A method for harvesting viable cells from wound dressings.

Wound fluid has been well studied for exploring protein biomarkers contained in it. However, cells in wound fluid have not received much attention due to the difficulty in their collection. Our study aimed to establish a method for collecting viable cells from discarded wound dressings. A protocol was designed to wash out nonadherent cells and detach adherent cells from silicone-faced foam wound dressings using trypsin-EDTA. The optimal concentration and incubation time of trypsin-EDTA for collecting equivalent proportions of different cell types to the original cell population were determined in vitro. Cell composition and gene expression changes in monocytes, lymphocytes, neutrophils, fibroblasts and keratinocytes were confirmed using immunocytochemistry and RNA-sequencing ex vivo. Full-thickness wounds were created on 9-week-old male C57BL/6J mice. Wound fluid was collected, and half of it was applied to the wound dressings. The original cell population in the wound fluid and the cell population collected from wound dressings were compared. In the in vitro study, 0.25% trypsin-EDTA and 2.5-min incubation time were considered optimal for collecting adherent cells from wound dressings. In the ex vivo study, among all cell types, only CD3+ lymphocytes showed a significantly higher cell proportion in the collected group. The relative gene expression of the five selected cells showed no significant changes (p-value >0.05, |log2 fold change| < 1.5, differential gene expression analysis). Viable nonadherent and adherent cells were collected from wound dressings without altering gene expression and could be used in future studies for cellular analysis of wound fluid.

Relationship between healing status and microbial dissimilarity in wound and peri-wound skin in pressure injuries.

AIM: Wound infection is the most serious cause of delayed healing for patients with pressure injuries. The wound microbiota, which plays a crucial role in delayed healing, forms by bacterial dissemination from the peri-wound skin. To manage the bioburden, wound and peri-wound skin care has been implemented; however, how the microbiota at these sites contribute to delayed healing is unclear. Therefore, we investigated the relationship between healing status and microbial dissimilarity in wound and peri-wound skin. METHODS: A prospective cohort study was conducted at a long-term care hospital. The outcome was healing status assessed using the DESIGN-R® tool, a wound assessment tool to monitor the wound healing process. Bacterial DNA was extracted from the wound and peri-wound swabs, and microbiota composition was analyzed using 16S rRNA gene analysis. To evaluate microbial similarity, the weighted UniFrac dissimilarity index between wound and peri-wound microbiota was calculated. RESULTS: Twenty-two pressure injuries (7 deep and 15 superficial wounds) were included in the study. For deep wounds, the predominant bacteria in wound and peri-wound skin were the same in the healing wounds, whereas they were different in all cases of hard-to-heal wounds. Analysis based on the weighted UniFrac dissimilarity index, there was no significant difference for healing wounds (p = 0.639), while a significant difference was found for hard-to-heal wounds (p = 0.047). CONCLUSIONS: Delayed healing is possibly associated with formation of wound microbiota that is different in composition from that of the skin commensal microbiota. This study provides a new perspective for assessing wound bioburden.

An in vivo critically colonised wound model with dysbiotic wound microbiota.

In critically colonised wounds, many of the signs of infection are often absent, and delayed healing may be the only clinical sign. The prevention of critical colonisation is important, but its pathophysiology has not yet been elucidated. We have previously reported that dysbiotic microbiota dissimilar to the peri-wound skin microbiota may develop in critically colonised wounds. To investigate the role of dysbiotic microbiota, this study aimed to develop a critically colonised wound model by transplantation of dysbiotic microbiota. To transplant microbiota, a bacterial solution (dysbiosis group) or with Luria-Bertani medium (commensal group) was inoculated to full-thickness wounds of rats. The bacterial solution was prepared by anaerobically culturing bacteria from donor rats on an artificial dermis in Luria-Bertani medium for 72 hours. As a result, the degree of the change in the microbial similarity between pre- and post-transplantation of microbiota was significantly higher in the dysbiosis group (P < .001). No signs of infection were observed in any rat in either group. The wound area in the dysbiosis group was significantly larger (P < .001), and there was a significant infiltration of neutrophils (P < .001). All rats of the dysbiosis group represented the clinical features of critically colonised wounds. Furthermore, there were significantly fewer regulatory T cells in the wounds of the dysbiosis group. This is the first study to develop a novel animal model that represents the clinical features of critically colonised wounds and will be useful in investigating the pathogenesis of critical colonisation via regulatory T cells.

