Publications by authors named "Joseph C Wenke"

180 Publications

Systemic T Cell Exhaustion Dynamics Is Linked to Early High Mobility Group Box Protein 1 (HMGB1) Driven Hyper-Inflammation in a Polytrauma Rat Model.

Cells 2021 Jun 30;10(7). Epub 2021 Jun 30.

Combat Wound Care, US Army Institute of Surgical Research, JBSA Ft Sam Houston, San Antonio, TX 78234, USA.

We previously reported an early surge in high mobility group box protein 1 (HMGB1) levels in a polytrauma (PT) rat model. This study investigates the association of HMGB1 levels in mediating PT associated dysregulated immune responses and its influence on the cellular levels of receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). Using the same PT rat model treated with anti-HMGB1 polyclonal antibody, we evaluated changes in circulating inflammatory cytokines, monocytes/macrophages and T cells dynamics and cell surface expression of RAGE and TLR4 at 1, 3, and 7 days post-trauma (dpt) in blood and spleen. Notably, PT rats demonstrating T helper (Th)1 and Th2 cells type early hyper-inflammatory responses also exhibited increased monocyte/macrophage counts and diminished T cell counts in blood and spleen. In blood, expression of RAGE and TLR4 receptors was elevated on CD68 monocyte/macrophages and severely diminished on CD4 and CD8 T cells. Neutralization of HMGB1 significantly decreased CD68 monocyte/macrophage counts and increased CD4 and CD8 T cells, but not γδTCR T cells in circulation. Most importantly, RAGE and TLR4 expressions were restored on CD4 and CD8 T cells in treated PT rats. Overall, findings suggest that in PT, the HMGB1 surge is responsible for the onset of T cell exhaustion and dysfunction, leading to diminished RAGE and TLR4 surface expression, thereby possibly hindering the proper functioning of T cells.
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http://dx.doi.org/10.3390/cells10071646DOI Listing
June 2021

Effects of nanocrystalline hydroxyapatite concentration and skeletal site on bone and cartilage formation in rats.

Acta Biomater 2021 Jun 12. Epub 2021 Jun 12.

Department of Biomedical Engineering, Vanderbilt University, 2201 West End Ave, Nashville TN 37235, United States; Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2201 West End Ave, Nashville TN 37235, United States; Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, 1211 Medical Center Dr., Nashville, TN 37212, United States; Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 1211 Medical Center Dr., Nashville, TN 37217, United States. Electronic address:

Most fractures heal by a combination of endochondral and intramembranous ossification dependent upon strain and vascularity at the fracture site. Many biomaterials-based bone regeneration strategies rely on the use of calcium phosphates such as nano-crystalline hydroxyapatite (nHA) to create bone-like scaffolds. In this study, nHA was dispersed in reactive polymers to form composite scaffolds that were evaluated both in vitro and in vivo. Matrix assays, immunofluorescent staining, and Western blots demonstrated that nHA influenced mineralization and subsequent osteogenesis in a dose-dependent manner in vitro. Furthermore, nHA dispersed in polymeric composites promoted osteogenesis by a similar mechanism as particulated nHA. Scaffolds were implanted into a 2-mm defect in the femoral diaphysis or metaphysis of Sprague-Dawley rats to evaluate new bone formation at 4 and 8 weeks. Two formulations were tested: a poly(thioketal urethane) scaffold without nHA (PTKUR) and a PTKUR scaffold augmented with 22 wt% nHA (22nHA). The scaffolds supported new bone formation in both anatomic sites. In the metaphysis, augmentation of scaffolds with nHA promoted an intramembranous healing response. Within the diaphysis, nHA inhibited endochondral ossification. Immunohistochemistry was performed on cryo-sections of the bone/scaffold interface in which CD146, CD31, Endomucin, CD68, and Myeloperoxidase were evaluated. No significant differences in the infiltrating cell populations were observed. These findings suggest that nHA dispersed in polymeric composites induces osteogenic differentiation of adherent endogenous cells, which has skeletal site-specific effects on fracture healing. STATEMENT OF SIGNIFICANCE: Understanding the mechanism by which synthetic scaffolds promote new bone formation in preclinical models is crucial for bone regeneration applications in the clinic where complex fracture cases are seen. In this study, we found that dispersion of nHA in polymeric scaffolds promoted in vitro osteogenesis in a dose-dependent manner through activation of the PiT1 receptor and subsequent downstream Erk1/2 signaling. While augmentation of polymeric scaffolds with nHA enhanced intramembranous ossification in metaphyseal defects, it inhibited endochondral ossification in diaphyseal defects. Thus, our findings provide new insights into designing synthetic bone grafts that complement the skeletal site-specific fracture healing response.
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http://dx.doi.org/10.1016/j.actbio.2021.05.056DOI Listing
June 2021

Early application of topical antibiotic powder in open-fracture wounds: A strategy to prevent biofilm formation and infections.

OTA Int 2020 Dec 12;3(4):e091. Epub 2020 Oct 12.

US Army Institute of Surgical Research.

Despite meticulous surgical care and systemic antibiotics, open fracture wounds have high rates of infection leading to increased morbidity. To reduce infection rates, orthopaedic surgeons may administer local antibiotics using various carriers that may be ineffective due to poor antibiotic release from carriers, subsequent surgery to remove nondegradable carriers, and mismatch between release kinetics and material degradation. Biofilms form rapidly as bacteria that are within the wound multiply quickly and transform from the antibiotic-susceptible planktonic phenotype to the antibiotic-tolerant biofilm phenotype. This tolerance to antibiotics can occur within hours. Currently, local antibiotics are placed in the wounds using a carrier such as polymethylmethacrylate beads; however, this occurs after surgical debridement that can be hours to even a day after initial injury allowing bacteria enough time to form a biofilm that makes the antibiotic containing polymethylmethacrylate beads less effective. In contrast, emerging practices in elective surgical procedures, such as spine fusion, place antibiotic powder (e.g. vancomycin) in the wound at the time of closure. This has been shown to be extremely effective, presumably because of the very small-time period between potential contamination and local antibiotic application. There is evidence that suggests that the ineffectiveness of local antibiotic use in open fractures is primarily due to the delay in application of local antibiotics from the time of injury and propose a concept of topical antibiotic powder application in the prehospital or emergency department setting.
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http://dx.doi.org/10.1097/OI9.0000000000000091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078147PMC
December 2020

Negative pressure wound therapy does not diminish efficacy of topical antibiotic powder in a preclinical contaminated wound model.

