Iatrogenic Electrocautery Damage and Cellular-Based Corrosion of Total Joint Arthroplasty Biomaterials

Introduction. The number of patients undergoing a Primary Total Knee Arthroplasty (PTKA) has been increasing steadily each year. Of those PTKA patients, 20% report long-term pain and/or some functional deficit. Cobalt-Chromium-Molybdenum (CoCrMo) alloy is one of the most used materials in Total Joint Arthroplasty (TJA) implants due the material’s high strength, high corrosion resistance, and biocompatibility. The release of metal ions and potential occurrence of metallosis in TJA has been shown to be detrimental to the longevity of the implant. The mechanisms leading to this increase in metal ion concentrations have been up for debate, with some believing it is caused by Electrocautery (EC) damage at the time of surgery and others believing it is caused by inflammatory cells attacking the implant surface. The purpose of this thesis is to identify to what degree Electrocautery damage can alter the implant surface and if inflammatory cells are able to alter the implant surface and ingest metal particles. Methodology. To better understand how EC damage can alter implant surfaces, three different types of femoral component bearing surfaces were selected and intentionally damaged in the operating room using the plasma arc from both monopolar (MP) (Bovie) and Bipolar (BP) (Aquamantys) sources. MP and BP EC damage was done at varying power levels using a 3-second hover method 3 mm from the implant surface. Scanning electron microscopy (SEM) (Zeiss, Oberkochen, Germany) was used to obtain a detailed microscopic analysis of the damaged areas. Energy-dispersive X-ray spectrometry (EDS) (Oxford, High Wycombe, UK) was utilized to assess the elements present in pits found in the corroded areas. Surface Topography was analyzed using a profilometer (DektakXT; Bruker, Tucson, AZ) in the central portion of the damaged area for each MP and BP energy setting. Each damaged area was evaluated with the aid of TalyMap (Mountains software; Digital Surf, Besançon, France) using ISO 4287 measurements for Arithmetic Average height (Ra), Kurtosis (Rk), Heighest Peak to Lowest Valley (Rz), and Skewness (Rsk). SEM, EDS, and Surface Topography were also used to look at undamaged areas of the implants. In a separate experiment, IC-21 ATCC murine peritoneal macrophages were cultured with RPMI 1640 growth medium of supplemented with 10% fetal bovine serum (FBS), L-glutamine, and gentamicin. Select groups of cells were then activated using Interferon Gamma (IFNγ) and Lipopolysaccharide (LPS). CoCrMo alloy disks were cut, polished, passivated, and placed into 96 well plates and a select number intentionally damaged in the operating room with a MP EC device. After the cells were allowed to attach to the surface for 24 hours, culture medium was replaced every 12 hours and supernatant fluid was collected every 4 days starting on the second day of the experiment. After 30 days, cells were removed from the surface, counted and digested. The metal concentrations found in the supernatant and digested cell mixture were assessed using inductively coupled plasma spectrometry (ICP-MS), conducted at Brooks Applied Labs (Bothwell, WA). Statistical analysis was conducted using SigmaPlot (Systat Software, Chicago, IL) and Microsoft Excel (Microsoft, Redmond, WA). Results. Surface Profilometry quantified the topographical changes due to the damage form the MP and BP EC devices. The median Ra and Rz measurements were larger for the BP damaged areas compared to the MP for all bearing surfaces. The Oxinium surface displayed the greatest increase in roughness parameters compared to the undamaged regions. The CoCr surface displayed the greatest Rsk for the BP damaged areas. The ZrN had the smallest differences in Rz and Ra for both MP and BP damage areas compared to undamaged areas. SEM imaging displayed pitting in the regions intentionally damage with a MP or BP EC device. Backscatter EDS analysis found significant changes in the elemental profile for the BP damage compared to the MP damage. Cellular corrosion of the CoCr disks was quantified by measuring the concentration of Co, Cr, and Mo in the supernatant fluid collected off of the culture over the course of the 30-day experiment. The Co supernatant concentration was higher in the Undamaged Disks with Activated Cells versus its control which contained medium with no cells. The Cr concentration was higher in the supernatant fluid of the EC Damaged Disks with Standard Cells versus its control which contained medium with no cells. Between experimental groups, higher concentrations of Co and Mo was found in the supernatant of the Undamaged Disks with Standard Cells versus the EC Damaged Disks with Standard Cells. There was also a higher Co supernatant metal concentration when comparing the Undamaged Disks with Activated Cells versus the EC Damaged Disks with Activated Cells. A higher Cr supernatant metal concentration was found in the EC Damaged Disks with Activated Cells versus the EC Damaged Disks with Standard Cells. Following the end of the 30-day experiment, cells were digested to determine their inner metal ion concentration. There was a significantly higher intracellular Co and Mo concentration in the Undamaged Disks with Activated Cells versus the Undamaged Disks with Standard Cells. As well as a higher intracellular Co concentration in the EC Damaged Disks with Activated Cells versus the EC Damaged Disks with Standard Cells. SEM imaging displayed microscopic pitting on the surface exposed to macrophages and EC damage. Backscatter EDS analysis found significant differences in the elemental concentration of Carbon, Oxygen, Iron and Nickel between the experimental groups. From the EDS Backscatter analysis, the disks with EC damage displayed a higher Fe/C ratio compared to the undamaged disks. Showing evidence that EC damage alters the chemical profile of the CoCr disk surface.

