Intravenous Cefazolin Achieves Sustained High Interstitial Concentrations in Open Lower Extremity Fractures.

BACKGROUND: Infection remains a serious clinical concern in patients with open fractures, despite timely antibiotic administration and surgical debridement. Soft tissue and periosteal stripping may alter local tissue homeostasis and antibiotic pharmacokinetics in the injured limb. The tissue (interstitial) concentration of intravenously administered antibiotics at an open fracture site has not been characterized using direct sampling techniques. QUESTION/PURPOSE: We performed this study to evaluate the concentration and pharmacokinetics of intravenously delivered cefazolin at an open fracture site after surgical debridement. METHODS: Twelve patients with an open fracture distal to the knee who presented at a regional Level I trauma center were approached for enrollment in this nonrandomized, observational study. Of the 12 patients, eight adults (one female, seven male) with a median age of 32 years (range 23 to 51 years) were enrolled and underwent successful sample collection for analysis. Three patients had incomplete datasets because of equipment malfunction and one elected not to participate. Seven patients had open tibia fractures, and one patient had an open fibula fracture associated with a closed tibia fracture. There were six Gustilo-Anderson Type II injuries and two Type IIIA injuries. Empiric antibiotics were administered in the prehospital setting or in the emergency department according to institutional protocol. When patients were taken to the operating room, a 2-g intravenous dose of cefazolin was administered. After surgical debridement, fracture stabilization, and wound closure, a microdialysis catheter was placed transdermally into the injury zone (within 5 cm of the fracture site) and a second catheter was placed in the contralateral uninjured (control) limb. Additional doses of cefazolin were administered every 8 hours postoperatively. Baseline and periodic interstitial fluid and whole blood (plasma) samples were collected in the operating room and at prespecified times for 24 hours postoperatively. Free cefazolin in the interstitial fluid and plasma samples were analyzed by ultra-high-performance liquid chromatography using C 18 column separation with quadrupole time-of-flight mass spectrometry detection. Data from the second postoperative dose of cefazolin were used to characterize pharmacokinetic parameters through a noncompartmental analysis using time-concentration curves of free cefazolin and assuming first-order elimination. For pharmacodynamic analyses, the modal cefazolin minimum inhibitory concentration (MIC) of Staphylococcus aureus (1 µg/mL) was used. RESULTS: With the samples available, no difference was observed in the median free cefazolin exposure over 24 hours ( f area under the curve [AUC] 0→24hrs ) between injured limbs (352 µg∙hr/mL [IQR 284 to 594 µg∙hr/mL]) and uninjured limbs (341 µg∙hr/mL [IQR 263 to 438 µg∙hr/mL]; p = 0.64). The median time to achieve the maximum concentration of free cefazolin ( f T max ) for injured limbs was delayed (2.7 hours [IQR 2.2 to 3.1 hours]) compared with control limbs (1.7 hours [IQR 1.2 to 2.0 hours]; p = 0.046). The time to the maximum concentration for plasma was not different from that of control limbs (p = 0.08). The time the cefazolin concentration was above the modal S. aureus MIC (T > MIC) in the injured and control limbs over 24 hours was 100% (IQR 100% to 100%) and 100% (IQR 97% to 100%), respectively. CONCLUSION: These preliminary findings suggest that current prophylactic cefazolin dosing regimens result in successful antibiotic delivery to the traumatized limb in moderately severe open fractures. Although cefazolin delivery to open-fracture wound beds was delayed compared with healthy tissues, the cefazolin concentration was sustained above the European Union Committee Antimicrobial Susceptibility Testing modal MIC for S. aureus , demonstrating a high likelihood of a prophylactic antimicrobial environment at an open fracture site with this empiric antimicrobial regimen. Importantly, patients in this analysis had Gustilo-Anderson Types II and IIIA injuries. Further research with a larger patient cohort is needed to determine whether antibiotic delivery to traumatized soft tissues in patients with higher-grade open fractures (Gustilo-Anderson Types IIIB and IIIC) demonstrates similar pharmacokinetic characteristics. LEVEL OF EVIDENCE: Level II, therapeutic study.

