Fabrizio Billi

A Mouse Model of Post-Arthroplasty Staphylococcus aureus Joint Infection to Evaluate In Vivo the Efficacy of Antimicrobial Implant Coatings

Background Post-arthroplasty infections represent a devastating complication of total joint replacement surgery, resulting in multiple reoperations, prolonged antibiotic use, extended disability and worse clinical outcomes. As the number of arthroplasties in the U.S. will exceed 3.8 million surgeries per year by 2030, the number of post-arthroplasty infections is projected to increase to over 266,000 infections annually. The treatment of these infections will exhaust healthcare resources and dramatically increase medical costs. Methodology/Principal Findings To evaluate novel preventative therapeutic strategies against post-arthroplasty infections, a mouse model was developed in which a bioluminescent Staphylococcus aureus strain was inoculated into a knee joint containing an orthopaedic implant and advanced in vivo imaging was used to measure the bacterial burden in real-time. Mice inoculated with 5×103 and 5×104 CFUs developed increased bacterial counts with marked swelling of the affected leg, consistent with an acute joint infection. In contrast, mice inoculated with 5×102 CFUs developed a low-grade infection, resembling a more chronic infection. Ex vivo bacterial counts highly correlated with in vivo bioluminescence signals and EGFP-neutrophil fluorescence of LysEGFP mice was used to measure the infection-induced inflammation. Furthermore, biofilm formation on the implants was visualized at 7 and 14 postoperative days by variable-pressure scanning electron microscopy (VP-SEM). Using this model, a minocycline/rifampin-impregnated bioresorbable polymer implant coating was effective in reducing the infection, decreasing inflammation and preventing biofilm formation. Conclusions/Significance Taken together, this mouse model may represent an alternative pre-clinical screening tool to evaluate novel in vivo therapeutic strategies before studies in larger animals and in human subjects. Furthermore, the antibiotic-polymer implant coating evaluated in this study was clinically effective, suggesting the potential for this strategy as a therapeutic intervention to combat post-arthroplasty infections.

Mouse model of chronic post-arthroplasty infection: noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study

Post‐arthroplasty infections are a devastating problem in orthopaedic surgery. While acute infections can be treated with a single stage washout and liner exchange, chronic infections lead to multiple reoperations, prolonged antibiotic courses, extended disability, and worse clinical outcomes. Unlike previous mouse models that studied an acute infection, this work aimed to develop a model of a chronic post‐arthroplasty infection. To achieve this, a stainless steel implant in the knee joints of mice was inoculated with a bioluminescent Staphylococcus aureus strain (1 × 102–1 × 104 colony forming units, CFUs) and in vivo imaging was used to monitor the bacterial burden for 42 days. Four different S. aureus strains were compared in which the bioluminescent construct was integrated in an antibiotic selection plasmid (ALC2906), the bacterial chromosome (Xen29 and Xen40), or a stable plasmid (Xen36). ALC2906 had increased bioluminescent signals through day 10, after which the signals became undetectable. In contrast, Xen29, Xen40, and Xen36 had increased bioluminescent signals through 42 days with the highest signals observed with Xen36. ALC2906, Xen29, and Xen40 induced significantly more inflammation than Xen36 as measured by in vivo enhanced green fluorescence protein (EGFP)‐neutrophil flourescence of LysEGFP mice. All four strains induced comparable biofilm formation as determined by variable‐pressure scanning electron microscopy. Using a titanium implant, Xen36 had higher in vivo bioluminescence signals than Xen40 but had similar biofilm formation and adherent bacteria. In conclusion, Xen29, Xen40, and especially Xen36, which had stable bioluminescent constructs, are feasible for long‐term in vivo monitoring of bacterial burden and biofilm formation to study chronic post‐arthroplasty infections and potential antimicrobial interventions.

Nanotoxicology of metal wear particles in total joint arthroplasty: a review of current concepts

Metal-on-metal (M-M) joint replacement has raised concerns about the long-term effects of metal wear debris and corrosion products. This review summarizes the current concepts in biological reactivity to metal wear particles, ions, and corrosion products. Attention is focused on Co-Cr-Mo alloy since it is the most diffused and discussed material in arthroplasty.

The John Charnley Award: an accurate and extremely sensitive method to separate, display, and characterize wear debris: part 2: metal and ceramic particles.

