Staphylococcus aureus and Acinetobacter baumannii Inhibit Osseointegration of Orthopedic Implants.

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

Mapping of common rib fracture patterns and the subscapular flail chest associated with operative scapula fractures.

BACKGROUND: Rib fractures occur in approximately 10% of trauma patients and are associated with more than 50% of patients with scapula fractures. This study investigates the location and patterns of rib fractures and flail chest occurring in patients with operatively treated scapula fractures. Novel frequency mapping techniques of rib fracture patterns in patients who also injure the closely associated scapula can yield insight into surgical approaches and fixation strategies for complex, multiple injuries patients. We hypothesize that rib fractures have locations of common occurrence when presenting with concomitant scapula fracture that requires operative treatment. METHODS: Patients with one or more rib fractures and a chest computed tomography scan between 2004 and 2018 were identified from a registry of patients having operatively treated scapula fractures. Unfurled rib images were created using Syngo-CT Bone Reading software (Siemens Inc., Munich, Germany). Rib fracture and flail segment locations were marked and measured for standardized placement on a two-dimensional chest wall template. Location and frequency were then used to create a gradient heat map. RESULTS: A total of 1,062 fractures on 686 ribs were identified in 86 operatively treated scapula fracture patients. The mean ± SD number of ribs fractured per patient was 8.0 ± 4.1 and included a mean ± SD of 12.3 ± 7.2 total fractures. Rib fractures ipsilateral to the scapula fracture occurred in 96.5% of patients. The most common fracture and flail segment location was ipsilateral and subscapular; 51.4% of rib fractures and 95.7% of flail segments involved ribs 3 to 6. CONCLUSION: Patients indicated for operative treatment of scapula fractures have a substantial number of rib fractures that tend to most commonly occur posteriorly on the rib cage. There is a pattern of subscapular rib fractures and flail chest adjacent to the thick bony borders of the scapula. This study enables clinicians to better evaluate and diagnose scapular fracture patients with concomitant rib fractures. LEVEL OF EVIDENCE: Diagnostic test, level IV.

Radiographic correlation of clinical shoulder deformity and patient perception following scapula fracture.

BACKGROUND: Interest in operative management of scapular fractures is increasing based upon defined radiographic displacement criteria and growing awareness that certain extra-articular fractures will not do well and result in dysfunction and deformity (slumped shoulder). We intend to quantify clinical deformity, analyze correlations of these novel measures with defined radiographic measures of fracture displacement and with the patients' reported perception of their deformity. METHODS: Prospectively enrolled patients underwent standardized questioning regarding their perception of the deformity. Radiographs were utilized to measure glenopolar angle medial/lateral displacement, and angulation of the displaced scapula fracture. Novel measurements of clinical deformity (shoulder area, shoulder angle and shoulder height difference) were calculated. All measurements were repeated post-operatively for patients undergoing operative treatment. RESULTS: Fifty-one patients (39 operative) were examined within 30 days of injury. Follow-up (≥2 months post-op) was obtained for 31/39 (79%). Medial-lateral displacement significantly correlated with all measures of clinical deformity and with patient reported shoulder appearance bothersome score. Angulation significantly correlated with patient perception and two clinical measures (shoulder area and shoulder angle difference). All post-operative radiographic measures, clinical measures of deformity, and patient reported scores statistically improved from baseline measures. DISCUSSION: Patients with scapula fracture do perceive deformity, and there is a significant correlation between the patients' perception, radiographic and clinical measurements of deformity after scapula fracture. All measures statistically improved in patients with operative treatment compared to baseline measurements. This study reinforces the importance of the clinicians' clinical examination and observation of shoulder deformity in the scapula fracture patient. LEVEL OF EVIDENCE: IV.

Injury mechanism, epidemiology, and Hospital trends of scapula fractures: A 10-year retrospective study of the National Trauma Data Bank.

