Aim. Fungal periprosthetic joint infections are difficult to treat and often associated with a limited outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules for bacteria. However, in vivo modeling of biofilm-associated fungi models are very rare. Furthermore, due to ethical restrictions, mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections with bacteria. This model organism was not used for fungi biofilm infection yet. Thus, we aimed to establish G. mellonella as in vivo model to study fungal implant infections using Candida albicans as model organism and to test anti-fungal medication. Method. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. For the infection process, implants were pre-incubated in specified fungal growth culture Candida albicans at 1×10. 7. CFU/ml for 30 min at 150 rpm shaking conditions. Later, these implants were washed with 10ml PBS and implanted in the larvae as mentioned. To analyze the susceptibility of the implant-associated fungal infections towards anti fungal compounds, the larvae were treated with amphotericin B, fluconazole and voriconazole after 24h of implantation. The effect of anti-fungal compounds was measured in terms of survival observation for 5 days and fungal load in larvae on 2. nd. day. To reveal the fungal biofilm formation on implant, the implants were removed on day 3 and processed for SEM analysis. Results. Pre-incubated K-wire caused the Candida infection and observed the death of the larvae. The treatment with antifungal compounds recovered the larvae from the implant-infection, except in case of Voriconazole. However, the recovery with treatment of anti fungal compounds was not effective as the larvae with planktonic infection, which highlights typical biofilm phenotype. Further, the treatment with anti-fungal compounds with Amphotericin B and Fluconazole reduced the fungal load in larvae tissue. The SEM analysis revealed the formation fungal biofilm with hyphae and spores associated with larvae tissue on implant surface. Conclusions. The results from survival analysis, antifungal treatment and SEM analysis are very promising to use of G. mellonella as in vivo model to study fungal infections on implanted materials. Our study highlights the use of G. mellonella larvae as alternative in vivo model to study implant-associated fungal infections that reduces the use of the higher mammals

ZrN-multilayer coating is clinically well established in total knee arthroplasty [1-3] and has demonstrated significant reduction in polyethylene wear and metal ion release [4,5]. The goal of our study was to analyze the biotribological behaviour of the ZrN-multilayer coating on a polished cobalt-chromium cemented hip stem. CoCr28Mo6 alloy hip stems with ZrN-multilayer coating (CoreHip®AS) were tested versus an un-coated version. In a worst-case-scenario the stems with ceramic heads have been tested in bovine serum in a severe cement interface debonding condition under a cyclic load of 3,875 N for 15 million cycles. After 1, 3, 5, 10 & 15 million cycles the surface texture was analysed by scanning-electron-microscopy (SEM) and energy-dispersive x-ray (EDX). Metal ion concentration of Co,Cr,Mo was measured by inductively coupled plasma mass spectroscopy (ICP-MS) after each test interval. Based on SEM/EDX analysis, it has been demonstrated that the ZrN-multilayer coating keeps his integrity over 15 million cycles of severe stem cemented interface debonding without any exposure of the CoCr28Mo6 substrate. The ZrN-multilayer coated polished cobalt-chromium cemented hip stem has shown a reduction of Co & Cr metal ion release by two orders of a magnitude, even under severe stem debonding and high interface micro-motion conditions. ZrN-multilayer coating on polished cobalt-chromium cemented hip stems might be a suitable option for further minimisation of Co & Cr metal ion release in total hip arthroplasty. Clinical evidence has to be proven during the next years

Aims. This study aims to enhance understanding of clinical and radiological consequences and involved mechanisms that led to corrosion of the Precice Stryde (Stryde) intramedullary lengthening nail in the post market surveillance era of the device. Between 2018 and 2021 more than 2,000 Stryde nails have been implanted worldwide. However, the outcome of treatment with the Stryde system is insufficiently reported. Methods. This is a retrospective single-centre study analyzing outcome of 57 consecutive lengthening procedures performed with the Stryde nail at the authors’ institution from February 2019 until November 2020. Macro- and microscopic metallographic analysis of four retrieved nails was conducted. To investigate observed corrosion at telescoping junction, scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDX) were performed. Results. Adjacent to the nail’s telescoping junction, osteolytic changes were observed in bi-planar radiographs of 20/57 segments (35%) after a mean of 9.5 months (95% confidence interval 7.2 to 11.9) after surgery. A total of 8/20 patients with osseous alterations (40%) reported rest and ambulation pain of the lengthened segment during consolidation. So far, 24 Stryde nails were retrieved and in 20 (83%) macroscopic corrosion was observed at the nail’s telescoping junction. Before implant removal 11/20 radiographs (55%) of lengthened segments with these 20 nails revealed osteolysis. Implant retrieval analysis by means of SEM showed pitting and crevice corrosion. EDX detected chromium as the main metallic element of corrosion. Conclusion. Patients are exposed to the risk of implant-related osteolysis of unclear short- and long-term clinical consequences. The authors advocate in favour of an early implant removal after osseous consolidation. Cite this article: Bone Joint Res 2021;10(7):425–436

