A novel spinal implant infection model in rabbits.

STUDY DESIGN: A new spinal implant model was designed to study device-centered infection with methicillin-resistant Staphylococcus aureus in multiple noncontiguous surgical sites in the lumbar spine region of a rabbit. OBJECTIVE: To develop a multiple-site spinal implant device-centered infection model in rabbits. SUMMARY OF BACKGROUND DATA: Results in many recent studies show that postoperative wound infection after spinal implant surgery and the increase in antibiotic-resistant bacteria are a concern. Anti-infection strategies must be tested in relevant animal models that will lead to appropriate clinical studies. METHODS: Eight anesthetized New Zealand White rabbits underwent completely isolated partial laminectomy and subsequent stainless steel Kirschner wire implantation directly into the transverse processes of vertebrae T13, L3, and L6. The middle sites (L3) were used as sterile control sites, and the outer sites (T13, L6) were challenged with different amounts of methicillin-resistant Staphylococcus aureus. Rabbits were killed after 7 days, and biopsies were performed to provide evidence for device-centered infection. Bacterial growth on the implant surfaces and in surrounding tissues and bone was assayed. RESULTS: Overall device-centered infection was established after 7 days in 100% of the sites challenged with 10(3) colony-forming units methicillin-resistant Staphylococcus aureus or higher. No infection was seen in any of the control sites located between infected vertebrae. Multiple blood and liver samples showed that the separate localized infections did not become systemic after 7 days. CONCLUSIONS: This new animal model demonstrates that multiple biomaterial implants can be evaluated in the same animal and provides a technique for investigating postoperative device-centered infection of the spine. Infection was demonstrated in noncontiguous lumbar sites of the spine, whereas adjacent control sites remained sterile. Because there was no cross contamination or systemic spread of the infection, multiple anti-infection strategies or implant materials can now be tested for efficacy in a single animal to combat dramatic and costly postoperative implant infections.

Biomaterial-induced dysfunction in the capacity of rabbit alveolar macrophages to kill Staphylococcus epidermidis RP12.

The effect of poly(methyl methacrylate) (PMMA), titanium alloy, and silicone discs on the capacity of rabbit alveolar macrophages (AM) to kill RP12 strain of Staphylococcus epidermidis (RP12) was studied in vitro. When freshly harvested AM were preincubated with PMMA discs for 3 h and subsequently assayed for RP12 killing, there was no change in the RP12 killing capacity of AM. However, when AM were incubated with PMMA discs for 6 or 18 h at 37 degrees C in 5% CO2, the RP12 killing capacity of AM was reduced to 15% and 4%, respectively. Preincubation of AM with titanium alloy for 6 h reduced RP12 killing capacity of AM to 30%, and to 21% in 18-h incubation. Silicone discs did not affect the RP12 killing by AM at 6 h of preincubation, but reduced RP12 killing (35%) by AM when preincubated for 18 h. Preincubation of AM with PMMA discs for 3 or 6 h did not affect the level of PMA-elicited oxidative burst of AM as measured by a luminol-enhanced chemiluminescent assay. Superoxide dismutase, which eliminated the oxidative burst of AM by 90%, did not affect the RP12 killing by AM.

Simple technique for the preparation of silicone gel particles: the effect of silicone gel particles on oxidative responses of macrophages.

A simple technique was developed to prepare phagocytosable-size particles from the silicone gel used in breast implants. Sonication of silicone gel (1 g) in 5 ml of 20 mM sodium phosphate buffer (pH 7.2) containing 1% (wt/vol) polyoxypropylene-polyethylene block surfactant (F-68 or F-108) produced silicone gel particles ranging from 1-50 microns in diameter. Passage of the suspension through a series of filters yielded phagocytosable particles (1-5 microns in diameter) at a concentration of ca. 2 x 10(9) particles/ml. The particles remained as individual particles, did not coalesce to form large clumps, and were not pelleted by centrifugation (2000 x g, 20 min). They were not toxic for rabbit alveolar macrophages (AM) during 24 h of incubation at 37 degrees C, did not elicit an oxidative burst from AM in vitro in a luminol-enhanced chemiluminescent assay, and did not significantly increase the phorbol myristate acetate (PMA)-elicited oxidative burst by AM. AM isolated from rabbits 2 days after the intravenous injection of silicone particles were not primed or activated (i.e., the AM did not show an enhanced oxidative burst when elicited with PMA in vitro). However, AM isolated from rabbits 2 days after intratracheal injection of the particles were primed but only exhibited a 4-6-fold increase in the oxidative burst elicited with PMA.

