A new model of implant-related osteomyelitis in the metaphysis of rat tibiae

The implant used in the present study was a custom-made titanium (Ti6Al4V) screw (Fig. 1). The screw was coated with HA and HA-Ag (Atesos medical AG, Aarau, Switzerland; Medicoat AG, Mähenwil, Switzerland) according to a manufacturing process previously described (Fig. 2) [14]. Briefly summarized, a modified technique of Vacuum Plasma Spraying (VPS) coating was used so that a thickness of ~100 μm was achieved for the coatings. The HA-Ag coating contained low amounts of Ag (45 ppb). The morphology of the surface was analyzed by Scanning electron microscope (Hitachi TM-3030Plus) using secondary electron detector. The surface roughness was determined on the apical position of the implant by Confocal Laser Scanning Microscope (Lext OLS 3100, Olympus) equipped with a 50× objective. The R_a-1D-values were determined by averaging n = 10 profiles of length 260 μm, using a cut-off wave-length of 26 μm. The SR_a and SR_z-2D-values were defined of n = 5 areas of 295 × 20 μm² also applying a cut-off wave-length of 26 μm. Due to the HA-VPS-coating, the surface of both implants showed a rough surface morphology with consistent arithmetic average roughness R_a of 2.49 ± 0.62 μm for group I (HA) and 2.21 ± 0.47 μm for group II (HA-Ag) surface moodification. The 2D surface rouhness parameters SR_a (arithmetic average roughness) and SR_z (maximum height) listed in Table 1 were also identical for both groups. The coarse surface topography was observed in the SEM image showing a homogeneous VPS-coating for either groups (Fig. 3).

The bacterial strain selected in the study was one of the most common causative pathogens associated with PII, namely S. aureus (ATCC25923, LGC Standards GmbH, Wesel, Germany). The strain was routinely cultured in Columbia Agar with 5 % sheep blood (Becton Dickinson, Heidelberg, Germany) at 37 °C overnight before testing. Bacteria were then harvested by centrifugation, rinsed, suspended, diluted in sterile phosphate buffered saline (PBS) and adjusted by densitometry (MacFarland Densimat™, BioMérieux, Marcy l’Etoile, France). For the study every suspension with its known bacterial concentration was diluted with NaCl to reach the targeted value for bacterial concentration (Group I-IIA: 10² CFU/10 μl, Group I-IIB: 10³ CFU/10 μl). In order to obtain log-phase growth over-night freezing of strains was strictly avoided and incubation of bacteria at 37 °C throughout the preparation time was conducted. To quantify the bacterial load prior to inoculation serial dilutions of the residual suspensions were incubated on agar plates for 48 h at 37 °C, and the number of inoculated viable cells was then calculated.

The study was approved by the Animal Experimentation Ethics Committee of Bavaria (Reg. No. 146-14) and was conducted with reference to the OECD Principles of Good Laboratory Practice. According to the committee’s recommendation that the number of experimental animals be minimized, the sample was limited to 12 rats per group and both tibiae were used for study purposes. Hence, 24 male, 5-month-old Wistar rats (Charles River Laboratories, Sulzfeld, Germany) with a mean body weight (BW) of 378.6 g (range: 353–401 g) were used. For acclimatization, the animals were delivered to the animal facility at least 1 week prior to treatment. Animals were housed in cages (2-5 animals) at normal room temperature and daylight illumination with ad libitum access to food and water.

Surgery was performed under general anesthesia induced by weight-adapted intramuscular injection of Medetomidin (150 μg/kg BW), Midazolam (200 μg/kg BW) and Fentanyl (5 μg/kg BW). Blood was collected by puncture of the tail vein prior to surgery.

Animals were prepared for surgery as follows (Fig. 4): Both hind legs were shaved, antisepticized with povidone–iodine, and dried. Bodies were covered with sterile sheets except for the hind legs. A skin incision (length, 1–2 cm) was made over the proximal lateral tibial metaphysis. A unicortical hole with a depth of ~8 mm was drilled using a 1.8 mm diameter drill bit. The drill hole was then tapped with a custom-made stainless steel tap and dried with gauze. After removal of the gauze 10 μl of either sterile PBS (left tibia = control) or PBS/S. aureus in the entitled concentrations (right tibia = infected) were injected into the hole with a 25 μl microsyringe (Hamilton, Reno, NV). The implant was inserted into the cavity immediately after injection of the solutions using a dedicated instrument. Soft tissue was irrigated with saline solution, the fascia was closed using absorbable suture material (Vicryl rapid, Ethicon Inc., Cincinnati, USA; size 6-0), the skin was closed by continuous intracutaneous (4-0 Monocryl, Fa. Ethicon, Norderstedt, Germany) and interrupted sutures (4-0 Prolene, Fa. Ethicon, Norderstedt, Germany). Anesthesia was antagonized after surgery by a subcutaneous injection of 750 μg/kg BW atipamezol (Alzane, Pfizer, Berlin, Germany), 200 μg/kg BW flumazenil (Flumazenil-ratiopharm, Ratiopharm, Ulm, Germany) and 120 μg/kg BW naloxone (Naloxon-Ratiopharm, Ratiopharm, Ulm, Germany). Metamizole (110 mg/kg BW) was administered subcutaneously (s.c.) at the beginning of the surgery. Additionally, buprenorphine (0.05 mg/kg BW) was administered at the end of the surgical procedure immediately before the wound closure was completed. Postoperative pain control was also carried out with buprenorphine (0.05 mg/kg BW s.c., 2 x daily) for 2 days. In addition, meloxicam was given s.c. immediately postoperatively and then followed for days 1–3 every 24 h at a dose of 1 mg/kg BW.

