The Dysregulated Galectin Network Activates NF-κB to Induce Disease Markers and Matrix Degeneration in 3D Pellet Cultures of Osteoarthritic Chondrocytes.

This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8: 5 µg/ml, Gal-3: 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-κB activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression.

Detection and differentiation of cerebral microemboli in patients undergoing major orthopaedic surgery using transcranial Doppler ultrasound.

Background: Cerebral microemboli (ME) are frequently generated during orthopaedic surgery and may impair cerebral integrity. However, the nature of cerebral ME, being either of solid or gaseous origin, is poorly investigated. Our primary aim was to determine both the frequency and nature of cerebral ME in generally anaesthetised patients undergoing major orthopaedic surgery. Methods: Fifty patients (hip/knee/shoulder prosthesis, spine surgery) were enrolled. Cerebral ME and cerebral blood flow velocity (CBFV) were determined in both middle cerebral arteries for 15 min preoperatively and postoperatively, using transcranial Doppler ultrasound. Cerebral tissue oxygen index, determined by near-infrared spectroscopy, was further examined. Statistical analysis was carried out using the Wilcoxon matched-pairs signed-ranks test (median (25 th ; 75 th percentile), P < 0.05). Results: Overall the frequency of postoperative cerebral ME rose to 600% of preoperative values. Primarily gaseous ME occurred preoperatively and postoperatively [19 (6; 63) vs 116 (24; 373), P < 0.001], while the number of solid ME was negligibly small [1 (0; 2) vs 2 (0; 6), P < 0.001]. CBFV and cerebral tissue oxygen index remained unaltered bilaterally before and after surgery. Conclusions: Our findings indicate that cerebral ME considerably increase after major orthopaedic surgery under general anaesthesia. The predominant accumulation of gaseous ME and their preoperative occurrence, suggest that the general anaesthesia and individual patient factors may contribute to the embolic load in addition to orthopaedic surgery. Clinical trial registration: . NCT02340416.

Performance of automated multiplex polymerase chain reaction (mPCR) using synovial fluid in the diagnosis of native joint septic arthritis in adults.

AIMS: This study aimed to assess the performance of an automated multiplex polymerase chain reaction (mPCR) technique for rapid diagnosis of native joint septic arthritis. PATIENTS AND METHODS: Consecutive patients with suspected septic arthritis undergoing aseptic diagnostic joint aspiration were included. The aspirate was used for analysis by mPCR and conventional microbiological analysis. A joint was classed as septic according to modified Newman criteria. Based on receiver operating characteristic (ROC) analysis, the area under the ROC curve (AUC) values of the mPCR and the synovial fluid culture were compared using the z-test. A total of 72 out of 76 consecutive patients (33 women, 39 men; mean age 64 years (22 to 92)) with suspected septic arthritis were included in this study. RESULTS: Of 72 patients, 42 (58%) were deemed to have septic joints. The sensitivity of mPCR and synovial fluid culture was 38% and 29%, respectively. No significant differences were found between the AUCs of both techniques (p = 0.138). A strong concordance of 89% (Cohen's kappa: 0.65) was shown. The mPCR failed to detect Staphylococcus aureus (n = 1) and Streptococcus pneumoniae (n = 1; no primer included in the mPCR), whereas the synovial fluid culture missed six microorganisms (positive mPCR: S. aureus (n = 2), Cutibacterium acnes (n = 3), coagulase-negative staphylococci (n = 2)). CONCLUSION: The automated mPCR showed at least a similar performance to the synovial fluid culture (the current benchmark) in diagnosing septic arthritis, having the great advantage of a shorter turnaround time (within five hours). Cite this article: Bone Joint J 2019;101-B:288-296.

Qualitative α-defensin test (Synovasure) for the diagnosis of periprosthetic infection in revision total joint arthroplasty.