A Retrospective Analysis of Combat Injury Patterns and Prehospital Interventions Associated with the Development of Sepsis.

BACKGROUND: Combat injury related wound infections are common. Untreated, these wound infections may progress to sepsis and septic shock leading to increased morbidity and mortality rates. Understanding infectious complications, patterns, progression, and correlated prehospital interventions are vital to understand the development of sepsis. We aim to analyze demographics, injury patterns, and interventions associated with sepsis in battlefield settings. MATERIALS AND METHODS: This is a secondary analysis of previously published data from the Department of Defense Trauma Registry (DoDTR) from 2007 to 2020. We searched for casualties diagnosed with sepsis (excluding line-sepsis) throughout their initial hospitalization. Regression models were used to seek associations. RESULTS: Our initial request yielded 28,950 encounters, of which 25,654 (88.6%) were adults that met inclusion, including 243 patients (0.9%) diagnosed with sepsis. Patients included US military (34%), non-North Atlantic Treaty Organization (NATO) military (33%) and humanitarian (30%) groups. Patients diagnosed with sepsis had a significantly lower survival rate than non-septic patients (78.1% vs. 95.7%, p < 0.001). There was no significant difference in administration of prehospital antibiotics between septic and the general populations (10.6% vs. 12.3%, p = 0.395). Prehospital intraosseous access (OR 1.56, 95% CI 1.27-1.91, p = 0.207) and packed red cell administration (1.63, 1.24-2.15, 0.029) were the interventions most associated with sepsis. CONCLUSIONS: Sepsis occurred infrequently in the DoDTR when evacuation from battlefield is not delayed, but despite increased intervention frequency, developing sepsis demonstrates a significant drop in survival rates. Future research would benefit from the development of risk mitigation measures.

FK506 increases susceptibility to musculoskeletal infection in a rodent model.

BACKGROUND: Delayed fracture healing caused by soft tissue loss can be resolved by the administration of a Th1 immunosuppressant, such as FK506. Additionally, open fractures are at high risk for infection. We hypothesized that the inclusion of an immunosuppressant to a subject at risk for a musculoskeletal infection will increase the likelihood of infection. METHODS: A rat model of musculoskeletal infection was used. Sprague Dawley rats received a stabilized femur defect and were inoculated with 104 CFU Staphylococcus aureus via a collagen matrix. Six hours after inoculation, the wounds were debrided of collagen and devitalized tissue and irrigated with sterile saline. The animals were randomized into two groups: carrier control and FK506, which were administered daily for 14 days and were euthanized and the tissues harvested to measure local bioburden. RESULTS: The dosing regimen of FK506 that restored bone healing increased the bioburden in the bone and on the fixation implant compared to the carrier control animals. As expected, the administration of FK506 decreased circulating white blood cells, lymphocytes, neutrophils, and monocytes. Additionally, the red blood cell count, hematocrit, and body weight were lower in those animals that received FK506 compared to carrier control. CONCLUSIONS: FK506 administration decreased the systemic immune cell counts and increased the bacterial bioburden within a model of musculoskeletal infection. Collectively, these outcomes could be attributed to the overall T cell suppression by FK506 and the altered antimicrobial activity of innate cells, thereby allowing S. aureus to thrive and subsequently leading to infection of severe, musculoskeletal injuries. These observations reveal the crucial continued investigation for the clinical use of FK506, and other immunosuppressant compounds, in trauma patients who are at increased risk of developing infections.

Orthopaedic Implant Coatings: Recent Approaches and Clinical Translation.