Inhibition of Staphylococcus adherence to biomaterials by extracellular slime of S. epidermidis RP12.

Adherence of selected strains of coagulase-negative staphylococci to various biomaterials, and the inhibition of their adherence by extracellular slime obtained from the RP12 strain of Staphylococcus epidermidis were studied in vitro. S. epidermidis RP12 adhered considerably more to polymethylmethacrylate (PMMA) discs than did the SP2 strain of S. hominis and the SE-360 strain of S. hyicus. Strain RP12 was less adherent to titanium alloy, ultrahigh molecular weight polyethylene (UHMWPE), and Teflon discs than to PMMA discs. Exposure of PMMA discs to extracellular slime extracted from strain RP12 greatly reduced adherence of strain RP12, SP2, SE-360, and S. epidermidis RP-62A. The active component(s) was present in the > 10 kD mol wt fraction obtained by Amicon YM10 ultrafiltration of crude slime; heat treatment of the fraction did not affect its inhibitory activity. When the bacteria and RP12 slime fractions were added simultaneously to the PMMA discs, the > 10 kD mol wt fraction of slime competitively inhibited adherence of strain RP12 to PMMA discs; in contrast, the < 10 kD mol wt fraction enhanced adherence of strain RP12 to PMMA discs.

The glycocalyx, biofilm, microbes, and resistant infection.

Biomaterial implants, traumatized tissues and bone are susceptible to immediate and delayed infections because microbes preferentially adhere to "inert biomaterials" or to damaged tissue surfaces. This type of infection is resistant to antibiotic therapy and most often requires removal of the prosthesis or infected tissue. This article discusses glycocalyx, biofilm, microbes, and resistant infection in prosthesis or infected tissue.

Site-specific adhesion of Staphylococcus epidermidis (RP12) in Ti-Al-V metal systems.

Staphylococcus epidermidis (RP12) adhesion patterns were studied on the following titanium (Ti)-aluminium (Al)-vanadium (V) metal systems: (i) microfabricated samples consisting of Ti, Al and V islands deposited onto Ti or V substrata, (ii) pure Ti, Al and V metals, and (iii) medical grade Ti6Al4-V alloy. All of these surfaces were covered with their respective oxides formed upon exposure of the metals to air. Quantitative analysis of the number of cells bound per unit area indicates that S. epidermidis (RP12) exhibits greatest adhesion to pure V surfaces. When exposed to surfaces having controlled spatial variations in chemical composition on the 10 microns scale (microfabricated samples), the bacteria preferentially populate V islands versus Ti or Al substrata. In the case of the biphasic Ti6Al4V alloy, the bacteria tend to adhere to V-rich, mixed phase regions and phase boundaries. These findings demonstrate that enhanced and preferential adhesion of S. epidermidis (RP12) occurs on V surfaces in Ti-Al-V metal systems and suggest that bacterial interactions are influenced by surface oxide composition.

Implant failure and the immuno-incompetent fibro-inflammatory zone.

Biomaterial implants are surrounded by an immuno-incompetent, fibro-inflammatory, integration-deficient zone within which stimulation of cellular immune responses results in superoxide radical and cytokine-mediated tissue damage with increased susceptibility to infection or aseptic loosening. Three important questions that pertain to surgical implants are (1) What are the mechanisms that cause abnormal inflammatory responses in the absence of infection and result in interface cellular disorganization and device failure? (2) What causes host defenses to be compromised to the extent that normal flora organisms like Staphylococcus epidermidis, with little or no virulence potential, can cause life-threatening infections at the implant-host interface? (3) What is the nature of surface regions of biomaterials that facilitate bacterial adherence? Pathogenic strains of S. epidermidis and Staphylococcus aureus have an affinity for biomaterial surfaces and are capable of initiating infection. Binding may be nonspecific and glue-like rather than a receptor-ligand event as for S. aureus and matrix proteins. This study indicates bacterial binding to sites of higher vanadium concentration at grain boundaries and mixed phases in titanium alloys. Repeated macrophage priming by biomaterial particulates results in the production of reactive oxygen intermediates, macrophage exhaustion, and adjacent tissue damage. A cytokine cascade is also initiated. A self-perpetuating enlarging immuno-incompetent fibro-inflammatory zone develops about implants, which features tissue cell damage, increased susceptibility to infection, and results in septic or aseptic failure of the implant. These effects are clearly exemplified by fibrosis about breast implants and osteolysis at the interface of total joint replacements.

