Association of ABCB1 gene polymorphisms rs1128503, rs2032582, rs4148738 with anemia in patients receiving dabigatran after total knee arthroplasty.

PURPOSE: Dabigatran is usually prescribed in recommended doses without monitoring of the blood coagulation for the prevention of venous thromboembolism after joint arthroplasty. ABCB1 is a key gene in the metabolism of dabigatran etexilate. Its allele variants are likely to play a pivotal role in the occurrence of hemorrhagic complications. METHODS: The prospective study included 127 patients with primary knee osteoarthritis undergoing total knee arthroplasty. Patients with anemia and coagulation disorders, elevated transaminase and creatinine levels as well as already receiving anticoagulant and antiplatelet therapy were excluded from the study. The association of ABCB1 gene polymorphisms rs1128503, rs2032582, rs4148738 with anemia as the outcome of dabigatran therapy was evaluated by single-nucleotide polymorphism analysis with a real-time polymerase chain reaction assay and laboratory blood tests. The beta regression model was used to predict the effect of polymorphisms on the studied laboratory markers. The probability of the type 1 error (p) was less than 0.05 was considered statistically significant. BenjaminiHochberg was used to correct for significance levels in multiple hypothesis tests. All calculations were performed using Rprogramming language v3.6.3. RESULTS: For all polymorphisms there was no association with the level of platelets, protein, creatinine, alanine transaminase, prothrombin, international normalized ratio, activated partial thromboplastin time and fibrinogen. Carriers of rs1128503 (TT) had a significant decrease of hematocrit (p = 0.001), red blood count and hemoglobin (p = 0.015) while receiving dabigatran therapy during the postoperative period compared to the CC, CT. Carriers of rs2032582 (TT) had a significant decrease of hematocrit (p = 0.001), red blood count and hemoglobin (p = 0.006) while receiving dabigatran therapy during the postoperative period compared to the GG, GT phenotypes. These differences were not observed in carriers of rs4148738. CONCLUSION: It might be necessary to reconsider thromboprophylaxis with dabigatran in carriers of rs1128503 (TT) or rs2032582 (TT) polymorphisms in favor of other new oral anticoagulants. The long-term implication of these findings would be the reduction of bleeding complications after total joint arthroplasty.

Mesenchymal Cells Retain the Specificity of Embryonal Origin During Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo.

Guideline for management of septic arthritis in native joints (SANJO).

This clinical guideline is intended for use by orthopedic surgeons and physicians who care for patients with possible or documented septic arthritis of a native joint (SANJO). It includes evidence and opinion-based recommendations for the diagnosis and management of patients with SANJO.

Oligopeptide Sortase Inhibitor Modulates Staphylococcus aureus Cell Adhesion and Biofilm Formation.

Prevention of bacterial adhesion is one of the most important antivirulence strategies for meeting the global challenge posed by antimicrobial resistance. We aimed to investigate the influence of a peptidic S. aureus sortase A inhibitor on bacterial adhesion to eukaryotic cells and biofilm formation as a potential method for reducing S. aureus virulence. The pentapeptide LPRDA was synthesized and characterized as a pure individual organic compound. Incubation of MSSA and MRSA strains with LPRDA induced a subsequent reduction in staphylococcal adhesion to Vero cells and biofilm formation, as visualized by microscopic and spectrophotometric methods, respectively. LPRDA did not have a cytotoxic effect on eukaryotic or bacterial cells. The pentapeptide LPRDA deserves further investigation using in vitro and in vivo models of Gram-positive bacteriemia as a potential antibacterial agent with an antiadhesive mechanism of action.

Isolation of Human Osteoblast Cells Capable for Mineralization and Synthetizing Bone-Related Proteins In Vitro from Adult Bone.

The culture of osteoblasts (OB) of human origin is a useful experimental model in studying bone biology, osteogenic differentiation, functions of bone proteins, oncological processes in bone tissue, testing drugs against bone desires, and many other fields. The purpose of the present study is to share a workflow that has established the conditions to efficiently isolate and grow OB cells obtained from surgically removed bones from human donors. The protocol described here also shows how to determine cell phenotype. Here we provide characteristics of cells isolated by this protocol that might help researchers to decide if such OB are suitable for the purposes of their study. Osteoblasts isolated from collagenase-treated explants of adult bones are able to proliferate and keep their phenotype in culture. OB cells have high synthetic properties. They express osteomarkers, such as RUNX2, osteocalcin, BMP2, and osteopontin both in control conditions and in an osteogenic medium that could be estimated by qPCR and immunocytochemical staining and by Western blotting. Induction of osteogenic differentiation does not dramatically influence the synthetic properties of OB cells, while the cells gain the ability to extracellular mineralization only in an osteogenic medium.