Aim. Prosthetic joint infections pose a major clinical challenge. Developing novel material surface technologies for orthopedic implants that prevent bacterial adhesion and biofilm formation is essential. Antimicrobial coatings applicable to articulating implant surfaces are limited, due to the articulation mechanics inducing wear, coating degradation, and toxic particle release. Noble metals are known for their antimicrobial activity and high mechanical strength and could be a viable coating alternative for orthopaedic implants [1]. In this study, the potential of thin platinum-based metal alloy coatings was developed, characterized, and tested on cytotoxicity and antibacterial properties. Method. Three platinum-based metal alloy coatings were sputter-coated on medical-grade polished titanium discs. The coatings were characterized using optical topography and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Ion release was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). Cytotoxicity was tested according to ISO10993-5 using mouse fibroblasts (cell lines L929 and 3T3). Antibacterial surface activity, bacterial adhesion, bacterial proliferation, and biofilm formation were tested with gram-positive Staphylococcus aureus ATCC 25923 and gram-negative Escherichia coli ATCC 25922. Colony forming unit (CFU) counts, live-dead fluorescence staining, and SEM-EDS images were used to assess antibacterial activity. Results. Three different platinum-based metal alloys consisting of platinum-iridium, platinum-copper, and platinum-zirconium. The coatings were found 80 nm thick, smooth (roughness average < 60 nm), and non-toxic. The platinum-copper coating showed a CFU reduction larger than one logarithm in adherent bacteria compared to uncoated titanium. The other coatings showed a smaller reduction. This data was confirmed by SEM and live-dead fluorescence images, and accordingly, ICP-OES measurements showed low levels of metal ion release from the coatings. Conclusions. The platinum-copper coating showed low anti-adhesion properties, even with extremely low metal ions released. These platinum-based metal alloy coatings cannot be classified as antimicrobial yet. Further optimization of the coating composition to induce a higher ion release based on the galvanic principle is required and copper looks most promising as the antimicrobial compound of choice. Acknowledgments. This publication is supported by the DARTBAC project (with project number NWA.1292.19.354) of the research program NWA-ORC which is (partly) financed by the Dutch Research Council (NWO); and the AMBITION project (with project number NSP20–1-302), co-funded by the PPP Allowance made available by Health-Holland, Top Sector Life Sciences & Health to ReumaNederland

Title. Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model. Aim. To assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model. Method. All animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 10. 5. CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours. Results. 1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the mouse femur. Proper placement of all implants were confirmed with in vivo X-rays, and ex vivo photos. We empirically derive the ROI during each imaging session by aggregating the imaged volume which ranges from (636.4um × 636.4um × 151um) = 0.625 +/- 0.014 mm. 3. of bone marrow in a global GFP-transgenic mouse. 2) IV-MLSM imaging acquisition of the “race for the surface”.In vitro MPLSM images of implants partially coated with USA300LAC (RFP-MRSA) were verified by SEM image. Results from IV-MLSM of RFP-MRSA and GFP. +. host cell colonization of the contaminated implants illustrated the mutually exclusive surface coating at 3hrs, which to our knowledge is the first demonstration of “the race for the surface” between bacteria and host cells via intravital microscopy. 3) Quantifying the “race for the surface” with CFU verification of S. aureus on the implant. 3D volumetric rendering of the GFP. +. voxels and RFP+ voxels within the ROI were generated in Imaris. The voxel numbers suggeste that the fight for the surface concludes ∼3hrs post-infection, and then transitions to an aggressive MRSA proliferation phase. The results of WT control demonstrate a significant increase in CFU by 12hrs post-op for both stainless steel (P<0.01) and titanium (P<0.01). Conclusions. We developed IV-MLSM to quantify the “Race for the Surface” between host cells and contaminating S. aureus in a murine femur implant model. This race is remarkably fast, as the implant surface is completely covered with 3hrs, peak bacterial growth on the implant occurs between 2 and 12 hours and is complete by 12hrs

Aim. A novel anti-infective biopolymer implant coating was developed to prevent bacterial biofilm formation and allow on-demand burst release of anti-infective silver (Ag) into the surrounding of the implant at any time after surgery via focused high-energy extracorporeal shock waves (fhESW). Method. A semi-crystalline Poly-L-lactic acid (PLLA) was loaded with homogeneously dissolved silver (Ag) applied onto Ti6Al4V discs. A fibroblast WST-1 assay was performed to ensure adequate biocompatibility of the Ag concentration at 6%. The prevention of early biofilm formation was investigated in a biofilm model with Staphylococcus epidermidis RP62A after incubation for 24 hours via quantitative bacteriology. In addition, the effect of released Ag after fhESW (Storz DUOLITH SD1: 4000 impulses, 1,24 mJ/mm. 2. , 3Hz, 162J) was assessed via optical density of bacterial cultures (Escherichia coli TG1, Staphylococcus epidermidis RP62A, Staphylococcus aureus 6850) and compared to an established electroplated silver coating. The amount of released Ag after the application of different intensities of fhESW was measured and compared to a control group without fhESW via graphite furnace atomic absorption spectrometry (GF-AAS), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Results. The coating with 6% Ag reduced Staphylococcus epidermidis biofilm formation by 99.7% (mean±SD: 2.1×10^5 ± 3,9×10^5 CFU/µL) compared to uncoated controls (6.8×10^7 ± 4.9×10^7 CFU/µL); (p=0.0001). After applying fhESW the commercially available electroplated silver coating did not prevent the growth of all tested bacterial strains. Bacterial growth is delayed with 4% Ag and completely inhibited with 6% Ag in the novel coating, except for a small increase of S. aureus after 17 hours. SEM and EDS confirmed a local disruption of the coating after fhESW. Conclusions. This novel anti-infective implant coating has the potential to prevent bacterial biofilm formation. The on-demand burst release of silver via fhESW could be an adjunctive in the treatment of implant related infection and is of particular interest in the concept of single stage revision surgery

Aim. Galleria mellonella larvae is a well-known insect infection model that has been used to test the virulence of bacterial and fungal strains as well as for the high throughput screening of antimicrobial compounds against infections. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections using small K-wire as implant material. Here, we aimed to further expand the use of G. mellonella to test other materials such as bone cement with combination of gentamicin to treat implant-associated infections. Method. The poly methyl methacrylate (PMMA) with and without gentamicin and liquid methyl methacrylate (MMA) were kindly provided by Heraeus Medical GmbH, Wehrheim. To make the bone cement implants as cubes, Teflon plate (Karl Lettenbauer, Erlangen) with specified well size was used. The Radiopaque polymer and monomer were mixed well in a bowl, applied over on to the Teflon plate and pressed with spatula to form fine and uniform cubes. After polymerization, the bone cement implants were taken out of the Teflon well plate with the help of pin. For the infection process, bone cement cubes were pre-incubated with S. aureus EDCC 5055 culture at 5×10. 6. CFU/ml for 30 min at 150 rpm shaking conditions. Later, these implants were washed with 10ml PBS and implanted in the larvae as mentioned. Survival of the larvae were observed at 37°C in an incubator. To analyze the susceptibility of the bacterial infections towards gentamicin, survival of the larvae compared with control group implanted only with bone cement. The effect of gentamicin was also measured in terms of S. aureus load in larvae on 2. nd. day. SEM analysis was performed to see the effect of gentamicin on biofilm formation on bone cement. Results. Our experiments established the G. mellonella as an excellent model to screen bone cement with antimicrobial compounds against bacterial infections. The gentamicin bone cement samples showed excellent S. aureus bacterial load reduction after the implantation in G. mellonella model. The bone cement with gentamicin showed better survival of larvae infected with S. aureus compared to control. Finally, the gentamicin also affected the biofilm formation on the bone cement surface with S. aureus. Conclusions. Thus, our work showed G. mellonella is a rapid, cheap economical pre-clinical model to study the bone cement associate bacterial infections as well as screening of the various antimicrobial compounds

