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BackgroundBacterial biofilms readily develop on all medical implants, including percutaneous osseointegrated (OI) implants. With the growing rate of antibiotic resistance, exploring alternative options for managing biofilm-related infections is necessary. Antimicrobial blue light (aBL) is a unique therapy that can potentially manage biofilm-related infections at the skin-implant interface of OI implants. Antibiotics are known to have antimicrobial efficacy disparities between the planktonic and biofilm bacterial phenotypes, but it is unknown if this characteristic also pertains to aBL. In response, we developed experiments to explore this aspect of aBL therapy.MethodsWe determined minimum bactericidal concentrations (MBCs) and antibiofilm efficacies for aBL, levofloxacin, and rifampin against Staphylococcus aureus ATCC 6538 planktonic and biofilm bacteria. Using student t-tests (p < 0.05), we compared the efficacy profiles between the planktonic and biofilm states for the three independent treatments and a levofloxacin + rifampin combination. Additionally, we compared antimicrobial efficacy patterns for levofloxacin and aBL against biofilms as dosages increased.ResultsaBL had the most significant efficacy disparity between the planktonic and biofilm phenotypes (a 2.5 log10 unit difference). However, further testing against biofilms revealed that aBL had a positive correlation between increasing efficacy and exposure time, while levofloxacin encountered a plateau. While aBL efficacy was affected the most by the biofilm phenotype, its antimicrobial efficacy did not reach a maximum.Discussion/conclusionWe determined that phenotype is an important characteristic to consider when determining aBL parameters for treating OI implant infections. Future research would benefit from expanding these findings against clinical S. aureus isolates and other bacterial strains, as well as the safety of long aBL exposures on human cells.

Negative-pressure wound therapy (NPWT) is commonly utilized to treat traumatic injuries sustained on the modern battlefield. However, NPWT has failed to decrease the incidence of deep tissue infections experienced by Wounded Warriors, despite attempts to integrate common antimicrobials, like Ag+ nanoparticles, into the wound dressing. The purpose of this study was to incorporate a unique antibiofilm compound (CZ-01179) into the polyurethane matrix of NPWT foam via lyophilized hydrogel scaffolding. Foam samples with 2.5%, 5.0%, and 10.0% w/w CZ-01179 were produced and antibiofilm efficacy was compared to the current standards of care: V.A.C.® GRANUFOAM SILVER™ and V.A.C.® GRANUFOAM™. Gravimetric analysis and elution kinetics testing confirmed that this loading technique was both repeatable and controllable. Furthermore, zone of inhibition and antibiofilm efficacy testing showed that foam loaded with CZ-01179 had significantly increased activity against planktonic and biofilm phenotypes of methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii compared to the clinical standards. These findings motivate additional ex vivo and in vivo work with NPWT foam loaded with CZ-01179 with the overall objective of reducing NPWT-associated infections that complicate battlefield-related and other wounds.

Periprosthetic joint infection (PJI) is a devastating complication of orthopedic implant surgeries, such as total knee and hip arthroplasties. Treatment requires additional surgeries because antibiotics have limited efficacy due to biofilm formation and resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA). A non-pharmaceutical approach is needed, and examples of this are found in nature; dragonfly and cicada wings are antibacterial because of their nanopillar surface structure rather than their chemistry. Carbon-infiltrated carbon nanotube (CICNT) surfaces exhibit a similar nanopillar structure, and have been shown to facilitate osseointegration, and it is postulated that they might provide a structurally-derived resistance to bacterial proliferation and biofilm formation. The objective of this study was to test the biofilm resistance of CICNT coatings. Two types of CICNT were produced: a vertically aligned CNT forest on a silicon substrate using a layer of iron as the catalyst (CICNT-Si) and a random-oriented CNT forest on stainless steel (SS) substrate using the substrate as the catalyst (CICNT-SS). These were tested against SS and carbon controls. After 48 h in an MRSA biofilm reactor, samples demonstrated that both types of CICNT coatings significantly (p

Clinical preoperative skin preparations (PSPs) do not eradicate skin flora dwelling in the deepest dermal regions. Survivors constitute a persistent infection risk. In search of solutions, we created a porcine model intended for PSP developmental testing. This model employed microbiological techniques sensitive to the deep-dwelling microbial flora as these microorganisms are frequently overlooked when using institutionally-entrenched testing methodologies. Clinical gold-standard PSPs were assessed. Ten Yorkshire pigs were divided into two groups: prepared with either povidone iodine (PVP-I) or chlorhexidine gluconate (CHG) PSP. Bioburdens were calculated on square, 4 cm by 4 cm, full-thickness skin samples homogenized in neutralizing media. Endogenous bioburden of porcine skin (3.3 log10 CFU/cm2) was consistent with natural flora numbers in dry human skin. On-label PSP scrub kits with PVP-I (n = 39) or CHG (n = 40) failed the 2–3 log10-reduction criteria established for PSPs by the Food and Drug Administration (FDA), resulting in a 1.46 log10 and 0.58 log10 reduction, respectively. Porcine dermal microbiota mirrored that of humans, displaying abundant staphylococcal species. Likewise, histological sections showed similarity in hair follicle depths and sebaceous glands (3.2 ± 0.7 mm). These shared characteristics and the considerable fraction of bacteria which survived clinical PSPs make this model useful for developmental work.

Prosthetics increase the risk of deep surgical site infections in procedures intended to restore function. In orthopaedics, prosthetic joint infections can lead to repetitive surgeries, amputation, or worse. Biofilm formation both in vitro and in vivo involves stages of attachment, accumulation, and maturation. The level of maturation affects susceptibility to antibiotics, the immune system, and the success of surgical interventions. A review of the literature indicates that orthopedic publications are less likely to mention biofilm. We have reviewed animal models of infection to assess in vivo models of prosthetic infection. Although most prosthetic infections seem to originate from local skin microbiota, clinically representative biofilm inocula are unusual. Biofilm-related end points are more widely adopted, but studies rarely include both quantification of adherent microbial burden and imaging of the in vivo biofilm. Failure to differentiate between planktonic and biofilm infections can skew research away from needed chronic disease models. In this review, we address prosthetic joint infections as an important model for chronic biofilm infection research, identify critical requirements for in vivo models of chronic infection, and propose that resistance to the terminology of biofilm research exists within both research and regulation, which could limit progress toward important orthopaedic targets.