Despite improved surgical techniques and prophylactic procedures, orthopaedic implant-associated infections remain high with complications that can lead to devastating outcomes for the patient. Implant coatings and associated surface modification techniques represent a promising means to prevent infections. Various approaches have emerged to address the challenges associated with implant infections, such as antibacterial resistance, biofilm prevention, and appropriate efficacy kinetics. Methods including antibiotic and antimicrobial peptide surface tethering, use of osteo-conductive and -inductive materials, and altering hydrophobicity and hydrophilicity of the implant surface, have all demonstrated efficacy toward diminished infection risk. Though many of these techniques have shown great potential in in vitro and in vivo studies, clinical translation remains limited with very few commercially available implant coatings globally. This review summarizes recent advancements in orthopaedic implant coatings, pre-clinical studies, and clinical translation, as well as potential future marketed products. (Journal of Surgical Orthopaedic Advances 31(3):169-176, 2022).

Can Dynamic Contrast-Enhanced CT Quantify Perfusion in a Stimulated Muscle of Limited Size? A Rat Model.

BACKGROUND: Muscle injury may result in damage to the vasculature, rendering it unable to meet the metabolic demands of muscle regeneration and healing. Therefore, therapies frequently aim to maintain, restore, or improve blood supply to the injured muscle. Although there are several options to assess the vascular outcomes of these therapies, few are capable of spatially assessing perfusion in large volumes of tissue. QUESTIONS/PURPOSES: Can dynamic contrast-enhanced CT (DCE-CT) imaging acquired with a clinical CT scanner be used in a rat model to quantify perfusion in the anterior tibialis muscle at spatially relevant volumes, as assessed by (1) the blood flow rate and tissue blood volume in the muscle after three levels of muscle stimulation (low, medium, and maximum) relative to baseline as determined by the non-stimulated contralateral leg; and (2) how do these measurements compare with those obtained by the more standard approach of microsphere perfusion? METHODS: The right anterior tibialis muscles of adult male Sprague Dawley rats were randomized to low- (n = 10), medium- (n = 6), or maximum- (n = 3) level (duty cycles of 2.5%, 5.0%, and 20%, respectively) nerve electrode coupled muscle stimulation directly followed by DCE-CT imaging. Tissue blood flow and blood volume maps were created using commercial software and volumetrically measured using NIH software. Although differences in blood flow were detectable across the studied levels of muscle stimulation, a review of the evidence suggested the absolute blood flow quantified was underestimated. Therefore, at a later date, a separate set of adult male Sprague Dawley rats were randomized for microsphere perfusion (n = 7) to define blood flow in the animal model with an accepted standard. With this technique, intra-arterial particles sized to freely flow in blood but large enough to lodge in tissue capillaries were injected. Simultaneously, blood sampling at a fixed flow rate was simultaneously performed to provide a fixed blood flow rate