Introduction. Titanium (Ti) alloys are used as porous bone ingrowth materials on non-cemented knee arthroplasty tibial tray implants. Nano-surface mechanism that increase the osseointegration rate between Ti alloys, and surrounding tissue has been recognized to improve the interface to ultimately allow patients to weight bear on non-cemented arthroplasty implants sooner. Bioactive TiO. 2. nanotube arrays has been shown to accelerate osseointegration. Ideally, these surfaces would both increase the adhesion of bone to the implant and help to reduction of infection to substitute for antibiotic bone cement. This study examines a combination treatment of both TiO. 2. nanotubes combined with silver nano-deposition, that simultaneously enhances osseointegration while improving infection resistance, by testing ex vivo implantation stability in an equine cadaver bone followed by in vitro and in vivo analysis to understand the biocompatibility and early stage osseointegration. Methods. 100nm diameter and 300nm length TiO. 2. nanotubes were formed on a CP titanium surface using anodization method at 20V for 45mins using 1% HF electrolyte. Silver deposition on TiO. 2. nanotubes were performed using 0.1M AgNO. 3. solution at 3V for 45s. Figure 1 shows SEM images showing (a) TiO. 2. nanotubes of 300nm length and (b) nanotubes with silver coating). Ti anodized samples with and without silver nanotubes implanted into an equine cadaver bone in an ex vivo manner to study the stability of nanotubes and the adherence of silver deposition. Silver release study was performed for a period of 14 days in a similar ex vivo manner. Dimensions for implantation samples: 2.5 mm diam. × 15 mm. For cell culture, circular disc samples 12.5mm in diameter and 3 mm in thickness were used to study the bone cell-material interactions using human fetal osteoblast (hFOB) cells. To evaluate the cell proliferation, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay was used. The in vitro cell-materials interaction study was performed for a period of 4 and 7 days. In vivo study was performed using rat distal femur model for a period of 12 weeks with dense Ti samples as control (Sample dimensions: 3mm diam. × 5mm). At the end of 12 weeks, the samples were analyzed for early stage osseointegration using histological analysis and SEM imaging. Results. No significant changes in the morphology of nanotubes was observed due to the implantation process which signifies the damage resistance these nanotubes can endure during implantation and explantation. Figure 2 shows SEM images of (a) & (b) nanotubes without silver coating before and after implantation and (c) & (d) nanotubes with silver coating before and after implantation respectively. Silver nanocoatings can be observed after implantation which shows the adherence of the antimicrobial nano-coating on the surface of nanotubes. Cumulative release profiles of silver ions after 14 days showed the total release was in the effective range for antimicrobial characteristics and was well below the toxic limit specified for human cells (10 ppm) Figure 3(a) shows cumulative release profile of silver after 14 days. MTT assay and SEM images show good cell proliferation, antimicrobial effect, and increase in cell density after 7 days for samples with nanotubes and silver with no cytotoxic effects and good cell attachment on the samples as shown in Figure 3(b) MTT assay results showing cell densities after 4 and 7 days and Figure 3(c) SEM images showing cell attachment after 4 and 7 days on samples. Histological analysis and SEM images showed osteoid formation around the implant with improved bonding towards the implant and bone showing signs of early stage osseointegration. Figure 4 shows histological and SEM images showing bonding between bone and implant surface for respective samples after 12 weeks. Conclusions. Mechanically stableTiO. 2. nanotubes with strongly adhered antimicrobial silver coating were grown on the surface of titanium which were biocompatible and non-toxic. In vitro and in vivo tests indicate improved cell-materials interaction with signs of early stage osseointegration. This nano-surface treatment shows promise towards simultaneously improving early stage osseointegration and providing an infection barrier on bone ingrowth materials