A standardized method for the assessment of shoulder function.

The American Shoulder and Elbow Surgeons have adopted a standardized form for assessment of the shoulder. The form has a patient self-evaluation section and a physician assessment section. The patient self-evaluation section of the form contains visual analog scales for pain and instability and an activities of daily living questionnaire. The activities of daily living questionnaire is marked on a four-point ordinal scale that can be converted to a cumulative activities of daily living index. The patient can complete the self-evaluation portion of the questionnaire in the absence of a physician. The physician assessment section includes an area to collect demographic information and assesses range of motion, specific physical signs, strength, and stability. A shoulder score can be derived from the visual analogue scale score for pain (50%) and the cumulative activities of daily living score (50%). It is hoped that adoption of this instrument to measure shoulder function will facilitate communication between investigators, stimulate multicenter studies, and encourage validity testing of this and other available instruments to measure shoulder function and outcome.

Cell biology and molecular mechanisms in artificial device infections.

Biomaterials are being used with increasing frequency for tissue substitution. Complex devices such as total joint replacement and the total artificial heart represent combinations of polymers and metal alloys for system and organ replacement. The major barrier to the extended use of these devices is bacterial adhesion to biomaterials, which causes biomaterial-centered infection, and the lack of successful tissue integration or compatibility with biomaterial surfaces. Adhesion-mediated infections are extremely resistant to antibiotics and host defenses and frequently persist until the biomaterial or foreign body is removed. The pathogenesis of adhesive infections is related, in part, to preferential colonization of "inert" substrate whose surfaces are not integrated with healthy tissues composed of living cells and intact extracellular polymers. Tissue integration is an interesting parallel to microbial adhesion and is a desired phenomenon for the biocompatibility of certain implants and biomaterials. Tissue integration requires a form of eukaryocytic adhesion or compatibility with possible chemical integration to an implant surface. Many of the fundamental principles of interfacial science apply to both microbial adhesion and to tissue integration and are general to and independent of the substratum materials involved. Interactions of biomaterials with bacteria and tissue cells are directed not only by specific receptors and outer membrane molecules on the cell surface, but also by the atomic geometry and electronic state of the biomaterial surface. An understanding of these mechanisms is important to all fields of medicine and is derived from and relevant to studies in microbiology, biochemistry, and physics.(ABSTRACT TRUNCATED AT 250 WORDS)

Particle-induced in vivo priming of alveolar macrophages for enhanced oxidative responses: a novel system of cellular immune augmentation.

A novel system for priming adult rabbit alveolar macrophages (AMs) in vivo for markedly enhanced oxidative responses is described. When adult rabbits were injected intravenously (i.v.) with 1- to 5-microns particles such as zymosan, latex particles, or heat-killed bacille Calmette-Guérin, AMs were primed in 1-3 days for greatly enhanced phorbol myristate acetate (PMA)- or opsonized zymosan (Op-zym)-elicited chemiluminescent (CL) responses. Intratracheal (i.t.) injection of zymosan particles also primed AMs for enhanced PMA- or Op-zym-elicited CL responses. AMs obtained from particle-injected rabbits showed up to 100-fold higher levels of PMA-elicited CL responses than AMs from normal rabbits. In contrast, Op-zym failed to prime normal AMs in vitro for enhanced CL responses. Whereas AMs could not be primed in vivo with an i.v. injection of particles of approximately 24 microns diameter. AMs could be primed if the particles were administered by the i.t. route. The priming appears to be independent of particle types. The priming effect was of short duration and declined after 5 to 7 days. The possibility that this system represents the primitive cellular immune response found in invertebrates is discussed. The potential use of this system as a means of immune augmentation prompts further investigation.

Altered oxidative responses and antibacterial activity of adult rabbit alveolar macrophages exposed to poly(methyl methacrylate).