Specificities of Scanning Electron Microscopy and Histological Methods in Assessing Cell-Engineered Construct Effectiveness for the Recovery of Hyaline Cartilage.

Damage to the hyaline layer of the articular surface is an urgent problem for millions of people around the world. At present, a large number of experimental methods are being developed to address this problem, including the transplantation of a cell-engineered construct (CEC) composed of a biodegradable scaffold with a premixed cell culture into the damaged area of the articular surface. However, current methods for analyzing the effectiveness of such CECs have significant limitations. This study aimed to compare the SEM technique, classical histology, and cryosectioning for the analysis of CECs transplanted to hyaline cartilage.

Methods of Modification of Mesenchymal Stem Cells and Conditions of Their Culturing for Hyaline Cartilage Tissue Engineering.

The use of mesenchymal stromal cells (MSCs) for tissue engineering of hyaline cartilage is a topical area of regenerative medicine that has already entered clinical practice. The key stage of this procedure is to create conditions for chondrogenic differentiation of MSCs, increase the synthesis of hyaline cartilage extracellular matrix proteins by these cells and activate their proliferation. The first such works consisted in the indirect modification of cells, namely, in changing the conditions in which they are located, including microfracturing of the subchondral bone and the use of 3D biodegradable scaffolds. The most effective methods for modifying the cell culture of MSCs are protein and physical, which have already been partially introduced into clinical practice. Genetic methods for modifying MSCs, despite their effectiveness, have significant limitations. Techniques have not yet been developed that allow studying the effectiveness of their application even in limited groups of patients. The use of MSC modification methods allows precise regulation of cell culture proliferation, and in combination with the use of a 3D biodegradable scaffold, it allows obtaining a hyaline-like regenerate in the damaged area. This review is devoted to the consideration and comparison of various methods used to modify the cell culture of MSCs for their use in regenerative medicine of cartilage tissue.

Comparative analysis of pathogen structure in patients with PJI after primary total hip and knee arthroplasty.

BACKGROUND: Microbiological profile of pathogens causing periprosthetic joint infection (PJI) after primary total hip (THA) and knee (TKA) arthroplasty varies in different regions, clinics and even departments. The objective of this study was to analyze the pathogen structure in patients with PJI after primary THA and TKA and its influence on the effectiveness of the infection eradication after two-stage reimplantation. MATERIALS AND METHODS: We collected the retrospective data of 364 patients-161 with PJI after primary TKA (113 treated in two stages 48 with failure after spacer implantation) and 203 patients with infected THA (127 after successful two-stage reimplantation and 76 with PJI recurrence after the first stage) within the time period from January 2012 to December 2017, treated with two-stage protocol in the single center. A comparative analysis of pathogen structure was performed between cohorts of patients with hip and knee PJI. A subanalysis was made between the subsets comprised from patients with successful two-stage treatment and the subsets with failure to treat the infection. RESULTS: Staphylococcus epidermidis was the most commonly identified pathogen in the full hip and knee cohorts: 30.1% and 32.5%, respectively. However, the percentage of methicillin-resistant Staphylococcus epidermidis (MRSE) among all S. epidermidis isolates was higher in the hip cohort-50% compared with 35% in the knee cohort (p = 0.073). Other coagulase-negative Staphylococci were more common to patients with PJI after primary TKA-10.3% compared with 5% (p < 0.02). Streptococcus sp. caused hip PJI in a larger percentage of cases than in knee PJI (p < 0.01)-7% and 2%, respectively (p < 0.01). Polymicrobial associations were significantly more common in hip PJI compared to knee PJI: 45.3% and 14% of cases, respectively (p < 0.001). The presence of polymicrobial infection significantly raised the risk of PJI recurrence [OR 2 (95% CI from 1.24 to 3.24)] in knee PJI patients and reduced the effectiveness of infection eradication from 73.9% to 47.8%. CONCLUSION: Comparative analysis showed significant differences in the structure of PJI pathogens in the hip and knee. These findings are useful when choosing treatment strategies and empirical antibiotics regimens, in the management of patients with PJIs after primary hip and knee arthroplasty.

International Organism Profile of Periprosthetic Total Hip and Knee Infections.

