Strains isolation and culture conditions
One patient with clinical infection after fracture at the Xinhua Hospital was selected to isolate the clinical pathogenic bacteria. Bacterial specimens were obtained after washing the wound’s surface vigorously by saline solution, followed by debridement of superficial exudates. The microbiological culture was carried out under microaerophilic conditions for seven days. The bacterial specimens were tested by pyrosequencing analysis of bacterial diversity, and antimicrobial susceptibility tests were tested by the disk diffusion method.
OP-145 (acetyl-IGKEFKRIVERIKRFLRELVRPLR-amide) was synthesized by Shanghai Apeptide Co. Ltd. (Shanghai, China). OP-145 was puriﬁed by high performance liquid chromatography, and the identity was verified by SDS-PAGE. In addition, the purity of OP-145 (>95%) and mass were conﬁrmed by electrospray ionization mass spectrometry.
Preparation of PLGA microspheres
OP-145 containing PLGA microspheres was prepared by previously described methods. Briefly, 200 mg of PLGA was dissolved in methylene chloride (45%, w/v). The OP-145 solution was then added to the polymer solution to form the organic phase and mixed via vortexing. The organic phase containing both the polymer and drug was then added at once to 100 mL of 0.35% (w/v) PVA solution (0.22-μm membrane filtered) to form an oil-in-water (o/w) emulsion using a homogenizer set at 14,000 rpm for 1 min. Next, leuprolide acetate microspheres with large particle sizes were prepared using a lower emulsification/size reduction force (9000 rpm for 1 min). The resultant o/w emulsion was stirred at 400 rpm for 3 h under vacuum at room temperature to allow microsphere solidification and solvent evaporation. The resultant microspheres were collected by centrifugation, washed using distilled water, and freeze-dried using a vacuum manifold. The drug release of OP-145 from PLGA microspheres was calculated by the methods of Garner J . To investigate the morphology of the PLGA microparticles, the PLGA microparticles were first treated by freeze-drying, observed under a scanning electron microscope (SEM, NNS-450, FEI, USA), and analyzed by x-ray photoelectron spectroscopy (XPS) .
Sixty adult female Sprague‐Dawley (SD) rats (weighing 260–320 g) were used in this study. Twenty rats in the control group were treated with physiologic saline solution after surgery, and 20 rats in the treatment group were treated with OP-145 PLGA microspheres and vancomycin after surgery. All surgery was performed under sodium pentobarbital anesthesia, and euthanasia was accomplished with CO2
The rats were anesthetized via inhalation of 2% isoflurane. The surgical procedure was as described in the previous work with few modifications. The skin was incised at the proximal end of the specimen to facilitate loading directly and longitudinally over the lateral upper forelimb. Dissection continued up to the fascia of the biceps and brachialis, which were then retracted to reveal the midshaft humerus. A femoral fracture was created with an electric drill, and normal saline was not ejected after the drill stopped.
An inoculum of clinically isolated bacteria (1×105 CFU/mL) in 2 mL of normal saline solution was pipetted into the femur space. The surgical site was closed with Dexon 5-0 sutures. The weights of the rats were recorded at 0, 1, 2, 3, 7, and 14 d. C-reactive protein (CRP) levels were measured by enzyme-linked immunosorbent assay (ELISA) at 12, 24, 36, 48, and 72 h.
Detection of biofilm formation
The biofilms were harvested from euthanized rats on days 1, 2, and 3 to test biofilm formation in the present rat model. The specimens were prepared according to the previous methods. The specimens were fixed in 2.5% glutaraldehyde at 4°C for 1 h and stained with LIVE/DEAD staining . The biofilms were observed under a Nikon 80i microscope equipped with an argon laser with an excitation wavelength of 488 nm (green fluorescence). All the images were captured and saved using Nis-Elements AR software (Nikon, Tokyo, Japan). The integrated optical density (IOD) of biofilm was evaluated using the Image-Pro-Plus.
The cytotoxicity of OP-145 (0.5 and 1 µg/mL) to bone marrow stromal cells (BMSCs) was assessed using an MTT cell proliferation kit (Roche Applied Science). The BMSCs were incubated at 37°C under 5% CO2 for 1 and 2 days after cell inoculation as described previously. Next, a 50-mL volume of MTT working solution was added to each well, and the mixture was incubated for another 4 h. Purple crystal formazan was observed around cells at ×40 magnification under a microscope. The cell medium was carefully removed, and then 100 mL of dimethyl sulfoxide was added to each well to dissolve formazan. After 15 minutes of incubation at 37°C to completely dissolve formazan, the absorbance was measured at 490 nm on an enzyme-linked immunosorbent assay (ELISA) plate reader, and the results were expressed as optical density (OD) values. The OD values were calculated from concentration-response curves and used as a measure of cellular sensitivity.
The data were analyzed using GraphPad Prism 5.0. One-way ANOVA and Tukey multiple comparison tests were used to compare different treatments. A P-value<0.05 was considered statistically significant.