Currently, no clinical options are available to prevent infections on uncemented orthopedic implants. Therefore we investigated the efficacy of DAC-hydrogel (disposable antibacterial coating(1), Novagenit, Italy) as carrier for various agents to prevent infections in an in vivo implant-model.

Titanium rods were implanted in the left tibiae in New Zealand White rabbits. Prior to implantation, the implant bed was contaminated with 10∧5 colony forming units S. aureus.

In the experimental groups, the hydrogel was loaded prior to be coated on the rods with: 2%(w/v) vancomycin (Van2 group, N=6), 5%(w/v) vancomycin (Van5 group, N=6), 10%(w/v) bioactive glass (BonAlive, Finland) (BAG group, N=6), which is antibacterial(2) and osteoconductive(3), or 0.5%(w/v) N-acetyl cysteine (NAC group, N=6), which inhibits bacterial growth and decreases biofilm formation(4). In the control group, empty hydrogel was applied (Gel group, N=12)

Blood values were measured weekly. Following explantation on day 28, the anterior tibia was processed for bacterial culture. The posterior tibia and rod were used for measuring bone-implant contact using micro-CT and for histopathology.

Results of the experimental groups were compared to the Gel group results. The blood values in the Van2 and Van5 groups were lower on day 7. Moreover, culture results demonstrated less animals with an infection in both groups at day 28. In accordance, these groups showed lower grades for infection. Further, the Van2 group demonstrated more bone-implant contact. These results suggest that infection was reduced in the Van2 and Van5 groups. In contrast, blood values, histological grades, and bone-implant contact of the BAG and NAC groups were comparable with the Gel group. These results suggest that infection was not prevented in the BAG and NAC groups.

Local application of vancomycin-loaded DAC-hydrogel successfully reduced implant-related infections. Loading of the hydrogel with BAG or NAC did not prevent infection. It is possible that BAG in powder form, as used in the present study, dissolved before the antibacterial effect could take place. Instead, BAG granules may be a viable alternative. Next, it is possible that the NAC concentration was too low to prevent infections in an in vivo environment, although this concentration was proven effective in vitro for its antibacterial properties.

Correct diagnosis of infection is crucial for an adequate treatment of orthopedic implant-related infections. In the orthopedic field, infections can be difficult to diagnose(1). As a consequence, patients may suffer from an undiagnosed and untreated implant-related infection.

To solve this problem, we are searching for a diagnostic method to detect these so-called low-grade infections. The technique fluorescence in situ hybridization (FISH) can detect slow-growing and even dead bacteria. Further, as FISH results are available within an hour after tissue collection it is an ideal candidate for diagnostic purposes.

AIM: to evaluate the FISH technique for its potential to detect and identify orthopedic infections.

Sonication fluid (SF) was collected by sonicating retrieved implants(2) from 62 patients. All samples were subjected to bacterial culture for clinical diagnostics. In addition, a commercially available FISH kit (miacom diagnostics, Germany), specifically designed for blood analysis (hemoFISH Masterpanel), was used. The kit contained 16S rRNA probes (positive control), non-sense probes (negative control), probes for Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acetinobacter spp., and Stenotrophomonas maltophilia. All FISH analyses were performed according to the protocol provided with the kit.

Culture and FISH results were compared, considering culture as the gold standard. Culture resulted in 27 positive and 35 negative samples. Comparing FISH (16S rRNA probe) with culture, 24 samples tested true-positive and 32 samples true-negative. Furthermore, 3 samples tested false-negative and 3 samples false-positive.

The species cultured with the highest incidence were Propionibacterium acnes and Staphylococcus epidermidis, both from 8 SF samples. As the kit did not contain a probe for Propionibacterium acnes, these strains were only detected by the 16S rRNA probe. In addition, the latter samples tested positive with the Staphylococcus spp. probe.

Interestingly, 3 samples tested positive with FISH that were culture negative. This result could indicate a higher sensitivity for detection of bacteria with FISH than with culture.

Before FISH can be used for diagnostic purposes, the technique needs to be optimized to prevent false-negative results, for use on other patient materials and for detection of bacterial strains relevant for the orthopedic field like Propionibacterium acnes.

In conclusion, FISH holds promise to be used as a diagnostic tool for identifying orthopedic infections.

Summary Statement

Prolonged presence of VEGF (released from gelatin microspheres) led to a significant increase in scaffold vascularization when applied in vivo. Bioprinted scaffolds with regional VEGF presence retained their architecture and regional vessel formation occurred.

Background

Both from experimental studies and the large arthroplasty registries there is evidence that bacteria are more often involved in implant loosening then is currently reported. To further elucidate this potential problem, the current study investigated the hypothesis that many total hip arthroplasty revisions, classified as aseptic, are in fact low-grade infections missed with routine diagnostics.

