This study investigates the fabrication, characterization and biocompatibility of a Ti-6Al-4V-4Cu alloy produced from spherical Ti-6Al-4V and Cu powders using the directed energy deposition (DED-LB) process with an annular laser beam (ALB). Based on process stability related wall rectangularity, and interlayer bonding, the optimal build strategy of the wall samples was achieved by a bi-directional path with reducing ALB power and initial power of 1250 W. Microstructural characterization included optical microscopy for grain morphology, SEM with analysis for microstructure and elemental distribution, and XRD for phase identification. Electrochemical performance was assessed in a simulated physiological environment, demonstrating passive behavior and corrosion resistance. To evaluate in vivo biocompatibility, disc-shaped implants were inserted subcutaneously in mice and monitored for physiological, hematological, biochemical, and histological responses over a period of 7 or 56days. Mice exhibited no signs of adverse effects with stable feed and water intake, normal body weight, and typical behavior. Hematological and metabolic profiles revealed no significant differences between the Ti-6Al-4V-4Cu, Ti-6Al-4V, sham, and untreated groups. Histological analysis revealed that the implants were well-integrated with surrounding tissues, with no evidence of granulomatous inflammation, immune cell infiltration, or abnormal tissue morphology. ICP-MS analysis of the mouse serum revealed stable concentrations of Cu, Al, V, and Ti, indicating no systemic metal release. These findings confirm that DED-LB fabricated Ti-6Al-4V-4Cu alloy exhibits favorable in vivo biocompatibility and systemic safety in mice. The results support its potential for biomedical applications that require corrosion resistance and biocompatibility, and warrant further in vivo evaluation of its antibacterial properties.

Cancer continues to represent a substantial burden in terms of its morbidity and mortality, underscoring the imperative for the development of novel and efficacious treatment modalities. Recent advances in cancer immunotherapy have highlighted the importance of identifying tumour-specific antigens, which can assist the immune system in targeting malignant cells effectively. Phage display technology has emerged as an effective tool for the discovery of novel antigens through cDNA library screening, representing a significant advancement in the field of immunological research. This review examines the discovery of tumour antigens using phage display technology, emphasising the construction of cDNA libraries, their subsequent display on bacteriophages and the utilisation of diverse biopanning techniques. These elements play a pivotal role in advancing the discovery of novel tumour antigens and the development of targeted cancer therapies. This review addresses the challenges associated with the filamentous phage display of cDNA libraries and proposes strategies to improve the effectiveness of this approach, encouraging further research for clinical applications.

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.

Prosthetic joint infections (PJIs) are commonly diagnosed via culture-based methods, which may miss hard-togrow pathogens. This study contrasts amplicon metagenomic sequencing (16S AS) with traditional culture techniques for enhanced clinical decision-making. We analyzed sonicate fluid from 27 patients undergoing revision arthroplasty using both methods, emphasizing the distinction between contaminants and true positives. Our findings show moderate agreement between the two methods, with a Cohen's kappa of 0.490, varying across bacterial genera (Cohen's kappa -0.059 to 1). The sensitivity of 16S AS compared to culture was 81% (95% CI, 68% to 94%). Sequencing revealed greater microbial diversity, including anaerobic genera like Anaerococcus and Citrobacter. Interestingly, several culture-negative PJI samples showed diverse bacteria via 16S AS. Despite rigorous controls and algorithms to eliminate contaminants, confirming bacteria presence with 16S AS remains a challenge. This highlights the need for improved PJI diagnostic methods, while also pointing out the limitations of next-generation sequencing (NGS) as a clinical diagnostic tool.

Tranexamic acid (TXA) is a synthetic derivative of the amino acid lysine and is commonly used as an antifibrinolytic agent in large joint surgery, such as knee arthroplasty, to minimize blood loss and its associated complications. Some studies have suggested that perioperative intravenous TXA application lowers the incidence of postoperative infections after hip and knee arthroplasty. While its effects on bacteria are not its primary purpose, more research is needed further to elucidate the extent of TXA's impact on bacterial inhibition and its clinical relevance. In order to determine possible inhibitory effects, 22 clinically relevant bacterial strains were exposed to different TXA concentrations. Two different assays were performed: a microdilution assay with traditional endpoint measurements and a kinetic growth assay for a time-dependent susceptibility assessment. With the microdilution technique, a growth inhibitory effect became evident at higher TXA concentrations (50 and 100 mg/mL) on eight tested bacterial strains, of which four demonstrated absence of growth at 100 mg/mL, suggesting minimal inhibitory concentration. However, the kinetic growth assay revealed more specific TXA-induced dynamic changes in the bacterial growth of all tested bacterial strains. This study showed that TXA affects the growth dynamics of bacteria by altering their lag phase duration, exponential growth rate, and population density and may contribute to the prevention of implant-associated infections.

