Bone infection: a clinical priority for clinicians, scientists and educators.

Bone infection has received increasing attention in recent years as one of the main outstanding clinical problems in orthopaedic-trauma surgery that has not been successfully addressed. In fact, infection may develop across a spectrum of patient types regardless of the level of perioperative management, including antibiotic prophylaxis. Some of the main unknown factors that may be involved, and the main targets for future intervention, include more accurate and less invasive diagnostic options, more thorough and accurate debridement protocols, and more potent and targeted antimicrobials. The underlying biology dominates the clinical management of bone infections, with features such as biofilm formation, osteolysis and vascularisation being particularly influential. Based on the persistence of this problem, an improved understanding of the basic biology is deemed necessary to enable innovation in the field. Furthermore, from the clinical side, better evidence, documentation and outreach will be required to translate these innovations to the patient. This review presents the findings and progress of the AO Trauma Clinical Priority Program on the topic of bone infection.

Revisiting the "race for the surface" in a pre-clinical model of implant infection.

Orthopaedic implant use increases infection risk. Implant infection risk can be explained by the "race for the surface" concept, where there is competition between host-cell integration and bacterial colonisation. Although generally accepted, the temporal dynamics have not been elucidated in vivo. Using a bilateral intramedullary rat model, Staphylococcus aureus was injected into the tail vein either immediately after or 1, 3 and 7 d following implant placement. This allowed assessment of the temporal interplay between bacterial colonisation and host-cell adhesion by uncoupling implant placement and bacterial challenge. 2 weeks following inoculation, animals were anaesthetised, euthanised and implants and tissues harvested for bacterial enumeration. To assess host participation in implant protection, additional animals were not inoculated but euthanised at 1, 3 or 7 d and the host cells adhered to the implant were evaluated by flow cytometry and microscopy. As time between implant placement and bacterial challenge increased, infection rate and bioburden decreased. All implants had measurable bioburden when challenged at day 1, but only two implants had recoverable bacteria when inoculated 7 d after implant placement. This protection against infection corresponded to a shift in host cell population surrounding the implant. Initially, cells present were primarily non-differentiated stem cells, such as bone marrow mesenchymal stem cells, or immature haematopoietic cells. At day 7, there was a mature monocyte/macrophage population. The present study illustrated a direct relationship between host immune cell attachment and decrease in bacterial colonisation, providing guidance for antimicrobial release devices to protect orthopaedic implants against bacterial colonisation.

Chlorhexidine-releasing implant coating on intramedullary nail reduces infection in a rat model.

The use of internal intramedullary nails for long bone fracture fixation is a common practice among surgeons. Bacteria naturally attach to these devices, increasing the risk for wound infection, which can result in non- or malunion, additional surgical procedures and extended hospital stays. Intramedullary nail surface properties can be modified to reduce bacterial colonisation and potentially infectious complications. In the current study, a coating combining a non-fouling property with leaching chlorhexidine for orthopaedic implantation was tested. Coating stability and chlorhexidine release were evaluated in vitro. Using a rat model of intramedullary fixation and infection, the effect of the coating on microbial colonisation and fracture healing was evaluated in vivo by quantitative microbiology, micro-computed tomography, plain radiography, three-point bending and/or histology. Low dose systemic cefazolin was administered to increase the similarities to clinical practice, without overshadowing the effect of the anti-infective coating. When introduced into a contaminated wound, the non-fouling chlorhexidine-coated implant reduced the overall bacteria colonisation within the bone and on the implant, reduced the osteolysis and increased the radiographic union, confirming its potential for reducing complications in wounds at high risk of infection. However, when implanted into a sterile wound, non-union increased. Further studies are required to best optimise the anti-microbial effectiveness, while not sacrificing fracture union.

Resorbable Nanocomposites with Bone-Like Strength and Enhanced Cellular Activity.

