Human wound samples
Six wound tissue samples from five diabetic patients (age: 68.7±9.0 years old; HbA1c(NGSP): 7.02±2.15%) and six wound tissue samples from six non-diabetic patients (age: 58.0±21.7 years old) were used in the study. The tissues used in the study were debrided and disposed during surgery for treatment, and no additional excisions were made for the study. Samples were recruited after agreement was obtained through informed consent. Detailed information about these subjects is given in Table 1. The Ethical Review Board at the Sapporo Medical University in Japan approved the study.
The Committee of the Animal Experimentation Center at the Sapporo Medical University School of Medicine approved all animal protocols. Mice were fed a standard chow diet and were maintained on a 12 h light / 12 h dark cycle with free access to food and water at all times. Male C57BLKS/JIar-+Leprdb/+Leprdb (Leprdb/db), male C57BLKS/JIar-m+/+Leprdb (Leprdb/＋), and female C57BL/6 mice (age>11 weeks; Sankyo Lab Service, Tokyo, Japan) were used in the experiments. At 10–11 weeks of age, blood samples were taken from the tail to measure blood glucose, which was confirmed to be above 300 mg/dL in the diabetic mice and below 200 mg/dL in the control mice. Nipro Stat Strip XP2 (Nova Biomedical, Tokyo, Japan) was used for blood glucose level measurements.
Mice were anesthetized using mixed anesthetic agents (medetomidine, midazolam, butorphanol)69. To create the skin ulcer model, a 10 mm diameter, full-thickness circle excision was made on the back of Leprdb/db and Leprdb/＋mice. Wounds were photographed with a digital camera (COLPIX S9700; Nikon, Tokyo, Japan). Images were analyzed by tracing the wound margin, and the enclosed pixel area was calculated using the Image J software, version 1.5 (National Institutes of Health, Bethesda, MD, USA). The wound areas were standardized by measuring the captured image. Using the original wound size for comparison, the percentage of wound closure was calculated as follows: day n area / day 0 area × 100 (%).
Adipose tissue transplantation
To perform adipose tissue transplantation, 100–120 mg of subcutaneous adipose tissue was collected from the back of Leprdb/db and Leprdb/+ mice and labeled with Cell Tracker Vybrant CM-DiI Cell-Labeling Solution (V22888; Thermo Fisher Scientific, MA, USA). Labeling of adipose tissue was performed according to the protocol. Briefly, tissue was incubated with CM-DiI at 37°C for 5 minutes and at 4°C for 15 minutes. The labeled adipose tissue was transplanted onto the back of Leprdb/+ mice, and the wound was sutured with nylon thread.
Histological analysis and wound healing scoring
Wound tissue and adipose tissue were harvested with marginal skin and fixed in 4% paraformaldehyde at 4℃ overnight. The following day, the tissue was cut into 5 mm sections and was paraffin-embedded. Each slide was stained with H&E and Masson trichrome, which were used for histological wound-healing scoring. For the scoring, an examiner assesses the progression of wound healing on a 12-point scale for inflammation, granulation, and collagen deposition33,35. In brief, each sample was given a score from 1 to 12: 1–3, none to minimal cell accumulation and granulation tissue or epithelial migration; 4–6, thin, immature granulation tissue dominated by inflammatory cells but with few fibroblasts, capillaries, or collagen deposition and minimal epithelial migration; 7–9, moderately thick granulation tissue, dominated by inflammatory cells and more fibroblasts and collagen deposition; and 10–12, thick, vascular granulation tissue dominated by fibroblasts and extensive collagen deposition. Because the wound was sutured, epithelialization was excluded from the evaluation criteria. All images were captured using a BZ-X700 fluorescence microscope (KEYENCE, Osaka, Japan).