Introduction. The ability to create patient-specific implants (PSI) at the point-of-care has become a desire for clinicians wanting to provide affordable and customized treatment. While some hospitals have already adopted extrusion-based 3D printing (fused filament fabrication; FFF) for creating non-implantable instruments, recent innovations have allowed for the printing of high-temperature implantable polymers including polyetheretherketone (PEEK). With interest in FFF PEEK implants growing, it is important to identify methods for printing favorable implant characteristics such as porosity for osseointegration. In this study, we assess the effect of porous geometry on the cell response and mechanical properties for FFF-printed porous PEEK. We also demonstrate the ability to design and print customized porous implants, specifically for a sheep tibial segmental defect model, based on CT images and using the geometry of triply periodic minimal surfaces (TPMS). Methods. Three porous constructs – a rectilinear pattern and gyroid/diamond TPMSs – were designed to mimic trabecular bone morphology and manufactured via PEEK FFF. TPMSs were designed by altering their respective equation approximations to achieve desired porous characteristics, and the meshes were solidified and shaped using a CAD workflow. Printed samples were mCT scanned to determine the resulting pore size and porosity, then seeded with pre-osteoblast cells for 7 and 14 days. Cell proliferation and alkaline phosphatase activity (ALP) were evaluated, and the samples were imaged via SEM. The structures were tested in compression, and stiffness and yield strength values were determined from resulting stress-strain plots. Roughness was determined using optical profilometry. Finally, our process of porous structure design/creation was modified to establish a proof-of-concept workflow for creating PSIs using geometry established from segmented sheep tibia CT images. Results. ALP activity measurements of the porous PEEK samples at 7 and 14 days were significantly greater than for solid controls (p < 0.001 for all three designs, 14 days). No difference between the porous geometries was found. SEM imaging revealed cells with flat, elongated morphology attached to the surface of the PEEK and into the pore openings, with filopodia and lamellipodia extensions apparent. mCT imaging showed average pore size to be 545 ± 43 µm (porosity 70%), 708 ± 64 µm (porosity 68%), and 596 ± 94 µm (porosity 69%) for the rectilinear, gyroid, and diamond structures, respectively. The average error between the theoretical and actual values was −16.3 µm (pore size) and −3.3 % (porosity). Compression testing revealed elastic moduli ranging from 210 to 268 MPa for the porous samples. Yield strengths were 6.6 ± 1.2 MPa for lattice, 14.8 ± 0.7 MPa for gyroid, and 17.1 ± 0.6 for diamond. Average roughness ranged from 0.8 to 3 µm. Finally, we demonstrated the ability to design and print a fully porous implant with the geometry of a sheep tibia segment. Assessments of implant geometrical accuracy and mechanical performance are ongoing. Discussion. We created porous PEEK with TPMS geometries via FFF and demonstrated a positive cellular response and mechanical characteristics similar to trabecular bone. Our work offers an innovative approach for advancing point-of-care 3D printing and PSI creation

Aim. Periprosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty. While research has focused on developing better tests for disease diagnosis, treatment options have stayed relatively constant over the years with high failure rates ranging from 30%–50% and are due in part to the protective biofilm produced by some bacterial species. Current treatment options are compromised by the presence of biofilm, emphasizing the need for novel treatment strategies to be developed. Our group has developed a novel treatment (PhotothermAA) which has demonstrated in vitro its ability to target bacterial biofilm. The purpose of this study was to test this PhotothermAA technology in vivo in a rabbit model of PJI for its efficacy in eradicating biofilm. Method. Rabbits were fitted with a titanium implant into the tibial plateau and inoculated with 5×10. 6. CFU Xen36 (luminescent Staphylococcus aureus). At two weeks, rabbits underwent irrigation and debridement and treatment with PhotothermAA gel for two hours and subsequently laser heated using an 808 nm laser for 10 minutes. Gel was washed out and implant was removed for quantitative biofilm coverage analysis via scanning electron microscopy (SEM, n=3 for control and n=2 for PhotothermAA treated). Periprosthetic tissue was collected before and after treatment for toxicity studies via hemotoxylin and eosin (H&E) staining and scored for necrosis by three blinded reviewers (n=5 per group). Student's t-test was used for statistical analysis. Results. Implants isolated after PhotothermAA gel treatment had less biofilm coverage on the surface of the implant compared to non-treated control via SEM analysis (36.9% vs. 55.2%, p<0.14). PhotothermAA gel treatment and subsequent laser treatment was not harmful to surrounding tissue as no increase in necrotic tissue was observed. Conclusions. PhotothermAA gel and laser treatment safely decreases biofilm coverage on infected knee implants in a rabbit PJI model

Aim. Quadrupled hamstring anterior cruciate ligament plasties (4xHp) have been described as having a higher risk of infection than bone patellar tendon bone plasties (BPTBp). There are 2 theories that might explain this phenomenon. One is the presence of sutures in a 4xHp that could act as a foreign body, The other is the more complex preparation of a 4xHp that might lead to higher contamination rates during the process. The objective of the present study was to evaluate the formation of biofilm in these plasties and to compare it between a 4xHp and a BPTBp. The hypothesis was that the presence of sutures in 4xHp would increase the amount of biofilm present in them in comparison to BPTBp. Method. A descriptive in vitro study was conducted. One 4xHp and one BPTBp were prepared. They were subsequently divided into 8 fragments. Three of them were reserved for negative control, and the rest were contaminated with a strain of S. Epidermidis (ATCC 35984) 10–5. Finally, a quantitative analysis was carried out by means of microcalorimetry and sonication with plating. Additionally, a qualitative analysis was carried out by means of electron microscopy. Results. In isothermal microcalorimetry, both contaminated plasties showed the same growth dynamics with a population peak (200uW) at 8h. No significant differences were found between the bacterial growth profiles of 4xHp and BPTBp. The product of sonication was plated and the number of colony forming units per milliliter (CFU/ml) was counted at 24 hours. No significant differences were detected between the 4×Hp (mean +/− sem = 3,5×107 +/− 3450000) and the BPTBp (4,6 ×107 +/− 1,455e+7). With a p value of 0.6667, there were no differences of significance (Mann-Whitney test). In the samples analyzed with electron microscopy, no specific biofilm growth pattern was identified upon comparing BPTBp with 4xHp. Conclusions. There were no significant differences at either the quantitative or qualitative level when comparing bacterial growth in BPTBp and 4xHp. Therefore, the presence of sutures in 4xHp cannot be established as a predisposing factor to higher infection rates. These findings may be justified in the sense that the plasties themselves already behave like foreign bodies. Therefore, the presence of sutures does not increase the possibility of biofilm forming on their surface

