Bioshell calcium oxide (BiSCaO) for cleansing and healing Pseudomonas aeruginosa –infected wounds in hairless rats

2.1. Preparations of BiSCaO suspension and HClO solution

Thoroughly cleansed scallop shell was heated at 1450 °C for 2–6 h, and cooled naturally. The powders were pulverized by Nano Jetmizer (NJ-300-D, Aishin Nano Technologies Co. Ltd., Saitama, Japan) to produce BiSCaO with about 6 μm in diameter [20,33]. The contents of CaO and Ca(OH)₂ in BiSCaO determined using X-ray diffractometer system (Phillips X’Pert-PRO; Phillips Japan, Ltd., Japan) were 99.6 % and 0.2 %, respectively. BiSCaO (1 g) was added to 1 L of pure water and centrifuged to prepare 1000 ppm (0.1 w%) of BiSCaO suspension (pH 12.2), which was used within 1 hour after the preparation.

HClO solution (500 ppm, pH 6.5) was prepared by adding 1/10 (vol/vol) of 0.5% NaClO (Yoshida Pharmaceutical Corp., Tokyo, Japan) to sterilized pure water. The pH of 200 ppm HClO then was adjusted to pH 6.5 with 1 N HCl. The concentration of HClO was measured as residual chlorides or free available chloride using ClO (HClO and ClO⁻)-selective test papers (high concentrations, 25–500 ppm; low concentrations, 1–25 ppm; Kyoritu Check Lab. Corp., Tokyo, Japan) [24].

2.2. Bactericidal activity of BiSCaO suspension, HClO, and povidone iodine solutions in vitro

P. aeruginosa (American Type Culture Collection 27853, Manassas, USA) colonies were stored at −80 °C in Luria–Bertani broth containing 50% sterile glycerol and were freshly grown at a density of 1.0 × 10⁶ colony forming units (CFU)/mL. Various concentrations of BiSCaO suspension, HClO, and providone iodine solutions (Isodine solution^®) were added into the P. aeruginosa suspension, each of which was incubated for 15 minutes at room temperature. Each P. aeruginosa suspension was plated onto 90 × 15 mm petri plates of Pseudomonas Isolation Agar (Neogen Ltd., Michigan, USA) and incubated at 37 °C for 24 hours. After incubation, the generated colonies were counted, and the disinfectants were evaluated for in vitro bactericidal activity against P. aeruginosa.

2.3. Cleansing P. aeruginosa–infected wounds with BiSCaO suspension, HClO, and povidone iodine solutions in vivo

All animal experiments were approved by the National Defense Medical College, Tokorozawa, Saitama, Japan and were carried out following the relevant guidelines for animal experimentation. Hairless rats (male, 300–350 g) were obtained from Japan SLC, Inc. (Shizuoka, Japan) and were maintained under appropriate conditions (i.e., 26 °C, 55% humidity). On the nominal day 0 of the study, the rats were placed under general anesthesia by intraperitoneal injection of pentobarbital sodium (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan). Full-thickness round wounds were created on the back of each rat using a sterile 8 mm dermal punch (Kai Industries Co., Ltd., Oyana, Japan) and a pair of sterilized sharp scissors. To generate infected wounds, 100 μL of freshly growing P. aeruginosa colonies was applied to the surface of each freshly generated wound, and each wound was covered first with a piece of chitin nanofiber sheets (CNFS), which had approximately 30% degree of deacetylation and were obtained as a commercial product (BeschitinW, Nipro Corp., Osaka, Japan). The animals were returned to their cages and 24 hours later, each infected wound was formed. P. aeruginosa–infected wounds on the hairless rats were cleansed once daily by gentle rubbing for 5 times using gauze dipped in 3 mL of either BiSCaO suspension, HClO, povidone iodine, or saline (total 15 mL), followed by covering with CNFS for the first 3 days. On days 4–9, all wounds in the 4 groups were cleansed with saline and covered with CNFS daily. The infected wounds in the non-treatment group were only covered with CNFS without cleansing for 9 days of the experimental period. After cleansing the wounds on days 1, 2, 3, 6, and 9, the bioburden was collected from each infected wound by wiping with a strip of sterile 1 cm² gauze. In the non-treatment group, the viable cells were counted after removal of the CNFS. The resulting cell suspension was subjected to 10-fold serial dilutions, and 100 μL samples of the diluted suspensions were plated onto 90 mm × 15 mm petri plates of Pseudomonas Isolation Agar. The plates were then incubated at 37 °C for 24 hours; the viable cells that were confirmed to harbor P. aeruginosa by observation of the morphology were counted [26]. Furthermore, digital photographs were recorded on days 0, 1, 2, 3, 6, and 9; these images were used to measure the rate of wound closure and to confirm the absence of complications, including acute inflammation, abscess formation, and seroma accumulation.