Bacterial invasion into the epidermis of rats with sodium lauryl sulphate-irritated skin increases damage and induces incontinence-associated dermatitis.

Incontinence-associated dermatitis (IAD) is caused by prolonged exposure to urine/liquid stool. It is a common and often painful skin condition in older incontinent adults because of poor prevention. Patients with urinary infections are at risk of developing IAD, and to guide the development of novel prevention strategies, we aimed to develop an animal model of IAD by urine and bacteria. First, contralateral sites on the dorsal skin of Sprague-Dawley rats were compromised by sodium lauryl sulphate (SLS), simulating frequent cleansing with soap/water. Filter discs were then placed inside ring-shaped chambers on foam dressings, inoculated with or without Pseudomonas aeruginosa, covered with agarose gels immersed in cultured filtrated urine, and secured in place with an occlusive dressing for 3 days. Untreated and SLS-compromised sites served as controls. The IAD was developed at bacteria-inoculated sites, characterised by severe IAD-like redness that persisted for up to 3 days post-exposure and higher disruption of the skin barrier function compared with non-inoculated sites. Pathological changes included epidermal thickening, partial skin loss, inflammatory cell infiltration, accumulation of red blood cells, and invasion of bacteria into the epidermis. This novel, clinically relevant IAD rat model can serve for future prevention developments.

Expression levels of NPPB, ITGB6, CPNE4, EML5, and ITSN1 in fresh exudates swabbed from critically colonised and infected full-thickness wounds in rats.

Pressure injury management requires reliable identification of critical colonisation due to lack of infection signs. Our research group previously proposed the mRNAs natriuretic peptide B (Nppb), integrin subunit beta 6 (Itgb6), copine 4 (Cpne4), echinoderm microtubule-associated protein like 5, and intersectin 1 as candidate markers in pooled exudates of critically colonised wounds. However, it is unclear whether mRNAs or proteins of the candidate genes would be suitable as biomarkers in fresh exudate. Therefore, this study aimed to evaluate the validity of the mRNAs and proteins as fresh exudate markers for critical colonisation. Three wound models of normal healing, critical colonisation, and infection were created in rats. Fresh swab-collected exudates were collected, and mRNA and protein expression levels were measured. In the fresh wound exudates, the detection frequency of Itgb6 tended to decrease in the critically colonised and infected wounds (P = .067), and those of Cpne4 and Nppb tended to be lower in the infected wounds than in the normal healing and critically colonised wounds (P = .006 and .067, respectively). In contrast, there was no difference in protein expression in the exudates. This study suggests that Itgb6 mRNA in fresh exudates is a promising biomarker for critical colonisation.

Identification of microRNAs responsive to shear loading in rat skin.

Pressure injuries (PIs) are localised skin injuries that result from pressure with or without shear force. Shear force is more destructive than pressure in clinical settings. Therefore, determining the critical external forces is important for selecting the appropriate care to prevent PIs. To quantitatively distinguish pressure and shear loading with high specificity, we focused on microRNAs (miRs). This study aimed to identify the miRs that are distinguishable between pressure with and without shear loading in rat skin. Microarray analysis identified six candidate miRs from the comparisons among the pressure, shear, and unloaded groups. We analysed the expression levels of the candidate miRs in the process of PI development using real-time reverse transcriptase polymerase chain reaction. In the pressure and shear groups, miR-92b expressions at 6 hours after loading were 2.3 ± 1.3 and 2.9 ± 1.0, respectively, which were significantly higher than those in the control group (P = .014 and .004, respectively). miR-877 expression at 6 hours after loading was significantly increased only in the shear group (2.8 ± 0.9) compared with the control group (P = .016). These results indicate that miR-92b and miR-877 are promising biomarkers to determine for which external force healthcare professionals should intervene.