sample. The tissues of interest were then explanted and assessed for the total number of particles per tissue volume. Tissue blood flow rate was then calculated based on the particle count ratio within the reference sample. Note that a tissue's blood volume cannot be calculated with this method. Comparison analysis to the non-stimulated baseline leg was performed using two-tailed paired student t-test. An ANOVA was used to compare difference between stimulation groups. RESULTS: DCE-CT measured (mean ± SD) increasing tissue blood flow differences in stimulated anterior tibialis muscle at 2.5% duty cycle (32 ± 5 cc/100 cc/min), 5.0% duty cycle (46 ± 13 cc/100 cc/min), and 20% duty cycle (73 ± 3 cc/100 cc/min) compared with the paired contralateral non-stimulated anterior tibialis muscle (10 ± 2 cc/100 cc/min, mean difference 21 cc/100 cc/min [95% CI 17.08 to 25.69]; 9 ± 1 cc/100 cc/min, mean difference 37 cc/100 cc/min [95% CI 23.06 to 50.11]; and 11 ± 2 cc/100 cc/min, mean difference 62 cc/100 cc/min [95% CI 53.67 to 70.03]; all p < 0.001). Similarly, DCE-CT showed increasing differences in tissue blood volumes within the stimulated anterior tibialis muscle at 2.5% duty cycle (23.2 ± 4.2 cc/100 cc), 5.0% duty cycle (39.2 ± 7.2 cc/100 cc), and 20% duty cycle (52.5 ± 13.1 cc/100 cc) compared with the paired contralateral non-stimulated anterior tibialis muscle (3.4 ± 0.7 cc/100 cc, mean difference 19.8 cc/100 cc [95% CI 16.46 to 23.20]; p < 0.001; 3.5 ± 0.4 cc/100 cc, mean difference 35.7 cc/100 cc [95% CI 28.44 to 43.00]; p < 0.001; and 4.2 ± 1.3 cc/100 cc, mean difference 48.3 cc/100 cc [95% CI 17.86 to 78.77]; p = 0.010). Microsphere perfusion measurements also showed an increasing difference in tissue blood flow in the stimulated anterior tibialis muscle at 2.5% duty cycle (62 ± 43 cc/100 cc/min), 5.0% duty cycle (89 ± 52 cc/100 cc/min), and 20% duty cycle (313 ± 269 cc/100 cc/min) compared with the paired contralateral non-stimulated anterior tibialis muscle (8 ± 4 cc/100 cc/min, mean difference 55 cc/100 cc/min [95% CI 15.49 to 94.24]; p = 0.007; 9 ± 9 cc/100 cc/min, mean difference 79 cc/100 cc/min [95% CI 33.83 to 125.09]; p = 0.003; and 18 ± 18 cc/100 cc/min, mean difference 295 cc/100 cc/min [95% CI 8.45 to 580.87]; p = 0.023). Qualitative comparison between the methods suggests that DCE-CT values underestimate tissue blood flow with a post-hoc ANOVA showing DCE-CT blood flow values within the 2.5% duty cycle group (32 ± 5 cc/100 cc/min) to be less than the microsphere perfusion value (62 ± 43 cc/100 cc/min) with a mean difference of 31 cc/100 cc/min (95% CI 2.46 to 60.23; p = 0.035). CONCLUSIONS: DCE-CT using a clinical scanner is a feasible modality to measure incremental changes of blood flow and tissue blood volume within a spatially challenged small animal model. Care should be taken in studies where true blood flow values are needed, as this particular small-volume muscle model suggests true blood flow is underestimated using the specific adaptions of DCE-CT acquisition and image processing chosen. CLINICAL RELEVANCE: CT perfusion is a clinically available modality allowing for translation of science from bench to bedside. Adapting the modality to fit small animal models that are relevant to muscle healing may hasten time to clinical utility.

