Potential of gelatin hydrogel nonwoven fabrics (Genocel) as a skin substitute in a diabetic mouse skin defect model.

Background: Gelatin hydrogel nonwoven fabrics (Genocel) are three-dimensional gelatin scaffolds that provide cells with space for proliferation, migration, and differentiation. They are expected to be an effective wound healing modality to treat intractable wounds, such as diabetic foot ulcers, because they enhance early neovascularization when used as a skin substitute. In this study, we explored the healing process of Genocel applied to skin defects in diabetic mice and compared it with that of a conventional skin substitute, Pelnac. Methods: Genocel and Pelnac sheets were used to treat skin defects on the backs of diabetic mice. On days 7 and 14, the remaining wound area was evaluated and specimens were harvested for HE, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue formation, capillary formation, and macrophage infiltration. Results: Wounds treated with Genocel showed a wound healing process comparable to that of wounds treated with Pelnac. No significant differences were observed in the remaining wound area, neoepithelial length, granulation formation, number of pan-macrophages, or M2 ratio on days 7 and 14. The only significant difference was the number of induced M2 macrophages, which was higher in Pelnac group than in the Genocel group on day 7 (p < 0.05). Conclusions: Genocel showed similar healing effects in diabetic wounds as Pelnac and is considered an effective wound management modality for diabetic ulcers.

Comparisons of the effects of silk elastin and collagen sponges on wound healing in murine models.

Introductions: Silk elastin, a recombinant protein with repeats of elastin and silk fibroin, possesses a self-gelling ability and is a potential wound dressing material. The aim of this study is to elucidate the mechanism of the wound healing-promoting effect of silk elastin by comparing its in vivo behavior in a mouse wound model with that of a collagen sponge. Methods: Skin defects (8 mm in diameter) were created on the backs of C57BL/6J and BKS.Cg- + Lepr/+Lepr db male mice. Silk elastin sponges of 2.5 or 5.0 mm thickness, as well as collagen sponges, were placed on the wounds and secured with a polyurethane film. In the control group, only the polyurethane film was applied. The remaining wound area was grossly evaluated, and tissue samples were collected after 7, 14, and 21 days for histological evaluation, including neoepithelialization, wound contraction, granulation tissue formation, newly formed capillaries, and macrophages. Genetic analysis was conducted using real-time polymerase chain reaction. Results: In the study with C57BL/6J, there were no significant differences between the silk elastin and collagen sponge groups. Similarly, in the study using BKS.Cg- + Lepr/+Lepr db, no significant differences were found in the remaining wound area and granulation tissue formation between the silk elastin and collagen sponge groups. However, on day 14, the 5.0-mm-thick silk elastin sponge group showed increased macrophages, longer neoepithelialization, and more frequent angiogenesis compared to other groups. Gene expression of inducible nitric oxide synthase and arginase-1 was also higher in the 5.0 mm thick silk elastin sponge group. Conclusions: Silk elastin sponges demonstrated superior neoepithelialization and angiogenesis compared to collagen sponges. The results suggest that silk elastin and collagen sponges promote wound healing through different mechanisms, with silk elastin possibly enhancing wound healing by facilitating increased macrophage migration. Further studies are needed, but silk elastin shows great potential as a versatile wound dressing material.

Development of new bioabsorbable implants with de novo adipogenesis.

Poly-L-lactic acid (PLLA) mesh implants containing collagen sponge (CS) were replaced with autologous adipose tissue regeneration in vivo. Herein, we investigated the optimal external frames and internal fillings using poly (lactic-co-ε-caprolactone) (P (LA/CL)), PLLA, and low-molecular-weight PLLA (LMW-PLLA) as the external frame and polyglycolic acid (PGA) nanosheets and CS as the internal filling. We prepared six implants: P (LA/CL) with PGA nano, PLLA with PGA nano, PLLA with CS, PLLA with 1/2 CS, PLLA with 1/4 CS, and LMW-PLLA with CS, and evaluated adipogenesis at 6 and 12 months using a rat inguinal model. The internal spaces in the P (LA/CL) and LMW-PLLA implants collapsed at 6 months, whereas those in the other four implants collapsed at 12 months. Adipose tissue regeneration was not significantly different between the PLLA-implanted groups at 6 and 12 months and was greater than that in the P (LA/CL) with PGA nano and LMW-PLLA with CS groups. The PGA nanosheet inside PLLA was comparable to the CS inside PLLA in the regeneration of adipose tissue and macrophage infiltration. In summary, PLLA is a promising external frame material in which the internal space can be replaced with adipose tissue. Thus, PGA nanosheets are an alternative internal filling material for adipose tissue regeneration.

