Animals
Forty ten-week-old male C57BL/6 mice, with body weights ranging from 25 to 30 g, were utilized in this study. These mice were housed under consistent and controlled laboratory conditions and provided with standard mouse feed and access to water. An acclimation period of one week was observed prior to the initiation of any experiments to ensure that the mice adapted to their environment. Throughout the entire experimental duration, the mice were maintained under conditions of constant temperature and relative humidity. Twenty animals were assigned to each of the two experimental groups: the wound healing model (restricted contracture) group and the full-thickness skin graft model group. Biopsies were performed on specific days following the surgical procedures, namely, on days 4, 7, 11, and 14 after the operation, designated as post-operative day (POD) 4, 7, 11, and 14, respectively. All procedures involving animals in this study were conducted in strict accordance with ethical guidelines and were approved by the Ethics Committee of the Daegu-Gyeongbuk Medical Innovation Foundation. These procedures adhered to the principles outlined in the Declaration of Helsinki, ensuring the ethical treatment and care of the animals involved.
Wound model-restricted contracture
Anesthesia and preparation The procedure began with the administration of inhaled anesthesia using isoflurane obtained from Hana Pharm, Seoul, Korea. Once the mice were appropriately anesthetized, the dorsal area of each animal was carefully shaved to ensure a clean surgical site. Subsequently, the shaved area was sterilized using a 7.5% povidone-iodine solution and alcohol to minimize the risk of infection.
Creating Circular Wounds A circular marking with a diameter of 15 mm was made on the dorsal area of the mice using a stamp. Two full-thickness skin defect models were generated within this marked circular area using a #15 surgical blade. The two wounds, each measuring 15 mm in size, were precisely created by removing the entire skin layer down to the level of the panniculus carnosus, which is approximately 0.2 cm thick. This ensured uniformity in the wound creation process. To prevent contamination from surrounding areas, sterile drapes were meticulously applied to cover regions other than the dorsal area.
Preventing Contracture To prevent wound healing through contracture, a donut-shaped silicone plate, 0.5 mm thick, was employed. This silicone plate featured a central hole with a diameter of 18 mm. It was securely fixed in place using Ethilon #5 sutures [9]; (Fig. 10). This step was crucial in maintaining the wound’s intended characteristics.
Production of a wound healing-restricted contracture model: A stamp, 15 mm in diameter, was used to create two full-thickness defects on the dorsal area. A donut-shaped silicon plate (0.5 mm thick) was placed near the wound site and fixed by suturing to prevent the wound from contracting
Experimental and Control Groups Among the two defect sites created, one was randomly designated as the experimental group, while the other served as the control group. To distinguish between the two, the ear on the same side as the experimental group was marked accordingly.
Application of A7-1 Protein In the experimental group, the novel A7-1 protein (100 µM, 2 cc) was applied to the wound site at 24-h intervals after the surgical operation. This application was performed to assess the protein’s impact on wound healing. In contrast, the control group’s wound received applications of normal saline (2 cc) using the same schedule. This distinction allowed for a comparative evaluation of the effects of A7-1 protein on wound healing.
Production of full-thickness skin graft (FTSG) model
Anesthesia and preparation The procedure commenced with the administration of inhaled anesthesia using isoflurane, supplied by Hana Pharm in Korea. Following anesthesia, the dorsal area of each mouse was meticulously shaved to provide a clean surgical site. Subsequently, the shaved area was thoroughly sterilized using a 7.5% povidone-iodine solution and alcohol to minimize the risk of infection.
Creating full-thickness excision sites to establish the graft model, a circular mark with a diameter of 15 mm was carefully drawn on the dorsal area of the mice. This marked area served as the site for the creation of two full-thickness skin defect models. Two full-thickness skin defect models were generated within the marked circular area. These excisions ensured that the entire thickness of the skin was removed.
Experimental and control groups After the creation of the skin defects, one side was randomly assigned as the experimental group, while the other served as the control group. To distinguish between the two groups, the ear on the same side as the experimental group was marked accordingly.
Ensuring uniform graft thickness It was essential to maintain uniformity in the thickness of the skin grafts harvested. To achieve this, an even amount of tension was applied to the donor surface.
