High frequency of BCP, but less CPP crystal-mediated calcification in cartilage and synovial membrane of osteoarthritis patients.

OBJECTIVE: Ectopic articular calcification is a common phenomenon of osteoarthritic joints, and closely related to disease progression. Identification of the involved calcium crystal types represents an important topic in research and clinical practice. Difficulties in accurate detection and crystal type identification have led to inconsistent data on the prevalence and spatial distribution of Basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) deposition. METHOD: Combining multiple imaging methods including conventional radiography, histology and Raman spectroscopy, this study provides a comprehensive analysis of BCP and CPP-based calcification, its frequency and distribution in cartilage and synovial membrane samples of 92 osteoarthritis patients undergoing knee replacement surgery. RESULTS: Conventional radiography showed calcifications in 35% of patients. Von Kossa staining detected calcified deposits in 88% and 57% of cartilage and synovial samples, respectively. BCP crystals presented as brittle deposits on top of the cartilage surface or embedded in synovial tissue. CPP deposits appeared as larger granular needle-shaped clusters or dense circular pockets below the cartilage surface or within synovial tissue. Spectroscopic analysis detected BCP crystals in 75% of cartilage and 43% of synovial samples. CPP deposition was only detected in 18% of cartilage and 15% of synovial samples, often coinciding with BCP deposits. CONCLUSION: BCP is the predominant crystal type in calcified cartilage and synovium while CPP deposition is rare, often coinciding with BCP. Distinct and qualitative information on BCP and CPP deposits in joint tissues gives rise to the speculation that different disease entities are involved that might need different treatment strategies.

Collagen 1 gel may improve the regenerative capacity of minced adult and preosteoarthritic cartilage.

PURPOSE: Minced cartilage implantation (MCI) is an evolving technique for the treatment of osteochondral lesions. It was hypothesised that mincing of cartilage may affect chondrocyte viability and phenotype and that embedding in collagen 1 gel results in an improved outcome. The objective of this study was to evaluate the impact of cartilage mincing and whether collagen 1 gel mediates beneficial effects on the chondrocyte phenotype and viability. METHODS: Human cartilage samples from 11 patients undergoing total knee arthroplasty were collected and minced according to the MCI protocol. Minced cartilage was cultured for 1 week with and without embedding in collagen 1 gel and was compared with unminced cartilage flakes as control. Quantitative reverse transcription-PCR and immunohistochemical staining for the chondrocyte marker genes SOX9, COL2, ACAN, COL10 and MMP13 were used to examine the chondrocyte phenotype. Cell death was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. RESULTS: Increased chondrocyte cell death of cultured cartilage after mincing was observed. Chondrocytes from minced cartilage exhibited significantly decreased expression and protein levels of homeostatic and hypertrophic chondrocyte markers. Embedding in collagen 1 gel showed no positive effect on viability. However, remarkable is the increased expression of ACAN and the preserved protein level of SOX9 in the collagen 1-embedded minced cartilage. CONCLUSIONS: This study shows that the mincing of cartilage leads to increased chondrocyte death and decreased expression of chondrocyte phenotypic marker genes after 7 days. The use of collagen 1 gel may improve the stability of the phenotype, which needs to be further elucidated. LEVEL OF EVIDENCE: Level III (therapeutic).

Loose Bodies Found in the Human Intra-Articular Space Showed Characteristics Similar to Endochondral Bone Formation.