For the improvement of surface roughness, titanium joint arthroplasty (TJA) components are grit-blasted with Al2O3 (corundum) particles during manufacturing. There is an acute concern, particularly with uncemented implants, about polymeric, metallic, and corundum debris generation and accumulation in TJA, and its association with osteolysis and implant loosening. The surface morphology, chemistry, phase analysis, and surface chemistry of retrieved and new Al2O3 grit-blasted titanium alloy were determined with scanning electron microscopy (SEM), X-ray energy-dispersive spectroscopy (EDS), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and confocal laser fluorescence microscopy, respectively. Peri-prosthetic soft tissue was studied with histopathology. Blasted retrieved and new stems were exposed to human mesenchymal stromal stem cells (BMSCs) for 7 days to test biocompatibility and cytotoxicity. We found metallic particles in the peri-prosthetic soft tissue. Ti6Al7Nb with the residual Al2O3 particles exhibited a low cytotoxic effect while polished titanium and ceramic disks exhibited no cytotoxic effect. None of the tested materials caused cell death or even a zone of inhibition. Our results indicate a possible biological effect of the blasting debris; however, we found no significant toxicity with these materials. Further studies on the optimal size and properties of the blasting particles are indicated for minimizing their adverse biological effects.

Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLightTM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.

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Introduction The main aim was to analyse the series of 29 collected cemented Charnley–Muller Alivium retrievals with the meantime in situ of 27 years. In addition, the revision rate of 1425 Alivium prostheses implanted at our institution between 1977 and 1992 was calculated. Materials and methods The revision percentage of the Alivium cohort was calculated up to 45 years of follow-up and compared to that of all total hip arthroplasties (THAs) implanted in the same period (No. 5535). Metal and polyethylene retrieved components were inspected in 29 cases for wear damage and roughness. Wear particles were retrieved from periprosthetic tissue using digestion protocols and their composition, morphology, and size distribution were investigated. Periprosthetic tissue was analysed histologically. Results The revision percentage of the Alivium cohort was 16% at 45 years of follow-up. It was comparable to all the THAs implanted at the same time (18%). The shape of polyethylene particles isolated from periprosthetic tissue corresponded to the wear pattern on polyethylene cups. Polyethylene particles were the main wear product, with the majority (68%) of particles smaller than 0.1 µm. Metal particles were rare with two types: CoCr and Cr based. Histological analysis showed that in 14 out of 18 specimens, the metal particles were graded + 1, reflecting that the metal loading in the periprosthetic tissue was low. Conclusions Our study represents valuable data not reported previously on the survival rate of Charnley–Muller prostheses at 45 years of follow-up and a unique insight into the collected retrievals from the materials’ point of view.

Infection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo. Two monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models. Characterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines. In conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.

The number of prosthetic joint infection (PJI) cases is increasing along with total joint arthroplasties. There is currently no diagnostic test available with 100% sensitivity to identify PJI. The aim of the study was to assess and compare two different bacteriophage K-based methods with standard microbiological culturing methods to detect staphylococci. Samples were retrieved from 104 patients undergoing revision surgery due to suspected PJI. Implants were subjected to sonication and sonicate fluid (SF) was assessed with the methods of qPCR detection of bacteriophage K DNA and adenosine triphosphate (ATP) detection after bacteriophage K lysis. The results were compared with the results of standard microbiological culturing methods. PJI was confirmed in 33 cases according to the PJI definition. Using the methods of ATP and bacteriophage K DNA detection 100% specificity and predictive value were achieved. The sensitivity of qPCR detection was higher (81.25%) than the sensitivity of ATP detection (62.50%) when analyzing SF directly. The sensitivity of the methods significantly improved (to 94.12%) with SF pre-cultivation. Importantly, both methods provided results in 3-4 h when analyzing SF directly, while results from pre-cultivated SF were obtained 19-20 h after sample collection. Our results suggest that bacteriophage-based methods are specific and sensitive and importantly, faster than standard culturing methods. The addition of new bacteriophages to expand the bacterial detection spectrum could lead to the development of a faster, more sensitive, specific, and also economical, and handy method for PJI diagnosis.