Mechanisms by which hydrogen sulfide attenuates muscle function following ischemia-reperfusion injury: effects on Akt signaling, mitochondrial function, and apoptosis.

Ischemia-reperfusion injury is caused by a period of ischemia followed by massive blood flow into a tissue that had experienced restricted blood flow. The severity of the injury is dependent on the time the tissue was restricted from blood flow, becoming more severe after longer ischemia times. This can lead to many complications such as tissue necrosis, cellular apoptosis, inflammation, metabolic and mitochondrial dysfunction, and even organ failure. One of the emerging therapies to combat ischemic reperfusion injury complications is hydrogen sulfide, which is a gasotransmitter that diffuses across cell membranes to exert effects on various signaling pathways regulating cell survival such as Akt, mitochondrial activity, and apoptosis. Although commonly thought of as a toxic gas, low concentrations of hydrogen sulfide have been shown to be beneficial in promoting tissue survival post-ischemia, and modulate a wide variety of cellular responses. This review will detail the mechanisms of hydrogen sulfide in affecting the Akt signaling pathway, mitochondrial function, and apoptosis, particularly in regards to ischemic reperfusion injury in muscle tissue. It will conclude with potential clinical applications of hydrogen sulfide, combinations with other therapies, and perspectives for future studies.

In Vitro activity of Manuka Honey and polyhexamethylene biguanide on filamentous fungi and toxicity to human cell lines.

Soft-tissue invasive fungal infections are increasingly recognized as significant entities directly contributing to morbidity and mortality. They complicate clinical care, requiring aggressive surgical debridement and systemic antifungal therapy. To evaluate new topical approaches to therapy, we examined the antifungal activity and cytotoxicity of Manuka Honey (MH) and polyhexamethylene biguanide (PHMB). The activities of multiple concentrations of MH (40%, 60%, 80%) and PHMB (0.01%, 0.04%, 0.1%) against 13 clinical mould isolates were evaluated using a time-kill assay between 5 min and 24 h. Concentrations were selected to represent current clinical use. Cell viability was examined in parallel for human epidermal keratinocytes, dermal fibroblasts and osteoblasts, allowing determination of the 50% viability (LD50) concentration. Antifungal activity of both agents correlated more closely with exposure time than concentration. Exophiala and Fusarium growth was completely suppressed at 5 min for all PHMB concentrations, and at 12 and 6 h, respectively, for all MH concentrations. Only Lichtheimia had persistent growth to both agents at 24 h. Viability assays displayed concentration-and time-dependent toxicity for PHMB. For MH, exposure time predicted cytotoxicity only when all cell types were analyzed in aggregate. This study demonstrates that MH and PHMB possess primarily time-dependent antifungal activity, but also exert in vitro toxicity on human cells which may limit clinical use. Further research is needed to determine ideal treatment strategies to optimize antifungal activity against moulds while limiting cytotoxicity against host tissues in vivo.

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections.

Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both tissues and individual cell lines. Collectively our findings demonstrate that, similar to other polyamines, norspermidine displays both inhibitory and dispersive activities on biofilms of clinical multidrug-resistant bacterial isolates, in particular for strains of A. baumannii. Additionally our findings suggest that direct application may be considered on tissues, albeit for limited exposure times.

Impairment of early fracture healing by skeletal muscle trauma is restored by FK506.

BACKGROUND: Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration. METHODS: We hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle). RESULTS: The salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque production CONCLUSION: Collectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.

Risk of Obtaining Routine Cultures During Presumed Aseptic Orthopaedic Procedures.

Treating patients with antibiotics that are selected based on routine cultures obtained from presumed aseptic orthopaedic procedures may lead to an increased risk of antibiotic-related complications without reducing the rate of late deep infection. Routine cultures obtained from 60 of 169 procedures resulted in 23 (38.3%) positive and 37 (61.7%) negative results. Twenty-two patients (13.5%) developed late infections. Seven of 14 patients with positive cultures, who were treated with antibiotics, developed a late infection, while two of nine patients with routine cultures, who received no antibiotic treatment, developed a late infection. Six of 37 patients with negative cultures and seven of 109 patients with no cultures developed a late infection. In patients who developed late deep infection, the microorganism isolated on routine culture only corresponded to the microorganism causing late infection 55.5% of the time. Of all patients treated with antibiotics, seven (29%) experienced an antibiotic-related complication (p = .01). (Journal of Surgical Orthopaedic Advances 26(4):239-245, 2017).

Pathophysiology of Volumetric Muscle Loss Injury.