BACKGROUND: There is scarce literature describing pathogens responsible for periprosthetic joint infections (PJIs) around the world. Therefore, we sought to describe periprosthetic joint infection causative organisms, rates of resistant organisms, and polymicrobial infections at 7 large institutions located in North/South America and Europe. METHODS: We performed a retrospective study of 654 periprosthetic hip (n = 361) and knee (n = 293) infections (January 2006 to October 2019) identified at Cleveland Clinic Ohio/Florida in the United States (US) (n = 159), Hospital Italiano de Buenos Aires in Argentina (n = 99), Hospital Asociación Española in Uruguay (n = 130), Guy's and St Thomas' Hospital in the United Kingdom (UK) (n = 103), HELIOS Klinikum in Germany (n = 59), and Vreden Institute for Orthopedics in St. Petersburg, Russia (n = 104). Analyses were performed for the entire cohort, knees, and hips. Alpha was set at 0.05. RESULTS: Overall, the most frequent organisms identified were Staphylococcus aureus (24.8%) and Staphylococcus epidermidis (21.7%). The incidence of organisms resistant to at least one antibiotic was 58% and there was a significant difference between hips (62.3%) and knees (52.6%) (P = .014). Rates of resistant organisms among countries were 37.7% (US), 66.7% (Argentina), 71.5% (Uruguay), 40.8% (UK), 62.7% (Germany), and 77.9% (Russia) (P < .001). The overall incidence of polymicrobial infections was 9.3% and the rates across nations were 9.4% in the US, 11.1% in Argentina, 4.6% in Uruguay, 4.9% in UK, 11.9% in Germany, and 16.3% in Russia (P = .026). CONCLUSION: In the evaluated institutions, S aureus and S epidermidis accounted for almost 50% of all infections. The US and the UK had the lowest incidence of resistant organisms while Germany and Russia had the highest. The UK and Uruguay had the lowest rates of polymicrobial infections.

The W.A.I.O.T. Definition of Peri-Prosthetic Joint Infection: A Multi-center, Retrospective Validation Study.

Peri-prosthetic joint infection (PJI) definition plays an important role in diagnostic and therapeutic decisions. However, while several criteria have been proposed by eminent institutions to define a PJI in the last decade, their clinical validation has been rarely performed. Aim of the present multicenter, international, retrospective study was to validate the World Association against Infection in Orthopedics and Trauma (WAIOT) pre/intra-operative PJI definition with post-operative confirmatory tests. A total of 210 patients, undergoing hip (n = 86) or knee (n = 124) revision surgery for any reason in six orthopedic centers in Africa, Asia, Europe and South America, were retrospectively evaluated at a two years minimum follow-up after surgery. All the available pre-, intra- and post-operative findings were collected and analyzed according to the WAIOT criteria, which include a set of tests to confirm (Rule In) or to exclude (Rule Out) a PJI. On average, patients were investigated pre/intra-operatively with 3.1 ± 1.1 rule out and 2.7 ± 0.9 rule in tests; the presence of a fistula or exposed implant was reported in 37 patients (17.6%). According to pre/intraoperative findings, 36.2% of the patients were defined as affected by high-grade PJI (n = 76; average score: 2.3 ± 0.8), 21.9% by low-grade PJI (n = 46; average score: 0.8 ± 0.8), 10.5% by biofilm-related implant malfunction (n = 22; average score: -1.6 ± 0.8), 2.9% as contamination (n = 6; average score: -3.5 ± 1.0), and 28.6% as no infection (n = 60; average score: -3.0 ± 1.4). Pre/intra-operative PJI definitions matched post-operative confirmatory tests, in 97.1% of the patients. This is, to our knowledge, one of the largest study ever conducted to validate a PJI definition The retrospective analysis in different centers was greatly facilitated by the structure of the WAIOT definition, that allows to include different tests on the basis of their sensitivity/specificity, while the comparison between pre/intra-operative and post-operative findings offered the internal validation of the scoring system. Our results authenticate the WAIOT definition as a reliable, simple tool to identify patients affected by PJI prior to joint revision surgery.

The World Association against Infection in Orthopaedics and Trauma (WAIOT) procedures for Microbiological Sampling and Processing for Periprosthetic Joint Infections (PJIs) and other Implant-Related Infections.