Factors related to the composition and diversity of wound microbiota investigated using culture-independent molecular methods: a scoping review.

All open wounds are often colonized by commensal microbes as a loss of skin can provide a ready portal of entry for microorganisms. Although the wound microbiota is known to be associated with wound infection and with delayed healing, the factors related to the formations of wound microbiota contributing to such poor clinical outcomes are not clear and have not led to effective infection prevention interventions. This review aimed to scope the factors related to the composition and diversity of wound microbiota that have been investigated using culture-independent molecular methods. Original articles on wound microbiota published from January 1986 to February 2020 were included in this review. Thirty-one articles met the inclusion criteria and were grouped according to wound types: chronic, acute, and animal model wounds. The factors identified were categorized according to patient characteristics, wound characteristics, treatment, and sampling. Although some studies reported the effect size of the factors, the values were small. No studies elucidated the mechanism of wound microbiota formation. The results of this scoping review highlight that the factors associated with the diversity of wound microbiota are poorly understood and that further studies are needed.

Risk scoring tool for forearm skin tears in Japanese older adults: A prospective cohort study.

[Aim] Because painful skin tears frequently occur in older patients, the prevention of skin tears is fundamental to improve their quality of life. However, a risk assessment tool for skin tears has not been established yet in Japan. Therefore, we aimed to propose a risk scoring tool for skin tears in Japanese older adult. [Methods] We conducted a prospective cohort study with 6-month follow-up in two long-term care hospitals in Japan. A total of 257 inpatients were recruited. Patient and skin characteristics were collected at baseline, and the occurrence of forearm skin tears were examined during follow-up. To develop a risk scoring tool, we identified risk factors, and converted their coefficients estimated in the multiple logistic regression analysis into simplified scores. The predictive accuracy of the total score was evaluated. [Results] Of 244 participants, 29 developed forearm skin tears during the follow-up period, a cumulative incidence of 13.5%. Senile purpura, pseudoscar, contracture, and dry skin were identified as risk factors for skin tears. Their weighted scores were 6, 4, 5, and 6, respectively. The area under the receiver operating characteristic curve of the total score was 0.806. At a cut-off score of 12, the sensitivity was 0.86, and the specificity was 0.67. [Conclusion] Our forearm skin tear risk scoring tool showed high accuracy, whereas specificity was low. This tool can contribute to prevent forearm skin tears in Japanese older adults.

Effectiveness of ultrasonic debridement on reduction of bacteria and biofilm in patients with chronic wounds: A scoping review.

Chronic wounds are defined as "hard-to-heal" wounds that are caused by disordered mechanisms of wound healing. Chronic wounds have a high risk of infection and can form biofilms, leading to the release of planktonic bacteria, which causes persistent infections locally or remotely. Therefore, infection control and removal of the biofilm in chronic wounds are essential. Recently, ultrasonic debridement was introduced as a new method to reduce infection and promote the healing of chronic wounds. This scoping review aimed to evaluate the effectiveness of ultrasonic debridement on the changes in bacteria and biofilms, and consequently the wound healing rate of chronic wounds. A total of 1021 articles were identified through the database search, and nine papers were eligible for inclusion. Findings suggest that non-contact devices are useful for wound healing as they reduce the inflammatory response, although the bacterial load is not significantly changed. Ultrasonic debridement devices that require direct contact with the wound promote wound healing through reduction of biofilm or bacterial load. The optimum settings for ultrasonic debridement using a non-contact device are relatively consistent, but the settings for devices that require direct contact are diverse. Further studies on ultrasonic debridement in chronic wounds are required.