Plume height and surface coverage analysis of methicillin-resistant Staphylococcus aureus isolates grown in a CDC biofilm reactor.

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.

Determining Which Combinatorial Combat-Relevant Factors Contribute to Heterotopic Ossification Formation in an Ovine Model.

Traumatic heterotopic ossification (HO) is frequently observed in Service Members following combat-related trauma. Estimates suggest that ~65% of wounded warriors who suffer limb loss or major extremity trauma will experience some type of HO formation. The development of HO delays rehabilitation and can prevent the use of a prosthetic. To date there are limited data to suggest a standard mechanism for preventing HO. This may be due to inadequate animal models not producing a similar bone structure as human HO. We recently showed that traumatic HO growth is possible in an ovine model. Within that study, we demonstrated that 65% of sheep developed a human-relevant hybrid traumatic HO bone structure after being exposed to a combination of seven combat-relevant factors. Although HO formed, we did not determine which traumatic factor contributed most. Therefore, in this study, we performed individual and various combinations of surgical/traumatic factors to determine their individual contribution to HO growth. Outcomes showed that the presence of mature biofilm stimulated a large region of bone growth, while bone trauma resulted in a localized bone response as indicated by jagged bone at the linea aspera. However, it was not until the combinatory factors were included that an HO structure similar to that of humans formed more readily in 60% of the sheep. In conclusion, data suggested that traumatic HO growth can develop following various traumatic factors, but a combination of known instigators yields higher frequency size and consistency of ectopic bone.

Development and validation of a large animal ovine model for implant-associated spine infection using biofilm based inocula.

Postoperative implant-associated spine infection remains poorly understood. Currently there is no large animal model using biofilm as initial inocula to study this challenging clinical entity. The purpose of the present study was to develop a sheep model for implant-associated spine infection using clinically relevant biofilm inocula and to assess the in vivo utility of methylene blue (MB) for visualizing infected tissues and guiding debridement. This 28-day study used five adult female Rambouillet sheep, each with two non-contiguous surgical sites- in the lumbar and thoracic regions- comprising randomized positive and negative infection control sites. A standard mini-open approach to the spine was performed to place sterile pedicle screws and Staphylococcus aureus biofilm-covered (positive control), or sterile (negative control) spinal fusion rods. Surgical site bioburden was quantified at the terminal procedure. Negative and positive control sites were stained with MB and staining intensity quantified from photographs. Specimens were analyzed with x-ray, micro-CT and histologically. Inoculation rods contained ∼10.44 log10 colony forming units per rod (CFU/rod). Biofilm inocula persisted on positive-control rod explants with ∼6.16 log10 CFU/rod. There was ∼6.35 log10 CFU/g of tissue in the positive controls versus no identifiable bioburden in the negative controls. Positive controls displayed hallmarks of deep spine infection and osteomyelitis, with robust local tissue response, bone resorption, and demineralization. MB staining was more intense in infected, positive control sites. This work presents an animal-efficient sheep model displaying clinically relevant implant-associated deep spine infection.

Assessing the effect of physeal biopsy on angular deformity in a rabbit model.

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.

Assessing the safety of an epiphyseal plate biopsy in a translational lamb model.

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.

The effect of estrogen-like compound on rotator cuff tendon healing in a murine model.

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.

Developing a combat-relevant translatable large animal model of heterotopic ossification.

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.

Bone ingrowth comparison of irregular titanium and cobalt-chromium coatings in a translational cancellous bone model.

The use of cobalt-chromium (CoCr)-bearing surfaces in total joint replacement (TJR) remains the predominate bearing surface. The conundrum with using this biomaterial has been selecting an ideal porous coating to assure reproducible skeletal attachment. There has been evidence that smooth CoCr beads may be inferior for skeletal attachment compared to identically shaped titanium (Ti) beads. Recent in vitro studies have demonstrated that an increased surface area and roughness favors osteoblast adhesion to metallic biomaterials. Therefore, we hypothesized that an irregular shape CoCr bead with an increased surface texture would help correct the negative bone responses that have been reported with smooth beaded CoCr coatings and thus allowing for bone ingrowth equivalently as an irregular commercially pure Ti porous coating with similar porosity. This investigation employed a weight-bearing translational sheep cancellous bone model to accurately simulate a cancellous bone response as it would be clinically in a human TJR. The data analyses obtained from this investigation revealed similar bone responses between the porous coatings. By 12 weeks the irregular shape CoCr coating was able to achieve similar bone ingrowth with skeletal interlock when compared to a clinically proven Ti porous coating.