BACKGROUND: This 10 year retrospective study of the NTDB is the first to describe trends in scapula fracture diagnosis frequency, epidemiology, injury mechanisms and the type of hospital where the condition is treated. METHODS: Demographics, ISS scores, hospital data, mechanism of injury, complications, and hospital length of stay were recorded for patients with diagnosed scapula fractures (ICD-9, 811.0) recorded in the NTDB, v7.2 (2002-2012). Mean and standard deviation for continuous variables and proportions for binary variables are calculated. RESULTS: The prevalence of scapula fractures in all patients submitted to the NTDB (2002-2012) was 1.74%. Between 2006-2007, the reported incidence doubled from 1% to 2.2%. There was a predominance of injury to white males (75% and 78% respectively). Forty-one percent were treated at a Level 1 trauma center and had a mean ISS of 20.1 (SD-11.8). Scapula fracture rates declined in patients 0-19 years and increased in the 60-79 and 80+ age groups. The increasing incidence of the aged population is also reflected in the increase of falls as the mechanism of injury in the elderly population. CONCLUSION: This study is the first to describe a full decade of scapula fracture epidemiology on a national scale. The number of diagnosed scapula fractures increased substantially in the NTDB between 2002-2012. Scapula fractures diagnosed in the geriatric demographic and fractures resulting from falls are both on the rise, whereas the reported incidence is decreasing in the younger demographic. Additionally, fractures as a result of motor vehicle accidents also decreased precipitously during the reported decade.

Five to Ten-Year Outcomes of Operatively Treated Scapular Fractures.

BACKGROUND: The purpose of this study was to assess the 5 to 10-year clinical and patient-reported functional outcomes after open reduction and internal fixation (ORIF) of intra-articular and extra-articular scapular fractures. METHODS: We conducted a retrospective review of prospectively collected data on 106 patients who underwent ORIF of a scapular fracture at a single level-I trauma center between January 2005 and December 2010. Eight patients were excluded from the study because they had either severe neurologic injury or an isolated process fracture, and 66 patients (37 with an isolated extra-articular fracture and 29 with an intra-articular fracture) participated in the 5 to 10-year follow-up, yielding a follow-up rate of 67%. A physical examination including a strength assessment and range-of-motion measurements was performed on 89% of the follow-up cohort. Disabilities of the Arm, Shoulder and Hand (DASH) and Short Form-12 version 2 (SF-12v2) or SF-36v2 questionnaires were completed by all participating patients. Intra-articular and extra-articular fractures were analyzed in separate groups. RESULTS: The mean follow-up was 7.8 years in the extra-articular group and 7.3 years in the intra-articular group, with a range of 4.7 to 10.3 years. The mean DASH score was 8.9 in the extra-articular group and 9.1 in the intra-articular group (normal population = 10.1). Strength examination revealed no significant differences between the injured and uninjured shoulders for any movement (p > 0.05), while the range of external rotation was slightly decreased in both the extra-articular (p = 0.01) and the intra-articular (p = 0.01) group. The abduction range of motion was also slightly decreased in the intra-articular cohort (p = 0.03). Arthroplasty was indicated as a subsequent procedure for 2 patients in the intra-articular cohort. Sixty-one of the 66 patients returned to their original occupation or changed occupations for reasons unrelated to the shoulder injury. CONCLUSIONS: At 5 to 10 years after ORIF of a scapular fracture, patients have excellent functional outcomes albeit with a small decrease in external rotation motion relative to the contralateral, normal shoulder. Interestingly, we found the outcomes after intra-articular and extra-articular fractures to be comparable. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Particle-Induced Osteolysis Is Mediated by TIRAP/Mal in Vitro and in Vivo: Dependence on Adherent Pathogen-Associated Molecular Patterns.

