulcer treatment. Jpn J. P.U., 5, 48–51 (2003)
• 13) Sakamoto M, Morimoto N, Ogino S, Jinno C, Taira T, Suzuki S. Efficacy of gelatin gel sheets in sustaining the release of basic fibroblast growth factor for murine skin defects. J. Surg. Res., 201, 378–387 (2016).
• 14) Wang C, Zhu F, Cui Y, Ren H, Xie Y, Li A, Ji L, Qu X, Qiu D, Yang Z. An easy-to-use wound dressing gelatin-bioactive nanoparticle gel and its preliminary in vivo study. J. Mater. Sci. Mater. Med., 28, 10 (2017).
• 15) York WS, van Halbeek H, Darvill AG, Albersheim P. Structural analysis of xyloglucan oligosaccharides by 1H-n.m.r; spectroscopy and fast-atom-bombardment mass spectrometry. Carbohydr. Res., 200, 9–31 (1990).
• 16) Gidley MJ, Lillford PJ, Rowlands DW, Lang P, Dentini M, Crescenzi V, Edwards M, Fanutti C, Grant Reid JS. Structure and solution properties of tamarind-seed polysaccharide. Carbohydr. Res., 214, 299–314 (1991).
• 17) Phillips GO, Williams PA. Handbook of hydrocolloids (2nd ed.), Woodhead Publishing, Cambridge (2009).
• 18) Yamanaka S, Yuguchi Y, Urakawa H, Kajiwara K, Shirakawa M, Yamatoya K. Gelation of tamarind seed polysaccharide xyloglucan in the presence of ethanol. Food Hydrocoll., 14, 125–128 (2000).
• 19) Shirakawa M, Yamatoya K, Nishinari K. Tailoring of xyloglucan properties using an enzyme. Food Hydrocoll., 12, 25–28 (1998).
• 20) Suisha F, Kawasaki N, Miyazaki S, Shirakawa M, Yamatoya K, Sasaki M, Attwood D. Xyloglucan gels as sustained release vehicles for the intraperitoneal administration of mitomycin C. Int. J. Pharm., 172, 27–32 (1998).
• 21) Tanaka A, Nagate T, Matsuda H. Acceleration of wound healing by gelatin film dressings with epidermal growth factor. J. Vet. Med. Sci., 67, 909–913 (2005).
• 22) Kanokpanont S, Damrongsakkul S, Ratanavaraporn J, Aramwit P. An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing. Int. J. Pharm., 436, 141–153 (2012).
• 23) Shigeyama M, Ohgaya T, Takeuchi H, Hino T, Kawashima Y. Formulation design of ointment base suitable for healing of lesions in treatment of bedsores. Chem. Pharm. Bull., 49, 129–133 (2001).
• 24) Miyazaki S, Takahashi A, Itoh K, Ishitani M, Dairaku M, Togashi M, Mikami R, Attwood D. Preparation and evaluation of gel formulations for oral sustained delivery to dysphagic patients. Drug Dev. Ind. Pharm., 35, 780–787 (2009).
• 25) Burgalassi S, Raimondi L, Pirisino R, Banchelli G, Boldrini E, Saettone MF. Effect of xyloglucan (tamarind seed polysaccharide) on conjunctival cell adhesion to laminin and on corneal epithelium wound healing. Eur. J. Ophthalmol., 10, 71–76 (2000).
• 26) Miyazaki S, Suisha F, Kawasaki N, Shirakawa M, Yamatoya K, Attwood D. Thermally reversible xyloglucan gels as vehicles for rectal drug delivery. J. Control. Release, 56, 75–83 (1998).
• 27) Shailaja T, Latha K. Sasib hushan P, Alkabab AM, Uhumwangho M U. A novel bioadhesive polymer: grafting of tamarind seed polysaccharide and evaluation of its use in buccal delivery of metoprolol succinate. Der Pharmacia Lettre, 4, 487–508 (2012).
• 28) Mahajan HS, Tyagi V, Lohiya G, Nerkar P. Thermally reversible xyloglucan gels as vehicles for nasal drug delivery. Drug Deliv., 19, 270–276 (2012).
• 29) Patil BS, Mahajan HS, Surana SJ. Development of suppositories containing flutamide-loaded alginate-tamarind microparticles for rectal administration: in vitro and in vivo studies. Chem. Pharm. Bull., 63, 851–857 (2015).
• 30) Ajovalasit A, Sabatino MA, Todaro S, Alessi S, Giacomazza D, Picone P, Di Carlo M, Dispenza C. Xyloglucan-based hydrogel film for wound dressing: Structure–property relationships. Carbohydr. Polym., 179, 262–272 (2018).
• 31) Picone P, Sabatino MA, Ajovalasit A, Giacomazza D, Dispenza C, Di Carlo M. Biocompatibility, hemocompatibility and antimicrobial properties of xyloglucan-based hydrogel film for wound healing application. Int. J. Biol. Macromol., 121, 784–795 (2019).
• 32) Society of Japanese Pharmacopoeia. Adhesive Plaster, The 12th edition of the Japanese Pharmacopoeia (1992).
• 33) Japanese Society of Hospital Pharmacists, Frandol tape interview form (2014).
• 34) Nakamura F, Ohta R, Machida Y, Nagai T. In vitro and in vivo nasal mucoadhesion of some water-soluble polymers. Int. J. Pharm., 134, 173–181 (1996).
• 35) Machida Y, Yamamoto N, Furuta K, Takahashi Y, Iwata M, Shirotake S, Onishi H. Preparation of a novel patch for the treatment of deep wounds and evaluation of the therapeutic effect on rats. Yakuzaigaku, 57, 57–63 (1997).
• 36) Klode J, Schöttler L, Stoffels I, Körber A, Schadendorf D, Dissemond J. Investigation of adhesion of modern wound dressings: a comparative analysis of 56 different wound dressings. J. Eur. Acad. Dermatol. Venereol., 25, 933–939 (2011).
• 37) Tustanowski J. Effect of dressing choice on outcomes after hip and knee arthroplasty: a literature review. J. Wound Care, 18, 449–450, 452, 454 passim (2009).
• 38) Furuta K. Selection and technical aspects of topical drugs. Jpn. J. P.U., 11, 92–100 (2009).
• 39) Kowa Co., Ltd., Package insert: UPASTA KOWA OINTMENT·oint. 8g pack (2013).