Bone Joint Res 2021 Feb;10(2):149-155

Orthopaedic Trauma Research Department, U.S. Army Institute of Surgical Research, San Antonio, Texas, USA.

Aims: High-energy injuries can result in multiple complications, the most prevalent being infection. Vancomycin powder has been used with increasing frequency in orthopaedic trauma given its success in reducing infection following spine surgery. Additionally, large, traumatic injuries require wound coverage and management by dressings such as negative pressure wound therapy (NPWT). NPWT has been shown to decrease the ability of antibiotic cement beads to reduce infection, but its effect on antibiotic powder is not known. The goal of this study was to determine if NPWT reduces the efficacy of topically applied antibiotic powder.

Methods: Complex musculoskeletal wounds were created in goats and inoculated with a strain of modified to emit light. Six hours after contaminating the wounds, imaging, irrigation, and debridement and treatment application were performed. Animals received either vancomycin powder with a wound pouch dressing or vancomycin powder with NPWT.

Results: There were no differences in eradication of bacteria when vancomycin powder was used in combination with NPWT (4.5% of baseline) compared to vancomycin powder with a wound pouch dressing (1.7% of baseline) (p = 0.986), even though approximately 50% of the vancomycin was recovered in the NPWT exudate canister.

Conclusion: The antimicrobial efficacy of the vancomycin powder was not diminished by the application of NPWT. These topical and locally applied therapies are potentially effective tools that can provide quick, simple treatments to prevent infection while providing coverage. By reducing the occurrence of infection, the recovery is shortened, leading to an overall improvement in quality of life. Cite this article:  2021;10(2):149-155.
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http://dx.doi.org/10.1302/2046-3758.102.BJR-2020-0171.R1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937412PMC
February 2021

Localized mandibular infection affects remote in vivo bioreactor bone generation.

Biomaterials 2020 10 23;256:120185. Epub 2020 Jun 23.

Department of Bioengineering, Rice University, Houston, TX, USA. Electronic address:

Mandibular reconstruction requires functional and aesthetic repair and is further complicated by contamination from oral and skin flora. Antibiotic-releasing porous space maintainers have been developed for the local release of vancomycin and to promote soft tissue attachment. In this study, mandibular defects in six sheep were inoculated with 10 colony forming units of Staphylococcus aureus; three sheep were implanted with unloaded porous space maintainers and three sheep were implanted with vancomycin-loaded space maintainers within the defect site. During the same surgery, 3D-printed in vivo bioreactors containing autograft or xenograft were implanted adjacent to rib periosteum. After 9 weeks, animals were euthanized, and tissues were analyzed. Antibiotic-loaded space maintainers were able to prevent dehiscence of soft tissue overlying the space maintainer, reduce local inflammatory cells, eliminate the persistence of pathogens, and prevent the increase in mandibular size compared to unloaded space maintainers in this sheep model. Animals with an untreated mandibular infection formed bony tissues with greater density and maturity within the distal bioreactors. Additionally, tissues grown in autograft-filled bioreactors had higher compressive moduli and higher maximum screw pull-out forces than xenograft-filled bioreactors. In summary, we demonstrated that antibiotic-releasing space maintainers are an innovative approach to preserve a robust soft tissue pocket while clearing infection, and that local infections can increase local and remote bone growth.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423761PMC
October 2020

Time Course of Immune Response and Immunomodulation During Normal and Delayed Healing of Musculoskeletal Wounds.

Front Immunol 2020 4;11:1056. Epub 2020 Jun 4.

Orthopaedic Trauma Research Department, US Army Institute of Surgical Research, Fort Sam Houston, TX, United States.

Single trauma injuries or isolated fractures are often manageable and generally heal without complications. In contrast, high-energy trauma results in multi/poly-trauma injury patterns presenting imbalanced pro- and anti- inflammatory responses often leading to immune dysfunction. These injuries often exhibit delayed healing, leading to fibrosis of injury sites and delayed healing of fractures depending on the intensity of the compounding traumas. Immune dysfunction is accompanied by a temporal shift in the innate and adaptive immune cells distribution, triggered by the overwhelming release of an arsenal of inflammatory mediators such as complements, cytokines and damage associated molecular patterns (DAMPs) from necrotic cells. Recent studies have implicated this dysregulated inflammation in the poor prognosis of polytraumatic injuries, however, interventions focusing on immunomodulating inflammatory cellular composition and activation, if administered incorrectly, can result in immune suppression and unintended outcomes. Immunomodulation therapy is promising but should be conducted with consideration for the spatial and temporal distribution of the immune cells during impaired healing. This review describes the current state of knowledge in the spatiotemporal distribution patterns of immune cells at various stages during musculoskeletal wound healing, with a focus on recent advances in the field of Osteoimmunology, a study of the interface between the immune and skeletal systems, in long bone fractures. The goals of this review are to (1) discuss wound and fracture healing processes of normal and delayed healing in skeletal muscles and long bones; (2) provide a balanced perspective on temporal distributions of immune cells and skeletal cells during healing; and (3) highlight recent therapeutic interventions used to improve fracture healing. This review is intended to promote an understanding of the importance of inflammation during normal and delayed wound and fracture healing. Knowledge gained will be instrumental in developing novel immunomodulatory approaches for impaired healing.
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http://dx.doi.org/10.3389/fimmu.2020.01056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287024PMC
March 2021

Nanocrystalline hydroxyapatite-poly(thioketal urethane) nanocomposites stimulate a combined intramembranous and endochondral ossification response in rabbits.

ACS Biomater Sci Eng 2020 01 10;6(1):564-574. Epub 2019 Dec 10.

Department of Biomedical Engineering, Vanderbilt University, 2201 West End Ave, Nashville, TN 37235.

Resorbable bone cements are replaced by bone osteoclastic resorption and osteoblastic new bone formation near the periphery. However, the ideal bone cement would be replaced by new bone through processes similar to fracture repair, which occurs through a variable combination of endochondral and intramembranous ossification. In this study, nanocrystalline hydroxyapatite (nHA)-poly(thioketal urethane) (PTKUR) cements were implanted in femoral defects in New Zealand White rabbits to evaluate ossification at 4, 12, and 18 months. Four formulations were tested: an injectable, flowable cement and three moldable putties with varying ratios of calcium phosphate to sucrose granules. New bone formation and resorption of the cement by osteoclasts occurred near the periphery. Stevenel's Blue and Safranin O staining revealed infiltration of chondrocytes into the cements and ossification of the cartilaginous intermediate. These findings suggest that nHA-PTKUR cements support combined intramembranous and endochondral ossification, resulting in enhanced osseointegration of the cement that could potentially improve patient outcomes.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220073PMC
January 2020

Adjuvant antibiotic-loaded bone cement: Concerns with current use and research to make it work.