The effect of poly(methyl methacrylate) (PMMA) on the oxidative responses and antibacterial activity of adult rabbit alveolar macrophages (AM) was studied. PMMA beads (ca. 0.3 micron diameter) elicited an acute respiratory burst within 6-8 min after the addition of the beads. In contrast. Teflon beads of comparable size (ca. 0.2 micron diameter) did not elicit an oxidative burst of AM. An oxidative response was elicited only by those PMMA samples that had affinity for AM adherence. Incubation of AM with PMMA beads reduced the subsequent phorbol myristate acetate (PMA)-elicited oxidative burst by more than 80%. The Staphylococcus epidermidis--RP12 killing capacity of AM was greatly increased when PMMA beads (ca. 0.3 micron) were added to the challenge dose of bacteria. Pre-incubation of freshly harvested AM with PMMA beads, which greatly reduced subsequent PMA-elicited chemiluminescent (CL) responses did not significantly affect the RP12 killing capacity of AM. Our data also suggest that killing of the RP12 strain of S. epidermidis does not involve reactive oxygen intermediates.

A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage.

Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187-amino-acid repeat motif. Collagen receptor-positive strains adhered to both collagen substrata and cartilage in a time-dependent process. Collagen receptor-specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.

The inhibition of bacterial adhesion to a tobramycin-impregnated polymethylmethacrylate substratum.

Tobramycin sulfate powder (1.2 g) was mixed with Palacos polymethylmethacrylate (PMMA) bone cement (40 g) to produce 100 discs containing 5.9 mg tobramycin per disc. These discs were used to evaluate the inhibition of bacterial adhesion to an antibiotic-laden biomaterial. Tobramycin-impregnated PMMA discs and control discs containing no tobramycin were exposed in vitro to Staphylococcus epidermidis. Colonization was quantitated using plate count techniques and electron microscopy. Tobramycin-impregnated surfaces reduced adhesive bacteria colonization by 1 log relative to control discs. These observations suggest that tobramycin-impregnated PMMA may not be significantly effective in preventing colonization of the biomaterial substratum and PMMA may be a poor choice as a drug delivery vehicle in biomaterial and compromised tissue-centered infections.

Mechanisms of musculoskeletal sepsis.

Musculoskeletal infection is extraordinarily resistant to treatment. Bacterial colonization is discussed as well as host defense systems and antibiotic resistance.

Musculoskeletal infection, microbial adhesion, and antibiotic resistance.

Osteomyelitis and intra-articular infection are septic diseases that present pathogenic features characteristic of molecular mechanisms involving adhesion to substrata. In this review, mechanisms of microbial adhesion to bone and cartilage as substrata are presented and related to host tissue response and to antibiotic treatment.

In vitro and in vivo comparative colonization of Staphylococcus aureus and Staphylococcus epidermidis on orthopaedic implant materials.

Clinically, Staphylococcus aureus appears to be the dominant organism associated with infected metal implants, whereas coagulase-negative staphylococcal strains are more frequently isolated from infected polymer implants. We reproduced this trend experimentally in vitro and in vivo. Discs of a titanium alloy, poly(methyl methacrylate) and ultra-high molecular weight polyethylene were exposed to a clinical isolate of Staphylococcus aureus or either of two strains of Staphylococcus epidermidis. Within 1 h Staphylococcus aureus was always the most rapid colonizer regardless of biomaterial. However, after 8 to 24 h, Staphylococcus aureus was present in higher numbers on metal and Staphylococcus epidermidis on polymers. Moreover, the exopolysaccharide produced by Staphylococcus epidermidis appeared to offer an effective protection against host defences in vivo.

Comparative in vitro antibiotic resistance of surface-colonizing coagulase-negative staphylococci.

The MBCs of nafcillin, vancomycin, gentamicin and daptomycin (LY146032) were determined for three clinical isolates of coagulase-negative staphylococci grown in suspension and adherent to biomaterials. Strains studied were the slime-producing strain Staphylococcus epidermidis RP-12 (ATCC 35983), S. hyicus SE-360, and the non-slime-producing strain S. hominis SP-2 (ATCC 35982). All three strains were allowed to colonize surgical-grade disks of stainless steel, polymethylmethacrylate, and ultrahigh-molecular-weight polyethylene for 24 h, and the disks were then exposed to various concentrations of antibiotics for 24 h. Surviving adherent bacteria were mechanically dislodged from the disks and quantitated by standard broth dilution plating techniques. Biomaterial-adherent RP-12 and SE-360 yielded approximately 10 times more CFU per disk than non-slime-producing SP-2 did. For all organisms, 10 times more bacteria bound to polymethylmethacrylate disks than to the other biomaterials. In general, bacteria adherent to biomaterials exhibited greater resistance to antibiotics than the same strains in suspension did. Resistance was independent of bacterial slime-producing characteristics and was related to the biomaterial colonized.