Bone cements for treatment of fractures at weight-bearing sites are subjected to dynamic physiological loading from daily activities. An ideal bone cement rapidly sets after injection, exhibits bone-like strength, stimulates osteogenic differentiation of endogenous cells, and resorbs at a rate aligned with patient biology. However, currently available materials fall short of these targeted properties. Nanocrystalline hydroxyapatite (nHA) enhances osteogenic differentiation, new bone formation, and osteoclast differentiation activity compared to amorphous or micron-scale crystalline hydroxyapatite. However, the brittle mechanical properties of nHA precludes its use in treatment of weight-bearing bone defects. In this study, we report settable nHA-poly(ester urethane) (PEUR) nanocomposites synthesized from nHA, lysine triisocyanate (LTI), and poly(caprolactone) triol via a solvent-free process. The nanocomposites are easily mixed and injected using a double-barrel syringe, exhibit mechanical properties exceeding those of conventional bone cements, enhance mineralization of osteoprogenitor cells in vitro, and undergo osteoclast-mediated degradation in vitro. This combination of properties cannot be achieved using other technologies, which underscores the potential of nHA-PEUR nanocomposites as a new approach for promoting bone healing at weight-bearing sites.

Antibiotic-loaded bone void filler accelerates healing in a femoral condylar rat model.

AIMS: Demineralised bone matrix (DBM) is rarely used for the local delivery of prophylactic antibiotics. Our aim, in this study, was to show that a graft with a bioactive glass and DBM combination, which is currently available for clinical use, can be loaded with tobramycin and release levels of antibiotic greater than the minimum inhibitory concentration for Staphylococcus aureus without interfering with the bone healing properties of the graft, thus protecting the graft and surrounding tissues from infection. MATERIALS AND METHODS: Antibiotic was loaded into a graft and subsequently evaluated for drug elution kinetics and the inhibition of bacterial growth. A rat femoral condylar plug model was used to determine the effect of the graft, loaded with antibiotic, on bone healing. RESULTS: We found that tobramycin loaded into a graft composed of bioglass and DBM eluted antibiotic above the minimum inhibitory concentration for three days in vitro. It was also found that the antibiotic loaded into the graft produced no adverse effects on the bone healing properties of the DBM at a lower level of antibiotic. CONCLUSION: This antibiotic-loaded bone void filler may represent a promising option for the delivery of local antibiotics in orthopaedic surgery. Cite this article: Bone Joint J 2016;98-B:1126-31.

Severe muscle trauma triggers heightened and prolonged local musculoskeletal inflammation and impairs adjacent tibia fracture healing.

OBJECTIVES: Complicated fracture healing is often associated with the severity of surrounding muscle tissue trauma. Since inflammation is a primary determinant of musculoskeletal health and regeneration, it is plausible that delayed healing and non-unions are partly caused by compounding local inflammation in response to concomitant muscle trauma. METHODS AND RESULTS: To investigate this possibility, a Lewis rat open fracture model [tibia osteotomy with adjacent tibialis anterior (TA) muscle volumetric muscle loss (VML) injury] was interrogated. We observed that VML injury impaired tibia healing, as indicated by diminished mechanical strength and decreased mineralized bone within the fracture callus, as well as continued presence of cartilage instead of woven bone 28 days post-injury. The VML injured muscle presented innate and adaptive immune responses that were atypical of canonical muscle injury healing. Additionally, the VML injury resulted in a perturbation of the inflammatory phase of fracture healing, as indicated by elevations of CD3(+) lymphocytes and CD68+ macrophages in the fracture callus at 3 and 14d post-injury, respectively. CONCLUSIONS: These data indicate that heightened and sustained innate and adaptive immune responses to traumatized muscle are associated with impaired fracture healing and may be targeted for the prevention of delayed and non-union following musculoskeletal trauma.

Combined local and systemic antibiotic delivery improves eradication of wound contamination: An animal experimental model of contaminated fracture.