J Orthop Res 2021 02 2;39(2):227-239. Epub 2020 Mar 2.

Orthopaedic Trauma Department, U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas.

Antibiotic-loaded bone cement (ALBC) is broadly used to treat orthopaedic infections based on the rationale that high-dose local delivery is essential to eradicate biofilm-associated bacteria. However, ALBC formulations are empirically based on drug susceptibility from routine laboratory testing, which is known to have limited clinical relevance for biofilms. There are also dosing concerns with nonstandardized, surgeon-directed, hand-mixed formulations, which have unknown release kinetics. On the basis of our knowledge of in vivo biofilms, pathogen virulence, safety issues with nonstandardized ALBC formulations, and questions about the cost-effectiveness of ALBC, there is a need to evaluate the evidence for this clinical practice. To this end, thought leaders in the field of musculoskeletal infection (MSKI) met on 1 August 2019 to review and debate published and anecdotal information, which highlighted four major concerns about current ALBC use: (a) substantial lack of level 1 evidence to demonstrate efficacy; (b) ALBC formulations become subtherapeutic following early release, which risks induction of antibiotic resistance, and exacerbated infection from microbial colonization of the carrier; (c) the absence of standardized formulation protocols, and Food and Drug Administration-approved high-dose ALBC products to use following resection in MSKI treatment; and (d) absence of a validated assay to determine the minimum biofilm eradication concentration to predict ALBC efficacy against patient specific micro-organisms. Here, we describe these concerns in detail, and propose areas in need of research.
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http://dx.doi.org/10.1002/jor.24616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390691PMC
February 2021

Pharmacological Mitigation of Fibrosis in a Porcine Model of Volumetric Muscle Loss Injury.

Tissue Eng Part A 2020 06 22;26(11-12):636-646. Epub 2020 Jan 22.

Extremity Trauma and Regenerative Medicine Task Area, United States Army Institute of Surgical Research, Fort Sam Houston, Texas.

Volumetric muscle loss (VML) resulting from extremity trauma presents functional deficits and fibrosis, ultimately manifesting disability. The extensive fibrotic accumulation is expected to interfere with neural, trophic, vascular, and mechanical connectivity of any possible regenerative medicine approaches. Our objective was to quantify the muscle properties and stiffness following injury and investigate if the fibrotic deposition could be mitigated using an antifibrotic agent; we hypothesized that antifibrotic treatment would prevent the overwhelming fibrotic response. Yorkshire Cross pigs ( = 10) were randomized to sham or a nontreated ∼20% VML injury. Immediately following surgery, injured animals were further randomized to nintedanib (Ofev; 300 mg/day) or no treatment for 30 days. Longitudinal analysis of muscle function via peroneal nerve stimulation, compartment volume, and quantitative muscle stiffness using shearwave elastography were conducted. Terminally comprehensive histopathologic, biochemical, and genetic investigations were conducted on the skeletal muscle and fibrosis. Through 4 weeks post-VML, nontreated muscles presented a significant deficit (23%) in maximal torque compared to the sham operated ( < 0.01). The stiffness in the VML defect area increased significantly (7-fold) in the VML-nontreated leg than the VML antifibrotic-treated legs by 4 weeks postinjury, which was coupled with the nontreated muscle having ∼40% more hydroxyproline per mg of tissue than those receiving antifibrotic treatment ( = 0.01). This work indicates that VML injury progressively induces fibrosis and muscle stiffness. Antifibrotic treatment can mitigate the pathologic development of fibrosis. Future work should evaluate optimal timing and duration of treatments combined with regenerative medicine approaches in efforts to improve function. Impact statement This work primarily evaluated the effect of a clinically available antifibrotic therapy (nintedanib) on the development of fibrosis after volumetric muscle loss (VML) injury in a large animal model. As a primary outcome measure of fibrosis, skeletal muscle stiffness was repeatedly measured and noninvasively using a quantitative ultrasound device with shearwave elastography capability. The most salient finding of the study is that the antifibrotic nintedanib significantly reduced the development of VML injury-induced fibrous tissue deposition and stiffness.
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http://dx.doi.org/10.1089/ten.TEA.2019.0272DOI Listing
June 2020

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

Clin Orthop Relat Res 2020 01;478(1):179-188

T. J. Walters, J. C. Wenke, Extremity Trauma and Regenerative Medicine, United States Army Institute of Surgical Research, Fort Sam Houston, TX, USA.

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.
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http://dx.doi.org/10.1097/CORR.0000000000001045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000042PMC
January 2020

Duration of extremity tourniquet application profoundly impacts soft-tissue antibiotic exposure in a rat model of ischemia-reperfusion injury.

Injury 2019 Dec 20;50(12):2203-2214. Epub 2019 Sep 20.

United States Army Institute of Surgical Research, 3698 Chambers Pass, Building 3611, Fort Sam Houston, TX, USA.

Introduction: Extremity tourniquet (TNK) application is an effective means of achieving compressible hemorrhage control in the emergency prehospital and clinical trauma setting. Modern United States military medical doctrine recommends TNK use to prevent lethal hemorrhage from extremity injury, followed by systemic prophylactic antibiotics to prevent wound infection. Because tissue pharmacokinetics of prophylactic antimicrobials during and after TNK-induced limb ischemia are largely unknown, this study was conducted to empirically determine the relationship between TNK application time and soft tissue antibiotic exposure in order to guide medical personnel in the management of extremity trauma.

Materials And Methods: Hind limbs of anesthetized male Sprague Dawley rats were exsanguinated, and ischemia maintained by a pneumatic cuff placed at the level of the mid femur on one limb; the non-ischemic contralateral limb served as comparison tissue. Systemic prophylactic antibiotics (cefazolin, moxifloxacin, or ertapenem) were administered intravenously before or after TNK release following 2 or 4 h of ischemia with subsequent re-dosing every 12 h for 3 days. Free antibiotic in the interstitial fluid (ISF) of the tibialis anterior muscle of both hind limbs was recovered via microdialysis during ischemia and over three periods during reperfusion: immediately following TNK release, at 24 h post TNK release, and at 72 h post TNK release. Plasma and ISF free drug concentrations were determined by high-performance liquid chromatography.

