Fibrinogen from human plasma

For taking nasal swabs, the transystem^® from Hain- Lifescienes, 72,147 Nehren, Germany, was used. Both nostrils of each participant were sampled by the same swab. After non-selective enrichment in cation-adjusted Mueller–Hinton broth, aliquots were streaked on CHROMagar™ MRSA from Becton Dickinson (Heidelberg, Germany) and in parallel on Mueller–Hinton blood agar plates (Oxoid, Wesel, Germany). After incubation at 37 °C for 24 h, one suspicious colony was subcultured on sheep blood agar, except differences in colonial morphology and haemolysis. Confirmation of S. aureus was performed by demonstration of the clumping factor and additionally by the tube coagulase test. For the detection of the clumping factor, we used a solution of fibrinogen from human plasma (Sigma-Aldrich, Taufkirchen, Germany) of 2 mg/mL 0.85% NaCl). For the tube coagulase test, we used fresh ready to use human plasma provided by DRK blood donation service, 31,831 Springe, Germany. In the case of negative results, we performed PCR for the S. aureus specific region of tuf by use of primers and PCR conditions according to Reference [46 (link)]. For PCR, genomic DNA was extracted from S. aureus grown in nutrient broth by use of the DNeasy tissue kit (Qiagen, Hilden, Germany), and lysostaphin (100 mg/L; Sigma, Taufkirchen, Germany) for bacterial lysis.

Bacteria were cultured in two media:

Standard microbiological Tryptic Soy Broth marked TSB. The detailed composition of TSB is presented in Table 4.

Medium prepared according to the formula presented by Kadam et al.^{22 (link)}, marked IVWM (In vitro Wound Milieu).

Sterile Fetal Bovine Serum (Biowest, France, cat. No. S181H) was used as the base component of IVWM. Firstly, stock solutions of the components were prepared as follows:

fibronectin (Human plasma fibronectin, Sigma-Aldrich, USA, cat. No. FC010) 1 mg/mL solution in autoclaved distilled water,

fibrinogen (fibrinogen from human plasma, Sigma-Aldrich, USA, cat. No. F3879) 10 mg/mL solution in saline (Stanlab, Poland),

lactoferrin (lactoferrin human, Sigma-Aldrich, USA, cat. No. L4040) 2 mg/mL solution in Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, USA),

lactic acid (Sigma-Aldrich, USA, cat. No. W261114) 11.4 M solution,

and they were filtered using a 0.22 ​μm syringe filter. Collagen (Collagen solution from bovine skin, concentration 2.9–3.2 mg/mL, Sigma-Aldrich, USA, cat. No. C4243) was purchased sterile. The IVWM was obtained by combining the ingredients at concentrations presented in Table 4. The medium was stored for a maximum of seven days at 2–8 °C and was protected from light.

Fibrinogen from human plasma (#F3879) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent (#M5655) were purchased from Sigma-Aldrich, St. Louis, MO. DRP1 shRNA(h) lentiviral particles (#sc-43732-V), Control shRNA lentiviral particles-A (#sc-108080), and Polybrene® (#sc-134220) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Antibodies were purchased from the following suppliers: against ZO-2 (#2847), phospho-DRP1[S616] (#3455), phospho-DRP1[S636] (#4867), and claudin-5 (#49,564) from Cell Signaling Technology, Danvers MA, USA; against JAM-A (#sc-53623) and occludin (#sc-133256) from Santa Cruz Biotechnology, Dallas, TX, USA; against β-actin (#A5441), and MFN2 (#M6444) from Sigma-Aldrich, St. Louis, MO, USA; against total DRP1 (#611,112) and OPA1 (#612,606) from BD Transduction Laboratory, San Jose, CA, USA; against PECAM-1 (#01004) from BiCell Scientific, Maryland Heights, MO, USA; against FIS1 (#ALX-210–1037) from Enzo Life Sciences, Inc, Farmingdale, NY, USA; and against VE-cadherin (#36–1900) from Invitrogen, Frederick, MD, USA.

Cells were collected and prepared in clots according to the procedure developed by Dr. Elizabeth Hyjek in the Department of Pathology at the Unversity of Chicago (unpublished) for formalin fixation and paraffin embedding for use in immunohistochemical (IHC) studies. After washing cells in Ca²⁺/Mg²⁺ deficient PBS, cells were collected and pelleted. Cells were resuspended in fibrinogen from human plasma (Sigma, St. Louis, MO) in complete media and clotted using thrombin (bovine origin) (King Pharmaceuticals, Bristol, TN). After overnight formalin fixation, the cell clots were paraffin embedded and mounted on slides in 4µm sections for IHC, as described below.

PLLA (average Mw: 230 kDa) was provided by Total Corbion (Amsterdam, Netherlands). L-Lactide was purchased from DURECT Co. (Birmingham, AL, USA). DL-lactide was obtained from Tokyo Chemical Ind. (Tokyo, Japan). Magnesium hydroxide (MH, 99.5%), ε-caprolactone, fibrinogen from human plasma, stannous octoate, sodium dodecyl sulfate (SDS), L-(+)-lactic acid (L-Lac), and 1-octanol were obtained from Sigma Aldrich (Burlington, MA, USA). Platelet concentrates (5 × 10⁴ platelets per μL) were purchased from the Korean National Red Cross (Seoul, Korea). IL-6 and IL-8 enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D Systems (Minneapolis, MN, USA).
HCAECs were acquired from Cambrex (Walkersville, MD, USA) and EGM-2 media with an MV bullet kit (Lonza, Basel, Switzerland). A cell-counting kit (CCK-8) was obtained from Dongin LS (Seoul, Korea). Phosphate-buffered saline (PBS) solution was supported from Hyclone (GE Healthcare Life Sciences, Boston, MA, USA). A Micro BCA^TM Protein assay kit was acquired from Thermo Scientific TM (Waltham, MA, USA). All chemicals were laboratory reagent grade and used without purification.