BACKGROUND: Proinflammatory signaling by toll-like receptors (TLRs) likely contributes to biologic responses to wear particles causing aseptic loosening. We recently reported associations with aseptic loosening in patients with polymorphisms in the locus encoding an adapter protein specific for TLR-2 and TLR-4 known as toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal). To directly examine the contribution of TIRAP/Mal, we tested the hypothesis that TIRAP/Mal deficiency reduces the activity of wear particles. Signaling by TLR-2 and TLR-4 through TIRAP/Mal can be activated by bacterial pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide or endogenous alarmins. To distinguish between those possibilities, we tested the hypothesis that the effects of TIRAP/Mal depend on the adherence of bacterial PAMPs to the particles. METHODS: In vitro mRNA levels and secretion of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 were measured after incubating wild-type and TIRAP/Mal(-/-) macrophages in the presence or absence of titanium particles with adherent bacterial debris, so-called endotoxin-free particles, or particles with adherent lipopolysaccharide. In vivo osteolysis was measured after implanting titanium particles on the calvaria of wild-type and TIRAP/Mal(-/-) mice. RESULTS: TIRAP/Mal deficiency significantly inhibited the activity of titanium particles with adherent bacterial debris to stimulate in vivo osteolysis and in vitro cytokine mRNAs and secretion. Those effects are dependent on adherent PAMPs because removal of >99% of the adherent bacterial debris from the particles significantly reduced their activity and the remaining activity was not dependent on TIRAP/Mal. Moreover, adherence of highly purified lipopolysaccharide to the endotoxin-free particles reconstituted the activity and the dependence on TIRAP/Mal. CONCLUSIONS: TIRAP/Mal deficiency reduces inflammatory responses and osteolysis induced by particles with adherent PAMPs. CLINICAL RELEVANCE: Our results, coupled with the genetic associations between aseptic loosening and polymorphisms within the TIRAP/Mal locus, support TLR signaling through TIRAP/Mal as one of the factors that enhances the activity of wear particles and further support the hypothesis that bacterial PAMPs likely contribute to aseptic loosening in a subset of patients.

Surface contaminants inhibit osseointegration in a novel murine model.

Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopedic implants after sterilization procedures and affect osseointegration. The goals of this study were to develop a murine model of osseointegration in order to determine whether removing surface contaminants enhances osseointegration. To develop the murine model, titanium alloy implants were implanted into a unicortical pilot hole in the mid-diaphysis of the femur and osseointegration was measured over a five week time course. Histology, backscatter scanning electron microscopy and X-ray energy dispersive spectroscopy showed areas of bone in intimate physical contact with the implant, confirming osseointegration. Histomorphometric quantification of bone-to-implant contact and peri-implant bone and biomechanical pullout quantification of ultimate force, stiffness and work to failure increased significantly over time, also demonstrating successful osseointegration. We also found that a rigorous cleaning procedure significantly enhances bone-to-implant contact and biomechanical pullout measures by two-fold compared with implants that were autoclaved, as recommended by the manufacturer. The most likely interpretation of these results is that surface contaminants inhibit osseointegration. The results of this study justify the need for the development of better detection and removal techniques for contaminants on orthopedic implants and other medical devices.

PI3Kγ deletion reduces variability in the in vivo osteolytic response induced by orthopaedic wear particles.

Orthopedic wear particles activate a number of intracellular signaling pathways associated with inflammation in macrophages and we have previously shown that the phosphoinositol-3-kinase (PI3K)/Akt pathway is one of the signal transduction pathways that mediates the in vitro activation of macrophages by orthopedic wear particles. Since PI3Kγ is primarily responsible for PI3K activity during inflammation, we hypothesized that PI3Kγ mediates particle-induced osteolysis in vivo. Our results do not strongly support the hypothesis that PI3Kγ regulates the overall amount of particle-induced osteolysis in the murine calvarial model. However, our results strongly support the conclusion that variability in the amount of particle-induced osteolysis between individual mice is reduced in the PI3Kγ(-/-) mice. These results suggest that PI3Kγ contributes to osteolysis to different degrees in individual mice and that the mice, and patients, that are most susceptible to osteolysis may be so, in part, due to an increased contribution from PI3Kγ.

Bacterial pathogen-associated molecular patterns stimulate biological activity of orthopaedic wear particles by activating cognate Toll-like receptors.

Aseptic loosening of orthopaedic implants is induced by wear particles generated from the polymeric and metallic components of the implants. Substantial evidence suggests that activation of Toll-like receptors (TLRs) may contribute to the biological activity of the wear particles. Although pathogen-associated molecular patterns (PAMPs) produced by Gram-positive bacteria are likely to be more common in patients with aseptic loosening, prior studies have focused on LPS, a TLR4-specific PAMP produced by Gram-negative bacteria. Here we show that both TLR2 and TLR4 contribute to the biological activity of titanium particles with adherent bacterial debris. In addition, lipoteichoic acid, a PAMP produced by Gram-positive bacteria that activates TLR2, can, like LPS, adhere to the particles and increase their biological activity, and the increased biological activity requires the presence of the cognate TLR. Moreover, three lines of evidence support the conclusion that TLR activation requires bacterially derived PAMPs and that endogenously produced alarmins are not sufficient. First, neither TLR2 nor TLR4 contribute to the activity of "endotoxin-free" particles as would be expected if alarmins are sufficient to activate the TLRs. Second, noncognate TLRs do not contribute to the activity of particles with adherent LPS or lipoteichoic acid as would be expected if alarmins are sufficient to activate the TLRs. Third, polymyxin B, which inactivates LPS, blocks the activity of particles with adherent LPS. These results support the hypothesis that PAMPs produced by low levels of bacterial colonization may contribute to aseptic loosening of orthopaedic implants.