Volumetric muscle loss (VML) injuries are prevalent in civilian and military trauma patients and are known to impart chronic functional deficits. The frank loss of muscle tissue that defines VML injuries is beyond the robust reparative and regenerative capacities of mammalian skeletal muscle. Given the nature of VML injuries, there is a clear need to develop therapies that promote de novo regeneration of skeletal muscle fibers, which can integrate with the remaining musculature and restore muscle strength. However, the pathophysiology of VML injuries is not completely defined, and, therefore, there may be other opportunities to improve functional outcomes other than de novo regeneration. Herein, clinical and preclinical studies of VML were reviewed to ascertain salient manifestations of VML injury that can impair limb function and muscle strength. The limited clinical data available highlighted proliferative fibrosis secondary to VML injury as a viable target to improve limb range of motion. Selected preclinical studies that used standardized neuromuscular functional assessments broadly identified that the muscle mass remaining after VML injury is performing suboptimally, and, therefore, percent VML strength deficits are significantly worse than can be explained by the initial frank loss of contractile machinery. Potential mechanisms of suboptimal strength of the remaining muscle mass suggested within the literature include intramuscular nerve damage, muscle architectural perturbations, and diminished transmission of force. Collectively, both clinical and preclinical data indicate a complex pathophysiology after VML that presents multiple therapeutic targets. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel.

Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization.

Tissue-engineering scaffolds are often seeded with a single type of cell, but there has been more focus on cocultures to improve angiogenesis and bone formation for craniofacial applications. Investigation of bone-derived osteoblasts (OBs) is important because of the use of bone grafts and migration of OBs from native bone into constructs in vivo and therefore, their contribution to bone formation in vivo. The limitation of primary OBs has been their inability to mineralize without osteogenic factors in vitro. Through coculture of OBs and endothelial cells (ECs) and manipulation of the coculture ratio, mineralization can be achieved without osteogenic media or additional growth factors, thus enhancing their utility for tissue-engineering applications. An optimal ratio of EC/OB for vasculogenesis and mineralization has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios, namely, 10:1, 5:1, 1:1, 1:5, and 1:10 with EC and OB monocultures as controls. The number of vasculogenic networks in a collagen matrix was highest in ratios of 5:1 and 1:1. ECs lined up and formed capillary-like networks by day 10, which was not seen in the other groups. On polystyrene, cells were cocultured with ECs and OBs in direct contact (direct coculture) or separated by a transwell membrane (indirect coculture). At day 21, Alizarin Red staining showed mineralization on the 1:5 and 1:10 direct coculture ratios, with 1:5 having more mineralization nodules present than 1:10. No mineralization was seen in other direct coculture ratios or in any of the indirect coculture ratios. Alkaline phosphatase secretion was highest in the 1:5 direct coculture group. Vascular endothelial growth factor secretion from OBs was present in the 1:5 and 1:10 direct coculture ratios at all time points and inhibited after day 1 in other coculture groups. To improve vasculogenesis, cocultures of primary human ECs and OBs in ratios of 5:1 should be used, but to improve bone formation, the 1:5 direct coculture ratio results in most mineralization.

Alternatives to autograft evaluated in a rabbit segmental bone defect.

PURPOSE: This study was designed to identify strategies for treating bone defects that can be completed on the day of surgery. METHODS: Forty New Zealand white rabbits with unilateral rabbit radius segmental defects (15 mm) were treated with commercially available scaffolds containing either demineralised bone matrix (DBM) or a collagen/beta-tricalcium phosphate composite (Col:ß-TCP); each scaffold was combined with either bone marrow aspirate (BMA) or concentrated BMA (cBMA). Bone regeneration was assessed through radiographic and histological analyses. RESULTS: The concentration of nucleated cells, colony-forming unit-fibroblasts and platelets were increased and haematocrit concentration decreased in cBMA as compared to BMA (p < 0.05). Radiographic analyses of bone formation and defect bridging demonstrated significantly greater bone regeneration in the defects treated with DBM grafts as compared to Col:ß-TCP grafts. The healing of bones treated with Col:ß-TCP was improved when augmented with cBMA. CONCLUSIONS: Scaffolds containing either DBM or Col:ß-TCP with BMA or cBMA are effective same-day strategies available to clinicians for the treatment of bone defects; the latter scaffold may be more effective if combined with cBMA.

Military Orthopaedic Trauma Registry: Quality Data Now Available.