OBJECTIVE: Loose bodies are free-floating tissues of cartilage and bone that can cause pain, swelling, the inability to straighten the knee, or intermittent locking of the knee. Loose bodies can arise from degenerative joint disease, flake fractures, osteochondritis dissecans, or chondromatosis. We hypothesized that loose bodies can be classified in stages with tissue characteristics similar to endochondral ossification. DESIGN: Loose bodies were harvested from patients undergoing joint replacement. Samples were processed for histology, gene expression analysis, and micro-computed tomography (µCT). Cartilage- and bone-related genes and proteins were selected for immunofluorescence stainings (collagen type I, II, and X, SOX9 [SRY-box transcription factor 9], and MMP13 [matrix metalloproteinase 13]) and gene expression analysis (FN [fibronectin], COL1A1, COL2A1, COL10A1, SOX9, MMP13, and aggrecan [ACAN]). RESULTS: Loose bodies were grouped in 4 stages: fibrous, (mineralized) cartilaginous, cartilage and bone, and bone. Hyaline-like cartilage tissue with Benninghoff arcades was present in stages 2 and 3. A transition from cartilaginous to mineralized tissue and bone trabecula was defined by an increase in COL1A1 and COL10A1 (stage 3 vs. 4: p = 0.047) positive area. Stage 4 showed typical trabecular bone tissue. The relative volume of calcified tissue (mineralized cartilage and bone tissue) decreased with stages (stages 1-2 vs. 3: p = 0.002; stage 1-2 vs. 4: p = 0.012). COL2A1 expression and stained area decreased from stages 1-2 to 4 (p = 0.010 and p = 0.004). ACAN expression decreased from stage 1-2 to stage 3 (p = 0.049) and stage 4 (p = 0.002). CONCLUSION: Loose bodies show tissue characteristics similar to endochondral ossification. They are probably a relevant substrate for regenerative therapeutic interventions in joint disease.

Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles.

Calcified aortic valve disease in its final stage leads to aortic valve stenosis, limiting cardiac function. To date, surgical intervention is the only option for treating calcific aortic valve stenosis. This study combined controlled drug delivery by nanoparticles (NPs) and active targeting by antibody conjugation. The chelating agent diethylenetriaminepentaacetic acid (DTPA) was covalently bound to human serum albumin (HSA)-based NP, and the NP surface was modified using conjugating antibodies (anti-elastin or isotype IgG control). Calcification was induced ex vivo in porcine aortic valves by preincubation in an osteogenic medium containing 2.5 mM sodium phosphate for five days. Valve calcifications mainly consisted of basic calcium phosphate crystals. Calcifications were effectively resolved by adding 1-5 mg DTPA/mL medium. Incubation with pure DTPA, however, was associated with a loss of cellular viability. Reversal of calcifications was also achieved with DTPA-coupled anti-elastin-targeted NPs containing 1 mg DTPA equivalent. The addition of these NPs to the conditioned media resulted in significant regression of the valve calcifications compared to that in the IgG-NP control without affecting cellular viability. These results represent a step further toward the development of targeted nanoparticular formulations to dissolve aortic valve calcifications.

Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression.

Selective Internal Radiotherapy Alters the Profiles of Systemic Extracellular Vesicles in Hepatocellular Carcinoma.

Incidence of hepatocellular carcinoma (HCC) is increasing globally. Radioembolization (RE)/selective internal radiotherapy (SIRT) is a promising treatment for inoperable HCC. RE triggers an immune response, involving extracellular vesicles (EVs) which are crucial for cell communication and tumor development. This study explores EV immune profiles and origins in patients with inoperable HCC before and after SIRT/RE. Blood samples from 50 HCC-patients treated with SIRT/RE were collected before and after therapy to determine cytokines and isolate EVs using size exclusion chromatography. The dynamic range and EV quality required for detecting variations in surface markers were assessed. Thirty-seven EV surface markers were analyzed using flow cytometry and correlated with clinical parameters. Several immunological markers (CD4, CD2, CD40, CD45, CD49e, CD69, CD209-EVs) were present in the circulation of HCC patients. These markers positively correlated with therapy response and survival. Conversely, B cell CD20, endothelial cell CD146, platelet CD49e, and CD41b EV markers negatively correlated with 60-day survival. Elevated levels of IL-6 and IL-8 before therapy correlated negatively with patient survival, coinciding with a positive correlation with CD20-positive EVs. Plasma EVs from HCC patients exhibit immunological, cancer, and coagulation markers, including potential biomarkers (CD4, CD20, CD49e, CD146). These may enhance our understanding of cancer biology and facilitate SIRT therapy monitoring.

In vivo corrosion on retrieved hip endoprostheses and in vitro effects of corrosion products on bone mineralization.