Results: Tourniquet application prevented delivery of prophylactic antibiotics into distal soft tissue for the duration of ischemia, and caused a profound reduction in skeletal muscle drug exposure for up to 72 h following TNK release. A progressive decline in tissue antibiotic exposure during reperfusion was observed as TNK times increased from 2 h to 4 h. The timing and severity of reduced drug distribution in post-ischemic skeletal muscle varied substantially among the three antibiotic classes evaluated.

Conclusions: Prolonged tourniquet application can significantly reduce distribution of prophylactic antibiotics into soft tissue during and after ischemia, potentially impairing prophylaxis of extremity wound infection. Our findings support the examination of alternative approaches to wound infection prophylaxis under conditions of delayed casualty evacuation when occlusive hemorrhage control measures are utilized.
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http://dx.doi.org/10.1016/j.injury.2019.09.025DOI Listing
December 2019

Burn and thoracic trauma alters fracture healing, systemic inflammation, and leukocyte kinetics in a rat model of polytrauma.

J Orthop Surg Res 2019 Feb 19;14(1):58. Epub 2019 Feb 19.

Extremity Trauma and Regenerative Medicine, US Army Institute of Surgical Research, San Antonio Military Medical Center, JBSA Ft Sam Houston, San Antonio, TX, USA.

Background: Singular traumatic insults, such as bone fracture, typically initiate an appropriate immune response necessary to restore the host to pre-insult homeostasis with limited damage to self. However, multiple concurrent insults, such as a combination of fracture, blunt force trauma, and burns (polytrauma), are clinically perceived to result in abnormal immune response leading to inadequate healing and resolution. To investigate this phenomenon, we created a model rat model of polytrauma.

Methods: To investigate relationship between polytrauma and delayed healing, we created a novel model of polytrauma in a rat which encompassed a 3-mm osteotomy, blunt chest trauma, and full-thickness scald burn. Healing outcomes were determined at 5 weeks where the degree of bone formation at the osteotomy site of polytrauma animals was compared to osteotomy only animals (OST).

Results: We observed significant differences in the bone volume fraction between polytrauma and OST animals indicating that polytrauma negatively effects wound healing. Polytrauma animals also displayed a significant decrease in their ability to return to pre-injury weight compared to osteotomy animals. Polytrauma animals also exhibited significantly altered gene expression in osteogenic pathways as well as the innate and adaptive immune response. Perturbed inflammation was observed in the polytrauma group compared to the osteotomy group as evidenced by significantly altered white blood cell (WBC) profiles and significantly elevated plasma high-mobility group box 1 protein (HMGB1) at 6 and 24 h post-trauma. Conversely, polytrauma animals exhibited significantly lower concentrations of plasma TNF-alpha (TNF-α) and interleukin 6 (IL-6) at 72 h post-injury compared to OST.

Conclusions: Following polytrauma with burn injury, the local and systemic immune response is divergent from the immune response following a less severe singular injury (osteotomy). This altered immune response that follows was associated with a reduced capacity for wound healing.
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http://dx.doi.org/10.1186/s13018-019-1082-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381742PMC
February 2019

Recommendations for design and conduct of preclinical in vivo studies of orthopedic device-related infection.

J Orthop Res 2019 02 21;37(2):271-287. Epub 2019 Feb 21.

AO Research Institute Davos, Clavadelerstrasse 8, 7270, Davos Platz, Switzerland.

Orthopedic device-related infection (ODRI), including both fracture-related infection (FRI) and periprosthetic joint infection (PJI), remain among the most challenging complications in orthopedic and musculoskeletal trauma surgery. ODRI has been convincingly shown to delay healing, worsen functional outcome and incur significant socio-economic costs. To address this clinical problem, ever more sophisticated technologies targeting the prevention and/or treatment of ODRI are being developed and tested in vitro and in vivo. Among the most commonly described innovations are antimicrobial-coated orthopedic devices, antimicrobial-loaded bone cements and void fillers, and dual osteo-inductive/antimicrobial biomaterials. Unfortunately, translation of these technologies to the clinic has been limited, at least partially due to the challenging and still evolving regulatory environment for antimicrobial drug-device combination products, and a lack of clarity in the burden of proof required in preclinical studies. Preclinical in vivo testing (i.e. animal studies) represents a critical phase of the multidisciplinary effort to design, produce and reliably test both safety and efficacy of any new antimicrobial device. Nonetheless, current in vivo testing protocols, procedures, models, and assessments are highly disparate, irregularly conducted and reported, and without standardization and validation. The purpose of the present opinion piece is to discuss best practices in preclinical in vivo testing of antimicrobial interventions targeting ODRI. By sharing these experience-driven views, we aim to aid others in conducting such studies both for fundamental biomedical research, but also for regulatory and clinical evaluation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:271-287, 2019.
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http://dx.doi.org/10.1002/jor.24230DOI Listing
February 2019

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

J Transl Med 2019 01 21;17(1):33. Epub 2019 Jan 21.

US Army Institute of Surgical Research, Extremity Trauma and Regenerative Medicine, 3698 Chambers Pass BLDG 3611, Ft. Sam Houston, San Antonio, TX, 78234, USA.

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.
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http://dx.doi.org/10.1186/s12967-018-1753-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340183PMC
January 2019

Infection Precedes Heterotopic Ossification in Combat Wounded.

US Army Med Dep J 2018 Jul-Dec(2-18):1-5

US Army Institute of Surgical Research.

Heterotopic ossification is the formation of ossified bone in soft tissue, particularly after soft tissue trauma. Heterotopic ossification is known cause of pain, prosthetic/orthotic malfit, and reoperation following combat extremity injury. The purpose of this research was to examine injury and treatment characteristics that are associated with heterotopic ossification in a broader population of deployment-injured subjects. The Department of Defense Trauma Registry and Military Orthopaedic Trauma Registry was queried for a sample of deployment-injured subjects and the complication of heterotopic ossification. Heterotopic ossification was identified in 15% of subjects following 5% of all injuries. Symptoms attributed to the heterotopic bone were present in 40% of subjects with diagnosed with heterotopic ossification. Heterotopic ossification was not associated with injury severity or aggressiveness of open wound treatment. However, infection was the only positive predictor of heterotopic ossification resulting in two-times greater odds of heterotopic bone formation. This finding is consistent with prior research suggesting that heterotopic ossification requires persistent inflammation to be present in at-risk soft tissue. Among all wounds sustained during deployment injury, heterotopic may not be abundantly common; however, the risk may be further minimized by focused infection control.
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April 2019

Poly(Thioketal Urethane) Autograft Extenders in an Intertransverse Process Model of Bone Formation.