Of 960 first-revision total hip replacements (THR) because of deep infection identified in the Swedish Hip Arthroplasty Registry, 16.9% were treated with a permanent implant extraction, while a staged or direct reconstruction revision protocol were employed in 56.2% and 26.9% respectively. The majority of the interventions were performed more than one year after index THR, and the dominating pathogen was coagulase negative staphylococci (CNS). We found a significant shift in types of bacteria over the years (Chi-square test, p smaller than 0.001): an increase in the CNS group and a decrease in Gram-negative aerobes. Patients treated with a permanent resection were generally older (p< 0.001), had more often a previous ipsilateral hip fracture (p< 0.001), and had more frequently Gram-negative infections (p=0.02). No systematic differences in patient characteristics or pathogens were detected between one-stage or two-stage procedures, of which the latter had a median re-implantation time of 2 (range: 0.2–62) months. Of 798 (one- or two-stage) revisions, 60 (7.5%) were revised again due to recurrent infection, with no difference between the two methods, and implying a 10-year survival of 90%; 95% confidence interval (CI95%) 88.2–93.0. Previous surgery for soft-tissue problems (RR 3.2 (CI95% 1.3–7.2)) predicted a worse outcome for one-stage procedures. The prognosis of two-staged revisions improved with increasing re-implantation interval (RR 0.8 (CI95% 0.7–1.0)) per month, and a 6 month interval carried the lowest risk of repeat revision due to infection; RR 0.1 (CI95% 0.0–0.9). Staged revisions in female patients (RR 2.3, (CI95% 0.9–5.7)) and with Staphylococcus aureus infections (RR 2.3 (CI95% 0.9–5.5)) predicted a worse outcome. Ten-year survival with repeat revision for aseptic loosening as end-point was 89% (95%CI 85.7–92.0), but decreased to 79% when all reasons for revision were taken into account (95%CI 75.0–82.3) mainly because of revision for peri-prosthetic fractures.

The results suggest that direct and staged revision protocols can have a good prognosis on a national level, but efforts must be made to counteract periprosthetic fractures and the high incidence of permanent implant extraction in elderly patients.

The use of platelet-leukocyte gel (PLG), made from platelet rich plasma, to stimulate bone formation and wound healing has been investigated extensively. As leukocytes play an important role in the innate host-defence, we hypothesised that PLG might also have antimicrobial properties.

The purpose of this study was to investigate the antimicrobial activity of PLG against Staphylococcus aureus in an in vitro experiment. To determine the contribution of myeloperoxidase (MPO), present in leukocytes, in this process, MPO release was measured.

Platelet rich plasma (PRP) was prepared from whole blood of 6 donors. In this process platelet poor plasma (PPP) was obtained as well. PLG was prepared by mixing PRP with either autologous (PLG-AT) or bovine thrombin (PLG-BT). The antimicrobial activity of PLG-AT, PLG-BT, PRP and PPP was determined in a bacterial kill assay, containing 1x106 CFU/ml of Staphylococcus aureus, during a 24-hour period. MPO release was measured by ELISA.

Cultures showed a rapid decrease in the number of bacteria in the presence of both PLG-AT and PLG-BT, which was maximal between 4 and 8 hours, to approximately 1% of the bacteria in controls. Also PRP and PPP induced a statistically significant bacterial kill, but the effect of PLG-AT was the largest (p=0.093 vs. PLG-BT; p=0.004 vs. PRP and p< 0.001 vs. PPP). PLG-AT, PLG-BT and PRP showed a comparable, gradually increasing MPO release for 8 to 12 hours. Some MPO was also measured in the PPP samples. No correlation between MPO release and bacterial kill could be found.

PLG appears to have potent antimicrobial capacity, but the role of MPO in this activity is questionable. PLG might represent a useful strategy against postoperative infections. Further research should investigate its antimicrobial capacity in the in vivo situation.

A new type of metallic silver bone cement was previously shown to be effective against both antibiotic sensitive and resistant bacteria.

In this study the efficacy of silver bone cement in preventing methicillin- sensitive Staphylococcal infections was compared with plain and tobramycin-containing bone cement, in a rabbit contaminated implant bed model.

In 48 rabbits 0.6%-silver, 1%-silver, plain or tobramycin-loaded (tobra) PMMA bone cement (Simplex^®P; Howmedica, Ireland) was injected into the medullary canal of the right femur after contamination of the implant bed with 105, 106 or 107 colony forming units (CFU) of Staphylococcus aureus. After 14 days bone was collected, homogenised and plated on blood agar plates. After an overnight incubation the number of CFU’s was counted. Bone was also collected for pathological analysis.

The plain and silver cement rabbits were all infected, whereas with tobra cement only 2 rabbits (17%) were infected (p< 0.001). The number of bacteria cultured from bone adjacent to the cement, was 6.4±0.3 and 6.1±0.3 for the 0.6% and 1%-silver rabbits. For the rabbits with plain and tobra cement this was 6.2±0.2 (p> 0.95) and 0.0±0.0 (p< 0.001). Two tobra rabbits had a positive culture of a distal bone sample. Histological sections of plain, 0.6% and 1%-silver cement rabbits all showed signs of infection; these signs were absent in the tobra rabbits.

Silver cement was not effective in preventing infection. However, in the current model bacteria are present directly at and distant from the implant surface, whereas silver cement predominantly exhibits an antimicrobial effect at the direct cement surface. The non-eluting silver cement seems less useful in situations where there are also bacteria present in surrounding tissues, like revision surgery. Whether silver cement has relevance in preventing bacterial colonization of cement, for instance in late haematogenous infections, or not remains to be seen.