In vivo corrosion of modular endoprostheses remains a great concern, as the release of heavy metal ions can impair the implant's service life and the wellbeing of the patient. The detailed corrosion mechanisms that occur in vivo are so far not completely understood. In this context, the effects of implant released cobalt (Co) and chromium (Cr) ions on osteoblast mineralization and gene expression have not been investigated extensively. This comprehensive study aimed at furthering the understanding of in vivo implant corrosion from the clinical signs via prosthesis retrievals and histology of the synovial membranes down to the molecular processes instigated by corrosion products and its effects on bone mineralization. A detailed in vivo failure analysis was performed investigating 22 retrieved hip endoprostheses from different manufacturers and taper material combinations. The aim was to find a correlation of taper damage and especially corrosion to susceptible biomedical alloys and its effect on periprosthetic tissue as well as the clinical implant performance with regard to revision diagnosis and presence of radiolucent lines (RLL). A second part investigated the effects of Co and Cr ions on the in vitro mineralization process of osteoblasts. Cell cultures were exposed to relevant concentrations of CoCl2 and CrCl3 (0 µM, 100 µM, 200 µM) with and without addition of phosphate. Mineralization behavior was analyzed with Alizarin Red assay and Von Kossa staining of calcium depots, alkaline phosphatase activity of osteoblasts and gene expression was analyzed with real time quantitative PCR. The retrieval study provides evidence of in vivo fretting and crevice corrosion on all metallic tapers combined with either ceramic or metal femoral heads. Within the modular taper junctions, selective dissolution of the α phase occurred in wrought TiAl6V4 alloys, and etching of the fine-grained wrought CoCr28Mo6 alloy implants was observed in formed crevices. In addition, significant amounts of wear particles and corrosion products were detected in retrieved synovial membranes. An increased risk for the occurrence of a RLL in the proximal zones was determined for patients with a corroded mixed metal taper. Whereas Co ions have hardly any effects on mineralization, Cr ions cause a significant concentration dependent decrease in mineralization rate of osteoblasts. However, this effect is alleviated by addition of a phosphate source. Our data reveal that Cr ions depleted dissolved phosphates by forming an insoluble complex (CrPO4), which inhibits the phosphate dependent mineralization process. No significant effect of the heavy metal ions on osteoblast activity by means of alkaline phosphate activity as well as on gene expression is determined. This study broadens the understanding of in vivo corrosion of metallic modular implants and its clinically relevant effects on mineralization. Based on these findings, in vivo corrosion of CoCr28Mo6 endoprostheses should be limited to avoid inhibitory effects of Cr3+ on bone mineralization which can contribute to premature implant failure.

Vibro-acoustic sensing of tissue-instrument-interactions allows a differentiation of biological tissue in computerised palpation.

BACKGROUND: The shift towards minimally invasive surgery is associated with a significant reduction of tactile information available to the surgeon, with compensation strategies ranging from vision-based techniques to the integration of sensing concepts into surgical instruments. Tactile information is vital for palpation tasks such as the differentiation of tissues or the characterisation of surfaces. This work investigates a new sensing approach to derive palpation-related information from vibration signals originating from instrument-tissue-interactions. METHODS: We conducted a feasibility study to differentiate three non-animal and three animal tissue specimens based on palpation of the surface. A sensor configuration was mounted at the proximal end of a standard instrument opposite the tissue-interaction point. Vibro-acoustic signals of 1680 palpation events were acquired, and the time-varying spectrum was computed using Continuous-Wavelet-Transformation. For validation, nine spectral energy-related features were calculated for a subsequent classification using linear Support Vector Machine and k-Nearest-Neighbor. RESULTS: Indicators derived from the vibration signal are highly stable in a set of palpations belonging to the same tissue specimen, regardless of the palpating subject. Differences in the surface texture of the tissue specimens reflect in those indicators and can serve as a basis for differentiation. The classification following a supervised learning approach shows an accuracy of >93.8% for the three-tissue classification tasks and decreases to 78.8% for a combination of all six tissues. CONCLUSIONS: Simple features derived from the vibro-acoustic signals facilitate the differentiation between biological tissues, showing the potential of the presented approach to provide information related to the interacting tissue. The results encourage further investigation of a yet little-exploited source of information in minimally invasive surgery.