Introduction. The ability to manufacture implants at the point-of-care has become a desire for clinicians wanting to provide efficient patient-specific treatment. While some hospitals have adopted extrusion-based 3D printing (fused filament fabrication; FFF) for creating non-implantable instruments with low-temperature plastics, recent innovations have allowed for the printing of high-temperature polymers such as polyetheretherketone (PEEK). Due to its low modulus of elasticity, high yield strength, and radiolucency, PEEK is an attractive biomaterial for implantable devices. Though concerns exist regarding PEEK for orthopaedic implants due to its bioinertness, the creation of porous networks has shown promising results for bone ingrowth. In this study, we endeavor to manufacture porous PEEK constructs via clinically-used FFF. We assess the effect of porous geometry on cell response and hypothesize that porous PEEK will exhibit greater preosteoblast viability and activity compared to solid PEEK. The work represents an innovative approach to advancing point-of-care 3D printing, cementless fixation for total joint arthroplasty, and additional applications typically reserved for porous metal. Methods. Three porous constructs – a rectilinear pattern and two triply period minimal surface (TPMSs) - were designed to mimic the morphology of trabecular bone. The structures, along with solid PEEK samples for use as a control, were manufactured via FFF using PEEK. The samples were mCT scanned to determine the resulting pore size and porosity. The PEEK constructs were then seeded with pre-osteoblast cells for 7 and 14 days. Cell proliferation and alkaline phosphatase activity (ALP) were evaluated at each time point, and the samples were imaged via SEM. Results. mCT imaging showed the pores in the PEEK constructs to be open and interconnected. The average pore size was 535 ± 92 µm for the rectilinear, 484 ± 237 µm for the diamond, and 669 ± 216 µm for the gyroid. Porosity was 71% for the rectilinear, 76% for the diamond, and 68% for the gyroid. The average error between the theoretical and actual values was −37.3 µm for pore size and −2.3 % for porosity. Normalized ALP activity of the three porous PEEK samples at 7 days were found to be significantly greater than the solid sample (p < 0.05 rectilinear, p < 0.005 gyroid, p < 0.001 diamond). At 14 days, the same relationships were observed (p < 0.001 for all three designs). No difference between the three geometries was found. SEM imaging revealed cells with flat, elongated morphology attached to the surface of the PEEK. The 14-day samples appeared to have proliferated well and spread along the PEEK pores. Extensions of filopodia and lamellipodia were observed along with large blankets of cells covering the PEEK surface. Discussion. We demonstrated the ability of FFF printed porous PEEK surfaces to promote cellular processes necessary for bone-implant fixation. While all porous structures showed promising results, more investigation into their material characteristics and osteogenic potential are necessary to determine which geometry may be suitable for orthopaedic use. Our work offers an innovative approach to advancing point-of-care 3D printing, cementless implant fixation, and additional applications typically reserved for porous metal

Introduction. Implant associated infections are responsible for over 10 % of recorded orthopaedic revision surgeries across the UK, with higher infection rates commonly observed for other endoprostheses such as cranioplasties. To prevent colonization and biofilm formation on implant surfaces, the use of silver coatings has shown positive results in clinical setting due to its synergistic function with conventional antibiotic prophylaxes. Additive manufacturing allows manufacture of entirely new implant geometries such as lattice structures to enhance osseointegration, however this limits the ability to uniformly coat implants. Direct integration of silver into the powder feedstock for selective laser melting (SLM) may allow manufacture of a biomedical alloy with innate, long lasting antimicrobial properties without compromising possible geometries and with no coating process necessary. Methods. Feedstock powders of 15–45 micron Grade 5 Ti-64 (Renishaw Plc) and Ag-999 powder (CooksonGold) were characterized using laser particle size analysis, ICP-OES, LECO-ONH, and morphological analysis in SEM. A blend of Ti-64 with 3 wt% Ag-999 powder (Ti-643) was produced by tumble blending, and validated by SEM and EDS. Parameters for manufacture were established using a 17 point design of experiment (DoE) exploring a 2D parameter space of applied laser power and laser scanning speed. Samples were manufactured using a ConceptLaser M2 LaserCusing SLM. Density was assessed by He pycnometry, and cross-sections analysed for defects by optical microscopy. Silver distribution was mapped by micro X-Ray Fluoroscopy (µXRF) and energy-dispersive X-ray spectroscopy (EDS). Optimum parameters were identified and used to manufacture all subsequent samples. Cylindrical Ti-643 samples were manufactured for further physical characterization and bacterial investigation, alongside control Ti-64 samples manufactured using existing optimum parameters. Samples were polished using silicon carbide papers to a 4000-grit surface finish. Contact angle measurements were made by goniometry. Silver elution characteristics were assessed by immersion in water refreshed on a daily basis, and sampled over a 14 day period using ICP-OES. Viability of S. aureus was compared to control samples according to the Japanese standard test method, JIS Z 2801:2000. Results. Across the entire parameter space tested, selective laser melting (SLM) of all 17 samples was successful, with no delamination. An increased recoater blade speed was required to achieve uniform spreading in process versus pure Ti-6Al- 4V powder, indicating an increased cohesivity of the Ti-643 blend. The presence of silver in all samples was confirmed by µXRF, indicating that there was no excessive evaporation of silver in-process. Laser parameters were found to alter the defect density and microstructure scale, though sample density was tightly clustered in a range from 4.415 to 4.453 gcm-3, showing relatively low process variation. No significant difference in bacterial survival was found between control and Ti-643 samples, indicating that further microstructural optimization is needed to guarantee efficacy