2.4. Histological examination

Following the post-cleansing collection of wound contents on day 9, the animals were euthanized by pentobarbital sodium. On day 9 after wound creation, the skin surrounding each infected wound, including wound tissue, was removed from each mouse (N = 6) for histological examination. The skin samples from each treatment group were fixed in 10% formaldehyde solution, embedded in paraffin, and sectioned in 4 μm increments (Yamato Kohki Inc., Asaka, Saitama, Japan). The 10 × 1.5 mm sections were made perpendicular to the anterior-posterior axis and to the surface of the wound and were transferred to glass slides for staining with hematoxylin–eosin (H&E) reagent. After placement of the cover slip, the tissues were evaluated microscopically. In each section (N = 8), the microscopic field showing the wound was photographed; the number of capillary lumens that measured ≥10 μm in diameter or contained ≥5 erythrocytes were microscopically counted in the microphotograph.

2.5. Statistical analyses

Results were expressed as mean ± SDs. Tukey’s test was used to compare the disinfecting solutions. The statistical software JMP^® (SAS Institute Inc., Tokyo, Japan) was used for the analyses. A value ofp < 0.05 was considered to be statistically significant.

3.1. Bactericidal activities of BiSCaO suspension, HClO, and povidone iodine solutions in vitro

The in vitro bactericidal activities of different concentrations of BiSCaO suspension, HClO, and povidone iodine solution against P. aeruginosa were tested by counting the viable bacterial colonies after treatment. The colonies of P. aeruginosa were completely eradicated after exposure to disinfectants that contained ≥1000 ppm of BiSCaO, HClO, and povidone iodine. In addition, the 250 ppm concentrations of BiSCaO and HClO significantly decreased the cell number in log 10 CFU/mL in a concentration-dependent manner (Fig. 1).

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Fig. 1.

In vitro bactericidal activity of each disinfectant. Pseudomonas aeruginosa was exposed to each diluted solution of BiSCaO suspension, HClO, and povidone iodine for 15 min. To measure the minimal bactericidal concentration of each disinfectant, the bacteria were counted as colonies (N = 6).

All animals tolerated the creation of P. aeruginosa-infected wounds and the daily wound cleansing without any complications. No signs of acute inflammation, abscess formation, or seroma accumulation were seen on the infected wound sites until the 9th day after surgery and infection.

On day 1 before cleansing, more than 1.0 × 10⁵ CFU of P. aeruginosa were detected from each infected wound. On day 1 after wound cleansing with BiSCaO, HClO, povidone iodine, and saline, the mean viable cell counts were 1.8 × 10³, 9.8 × 10³, 1 × 10⁴, and 9.5 × 10⁴ CFU, respectively. The results showed that the bioburden effectively decreased by cleansing with BiSCaO starting from the first cleansing, but slightly increased in the no cleansing group. On Day 3, the mean viable cell counts in the BiSCaO, HClO, povidone iodine, and saline groups were 80, 8.5 × 10³, 9 × 10³, and 4.5 × 10⁴ CFU, respectively. On Day 6, the P. aeruginosa colonies in the BiSCaO group were completely eradicated, whereas the mean viable cell counts that survived in the HClO, povidone iodine, and saline groups were 6.8 × 10³, 6.8 × 10³, and 1.7 × 10³ CFU, respectively; there were no significant differences in the mean viable cell counts among the 3 groups. On day 9, the P. aeruginosa colonies in the HClO, povidone iodine, and saline groups were completely eradicated, whereas the mean viable cell count in no cleansing group was 8 × 10⁴ CFU (Fig. 2).