Tissue Eng Part A 2019 07 9;25(13-14):949-963. Epub 2019 Jan 9.

1Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee.

Impact Statement: The development of autograft extenders is a significant clinical need in bone tissue engineering. We report new settable poly(thioketal urethane)-based autograft extenders that have bone-like mechanical properties and handling properties comparable to calcium phosphate bone cements. These settable autograft extenders remodeled to form new bone in a biologically stringent intertransverse process model of bone formation that does not heal when treated with calcium phosphate bone void fillers or cements alone. This is the first study to report settable autograft extenders with bone-like strength and handling properties comparable to ceramic bone cements, which have the potential to improve treatment of bone fractures and other orthopedic conditions.
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http://dx.doi.org/10.1089/ten.TEA.2018.0223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648232PMC
July 2019

Topical rifampin powder for orthopaedic trauma part II: Topical rifampin allows for spontaneous bone healing in sterile and contaminated wounds.

J Orthop Res 2018 12 29;36(12):3142-3150. Epub 2018 Oct 29.

U.S. Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston, Texas, 78234.

Infectious complications can reduce fracture healing rate. Broad spectrum antibiotics are commonly administered to prevent and treat musculoskeletal infections. Local antibiotics are applied to the wound site to increase therapeutic concentrations without increasing systemic toxicity, however, may hinder local tissue recovery. Rifampin has been shown to eradicate mature Staphylococcal biofilms and its use proven for treating musculoskeletal infections. In this study, a spontaneously healing defect model in a rat was used to investigate the impact rifampin powder has on endogenous bone healing in both a sterile and contaminated wound. No significant differences were identified in bone volume fraction via microcomputed tomography, radiological scoring, or histology between an empty defect and animals that received vancomycin or rifampin powder in a sterile wound. When applied to a contaminated musculoskeletal wound, the rifampin powder had significantly greater bone formation compared to the control, as measured by microcomputed tomography, plain radiology, and histology. In addition, the animals treated with rifampin powder had reduced bacteria, reduced white blood cell count and reduced number of clinical indications of infection. Interestingly, while the vancomycin group still displayed signs of infection via quantitative microbiology, plain radiology, and histology, there was significant bone formation within the defect and reduction of systemic signs of infection. We demonstrated that the use of rifampin powder allows bone to heal in both a sterile and contaminated model of musculoskeletal infection. To our knowledge, this is the first time the direct impact of local antibiotics on bone healing has been investigated. Published 2018. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:3142-3150, 2018.
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http://dx.doi.org/10.1002/jor.24155DOI Listing
December 2018

Rapid degradation and non-selectivity of Dakin's solution prevents effectiveness in contaminated musculoskeletal wound models.

Injury 2018 Oct 6;49(10):1763-1773. Epub 2018 Aug 6.

Extremity Trauma and Regenerative Medicine, United States Army Institute of Surgical Research, Fort Sam Houston, TX, USA.

Background: Dakin's solution (buffered sodium hypochlorite) has been used as a topical adjunct for the treatment of invasive fungal infections in trauma patients. Prudent use of Dakin's solution (DS) for complex musculoskeletal wound management implies balancing antimicrobial efficacy and human tissue toxicity, but little empirical evidence exists to inform clinical practice. To identify potentially efficacious DS concentrations and application methods, we conducted two animal studies to evaluate the ability of DS to reduce bacterial burden in small and large animal models of contaminated musculoskeletal wounds.

Methods: An established rat (Rattus norvegicus) contaminated femoral defect model was employed to evaluate the antimicrobial efficacy of DS as a topical adjunctive treatment for Staphylococcus aureus infection. A range of clinically-relevant DS concentrations (0.00025%-0.125%) were tested, both with and without periodic replenishment during treatment. Next, an established goat (Capra hircus) musculoskeletal wound model, consisting of a Pseudomonas aeruginosa contaminated proximal tibia cortical defect, muscle crush, and thermal injury, was utilized to evaluate the antimicrobial efficacy of dilute DS (0.0025% and 0.025%) as a surgical irrigant solution. In situ reactive chlorine concentrations were monitored throughout each treatment using an automated iodometric titration approach.

Results: In a rat wound model, DS treatment did not significantly reduce S. aureus bioburden after 14 days as compared to saline control. Two treatment groups (0.01% single application and 0.025% multiple application) exhibited significantly higher bacterial burden than control. In a goat musculoskeletal wound model, neither 0.0025% nor 0.025% DS significantly altered P. aeruginosa bioburden immediately following treatment or at 48 h post-treatment. Overall, DS applied to exposed soft tissue exhibited rapid degradation, e.g., 0.125% DS degraded 32% after 5 s progressing to 86% degradation after 15 min following single application.

Conclusions: We did not observe evidence of a therapeutic benefit following Dakin's solution treatment for any tested concentration or application method in two contaminated musculoskeletal wound models. Despite confirmation of robust bactericidal activity in vitro, our findings suggest DS at current clinically-used concentrations does not kill tissue surface-attached bacteria, nor does it necessarily cause host tissue toxicity that exacerbates infection in the setting of complex musculoskeletal injury.
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http://dx.doi.org/10.1016/j.injury.2018.08.004DOI Listing
October 2018

Settable polymer/ceramic composite bone grafts stabilize weight-bearing tibial plateau slot defects and integrate with host bone in an ovine model.

Biomaterials 2018 10 26;179:29-45. Epub 2018 Jun 26.

Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, 37235, USA; Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, 37235, USA; Center for Bone Biology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37235, USA. Electronic address:

Bone fractures at weight-bearing sites are challenging to treat due to the difficulty in maintaining articular congruency. An ideal biomaterial for fracture repair near articulating joints sets rapidly after implantation, stabilizes the fracture with minimal rigid implants, stimulates new bone formation, and remodels at a rate that maintains osseous integrity. Consequently, the design of biomaterials that mechanically stabilize fractures while remodeling to form new bone is an unmet challenge in bone tissue engineering. In this study, we investigated remodeling of resorbable bone cements in a stringent model of mechanically loaded tibial plateau defects in sheep. Nanocrystalline hydroxyapatite-poly(ester urethane) (nHA-PEUR) hybrid polymers were augmented with either ceramic granules (85% β-tricalcium phosphate/15% hydroxyapatite, CG) or a blend of CG and bioactive glass (BG) particles to form a settable bone cement. The initial compressive strength and fatigue properties of the cements were comparable to those of non-resorbable poly(methyl methacrylate) bone cement. In animals that tolerated the initial few weeks of early weight-bearing, CG/nHA-PEUR cements mechanically stabilized the tibial plateau defects and remodeled to form new bone at 16 weeks. In contrast, cements incorporating BG particles resorbed with fibrous tissue filling the defect. Furthermore, CG/nHA-PEUR cements remodeled significantly faster at the full weight-bearing tibial plateau site compared to the mechanically protected femoral condyle site in the same animal. These findings are the first to report a settable bone cement that remodels to form new bone while providing mechanical stability in a stringent large animal model of weight-bearing bone defects near an articulating joint.
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http://dx.doi.org/10.1016/j.biomaterials.2018.06.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065109PMC
October 2018

Topical rifampin powder for orthopedic trauma part I: Rifampin powder reduces recalcitrant infection in a delayed treatment musculoskeletal trauma model.

J Orthop Res 2018 12 5;36(12):3136-3141. Epub 2018 Oct 5.

US Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston 78234, Texas.

Open fractures become infected despite meticulous debridement and care. Locally applied antibiotics, commonly embedded in polymethylmethacrylate, deliver high doses of drug directly to the fracture site. Direct application of antibiotic powder, which is being applied prophylactically in spine surgery, is a recent interest in the trauma sector, where bacterial biofilms are more prevalent. Traditional antibiotics, such as vancomycin, are poor performers against bacterial biofilms thus are ineffective in delayed treatment. Rifampin is an effective eradicator of Staphylococcal biofilms. Here, a rat model of musculoskeletal trauma was used to evaluate the utility of locally applied rifampin powder for reducing established orthopedic Staphylococcal infections in a delayed treatment scenario that previously indicated the limited use of local vancomycin. By applying rifampin powder directly to the contaminated segmental defect, the number of bacteria, as well as clinical indications of infection, were significantly reduced compared to vancomycin and daptomycin. Considering the Infectious Disease Society of America's recommendation to use rifampin in combination with another antibiotic to reduce the onset of rifampin resistance, rifampin powder was also applied in combination with vancomycin or daptomycin with insignificant changes in eradication performance. No indications of rifampin resistance were identified. Clinical Significance: The use of locally applied rifampin is a promising therapy for mature and tolerant musculoskeletal infections. Published 2018. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:3136-3141, 2018.
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http://dx.doi.org/10.1002/jor.24055DOI Listing
December 2018

Current therapies in treatment and prevention of fracture wound biofilms: why a multifaceted approach is essential for resolving persistent infections.

J Bone Jt Infect 2018 12;3(2):50-67. Epub 2018 Apr 12.

US Army Institute of Surgical Research, Ft Sam Houston, TX.

Traumatic orthopedic injuries, particularly extremity wounds, are a significant cause of morbidity. Despite prophylactic antibiotic treatment and surgical intervention, persistent infectious complications can and do occur. Persistent bacterial infections are often caused by biofilms, communities of antibiotic tolerant bacteria encased within a matrix. The structural and metabolic differences in this mode of growth make treatment difficult. Herein, we describe both established and novel, experimental treatments targeted at various stages of wound healing that are specifically aimed at reducing and eliminating biofilm bacteria. Importantly, the highly tolerant nature of these bacterial communities suggests that most singular approaches could be circumvented and a multifaceted, combinatorial approach will be the most effective strategy for treating these complicated infections.
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http://dx.doi.org/10.7150/jbji.23423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949568PMC
April 2018

Treatments and Preventative Measures for Trauma-Induced Heterotopic Ossification: A Review.

Clin Transl Sci 2018 07 26;11(4):365-370. Epub 2018 Apr 26.

Unites States Army Institute of Surgical Research, Joint Base Fort Sam Houston, Texas, USA.

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http://dx.doi.org/10.1111/cts.12552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039201PMC
July 2018

Antibiotic Treatment - What Can Be Learned from Point of Injury Experience?

Mil Med 2018 03;183(suppl_1):466-471

Israel Defense Forces, Medical Corps, Tel Hasomer, Ramat Gan 02718, Israel.

Introduction: Early antibiotic administration after trauma reduces infection rates of open wounds. A clinical practice guideline (CPG) was created to ensure that wounded personnel who are not expected to arrive at the hospital within an hour receive antibiotic treatment in the field. This study evaluated how well-advanced life saver (ALS) providers complied with Israeli Defense Force (IDF) CPG.

Materials And Methods: A retrospective review of all trauma patients between November 2011 and January 2015 was conducted. All casualties who suffered from penetrating injuries with evacuation times greater than 60 min were examined. Casualties who should have received antibiotic treatment in accordance with the IDF CPG were further divided into those who received antibiotics (i.e., "Antibiotic" group) and those who did not receive antibiotic treatment (i.e., "No Antibiotics" group).

Results: For a 3-yr period, a total of 5,142 casualties occurred in the pre-hospital environment. According to parameters established in the CPG, 600 casualties should have received antibiotic treatment. Of these patients, only 49 (8.2%) received antibiotic treatment. Comparative analysis between these groups revealed no significant differences in regards to gender, age, and time to MTF; however, significant differences were found in regards to injury severity score (ISS) (p < 0.01), care under fire (i.e., treatment at a combat zone) criteria (p < 0.00001), and life-saving interventions (p < 0.005).

Discussion: Although the reasons for poor adherence to IDF CPG's are not entirely clear, the data suggest that the severity of the injuries sustained by these casualties requiring a greater number of LSIs, longer evacuation distances, and a more hostile battlefield environment may each contribute to poor adherence. Since this has been identified as a training gap, the importance of antibiotic administration at point of injury in delayed evacuation scenarios has been reinforced.
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http://dx.doi.org/10.1093/milmed/usx144DOI Listing
March 2018

Local control of polymicrobial infections via a dual antibiotic delivery system.

J Orthop Surg Res 2018 Mar 15;13(1):53. Epub 2018 Mar 15.

United States Army Institute of Surgical Research, 3855 Roger Brooke Drive, Fort Sam Houston, San Antonio, TX, 78234, USA.