INTRODUCTION. The lifetime of total hip replacements (THR) is often limited by adverse local tissue reactions to corrosion products generated from modular junctions. Two prominent damage modes are the imprinting of the rougher stem topography into the smoother head taper topography (imprinting) and the occurrence of column-like troughs running parallel to the taper axis (column damage). It was the purpose of this study to identify mechanisms that lead to imprinting and column damage based on a thorough analysis of retrieved implants. METHODS. 776 femoral heads were studied. Heads were visually inspected for imprinting and column damage. Molds were made of each head taper and scanned with an optical coordinate measuring machine. The resulting intensity images were used to visualize damage on the entire surface. In selected cases, implant surfaces were further analyzed by means of scanning electron microscopy (SEM) and white light interferometry. The alloy microstructure was characterized for designs from different manufactures. RESULTS. 165 heads exhibited moderate to severe damage (modified Goldberg scale). Out of those heads 83% had imprinting and 28% exhibited column damage. In most cases with imprinting, the entire contact area between stem and head was affected (Figure 1). Several cases exhibited early signs of imprinting, usually starting on the distal-inferior and distal superior side. High resolution SEM imaging revealed that imprinting was a fretting driven process that was independent of the hardness and material of the stem and head. The SEM images showed that the main mechanism was surface fatigue under partial slip fretting. The generated wear debris was the primary driver of imprinting by three-body fretting. The effect was detrimental on the smoother head surface, but less severe on the rougher stem, where debris was pushed into the troughs of the machining mark topography. 90% of cases with column damage also exhibited imprinting. The other ten percent were either cases in which column damage was too extensive to identify imprinting, or the stem taper was smooth and therefore could not induce imprinting. Metallographic analysis showed that column damage was dictated by the alloy microstructure. Wrought alloy heads frequently exhibited banding related to slight alloy segregations. The process of column damage was entirely chemically driven with etching occurring along the banded microstructure eventually resulting in troughs that were several tens of micrometers deep (Figure 2). DISCUSSION. Imprinting and column damage are common damage modes in THR femoral heads. Imprinting is fretting (miro-motion) driven while column damage is caused chemically, but is also dictated by the alloy micro-structure. However, the results suggest that these two damage modes may be related. The damage process starts with local fretting slowly progressing to a large area of imprinting. The imprinting process leads to widening of the crevice, enabling joint fluid and biological constituents (protein, cells, etc.) to enter the taper interface. This change in local chemistry within a confined crevice environment can cause an etching process that leads to column damage, but only if the femoral head alloy has a banded microstructure

Introduction. Little is known about the relationship between head-neck corrosion and its effect on periprosthetic tissues and distant organs in the majority of patients hosting apparently well-functioning devices. We studied the degree and type of taper damage and the histopathologic response in periprosthetic tissue and distant organs. Methods. A total of 50 contemporary THRs (34 primary, 16 revision) retrieved postmortem from 40 patients after 0.4–26 years were studied. Forty-three femoral stems were CoCrMo and 7 were Ti6Al4V. In every case, a CoCrMo-alloy head articulated against a cementless polyethylene cup (19 XLPE and 31 UHMWPE). H&E and IHC sections of the joint pseudocapsules and liver were graded 1–4 for the intensity of various inflammatory cell infiltrates and tissue necrosis. The nature of the tissue response in the joint capsule, liver, spleen, kidneys and lymph nodes was assessed. Wear and corrosion products in the tissues were identified using SEM and EDS. Taper surfaces were graded for corrosion damage using modified Goldberg scoring and examined by SEM to determine the acting corrosion mode. Correlations between damage scores and the histologic variables were generated using the Spearman test. Results. No correlation was seen between taper damage scores and the macrophage response in the joint pseudocapsule. The distribution of corrosion scores for heads and femoral trunnions is shown in Figure 1. Moderate or severe corrosion of the head and/or trunnion was present in 9 hips (8 CoCr/CoCr and 1 CoCr/TiAlV). One patient with bilateral hips had local ALVAL-like lymphocyte-dominated tissue reactions (Figure 2) and mild focal lymphocytic infiltrates in the liver and kidneys (Figure 3). This was associated with severe intergranular corrosion of the CoCrMo trunnion and column damage on the head taper. Particle-laden macrophages in pseudocapsules were significantly correlated with liver macrophages (r=.382, p=0.012) and liver lymphocytes (r=.367, p=0.013). Pseudocapsule macrophage responses to metallic and/or polyethylene wear particles ranged widely from minimal to marked. Focal tissue necrosis was related to high concentrations of particulate wear debris. A minimal number of metallic particle-laden macrophages were also detected in the liver and spleen; and macrophage granulomas were present in para-aortic lymph nodes, especially in revision cases. DISCUSSION. The generation of metal ions and particulates at corroded CoCrMo heads and CoCrMo or Ti6Al4V trunnions was a significant contributor to the presence of perivascular lymphocytes within the joint pseudocapsule, with 1 patient showing a histologic pattern consistent with ALVAL. Patient factors and the rate of corrosion are among variables influencing whether an ALVAL-type reaction will develop and whether or not it will become symptomatic. Macrophages in the joint pseudocapsules were positively correlated with inflammatory cells in the liver. In this study, the intensity of inflammatory infiltrates in distant organs was mild. However, several cases of organ dysfunction have been reported in association with catastrophic wear of CoCrMo components. It continues to be essential to minimize the generation of metal ions and particulates and to improve strategies for identifying and managing patients exposed to high levels of degradation products. For any figures or tables, please contact the authors directly

Introduction. Dislocation of an uncemented total hip replacement (THR) can cause damage to the femoral hear, when it passes through the rim of metal acetabular shell. This can lead to metal transfer on the surface of the head or chipping of bulk head material. Although dislocation is one of most common complications in total hip arthroplasty (THA), little is known if causes any further damage to the articulating surface of ceramic heads in long term observations. Aim of the study. To evaluate, if dislocations of THR with ceramic on polyethylene bearing causes structural damage to the articulating surface of the femoral head in a follow-up of minium 10 years. Materials and methods. MATERIAL. This study included four groups of third generation (CeramTec Biolox forte) ceramic femoral heads with a diameter of 28 mm:. 8 heads dislocated in the first year(managed nonsurgically) where implants functioned for at least 10 years. 9 heads removed within 12 months after implantation because of recurrent dislocations. 11 heads retrieved after a minimum of 10 years from hips where no dislocation occurred. 8 unused heads. METHODS. Surface topography of retrieved heads was evaluated using Scanning Electron Microscopy with Energy – Dispersive X-ray Spectroscopy; roughness measurements were performed in three directions (0, 45,90 degrees) over a distance of 1,5 mm using a contact profilometer. Results. On the dislocated heads dark stained scratches were present in the lower, non weight-bearing part; SEM studies demonstrated presence of multidirectional smear-like metal deposits (Fig 1) with a small number of chipped ceramic grains. EDS analysis of scratches indicated presence of Ti or Ti,Al,V - dependant on construction alloy of the acetabular component. There were no differences in morphology of scratched areas between heads removed within a few months after dislocation and components retrieved after at least 10 years. Interestingly SEM findings within the weight-bearing part of dislocated heads were identical within corresponding areas of femoral heads articulating for 10 or more years as well as unused components (Fig 2). In these areas we observed a small number of pinholes and scratches, which we believe resulted from machining and subsequent polishing. Roughness measurements were consistent with SEM findings (Table 1). Areas covered by metal deposits had a significantly higher roughness compared to undamaged area. We did not observe statistical differences between roughness of weight-bearing area in all examined groups. Conclusions. Our study indicates, that dislocation of femoral head in a uncemented total hip replacement with ceramic on polyethylene bearing results in contained damage in the area which contacted the metal acetabular shell. Dislocation does not affect the weight-bearing surface of the head, even in a long-term follow-up. This study was financed by a National Science Centre grant No. 2012/05/D/NZ5/01840