Multimodal treatment approaches, such as radio-immunotherapy, necessitate regimen optimization and the investigation of the interactions of different modalities. The aim of this study was two-fold. Firstly, to select the most effective combination of irradiation and the previously developed tumor cell-based vaccine and then to provide insight into the immune response to the selected combinatorial treatment. The study was performed in immunologically different murine tumor models: B16F10 melanoma and CT26 colorectal carcinoma. The most effective combinatorial treatment was selected by comparing three different IR regimens and three different vaccination regimens. We determined the local immune response by investigating immune cell infiltration at the vaccination site and in tumors. Lastly, we determined the systemic immune response by investigating the amount of tumor-specific effector lymphocytes in draining lymph nodes. The selected most effective combinatorial treatment was 5× 5 Gy in combination with concomitant single-dose vaccination (B16F10) or with concomitant multi-dose vaccination (CT26). The combinatorial treatment successfully elicited a local immune response at the vaccination site and in tumors in both tumor models. It also resulted in the highest amount of tumor-specific effector lymphocytes in draining lymph nodes in the B16F10, but not in the CT26 tumor-bearing mice. However, the amount of tumor-specific effector lymphocytes was intrinsically higher in the CT26 than in the B16F10 tumor model. Upon the selection of the most effective combinatorial treatment, we demonstrated that the vaccine elicits an immune response and contributes to the antitumor efficacy of tumor irradiation. However, this interaction is multi-faceted and appears to be dependent on the tumor immunogenicity.

INTRODUCTION:Revisions due to the fracture of ceramic-on-ceramic (CoC) bearing are rare, however when they occur, they represent a major challenge to an orthopedic surgeon for ensuring safe and long-term survival of the replaced bearing.CASE REPORT:We present a case of fractured ceramic liner of total hip prosthesis that underwent revision to a metal-on-polyethylene (MoP) bearing couple, with consequent huge periprosthetic metallosis. Shortly after, the second revision operation followed using the third bearing couple of ceramic-on-polyethylene (CoP). At 10 years follow-up after the operation due to ceramic fracture, the patient is now pain free with full range of motion of the revised hip.CONCLUSION:Establishment of diagnostic routes and recommended protocols for CoC bearing fracture would allow easier recognition of potential fracture and diminish its consequences for the patients.

Prosthetic joint infections (PJI) represent the most serious cause of prosthetic joint loosening, with high impact on patient life and health economics. Although not entirely reliable, the cultivation of intraoperative prosthetic tissue or synovial fluid remains the gold standard for determining the cause of PJI. Therefore, molecular methods are increasingly being introduced. The aim of this study was to optimize and assess an alternative molecular approach with the use of bacteriophage K for more rapid and specific detection of staphylococci in sonicate fluid (SF) of PJI. The best results with the method were obtained after 180 min of sample incubation with 104 PFU/mL of bacteriophage K. DNA isolation prior to qPCR analysis was confirmed unnecessary, while chloroform addition to samples after incubation with bacteriophage K improved bacterial detection by 100×. The method had a limit of detection of 6.8×102 CFU/mL and was found suitable for the detection of staphylococci in SF of removed prosthetic joints, giving results comparable to standard microbiological methods in just four hours. The optimized method was found fit for the purpose, offering potential advantages over the use of molecular detection methods to detect bacterial DNA.

The ultimate challenge for orthopedic surgeons who implant hip prosthesis is providing long-term function of the joint without pain. The most common cause of total hip arthroplasty failure is aseptic loosening mostly initiated by wear particles.1 The current paradigm explains aseptic loosening as an inflammatory response to the wear debris particles produced by prosthetic implants. Wear particles are phagocytosed by macrophages and induce a variety of cytokines that mediate inflammatory response leading to the recruitment and activation of osteoclasts, and eventual implant loosening.2 For over 40 years, the metal-on-polyethylene (MoPE) bearing has dominated the field of total hip replacement (THR). The second generation of metal on metal (MoM) THRs made of CoCrMo alloy was introduced into the market in early 1990s with the aim to eliminate the wear of polyethylene (PE) and therefore, to achieve better long-term clinical results compared to conventional MoPE THRs. MoM artificial hip prostheses showed an extremely low rate of wear of the articulation surfaces. In addition, metal particles that are produced are smaller than PE particles and hence, they may induce less tissue reaction.3–5 However, patients with M-M articulations have increased levels of Co and Cr in the serum and urine, and this has raised concerns about toxicity, mutagenesis and hypersensitivity.6 There are reports from orthopedic registers about increase complications and potential problems of early failure of MoM, often require revision surgery.7,8 Several studies reported soft tissue damages with adverse reaction to metal debris.9

Background Currently, the gold standard for the microbiological diagnosis remains the culturing of preoperative aspirated joint fluid and intraoperative periprosthetic tissue samples, which give fa...