Background: Contaminated traumatic open orthopedic wounds are frequently complicated by polymicrobial contamination and infection. In high-risk wounds, the standard of care comprises debridement and irrigation combined with antibiotics which can be applied directly or combined with systemic antibiotics. Recently, bioabsorbable chitosan sponges have been shown to be an effective single-agent delivery device for local antibiotics with and without negative pressure wound therapy (NPWT). Severely contaminated orthopedic wounds, however, are often complicated by polymicrobial infections, necessitating multiple antibiotic agents. As such, the purpose of this study was to determine if a chitosan sponge would provide a suitable delivery vehicle for multiple antibiotics for the treatment of a polymicrobial infection in a large animal polytraumatic extremity wound model.

Methods: A complex polytraumatic extremity wound was created in 11 adult male Boer goats. Each wound was contaminated with a bioluminescent strain of S. aureus (1 ml of 10 colony forming units/ml) and of P. aeruginosa (1 ml of 10 CFU/ml) which are genetically engineered to allow quantification with a photon-counting camera. Six hours following initial wound creation and contamination, wounds were debrided and irrigated with low-pressure normal saline. The animals were randomized into one of two treatments: wet-to-dry dressings alone or a commercially available chitosan sponge loaded with 1 g vancomycin and 1.2 g of tobramycin. Each animal was then recovered and reimaged 48 h later for total bacteria content; tissue samples were taken from the wound bed to determine relative bacterial colonization.

Results: All animals in the chitosan sponge group saw significant reductions in overall bacterial load of S. aureus and P. aeruginosa (p = 0.001). The bioluminescence was also significantly reduced compared to the wet-to-dry dressing group (p = 0.0001). Furthermore, whereas the antibiotic sponge group displayed near complete eradication of bacteria, the wounds treated with the wet-to-dry dressings alone displayed a significant 2-log increase in total bacteria at 48 h p = 0.0001). S. aureus was the predominant species found in the wounds, comprising 95 and 99% of all bacteria found in the chitosan sponge and wet-to-dry, respectively.

Conclusion: Dual antimicrobial therapy loaded in a chitosan sponge is an effective way to reduce polymicrobial infections traumatic extremity wound.
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http://dx.doi.org/10.1186/s13018-018-0760-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856197PMC
March 2018

Risk of Obtaining Routine Cultures During Presumed Aseptic Orthopaedic Procedures.

J Surg Orthop Adv 2017 WINTER;26(4):239-245

Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, North Carolina.

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).
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May 2018

Fresh whole blood resuscitation does not exacerbate skeletal muscle edema and long-term functional deficit after ischemic injury and hemorrhagic shock.

J Trauma Acute Care Surg 2018 05;84(5):786-794

From the Extremity Trauma and Regenerative Medicine (A.A., J.L.R., N.A.U., J.C.W., T.J.W.), Clinical Research Support Branch (A.A.), and Damage Control Resuscitation (M.D.), Institute of Surgical Research, JBSA, Ft Sam Houston, San Antonio, TX.

Background: Hemorrhagic shock caused by extremity vascular injuries is common in combat injuries. Fluid resuscitation is the standard treatment for severe hemorrhage (HEM). Tourniquets (TKs) used for HEM control cause ischemia-reperfusion (I/R) injury that induces edema formation in the injured muscle. Resuscitation fluids affect edema formation; however, its effect on long-term functional response remains unknown. The objectives of this study are to (1) compare acute muscle damage; (2) determine long-term functional recovery of ischemic muscle; and (3) compare local and systemic inflammatory response including the expression of junctional proteins following early resuscitation with Hextend and fresh whole blood using a rodent model of combined HEM and TK-induced limb I/R.

Methods: Anesthetized Sprague-Dawley rats underwent 42.5% arterial HEM, followed by 3 hours of TK application. Animals were either not resuscitated or resuscitated with Hextend or fresh whole blood. Two time points were evaluated, 2 and 28 days. Plasma cytokine concentrations were determined at baseline and end resuscitation. At 2 days, edema formation, expression of junctional proteins, and tissue level cytokines concentrations were evaluated. At 28 days, in vivo muscle contractile properties were determined. At both time points, routine histology was performed and graded using a semiquantitative grading system.

Results: All animals developed hemorrhagic hypovolemia; the mortality rate was 100% in nonresuscitated rats. Hextend resuscitation exacerbated muscle edema (~11%) and muscle strength deficit (~20%). Fresh whole blood resuscitation presented edema and muscle strength akin to TK only. Fresh whole blood resuscitation upregulated expression of junctional proteins including proangiogenic factors and dampened the inflammatory response.

Conclusion: Fresh whole blood resuscitation does not exacerbate either TK-induced edema or muscle strength deficit. Fresh whole blood resuscitation may reduce both acute and long-term morbidity associated with extremity trauma. To our knowledge, this is the first study to demonstrate the nature of the resuscitation fluid administered following HEM impacts short- and long-term indices of I/R in skeletal muscle.
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http://dx.doi.org/10.1097/TA.0000000000001806DOI Listing
May 2018

Tacrolimus as an adjunct to autologous minced muscle grafts for the repair of a volumetric muscle loss injury.

J Exp Orthop 2017 Nov 10;4(1):36. Epub 2017 Nov 10.

Extremity Trauma and Regenerative Medicine Task Area, United States Army Institute of Surgical Research, 3698 Chambers Pass, BHT1, Fort Sam Houston, TX, 78234, USA.

Background: Volumetric muscle loss (VML) following extremity orthopaedic trauma or surgery results in chronic functional deficits and disability. A current translational approach to address the devastating functional limitations due to VML injury is the use of an autologous minced muscle graft (~1 mm pieces of muscle tissue) replacement into the injured defect area, although limitations related to donor site morbidity are still unaddressed. This study was designed to explore adjunct pharmacological immunomodulation to enhance graft efficacy and promote muscle function following VML injury, and thereby reduce the amount of donor tissue required.

Findings: Using a validated VML porcine injury model in which 20% of the muscle volume was surgically removed, this study examined muscle function over 3 months post-VML injury. In vivo isometric torque of the peroneus teritus (PT) muscle was not different before surgery among sham, non-repaired, non-repaired with tacrolimus, graft-repaired, and graft-repaired with tacrolimus VML groups. Bi-weekly torque analysis of the VML injured musculature presented a significant strength deficit of ~26% compared to pre-injury in the non-repaired, non-repaired with tacrolimus, and graft-repaired groups. Comparatively, the strength deficit in the graft-repair with systemic tacrolimus was marginally improved (~19%; p = 0.056). Both of the minced graft repaired groups presented a greater proportion of muscle tissue in full-thickness histology specimen.