AIMS: The diagnosis of periprosthetic joint infection (PJI) remains demanding due to limitations of all the available diagnostic tests. The synovial fluid marker, α-defensin, is a promising adjunct for the assessment of potential PJI. The purpose of this study was to investigate the qualitative assessment of α-defensin, using Synovasure to detect or exclude periprosthetic infection in total joint arthroplasty. PATIENTS AND METHODS: We studied 50 patients (28 women, 22 men, mean age 65 years; 20 to 89) with a clinical indication for revision arthroplasty who met the inclusion criteria of this prospective diagnostic study. The presence of α-defensin was determined using the qualitative Synovasure test and compared with standard diagnostic methods for PJI. Based on modified Musculoskeletal Infection Society (MSIS) criteria, 13 cases were categorised as septic and 36 as aseptic revisions. One test was inconclusive. RESULTS: The Synovasure test achieved a sensitivity of 69% and a specificity of 94%. The positive and negative likelihood ratios were 12.46 and 0.33, respectively. A good diagnostic accuracy for PJI, with an area under the curve of 0.82, was demonstrated. Adjusted p-values using the method of Hochberg showed that Synovasure is as good at diagnosing PJI as histology (p = 0.0042) and bacteriology with one positive culture (p = 0.0327). CONCLUSION: With its ease of use and rapid results after approximately ten minutes, Synovasure may be a useful adjunct in the diagnosis of PJI. Cite this article: Bone Joint J 2017;99-B:66-72.

Anticancer effects of zoledronic acid against human osteosarcoma cells.

Based on neoadjuvant chemotherapy, the prognosis of osteosarcoma patients has improved dramatically. However, due to therapy resistance in patient subgroups, the development of new treatment strategies is still of utmost importance. The aim of our study was to test the effects of the nitrogen-containing bisphosphonate zoledronic acid (ZOL) on osteosarcoma cell lines (N = 9). Exposure to ZOL at low micromolar concentrations induced a dose- and time-dependent block of DNA synthesis and cell cycle progression followed by microfilament breakdown and apoptosis induction. The ZOL-induced cell cycle accumulation in S phase was accompanied by significant changes in the expression of cyclins and cyclin-dependent kinase inhibitors with a prominent loss of cyclin E and D1. ZOL not only inhibited growth but also migration of osteosarcoma cells. The mevalonate pathway intermediary geranyl-geraniol (GGOH) but not farnesol (FOH) significantly inhibited the anticancer effects of ZOL against osteosarcoma cells. Correspondingly, ZOL sensitivity correlated with the blockade of protein geranylgeranylation indicated by unprenylated Rap1. Overexpression of even high levels of P-glycoprotein, as frequently present in therapy-resistant osteosarcomas, did not impair the anticancer activity of ZOL. Summarizing, our data suggest that ZOL, which selectively accumulates in the bone, represents a promising agent to improve osteosarcoma therapy.

Review of the outcomes of complex acetabular reconstructions using a stemmed acetabular pedestal component.

AIM: Until now, there has been no consensus as to whether stemmed acetabular components are appropriate for use in patients undergoing revision total hip arthroplasty (THA) who have major acetabular defects or pelvic discontinuity. We wished to address this deficiency in the literature. PATIENTS AND METHODS: We carried out a retrospective study of 35 patients (six men and 29 women) with a mean age of 68 years (37 to 87), with major acetabular defects who underwent revision THA between 2000 and 2012. RESULTS: At a mean follow-up of 63 months (24 to 141), a total of 15 patients (43%) had required at least one further operation. Six implants (17%) loosened aseptically, four (11%) were further revised for infection and two (6%) for technical failure. By taking revision for any reason as the endpoint, the rate of survival of the implant was 61% after five years; by taking revision for aseptic loosening as the end point, it was 78%. The cumulative five-year survival for aseptic loosening was 94% in patients without pelvic discontinuity, and 56% in those with pelvic discontinuity. CONCLUSION: These results indicate a significantly worse survival in patients with pelvic discontinuity (p = 0.020) and we advise caution in the use of the pedestal component in patients with major acetabular defects and pelvic discontinuity who require revision THA. As a result of these findings we have stopped using this implant and prefer to use particulate bone grafts protected with an anti-protrusion cage and posterior column plate fixation, if necessary. TAKE HOME MESSAGE: Based on these poor results, we advise caution if using the pedestal component in patients with major acetabular defects with the presence of a pelvic discontinuity. Cite this article: Bone Joint J 2016;98-B:772-9.