Application of A7-1 protein and suturing In the experimental group, the novel A7-1 protein (100 µM, 2 cc) was applied to the graft site. Subsequently, the skin that had been removed was sutured back in place using 5–0 Ethilon sutures. This process aimed to assess the impact of A7-1 protein on the graft site. In the control group, normal saline (2 cc) was applied to the graft site using the same method. The skin was then sutured back into place using 5-0 Ethilon sutures [5].
Wound healing rate
Image acquisition After inducing the wounds in both the wound and full-thickness skin graft (FTSG) model, images were captured on specific days. These days included the day of the surgical operation and PODs 4, 7, 11, and 14. To capture high-quality images, a stereoscopic zoom microscope (SMZ 745 T, Nikon, Tokyo, Japan) was employed.
Image analysis The acquired images of the wound and graft sites were analyzed using an image analysis program called Image J (NIH, Bethesda, MD, USA). This software facilitated accurate measurement of the wound area and graft site.
Biopsy Tissue biopsies were conducted on select animals at different time points. Specifically, biopsies were performed on 5 animals from a total of 20 animals on POD 4, 15 animals on POD 7, 10 animals on POD 11, and 5 animals on POD 14. This biopsy strategy allowed for the collection of tissue samples at various stages of the healing process, providing valuable insights [4].
Consistency in image acquisition To ensure the reliability of the acquired images and measurements and eliminate potential variations in image quality, it was imperative that consistency was maintained. Therefore, all images were captured by the same researcher in the same location under identical lighting conditions.
Measurement parameters In the wound model, measurements focused on assessing the raw surface area of the wound site where healing had not occurred, reflecting the absence of re-epithelialization. In contrast, the FTSG model involved measuring the area where the graft had not healed, indicating a lack of graft acceptance, as well as assessing cluster areas.
Complete treatment point The point at which a wound or graft was considered completely treated was defined as the stage of reepithelialization where no further dressing application was required. This marked the successful conclusion of the healing process.
Laser-induced fluorescein fluoroscopy
To evaluate angiogenesis, laser-induced fluorescein fluoroscopy was employed, following this protocol:
Anesthesia On PODs 4, 7, 11, and 14, anesthesia was induced in 5 mice for each time point. This was achieved through the intraperitoneal injection of 0.1 cc of ketamine. Anesthesia ensured that the mice remained immobile during the imaging procedure.
Fluorescein injection Following anesthesia, 0.1 mm of a 10% fluorescein solution was injected into the tail vein of each mouse. The fluorescein solution used in this procedure consisted of 500 mg of 10% fluorescein sodium, obtained from Unimed Pharma, Inc. in Seoul, Korea.
Real-Time angiogenesis analysis One minute after the administration of fluorescein, the degree of angiogenesis was assessed through real-time analysis. This analysis was conducted using the HEIDELBERG Retina Angiogram Digital Angiography System (HRA). The HRA system facilitated the visualization and examination of angiogenesis at the wound site (Fig. 11).
Stereoscopic image of wound for analysis of Angiogenesis using Laser-induced fluorescein fluoroscopy
Image analysis For a thorough evaluation of the angiogenesis, Image J software was employed. This software enabled the comparison and analysis of the brightness of the images obtained during the angiography process [11]; (Fig. 12). This approach allowed for the quantification and assessment of angiogenesis levels at specific time points in the wound healing process. It provided valuable insights into the development of blood vessels in the wound area, aiding in the understanding of tissue repair and regeneration.
Wound site brightness after fluoroscopy was measured using Image J software
Confocal laser scanning microscopy
To further assess angiogenesis and observe the appearance and status of angiogenesis, a confocal laser scanning microscope was utilized. The procedure is as follows:
Post-Fluoroscopy angiogenesis assessment After the evaluation of angiogenesis through laser-induced fluorescein fluoroscopy on PODs 4, 7, 11, and 14, additional steps were taken to gain more detailed insights into angiogenesis [9].
Injection of fluorescein isothiocyanate-dextran On these specified days, 300 µL of a solution conjugated with fluorescein isothiocyanate-dextran was injected into the mouse tail vein. This injection aimed to label blood vessels and visualize their structure and density in the wound area.
Tissue Biopsy: 5 min after the injection of the fluorescein isothiocyanate-dextran solution, a skin biopsy was performed. The biopsy included not only the wound site but also a 4-mm margin of healthy skin surrounding the wound. This comprehensive sampling allowed for the examination of both the wound area and its immediate surroundings.