Systemic antibiotics reduce infection in open fractures. Local delivery of antibiotics can provide higher doses to wounds without toxic systemic effects. This study investigated the effect on infection of combining systemic with local antibiotics via polymethylmethacrylate (PMMA) beads or gel delivery. An established Staphylococcus aureus contaminated fracture model in rats was used. Wounds were debrided and irrigated six hours after contamination and animals assigned to one of three groups, all of which received systemic antibiotics. One group had local delivery via antibiotic gel, another PMMA beads and the control group received no local antibiotics. After two weeks, bacterial levels were quantified. Combined local and systemic antibiotics were superior to systemic antibiotics alone at reducing the quantity of bacteria recoverable from each group (p = 0.002 for gel; p = 0.032 for beads). There was no difference in the bacterial counts between bead and gel delivery (p = 0.62). These results suggest that local antibiotics augment the antimicrobial effect of systemic antibiotics. Although no significant difference was found between vehicles, gel delivery offers technical advantages with its biodegradable nature, ability to conform to wound shape and to deliver increased doses. Further study is required to see if the gel delivery system has a clinical role.

Muscle-bone interactions during fracture healing.

Although it is generally accepted that the rate and strength of fracture healing is intimately linked to the integrity of surrounding soft tissues, the contribution of muscle has largely been viewed as a vascular supply for oxygen and nutrient exchange. However, more is becoming known about the cellular and paracrine contributions of muscle to the fracture healing process. Research has shown that muscle is capable of supplying osteoprogenitor cells in cases where the periosteum is insufficient, and the muscular osteoprogenitors possess similar osteogenic potential to those derived from the periosteum. Muscle's secrotome includes proteins capable of inhibiting or enhancing osteogenesis and myogenesis following musculoskeletal injury and can be garnered for therapeutic use in patients with traumatic musculoskeletal injuries. In this review, we will highlight the current knowledge on muscle-bone interaction in the context of fracture healing as well as concisely present the current models to study such interactions.

A versatile model of open-fracture infection : a contaminated segmental rat femur defect.

OBJECTIVES: The purpose of this study was to refine an accepted contaminated rat femur defect model to result in an infection rate of approximately 50%. This threshold will allow examination of treatments aimed at reducing infection in open fractures with less risk of type II error. METHODS: Defects were created in the stablised femurs of anaethetised rats, contaminated with Staphylococcus aureus and then debrided and irrigated six hours later. After 14 days, the bone and implants were harvested for separate microbiological analysis. This basic model was developed in several studies by varying the quantity of bacterial inoculation, introducing various doses of systemic antibiotics with and without local antibiotics. RESULTS: The bacterial inoculation associated with a 50% infection rate was established as 1 × 10(2) colony forming units (CFU). With an initial bacterial inoculum of 1 × 10(5) CFU, the dose of systemic antibiotics associated with 50% infection was 5 mg/Kg of cafazolin injected sub-cutaneously every 12 hours, starting at the time of the first debridment and continuing for 72 hours (seven doses). The systemic dose of cafazolin was lowered to 2 mg/Kg when antibiotic polymethyl methacrylate beads were used concurrently with the same amount of bacterial inoculation. CONCLUSION: This model of open fracture infection has been further refined with potential for local and systemic antibiotics. This is a versatile model and with the concepts presented herein, it can be modified to evaluate various emerging therapies and concepts for open fractures. Cite this article: Bone Joint Res 2014;3:187-92.

Translational research to improve the treatment of severe extremity injuries.

OBJECTIVES: Severe extremity injuries are the most significant injury sustained in combat wounds. Despite optimal clinical management, non-union and infection remain common complications. In a concerted effort to dovetail research efforts, there has been a collaboration between the UK and USA, with British military surgeons conducting translational studies under the auspices of the US Institute of Surgical Research. This paper describes 3 years of work. METHODS: A variety of studies were conducted using, and developing, a previously validated rat femur critical-sized defect model. Timing of surgical debridement and irrigation, different types of irrigants and different means of delivery of antibiotic and growth factors for infection control and to promote bone healing were investigated. RESULTS: Early debridement and irrigation were independently shown to reduce infection. Normal saline was the most optimal irrigant, superior to disinfectant solutions. A biodegradable gel demonstrated superior antibiotic delivery capabilities than standard polymethylmethacrylate beads. A polyurethane scaffold was shown to have the ability to deliver both antibiotics and growth factors. DISCUSSION: The importance of early transit times to Role 3 capabilities for definitive surgical care has been underlined. Novel and superior methods of antibiotic and growth factor delivery, compared with current clinical standards of care, have been shown. There is the potential for translation to clinical studies to promote infection control and bone healing in these devastating injuries.