Introduction. Wear debris induced osteolysis and loosening continue to cause clinical failure in total knee replacement (TKR). To improve longevity and reduce wear alternative materials have been examined. Carbon-fibre-reinforced poly–ether-ether-ketone (CFR-PEEK) has shown promising results in wear studies [1–2]. The aim of this study was to explore the use of CFR-PEEK and PEEK as alternative bearing materials for polyethylene in TKR through experimental knee joint wear simulation. Methods. Two novel materials were studied as an alternative to polyethylene as the tibial bearing surface in a TKR configuration using a Cobalt chrome femoral bearing in current clinical use. Six right Sigma CR fixed bearing TKRs (DePuy Synthes, UK) were paired with either PEEK or CFR-PEEK custom-made flat inserts (Invibio, UK) in a Prosim knee simulator (Simulator Solutions, UK). The tibial inserts were 14mm thick, to give an equivalent thickness to existing insert designs. A flat geometry was selected as this has previously been shown to yield low wear in polyethylene bearings [3]. The tests were conducted under High Kinematics, with anterior-posterior and internal-external displacement control [4]. Tests were conducted for three million cycles, lubricated with 25% bovine serum, with wear assessed gravimetrically at 1 and 3Mc. Images of the wear scars were recorded at completion of the study. Visual inspection of the tibial inserts was used to identify regions of wear damage for SEM analysis (EVO MA15-Smart SEM, Zeiss, Germany). Results. Extremely large material loss and wear was observed for both PEEK and CFR-PEEK inserts at 3Mc (Figure 1). The PEEK material showed higher volumetric loss than the CFR-PEEK. Wear rates of >200mm. 3. /Mc compare with less than 4 mm. 3. /Mc for polyethylene under the same conditions. [3]. Large, deep wear scars were observed on all plates (Figure 2a and b). Evidence of mechanical material failure was observed through SEM including material cracking, deformation and fibre pull-out (Figure 3a and b). Discussion. Wear simulator testing of flat PEEK and CFR-PEEK as potential replacements for polyethylene materials in a TKR configuration produced high material loss after 3Mc. This is in contrast to promising data reported in another study of the knee [5]. The present study examined a low-conformity condition. Previous studies have included hip and highly-conforming TKR designs; therefore the contact pressures would be lower than the current study. This study highlights these new materials may undergo mechanical failure at higher contact stress conditions

INTRODUCTION. Rotator cuff tears are common injuries which often require surgical repair. Unfortunately, repairs often fail [1] and improved repair strength is essential. P2 Porous titanium (DJO Surgical, Austin TX) has been shown to promote osseointegration [2,3] and subdermal integration [4]. However, the ability of P2Porous titanium to aid in supraspinatus tendon-to-bone repair has not been evaluated. Therefore, the purpose of this study was to investigate P2 implants used to augment supraspinatus tendon-to-bone repair in a rat model [5]. We hypothesized that supraspinatus tendon-to-bone repairs with P2 implants would allow for ingrowth and increased repair strength when compared to standard repair alone. METHODS. Thirty-four adult male Sprague-Dawley rats were used (IACUC approved). Rats received bilateral supraspinatus detachment and repair with one limb receiving P2 implant. Animals were sacrificed at time 0 (n=3), 2 weeks (n=8), 4 weeks (n=9) and 12 weeks (n=14). Limbs were either dissected for histological and SEM analysis or mechanical testing as described previously [5]. Specimens for histology and SEM were embedded in PMMA for tissue-implant interface analysis. Specimens were first viewed in SEM under BSE to detect bony ingrowth, then stained with Sanderson's Rapid Bone Stain and viewed under transmitted and polarized light for tissue ingrowth. Comparisons were made using Student's t-tests with significance at p≤0.05. RESULTS. No differences in cross-sectional area were detected at any time point (Fig 1A). Percent relaxation was significantly increased in the P2 group at 2 weeks, but not at 4 and 12 weeks (Fig 1B). Maximum load was significantly increased in the P2 group at 2 weeks, but not at 4 weeks (Fig 1C – maximum load not reported due to failure at grip at 12 weeks). Modulus was significantly increased in the P2 group at 4 weeks, but not at 2 or 12 weeks (Fig 1D). No differences were detected in stiffness at any time point (data not shown). BSE analysis demonstrated bone ingrowth (Fig 2) and histological analysis showed soft tissue integration (Fig 3). DISCUSSION. Results indicate superior mechanical properties in the P2 group at 2 and 4 weeks, and tissue ingrowth at all time points. Importantly, at 2 weeks, the P2group had 76% increased maximum load compared to standard repair. As supraspinatus tendon re-tears are extremely common early [1] and occur at the tendon-to-bone interface, this finding supports the reduction of re-tear risk with the P2 implant. Although no differences were detected in maximum load at 4 weeks, the increase at 2 weeks denotes that P2 implants improved early tendon-to-bone healing. Additionally, at 4 weeks, the P2 implant group had significantly increased elastic modulus, further supporting increased mechanical properties due to the P2 implant. Clinically, improved early healing might allow faster rehabilitation and associated recovery. This study demonstrates that the P2 implant improves tendon-to-bone healing up to 4 weeks (with no detrimental effects at longer time points), suggesting that P2 porous titanium may be of benefit for use in clinical rotator cuff repairs