Dissociated forms of moraxella isolated from the affected eyes of cattle

The dissociation phenomenon of epizootic cultures of Moraxella was studied. The study was conducted in economic entities of Almaty region of the Republic of Kazakhstan for 233 heads of cattle with clinical signs of keratoconjunctivitis. Isolation of the causative agent of Moraxella was performed by bacteriological washes from the conjunctival sacs of the eyes of animals. The laboratory study was carried out according to the approved methodological guidelines. It was found that bacteria of the genus Moraxella dissociate when grown on a solid nutrient medium for more than 6 hours in a thermostat at 37 °C. The bacteria were studied by the following methods: staining according to White-Wilson, thermoagglutination and acriflavine assay. When evaluating the grown colonies according to White-Wilson, the optimal dilution for crystal violet was found to be 1 : 2000, and for gentian violet stain 1 : 1000. In this case, the colonies in the S-form have a dark purple color with a metallic tint, and the dissociated colonies in the R-form do not stain. In the presence of dissociated cells, precipitation (thermoagglutination), sediment formation and clearing of the supernatant fluid at 90 °C for 30 minutes were noted. The suspension of undissociated colonies remained cloudy. When weighing microbial cells isolated by a bacterial loop from individual grown colonies in a solution of acriflavine, dissociated bacteria stick together to form conglomerates. When studying the antigenic activity of the S-, R- forms of Moraxella, it was revealed that the activity of the S-antigen significantly exceeded that of the R-forms. Data on the dissociation of Moraxella cultures can be used for the development of diagnostic and prophylactic drugs against moraxellosis in cattle.

Detection of BRAF mutation in the cytocentrifugation supernatant fluid from fine-needle aspiration of thyroid lesions may enhance the diagnostic yield

Objective: BRAF mutations using cellular DNA from fine-needle aspiration (FNA) specimens are commonly used to support the diagnosis of papillary thyroid carcinoma (PTC). The goal of this study was to preliminarily evaluate the diagnostic utility of detecting BRAF mutations in the routinely discarded FNA specimen supernatant fluid. Materials and Methods: Seventy-eight FNAs of thyroid lesions were evaluated for BRAF mutations using both cellular and supernatant DNA. BRAF mutation data were correlated with cytology and surgical pathology. Results: Of the 78 samples evaluated, 68 (87%) had amplifiable DNA in the supernatant with 2 (3%) positive for BRAF mutations. These two samples showed no mutations in the cellular counterpart. Among the 11 samples showing morphologic findings (FNA/surgical pathology) suspicious/diagnostic of PTC, 6 (55%) samples (one supernatant and five cellulars) were positive for BRAF mutations. This suggests that testing supernatant DNA in FNA specimens may increase the diagnostic yield by 1/11 (9%) in this setting. Conclusions: The vast majority of routinely discarded FNA supernatants contain amplifiable DNA. In addition, profiling the mutations of BRAF and other genes using supernatant DNA may provide valuable diagnostic information to assist the diagnosis of PTC in patients with clinical/morphologic findings suspicious for malignancies and cellular DNA showing no mutations.