Paraffin-embedded sections were deparaffinized and rehydrated for immunostaining. Antigen retrieval was performed in a microwave oven (95–98℃ for 10 minutes) using citrate buffer (10 mM sodium citrate, pH 6.0). After cooling, the slides were washed twice with deionized water and once with 1X Tris-buffered saline with Tween-20 (TBST) for 5 minutes each. The sections were blocked with 1% bovine serum albumin (BSA) in TBST for 15 minutes at room temperature (RT) and were then incubated with primary antibodies overnight at 4°C or for 1 hour at RT. After washing three times with TBST for 5 minutes each, the sections were incubated with SignalStain Boost IHC Detection Reagent (HRP, Rabbit #8114; Cell Signaling Technology, Danvers, MA, USA) for 30 minutes at RT in the dark. The sections were then washed in TBST three times for 5 minutes each and treated with TSA Plus Working Solution (Fluorescein, Cyanine 3, and Cyanine 5; AKOYA BIOSCIENCES, Malborough, MA, USA) for 10 minutes at RT in the dark. For multiplex staining, stripping was performed in a microwave oven (95–98℃ for 10 minutes) using citrate buffer. After cooling, staining with different tyramide fluorescent labels was performed according to the above procedure. Nuclei were labeled with Cellstain DAPI solution (1:1000, 4′,6-diamidino-2-phenylindole; Dojindo, Kumamoto, Japan), and after further washes, the sections were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA). The following primary antibodies were used: rabbit anti-p15 (1:500; ab53034; Abcam, Cambridge, UK), rabbit anti–PDGFR-a (1:1000 (mouse) and 1:500 (human); D1E1E, XP; Cell Signaling Technology), rabbit anti–a-SMA (1:500; D4K9N, XP; Cell Signaling Technology), and rabbit anti–phospho-histone H2A.X(Ser139) (1:480; Ser139, 20E3; Cell Signaling Technology). These primary antibodies were used after dilution with SignalStain Antibody Diluent (Cell Signaling Technology). All images were captured using a BZ-X700 fluorescence microscope (KEYENCE).
RNA extraction and quantitative real-time PCR
Total RNA was isolated from skin wound tissues and subcutaneous adipose tissues using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) and was reverse-transcribed into cDNA using the iScript cDNA Synthesis Kit (1708891; Bio-Rad). Quantitative real-time PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following conditions: 95°C for 30 seconds and 40 amplification cycles of 95°C for 15 seconds and 60°C for 1 minute. Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or beta-actin (ACTB) levels. The primer sequences used for the PCR analysis are shown in Supplementary Table 1.
Organ culture and SASP characterization
Subcutaneous adipose tissue under the wound for five Leprdb/+ or Leprdb/db mice in each group [0 DPW (before wound), 2 DPW, and 8 DPW] was collected and put into phosphate-buffered saline (PBS) supplemented with 2% penicillin/streptomycin. The tissue was then washed and transferred to Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The volume of the culture medium was 1 mL for every 60 mg of adipose tissue, the medium was changed once at 24 hours post-extraction, and the culture medium was collected at 48 hours post-extraction for use in the following assay. The culture medium from adipose tissue culture was characterized using the Proteome Profiler Mouse XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA). Developed films were imaged and integrated density analysis was performed using Image J, version 1.5 (National Institutes of Health).
Cell preparation and in vitro wound-healing assays
Skin fibroblasts were collected from C57BL/6 mice. After euthanasia, skin was harvested and digested in Liberase TL (5401020001, Merck) for 120 minutes at 37°C. The digested skin slurries were filtered through a 100 µm cell strainer (EASYstrainer Cell; Greiner Bio-One, Kremsmuenster, Austria) and through a 70 µm cell strainer (Greiner Bio-One). Cells were suspended in DMEM supplemented with 10% PBS and 1% penicillin/streptomycin and were cultured in a T75 culture flask. Cells reached 80–90% confluence after incubation for 1–2 weeks, and the cells were passaged. Cells from passage 2 were used for the in vitro studies. In vitro wound healing was studied using 2-well Culture-Inserts (Ibidi, Bavaria, Germany). Mouse skin–derived fibroblasts were cultured in 2-well Culture-Inserts with adipose tissue–cultured media for 6 days, and phase contrast images were obtained every 24 hours and immediately after removing the 2-well Culture-Insert using Primovert and Axiocam208 microscopes (Carl Zeiss, Jena, Germany).
Mice were only excluded from the study if they had visible wounds from fighting. Statistical analyses were performed using R (The R Foundation for Statistical Computing, Vienna, Austria). Statistical significance between two groups was determined using an unpaired t-test. A one-way or two-way analysis of variance (ANOVA) was conducted to assess differences among three or more groups. Pairwise comparisons were made only when the ANOVA test identified a statistical significance. p-values for multiple comparisons were adjusted using the Tukey method. Statistical analyses were performed using EZR, which is a graphical user interface for R70. Two-sided p-values <0.05 were considered statistically significant. Quantitative data are presented as either the mean±standard error of the mean (SEM) or median with interquartile range (IQR) and 1.5 × IQR. Box-and-whisker plots and bar plots were generated using ggplot2, a plotting system for R based on The Grammar of Graphics (The R Foundation for Statistical Computing, Vienna, Austria). The R packages FactoMineR and factoextra were used to generate heat maps, Ward’s hierarchical agglomerative clustering, and principal component analyses.