Injection of autologous conditioned plasma combined with a collagen scaffold may improve the clinical outcome in shoulder impingement syndrome: a prospective study.

BACKGROUND: Shoulder impingement syndrome (SIS) is one of the most common diseases of the shoulder and can be addressed with various therapeutic concepts. Orthobiological agents such as platelet rich plasma with a low side effect rate gain importance in the conservative treatment of SIS. Currently, the knowledge about success rate influencing factors, such as the growth factors (GF) concentration or acromion type, is limited. The aim of this study was to analyze the clinical outcome in the therapy of external SIS using autologous conditioned plasma combined with recombinant human collagen scaffold (ACP/STR) injection in comparison with a corticosteroid-local anesthetic (CSA) injection. Additionally, the influence of potential limiting factors such as GF concentration, age and acromial morphology was proved. MATERIALS AND METHODS: This prospective pseudo-randomized trial recruited 58 patients with external SIS who received an ultrasound-guided subacromial injection either an ACP/STR or a CSA followed by physical therapy. Follow-up (FU) was performed at 6 weeks, 3 and 6 months. The outcome was assessed with Constant-Murley score, disability of arm, shoulder and hand score and simple shoulder test. The concentration of GF was measured using ELISA. RESULTS: During the FU, the improvement of outcome measures was observed with no differences between both groups. Shoulder force was significantly increased in the ACP/STR group (p < 0.01). We found no correlation between the amount of GF and age or gender in the ACP/STR patients. An acromion Bigliani type III predisposes for therapy failure (p < 0.001, OR = 56) in both treatment groups. CONCLUSIONS: Patients with SIS benefit regarding to PROMs after both ACP/STR and CSA injection and physical therapy. Patients who received ACP/STR obtained superior improvement in force. The quantity of GF did not vary depending on the age, so that ACP/STR can be a treatment option for SIS in elderly patients with multimorbidity. The presence of an acromion type III seems to be a predictive factor for limited effectivity of injections in the clinical management of SIS.

CoNiCrMo Particles, but Not TiAlV Particles, Activate the NLRP3 Inflammasome in Periprosthetic Cells.

Aseptic loosening is the main reason for arthroplasty failure. The wear particles generated at the tribological bearings are thought to induce an inflammatory tissue response, leading to bone loss and the subsequent loosening of the implant. Different wear particles have been shown to activate the inflammasome, thereby contributing to an inflammatory milieu in the direct vicinity of the implant. The aim of this study was to investigate whether the NLRP3 inflammasome is activated by different metal particles in vitro and in vivo. Three different cell lines representing periprosthetic cell subsets (MM6, MG63 and Jurkat) were incubated with different amounts of TiAlV or CoNiCrMo particles. The activation of the NLRP3 inflammasome was determined through the detection of the caspase 1 cleavage product p20 in a Western blot. The formation of the inflammasome was also investigated in vivo using immunohistological staining for ASC in primary synovial tissues as well as tissues containing TiAlV and CoCrMo particles and in vitro after the stimulation of the cells. The results show that the CoCrMo particles induced ASC more markedly, as a readout for inflammasome formation in vivo, compared to TiAlV particular wear. The CoNiCrMo particles also induced ASC-speck formation in all the tested cell lines, which was not induced by the TiAlV particles. The Western blot shows that NRLP3 inflammasome activation, measured through caspase 1 cleavage, was increased only by the CoNiCrMo particles in the MG63 cells. We conclude from our data that the activation of the inflammasome is mainly driven by CoNiCrMo particles and less by TiAlV particles, indicating that different inflammatory pathways are activated by the different alloys.

C9 immunostaining as a tissue biomarker for periprosthetic joint infection diagnosis.

