Bacteria and biofilm formation

MSSA strain ATCC29213 (American Type Culture Collection, Manassas, VA, USA) was used as it tends to form biofilms32,33. MSSA was streaked onto plates containing tryptic soy broth and Bacto agar (BD Biosciences, Franklin Lakes, NJ, USA) and grown overnight in 5 mL of tryptic soy broth at 37 C in a shaking incubator. MSSA cells in the incubation medium were grown to the early exponential growth phase (0.20.3 optical density at 600 nm), corresponding to 5.0 107 CFU/mL.

Adipose tissue (~1.5 g) was obtained from Wistar rats (female; 12 weeks old; Japan SLC Corp., Shizuoka, Japan). ADSCs were prepared by modifying previously reported methods34. Further details can be found in the Supplementary file. Cellular characteristics (i.e. expression of stem cell surface markers) were determined using flow cytometric analysis after labeling ADSCs with appropriate antibodies of cultivation.

Wistar rats (female; 12 weeks old; Japan SLC Corp.) were housed under specific pathogen-free conditions with a 12-h light/dark cycle and ad libitum access to a certified diet (CRF-1; Oriental Yeast Corp., Tokyo, Japan) and water (chlorine concentration; 10 ppm). The drinking, feeding behavior, and body weight of the rats were monitored regularly. The animals were acclimatized for 7 days before undergoing the implant operation.

Rats were anesthetized with midazolam (2.5 mg/kg; Astellas Pharma, Tokyo, Japan), medetomidine (0.5 mg/kg; Zenoaq, Fukushima, Japan), and butorphanol tartrate (2.5 mg/kg; Meiji Seika Pharma, Tokyo, Japan). To establish infection, A medical-grade K-wire (1.2 mm diameter; Synthes Inc., West Chester, PA, USA) was incubated in an overnight culture with MSSA strain ATCC29213 and then air-dried for 20min prior to insertion. This MSSA strain exposure coats the screw with 5107 CFU. The K-wire was surgically placed into the distal femur as previously described34,35,36. Briefly, the skin overlying the leg was shaved and cleaned with iodine solution. A medial parapatellar approach was used, and the patella was dislocated laterally to access the knee joint. The femoral medullary canal was reamed with an 18-gauge needle and the K-wire was placed in a retrograde fashion with 1 mm of the wire protruding into the joint space. The quadriceps-patellar complex was reduced to the anatomic position, and the wound was closed with nylon 4-0 sutures. Rats were randomly assigned and equally divided into three groups: no-treatment, antibiotic (ciprofloxacin [3.0 mg/kg per day intravenously]), and ADSCs [5.0 105 cells intravenously 30 min, 6 h, and 18 h after the surgical procedure]) with antibiotic (ciprofloxacin [3.0 mg/kg per day intravenously] groups. The ADSC dose, based on a previous report37, is considered to not induce adverse effects, including a high mortality rate. Additionally, a previous report showed that a ciprofloxacin dose of 3.0 mg/kg per day caused no adverse effects or unstable conditions in rats11. MSSA induced infection in 100% of the untreated rats with no significant differences in the initial body weights between the different groups.

After evaluating the general overall condition and soft tissue swelling, the rats were euthanized on POD 14 using thiopental sodium (100 mg/kg body weight). Tissues from the knee joint space, femur, and implant were harvested in a sterile manner for ex vivo analyses.

Weight change (n = 6 rats per group) was calculated as a percentage change based on the preoperative weight to quantitatively measure the systemic response to infection. Preoperative baseline measurements were performed on the day before surgery. The weight of the rats was also evaluated on PODs 1, 3, 7, and 14.

Soft tissue and bone damage (n = 6 rats per group) on POD14 was scored by three examiners (D.I, A.T. and T.K.) blinded to the rats according to a modified Rissing scoring38,39. Further details can be found in Supplementary file.