Prosthetic joint infection (PJI) is a well-known and persisting problem. Active release coatings have promise to provide early protection to an implant by eradicating small colony biofilm contaminants or planktonic bacteria that can form biofilm. Traditional antibiotics can be limited as active release agents in that they have limited effect against biofilms and develop resistance at sub-lethal concentrations. A unique first-in-class compound (CZ-01127) was assessed as the active release agent in a silicone (Si)-based coating to prevent PJI in a sheep model of joint space infection. Titanium (Ti) plugs contained a porous coated Ti (PCTi) region and polymer-coated region. Plugs were implanted into a femoral condyle of sheep to assess the effect of the Si polymer on cancellous bone ingrowth, the effect of CZ-01127 on bone ingrowth, and the ability of CZ-01127 to prevent PJI. Microbiological results showed that CZ-01127 was able to eradicate bacteria in the local region of the implanted plugs. Data further showed that Si did not adversely affect bone ingrowth. However, bacteria that reached the joint space (synovium) were not fully eradicated. Outcomes suggested that the CZ-01127 coating provided local protection to the implant system in a challenging model, the design of which could be beneficial for testing future antimicrobial therapies for PJI. STATEMENT OF SIGNIFICANCE: Periprosthetic joint infection (PJI) is now commonplace, and constitutes an underlying problem that patients and physicians face. Active release antibiotic coatings have potential to prevent these infections. Traditional antibiotics are limited in their ability to eradicate bacteria that reside in biofilms, and are more susceptible to resistance development. This study addressed these limitations by testing the efficacy of a unique antimicrobial compound in a coating that was tested in a challenging sheep model of PJI. The unique coating was able to eradicate bacteria and prevent infection in the environment adjacent to the implant. Bacteria that escaped into the joint space still caused infection, yet benchmark data can be used to optimize the coating and translate it toward clinical use.

Biofilm formation is a dynamic process that leads to mature communities over time. Despite a general knowledge of biofilm community formation and the resultant limitations of antibiotic therapy, there is a paucity of data describing specific plume heights, surface coverage and rates of maturation. Furthermore, little is published on the effect that the broth medium might have on the degree of biofilm maturation. In this study, three strains of methicillin-resistant Staphylococcus aureus (MRSA) (USA300, USA400 and a clinical isolate) were grown in brain heart infusion broth (BHI) or tryptic soy broth (TSB). Following growth, SEM images were captured for 3-D analysis to assess plume height. TSB produced significantly higher plume heights of USA300 and USA400 compared to BHI. Broth type was less influential on the clinical isolate. The data indicate that broth type and time may be important factors to consider when assessing maturation and plume height formation of MRSA biofilms.

Heterotopic ossification (HO) refers to ectopic bone formation, typically in residual limbs following trauma and injury. A review of injuries from Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) indicated that approximately 70% of war wounds involved the musculoskeletal system, largely in part from the use of improvised explosive devices (IED) and rocket-propelled grenades (RPG). HO is reported to occur in approximately 63%–65% of wounded warriors from OIF and OEF. Symptomatic HO may delay rehabilitation regimens since it often requires modifications to prosthetic limb componentry and socket size. There is limited evidence indicating a mechanism for preventing HO. This may be due to inadequate models, which do not produce HO bone structure that is morphologically similar to HO samples obtained from wounded warfighters injured in theatre. We hypothesized that using a high-power blast of air (shockwave) and simulated battlefield trauma (i.e. bone damage, tourniquet, bacteria, negative pressure wound therapy) in a large animal model, HO would form and have similar morphology to ectopic bone observed in clinical samples. Initial radiographic and micro-computed tomography (CT) data demonstrated ectopic bone growth in sheep 24 weeks post-procedure. Advanced histological and backscatter electron (BSE) analyses showed that 5 out of 8 (63%) sheep produced HO with similar morphology to clinical samples. We conclude that not all ectopic bone observed by radiograph or micro-CT in animal models is HO. Advanced histological and BSE analyses may improve confirmation of HO presence and morphology, which we demonstrated can be produced in a large animal model., Graphical abstract Unlabelled Image

Estrogen deficiency has been shown to negatively influence rotator cuff tendon healing. Therefore, the addition of an estrogen-like-compound (ELC) in a nonestrogen-deficient animal may improve the quality of a rotator cuff repair. The purpose of this study was to evaluate the effects of an ELC, diethylstilbestrol (DES), on tendon healing in a murine rotator cuff repair model. Thirty-three male wild-type mice (C57BL/6NJ) were randomly divided into three study groups. Group 1-unoperated mice with normal rotator cuff tendons. Groups 2 and 3 consisted of surgically repaired rotator cuff tendons; Group 2 (repair-only) was the standard repair group (no DES injected), whereas Group 3 (repair + DES) was the experimental repair group (injected with DES). Comparing the maximal thickness of calcified fibrocartilage to uncalcified fibrocartilage, the ratios for the control (intact tendon), repair-only, and repair + DES groups were 2:1, 0.9:1, and 1.7:1. RNA expression data demonstrated upregulation of chondrogenic, angiogenic, and tendon modulation genes in the repair- only group compared to the control (intact tendon) group (p < 0.04 for all), and that addition of DES further increased the osteogenic, angiogenic, and tendon modulation gene expression compared to the repair-only group (p < 0.02). Immunohistochemical analysis indicated that the addition of DES further increased osteogenic, angiogenic, and tendon maturation protein expression at the enthesis compared to standard repairs.