Effect of cell-seeded hydroxyapatite scaffolds on rabbit radius bone regeneration.

Highly porous hydroxyapatite (HA) scaffolds were developed as bone graft substitutes using a template coating process, characterized, and seeded with bone marrow-derived mesenchymal stem cells (BMSCs). To test the hypothesis that cell-seeded HA scaffolds improve bone regeneration, HA scaffolds without cell seeding (HA-empty), HA scaffolds with 1.5 × 10(4) BMSCs (HA-low), and HA scaffolds with 1.5 × 10(6) BMSCs (HA-high) were implanted in a 10-mm rabbit radius segmental defect model for 4 and 8 weeks. Three different fluorochromes were administered at 2, 4, and 6 weeks after implantation to identify differences in temporal bone growth patterns. It was observed from fluorescence histomorphometry analyses that an increased rate of bone infiltration occurred from 0 to 2 weeks (p < 0.05) of implantation for the HA-high group (2.9 ± 0.5 mm) as compared with HA-empty (1.8 ± 0.8 mm) and HA-low (1.3 ± 0.2 mm) groups. No significant differences in bone formation within the scaffold or callus formation was observed between all groups after 4 weeks, with a significant increase in bone regenerated for all groups from 4 to 8 weeks (28.4% across groups). Although there was no difference in bone formation within scaffolds, callus formation was significantly higher in HA-empty scaffolds (100.9 ± 14.1 mm(3) ) when compared with HA-low (57.8 ± 7.3 mm(3) ; p ≤ 0.003) and HA-high (69.2 ± 10.4 mm(3) ; p ≤ 0.02) after 8 weeks. These data highlight the need for a better understanding of the parameters critical to the success of cell-seeded HA scaffolds for bone regeneration.

Effect of coadministration of vancomycin and BMP-2 on cocultured Staphylococcus aureus and W-20-17 mouse bone marrow stromal cells in vitro.

In this study, we aimed to establish an in vitro bacterium/bone cell coculture model system and to use this model for dose dependence studies of dual administration of antibiotics and growth factors in vitro. We examined the effect of single or dual administration of the antibiotic vancomycin (VAN) at 0 to 16 µg/ml and bone morphogenetic protein-2 (BMP-2) at 0 or 100 ng/ml on both methicillin-sensitive Staphylococcus aureus and mouse bone marrow stromal cells (W-20-17) under both mono- and coculture conditions. Cell metabolic activity, Live/Dead staining, double-stranded DNA (dsDNA) amounts, and alkaline phosphatase activity were measured to assess cell viability, proliferation, and differentiation. An interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit was used to test the bone cell inflammation response in the presence of bacteria. Our results suggest that, when delivered together in coculture, VAN and BMP-2 maintain their primary functions as an antibiotic and a growth factor, respectively. Most interestingly, this dual-delivery type of approach has shown itself to be effective at lower concentrations of VAN than those required for an approach relying strictly on the antibiotic. It may be that BMP-2 enhances cell proliferation and differentiation before the cells become infected. In coculture, a dosage of VAN higher than that used for treatment in monoculture may be necessary to effectively inhibit growth of Staphylococcus aureus. This could mean that the coculture environment may be limiting the efficacy of VAN, possibly by way of bacterial invasion of the bone cells. This report of a coculture study demonstrates a potential beneficial effect of the coadministration of antibiotics and growth factors compared to treatment with antibiotic alone.

Early antibiotics and debridement independently reduce infection in an open fracture model.