Histological Analysis of a Retrieved Porous Tantalum Total Ankle Replacement: A Case Report.

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.

Ability of a wash regimen to remove biofilm from the exposed surface of materials used in osseointegrated implants.

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.

System Setup to Deliver Air Impact Forces to a Sheep Limb: Preparation for Model Development of Blast-Related Heterotopic Ossification.

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.

In vivo analysis of a first-in-class tri-alkyl norspermidine-biaryl antibiotic in an active release coating to reduce the risk of implant-related infection.

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.

Flash autoclave settings may influence eradication but not presence of well-established biofilms on orthopaedic implant material.

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.

Transcortical or intracondylar? Which model is accurate for predicting biomaterial attachment in total joint replacement?

Despite four decades of research on material and porous coatings intended for cementless fixation in total joint replacement (TJR), aseptic mechanical loosening unrelated to particulate disease remains a concern. One main question asked is how translational are the animal models used to screen material and porous coatings intended for TJR fixation? Another question is how specific are the translational models at targeting the cementless TJR components that have the highest loosening rates? The hypothesis tested was that the bone response would be different between the two bone types-cortical and cancellous-used in translational animal modeling. The osteoblastic jumping distance (OJD), percent ingrowth, and appositional bone response were measured to assess the response between cancellous and cortical bone at two different anatomical locations, within the same limb. With 500 µm inset, titanium porous coated implants and negative control dinosaur (coprolite) implants were investigated. The data demonstrated that cortical bone had 7 times OJD than cancellous bone. The bone ingrowth data demonstrated 16 times higher bone ingrowth than the cancellous bone. Light microscopy showed predominately fibrous tissue attachment (98%) in cancellous bone. Screening of materials intended for TJR require a translational model predictive of the clinical condition. The results demonstrated that the transcortical model rendered false-positive data. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 578-588, 2018.

Host bone response to polyetheretherketone versus porous tantalum implants for cervical spinal fusion in a goat model.

STUDY DESIGN: In vivo assessment of polyetheretherketone (PEEK) and porous tantalum (TM) cervical interbody fusion devices in a goat model. OBJECTIVE: Directly compare host bone response to PEEK and TM devices used for cervical interbody fusion. SUMMARY OF BACKGROUND DATA: PEEK devices are widely used for anterior cervical discectomy and fusion but are nonporous and have limited surface area for bone attachment. METHODS: Twenty-five goats underwent single-level anterior cervical discectomy and fusion and were alternately implanted with TM (n = 13) or PEEK devices (n = 12) for 6, 12, and 26 weeks. Both devices contained a center graft hole (GH), filled with autograft bone from the animal's own iliac crest. The percentage of bone tissue around the implant, percentage of the implant surface in direct apposition with the host bone, and evidence of bone bridging through the implant GH were assessed by using backscattered electron imaging. Bone matrix mineral apposition rate was determined through fluorochrome double labeling, and sections were stained for histological analysis. RESULTS: The TM-implanted animals had significantly greater volumes of bone tissue at the implant interface than the PEEK animals at all-time points. The TM animals also had a significantly greater average mineral apposition rate in the GH region at 6 and 12 weeks than the PEEK animals. No difference was observed at 26 weeks. A greater number of TM-implanted animals demonstrated connection between the autograft bone and both vertebrae compared with the PEEK implants. Histological staining also showed that the TM devices elicited improved host bone attachment over the PEEK implants. CONCLUSION: The TM implants supported bone growth into and around the implant margins better than the PEEK devices. TM's open cell porous structure facilitated host bone ingrowth and bone bridging through the device, which could be beneficial for long-term mechanical attachment and support in clinical applications.

In vivo efficacy of a silicone‒cationic steroid antimicrobial coating to prevent implant-related infection.

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.