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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.

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.

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.

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 underpin the disease etiology of nearly all opportunistic bacterial infections especially when integumentary barriers are surgically breached and foreign materials remain implanted. Endogenous spread from the patient’s own microbial flora is the likely source of most surgical site infections. Biomaterials potentiate infection by providing a substrate for biofilm formation. The biofilm protects these pathogens from both host immunity and clinical interventions in a variety of ways. Biofilm-forming bacteria excrete sticky exopolysaccharides to form cohesive communal aggregates and adhesive attachments to foreign surfaces like devitalized tissues and implanted biomaterials; this strategy deranges phagocytic clearance by host immune cells. Quiescent phenotypic variants in the biofilm cells are tolerant of antibiotic concentrations many orders of magnitude greater than would otherwise kill planktonic phenotypes, concentrations greatly exceeding toxic thresholds bounding safe systemic antibiotic concentrations. Biofilms are, thus, a nidus for infection as tolerant cells can outlast clinical antibiotic courses to subsequently reseed infection. Much emphasis has been placed on preventing biofilm infections from occurring as clinical strategies for eradicating established biofilm infections frequently fail. Biofilm infections usually require extensive surgical intervention to remove implanted biomaterials and debride affected tissues. These procedures are costly and usually accompanied by high patient mortality and morbidity. There is a pressing need for strategies that specifically target the biofilm because clinical measures to prevent infection still fail in this antibiotic era at great financial and physical expense.

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.

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.

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.

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.

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

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