The literature demonstrates that obtaining a biopsy of the physis may be beneficial for diagnostic purposes. A small biopsy of the epiphyseal plate may allow for earlier detection of certain conditions and be used to monitor the healing of diseased and/or damaged physes. However, due to the fear of a growth arrest in a growing child, biopsies are not currently performed. In this study, we investigated the effects of a biopsy of the epiphyseal plate in 3-month-old lambs. A total of 4.2 mm biopsy samples were captured in the proximal tibiae and distal femora physes. The lambs were monitored 12- and 24-week post-biopsy. Computed tomography (CT) and micro-CT scans were obtained to determine if any angular deformities occurred, while scanning electron microscope (SEM) and histological analysis were utilized to assess the bone response due to the biopsy. The contralateral limbs served as unaltered controls for direct comparison within each lamb. The data demonstrated no signs of angular deformities following a 4.2 mm biopsy of the physis. Bone growth/elongation was confirmed by CT, SEM, and fluorochrome analyses and indicated that the lambs were in fact immature and still growing at the time of the biopsy. Clinical Significance: This investigation demonstrated that a small biopsy of the epiphyseal plate can be obtained safely without the cause of growth arrest and angular deformities. The ability to precisely diagnose, treat, and/or monitor at-risk children at an earlier timepoint by way of a biopsy sample could be an important advancement in regard to researching diseased and/or damaged physes.

Biofilm-impaired tissue is a significant factor in chronic wounds such as diabetic foot ulcers. Most, if not all, anti-biotics in clinical use have been optimized against planktonic phenotypes. In this study, an in vitro assessment was performed to determine the potential efficacy of a unique first-in-class series of antibiofilm antibiotics and compare outcomes to current clinical standards of care. The agent, CZ-01179, was formulated into a hydrogel and tested against mature biofilms of a clinical isolate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa ATCC 27853 using two separate methods. In the first method, biofilms were grown on cellulose discs on an agar surface. Topical agents were spread on gauze and placed over the biofilms for 24 h. Biofilms were quantified and imaged with confocal and scanning electron microscopy. In the second method, biofilms were grown on bioabsorbable collagen coupons in a modified CDC biofilm reactor. Coupons were immersed in treatment for 24 h. The first method was limited in its ability to assess efficacy. Efficacy profiles against biofilms grown on collagen were more definitive, with CZ-01179 gel eradicating well-established biofilms to a greater degree compared to clinical standards. In conclusion, CZ-01179 may be a promising topical agent that targets the biofilm phenotype. Pre-clinical work is currently being performed to determine the translatable potential of CZ-01179 gel.

Flash autoclaving is one of the most frequently utilized methods of sterilizing devices, implants or other materials. For a number of decades, it has been common practice for surgeons to remove implantable devices, flash autoclave and then reimplant them in a patient. Data have not yet indicated the potential for biofilms to survive or remain on the surface of orthopaedic-relevant materials following flash autoclave. In this study, monomicrobial and polymicrobial biofilms were grown on the surface of clinically relevant titanium materials and exposed to flash autoclave settings that included varying times and temperatures. Data indicated that when the sterilization and control temperatures of an autoclave were the same, biofilms were able to survive flash autoclaving that was performed for a short duration. Higher temperature and increased duration rendered biofilms non-viable, but none of the autoclave settings had the ability to remove or disperse the presence of biofilms from the titanium surfaces. These findings may be beneficial for facilities, clinics, or hospitals to consider if biofilms are suspected to be present on materials or devices, in particular implants that have had associated infection and are considered for re-implantation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1543-1550, 2018.

Obtaining a biopsy of the physis in a pediatric/juvenile could provide the ability to diagnose and manage children with physeal abnormalities. However, it has not yet been determined whether a physeal biopsy procedure affects angular deformity. We employed a rabbit model to collect biopsies of the distal femoral and proximal tibial physes on anesthetized, 8-week old New Zealand rabbits. The contralateral limb served as a control. At 8 (n = 5) and 16 (n = 5) weeks postbiopsy, animals were euthanized. Micro-computed tomography (CT) was employed to estimate percentage of the physis biopsied and assess structural abnormalities resulting from biopsy. Bone samples were embedded in polymethylmethacrylate and analyzed. The percentage of physis sampled was ≤1.5% of the total femoral physis while all but one of the tibiae had ≤2.3% removed. There were no iatrogenic clinical or radiographic deformities (frontal or sagittal). Micro-CT and histological analysis suggested that physeal defects had signs of healing that did not lead to subsequent angular deviation. A defect caused by physeal biopsy may not lead to angular deformity. Long-term data could help determine the safety and efficacy of collecting biopsies for histological analyses. Advanced imaging may demonstrate a detailed picture of anatomic or structural alteration of a given physis, but provides no functional information. The diagnostic and therapeutic information that could be gleaned from one or more serial biopsy samples could be invaluable in decision making and clinical management (e.g. skeletal dysplasias and metabolic conditions), so long as subsequent deformity is not a future possibility.

A variety of polyamines have been synthesized from their corresponding amino alcohols through a 3-step process. The synthesis predicates that the most likely impurities are of higher molecular weight and that distillation is effective at producing highly pure materials. This sequence is scalable, requires no protecting groups or chromatography, and provides the requisite polyamines in moderate to good yields.