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Fig. 2.

Removal of P. aeruginosa colonies from each wound treated with disinfectant in vivo. The P. aeruginosa–infected wounds on hairless rats were cleansed once daily by gentle rubbing with gauze impregnated with each disinfectant or saline on days 1–3 and were covered with CNFS. The wounds that were cleansed with saline once daily on days 4–9 were covered with CNFS. Before cleansing the wounds on the indicated day after wound creation, the wound contents were collected and the viable cell counts were measured. Data are presented as mean ± SD (N = 7).

Examination of the digital photographs showed the absence of wound closure in all groups and no significant differences in the wound closure rates on days 1 and 2 among the groups (Fig. 3). On days 3–9, the wounds in the BiSCaO group exhibited significant stimulation of wound closure, compared to the other 4 groups. On day 3, the percentage of open wound in the BiSCaO, HClO, povidone iodine, saline, and no cleansing groups were 68, 95, 93, 98, and 105%, respectively.

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Fig. 3.

Digital photographs of the open areas of the infected wounds. The P. aeruginosa-infected wounds on hairless rats were cleansed once daily with each disinfectant or saline and were covered with CNFS. The wounds that were cleansed with saline on days 4–9 were covered with CNFS. After the wound creation, the wounds were photographed before cleansing on days 0, 1, 2, 3, 6, and 9 to allow assessment of the rate of open wound. The images represent the wounds (N = 7) of the indicated treatment group on the indicated day.

Histological examinations revealed that on day 9, granulation tissue formation in the wounds was significantly lower in the no cleansing group (<23%) than in the BiSCaO, HClO, povidone iodine, and saline groups (Fig. 4). Furthermore, granulation tissue formation on day 9 was significantly higher in the BiSCaO and HClO groups than in the other groups (Table1).

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Fig. 4.

Ratio of open wounds. The area of the open wounds on day 1 was defined as 100%, and the relative open wound in each group was calculated using the digital photographs in Fig. 3. Data are presented as mean ± SD (N = 7). *p < 0.05; Tukey’s t-test.

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Table 1

Histological examinations of the length of granulation tissue and capillary formation

Disinfectant	Length of granulation tissue	Capillary formation
	(μm)	(capillary number/image)
No cleansing	920 ± 95	7 ± 5
Saline	1010 ± 160	18 ± 5
Providone-iodine	930 ± 70	8 ± 3
HClO	1030 ± 50**	20 ± 6**
BiSCaO	1050 ± 50*,**	23 ± 6*,**

The data represent the mean ± SD. In length of granulation tissue, *P < 0.05 vs. saline, **P < 0.01 vs. no cleansing (n = 7), in capillary formation, *P < 0.05 vs. saline, **P < 0.01 vs. no cleansing and providone-iodine (n = 7).

Examination of vascularization in the microphotographs showed that capillary formation on day 9 was significantly higher in the BiSCaO and HClO groups than in the other groups (Table1, Fig. 5).

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Fig. 5.

Histological examination of granulation tissue formation in each treatment group. The skin surrounding the infected wounds in each treatment group on day 9 was harvested, processed for H&E staining, and microphotographed (N = 7) (magnification: ×100). The bars show the generated granulation tissues and the arrows show the blood vessels. The microphotographs represent the wounds for the indicated treatment group. These microphotographs were used for Table1.

