Systemic and Local Administration of Antimicrobial and Cell Therapies to Prevent Methicillin-Resistant Staphylococcus epidermidis-Induced Femoral Nonunions in a Rat Model

Abstract

S. epidermidis is responsible for biofilm-related nonunions. This study compares the response to S. epidermidis-infected fractures in rats systemically or locally injected with vancomycin or bone marrow mesenchymal stem cells (BMSCs) in preventing the nonunion establishment. The 50% of rats receiving BMSCs intravenously (s-rBMSCs) died after treatment. A higher cytokine trend was measured in BMSCs locally injected rats (l-rBMSCs) at day 3 and in vancomycin systemically injected rats (l-VANC) at day 7 compared to the other groups. At day 14, the highest cytokine values were measured in l-VANC and in l-rBMSCs for IL-10. µCT showed a good bony bridging in s-VANC and excellent both in l-VANC and in l-rBMSCs. The bacterial growth was lower in s-VANC and l-VANC than in l-rBMSCs. Histology demonstrated the presence of new woven bone in s-VANC and a more mature bony bridging was found in l-VANC. The l-rBMSCs showed a poor bony bridging of fibrovascular tissue. Our results could suggest the synergic use of systemic and local injection of vancomycin as an effective treatment to prevent septic nonunions. This study cannot sustain the systemic injection of BMSCs due to high risks, while a deeper insight into local BMSCs immunomodulatory effects is mandatory before developing cell therapies in clinics.

Figure 1

Treatments in the experimental groups. (a) All animals received MRSE locally. The picture represents the injection of the bacterial suspension within the site of the fracture. (b) Representative picture of the local treatment with rBMSCs in the site of the fracture 24 h after surgery. The picture represents the transcutaneous injection of rBMSCs after disinfection within the site of the fracture. (c) The l-VANC group received a vancomycin-enriched hydrogel locally layered on the plate surface before the fracture stabilization and (d) within the site of the osteosynthesis after the plate fixation. The pictures represent the distribution of 250 µL of the vancomycin-enriched hydrogel on the bottom (c) and top side of the plate (d).

Figure 2

Representative histological panel of the rat lungs reporting the effects of the acute intravenous administration of allogeneic BMSCs in the s-rBMSCs group. The lung sections are stained with haematoxylin and eosin. (a, b) Presence of acute and diffuse hyperemia within the lung parenchyma. (c) Presence of multifocal alveolar edema (∗). (a) scale bar 1000 µm; (b) scale bar 200 µm; (c) scale bar 50 µm.

Figure 3

Clinical data. (a) The histogram shows the relative changes in body weight in the experimental groups over time. (b) The histogram shows the systemic neutrophil count in the experimental groups at days 14 and 42 after surgery. Comparisons between groups and time points were analyzed with two-way ANOVA and Bonferroni's post hoc test. Statistical significance was p < 0.05 (∗), p < 0.01 (A, ∗∗), and p < 0.001 (#); n = 6, n = 3 s-rBMSCs.

Figure 4

Cytokine analysis. The histograms show the cytokine values of the experimental groups at 3, 7, and 14 days after surgery and bacterial injection. Comparisons between groups and time points were analyzed with two-way ANOVA and Bonferroni's post hoc test. Statistical significance was p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗); n = 4, n = 3 s-rBMSCs.

Figure 5

Qualitative µCT imaging, isosurface, and semiquantitative osteomyelitis score. The representative panel shows µCT images on the day of explantation. (a, c, e, g, i) Sagittal, coronal, and axial planes. (b, d, f, h, j) 3D isosurface reconstruction is presented for the s-VANC, s-rBMSCs, l-VANC, l-rBMSCs, and PC groups. Symbols indicate cortical reaction (white arrows); medullary reaction (yellow arrow); and loss of cortical wall and osteolysis (asterisks). (k) Osteomyelitis grading score based on Odekerken's scale is reported in the histogram. Comparisons among groups were analyzed with one-way ANOVA corrected with Bonferroni's post hoc test. Statistical significance was p < 0.05 (∗) and p < 0.001 (∗∗∗); n = 6, n = 3 s-rBMSCs.

Figure 6

µCT quantitative analyses of bone structure. (a) Bone volume (BV) and (b) tissue mineral density (TMD) quantitative analysis of the treated groups normalized on the PC group, reported as fold increase. Comparisons among groups were analyzed with one-way ANOVA corrected with Bonferroni's post hoc test. Statistical significance was p < 0.05 (∗) and p < 0.01 (∗∗); n = 6, n = 3 s-rBMSCs.

Figure 7

Microbiological detection of bacterial growth on the explanted specimens. The limit of detection (L.o.D.) was set at 0.18 Log (CFU/g)/explant. Comparisons among groups were analyzed with one-way ANOVA corrected with Bonferroni's post hoc test. Statistical significance was p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗); n = 6, n = 3 s-rBMSCs.

Figure 8

Histological analysis (H&E) at the day of explantation. The representative panel shows H&E staining of the femurs in all the experimental groups. The panels depict an overview of the samples, scale bar 1 mm. WB: woven bone; LB: lamellar bone; PR: periosteal reaction; FV: fibrovascular tissue; MH: myeloid hyperplasia; vascular infiltrates (black arrow); alteration of cortical bone (green arrow). For the l-rBMSCs group, (a) a specific area containing fibrovascular tissue and polymorphonucleated cells is reported in the big black box (scale bar 200 µm) and (b) the presence of giant cells in the small black box (scale bar 100 µm) is reported. For the PC group, (c) a specific area with fibrovascular tissue is reported in the big red box (scale bar 200 µm) and (d) the myeloid hyperplasia is shown in the small red box (scale bar 200 µm).

Figure 9

Gram staining of the femurs at the day of explantation. The representative panel shows the Gram staining of the femurs in all the experimental groups. The panels depict an overview of the samples, scale bar 1 mm. In each group, the white boxes have a scale bar 10 µm and represent (a) detail of the s-VANC group; (b) detail of the s-rBMSCs group; (c) detail of the l-VANC group; (d) detail of the l-rBMSCs group; (e, f) details of the PC group.