Representative micro-CT images and bone mineral density (BMD).

<p>A) Representative fluoroscopic image attests the correct placement of the implant within the femoral canal and the phantom calibration placed in the field of view; B) Magnified representative micro-CT images of the femurs containing metallic implants in transversal views in all the experimental groups. The images of groups I and III show an intact cortical bone profile of the whole femur. The image of group II shows a diffuse cortical and endosteal bone loss as signs of osteomyelitis; C) The histogram shows a high statistically significant difference in the BMD relative decrease of group II versus group III (unpaired t-test, ***P<0.0001; n = 5).</p

Histology of the femurs in the experimental groups (n = 4).

<p>Figures represent H&E staining in the left and middle panels and Gram-positive staining in the right panel. Legend: femur (F), tibia (T), cortical bone (CB), and medullary canal (MC). Group I - A) Normal aspect of the knee joint and absence of signs of infection (Magnification 2X, scale bar 1 mm); B) Absence of inflammatory cells in the medullary canal, of bone resorption or periosteal reaction (Magnification 4X, scale bar 0.5 mm) and presence of osteocytes within cortical bone lacunae (small box, Magnification 1000X); C) Absence of Gram-positive bacteria aggregates (Magnification 10X, scale bar 0.2 mm). Group II – D) Abscesses in the knee joint (black arrow) and in the medullary canal (Magnification 2X, scale bar 1 mm); E) Endosteal bone resorption (#), active osteoclasts (black arrows), marked periosteal reaction (*) (Magnification 4X, scale bar 0.5 mm) and diffuse enlargement of the medullary canal with osteoclastic resorption in the endosteal side (small box, Magnification 20X, scale bar 0.1 mm); F) Presence of numerous Gram-positive bacteria aggregates (Magnification 10X, scale bar 0.2 mm; small box, Magnification 1000X). Group III - G) Irregular surface of the articular cartilage of the knee joint and mild inflammatory changes of bone and joint (Magnification 2X, scale bar 1 mm); H) Diffuse increase of the vascular network and bone vessel enlargement (#) and areas of bone remodeling (black arrow) (Magnification 4X, scale bar 0.5 mm); large osteocytes embedded in cortical bone lacunae (small box, Magnification 1000X); I) Mild presence of dispersed Gram-positive bacteria within the medullary canal (Magnification 10X, scale bar 0.1 mm; small box, Magnification 1000X).</p

Bacterial load in bones of all the experimental groups.

<p>No colonies were detected in group I, the sham control (L.o.D.  =  limit of detection). With an infecting dose of 1×10³ CFU/mouse, a mean of 5.3±1.2 (Log CFU)/g of bone was found in the explants of group II, with a statistical difference in respect to group I. A mean of 3.6 ± 0.9 (Log CFU)/g of bone was found in the explants of group III, with a statistical difference in respect to group I (one-way ANOVA, *P<0.05; **P<0.01; n = 4).</p

Histological grading score histogram.

<p>The histogram shows a high statistically significant difference in the histological grading score in periosteum, cortex and medullary canal of group I versus group II and a difference in the score of the medullary canal of group I versus group III (two-way ANOVA, ***P<0.001; *P<0.05; n = 4).</p

Allocation of animals per group and investigations.

<p>Allocation of animals per group and investigations.</p

Bacterial load in bones of all experimental groups.

<p>The histogram compares the bacterial counts in the infected and control mice. After inoculation with an infecting dose of 1×10³ CFU/mouse, no colonies were detected in groups I, II and III. In contrast, a mean of 5.1±1.5 (Log CFU)/g of bone in the femoral canal was found in group IV (L.o.D.  =  limit of detection), which was statistically different from the other groups, as analyzed with one-way ANOVA and Bonferroni’s post-hoc (***P<0.0001; n = 4).</p

Gross appearance at explantation.

<p>Draining abscess in the knee joint (A) and the hip joint (B), involving the subcutaneous tissue in the mice in group IV (C).</p

Mouse surgical procedures.

<p>Anesthesia chamber with isoflurane inhalation (A); skin preparation (B); skin incision over the right knee (C); introduction of a 25-gauge needle retrograde into the femoral medullary canal (D); inoculation of 3 µl of <i>S. aureus</i> (E) and closure of the surgical site with interrupted sutures (F).</p

Representative micro-CT images and bone mineral density (BMD).

<p>Representative maximal intensity projection micro-CT images of the femurs with the pin implants in transverse (A, D) and sagittal (B, C, E, F) views in group I (upper panel) and group IV (lower panel). The upper panel shows the intact cortical bone profile without signs of infection (A), of the knee joint (B) and of the middle diaphysis section (C) in group I. The lower panel shows extensive bone loss, disruption of cortical integrity, cortical thinning and canal widening (D), acute septic arthritis of the knee joint (E) and bone loss in the middle diaphysis section (F) suggesting signs of established infection in group IV. The histogram shows a statistically significant difference in the BMD of group IV, as analyzed with one-way ANOVA and Bonferroni’s post-hoc (*P<0.05; n = 5).</p

Histology of the femur in groups I and III and in group II.

<p>H&E staining of longitudinal sections of femurs. Note the absence of inflammatory cells in the medullary canal, bone resorption and periosteal reaction in group I (A, B), group III (C, D), and group II (E, F) 28 days after surgery. Left panel magnification 4X (scale bars 0.5 mm); right panel magnification 10X (scale bars 0.2 mm).</p