Background: Culture-negative periprosthetic joint infections (PJI) are often false diagnosed as aseptic implant failure leading to unnecessary revision surgeries due to repeated infections. A marker to increase the security of e PJI diagnosis is therefore of great importance. The aim of this study was to test C9 immunostaining of periprosthetic tissue as a novel tissue-biomarker for a more reliable identification of PJI, as well as potential cross-reactivity. Method: We included 98 patients in this study undergoing septic or aseptic revision surgeries. Standard microbiological diagnosis was performed in all cases for classification of patients. Serum parameters including C-reactive protein (CRP) serum levels and white blood cell (WBC) count were included, and the periprosthetic tissue was immunostained for C9 presence. The amount of C9 tissue staining was evaluated in septic versus aseptic tissue and the amount of C9 staining was correlated with the different pathogens causing the infection. To exclude cross-reactions between C9 immunostaining and other inflammatory joint conditions, we included tissue samples of a separate cohort with rheumatoid arthritis, wear particles and chondrocalcinosis. Results: The microbiological diagnosis detected PJI in 58 patients; the remaining 40 patients were classified as aseptic. Serum CRP values were significantly increased in the PJI cohort. Serum WBC was not different between septic and aseptic cases. We found a significant increase in C9 immunostaining in the PJI periprosthetic tissue. To test the predictive value of C9 as biomarker for PJI we performed a ROC analyses. According to the Youden's criteria C9 is a very good biomarker for PJI detection with a sensitivity of 89% and a specificity of 75% and an AUC of 0.84. We did not observe a correlation of C9 staining with the pathogen causing the PJI. However, we observed a cross reactivity with the inflammatory joint disease like rheumatoid arthritis and different metal wear types. In addition, we did not observe a cross reactivity with chondrocalcinosis. Conclusion: Our study identifies C9 as a potential tissue-biomarker for the identification of PJI using immunohistological staining of tissue biopsies. The use of C9 staining could help to reduce the number of false negative diagnoses of PJI.

Hypersensitivity and lymphocyte activation after total hip arthroplasty.

In the last decades total hip arthroplasty (THA) has become a standard procedure with many benefits but also a few still unsolved complications, which can lead to surgical revision in 19-23% of cases. Thus, aseptic loosening and metal hypersensitivity remain challenges. The phenomenon of wear debris causes chronic inflammation, which produces osteolysis and aseptic loosening. Wear debris promotes osteoclast production and inhibits osteoblasts by secretion of pro-inflammatory cytokines. Micro-abrasions can be induced by abrasive, adhesive and fatigue wear and cause a liberation of metal ions, which lead to another immune response elicited mostly by macrophages. Another reaction in the neocapsule can be a type IV hypersensitivity reaction to various alloys, containing metals such as nickel, cobalt and chromium. Patch testing and the lymphocyte transformation test (LTT) are not the best diagnostic possibilities to exclude a postoperative hypersensitivity reaction, because of the different alignment of the epicutaneous cells compared to the periprosthetic deep tissue. This hypersensitivity reaction is mostly induced by cytokines, which are secreted by macrophages rather than lymphocytes. In cell cultures and in animal studies, multipotent mesenchymal stem cells (MSC) have been shown to play a role in improving initial implant integration, to limit periprosthetic osteolysis and also to reconstitute peri-implant bone stock during implant revision. Thus, MSC might be used in the future to prolong the durability of THA. A better understanding of the interactions between primary chronic inflammation, corrosion, osteolysis and hypersensitivity is mandatory to develop new therapeutic strategies, aiming at the reduction of the incidence of implant failures. In this article the underlying immunological mechanisms to aseptic loosening are presented.

Damage analysis of retrieved BioloxⓇdelta components used in hard and soft bearings.