Most animal studies indicate that early irrigation and debridement reduce infection after an open fracture. Unfortunately, these studies often do not involve antibiotics. Clinical studies indicate that the timing of initial debridement does not affect the rate of infection but these studies are observational and fraught with confounding variables. The purpose of this study was to control these variables using an animal model incorporating systemic antibiotics and surgical treatment. We used a rat femur model with a defect which was contaminated with Staphylococcus aureus and treated with a three-day course of systemic cefazolin (5 mg/kg 12-hourly) and debridement and irrigation, both of which were initiated independently at two, six and 24 hour time points. After 14 days the bone and hardware were harvested for separate microbiological analysis. No animal that received antibiotics and surgery two hours after injury had detectable bacteria. When antibiotics were started at two hours, a delay in surgical treatment from two to six hours significantly increased the development of infection (p = 0.047). However, delaying surgery to 24 hours increase the rate of infection, but not significantly (p = 0.054). The timing of antibiotics had a more significant effect on the proportion of positive samples than earlier surgery. Delaying antibiotics to six or 24 hours had a profoundly detrimental effect on the infection rate regardless of the timing of surgery. These findings are consistent with the concept that bacteria progress from a vulnerable planktonic form to a treatment-resistant biofilm.

Time-dependent effect of chlorhexidine surgical prep.

Despite continued advances in preoperative preventive measures and aseptic technique, surgical site infections remain a problem. The purpose of this study was to evaluate the time-dependent effectiveness of chlorhexidine, a common surgical preparation solution, at various concentrations. Agar plates containing a Mueller-Hinton medium were inoculated with Staphylococcus aureus (lux) bacteria. The bacteria are genetically engineered to emit photons, allowing for quantification with a photon-counting camera system. Standardized amounts of aqueous chlorhexidine at three different concentrations (group 1:4%; group 2:2%; group 3:0.4%) were applied to the agar plates and comparisons in bacterial reduction were made. After 2 min of contact time, groups 1 and 2 had similar reductions in bacterial load with 30% bacterial load remaining in each group (P=0.512), whereas group 3 had a significantly higher bacterial load (33%) when compared to both groups 1 and 2 (1 vs 3, P<0.0001; 2 vs 3, P=0.0002). The bacterial load in all three groups continued to decrease out to the final time point (1h) with group 1 having the least amount of bacterial load remaining, 9% (P<0.0001) and group 3 with the highest bacterial load remaining, 19% (P<0.0001). This study demonstrates two key results: first, dilution of chlorhexidine correlates directly with its bactericidal activity; second, its effectiveness is directly related to its contact time. Based on the results of this study, the authors recommend using 4% chlorhexidine for surgical site preparation and allowing a minimum of 2 min of contact time prior to making the skin incision.

Locally-administered antibiotics in wounds in a limb.

We used a goat model of a contaminated musculoskeletal defect to determine the effectiveness of rapidly-resorbing calcium-sulphate pellets containing amikacin to reduce the local bacterial count. Our findings showed that this treatment eradicated the bacteria quickly, performed as well as standard polymethylmethacrylate mixed with an antibiotic and had many advantages over the latter. The pellets were prepared before surgery and absorbed completely. They released all of the antibiotic and did not require a subsequent operation for their removal. Our study indicated that locally administered antibiotics reduced bacteria within the wound rapidly. This method of treatment may have an important role in decreasing the rate of infection in contaminated wounds.

Combat casualty care research at the U.S. Army Institute of Surgical Research.

The Institute of Surgical Research is the U.S. Army's lead research laboratory for improving the care of combat casualties. The Institute follows a rigorous process for analyzing patterns of injury and the burden of disease to determine where research can be conducted in order to positively impact care. These analyses led the ISR to focus research on: preventing death from bleeding; developing improved pain control techniques; developing improved vital signs analysis techniques; improving the treatment of extremity injuries; preventing burn injuries on the battlefield; and improving critical care for combat casualties. This process has resulted in numerous improvements in care on the battlefield. Highlights include development, fielding, and efficiency testing of tourniquets and improved dressings for bleeding control. Significant progress has also been made in the resuscitation of combat casualties using blood products instead of crystalloid or colloid solutions. Improvements in pain control include assessments of the effect of perioperative anaesthetics on the development of post-traumatic stress disorder [PTSD]. Novelvital signs analyses have been successful in identifying promising techniques which may improve the medic's ability to accurately triage patients. Current research in extremity injuries has focused on optimizing the use of negative pressure wound therapy for contaminated wounds. Burn research has focused on improving personnel protective equipment and implementing continuous renal replacement therapy. This research program is soldier focused and addresses care from self aid and buddy aid through all echelons of care. Many of these advances have been adopted in civilian medical centres as well, benefiting not only the military trauma patient, but also the civilian trauma patient.