The Military Orthopaedic Trauma Registry (MOTR) orginally began as part of the Department of Defense Trauma Registry (DoDTR) and became a live registry in 2013. As a quality improvement process, this study examined MOTR data for 20 female amputees compared with DoDTR data. The DoDTR provided diagnosis and procedure codes as a list but no details. The MOTR provided additional data, including specific limb, fracture classifications, and associated injuries per limb. The MOTR allowed for construction of a treatment time line for each limb, including number and timing of debridements, antibiotics, and implant types. Orthopaedic-specific complications were also coded more frequently in the MOTR and clearly identified with a specific injury and treatment. During initial quality control checks, the MOTR provides a greater volume and granularity of detail for orthopaedic-specific injury and treatment information, indicating that the MOTR is on track to provide a valuable repository for data-driven orthopaedic management of combat injury.

Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism.

Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 µg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity.

Soluble factors from biofilms of wound pathogens modulate human bone marrow-derived stromal cell differentiation, migration, angiogenesis, and cytokine secretion.

BACKGROUND: Chronic, non-healing wounds are often characterized by the persistence of bacteria within biofilms - aggregations of cells encased within a self-produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from planktonic, or culture-grown, bacterial phenotype, including diminished responses to antimicrobial therapy and persistence against host immune responses. Mesenchymal stromal cells (MSCs) are host cells characterized by their multifunctional ability to undergo differentiation into multiple cell types and modulation of host-immune responses by secreting factors that promote wound healing. While these characteristics make MSCs an attractive therapeutic for wounds, these pro-healing activities may be differentially influenced in the context of an infection (i.e., biofilm related infections) within chronic wounds. Herein, we evaluated the effect of soluble factors derived from biofilms of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on the viability, differentiation, and paracrine activity of human MSCs to evaluate the influence of biofilms on MSC activity in vitro. RESULTS: Exposure of MSCs to biofilm-conditioned medias of S. aureus and P. aeruginosa resulted in reductions in cell viability, in part due to activation of apoptosis. Similarly, exposure to soluble factors from biofilms was also observed to diminish the migration ability of cells and to hinder multi-lineage differentiation of MSCs. In contrast to these findings, exposure of MSCs to soluble factors from biofilms resulted in significant increases in the release of paracrine factors involved in inflammation and wound healing. CONCLUSIONS: Collectively, these findings demonstrate that factors produced by biofilms can negatively impact the intrinsic properties of MSCs, in particular limiting the migratory and differentiation capacity of MSCs. Consequently, these studies suggest use/application of stem-cell therapies in the context of infection may have a limited therapeutic effect.

In vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells.

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

Clinical infectious outcomes associated with biofilm-related bacterial infections: a retrospective chart review.

BACKGROUND: Biofilms are associated with persistent infection. Reports characterizing clinical infectious outcomes and patient risk factors for colonization or infection with biofilm forming isolates are scarce. Our institution recently published a study examining the biofilm forming ability of 205 randomly selected clinical isolates. This present study aims to identify potential risk factors associated with these isolates and assess clinical infectious outcomes. METHODS: 221 clinical isolates collected from 2005 to 2012 and previously characterized for biofilm formation were studied. Clinical information from the associated patients, including demographics, comorbidities, antibiotic usage, laboratory values, and clinical infectious outcomes, was determined retrospectively through chart review. Duplicate isolates and non-clinical isolates were excluded from analysis. Associations with biofilm forming isolates were determined by univariate analysis and multivariate analysis. RESULTS: 187 isolates in 144 patients were identified for analysis; 113 were biofilm producers and 74 were not biofilm producers. Patients were primarily male (78 %) military members (61 %) with combat trauma (52 %). On multivariate analysis, the presence of methicillin-resistant Staphylococcus aureus (p < 0.01, OR 5.09, 95 % CI 1.12, 23.1) and Pseudomonas aeruginosa (p = 0.02, OR 3.73, 95 % CI 1.46, 9.53) were the only characteristics more likely to be present in the biofilm producing isolate group. Infectious outcomes of patients with non-biofilm forming isolates, including cure, relapse/reinfection, and chronic infection, were similar to infectious outcomes of patients with biofilm-forming isolates. Mortality with initial infection was higher in the biofilm producing isolate group (16 % vs 5 %, p = 0.01) but attributable mortality was low (1 of 14). No characteristics examined in this study were found to be associated with relapse/reinfection or chronic infection on multivariate analysis. CONCLUSIONS: Bacteria species, but not clinical characteristics, were associated with biofilm formation on multivariate analysis. Biofilm forming isolates and non-biofilm forming isolates had similar infectious outcomes in this study.