While implant-related infections continue to play a relevant role in failure of implantable biomaterials in orthopaedic and trauma there is a lack of standardised microbiological procedures to identify the pathogen(s). The microbiological diagnosis of implant-related infections is challenging due to the following factors: the presence of bacterial biofilm(s), often associated with slow-growing microorganisms, low bacterial loads, previous antibiotic treatments and, possible intra-operative contamination. Therefore, diagnosis requires a specific set of procedures. Based on the Guidelines of the Italian Association of the Clinical Microbiologists (AMCLI), the World Association against Infection in Orthopaedics and Trauma has drafted the present document. This document includes guidance on the basic principles for sampling and processing for implant-related infections based on the most relevant literature. These procedures outline the main microbiological approaches, including sampling and processing methodologies for diagnostic assessment and confirmation of implant-related infections. Biofilm dislodgement techniques, incubation time and the role of molecular approaches are addressed in specific sections. The aim of this paper is to ensure a standardised approach to the main microbiological methods for implant-related infections, as well as to promote multidisciplinary collaboration between clinicians and microbiologists.

The W.A.I.O.T. Definition of High-Grade and Low-Grade Peri-Prosthetic Joint Infection.

The definition of peri-prosthetic joint infection (PJI) has a strong impact on the diagnostic pathway and on treatment decisions. In the last decade, at least five different definitions of peri-prosthetic joint infection (PJI) have been proposed, each one with intrinsic limitations. In order to move a step forward, the World Association against Infection in Orthopedics and Trauma (W.A.I.O.T.) has studied a possible alternative solution, based on three parameters: 1. the relative ability of each diagnostic test or procedure to Rule OUT and/or to Rule IN a PJI; 2. the clinical presentation; 3. the distinction between pre/intra-operative findings and post-operative confirmation. According to the WAIOT definition, any positive Rule IN test (a test with a specificity > 90%) scores +1, while a negative Rule OUT test (a test with a sensitivity > 90%) scores -1. When a minimum of two Rule IN and two Rule OUT tests are performed in a given patient, the balance between positive and negative tests, interpreted in the light of the clinical presentation and of the post-operative findings, allows to identify five different conditions: High-Grade PJI (score ≥ 1), Low-Grade PJI (≥0), Biofilm-related implant malfunction, Contamination and No infection (all scoring < 0). The proposed definition leaves the physician free to choose among different tests with similar sensitivity or specificity, on the basis of medical, logistical and economic considerations, while novel tests or diagnostic procedures can be implemented in the definition at any time, provided that they meet the required sensitivity and/or specificity thresholds. Key procedures to confirm or to exclude the diagnosis of PJI remain post-operative histological and microbiological analysis; in this regard, given the biofilm-related nature of PJI, microbiological investigations should be conducted with proper sampling, closed transport systems, antibiofilm processing of tissue samples and explanted biomaterials, and prolonged cultures. The proposed WAIOT definition is the result of an international, multidisciplinary effort. Next step will be a large scale, multicenter clinical validation trial.

Functional outcome of two-stage reimplantation in patients with periprosthetic joint infection after primary total knee arthroplasty.

INTRODUCTION: Two-stage reimplantation in patients with chronic periprosthetic joint infection (PJI) after total knee arthroplasty (TKA) with the use of either articulating or static antibiotic-loaded spacers during the first step is considered to be the golden standard in orthopaedics. THE AIM OF THE STUDY: The aim of the study was to evaluate the correlation of spacer type with the infection elimination rate as well as functional outcomes after two-staged revision TKA in patients with PJI. MATERIALS AND METHODS: The cohort comprised 161 patients who were treated for PJI after TKA during a period from January 2007 to December 2015. After the exclusion of patients with severe bone defects (AORI 2B or 3), 104 were left for the analysis: 72 patients with articulating and 32 with static spacers. The overall patient mean age was 62 years old (95% CI, 30-84): 73 for females, 31 for males. The outcomes were evaluated after three, six and 12 months using the American Knee Society Score (KSS) and EQ-5D. Only 92 patients were available for observation: 25 with static and 67 with articulating spacers. RESULTS: One year after the surgery, patients with articulating spacers demonstrated significantly higher mean KSS and function scores in comparison to patients with static spacers (90.4, 77.3 and 78.5, 67.8, respectively (p < 0.05)). The range of motion was also significantly better in patients with articulating spacers: 104.9° in comparison to 93.9° (р < 0.0001). The final EQ-5D score was comparable in both groups (0.82, 73.1 in articulating and 0.82, 72.6 in static spacers). CONCLUSION: The two-stage revision TKA for PJI using articulating spacers in comparison to the static ones provides better infection eradication rate as well as functional outcomes and improved quality of life.