A high-hydrostatic pressure device for nevus tissue inactivation and dermal regeneration for reconstructing skin defects after giant congenital melanocytic nevus excision: a clinical trial.

Background: A novel treatment has been developed to reconstruct large skin defects caused by the excision of giant congenital melanocytic nevi. It involves the reimplantation of high-hydrostatic pressurized nevus tissue as a cell-inactivated autologous scaffold for dermal regeneration, followed by the implantation of cultured epithelial autografts on the regenerated dermis. Because this treatment has shown promise in a first-in-human clinical trial which used a prototype pressure machine, a novel pressure device was specifically designed for clinical use. Methods: In a prospective investigator-initiated clinical trial involving three patients, we evaluated the safety and efficacy of the skin regeneration treatment using a pressure device. All three patients underwent surgical excision of the nevus tissue, primary reimplantation of the inactivated nevus tissue, and secondary implantation of cultured epithelial autografts. Results: Engraftment of inactivated nevus tissue and cultured epithelial autografts was successful in all three cases, with over 90% epithelialization at 8 weeks post-surgery. No serious adverse events or device malfunction were observed during the trial. Conclusion: The novel pressure device safely and effectively enabled dermal regeneration using the nevus tissue as an autologous scaffold. This innovative approach offers several advantages, including reduced invasiveness due to minimal sacrifice of normal skin for skin grafting and high curative potential resulting from full-thickness removal of the nevus tissue.

Modified gelatin hydrogel nonwoven fabrics (Genocel) as a skin substitute in murine skin defects.

Introduction: From previous research, an emerging material composed of gelatin hydrogel nonwoven fabric (Genocel) has shown potential as a skin substitute, by improving neovascularization promotion in the early phase of wound healing. However, Genocel was inferior in terms of granulation formation compared to Pelnac. To solve this problem, we modified the manufacturing process of Genocel to reduce its water content, extend the degradation time (Genocel-L), and evaluate its healing process as a skin substitute. Methods: Genocel with a low water content (Genocel-L) was prepared and the difference in water content compared to that of the conventional Genocel was confirmed. Degradation tests were performed using collagenase and compared among Genocel-L, Genocel, and Pelnac sheets. In the in vivo study, sheets of Genocel-L or Pelnac were applied to skin defects created on the backs of C57BL/6JJcl mice. On days 7, 14, and 21, the remaining wound area was evaluated and specimens were harvested for Hematoxylin and Eosin, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue, capillary formation, and macrophage infiltration. Results: Genocel-L had a lower water content than the conventional Genocel and a slower degradation than Genocel and Pelnac. In the in vivo experiment, no significant differences were observed between Genocel-L and Pelnac in relation to the wound area, neoepithelium length, granulation formation, and the number of newly formed capillaries. The area of newly formed capillaries in the Pelnac group was significantly larger than that in the Genocel-L group on day 21 (p < 0.05). Regarding macrophage infiltration, significantly more M2 macrophages were induced in the Pelnac group on days 14 and 21, and the M2 ratio was larger in the Pelnac group (p < 0.05) during the entire process. Conclusions: Genocel-L has a lower water content and slower degradation rate than the conventional Genocel. Genocel-L had equivalent efficacy as a skin substitute to Pelnac, and can therefore be considered feasible for use as a skin substitute. However, a manufacturing method that can further modify Genocel-L is required to recover its early angiogenic potential.

Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP).

Surgical resection of malignant bone tumors leads to significant defects in the normal surrounding tissues that should be reconstructed to avoid amputation. Our research aimed to inactivate osteosarcoma (OS)-affected bone to obtain autologous bone grafts for bone defect reconstruction using a novel therapy called high hydrostatic pressurization (HHP) therapy. The key points are complete tumor death and preservation of the non-denatured native extracellular matrix (ECM) and bone tissue by HHP. Previously, we found that HHP at 200 MPa for 10 min can completely inactivate cells in normal skin and skin tumors, including malignant melanoma and squamous cell carcinoma while maintaining their original biochemical properties and biological components. Based on our previous research, this study used HHP at 200 MPa for 10 min to eradicate OS. We prepared an OS cell line (LM8), pressurized it at 200 MPa for 10 min, and confirmed its inactivation through morphological observation, WST-8 assay, and live/dead assay. We then injected OS cells with or without HHP into the bone marrow of the murine tibia, after which we implanted tumor tissues with or without HHP into the anterior surface of the tibia. After HHP, OS cells did not proliferate and were assessed using a live/dead assay. The pressurized cells and tumors did not grow after implantation. The pressurized bone was well prepared as tumor-free autologous bone tissues, resulting in the complete eradication of OS. This straightforward and short-pressing treatment was proven to process the tumor-affected bone to make a transplantable and tumor-free autologous bone substitute.

Comparison of Wound Healing Effect of Skin Micrograft Impregnated into Two Kinds of Artificial Dermis in a Murine Wound Model.

A micrograft (MG) suspension produced by the Rigenera protocol has been used to stimulate tissue regeneration. Recently, a combination therapy of an artificial dermis and skin MG has been used to promote angiogenesis and granulation tissue formation in the artificial dermis. There are no reports comparing the differences in MG impregnation efficiency between different artificial dermis products. Therefore, we compared the impregnation of skin MG in Pelnac Gplus and Integra. Methods: Skin MG was prepared from the skin of C57BL/6JJcl mice using Rigeneracons and administered onto Pelnac Gplus and Integra sheets. The amount of MG suspension impregnated in Pelnac Gplus and Integra was evaluated. Pelnac Gplus and Integra sheets combined with MG were applied to murine defects, and wound area, neoepithelium length, granulation tissue formation, and newly formed capillaries were compared with the control groups on days 7 and 14. Results: The weight percentage of the MG absorbed by Pelnac Gplus and Integra was 88.8% ± 3.5% and 28.2% ± 7.0%, respectively (P < 0.05). In the in vivo experiment, the area of newly formed granulation tissue and both the number and area of newly formed capillaries in the PelnacG + MG group were significantly larger than those in the control group at 14 days after implantation (P < 0.05). Conclusions: Skin MG was successfully impregnated into Pelnac Gplus by simple administration but not into Integra. Administration of skin MG into the Pelnac Gplus promoted granulation formation and angiogenesis. Pelnac Gplus was more suitable than Integra in the combination therapy.

Group A Streptococcal Rapid Antigen Detection Test: A Clinical Study to Evaluate the Reproducibility Using Human Wound Samples.

Background: The early diagnosis of necrotizing soft tissue infection (NSTI) caused by group A streptococcus (GAS) for performing debridement surgery is key to the patient survival. In 1996, the diversional use of a GAS-rapid antigen detection test (RADT) for pharyngitis was reported, quickly spreading as a clinically useful method. However, no clinical study has evaluated the reliability of RADT by using wound samples. This is the first study using clinical wound samples to examine the reproducibility between GAS-RADT and wound culture. Methods: Patients in whom wound culture samples were clinically necessary were included in this study. Two samples were obtained simultaneously: one for isolation of bacteria as wound culture and the other for GAS-RADT, with written informed consent. The reproducibility between GAS-RADT and wound culture was statistically evaluated by Cohen's kappa coefficient. Results: One hundred samples from 94 patients were collected from 2020 to 2021. Two samples were GAS-positive on wound culture, and both were RADT-positive (positive reproducibility: 100%). Ninety-eight samples were GAS-negative on wound culture; of these, 97 were RADT-negative (negative reproducibility: 99%). Cohen's kappa coefficient was 0.80, indicating excellent agreement beyond chance. None of the bacteria showed cross-reactional influences. The only discrepant case (RADT-positive and wound culture-negative) was attributed to the administration of antibiotics for 2 days before the sampling. Conclusions: The reproducibility between GAS-RADT and wound culture was statistically excellent, underscoring the reliability of GAS-RADT for wounds.

