Tegaderm

Luciferase/GFP-labeled GMSCs were implanted in wound healing model as described previously [22 (link)–24 (link)]. In brief, 5-week-old immunocompromised mice were individually anesthetized using an intraperitoneal injection of ketamine (75 mg/kg) and rinsed with an alcohol swab and sterilely prepped with betadine and draped. A sterile 8 mm diameter full-thickness wound was created on the dorsum of the nude. A donut-shaped splint with a 10 mm inner diameter and 20 mm outer diameter was fashioned from a 0.5 mm-thick silicone sheet (Grace Bio-Laboratories, Bend, OR). An immediate-bonding adhesive (Tegaderm, 3 M) was used to fix the splint to the skin followed by interrupted 5–0 nylon (Ethicon, Inc,Somerville, NJ) sutures to ensure position. Mastisol (Fernadale, MI) was applied to the perimeter of the wound to improve adherence of the occlusive dressing (Tegaderm, 3 M) placed to cover the wounds. The animals were placed in individual cages under a warming lamp and allowed to recover fully from anesthesia. The wound dressings in each group were changed every 3 days according the above methods. 15 mice were randomly divided into three groups: Group A, hydrogel/control GMSCs; Group B, hydrogel/center-BRONJ GMSCs; Group C, hydrogel/peri-BRONJ GMSCs, n = 5. Figure 4 showed the experimental design and schematic representation of wound healing model in nude mice.

Wst-1 assay kit was purchased from Daeillab (Daeillab, Korea). LY294002 (PI3K/Akt inhibitor) and PD98059 (Erk inhibitor), L-Ascorbic acid were purchased from Sigma Aldrich (Sigma Aldrich, USA). BIO (GSK-3β inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as p-Akt, total-Akt, β-actin, Cyclin E, p-cdk2, p21 were obtained from Cell Signaling Technology (Beverly, USA). and p-GSK-3β, total-GSK-3β, p-Erk, total-Erk, Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse™ Muse™ Cell Cycle Kit (MCH100106) and Muse™ Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany). 3M™ Tegaderm (sterile barrier to external contaminants) was purchased from 3 M (3 M, USA).

The cream requirement for each participant in both groups was 0.025 mL/cm². The cream was applied topically on wound surface using a 1-mL syringe and was spread evenly with a spatula. The wound was subsequently dressed with 3 M^™ Tegaderm^™ (Maplewood, Minnesota, the USA).

Cellulose acetate (CA) (MW ~50,000) and polyethylene oxide (PEO) (MW 8,000,000) were purchased from Sigma-Aldrich (Darmstadt, Germany). Sodium alginate (SA) (MW 216,121) was purchased from Cellco Chemicals SA (Athens, Greece). Pinus halepensis bark was collected from Kaisariani forest, a suburb near Athens, Greece. A voucher specimen has been deposited at the Herbarium of the Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens (ATPH/TP0115). The bark was pulverized in a blender and extracted with dH₂O in a 1:10 ratio for 48 h at 40 °C. The extract was filtered and freeze-dried to afford a solid residue. All reagents used were of analytical grade. Sterile gauze (Asepta Gauze^®, Asepta, Athens, Greece) and adhesive tapes (Aseptafix^®, Asepta, Athens, Greece; Rolltex^® Skin, Master Aid, Capannori, Italy; Tegaderm^TM, 3M, St. Paul, MN, USA) were purchased from a local pharmacy store (Athens, Greece).

Nurses inserted and maintained all PIVCs following the existing hospital policy, being like CPG recommendations. In summary, skin preparation before insertion was carried out with 2% chlorhexidine in 70% isopropyl alcohol. All PIVCs were Introcan Safety^TM (non-winged) catheters (B. Braun), with a needle-free valve directly connected to 10cm of extension tubing ending in a three-way connector (Becton Dickinson). A transparent dressing with polyurethane borders (Tegaderm^TM, 3M) was applied at the insertion site to secure the PIVC in situ. Standard caps on all needleless connectors were in place to minimise accidental tubing disconnections.

The NPWT device was the CuraVAC™ Ag (Daewoong Pharm Co, Ltd., Seoul, Korea), which has a foam dressing that contains silver nanoparticles. The following procedure was applied to the mice wounds: First, a sterile foam dressing was placed on the wound. Next, a circular hole was cut in the Tegaderm^TM (3M, Saint Paul, MN, USA), and a silicone suction head was placed through the hole. Then, the foam and an additional 2–3 cm of surrounding intact skin were covered. Finally, the suction head tube was connected to the vacuum pump. Negative pressure was applied via the therapy unit, causing the dressing to collapse into the wound. The negative pressure was maintained for 4 h at 125 mm Hg, and NPWT was applied for 7 days.