Human bone graft cytocompatibility with mesenchymal stromal cells is comparable after thermal sterilization and washing followed by γ-irradiation: an in vitro study.

Human bone allografts present a better alternative to autografts in terms of minimization of the harvesting procedure complications. Prior to the use in clinical applications, they require sterilization which aims to reduce bioburden. This often comes at the expense of their biological properties as carriers of cells. In this study, we evaluated the cytocompatibility of human bone allografts processed and sterilized by three different methods with mesenchymal stromal cells. Bone morphology, biological and biochemical properties of the extracted bone-conditioned medium and viability of cells were assessed. We found that chemical sterilization had a strong negative effect on cell viability, whereas thermal sterilization and washing with subsequent γ-irradiation both resulted in a bone graft compatible with the progenitor cells. Moreover, washing of the bone prior to sterilization allowed solid removal of cell debris and other bone marrow components. Taken together, our findings demonstrate the importance of a proper choice of the bone graft processing method for the production of the biomaterial suitable for tissue engineering.

Failure of the first step of two-stage revision due to polymicrobial prosthetic joint infection of the hip.

BACKGROUND: The unsuccessful treatment of prosthetic joint infection (PJI) with two-stage revision leads to infection recurrence. The objectives of the study were to assess the clinical and demographic characteristics of patients with polymicrobial PJI, and to evaluate the role of the microbial profile involved in PJI in the risk of infection recurrence after the first step of two-stage revision surgery. MATERIALS AND METHODS: A retrospective analysis of 189 cases of culture-positive PJI following total hip replacement over a 5-year period was performed. The demographic characteristics of patients, clinical symptoms, microbiology cultures of intraoperative biopsies, laboratory values of C-reactive protein (CRP), white blood cell count and erythrocyte sedimentation rate were analyzed. Patients were divided into two groups-135 with monomicrobial and 54 with polymicrobial infection. RESULTS: Of all patients, 68.9 % in the monomicrobial and 83.3 % in the polymicrobial group had a body mass index >25 kg/m2 (p = 0.05). The median CRP values were 5.7 mg/L (IQR 4.0-10.0 mg/L) in the monomicrobial compared to 8.8 mg/L (IQR 5.0-27 mg/L) in the polymicrobial group (p = 0.01). The percentage of successful outcomes was 27.8 % in patients with microbial associations (p < 0.0001). Gram-negative pathogens caused polymicrobial PJI in 61.5 % of cases with infection recurrence (OR 4.4; 95 % CI 1.18-16.37; p = 0.03). CONCLUSIONS: Overweight and obese patients or those with elevated CRP had a greater risk of polymicrobial PJI. They were predisposed to recurrence of infection after the first step of two-stage revision. An unsuccessful outcome was more likely in cases with polymicrobial infection compared to those with monomicrobial infection. In addition, the presence of multidrug-resistant strains of Gram-negative bacteria substantially increased the risk of PJI treatment being unsuccessful. LEVEL OF EVIDENCE: Level III, therapeutic study.

Risk factors and a prognostic model of hip periprosthetic infection recurrence after surgical treatment using articulating and non-articulating spacers.

PURPOSE: The purpose of this study was evaluation of the efficacy of the first step of a two-stage procedure for treatment of hip prosthetic joint infection (PJI) using articulating and non-articulating spacers as well as development of a prediction model and prognostic score for infection recurrence. METHODS: In a cohort of 217 patients treated for PJI of the hip, demographic characteristics, clinical symptoms, body temperature, body mass index (BMI), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell count (WBC), microbiological cultures and the type of the spacer used were retrospectively analyzed for association with the recurrence of PJI. RESULTS: Patients with infection recurrence had increased levels of ESR and CRP (P < 0.001) together with higher BMI and shorter infection manifestation period after previous surgery (P < 0.05). Among these patients, there was no significant difference of clinical characteristics between subjects with articulating and non-articulating spacers. Microbial associations were more often identified in patients with recurrent infection (50 %, P < 0.01) where Gram-negative bacteria were predominant (61.5 %, P < 0.01). These patients had higher percentage of Acinetobacter sp. and P. aeruginosa isolates (28.2 %, P < 0.01). CONCLUSIONS: Efficacy of the first step of two-stage revision was 64.1 %. Placement of either articulating or non-articulating spacers did not influence recovery from PJI. Laboratory values of ESR, CRP, BMI and the type of previous surgery were identified as main factors that affect outcomes of the two-stage procedure. A prognostic model with the calculation of a total risk score for PJI recurrence was developed.