This retrieval study included 43 Biolox delta explants (18 CoC, 25 CoP). Implants were examined macroscopically, whereby damage was evaluated using a semi quantitative scoring system. Confocal microscopy was used to examine wear related damage patterns of the articulating surfaces. Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) was used to analyze wear marks on the implant surface and wear debris in periprosthetic tissue samples. Raman spectroscopy and X-ray diffraction (XRD) were used to quantify monoclinic zirconia fractions. On all components, in vivo wear resulted predominantly in different damage patterns caused by metal transfer. In CoC bearings stripe wear was additionally detected, and some implants underwent severe damage due to component breakage. The wear scores were higher for CoC components, with no differences between the scores for CoC heads and liners. Wear features caused comparable roughening on implants from CoC and CoP bearings. SEM studies demonstrated that most wear marks were caused by metal debris released from implant components. Grain pull-out was observed in stripe wear regions. Monoclinic phase shift was observed in a similar quantity on components from CoP and CoC bearings. The increase of monoclinic zirconia content around metal deposits was minimal and was more pronounced in areas of stripe wear. The results of this study indicate, that ZTA components in general undergo minimal wear in both, CoC and CoP bearings, however, it is more pronounced in the former. Metal deposits, as the most common wear feature, have no significant effect on monoclinic phase transition. STATEMENT OF SIGNIFICANCE: In this paper, we classify all damage patterns macroscopically according to an established scoring system and assess them regarding surface roughness (confocal microscopy) and monoclinic phase content (Raman spectroscopy) in order to derive the severity for patients. We compare hard-hard and hard-soft bearings and relate damage patterns with metal transfer based on SEM/EDS examinations. Furthermore, we work out correlations between patient-specific data, cause of revision and the physical condition of each individual sample Our cohort consists of 43 Biolox delta retrievals, a comparatively large quantity. In addition, we address current topics such as metal transfer and, based on the classification of damage patterns, provide incentives and/or meaningful focal points for further research.

Surface integrity modification of CoCrMo alloy by deep rolling in combination with sub-zero cooling as potential implant application.

Alloys made of CoCrMo are well established as implants materials since decades in orthopedic surgery. The good mechanical properties, biocompatibility and especially the corrosion resistance are important rationales for the use of these alloys. Nevertheless, retrieved implants from revision surgery showed the occurrence of abrasion and corrosion. The wear mechanisms and the occurring corrosion processes might be reduced with a functionalization of the surface. The hexagonal phase of the cobalt chromium matrix plays an important role in the surface functionalization. It can be specifically transformed and set during the manufacturing process. One possibility for the induction of the transformation is the use of a deep rolling process in combination with a novel "sub-zero" cooling strategy during machining. The influence of force and temperature during the deep rolling process on the formation of the hexagonal Co-phase is examined in this study. The results from the targeted setting of the hexagonal Co-phase in the subsurface are shown. For this purpose, EBSD studies have been carried out to detect and quantify the proportion of Co-hex phase in the subsurface of the modified alloys. To analyze the mechanical properties, we measured the residual stress and hardness in the near surface layer under conditions close to the application. Furthermore, we performed biological tests to show a potential influence of the modification on the biocompatibility when using the sub-zero cooling approach. We observed no negative effect on the osteoblastic cell line which attached similarly to all tested surfaces. The investigations provide first insights into the potential use of "sub-zero" cooling in modifying orthopedic implant materials, but also the respective limits with regard to the surface functionalization. Deep rolling in combination with an innovative cooling strategy has a great potential to improve the mechanical properties of CoCr28Mo6 wrought alloy, by subsurface hardening and phase transformation.

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients' quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

Phenotypic Discovery of Triazolo[1,5-c]quinazolines as a First-In-Class Bone Morphogenetic Protein Amplifier Chemotype.

Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.

Interaction of Masitinib with Organic Cation Transporters.

Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into targeted cells by the action of specific transport proteins, as they are hydrophilic and not able to cross cell membranes freely. Accordingly, the efficacy of TKI in target cells is closely dependent on the expression of their transporters. Specifically, Masitinib is an organic cation and is expected to interact with organic cation transporters (OCT and Multidrug and Toxin Extrusion proteins-MATE-). The aim of this work was to characterize the interaction of Masitinib with different OCTs. Human embryonic kidney 293 cells stably transfected with murine or human OCT were used for the experiments. The interaction of Masitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of this drug was quantified using high performance liquid chromatography. Our results identified interactions of Masitinib with almost all investigated mouse (m) and human (h) OCTs and hMATE1 and indicated OCT1 and hOCT2 to be especially potent Masitinib translocators across cell membranes. Interestingly, some important differences were observed for the interaction with murine and human OCTs. In the future, investigations concerning further in vitro and in vivo properties of Masitinib and its efficacy related to transporter-related uptake mechanisms under pathophysiological conditions should be performed. Clinical trials in humans and other animals with Masitinib have already shown promising results. However, further research is necessary to understand the disease specific transport mechanisms of Masitinib to contribute to a successful and responsible therapy employment.