The Impact of Personality Traits on the Outcome of Total Knee Arthroplasty.

Ten to twenty percent of patients with total knee arthroplasty (TKA) are dissatisfied with their clinical outcome. Aim of this study was to investigate the impact of personality traits on the subjective outcome of TKA. We investigated 80 patients with 86 computer navigated TKAs. We asked for patients satisfaction and divided patients into two groups (satisfied or dissatisfied). 12 personality traits were tested by the Freiburg Personality Inventory (FPI-R). Postoperative examination included Knee Society Score (KSS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC), and the Visual Analogue Scale (VAS). Radiologic investigation was done in all patients. 84% of our patients were satisfied, while 16% were not satisfied. The FPI-R showed statistical significant influence of four personality traits on patient satisfaction: life satisfaction (p = 0.006), performance orientation (p = 0.015), somatic distress (p = 0.001), and emotional stability (p = 0.002). All clinical scores (VAS, WOMAC, and KSS) showed significantly better results in the satisfied patient. Radiological examination showed optimal alignment of all TKAs. There were no complications requiring revision surgery. The results of our study show that personality traits may influence patients satisfaction and clinical outcome after TKA. Therefore patients personality traits may be a useful predictive factor for postoperative satisfaction after TKA.

Effect of granulocyte-macrophage colony-stimulating factor on natural-killer cell mediated cytotoxicity.

Previous studies demonstrated an enhancing effect of granulocyte-macrophage colony-stimulating-factor (GM-CSF) on natural cytotoxicity. It was the aim of this study to investigate if CD56(+) natural-killer (NK) cells are responsible for the increased natural cytotoxicity after GM-CSF treatment. NK-cells were incubated with or without GM-CSF and Interferon-alpha (IFN-alpha) at various concentrations. NK-activity was determined by their ability to lyse NK-sensitive tumor cells (K562) and by cell surface expression of activation markers (CD25 and CD69). In our experimental setting incubation of CD56(+) NK-cells with GM-CSF did not significantly alter NK-cell mediated cytotoxicity or the expression of activation markers. In contrast, pre-treatment with IFN-alpha, a well known stimulant of NK-activity enhanced cytotoxicity by 69.2%+/-13.2%, P<0.05, effector/target cell ratio (E/T) 10:1 and by 43.3%+/-17.3%, P<0.05, E/T 20:1 and increased the expression of CD69 and CD25. Our results suggest that GM-CSF treatment alone cannot enhance natural cytotoxicity mediated by CD56(+) NK-cells in vitro.

Hyperthermia increases the susceptibility of chondro- and osteosarcoma cells to natural killer cell-mediated lysis.

As an adjuvant to chemotherapy hyperthermia has proven to be successful as a treatment for osteo- and chondrosarcoma patients. The aim of this study was to investigate whether hyperthermia could increase cellular expression of heat-shock-protein 72 in human osteo- and chondrosarcoma cells and how heat treatment would affect their susceptibility to natural killer cell (NK-cell)-mediated lysis. About 5-10% of the peripheral mononuclear blood cells (PBMC) in the human peripheral blood are natural killer cells (NK-cells). Natural cytotoxicity, mediated by NK-cells, is believed to play an important role in host defense against cancer. The exact mechanisms of recognition of target cells and subsequent NK-cell activation are not yet known. NK-cells, isolated from the peripheral blood of healthy donors, were enriched by magnetic cell-separation to a purity of 85-97%, assessed by FACS-analysis. The susceptibility of heat-treated (42.5 degrees C, 90 minutes) and untreated osteosarcoma (MG63) and chondrosarcoma (HTB94) cell lines to NK-killing was determined by a release assay of lactate dehydrogenase (LDH). Lysis by NK-cells was increased by heat treatment of the target cells from 16.6% + 4.5% to 33% + 15%, p=0.035, for osteosarcoma cells, (E/T ratio of 5:1) and from 13.7% + 3.1% to 27.9% + 16.9%, p=0.021, (E/T ratio of 20:1) for chondrosarcoma cells. An increased expression of HSP72 of chondro- and osteosarcoma cells after heat treatment was detected by the Western blot technique. The results of this study show that hyperthermia increases HSP72 expression in osteo- and chondrosarcoma cells and their susceptibility to NK-cell-mediated lysis. These findings may lead to new therapeutic strategies, using hyperthermia to improve immunological defense against chondro- and osteosarcoma cells.