Confocal microscopy analysis The collected tissue samples were immediately analyzed using a confocal laser scanning microscope. This specialized microscope enabled researchers to obtain high-resolution images of the skin tissue, focusing specifically on the appearance and status of angiogenesis.
This confocal microscopy procedure provided detailed visual information about the development and characteristics of blood vessels within the wound and surrounding tissue. It offered a more in-depth perspective on angiogenesis, enhancing the overall understanding of vascular changes during the healing process.
Histological examination
After observing the blood vessels by confocal laser scanning microscopy, the same animals were sacrificed on PODs 4, 7, 11, and 14 and the skin area that included the skin defect and 4 mm of healthy skin around the wound site was excised up to the panniculus carnosus. The harvested tissue was fixed in formalin, and then the center of the wound site was cut cross-sectionally to prepare a paraffin block, which was sliced into 3-μm slices for hematoxylin and eosin and Masson’s trichrome staining. The degree of reepithelization, proliferation of granulation tissue, necrosis, degree of inflammation, and pattern of angiogenesis were measured by optical microscopy at 40 × and 100 × magnification.
Vascular endothelial growth factor(VEGF) western blot
Tissue harvest and storage Tissue samples were collected from mice that had been sacrificed on PODs 4, 7, 11, and 14. These tissue samples were immediately freeze-stored at −70 °C to preserve their protein content.
Tissue preparation To quantitatively analyze VEGF, the frozen tissue samples were removed from storage and added to 0.5 mL of cell lysis buffer. The cell lysis buffer contained the following components: 1% Triton, 1% cholic acid, 50 mM NaCl, and 20 mM Tris–HCl (pH 7.4). Additionally, protease inhibitors, including 1 µg leupeptin, 1 µg pepstatin A, 1 µg aprotinin, and 1 mM PMSF, were added to the buffer. The entire process was conducted at 4 °C to maintain protein stability.
Tissue homogenization a tissue homogenizer (Ultra-Turrax T25, IKA-Labor Technique, IKA Process, Wilmington, NC, USA) was used to mechanically disrupt and homogenize the tissue. This step was performed three times for 1 min each time to ensure thorough tissue decomposition.
Incubation After tissue homogenization, the samples were incubated at room temperature for 1 h. This incubation allowed for the proper extraction of proteins from the cells and tissue components.
Centrifugation Following incubation, the solution was subjected to centrifugation at 15,000 rpm and 15 °C for 30 min. This centrifugation step resulted in the separation of the supernatant, which contained the extracted proteins, from the cellular and tissue debris.
Reference standard Bovine serum albumin was used as the reference standard sample for quantification.
Polyacrylamide Gel Electrophoresis A portion of the extracted protein supernatant (25 µg) was separated by 10% polyacrylamide gel electrophoresis for 1 h. This separation step allowed the proteins to be separated based on their molecular weights.
Protein transfer Following electrophoresis, the proteins were transferred from the gel to a nitrocellulose membrane. This transfer step facilitated subsequent antibody detection.
Blocking The nitrocellulose membrane was treated with a blocking buffer for 1 h. The blocking buffer contained 5% nonfat dry milk in a buffer solution composed of 10 mM Tris HCl, 0.15 M NaCl, and 0.1% sodium azide. Blocking helped prevent the non-specific binding of antibodies.
Primary antibody incubation The primary mouse antibody for VEGF (sc-7269, Santa Cruz Biotechnology, Dallas, TX, USA) was diluted to a 1:1,000 concentration in the blocking buffer. The membrane was then incubated with the primary antibody for 1 day at 4 °C.
Secondary antibody incubation Following incubation with the primary antibody, a secondary antibody bound to horseradish peroxidase was applied to the membrane and allowed to react for 1 h at room temperature.
Washing the membrane was washed three times for 10 min each with Tris-buffered saline with Tween 20 (TTBS) to remove unbound antibodies.
Enhanced chemiluminescence (ECL) Staining ECL staining was performed to visualize and quantify the proteins of interest. ECL is a chemiluminescent reaction that produces light upon exposure to the target protein-antibody complex.
This comprehensive western blot analysis allowed for the quantitative assessment of VEGF protein levels in the tissue samples, providing valuable data on the expression of this critical growth factor during the wound healing process [9].
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