BackgroundMetal-on-metal and ceramic-on-ceramic bearings were introduced as alternatives to conventional polyethylene in hip arthroplasties to reduce wear. Characterization of wear particles has been particularly challenging due to the low amount and small size of wear particles. Current methods of analysis of such particles have shortcomings, including particle loss, clumping, and inaccurate morphologic and chemical characterization.Questions/purposesWe describe a method to recover and characterize metal and ceramic particles that (1) improves particle purification, separation, and display; (2) allows for precise particle shape characterization; (3) allows accurate chemical identification; and (4) minimizes particle loss.MethodsAfter enzymatic digestion, a single pass of ultracentrifugation cleaned and deposited particles onto silicon wafers or grids for imaging analysis. During centrifugation, particles were passed through multiple layers of denaturants and a metal-selective high-density layer that minimized protein and nucleic acid contamination. The protocol prevented aggregation, providing well-dispersed particles for chemical and morphologic analysis. We evaluated the efficacy and accuracy of this protocol by recovering gold nanobeads and metal and ceramic particles from joint simulator wear tests.ResultsThe new protocol recovered particles ranging in size from nanometers to micrometers and enabled accurate morphologic and chemical characterization of individual particles.ConclusionBoth polyethylene and metal wear debris can be simultaneously analyzed from the same sample by combining a silicon wafer display protocol for polyethylene and the metal and ceramics silicon wafer display protocol.Clinical RelevanceAccurate analysis of wear debris is essential in understanding the processes that produce debris and a key step in development of more durable and biocompatible implants.

Protective role of IL-1β against post-arthroplasty Staphylococcus aureus infection

MyD88 is an adapter molecule that is used by both IL‐1R and TLR family members to initiate downstream signaling and promote immune responses. Given that IL‐1β is induced after Staphylococcus aureus infections and TLR2 is activated by S. aureus lipopeptides, we hypothesized that IL‐1β and TLR2 contribute to MyD88‐dependent protective immune responses against post‐arthroplasty S. aureus infections. To test this hypothesis, we used a mouse model of a post‐arthroplasty S. aureus infection to compare the bacterial burden, biofilm formation and neutrophil recruitment in IL‐1β‐deficient, TLR2‐deficient and wild‐type (wt) mice. By using in vivo bioluminescence imaging, we found that the bacterial burden in IL‐1β‐deficient mice was 26‐fold higher at 1 day after infection and remained 3‐ to 10‐fold greater than wt mice through day 42. In contrast, the bacterial burden in TLR2‐deficient mice did not differ from wt mice. In addition, implants harvested from IL‐1β‐deficient mice had more biofilm formation and 14‐fold higher adherent bacteria compared with those from wt mice. Finally, IL‐1β‐deficient mice had ∼50% decreased neutrophil recruitment to the infected postoperative joints than wt mice. Taken together, these findings suggest a mechanism by which IL‐1β induces neutrophil recruitment to help control the bacterial burden and the ensuing biofilm formation in a post‐surgical joint.

Fracture of a cross-linked polyethylene liner: a multifactorial issue.

A limited number of reports have detailed the cause of fracture of a highly cross-linked polyethylene liner. Typically, the fractures have occurred in a region of thin and/or unsupported polyethylene, in association with superiorly directed edge loading conditions secondary to an excessively inclinated acetabular component. This case report details an unusual fracture mechanism of a 5-mrad cross-linked liner caused by horizontal loading conditions. The report details several factors that were felt to be etiologic including the specific liner locking mechanism. The treatment options are discussed.

Daptomycin and tigecycline have broader effective dose ranges than vancomycin as prophylaxis against a Staphylococcus aureus surgical implant infection in mice.

ABSTRACT Vancomycin is widely used for intravenous prophylaxis against surgical implant infections. However, it is unclear whether alternative antibiotics used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections are effective as prophylactic agents. The aim of this study was to compare the efficacies of vancomycin, daptomycin, and tigecycline as prophylactic therapy against a methicillin-sensitive S. aureus (MSSA) or MRSA surgical implant infection in mice. MSSA or MRSA was inoculated into the knee joints of mice in the presence of a surgically placed medical-grade metallic implant. The efficacies of low- versus high-dose vancomycin (10 versus 110 mg/kg), daptomycin (1 versus 10 mg/kg), and tigecycline (1 versus 10 mg/kg) intravenous prophylaxis were compared using in vivo bioluminescence imaging, ex vivo bacterial counts, and biofilm formation. High-dose vancomycin, daptomycin, and tigecycline resulted in similar reductions in bacterial burden and biofilm formation. In contrast, low-dose daptomycin and tigecycline were more effective than low-dose vancomycin against the implant infection. In this mouse model of surgical implant MSSA or MRSA infection, daptomycin and tigecycline prophylaxis were effective over a broader dosage range than vancomycin. Future studies in humans will be required to determine whether these broader effective dose ranges for daptomycin and tigecycline in mice translate to improved efficacy in preventing surgical implant infections in clinical practice.