Biofilm formation by clinical isolates and its relevance to clinical infections.

Reports of biofilms have increased exponentially in the scientific literature over the past two decades, yet the vast majority of these are basic science investigations with limited clinical relevance. Biofilm studies involving clinical isolates are most often surveys of isolate collections, but suffer from lack of standardization in methodologies for producing and assessing biofilms. In contrast, more informative clinical studies correlating biofilm formation to patient data have infrequently been reported. In this chapter, biofilm surveys of clinical isolates of aerobic and anaerobic bacteria, mycobacteria, and Candida are reviewed, as well as those pertaining to the unique situation of cystic fibrosis. In addition, the influence of host components on in vitro biofilm formation, as well as published studies documenting the clinical impact of biofilms in human infections, are presented.

Rifamycin Derivatives Are Effective Against Staphylococcal Biofilms In Vitro and Elutable From PMMA.

BACKGROUND: Local antimicrobial delivery through polymethylmethacrylate beads (PMMA), commonly vancomycin, is used for the treatment of contaminated open fractures but has limited activity against Staphylococcus aureus biofilms, which occur commonly in such fractures. Rifamycins have activity against biofilms and are an effective treatment for osteoarticular infections involving staphylococcal biofilms, but there are limited studies evaluating the activity of rifamycin derivatives, other than rifampin, against biofilms of S. aureus and evaluating incorporation of these drugs into PMMA for treatment of contaminated open fractures. QUESTIONS/PURPOSES: (1) Are rifamycin derivatives effective against established biofilms of clinical isolates of S. aureus? (2) Can PMMA be used as a carrier for rifamycin derivatives? METHODS: Biofilms were developed and evaluated for susceptibility to a panel of antimicrobials in vitro using the minimum biofilm eradication concentration high-throughput model. Susceptibility was assessed by measuring bacterial recovery at 6 and 24 hours after antimicrobial treatment. Activity of rifamycin derivatives against intracellular bacteria was also evaluated using a gentamicin protection assay. Evaluation of PMMA as a carrier for rifampin and rifamycin derivatives was determined by assessing the curing time subsequent to loading of rifamycins and characterizing the release kinetics of rifamycins at daily intervals for 14 days from PMMA by performing bioassays. RESULTS: Rifamycin derivatives between 1 and 8 µg/mL reduced bacteria within biofilms 5- to 9-logs and prevented bacterial recovery up to 24 hours post-treatment, indicating near to complete eradication of biofilms. Rifamycin derivatives at 32 µg/mL had activity against intracellular staphylococci, significantly reducing the number of internalized bacteria with limited effects on osteoblast viability. Rifampin was the only rifamycin observed to have a suitable release profile from PMMA, releasing 49% of the total antibiotic and maintaining a sustained released profile up to 14 days at a mean 28 ± 6 µg/mL. CONCLUSIONS: Rifampin can be incorporated into PMMA and eluted at concentrations effective against biofilms and intracellular staphylococci. CLINICAL RELEVANCE: Our in vitro findings suggest that local delivery of rifampin may be an effective strategy for the prevention and/or treatment of open fractures where S. aureus biofilms might develop. Clinical studies are needed to characterize what role this approach might have in the prevention and treatment of infections involving biofilms.