Anti-infective potential and biocompatibility in vivo

We here established a subcutaneous implantation model to evaluate the anti-infection performances of the sutures in vivo. All experimental procedures were approved and performed in accordance with the guidelines of the Animal Ethics Committee of Shanghai Ninth People’s Hospital. In brief, 24 6-week-old Sprague-Dawley male rats weighing approximately 300 g were assigned randomly to four independent groups: V, VP, GV, and HV. The rats were initially anaesthetised using an intraperitoneal injection of 1% pentobarbital sodium (100 mg/kg body wt) and both of the lateral thighs were shaved and cleaned with 2% iodine prior to the procedure. An approximately 3 cm longitudinal skin incision was made over the lateral thighs and reached the musculoaponeurotic layer, then the superficial muscle was incised at a length of approximately 2.5 cm. Prior to the suture of the incised superficial muscle in the left thigh, these tested sutures were incubated with the prepared ATCC43300 suspensions (1 × 10⁶ CFUs/mL in PBS) for 5 minutes. Meanwhile, sutures without bacteria inoculation were implanted in the right thigh to evaluate the in vivo biocompatibility. Then, the skin closure was performed using 4-0 Mersilk nonabsorbable sutures (Ethicon). The rats were housed in ventilated rooms and allowed to eat and drink ad libitum after surgery. The animals were sacrificed 21 days after implantation. No antibiotics were administered, and no mortality occurred during the experiment. All animal operations were performed by the same person under the same experimental condition.

The implanted sutures and the peri-implant tissues in each group were harvested at 21 days after implantation, and then immersed in 4% neutral-buffered formaldehyde for 48 hours. The collected specimens were then embedded into paraffin. The embedded specimens were cut into sections parallel to the cross-section of the sutures and prepared at a thickness of 5 μm (EXAKT-400 grinding equipment, SLEE Medical, Mainz, Germany). Haematoxylin and eosin and Giemsa staining were used to assess morphology and bacterial contamination, respectively. Meanwhile, immunohistochemical staining was also used to observe the inflammatory cells (lymphocyte and macrophage) surrounding the sutures according to the previous protocol [38]. In brief, prepared slices were incubated overnight at 4°C with anti-CD3 antibody (1:100, ab16669, Abcam, Cambridge, UK) or anti-CD68 antibody (1:400, ab955, Abcam) after deparaffinisation, rehydration with descending concentrations of ethanol, quenching of endogenous peroxidase, and blocking with 3% bovine serum albumin. Then, the sections were visualised using diaminobenzidine (DakoCytomation, Glostrup, Denmark) containing biotinylated anti-rat IgG secondary antibody. The histological images were captured on a Leica AF6000 (Leica Microsystems, Wetzlar, Germany).