CT imaging (n = 6 rats per group) was performed on POD14 to determine the degree of infection within the femoral region of interest. Considering that image artifacts from the K-wires may cause artifacts in the reconstructed CT images, isolated femurs from rats with the wire removed were subjected to CT scanning (LaTheta LCT-200; Hitachi Aloka Medical, Tokyo, Japan), operating at 50 kV and 0.5 mA (radiation exposure remained below 40 mSv). BMD was calculated automatically using LaTheta software (version 3.51). Reconstructed CT images were initially visualized in three dimensions (3D) to evaluate changes in bone morphology resulting from implant infection. A threshold-limited 3D rendering was created to visualize bone damage.

Implants were harvested (n = 6 rats per group) from each group. Based on a previous report, the bacterial burden on the implants was determined using a CFU assay40,41. To quantify living bacteria adherent to the implant within the biofilm, the removed implants were placed individually into 1.5-mL microtubes containing PBS (1 mL at 4C), vortexed for 15 s and sonicated for 5 min at 40 Hz in a water bath (Bransonic 5210; Branson Ultrasonics, Brookfield, CT, USA), followed by an additional 1 min of vortexing. The spread plate method was used to quantitatively evaluate the biofilm; the solution containing each bacterium from the biofilm was serially diluted 10-fold with PBS, followed by culturing on an agar plate at 37C for 24 h. MSSA was cultured on tryptic soy broth agar plates. The bacterial CFUs obtained from the implant were determined by counting the CFUs after culturing on plates overnight.

At the established endpoint (POD14), the femurs isolated from the rats were fixed in 10% neutralized formalin solution and dehydrated using an ethanol gradient (70%, 80%, 90%, and 100%). The fixed specimens were decalcified in 10% formic sodium citrate solution, embedded in paraffin, and sectioned in the coronal plane. The sections were stained with hematoxylin and eosin, and the slides were observed using an optical microscope (Biorevo BZ-9000; Keyence Corp., Osaka, Japan).

At the established endpoint (POD14), total RNA was extracted from the knee tissue of the rats (n = 6 rats per group). The mRNA expression of rCRAMP, TNF-, IL-6 and IL-1b was evaluated by quantitative PCR. All values were normalized to the level of the GAPDH gene, and relative gene expression levels were calculated using the 2Ct method42. Further details can be found in the Supplementary file (Supplementary Table S1).

Tissue sections were evaluated to determine the location of ADSCs following injection. To confirm the location of the injected ADSCs, they were labeled with the fluorescent dye DiI (Vybrant DiI Cell Labeling Solution; Life Technologies, Carlsbad, CA, USA) before injection. DiI binds to cellular thiols and has long-term stability, enabling the tracing of DiI-labeled transplanted cells in the host tissue. The concentration of ADSCs was adjusted to 5.0 105 cells/mL; DiI (5 L/mL) was dissolved in the cell culture media and incubated for 15 min at 37C in a 5% CO2 incubator for ADSCs labeling. The filtrate was centrifuged at 180g for 5 min at 25C and the supernatant was removed to separate the DiI from the filtrate. The ADSCs were centrifuged twice with Dulbeccos modified Eagle medium under the same conditions and the supernatant was removed. We used separate rats for this experiment (n = 3 rats per antibiotic group and DiI-labeled ADSCs with antibiotic group). On day 14 post-injection, a frozen section was prepared using Kawamotos film method in the sagittal plane43. For identification of tissues following DiI labeling, the gray-scale scale (16 bit) of the DiI-labeled section was used.

All continuous variables were assessed for normality using the ShapiroWilk test. Normally distributed data were expressed as the mean standard error. Data were analyzed using SPSS software (version 25.0; SPSS, Inc., Armonk, NY, USA). Multiple groups were compared using the Welch ANOVA followed by Tukey HSD or Games-Howell post-hoc test. For all analyses, results were considered statistically significant at p < 0.05.

The investigational protocol was approved by the Kanazawa University Advanced Science Research Centre (Approval Number: AP-194052), and all animals were treated in accordance with Kanazawa University Animal Experimentation Regulations. The study was carried out in compliance with the ARRIVE guidelines.

Visit link:
Combined adipose-derived mesenchymal stem cell and antibiotic ... - Nature.com