Comparison of the roles of IL-1, IL-6, and TNFalpha in cell culture and murine models of aseptic loosening.

Pro-inflammatory cytokines, such as IL-1, IL-6, and TNF, are considered to be major mediators of osteolysis and ultimately aseptic loosening. This study demonstrated that synergistic interactions among these cytokines are required for the in vitro stimulation of osteoclast differentiation by titanium particles. In contrast, genetic knock out of these cytokines or their receptors does not protect murine calvaria from osteolysis induced by titanium particles. Thus, the extent of osteolysis was not substantially altered in single knock out mice lacking either the IL-1 receptor or IL-6. Osteolysis also was not substantially altered in double knock out mice lacking both the IL-1 receptor and IL-6 or in double knock out mice lacking both TNF receptor-1 and TNF receptor-2. The differences between the in vivo and the cell culture results make it difficult to conclude whether the pro-inflammatory cytokines contribute to aseptic loosening. One alternative is that in vivo experiments are more physiological and that therefore the current results do not support a role for the pro-inflammatory cytokines in aseptic loosening. We however favor the alternative that, in this case, the cell culture experiments can be more informative. We favor this alternative because the role of the pro-inflammatory cytokines may be obscured in vivo by compensation by other cytokines or by the low signal to noise ratio found in measurements of particle-induced osteolysis.

The balance between endotoxin accumulation and clearance during particle-induced osteolysis in murine calvaria.

Bacterial endotoxin may contribute to aseptic loosening of orthopedic implants even in the absence of clinical or microbiological evidence of infection. One potential source of endotoxin during aseptic loosening is systemically circulating endotoxin, derived from intestinal flora, minor infections, or dental procedures, that may bind to wear particles. The current study demonstrates that systemically derived endotoxin accumulates when 'endotoxin-free' titanium and polyethylene particles are implanted on murine calvaria. Time-course experiments and experiments using germ-free mice rule out the possibility that the observed endotoxin accumulation may be due to bacterial contamination. In contrast, endotoxin is cleared from titanium particles that originally carry high amounts of adherent endotoxin. The mechanism of endotoxin clearance is not dependent on induction of a respiratory burst. Taken together, these results indicate that a balance between endotoxin accumulation and endotoxin clearance controls the steady-state level of endotoxin surrounding orthopedic wear particles implanted on murine calvaria. This balance may regulate the rate of osteolysis in the murine calvaria model as well as in patients with aseptic loosening.

Polyethylene and titanium particles induce osteolysis by similar, lymphocyte-independent, mechanisms.

Periprosthetic osteolysis is a major clinical problem that limits the long-term survival of total joint arthroplasties. Osteolysis is induced by implant-derived wear particles, primarily from the polyethylene bearing surfaces. This study examined two hypotheses. First, that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles. Second, that lymphocytes do not play a major role in particle-induced osteolysis. To test these hypotheses, we used the murine calvarial model that we have previously used to examine titanium-induced osteolysis. Polyethylene particles rapidly induced osteolysis in the murine calvaria 5-7 days after implantation. The polyethylene-induced osteolysis was associated with large numbers of osteoclasts as well as the formation of a thick periosteal fibrous tissue layer with numerous macrophages containing phagocytosed polyethylene particles. Polyethylene-induced osteolysis was rapidly repaired and was undetectable by day 21 after implantation. Lymphocytes were noted in the fibrous layer of wild-type mice. However, the amount of osteolysis and cytokine production induced by polyethylene particles was not substantially affected by the lack of lymphocytes in Pfp/Rag2 double knock out mice. All of these findings are similar to our observations of osteolysis induced by titanium particles. These results provide strong support for both of our hypotheses: that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles and that lymphocytes do not play a major role in particle-induced osteolysis.