Effectiveness of commercially-available antibiotic-impregnated implants.

The aim of this study was to determine the effectiveness of antibiotic-impregnated implants in the prevention of bone infection. We used a model of contaminated fracture in goats to evaluate four treatment groups: no treatment, hand-made tobramycin-impregnated polymethylmethacrylate beads, commercially-available tobramycin-impregnated calcium sulphate pellets and commercially-available tobramycin-impregnated polymethylmethacrylate beads. Three weeks after intraosseous inoculation with streptomycin-resistant Staphylococcus aureus tissue cultures showed no evidence of infection in any of the antibiotic-treated groups. All of the cultures were positive in the untreated group. These results show that effective local antibiotic delivery can be obtained with both commercially-available products and with hand-made polymethylmethacrylate beads. The calcium sulphate pellets have the advantage of being bioabsorbable, thereby obviating the need for a second procedure to remove them.

Suturing of lacerations of skeletal muscle.

Our aim was to compare the biomechanical properties of suturing methods to determine a better method for the repair of lacerated skeletal muscle. We tested Kessler stitches and the combination of Mason-Allen and perimeter stitches. Individual stitches were placed in the muscle belly of quadriceps femoris from a pig cadaver and were tensioned mechanically. The maximum loads and strains were measured and failure modes recorded. The mean load and strain for the Kessler stitches were significantly less than those for combination stitches. All five Kessler stitches tore out longitudinally from the muscle. All five combination stitches did not fail but successfully elongated. Our study has shown that the better method of repair for suturing muscle is the use of combination stitches.

Effects of eccentric exercise training on cortical bone and muscle strength in the estrogen-deficient mouse.

The purpose of this study was to determine whether eccentrically biased exercise training could attenuate changes in muscle and bone function associated with estrogen deficiency in the mouse model. Four groups of ICR mice were used: control (Con), sham ovariectomized (Sham), ovariectomized (OVX), and ovariectomized + high-force resistance training (OVX+Train). All groups except Con were implanted with a nerve cuff surrounding the peroneal nerve to stimulate the left ankle dorsiflexors. Training consisted of 30 stimulated eccentric contractions of the left ankle dorsiflexors at approximately 150% of peak isometric torque every third day for 8 wk. After the training period, groups were not significantly different with regard to peak torque or muscle size. However, the tibial midshaft of the trained leg in the OVX+Train mice exhibited greater stiffness (+15%) than that in the untrained OVX mice, which could not be explained by changes in cross-sectional geometry of the tibia. Scaling of bone mechanical properties to muscle strength were not altered by ovariectomy or training. These data indicate that eccentric exercise training in adult mice can significantly increase bone stiffness, despite the absence of ovarian hormones.

Adaptation to lengthening contraction-induced injury in mouse muscle.

Adaptations to repeated bouts of injury-inducing lengthening contractions were studied in mouse anterior crural muscles. Five bouts of 150 lengthening contractions were performed in vivo, with each bout separated by 2 wk of rest. Three primary observations were made. First, there was little, if any, attenuation in the immediate isometric torque losses after lengthening contractions at "physiological" stimulation frequencies (i.e., <125 Hz), although there was a pronounced decrease in torque loss at higher frequencies between the first and second bouts. Second, the immediate losses in strength that occurred after all five lengthening contraction bouts could be explained in part by excitation-contraction uncoupling. Third, the most important adaptation was a significant enhancement in the rate of recovery of strength after the lengthening contractions. It is probable that the accelerated rate of strength recovery resulted from the more rapid loss and subsequent recovery of myofibrillar protein observed after the fifth bout.