Microstructural Modification of TiAl6V4 Alloy to Avoid Detrimental Effects Due to Selective In Vivo Crevice Corrosion.

TiAl6V4 wrought alloy is a standard material used for endoprostheses due to its ideal characteristics in terms of osseointegration. However, the insufficient wear and crevice corrosion resistance of TiAl6V4 are limiting factors that can cause clinical problems. Therefore, the objective of this study was to analyze and identify suitable phases and microstructural states of TiAl6V4 alloy with advantageous implant properties by thermal treatments. By varying the temperature and cooling rate, four heat treatment strategies were derived that produced different microstructural states that differed in morphology, arrangement and proportions of phases present. All TiAl6V4 modifications were characterized regarding their microstructure, mechanical, corrosive and tribological properties, as well as cell adhesion. The acicular, martensitic microstructure achieves a significant hardness increase by up to 63% and exhibits improved corrosion and wear resistance compared to the forged condition. Whereas the modified microstructures showed similar electrochemical properties in polarization tests using different electrolytes (PBS with H2O2 and HCl additives), selective α or ß phase dissolution occurred under severe inflammatory crevice conditions after four weeks of exposure at 37 °C. The microstructurally selective corrosion processes resemble the damage patterns of retrieved Ti-based implants and provide a better understanding of clinically relevant in vivo crevice corrosion mechanisms. Furthermore, a microstructural effect on cell attachment was determined and is correlated to the size of the vanadium-rich ß phase. These key findings highlight the relevance of an adapted processing of TiAl6V4 alloy to increase the longevity of implants.

Selective Internal Radiotherapy Changes the Immune Profiles of Extracellular Vesicles and Their Immune Origin in Patients with Inoperable Cholangiocarcinoma.

The incidence of cholangiocellular carcinoma (CCA) is rising worldwide. As there are no specific early symptoms or specific markers of CCA, it is often diagnosed in later inoperable stages. Accumulating evidence underlines the importance of radiation therapy in the induction of antitumor immunity. The surface protein composition on extracellular vesicles (EVs) relates to originating cells and thus may play a role in vesicle function. We assessed immune profiles of EVs and their immune origin in patients with inoperable CCA prior and after selective internal radiotherapy (SIRT). A total of 47 CCA patients receiving SIRT and 12 healthy volunteers (HV) were included. Blood was withdrawn before therapy (pre T) and after T. EVs were purified from plasma by cluster of differentiation (CD)9-, CD63-, and CD81-immunobead isolation. To detect differently abundant surface markers, dynamic range and EVs input quality were assessed. A total of 37 EVs surface markers were measured by flow cytometry and correlated either with the administered activity dose (MBq) or with the interval until death (month). EVs phenotyping identified lymphocytes, B cells, NK cells, platelets, endothelial cells, leukocyte activation, B cell activation, T and B cell adhesion markers, stem/progenitor cells, and antigen-presenting cells (APC) as EVs-parenteral cells. CD4 and CD8 significantly declined, while other markers significantly increased in CCA patients pre T vs. HV. Platelets-deriving EVs significantly decreased, normalizing to levels of HV but still significantly increasing vs. HV post SIRT. B cells-deriving EVs significantly increased pre T vs. HV, positively correlating with administered activity dose. MHCII and CD40 EVs significantly increased pre SIRT and negatively correlated with administered activity dose, while EVs from antigen presenting cells and CD49e pre SIRT positively correlated with survival time after therapy. Increased levels of CD24 and CD44 in cancer pre T were significantly decreased post T. Among the heterogeneity of EVs that was demonstrated, in particular, B cells-deriving, MHCII, and CD40 positive or APC-deriving EVs need to be further studied for their diagnostic or prognostic relevance in clinical scenarios.