Wounds complicated by biofilms challenge even the best clinical care and can delay a return to duty for service members. A major component of treatment in wounded warriors includes infected wound management. Yet, all antibiotic therapy options have been optimized against planktonic bacteria, leaving an important gap in biofilm-related wound care. We tested the efficacy of a unique compound (CZ-01179) specifically synthesized to eradicate biofilms. CZ-01179 was formulated as the active agent in a hydrogel, and tested in vitro and in vivo in a pig excision wound model for its ability to treat and prevent biofilm-related wound infection caused by Acinetobacter baumannii. Data indicated that compared to a clinical standard—silver sulfadiazine—CZ-01179 was much more effective at eradicating biofilms of A. baumannii in vitro and up to 6 days faster at eradicating biofilms in vivo. CZ-01179 belongs to a broader class of newly-synthesized antibiofilm agents (referred to as CZ compounds) with reduced risk of resistance development, specific efficacy against biofilms, and promising formulation potential for clinical applications. Given its broad spectrum and biofilm-specific nature, CZ-01179 gel may be a promising agent to increase the pipeline of products to treat and prevent biofilm-related wound infections.

Biofilms were unknowingly observed centuries ago. Within the last 70 years, our understanding of biofilms, their morphology, and characteristics has grown. Yet despite an increased understanding, the presence of biofilms and their impact on healthcare are still often overlooked, or misunderstood. Antimicrobial technologies and applications are primarily focused on planktonic bacteria. A targeted approach against the biofilm phenotype is likely to improve infection outcomes wherein biofilms are the source of difficult-to-treat infections.

The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for futurein vitroandin vivoapplications.

Biofilm-related infection is among the worst complication to prosthetic joint replacement procedures; once established on the implant surface, biofilms show strong recalcitrance to clinical antibiotic therapy, frequently requiring costly revision procedures and prolonged systemic antibiotics for their removal. A well-designed active release coating might assist host immunity in clearing bacterial contaminants within the narrow perioperative window and ultimately prevent microbial colonization of the joint prosthesis. A first-in-class compound (CZ-01127) was tested as the active release agent in a silicone (Si) coating using an in vitro dynamic flow model of surgical site contamination and compared with analogous coatings containing clinical gold-standard antibiotics vancomycin and gentamicin; the CZ-01127 coating outperformed both vancomycin and gentamicin coatings and was the only to decrease the methicillin-resistant Staphylococcus aureus (MRSA) inocula below detectable limits for the first 3 days. The antimicrobial activity of CZ-01127, and for comparison vancomycin and gentamicin, were characterized against both planktonic and biofilm MRSA using the minimum inhibitory concentration (MIC) assay, serial passages, and serial dilution tests against established biofilms grown with a CBR 90 CDC biofilm reactor. Despite a similar MIC (1 µg/ml) and behavior in a 25-day serial passage analysis, CZ-01127 displayed much greater bactericidal activity against established biofilms and was the only to decrease biofilm colony forming units (CFUs) below detectable limits at the highest concentration tested (500 µg/ml). Coating release profiles were characterized using ATR-FTIR and displayed burst release kinetics within the decisive period of the perioperative window suggesting the silicon carrier is broadly useful for screening antibiotic compound for local delivery applications. Statement of Significance With an aging population, an increasing number of people are undergoing total joint replacement procedures in which diseased joint tissues are replaced with permanent metallic implants. Some of these procedures are burdened by costly and debilitating infections. A promising approach to prevent infections is the use of an antimicrobial coating on the surface of the implant which releases antibiotics into the surgical site to prevent infection. In this study, we tested a new antibiotic compound formulated in a silicone coating. Data showed that this compound was more effective at killing pathogenic methicillin resistant Staphylococcus aureus (MRSA) bacteria than two clinical gold-standard antibiotics—vancomycin and gentamicin—and could be a promising agent for antimicrobial coating technologies.

BACKGROUND Heterotopic ossification (HO) is a significant complication for wounded warriors with traumatic limb loss. Although this pathologic condition negatively impacts the general population, ectopic bone has been observed with higher frequency for service members injured in Iraq and Afghanistan due to blast injuries. Several factors, including a traumatic insult, bioburden, tourniquet and wound vacuum usage, and bone fractures or fragments have been associated with increased HO for service members. A large combat-relevant animal model is needed to further understand ectopic bone etiology and develop new pragmatic solutions for reducing HO formation and recurrence. OBJECTIVE This study outlines the optimization of a blast system that may be used to simulate combat-relevant trauma for HO and replicate percussion blast experienced in theater. METHODS We tested the repeatability and reproducibility of an air impact device (AID) at various pressure settings and compared it with a model of blunt force trauma for HO induction. Furthermore, we assessed the ability of the higher-power air delivery system to injure host tissue, displace metal particulate, and disperse bone chips in cadaveric sheep limbs. RESULTS Data demonstrated that the air delivery setup generated battlefield-relevant blast forces. When the AID was charged to 40, 80, and 100 psi, the outputs were 229 (SD 13) N, 778 (SD 50) N, and 1085 (SD 114) N, respectively, compared with the blunt force model which proposed only 168 (SD 11) N. For the 100-psi AID setup, the force equaled a 5.8-kg charge weight of trinitrotoluene at a standoff distance of approximately 2.62 m, which would replicate a dismounted improvised explosive device blast in theater. Dispersion data showed that the delivery system would have the ability to cause host tissue trauma and effectively disperse metal particulate and host bone chips in local musculature compared with the standard blunt force model (13 mm vs 2 mm). CONCLUSIONS Our data showed that a high-pressure AID was repeatable or reproducible, had the ability to function as a simulated battlefield blast that can model military HO scenarios, and will allow for factors including blast trauma to translate toward a large animal model.