Primary cementless total hip arthroplasty with second-generation metal-on-metal bearings: a concise follow-up, at a minimum of seventeen years, of a previous report.

BACKGROUND: Second-generation, metal-on-metal bearings were introduced in 1988, to reduce wear and avoid polyethylene particle-induced osteolysis from total hip arthroplasty. In 2007, we reported the long-term results of ninety-eight patients (105 hips) who underwent primary cementless total hip arthroplasty involving the use of a prosthesis with a high-carbide-concentration, metal-on-metal articulating surface between November 1992 and May 1994. The present study gives an update on this patient cohort. METHODS: At a minimum of seventeen years postoperatively, forty-nine patients (fifty-two hips) were available for follow-up examination. We retrospectively evaluated clinical and radiographic results as well as serum metal concentration. The mean patient age at the time of the index arthroplasty was fifty-six years. RESULTS: Three cups (6% of the hips) and one stem (2% of the hips) were revised because of aseptic loosening of the implants combined with focal osteolysis. At the time of the latest follow-up evaluation, the mean Harris hip score was 88.8 points, and the mean University of California Los Angeles (UCLA) activity score was 6.7 points. The cumulative rate of implant survival, with aseptic failure as the end point, was 93.0% at 18.8 years. The median serum cobalt concentration in patients whose hip implant was the only source of cobalt was 0.70 µg/L (range, 0.4 to 5.1 µg/L), showing no increase in the value as noted at a minimum of ten years of follow-up. CONCLUSIONS: The clinical and radiographic results of our study, which, to our knowledge, represent the longest duration of follow-up for a series of cementless total hip arthroplasties with use of a 28-mm metal-on-metal bearing, continue to be comparable with the results observed for other hard-on-hard bearings.

Effects of selenium coating of orthopaedic implant surfaces on bacterial adherence and osteoblastic cell growth.

The aim of this study was to evaluate whether coating titanium discs with selenium in the form of sodium selenite decreased bacterial adhesion of Staphylococcus aureus and Staph. epidermidis and impeded osteoblastic cell growth. In order to evaluate bacterial adhesion, sterile titanium discs were coated with increasing concentrations of selenium and incubated with bacterial solutions of Staph. aureus (ATCC 29213) and Staph. epidermidis (DSM 3269) and stained with Safranin-O. The effect of selenium on osteoblastic cell growth was also observed. The adherence of MG-63 cells on the coated discs was detected by staining with Safranin-O. The proportion of covered area was calculated with imaging software. The tested Staph. aureus strain showed a significantly reduced attachment on titanium discs with 0.5% (p = 0.011) and 0.2% (p = 0.02) selenium coating. Our test strain from Staph. epidermidis showed a highly significant reduction in bacterial adherence on discs coated with 0.5% (p = 0.0099) and 0.2% (p = 0.002) selenium solution. There was no inhibitory effect of the selenium coating on the osteoblastic cell growth. Selenium coating is a promising method to reduce bacterial attachment on prosthetic material.

The effect of a flexible pylon system on functional mobility of transtibial amputees. A prospective randomized study.

BACKGROUND: Prosthetic component selection strongly influences the functional mobility of transtibial amputees. Until now, little attention has been paid to the connection between the prosthetic socket and the foot component. AIM AND DESIGN: Aim of this study was to compare a novel flexible with a conventional rigid pylon system in a prospective randomized trial in transtibial amputees to determine effects on gait and mobility. SETTING: The study was performed in a rehabilitation centre for geriatric amputees. POPULATION: Twenty-eight geriatric unilateral transtibial amputees (8 female, 20 male) with low degree of mobility were included. METHODS: At admission to the rehabilitation centre objective and subjective parameters were assessed including demographic data and levels of daily activity using standardized questionnaires. Three months after prosthetic fitting a follow-up was performed and additional parameters such as walking speed, step length and vertical ground reaction forces were examined using a dynamic gait analysis. RESULTS: The study revealed a significant improvement in step length (P=0.03), in using mobility aids (P=0.04) and benefits concerning the self-selected gait speed, the mobility and gait disorders using the flexible device. CONCLUSION: The results demonstrate advantages for the flexible system, resulting in a positive effect on the patient's gait accomplishments in a geriatric population. CLINICAL REHABILITATION IMPACT: The advantages of the dynamic pylon may improve clinical rehabilitation, especially for geriatric patients with vascular disorders, as it provides comfort and better prosthetic performance during gait as a short-term benefit and thus gives a better quality of life in the long-term.

