Monitoring Endothelial and Tissue Responses to Cobalt Ferrite Nanoparticles and Hybrid Hydrogels - Fig 3

<p>(A) Endothelial cell number after incubation for 3 days with CMC alone, CoFe₂O₄-CMC or CoFe₂O₄-NH₂-CMC (0.5 mg/ml). Data are means±SEM. (B) ECs survival was evaluated by MTT test. Cells were exposed to CMC, CoFe₂O₄-CMC or CoFe₂O₄-NH₂-CMC (0.5 mg/ml) for 3 days and data are expressed as absorbance at 540 nm. (C-G) Evaluation by western blot of the expression of markers of apoptosis (C), cleaved caspase-3 and cell cycle arrest (E, p53, and F, p21) in HUVEC exposed to CMC, CoFe₂O₄-CMC or CoFe₂O₄-NH₂-CMC (0.5 mg/ml) for 24 h. Blots are representative of 3 experiments with overlapping results. (G) Data in the graph represent the quantification of the protein of interest vs total caspase-3 or beta actin, and are expressed as fold increase vs Ctr.</p

Histological analysis of skin and subcutaneous tissue around the implant.

<p>Panels a and b are representative images of dermis and epidermis near implants of CMC and CoFe₂O₄-NH₂-CMC, respectively. Panels c and d show tissue reactivity to the biomaterials. The CMC scaffold is no longer visible probably due to biodegradation. Representative images at 20X (A, C) and 63X (B,D) magnification. Legend: *epidermis, ♦ dermis, ■ hair follicles. Arrows indicate inflammatory cell infiltration (neutrophils, macrophages/monocytes, lymphocytes). Nanoparticles are clearly visible in phagocytes (white arrowheads).</p

Monitoring Endothelial and Tissue Responses to Cobalt Ferrite Nanoparticles and Hybrid Hydrogels - Fig 2

<p>(A) Immunofluorescence analysis of actin in HUVEC treated for 24 h with CoFe₂O₄ NPs or CoFe₂O₄-NH₂ NPs (0.25 mg/ml). (B) Permeability in HUVEC monolayers was detected as passage of FITC-dextran from upper to lower compartment of a transwell. Data are expressed as relative fluorescence units. **p<0.01 vs Ctr. (C) ROS measurement after 2 h of exposure with the different NPs. Data are expressed as relative fluorescence units. **p<0.01 vs Ctr. (D-E) Expression of COX-2 and iNOS in HUVEC exposed for 24 h to CoFe₂O₄ NPs or CoFe₂O₄-NH₂ NPs (0.25 mg/ml) measured by western blot. Blots are representative of 3 experiments with overlapping results. (E) Data in the graph represent the quantification of the protein of interest vs beta actin and are expressed as fold increase vs Ctr. *p<0.05 vs Ctr and **p<0.0 vs Ctr.</p

Macroscopic analysis of subcutaneous implants of hybrid hydrogels in mice.

<p>Sterilized biomaterials were implanted subcutaneously in C57 black mice under anaesthesia. Surgical implants were closed with suture thread. Animals were sacrificed 7 days after implant; the subcutaneous implant was exposed and photographed.</p