The skin/implant interface of osseointegrated (OI) implants is susceptible to infection, causing excess pain, increased morbidity, and possibly implant removal. Novel distal femoral OI implants with binary nitride coatings have been developed with little physiological modeling to collect microbiological evidence of resistance to bacterial attachment. This in vitro study evaluated a Ti-6Al-4V alloy coated with TiNbN and treated with low plasticity burnishing (LPB) to assess attachment and biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) under physiologically modeling conditions compared to standard Ti-6Al-4V alloy materials with a polished ("Color Buff") or non-polished finish ("Satin Finish"). Washability of the materials were also assessed and compared. It was hypothesized that the TiNbN/LPB treatments would resist bacterial adhesion and biofilm formation to a greater degree than the other two materials, and have a higher degree of bacterial removal following a clinically relevant wash regimen. Material types were exposed to a constant flow of broth containing MRSA and were analyzed using bacterial quantification, surface coverage analysis, and SEM imaging. Quantification data showed no difference in bacterial attachment among the varying material types both with and without the wash regimen. Surface coverage and SEM analysis confirmed results. The wash regimen led to an approximately 3 log10 reduction in bacteria for all material types. Though the results did not support the hypothesis that a TiNbN coating/LPB treatment might resist bacterial attachment/biofilm formation more than other alloys, or have less bacteria after cleaning, results did support the potential importance of a daily wound-hygiene regimen at the skin/implant interface of OI materials. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Flash autoclaving is one of the most frequently utilized methods of sterilizing devices, implants or other materials. For a number of decades, it has been common practice for surgeons to remove implantable devices, flash autoclave and then reimplant them in a patient. Data have not yet indicated the potential for biofilms to survive or remain on the surface of orthopaedic-relevant materials following flash autoclave. In this study, monomicrobial and polymicrobial biofilms were grown on the surface of clinically relevant titanium materials and exposed to flash autoclave settings that included varying times and temperatures. Data indicated that when the sterilization and control temperatures of an autoclave were the same, biofilms were able to survive flash autoclaving that was performed for a short duration. Higher temperature and increased duration rendered biofilms non-viable, but none of the autoclave settings had the ability to remove or disperse the presence of biofilms from the titanium surfaces. These findings may be beneficial for facilities, clinics, or hospitals to consider if biofilms are suspected to be present on materials or devices, in particular implants that have had associated infection and are considered for re-implantation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1543-1550, 2018.

Case We present a case report documenting the retrieval and histological analysis of a porous tantalum (P-Ta) total ankle replacement (TAR) from a 50-year-old woman after a below-knee transtibial amputation. This rare opportunity to examine an intact TAR may help to better understand the implant-bone relationship because it would be in situ. Conclusion In this case study, we demonstrate bone ingrowth to the first layer of the P-Ta and organized trabecular orientation, suggesting that equal bone load was achieved on the base and the rails in both components using a transfibular surgical approach.

Implant-related infections are becoming increasingly difficult to treat due to the formation of biofilms on implant surfaces. This study analyzed the in vitro efficacy of a novel antimicrobial coating against biofilms of Pseudomonas aeruginosa , using a flow cell system. Results indicated that P. aeruginosa biofilms were reduced by greater than 8 log 10 units in less than 24 h. Data indicated that this active-release coating may be promising for preventing biofilm implant-related infections.

BACKGROUND CONTEXT Tenacious bacterial biofilms pose a major challenge in treating deep spine infections. Biofilms provide bacteria substantial protection against antimicrobial agents, the host immune response, and are invisible to the naked eye. Biofilms are notoriously difficult to eradicate, and to that end, methylene blue has shown promise as a biofilm disclosing agent in the arthroplasty literature in both in vitro and in vivo settings. PURPOSE The objective of the present study was to assess the intensity of methylene blue staining at varying concentrations as a biofilm disclosing agent in vitro for common biofilm forming bacterial infections and to determine performance characteristics across a range of spine implant materials. STUDY DESIGN/SETTING Microbiology. METHODS S. aureus biofilms were grown to maturity in bioreactors according to established lab protocol on titanium, cobalt chromium, and polyetherketone (PEEK) wafers. Biofilms were stained with 0.05% and 0.01% methylene blue solutions for 5-minutes and then washed with normal saline for 1-minute. Gross images were obtained to compare the visual sensitivity of the blue dye at different dilutions. Scanning electron microscopy was performed to confirm the presence or absence of biofilm on methylene blue stained areas. Images were compared to controls. RESULTS S. aureus biofilms were grown for 7days on double sided titanium, cobalt chromium, and PEEK wafers (n=4 each). There appeared to be a visible dose – dependent relationship based on the staining and dye concentration. At each dilution, biofilms demonstrated visible blue staining after immersion in methylene blue solution; however, blue dye was visible only where biofilms were present as confirmed by SEM. There was no evidence that methylene blue was able to stain titanium, cobalt chromium, or PEEK. CONCLUSIONS Methylene blue functions as an effective disclosing agent for S. aureus biofilm in vitro. Currently, there are no techniques for identifying bacterial biofilm in vivo once it has formed. Given the low cost, favorable safety profile and FDA approval, methylene blue may be considered a first-in-class agent for disclosure of biofilm and may allow surgeons to see biofilms on implants and host tissue in vivo. In so doing, it may allow for efforts at eradicating these biofilms once visualized. Additional work is needed to further elucidate this concept.

Background Heterotopic ossification (HO) is a significant complication for wounded warriors with traumatic limb loss. Although this pathologic condition negatively impacts the general population, ectopic bone has been observed with higher frequency for service members injured in Iraq and Afghanistan due to blast injuries. Several factors, including a traumatic insult, bioburden, tourniquet and wound vacuum usage, and bone fractures or fragments have been associated with increased HO for service members. A large combat-relevant animal model is needed to further understand ectopic bone etiology and develop new pragmatic solutions for reducing HO formation and recurrence. Objective This study outlines the optimization of a blast system that may be used to simulate combat-relevant trauma for HO and replicate percussion blast experienced in theater. Methods We tested the repeatability and reproducibility of an air impact device (AID) at various pressure settings and compared it with a model of blunt force trauma for HO induction. Furthermore, we assessed the ability of the higher-power air delivery system to injure host tissue, displace metal particulate, and disperse bone chips in cadaveric sheep limbs. Results Data demonstrated that the air delivery setup generated battlefield-relevant blast forces. When the AID was charged to 40, 80, and 100 psi, the outputs were 229 (SD 13) N, 778 (SD 50) N, and 1085 (SD 114) N, respectively, compared with the blunt force model which proposed only 168 (SD 11) N. For the 100-psi AID setup, the force equaled a 5.8-kg charge weight of trinitrotoluene at a standoff distance of approximately 2.62 m, which would replicate a dismounted improvised explosive device blast in theater. Dispersion data showed that the delivery system would have the ability to cause host tissue trauma and effectively disperse metal particulate and host bone chips in local musculature compared with the standard blunt force model (13 mm vs 2 mm). Conclusions Our data showed that a high-pressure AID was repeatable or reproducible, had the ability to function as a simulated battlefield blast that can model military HO scenarios, and will allow for factors including blast trauma to translate toward a large animal model.