Paired cartilage and subchondral bone of subjects with no clinical history of joint disorders were analyzed to determine whether antioxidant enzymes, inflammatory cytokines and growth factors can be linked to a pre-osteoarthritis. Tissue explants were phenotyped according to Osteoarthritis Research Society International grading and micro-computed tomography, and also screened for the expression of several markers using quantitative polymerase chain reaction. The expression of these same genes was measured in SW1353 cells treated with hydrogen peroxide, to gain insight into the pathways involved with oxidative stress responses. Vascular endothelial growth factor A (VEGF-A) was up-regulated in the cartilage samples that showed early cartilage or bone degeneration. Oxidative stress in chondrocytes provoked up-regulation of interleukin-1β, interleukin-6, aggrecan, and SRY-box containing gene 9. Our results confirm the hitherto evidence of the deteriorating effects of the oxidative stress on cartilage and suggest the link between VEGF-A and pre-osteoarthritis.

Staphylococcus spp. accounts for up to two thirds of all microorganisms causing prosthetic joint infections, with Staphylococcus aureus and Staphylococcus epidermidis being the major cause. The present study describes a diagnostic model to detect staphylococci using a specific bacteriophage and bioluminescence detection, exploring the possibility of its use on sonicate fluid of orthopaedic artificial joints. Intracellular adenosine-5'-triphosphate release by bacteriophage mediated lysis of staphylococci was assessed to determine optimal parameters for detection. With the optimized method, a limit of detection of around 103 CFU/mL was obtained after incubation with bacteriophage for 2 h. Importantly, sonicate fluid did not prevent the ability of bacteriophage to infect bacteria and all simulated infected sonicate fluid as well as 6 clinical samples with microbiologically proven staphylococcal infection were detected as positive. The total assay took approximately 4 h. Collectively, the results indicate that the developed method promises a rapid, inexpensive and specific diagnostic detection of staphylococci in sonicate fluid of infected prosthetic joints. In addition, the unlimited pool of different existing bacteriophages, with different specificity for all kind of bacteria gives the opportunity for further investigations, improvements of the current model and implementation in other medical fields for the purpose of the establishment of a rapid diagnosis.

PURPOSE:The purpose of this study was to compare the results of revision total hip arthroplasty after fracture of primary ceramic components using different type of revision bearing surfaces.METHODS:We analysed the results of 16 patients with a follow-up more than 3 years after first revision. 6 were revised to ceramic-on-ceramic (CoC) bearing, 9 to metal-on-polyethylene (MoP) and 1 to ceramic-on-cross-linked polyethylene (CoXLP) bearing.RESULTS:The mean follow-up was 87 months. Patients with revision to CoC had higher Harris Hip Score (HHS) of 89 points in comparison to the patients with revision to MoP with 84 points. Radiographic examinations revealed visible eccentric polyethylene wear with osteolysis in 3 out of 9 patients revised to MoP. There were no detrimental x-ray changes in patients revised to CoC components.CONCLUSIONS:We consider CoC as the best option at revision operation for ceramic component fracture.

Background and purpose - The correct diagnosis of prosthetic joint infection (PJI) can be difficult because bacteria form a biofilm on the surface of the implant. The sensitivity of culture from sonication fluid is better than that from periprosthetic tissue, but no comparison studies using molecular methods on a large scale have been performed. We assessed whether periprosthetic tissue or sonication fluid should be used for molecular analysis. Patients and methods - Implant and tissue samples were retrieved from 87 patients who underwent revision operation of total knee or total hip arthroplasty. Both sample types were analyzed using broad-range (BR-) PCR targeting the 16S rRNA gene. The results were evaluated based on the definition of periprosthetic joint infection from the Workgroup of the Musculoskeletal Infection Society. Results - PJI was diagnosed in 29 patients, whereas aseptic failure was diagnosed in 58 patients. Analysis of sonication fluid using BR-PCR detected bacteria in 27 patients, whereas analysis of periprosthetic tissue by BR-PCR detected bacteria in 22 patients. In 6 of 7 patients in whom BR-PCR analysis of periprosthetic tissue was negative, low-virulence bacteria were present. The sensitivity and specificity values for periprosthetic tissue were 76% and 93%, respectively, and the sensitivity and specificity values for sonication fluid were 95% and 97%. Interpretation - Our results suggest that sonication fluid may be a more appropriate sample than periprosthetic tissue for BR-PCR analysis in patients with PJI. However, further investigation is required to improve detection of bacteria in patients with so-called aseptic failure.