Livin and Bcl-2 expression in high-grade osteosarcoma.

AIMS: The evaluation of prognosis in patients with osteosarcoma is limited to clinical parameters. Although numerous molecular markers have been studied, none are currently in routine clinical use. The aim of this study was to determine if Livin and Bcl-2, acting as antiapoptotic proteins through different mechanisms, are expressed in osteosarcoma, and whether they can be used as prognostic markers in human osteosarcoma. METHODS: Tumor specimens of 29 patients with high-grade central osteosarcoma, with complete clinical follow-up for a minimum of 5 years, were studied. The localization and distribution of Livin and Bcl-2 were investigated using immunohistochemistry. Results were correlated with the histological response to chemotherapy, 5-year disease-free and 5-year overall survival. RESULTS: Bcl-2 was expressed only in the cytoplasm of 16/29 cases and there was no statistically significant correlation between expression and any of the studied parameters. Livin was detected in 17/29 cases, in the cytoplasm of all 17 and in the nucleus of only 3 cases. Nuclear expression was significantly correlated with a decreased overall survival (P < 0.0002) compared with those patients without nuclear expression. CONCLUSIONS: The results of this study indicate that Bc1-2 and Livin are highly expressed in osteosarcoma cells and that possibly, the evaluation of nuclear Livin expression might be a useful prognostic marker in osteosarcoma.

Effects of hyperthermia on heat shock protein expression, alkaline phosphatase activity and proliferation in human osteosarcoma cells.

Hyperthermia can be used as a possible adjuvant therapy in treatment of cancer patients. In this study, the direct effect of hyperthermia on osteosarcoma derived cell lines HOS85, MG-63 and SaOS-2 was investigated. Heat shock at 42 degrees C inhibited proliferation significantly in all three cell lines tested. Furthermore a sub-lethal heat shock (42 degrees C, 1 h) decreases alkaline phosphatase activity, the absolute marker for osteoblast-like cells, in all of the three cell lines. Hsp70 was expressed constitutively and was found to be upregulated in a time-dependent manner; by up to 150% in Western blot analysis. The results of this study indicate that heat shock has an inhibitory effect on human osteosarcoma cells. These data suggest that hyperthermia has an anti-tumour effect on cancers of the bone and might, therefore, become an adjuvant treatment option.

Effects of extracorporeal shock waves on human articular chondrocytes and ovine bone marrow stromal cells in vitro.

INTRODUCTION: We investigated the effects of extracorporeal shock waves on cytotoxicity and on the proliferation of human chondrocytes and ovine bone marrow stromal cells. MATERIALS AND METHODS: Isolated cells were cultured to confluence, and 500 shock waves were applied at energy flux densities of 0, 0.02, 0.06, and 0.17 mJ/mm(2 )for the cytotoxicity assay. The same energies at 100, 500, and 1000 impulses were used for the proliferation assay. RESULTS: Although bone marrow stromal cells revealed a dose- and impulse-dependent increase in the proliferation rate, no significant differences were found. Chondrocytes had less proliferative potential than untreated control groups. In the experimental set-up using 1000 impulses, proliferation was even higher in the control group. Both types of cells revealed a dose-dependent increase in cytotoxicity in the lactate dehydrogenase (LDH) assay. CONCLUSION: As femoral head necrosis, osteochondritis dissecans, and similar disorders are increasingly treated with shock waves, their effect on human cartilage and chondrocytes deserves attention. We recommend further in vitro experiments with bone marrow stromal cells, as the latter might play an important role in the presumed multifactorial osteogenetic mechanism of shock waves due to their pluripotent character.