Rats were divided into two groups according to the type of implant inserted (Table 2).

The animals were sacrificed after 42 days. Under general anesthesia 3 ml of blood was collected by puncture of the right atrium and a lethal dose of pentobarbital (80 mg/kg BW) was injected. Tibiae of both hind legs, starting with the control site (left) were dissected under sterile conditions and further investigations were conducted.

Radiographs were taken in two planes immediately after implantation and on the day of sacrifice. For X-rays, digital films (DLR Cassette, Digiscan 2H/2C, Siemens) and a Mobilett Plus X-ray unit (Siemens AG, Erlangen, Germany) were used. Three regions of interest (ROIs) were determined (R1, epiphysis; R2, metaphysis with implant; R3, proximal diaphysis) and investigated separately for [5]:

Parameters 1–4 ranged from 0 (absent), 1 (mild) to 2 (severe), and were evaluated separately for ROIs 1–3. Parameter 5 was evaluated for present or not present. If the implant was loose the average score for parameters 1–4 was doubled. The maximum score possible was 48 (3 ROIs × 4 parameters × 2 points × 2 for loose implant). We did not perform radiological evaluations in the acute phase (first weeks after operation) as most radiological signs of bone infection are not apparent in this phase and therefore an additional general anesthesia for the animals would be of minor scientific benefit [6].

The screw was removed manually using a sterile custom-made screwdriver and afterwards investigated macroscopically for attached bone remnants. Resistance-to-removal (RTR) was evaluated semiquantitatively, attached bone remnants (ABR) were scored quantitatively. The following scoring system ranging from 1–5 points for either items (RTR and ABR) was applied (Table 3, Fig. 5). Means of either values (RTR and ABR) were summarized and osseointegration-score was calculated accordingly (range: 2–10). Additionally, two sterilely inserted implants of group I (HA) and II (HA-Ag) were randomly chosen and not removed but histologically evaluated for osseointegration [15]. Specimens were sectioned by a diamond band saw (Exakt 300P) in the middle of the implant, glued to a support, and sectioned again, so that a 200 mm-thick sample from the middle was attached to the support. The thickness of this sample was further reduced to 40–60 mm by a grinding machine (Exakt 420 CS). To visualize tissues, the samples were surface stained by toluidine blue.

One randomly chosen tibia from each group (IA/B, IIA/B; one from the control site, one from the infected site = totally eight specimens) was fixed for two days in 5 % formaldehyde and decalcified in 5 % nitric acid solution. The tibia was embedded in paraffin and cut into 4 mm thick sections. Slices were stained with haematoxylin/eosin. Three ROIs according to the radiographical evaluation were analyzed on [7, 12]:

Parameters 1 to 5 were scored with 0 (absent) or 1 (present). Parameter 6 was scored from 0 (absent), 1 (mild) to 2 (severe). The maximum score was 21. (3 × ROIs (parameter 1–5) × max. 1 point + 3 × ROI (parameter 6) × max. 2 points).

The animals recovered quickly after surgery and showed no signs of discomfort. There was no statistically significant difference in mean weight gain between the groups if the inoculated bacterial load was considered (Group I/IIA: 30 ± 11 g, Group I/IIB: 23 ± 17 g).

The course of hemoglobin (day of surgery vs. day of sacrifice) showed no significant difference between the groups throughout the experimental period. Further, infect parameters (white blood cell count, platelets) showed no significant elevation at day of sacrifice (Table 4).

All retrieved specimens of the control sites of groups I-IIA/B (inoculated PBS) showed no signs of bone infection (Table 7). In these groups large amount of newly formed bone around the implants and in contact with the implant surface were observed suggesting good biocompatibility of the implants. All histological slices from infected sites showed typical signs of chronic bone infection (Fig. 6). Infection signs were only detectable in ROIs 1 (epiphysis) and 2 (metaphysis). Mean score value of control sites (PBS) was zero. Due to the small sample size per group no statistical analysis was conducted (Table 7).

X-rays of all infected sites revealed radiographic signs of osseous destruction in ROIs 1 (epiphysis) and 2 (metaphysis). No signs of infection were detected in ROI 3 (proximal diaphysis). Osteolysis, soft-tissue swelling and implant loosening were the most common findings, sequestrum formation and periosteal reaction on the other hand was never observed. In contrast to infected sites, X-rays of the control sites showed no signs of osteomyelitis (Fig. 7, Table 7).

All implants of the control site showed excellent osseointegration with average score values ranging from 4 to 5. All infected sides on the other hand showed poor resistance to screw-out. Values ranged from 2 to 3. The differences between the groups were statistically significant (Table 7). Evaluation of osseointegration by histology showed broad bone integration throughout the whole surface of HA and HA-Ag implants (Fig. 8).

The datasets supporting the conclusions of this article are included within the article.