Richard T. Epperson

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.

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 animals 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. TMs 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.

Link Between Clinical Predictors of Heterotopic Ossification and Histological Analysis in Combat-injured Service Members

BACKGROUND Heterotopic ossification (HO) is a debilitating condition that occurs following traumatic injury and may restrict range of motion and delay rehabilitation. The timing and efficacy of surgical resection have varied widely, and there is a gap in knowledge between clinical predictors of HO recurrence and histological analysis. METHODS Thirty-three service members seen at Walter Reed National Military Medical Center for symptomatic HO were enrolled in an institutional review board-approved study. Participants took oxytetracycline on four scheduled days prior to HO resection to determine the mineral apposition rate (bone growth rate). RESULTS Detailed histological analyses included scanning electron microscopy with backscattered electron imaging and light microscopy. Data indicated that the mineral apposition rate of trauma-induced HO was approximately 1.7 μm/day at the time of operative intervention, which was 1.7 times higher than the rate in non-pathological human bone. The mineral apposition rate and postoperative alkaline phosphatase values were demonstrated to be positively and significantly related (ρ = 0.509, p = 0.026, n = 19). When the analysis was limited to patients with no more than a two-year period from injury to excision (thereby removing outliers who had a longer time period than their counterparts) and traumatic brain injury and nonsteroidal anti-inflammatory drugs (known correlates with HO development) were controlled for in the statistical analysis, the mineral apposition rate and recurrence severity were significantly related (ρ = -0.572, p = 0.041, n = 11). CONCLUSIONS Data demonstrated a link between benchtop research and bedside care, with the mineral apposition rate elevated in patients with HO and correlated with recurrence severity; however, a larger sample size and more clinical factors are needed to refine this model. LEVEL OF EVIDENCE Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

The Significance of Calcified Fibrocartilage on the Cortical Endplate of the Translational Sheep Spine Model

To gain an understanding of the vertebral cortical endplate and factors that may affect the ability to achieve skeletal attachment to intervertebral implants and fusion, this study aimed to characterize the hypermineralized tissue on the cortical endplate of the vertebral body on a commonly used animal model. Skeletally mature sheep were injected with tetracycline prior to euthanasia and the C2‐C3, T5‐T6, and L2‐L3 spinal motion segments were excised and prepared. Vertebral tissues were imaged using backscatter electron (BSE) imaging, histology, and tetracycline labeling was used to assess bone remodeling within different tissue layers. It was determined that the hypermineralized tissue layer was calcified fibrocartilage (CFC). No tetracycline labels were identified in the CFC layer, in contrast to single and double labels that were present in the underlying bone, indicating the CFC present on the cortical endplate was not being actively remodeled. The average thickness of the CFC layer was 146.3 ± 70.53 µm in the cervical region, 98.2 ± 40.29 µm in the thoracic region, and 150.89 ± 69.25 µm in the lumbar region. This difference in thickness may be attributed to the regional biomechanical properties of the spine. Results from this investigation indicate the presence of a nonremodeling tissue on the cortical endplate of the vertebral body in sheep spines, which attaches the intervertebral disc to the vertebrae. This tissue, if not removed, would likely prevent successful bony attachment to an intervertebral device in spinal fusion studies and total disc replacement surgeries. Anat Rec, 296:736–744, 2013.

A new trichrome technique for PMMA embedded percutaneous implants for the study and characterization of epithelial integration

Abstract Poly(methylmethacrylate) (PMMA) embedding is routinely used in histomorphometry to analyze the periprosthetic tissue response to the presence of an implant in situ. However, the embedment process can limit the variety of stains that can be successfully used to highlight different tissue types within the periprosthetic tissue. In this study, a new staining protocol, the modified trichrome method, was developed to simultaneously label the collagen fiber matrix, epithelial tissue morphology, vascular network, granulation tissue, and fibrous capsule that surround the percutaneous implant. The new staining technique was compared to established histological evaluation protocols, namely Masson’s trichrome and hematoxylin and eosin (H&E) protocols. The newly developed staining protocol for analyzing implant–soft tissue integration was significantly more effective in identifying cellular and tissue structures than previously established techniques. The detail found with the modified trichrome allows a high degree of structural quantification. The ultimate goal of the present research is to improve the long term soft tissue integration of implants made from different biomaterials. The modified trichrome can benefit translational studies through a more accurate quantification of both skin attachment mechanism and cellular response to the presence of percutaneous biomaterials.

Deparaffinizing Soft–Tissue Sections for Elemental Analysis of Wear Particulate

Abstract Often after orthopaedic revision surgery, tissue samples are sent for histopathology and possible identification of wear particulate (polyethylene, metal, and ceramic). Cellular reaction and inflammation can be detected from standard paraffin embedded and hematoxylin and eosin-stained sections during light microscopy. Although it is also possible to detect the presence of wear particulate, sometimes metal and ceramic particulate can be falsely identified as artifact. Errors in reporting can lead to false assumptions about implant wear and particulate type. Scanning electron microscopes using backscattered electrons with a correlated energy dispersive X-ray spectrometer can identify metal and ceramic particulate matter based on their elemental composition. Thus, the presence of metal and ceramic particulate is ascertained and its origin can be determined. This technique enables orthopaedic researchers and clinicians to determine whether there are loose coatings, wear particles, or thirdbody inclusions in the articulation. Previously, energy dispersive X-ray spectrometry was performed from orthopaedic tissue sections that had already been embedded in polymethylmethacrylate after histopathological analysis. A new process was developed to confirm light microscopy findings following a simplified process of deparaffinizing an unstained section in preparation for correlated backscattered electrons and energy-dispersive X-ray analysis that would eliminate the need for polymethylmethacrylate embedment and decrease the time to diagnosis. (The J Histotechnol 29:233, 2006) Submitted August 21, 2006; accepted with revisions October 31, 2006

Growth substrate may influence biofilm susceptibility to antibiotics

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 future in vitro and in vivo applications.

Ability of a Wash Regimen to Remove Biofilm from the Exposed Surface of Materials Used in Osseointegrated Implants: Biofilm Growth/Removal from OI Materials

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 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.

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.