Over the past 200 years, there has been significant advancements in the fields of bioengineering and orthopaedics. Investigators, clinicians and manufactures are learning that the success of implant systems is not limited to a single factor, but a combination of variables that must work in unison to provide stability and high survivorship. Innovations continue to advance these fields and include: biomimetic alterations, three-dimensional, patient-specific reconstructions and novel coatings to mitigate aseptic loosening or other pathologies. However, implant systems continue to fail in clinical practice since they do not adhere to key fundamental principles. Therefore, this article is intended to highlight 5 hallmarks of biomaterials that should be considered during design, surgery, and post-operative rehabilitation.

Antibiotic resistant bacterial infections are a growing problem in patient care. These infections are difficult to treat and severely affect the patient's quality of life. The goal of this translational experiment was to investigate the antimicrobial potential of cationic steroidal antimicrobial-13 (CSA-13) for the prevention of perioperative device-related infections in vivo. It was hypothesized that when incorporated into a polymeric device coating, the release of CSA-13 could prevent perioperative device-related infection without inhibiting skeletal attachment. To test this hypothesis, 12 skeletally mature sheep received a porous coated titanium implant in the right femoral condyle. Group 1 received the titanium implant and an inoculum of 5 × 10(8) CFU of methicillin-resistant Staphylococcus aureus (MRSA). Group 2 received a CSA-13 coated implant and the MRSA inoculum. Group 3 received only the CSA-13 coated implant and Group 4 received only the implant-without the CSA-13 coating or MRSA inoculum. In conclusion, the CSA-13 combination coating demonstrated bactericidal potential without adversely affecting skeletal attachment. The CSA-13 containing groups exhibited no evidence of bacterial infection at the conclusion of the 12 week study and established skeletal attachment consistent with Group 4. In contrast, all of the Group 1 animals became infected and required euthanasia within 6-10 days. The significance of this finding is that this combination coating could be applied to implanted devices to prevent perioperative device-related infections. This method may facilitate significantly reduced incidences of device-related infections as well as a new method to treat and prevent resistant strain bacterial infections.

Biofilm implant-related infections cost the US healthcare system billions of dollars each year. For several decades, device coatings have been developed that actively release antimicrobial compounds in an attempt to prevent these infections from developing. To date, few coatings have been put into clinical use. These have shown limited to no efficacy in clinical trials. Recent data have shown the in vitro and in vivo efficacy of a novel active release coating that may address the limitations of coatings that are used clinically. In this study, the novel active release coating was characterized to gain an understanding of the effects of combining an antimicrobial additive, cationic steroid antimicrobial-13 (CSA-13), to a medical grade polydimethylsiloxane (PDMS) material. Results indicated that the addition of CSA-13 did influence the physical properties of the PDMS, but not with adverse effects to the desired material properties. Furthermore, there was no indication of chemical reactivity. It was shown that CSA-13 was uniformly dispersed as small particles throughout the PDMS matrix. These particles were able to dissolve and elute out of the PDMS within a 30-day period. The results of this work suggested that the PDMS with CSA-13 was thermally, chemically and physically stable when used as a device coating to treat local infection and/or prevent biofilm implant-related infections from developing.

Orthodontic retention has been proposed as a life-long commitment for patients who desire to maintain straight teeth. However, the presence of foreign material increases risk of bacterial colonization and caries formation, of which Streptococcus mutans is a key contributor. Multiple studies have assessed the ability of silver to be added to base plate material and resist attachment of S. mutans. However, it does not appear that long-term washout in connection with biofilm growth under physiologically relevant conditions has been taken into consideration. In this study, silver was added to base plate material and exposed to short- or long-term washout periods. Materials were then assessed for their ability to resist biofilm formation of S. mutans using a drip flow reactor that modeled the human oral environment. Data indicated that silver was able to resist biofilm formation following short-term washout, but long-term washout periods resulted in a lack of ability to resist biofilm formation. These data will be important for future development of base plate materials to achieve long-term antimicrobial efficacy to reduce risk of caries formation and benefit patients in the long term. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2632-2639, 2017.

Sustained local delivery of antibiotics from a drug reservoir to treat or prevent bacterial infections can avoid many of the drawbacks of systemic administration of antibiotics. Prolonged local release of high concentrations of antibiotics may also be more effective at treating bacteria in established biofilm populations that are resistant to systemic antibiotics. A double network hydrogel comprising an organic polyphosphate pre-polymer network polymerized within a polyacrylamide network de-swelled to about 50% of its initial volume when the polyphosphate network was crosslinked with polycationic tobramycin, an aminoglycoside antibiotic. The antibiotic-loaded hydrogels contained approximately 200 mg/ml of tobramycin. The hydrogels continuously released daily amounts of tobramycin above the Pseudomonas aeruginosa minimal bactericidal concentration for greater than 50 days, over the pH range 6.0–8.0, and completely eradicated established P. aeruginosa biofilms within 72 h in a flow cell bioreactor. The presence of physiological concentrations of Mg2+ and Ca2+ ions doubled the cumulative release over 60 days. The polyphosphate hydrogels show promise as materials for sustained localized tobramycin delivery to prevent post-operative P. aeruginosa infections including infections established in biofilms. Statement of Significance Polyphosphate hydrogels were loaded with high concentrations of tobramycin. The hydrogels provided sustained release of bactericidal concentrations of tobramycin for 50 days, and were capable of completely eradicating P. aeruginosa in established biofilms. The hydrogels have potential for localized prevention or treatment of P. aeruginosa infections.