Introduction. Orthopedic implants are subject to wear and release ultra-high molecular weight polyethylene (UHMWPE) debris. Analysis of UHMWPE wear particles is critical in determining the safety and effectiveness of novel orthopedic implants. Complete digestion of periprosthetic tissue and wear fluid is necessary to ensure accurate morphological and quantitative particle analysis. Acid digestion methods are more effective than enzymatic and base digestion approaches [Baxter+ 2009]. However, optimal digestion times, quantity, and type of acid are unclear for particle isolation. In addition, imaging and analysis techniques are critical to ensure accurate reporting of particle characteristics. Here, we 1) compared the efficacy of three acid-based digestion methods in isolating particles from a) bovine serum and b) animal/human tissue, and 2) analyzed the effects of imaging location on particle quantity/morphology results. Methods. 1a) UHMWPE (GUR 150) particles were generated by Mode I knee wear testing for 1 million cycles in bovine serum. Serum was digested in one of four solutions: 12.2M HCl, 15.8M HNO. 3. , a 1:1 volume ratio of HNO. 3. :HCl (aqua regia), or filtered H. 2. O (control). The serum:solution volume ratio was 1:5 [Niedzwiecki+ 2001, ISO 17853:2011]. Digestion occurred for 60min on a stir plate at 60°C. Each digest was combined with MeOH at a 1:5 digest:MeOH volume ratio and filtered using a 100 nm polycarbonate membrane. The particle-containing membranes were imaged (12 images/membrane) using scanning electron microscopy (SEM) to determine particle characteristics, including quantity, equivalent circular diameter (ECD) and aspect ratio (AR). 1b) Based on 1a, HNO. 3. was used to digest porcine and human tissue at concentrations of 1:40, 1:60, or 1:80 tissue:HNO. 3. volume ratios for either 1, 12, or 24 hours, followed by SEM analysis. 2) Particle characteristics were compared at nine locations (20 images/location) across a particle-containing membrane to determine the effects of imaging location. Results. 1a) HNO. 3. and aqua regia methods successfully digested the bovine serum, whereas the HCl and H. 2. O methods were unsuccessful (Fig.1A). Comparing HNO. 3. and aqua regia groups, particle characteristics and ECD frequency distribution were nearly identical (Fig.1B). 1b) Nitric acid did not fully digest porcine or human tissues. 2) Similar particle characteristics were observed in all nine locations analyzed across the polycarbonate membrane. The particle quantity, ECD, and AR for a representative center vs. intermediate location were 808 vs. 780 particles, 0.33±0.28 vs. 0.35±0.29 µm, and 1.57±0.56 vs. 1.51±0.4, respectively (Fig.2). Conclusions. Nitric acid and aqua regia are capable of digesting bovine serum using low quantities of acid for short duration, allowing precise analysis of UHMWPE particle debris from orthopedic implants. However, further optimization of digestion techniques for animal/human tissue is warranted. In addition, an accurate representation of particle distribution can be achieved without analyzing hundreds of images, because membrane location does not strongly influence particle results. Finally, ASTM F1877-16 – Standard Practice for Characterization of Particles – could benefit from adding software-based automated particle characterization as an optional method. An automated approach that uses k-means clustering image segmentation to identify particles and computer vision tools to extract relevant morphological features is under development and validation

The treatment of critical-sized bone defects still remains today a challenge, especially when the surrounding soft, vascularized and innervated tissues have been damaged - a lack of revascularization within the injured site leading to physiological disorders, from delayed healing to osteonecrosis. The axial insertion of a vascular bundle (e.g. arterio-venous loop, AVL) within a synthetic bone filler to initiate and promote its revascularization has been foreseen as a promising alternative to the current strategies (e.g., vascularized free flaps) for the regeneration of large bone defects. In a previous work, we showed that the insertion of a vein in a 3D-printed monetite scaffold induced its higher revascularization than AVL, thus a possible simplification of the surgical procedures (no microsurgery required). Going further, we investigate in this study whether or not the presence of a vein could stimulate the formation of mineralized tissue insides a synthetic scaffold filled with bone marrow and implanted in ectopic site. Monetite scaffolds were produced by additive manufacturing according to a reactive 3D-printing technique co-developed by the authors then thoroughly characterized. Animal study was performed on 14 male Wistar rats. After anesthesia and analgesia, a skin medial incision in rat thigh allowed the site on implantation to be exposed. Bone marrow was collected on the opposite femur through a minimally invasive procedure and the implant was soaked with it. For the control group (N=7), the implant was inserted in the incision and the wound was closed whereas the femoral bundle was dissected and the vein inserted in the implant for the experimental group (N=7). After 8 weeks animals were sacrificed, the implant collected and fixed in a 4% paraformaldehyde solution. Explants were characterized by µCT then embedded in poly-methyl methacrylate prior SEM, histology and immunohistochemistry. Images were analyzed with CT-Analyzer (Bruker) and ImageJ (NIH) and statistical analyses were carried out using SPSS (IBM). Implants were successfully 3D-printed with a +150 µm deviation from the initial CAD. As expected, implants were composed of 63%wt monetite and 37%wt unreacted TCP, with a total porosity of 44%. Data suggested that scaffold biodegradation was significantly higher when perfused by a vein. Moreover, the latter allowed for the development of a dense vascular network within the implant, which is far more advanced than for the control group. Finally, although mineralized tissues were observed both inside and outside the implant for both groups, bone formation appeared to be much more important in the experimental one. The ectopic formation of a new mineralized tissue within a monetite implant soaked with bone marrow seems to be highly stimulated by the simple presence of a vein alone. Although AVL have been studied extensively, little is known about the couple angiogenesis/osteogenesis which appears to be a key factor for the regeneration of critical-sized bone defects. Even less is known about the mechanisms that lead to the formation of a new bone tissue, induced by the presence of a vein only. With this in mind, this study could be considered as a proof of concept for further investigations