Conclusions: We demonstrate that adjunctive use of tacrolimus with an ~50% minced muscle graft replacement resulted in modest improvements in muscle function 3 months after injury and repair, but the magnitude of improvement is not expected to elicit clinically meaningful functional improvements.
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http://dx.doi.org/10.1186/s40634-017-0112-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681453PMC
November 2017

Determining potential of PMMA as a depot for rifampin to treat recalcitrant orthopaedic infections.

Injury 2017 Oct 16;48(10):2095-2100. Epub 2017 Aug 16.

Extremity Trauma and Regenerative Medicine Task Area, US Army Institute of Surgical Research, JBSA-Fort Sam Houston, TX, United States.

Background: Open fractures are often complicated by infection. In cases of severe soft tissue and vascular injury, systemic antibiotics may be ineffective due to their inability to reach and provide direct antimicrobial activity to the zone of injury. High antibiotic concentrations within the wound can be achieved with reduced systemic toxicity by using local antibiotic delivery. As bacteria associated with musculoskeletal injuries frequently form biofilms, antibiotic selection is important. Herein, the use of rifampin, an antibiotic with activity against biofilms, delivered via polymethylmethacrylate (PMMA) beads is evaluated for use in a traumatic musculoskeletal wound model.

Methods: PMMA beads loaded with rifampin, or combinations of rifampin and vancomycin, were prepared and evaluated for time to curing, drug release kinetics in vitro, and infection prevention in vivo using a well-established rat model of musculoskeletal infection. A segmental bone defect was created and contaminated with methicillin susceptible Staphylococcus aureus (UAMS-1). Wounds were debrided, irrigated, and treated with PMMA beads, containing rifampin or combinations of rifampin plus vancomycin, following a 6-h (early) or 24-h (delayed) treatment. After 14days, tissue, implants, and beads were removed for bacterial quantification and assessed for rifampin resistance.

Results: There was a direct association between loaded concentration and release kinetics of the rifampin and vancomycin from PMMA beads. Higher rifampin concentrations delayed PMMA curing times. The addition of vancomycin to PMMA resulted in more rapid release of rifampin from beads. However, the highest concentration of rifampin loaded PMMA beads (10% wt/wt) was the only treatment to significantly reduce bacterial counts. No rifampin resistance was observed.

Conclusion: Although higher concentrations of rifampin resulted in significant reductions of bacteria, these levels extended PMMA curing times and transformed PMMA material characteristics. While these characteristics make the material unsuitable for weight-bearing applications, such as total joint arthroplasty, the use of rifampin-loaded PMMA beads may be an effective intervention in a contaminated traumatic extremity wound due to its ability to eradicate biofilms.
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http://dx.doi.org/10.1016/j.injury.2017.08.021DOI Listing
October 2017

Autologous minced muscle grafts improve endogenous fracture healing and muscle strength after musculoskeletal trauma.

Physiol Rep 2017 Jul;5(14)

Extremity Trauma and Regenerative Medicine Task Area, US Army Institute of Surgical Research, Fort Sam Houston, Texas

The deleterious impact of concomitant muscle injury on fracture healing and limb function is commonly considered part of the natural sequela of orthopedic trauma. Recent reports suggest that heightened inflammation in the surrounding traumatized musculature is a primary determinant of fracture healing. Relatedly, there are emerging potential therapeutic approaches for severe muscle trauma (e.g., volumetric muscle loss [VML] injury), such as autologous minced muscle grafts (1 mm pieces of muscle; GRAFT), that can partially prevent chronic functional deficits and appear to have an immunomodulatory effect within VML injured muscle. The primary goal of this study was to determine if repair of VML injury with GRAFT rescues impaired fracture healing and improves the strength of the traumatized muscle in a male Lewis rat model of tibia open fracture. The most salient findings of the study were: (1) tibialis anterior (TA) muscle repair with GRAFT improved endogenous healing of fractured tibia and improved the functional outcome of muscle regeneration; (2) GRAFT repair attenuated the monocyte/macrophage (CD45CDllb) and T lymphocyte (CD3) response to VML injury; (3) TA muscle protein concentrations of MCP1, IL-10, and IGF-1 were augmented in a proregenerative manner by GRAFT repair; (4) VML injury concomitant with osteotomy induced a heightened systemic presence of alarmins (e.g., soluble RAGE) and leukocytes (e.g., monocytes), and depressed IGF-1 concentration, which GRAFT repair ameliorated. Collectively, these data indicate that repair of VML injury with a regenerative therapy can modulate the inflammatory and regenerative phenotype of the treated muscle and in association improve musculoskeletal healing.
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http://dx.doi.org/10.14814/phy2.13362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532491PMC
July 2017

An Effective Negative Pressure Wound Therapy-Compatible Local Antibiotic Delivery Device.

J Orthop Trauma 2017 Dec;31(12):631-635

US Army Institute of Surgical Research, Fort Sam Houston, TX.

Objectives: The current clinical standard for local antibiotic depot is polymethylmethacrylate (PMMA) beads. Unfortunately, these are not ideal and negative pressure wound therapy (NPWT) reduces their limited effectiveness. Recently, a chitosan sponge has been shown to be an effective carrier of antibiotics. Because it acts as a delivery vehicle with increased wound contact area instead of an antibiotic depot, it may be more effective. The objectives of this study were to determine if (1) a chitosan sponge would be more effective than PMMA beads as a local antibiotic delivery device and (2) the chitosan sponge remains an effective method of delivery when used in conjunction with NPWT.

Methods: Contaminated musculoskeletal wounds were created on the proximal tibia of goats; the animals were assigned to 1 of 4 groups (bead pouch, beads with NPWT, sponge pouch, and sponge with NPWT). The animals were survived for 48 hours, and the bacteria in the wound were quantified. The antibiotic levels in the blood and within the NPWT canisters were measured throughout the study period.

Results: After treatment, there were significantly fewer bacteria in wounds treated with antibiotic chitosan sponge delivery than antibiotic PMMA bead depot (P < 0.05), and NPWT did not reduce the effectiveness of the chitosan sponge even though large amounts of vancomycin was found in the canisters. The peak serum levels of vancomycin were well below what is considered safe levels.

Conclusions: Antibiotic delivery to the wound using a chitosan sponge is compatible with NPWT and is more effective than PMMA antibiotic depot. The chitosan sponge works in conjunction with NPWT and may improve the outcomes of open fracture wounds.
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http://dx.doi.org/10.1097/BOT.0000000000000988DOI Listing
December 2017