Development of gelatin hydrogel nonwoven fabrics (Genocel®) as a novel skin substitute in murine skin defects.

Introduction: Genocel is an emerging material, used in cell culture, with high mechanical strength and good cytocompatibility. Based on these characteristics, Genocel is considered a promising skin substitute for wound healing. In this study, we explored the possibility of using Genocel as a skin substitute for murine skin defects and compared it with a conventional skin substitute. Methods: Sheets of Genocel and Pelnac were applied to skin defects created on the backs of mice. On days 7, 14, and 21, the remaining wound area was evaluated and specimens were harvested for HE, Azan, anti-CD31, CD68, and CD163 staining to assess neoepithelialization, granulation tissue, capillary formation, and macrophage infiltration. Results: No significant differences in the wound area or neoepithelium length were observed between groups. The number of newly formed capillaries in the Genocel group was significantly higher than that in the Pelnac group on day 7 (p < 0.05). In contrast, granulation tissue formation in the Pelnac group was greater than that in the Genocel group on day 14 (p < 0.05). Regarding macrophage infiltration, the pan-macrophage number, M2 macrophage number, and M2 ratio in the Pelnac group were higher than those in the Genocel group on day 14 (p < 0.05). In other aspects, the two materials displayed comparable behavior. Conclusions: Genocel can be used as a skin substitute equivalent to the conventional one. In addition, Genocel accelerated capillary formation, which is more appropriate than conventional treatments for chronic skin ulcers, such as diabetic ulcers.

Clinical Utility of Silk-Elastin Sponge in Patients with Chronic and Acute Skin Ulcers: Study Protocol of a Multicenter Clinical Trial.

INTRODUCTION: Not only chronic but also some acute wounds have a risk of infection and become unhealed wounds. Silk-elastin sponge has been developed to treat chronic wounds that are susceptible to infection. Preclinical and clinical studies suggested that silk-elastin sponge is safe for humans and can promote granulation tissue formation by reducing bacterial growth in chronic wounds. The central aim of this trial is to evaluate the clinical utility and safety of silk-elastin sponge for the treatment of chronic and acute skin ulcers. METHODS: This study is a prospective, multicenter, single-arm, uncontrolled clinical trial. In this study, 20 patients with chronic ulcers and five with an acute one will be included; patients with wound infection will be excluded. Silk-elastin sponges are applied and covered with a dressing for 14 days. PLANNED OUTCOMES: The primary endpoint is the frequency of patients with chronic wounds in whom the investigator confirms the formation of a healthy wound bed at 14 days after the initial application of the study device. In addition, safety for acute wounds and handiness of the study device will be assessed. TRIAL REGISTRATION NUMBER: jRCT2052210072.

A Case of Concurrent Extramammary Paget Disease and Basal Cell Carcinoma of the Perianal Region.

An 83-year-old Japanese man who had been aware of a tumor near his anus for 2 years underwent tumor resection. Although he was diagnosed with basal cell carcinoma (BCC), extramammary Paget disease (EMPD) was also accidentally found in the same specimen. In the pathological histology of EMPD, there were large round cells with ample cytoplasm spread in the epidermis; these cells were positive for cytokeratin 7 and gross cystic disease fluid protein 15. No signs that are typically associated with EMPD, such as erythema or leukoderma, were observed near the anus. There have been only 4 reports in which BCC and EMPD developed in the same area, and the authors present the fifth case. In the reported case, no clear evidence was found for the corelative development of BCC and EMPD.