Active release antimicrobial coatings for medical devices have been developed to prevent and treat biofilm implant-related infections. To date, only a handful of coatings have been put into clinical use, with limited success. In this study, a novel antimicrobial compound was incorporated into a silicone (polydimethylsiloxane or PDMS) polymer to develop a novel active release coating that addressed several limitations of current device coatings. The efficacy of this coating was optimized using an in vitro flow cells system, then translated to an animal model of a simulated Type IIIB open fracture wherein well-established biofilms were used as initial inocula. Results indicated that the novel coating was able to prevent infection in 100% (9/9) of animals that were treated with biofilms and the novel coating (treatment group). In contrast, 100% (9/9) of animals that were inoculated with biofilms and not treated with the coating (positive control), did develop infection. Nine animals were used as negative controls, i.e., those that were not treated with biofilms, and showed a rate of infection of 11% (1/9). Eight animals were treated with the novel coating only to determine its effect on host tissue. Results indicated that the novel active release coating may have significant promise for future application to prevent biofilm implant-related infections in patients.

More than 400,000 primary hip and knee replacement surgeries are performed each year in the United States. From these procedures, approximately 0.5–3% will become infected and when considering revision surgeries, this rate has been found to increase significantly. Antibiotic resistant bacterial infections are a growing problem in patient care. This in vitro research investigated the antimicrobial potential of the polymer released, broad spectrum, Cationic Steroidal Antimicrobial-13 (CSA-13) for challenges against 5 × 108 colony forming units (CFU) of methicillin-resistant Staphylococcus aureus (MRSA). It was hypothesized that a weight-to-weight (w/w) concentration of 18% CSA-13 in silicone would exhibit potent bactericidal potential when used as a controlled release device coating. When incorporated into a polymeric device coating, the 18% (w/w) broad-spectrum polymer released CSA-13 antimicrobial eliminated 5 × 108 CFU of MRSA within 8 hours. In the future, these results will be utilized to develop a sheep model to assess CSA-13 for the prevention of perioperative device related infections in vivo.

Biofilm-related infections have become a major clinical concern. Typically, animal models that involve inoculation with planktonic bacteria have been used to create positive infection signals and examine antimicrobial strategies for eradicating or preventing biofilm-related infection. However, it is estimated that 99.9% of bacteria in nature dwell in established biofilms. As such, open wounds have significant potential to become contaminated with bacteria that reside in a well-established biofilm. In this study, a modified CDC biofilm reactor was developed to repeatably grow mature biofilms of Staphylococcus aureus on the surface of polyetheretherketone (PEEK) membranes for inoculation in a future animal model of orthopaedic implant biofilm-related infection. Results indicated that uniform, mature biofilms repeatably grew on the surface of the PEEK membranes.

Percutaneous osseointegrated implant technology provides a potential alternative to current socket prosthetics for individuals with limb loss. However, similar to other percutaneous devices, there remain concerns of periprosthetic infection. To understand this process of infection, bacterial isolates were collected and characterized from a sheep model of osseointegration. CSA-13, a novel cationic steroid antimicrobial, was used at the skin/implant interface in an attempt to reduce the rate of infection. Results indicated that in this application, normal flora and environmental organisms continued to colonize the skin/implant interface as well as cause infection in the presence of CSA-13. Two factors are believed to have contributed to this outcome: the delivery of CSA-13 and the lack of a skin seal at the skin/implant interface, which would create a biological barrier to infection.

The majority of animal studies that have been used to model biofilm-related infections that accompany the use of biomedical devices have primarily involved an initial inoculum of planktonic bacterial cells from batch cultures. Although valuable, data that has been derived from these experiments may not provide important clinical insight into how bacteria in well-established, mature biofilms impact device-related and other clinical infections when they initially contaminate a patient site or implanted device. In this chapter, a discussion is presented on the impact that a shift in biofilm research may have if initial inocula of well-established, mature biofilms are used to model device-related infections in animal models.

Biofilm implant-related infections cost the US healthcare system billions of dollars each year. For several decades, device coatings have been developed that actively release antimicrobial compounds in an attempt to prevent these infections from developing. To date, few coatings have been put into clinical use. These have shown limited to no efficacy in clinical trials. Recent data have shown the in vitro and in vivo efficacy of a novel active release coating that may address the limitations of coatings that are used clinically. In this study, the novel active release coating was characterized to gain an understanding of the effects of combining an antimicrobial additive, cationic steroid antimicrobial-13 (CSA-13), to a medical grade polydimethylsiloxane (PDMS) material. Results indicated that the addition of CSA-13 did influence the physical properties of the PDMS, but not with adverse effects to the desired material properties. Furthermore, there was no indication of chemical reactivity. It was shown that CSA-13 was uniformly dispersed as small particles throughout the PDMS matrix. These particles were able to dissolve and elute out of the PDMS within a 30-day period. The results of this work suggested that the PDMS with CSA-13 was thermally, chemically and physically stable when used as a device coating to treat local infection and/or prevent biofilm implant-related infections from developing.

Currently, the majority of animal models that are used to study biofilm-related infections utilize planktonic bacterial cells as initial inocula to produce positive signals of infection in biomaterials studies. However, the use of planktonic cells has potentially led to inconsistent results in infection outcomes. In this study, well-established biofilms of methicillin-resistant Staphylococcus aureus (MRSA) were grown and used as initial inocula in an animal model of a Type IIIB open fracture. The goal of the work was to establish, for the first time, a repeatable model of biofilm implant-related osteomyelitis wherein biofilms were used as initial inocula to test combination biomaterials. Results showed that 100% of animals that were treated with biofilms developed osteomyelitis, whereas 0% of animals not treated with biofilm developed infection. The development of this experimental model may lead to an important shift in biofilm and biomaterials research by showing that when biofilms are used as initial inocula, they may provide additional insights into how biofilm-related infections in the clinic develop and how they can be treated with combination biomaterials to eradicate and/or prevent biofilm formation.