4. Discussion and conclusion

In the clinical settings, infection of wounds with P. aeruginosa is a major complication. In this study, in vitro bactericidal test against P. aeruginosa showed complete eradication with 100 ppm BiSCaO, HClO, and 1000 ppm povidone iodine (Fig. 1). To evaluate bactericidal activity and wound healing in vivo, P. aeruginosa-infected wounds on hairless rats were cleansed with BiSCaO suspension (1000 ppm, pH 12.2); HClO solution (500 ppm, pH 6.5); and povidone iodine (1000 ppm) and were covered with CNFS for 3 days, followed by cleansing with saline and covering with CNFS daily for 6 days. The results suggested that BiSCaO treatment significantly enhanced disinfection and wound healing in vivo, in comparison to the effects of HClO, povidone iodine, and saline. The histological examinations showed significantly advanced formations of granulation tissue and capillaries with 3 days of BiSCaO treatment. Furthermore, treatment with BiSCaO, HClO, and povidone iodine for 3 days did not show signs of complications in the animals that harbored the wounds; this absence of complications was confirmed on histological analysis of wound skin harvested on day 9. The results suggested that limiting disinfection to 3 days with BiSCaO treatment may be appropriate in a clinical situation, such as in the prevention and treatment of infection in chronic wounds of healing-impaired patients.

The antiseptics, such as povidone iodine and HClO, which are used in the clinical setting, were shown to be cytotoxic to the cellular components of wound healing and are required in higher concentrations to have disinfectant activity [7–9]. Therefore, a topical disinfectant that can decrease the bacterial bioburden of chronic wounds without inhibiting the wound healing process is a therapeutic imperative. Although both BiSCaO and commercially available SSP-Ca(OH)₂ were poorly water-soluble at strong alkaline (pH ≧ 11.5), BiSCaO and SSP-Ca(OH)₂ suspensions in water generates a strong base and is the primary mechanism for their microbicidal activities. The high disinfection activity of suspensions of BiSCaO and SSP-Ca(OH)₂ particles might be caused by the higher OH⁻ concentration of the thin water layer of their particles than that in the bulk solvent [20,33]. On the other hand, we found that the disinfection activity of BiSCaO for both TC and CF was higher than that of SSP-Ca(OH)₂ at identical pH [20,33]. This suggested that BiSCaO has another mechanism for its high disinfection activity. In fact, several researches reported that heated shell powders composed of mainly CaO had relatively higher disinfection activity by deactivation and removal of biofilms of Escherichia coli [34] and Listeria species [19]. We hypothesized that a higher concentration of the OH⁻ group might cause damage to the bacterial cells in the thin aqueous surface layer of BisCaO particles and remove and kill various bacterial cells, when BiSCaO particles in the suspension come in contact with the contaminated surfaces of skin wound. Furthermore, heated SSP composed of mainly CaO but not Ca(OH)₂, that is BiSCaO, had been reported to generate reactive hydroxyl radical species, which may also contribute to a stronger disinfection activity than that of SSP-Ca(OH)₂ [19,34].

In comparison to the basic NaClO solution, weakly acidic HClO solution at lower concentrations (50–200 ppm) has been reported to have excellent in vitro bactericidal properties against Gram-positive organisms, such as Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis, and Gram-negative bacteria, such as P. aeruginosa [35,36]. However, HClO had been reported to react readily with various NH₂- or CHO-containing organic compounds (e.g., proteins, amino acids, and carbohydrates), which can result in rapid consumption of HClO molecules in the area of the infected wound [24,37]. One study demonstrated that HClO interacts with primary amino groups (–NH₂) in organic compounds, such as amino acids, thereby, generating chloramine groups (–NH₂Cl or –NHCl₂) that are known to have oxidizing properties and antimicrobial activity [38].

BiSCaO and HClO have not been approved by the Pharmaceuticals and Medical Devices Agency of Japan for the use as a pharmaceutical or medical device, despite the approval of NaClO for such purpose. Additional systemic studies on BiSCaO and HClO are required to establish their efficacy, safety, and stability for medical use.

In conclusion, the treatment of P. aeruginosa-infected wounds on hairless rats by cleansing them once daily with BiSCaO suspension (1000 ppm, pH 12.2) for 3 days and covering them with CNFS resulted in a significantly decreased P. aeruginosa bioburden and enhanced wound repair.

Footnotes

Acknowledgements

The authors would like to thank Forte Science Communication () for the English language review.

Conflict of interest

The authors have no conflict of interest to report.

Funding

This study was partially supported by the Ministry of Education, Culture, Sports, Science and Technology of the Government of Japan (grant no. 17K19861).

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