Material and surface factors influencing backside fretting wear in total knee replacement tibial components

Retrieval studies have shown that the interface between the ultra-high molecular weight polyethylene insert and metal tibial tray of fixed-bearing total knee replacement components can be a source of substantial amounts of wear debris due to fretting micromotion. We assessed fretting wear of polyethylene against metal as a function of metal surface finish, alloy, and micromotion amplitude, using a three-station pin-on-disc fretting wear simulator. Overall, the greatest reduction in polyethylene wear was achieved by highly polishing the metal surface. For example, highly polished titanium alloy surfaces produced nearly 20 times less polyethylene wear compared with blasted titanium alloy, whereas, decreasing the micromotion amplitude from 200 to 50microm produced approximately four times less polyethylene wear for the same blasted titanium alloy surface. Although the effect of the metal alloy was much smaller than the effect of metal surface roughness or the micromotion amplitude, CoCr discs produced slightly greater polyethylene fretting wear than titanium alloy discs under each condition. The results are essential in design and manufacturing decisions related to fixed-bearing total knee replacements.

Metal wear particles: What we know, what we do not know, and why

The importance of wear particle characterization for orthopaedic implants has long been established in the hip and knee arthroplasty literature. With the increasing use of motion preservation implants in the spine, the characterization of wear debris, particularly metallic nature, is gaining importance. An accurate morphological analysis of wear particles provides for both a complete characterization of the biocompatibility of the implant material and its wear products, and an in-depth understanding of the wear mechanisms, ion release, and associated corrosive activity related to the wear particles. In this paper, we present an overview of the most commonly-used published protocols for the isolation and characterization of metal wear particles, and highlight the limitations and uncertainties inherent to metal particle analysis.

Parathyroid hormone treatment improves the cortical bone microstructure by improving the distribution of type I collagen in postmenopausal women with osteoporosis

Although an important index, the level of bone mineral density (BMD) does not completely describe fracture risk. Another bone structural parameter, the orientation of type I collagen, is known to add to risk determination, independently of BMD, ex vivo. We investigated the Haversian system of transiliac crest biopsies from postmenopausal women before and after treatment with parathyroid hormone (PTH). We used the birefringent signal of circularly polarized light and its underlying collagen arrangements by confocal and electron microscopy, in conjunction with the degree of calcification by high‐resolution micro‐X‐ray. We found that PTH treatment increased the Haversian system area by 11.92 ± 5.82 mm2 to 12.76 ± 4.50 mm2 (p = 0.04); decreased bright birefringence from 0.45 ± 0.02 to 0.40 ± 0.01 (scale zero to one, p = 0.0005); increased the average percent area of osteons with alternating birefringence from 48.15% ± 10.27% to 66.33% ± 7.73% (p = 0.034); and nonsignificantly decreased the average percent area of semihomogeneous birefringent osteons (8.36% ± 10.63% versus 5.41% ± 9.13%, p = 0.40) and of birefringent bright osteons (4.14% ± 8.90% versus 2.08% ± 3.36%, p = 0.10). Further, lamellar thickness significantly increased from 3.78 ± 0.11 µm to 4.47 ± 0.14 µm (p = 0.0002) for bright lamellae, and from 3.32 ± 0.12 µm to 3.70 ± 0.12 µm (p = 0.045) for extinct lamellae. This increased lamellar thickness altered the distribution of birefringence and therefore the distribution of collagen orientation in the tissue. With PTH treatment, a higher percent area of osteons at the initial degree of calcification was observed, relative to the intermediate‐low degree of calcification (57.16% ± 3.08% versus 32.90% ± 3.69%, p = 0.04), with percentage of alternating osteons at initial stages of calcification increasing from 19.75 ± 1.22 to 80.13 ± 6.47, p = 0.001. In conclusion, PTH treatment increases heterogeneity of collagen orientation, a starting point from which to study the reduction in fracture risk when PTH is used to treat osteoporosis.