One of the most practical strategies that has been undertaken to fight biofilm implant-related infections has been the development of coatings on biomaterial devices that can elute antimicrobials into regions of patients' tissues. To date, the majority of animal studies that have been developed to model infections that accompany the use of these materials have primarily involved an initial inoculum of planktonic bacterial cells from batch cultures. Although valuable, data that have been derived from these experiments may not provide important clinical insight into how bacteria in well-established, mature biofilms impact device-related and other clinical infections when they contaminate a patient site or implanted device. In this review, a discussion is presented on the impact that a shift in biofilm research may have if initial inocula of well-established, mature biofilms are used to model biomaterial device-related infections in animal models. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2011.

Infections of percutaneous osseointegrated prostheses (POP) cause prolonged morbidity and device failure because once established, they are refractory to antibiotic therapy. To date, only limited translational animal studies have investigated the efficacy of POP designs in preventing infections. We developed an animal model to evaluate the efficacy of a porous-coated titanium (Ti) subdermal barrier to achieve skin–implant integration and to prevent periprosthetic infection. In a single-stage “amputation and implantation” surgery, 14 sheep were fitted with percutaneous devices with an attached porous-coated Ti subdermal barrier. Nine sheep were implanted with a smooth Ti subdermal barrier construct and served as controls, with one control sheep removed from the study due to a fractured bone. Clinical, microbiological, and histopathological data showed that the porous Ti barrier prevented superficial and deep tissue infections in all animals (14/14, 100%) at the 9-month endpoint. In contrast, animals with the smooth Ti implant construct had a 25% (2/8) infection rate. Survival analysis indicated a significant difference between the groups (log-rank test, p = 0.018). Data also indicated that although skin marsupialization was evident in both implant types, animals in the control group had a four times greater marsupialization rate. We concluded that osseointegrated implants incorporating porous-coated Ti subdermal barriers may have the ability to prevent infection by maintaining a healthy, biologically attached epithelial barrier at the skin–implant interface in load-bearing animals up to a 9-month terminus. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1304–1311, 2012

Bacteria flourish in nearly every environment on earth. Contributing to their ability to grow in many esoteric locations is their development into a biofilm structure. In an effort to more accurately model the growth environment of biofilms in nature, a Center for Disease Control and Prevention (CDC) biofilm reactor has been developed that mimics nature-like shear forces and renewable nutrient sources. To date, there has been no confirmation by scanning electron microscopy (SEM) that mature biofilms develop on a surface when grown using the CDC biofilm reactor. Three different SEM methods were used to collect images ofStaphylococcus epidermidisATCC 35984 that was to be grown using the CDC biofilm reactor. In addition, two different fixative techniques were used in each of the imaging methods. Results indicated that after 48 hours of growth in the reactor,S. epidermidisATCC 35984 does produce a significant network of matrix components and 3D mushroom- or pillar-like structures with signs of water channel development. In conclusion,S. epidermidisATCC 35984 grown using the CDC biofilm reactor does appear to display signs of mature biofilm development. These results could be important for studies wherein mature biofilms are needed forin vitroand/orin vivoapplications.

Background: Infection remains the main challenge to percutaneous, intramedullary osseointegrated implant technology. The purpose of this investigation was to determine if a broad spectrum antimicrobial, Ceragenin™ (CSA-13) could prevent pin track infections in a percutaneous tibial pin site in a sheep model. Methods: In 20 sheep, a smooth titanium alloy pin/implant was inserted percutaneously through the medial skin and both cortices of the proximal tibia. In 10 sheep, the pin/skin interface was treated with a CSA-13-embedded foam pad. Ten sheep served as controls receiving an untreated pad. At the end of 24 weeks, or if they presented with clinical signs of infection, the animals were euthanized. Histological stains were processed from soft tissue and bone, and bacterial cultures were taken from tissue, bone, and blood. In addition to clinical signs, sheep were considered infected if at least one tissue culture and/or histologically stained sample was positive. Results: Compared with the controls, CSA-13 did not prevent pin track infection (p = 0.88). Large gaps around the pin indicated a lack of skin-pin adhesion. Conclusions: In this application, CSA-13 was not effective in preventing pin track infections. This study suggests that maintaining skin attachment, at the implant surface of osseointegrated implants, is essential as a primary barrier to infection. Local antimicrobial treatments should be considered a secondary barrier to bacterial invasion of the pin/skin interface and deeper tissues. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010

Staphylococcus aureus is a common infecting agent of many surgical sites. As a commensal organism to humans and rabbits, the infection process may occur due to native or exogenous S. aureus. We applied exogenous S. aureus ATCC 49230 once weekly to the surgical site of an osseointegrated pin in 20 New Zealand white rabbits. Clinical signs of infection resulted in euthanasia and at necropsy samples were collected from putatively infected sites. The predominant organism cultured was S. aureus. We observed various beta-hemolysis patterns of S. aureus on culture media and used pulsed field gel electrophoresis (PFGE) to determine whether there were distinct strains of S. aureus collected from various sites of the rabbits. On the basis of PFGE results, we found that the exogenous S. aureus ATCC 49230 was not the S. aureus cultured during necropsy, but that S. aureus native to the rabbits was in fact the infecting agent. We conclude that this rabbit model for S. aureus infection, which has not been described previously, may contribute to understanding the pathogenesis of S. aureus infections in future studies with simulated osseointegrated pin infections secondary to S. aureus.