The first objective was to investigate new bone formation in a critical-size metaphyseal defect in the femur of ovariectomized rats filled with a strontium modified calcium phosphate cement (SrCPC) compared to calcium phosphate cement (CPC) and empty defects. Second, detection of strontium release from the materials as well as calcium and collagen mass distribution in the fracture defect should be targeted by time of flight secondary ion mass spectrometry (TOF-SIMS). 45 female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) SrCPC (n = 15), (2) CPC (n = 15), and (3) empty defect (n = 15). Bilateral ovariectomy was performed and three months after multi-deficient diet, the left femur of all animals underwent a 4 mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with SrCPC or CPC or was left empty. After 6 weeks, histomorphometric analysis showed a statistically significant increase in bone formation of SrCPC compared to CPC (p = 0.005) and the empty defect (p = 0.002) in the former fracture defect zone. Furthermore, there was a statistically significant higher bone formation at the tissue-implant interface in the SrCPC group compared to the CPC group (p < 0.0001). These data were confirmed by immunohistochemistry revealing an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin expression and a statistically significant higher gene expression of alkaline phosphatase, collagen10a1 and osteocalcin in the SrCPC group compared to CPC. TOF-SIMS analysis showed a high release of Sr from the SrCPC into the interface region in this area compared to CPC suggesting that improved bone formation is attributable to the released Sr from the SrCPC.

Staphylococcus aureus is the most clinically relevant pathogen regarding implant-associated bone infection and its capability to invade osteoblasts is well known. The aim of this study was to investigate firstly whether S. aureus is not only able to invade but also to proliferate within osteoblasts, secondly to delineate the mechanism of invasion and thirdly to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival of S. aureus. The SAOS-2 osteoblast-like cell line and human primary osteoblasts were infected with S. aureus EDCC5055 and S. aureus Rosenbach 1884. Both S. aureus strains were able to invade efficiently and to proliferate within human osteoblasts. Immunofluorescence microscopy showed intracellular invasion of S. aureus and transmission electron microscopy images could demonstrate bacterial division as a sign of intracellular proliferation as well as cytosolic bacterial persistence. Cytochalasin D, the major actin depolymerisation agent, was able to significantly reduce S. aureus invasion, suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 μg/mL of rifampicin was able to inhibit bacterial survival in SAOS-2 cells with almost complete elimination of bacteria after 4 h. Gentamicin could also kill intracellular S. aureus in a dose-dependent manner, an effect that was significantly lower than that observed using rifampicin. In conclusion, S. aureus is not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication of S. aureus whereas gentamicin only poorly eliminates intracellularly replicating bacteria.

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Management of bone and soft tissue infections generally includes surgical procedures as well as attendant treatment and prevention with gentamicin-loaded fleeces. Conventional gentamicin-containing collagen fleeces currently in use are strongly acidic and exhibit limited biocompatibility thereby adversely affecting wound healing. To improve the antibiotic delivery system, a new phosphate-buffered, gentamicin-loaded fleece with pH-neutral properties has been developed (Jason G). This study aimed at comparing the elution kinetics of gentamicin release and the antimicrobial efficacy of conventional fleeces with the newly developed fleece in vitro. In addition, degradation and microvasculature of implanted fleeces were examined in a rat model and assessed using histology, as well as detection of ED-1 and PECAM-expression using immunohistochemistry. We show that the phosphate-buffered fleeces have reduced release (p < 0.05) of the integrated gentamicin. However, all of the fleeces tested had a significant antimicrobial effect on the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains (p < 0.01). Among the fleeces tested, the new Jason G fleece had the weakest but nevertheless sufficient antimicrobial effectiveness. Evaluation of the antibiotic effect in the prevention of an infection showed no differences between the applied fleeces. Following surgical implantation of fleece in the backs of Wistar rats we observed, on day 5 after implantation, an increase in cell infiltration and microvascularization with the phosphate-buffered fleece as compared with conventional fleeces, which show necrotic cells on their surface. Unlike the acidic fleeces, on day 15 after implantation the pH-neutral fleece was resorbed widely. Here, we show that the new, pH-neutral, gentamicin-containing fleece Jason G exhibits good overall antimicrobial effectiveness against both gram-positive and gram-negative bacteria in vitro with improved degradation properties and microvasculature formation in vivo.