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Purpose: Periprosthetic joint infections (PJIs) are a very demanding complication of arthroplasty. Diagnosis of PJI and pathogen identification pose considerable challenges in clinical practice. We hypothesized that the pathogen-specific immune response to PJI reflects the infection process, provides clinically relevant information on disease course, and has the potential to further optimize antimicrobial therapy., Methods: We conducted a prospective matched cohort pilot study with 13 patients undergoing two-stage septic revision arthroplasty (PJI patients) between 06/2020 and 06/2021, as well as 11 control patients undergoing one-stage aseptic revision arthroplasty (Non-PJI patients). Pre-, intra- and postoperative serum samples were collected at standardized time points. We developed a custom Luminex®-based quantitative bead-based suspension array (Infection Array; IA), and used it for simultaneous measurement of antibody specificities against 32 pathogens commonly associated with PJI in 267 serum samples., Results: The IA was able to trace the dynamics of the pathogen-specific humoral immune response in all patients against PJI-related pathogens, prominently coagulase-negative staphylococci and streptococci. Pathogen-specific serum antibody titers declined in 62% of PJI patients over the course of treatment, while no changes in antibody titers were observed in 82% of Non-PJI patients during this study. Our serological data strongly suggested that antibody signatures reflect an immune response to microbial invasion., Conclusion: Our results provide insights into the pathophysiology of PJI and information on the individual disease courses. The IA is therefore a promising and novel serological tool of high resolution for monitoring the immunoproteomic footprints of infectious pathogens in the course of PJI., (© 2024. The Author(s).)

Primary leiomyosarcoma of bone (LMSoB) is extremely rare, comprising only <0.7% of primary malignant bone tumors, and is therefore considered an ultra-rare tumor entity. There is currently no consensus as to whether therapeutic strategies should be based on the biological characteristics of soft tissue leiomyosarcoma or on primary tumor localization in the bone. The use of perioperative chemotherapy and its effectiveness in this rare tumor entity remains unclear. We aimed to evaluate the impact of different treatment approaches in a multicenter setting with a total of 35 patients included. The 5-year overall survival (OS) was 74%. Patients with localized disease undergoing surgery had a significantly higher 5-year OS compared to patients who did not undergo surgical treatment (82% vs. 0%, p = 0.0015). Axial tumor localization was associated with worse event-free survival (EFS) probability ( p < 0.001) and OS ( p = 0.0082). A high proportion of our patients developed secondary metastases. Furthermore, the perioperative chemotherapy protocols applied to our patients were not associated with an improved EFS or OS. Therefore, the benefit of perioperative chemotherapy in LMSoB needs to be further investigated, and the choice of agents still needs to be clarified.

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research., (© 2024. The Author(s).)

The gadolinium-based contrast agent DOTA-Gd is clinically used in combination with local anesthetics for direct magnetic resonance arthrography. It remains unclear whether gadolinium uptake into cartilage is influenced by co-administration of bupivacaine or ropivacaine and whether DOTA-Gd alters their chondrotoxicity. Gadolinium quantification of chondrogenic spheroids revealed enhanced gadolinium uptake after simultaneous exposure to local anesthetics. Analyses of the spatial gadolinium distribution using synchrotron X-ray-fluorescence scanning indicates gadolinium exposed chondrocytes. In vitro exposure to DOTA-Gd does not alter viability and proliferation of human chondrocytes and the chondrotoxic potential of the anesthetics. Reduced viability induced by ropivacaine was found to be reversible, while exposure to bupivacaine leads to irreversible cell death. Our data suggest that ropivacaine is more tolerable than bupivacaine and that DOTA-Gd exposure does not alter the cytotoxicity of both anesthetics. Enhanced gadolinium uptake into cartilage due to co-administration of anesthetics should find attention., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Ewing's sarcoma (ES) is the second most common bone tumor in children and adolescents and is highly malignant. Although the new chemotherapy has significantly improved the survival rate for ES from about 10 to 75%, the survival rate for metastatic tumors remains around 30%. This treatment is often associated with various side effects that contribute to the suffering of the patients. Cold physical plasma (CPP), whether used alone or in combination with current chemotherapy, is considered a promising adjunctive tool in cancer treatment. This study aims to investigate the synergistic effects of CPP in combination with cytostatic chemotherapeutic agents that are not part of current ES therapy. Two different ES cell lines, RD-ES and A673, were treated with the determined IC 20 concentrations of the chemotherapeutic agents cisplatin and methotrexate (MTX) in combination with CPP. The effects on population doubling, cell viability, and apoptotic processes within these cell lines were assessed. This combination therapy has led to a reduction of population doubling and cell viability, as well as an increase in apoptotic activity in cells compared to CPP monotherapy. The results of this study provide evidence that combining CPP with non-common chemotherapy drugs such as MTX and CIS in the treatment of ES enhances the anticancer effects of these drugs. These findings open up new possibilities for the effective use of these drugs against ES., (© 2024. The Author(s).)

Cobalt-chromium-molybdenum (CoCrMo) alloys are routinely used in arthroplasty. CoCrMo wear particles and ions derived from arthroplasty implants lead to macrophage-driven adverse local tissue reactions, which have been linked to an increased risk of periprosthetic joint infection after revision arthroplasty. While metal-induced cytotoxicity is well characterized in human macrophages, direct effects on their functionality have remained elusive. Synchrotron radiation X-ray microtomography and X-ray fluorescence mapping indicated that peri-implant tissues harvested during aseptic revision of different arthroplasty implants are exposed to Co and Cr in situ. Confocal laser scanning microscopy revealed that macrophage influx is predominant in patient tissue. While in vitro exposure to Cr 3+ had only minor effects on monocytes/macrophage phenotype, pathologic concentrations of Co 2+ significantly impaired both, monocyte/macrophage phenotype and functionality. High concentrations of Co 2+ led to a shift in macrophage subsets and loss of surface markers, including CD14 and CD16. Both Co 2+ and Cr 3+ impaired macrophage responses to Staphylococcus aureus infection, and particularly, Co 2+ -exposed macrophages showed decreased phagocytic activity. These findings demonstrate the immunosuppressive effects of locally elevated metal ions on the innate immune response and support further investigations, including studies exploring whether Co 2+ and Cr 3+ or CoCrMo alloys per se expose the patients to a higher risk of infections post-revision arthroplasty.

In musculoskeletal surgery, the treatment of large bone defects is challenging and can require the use of bone graft substitutes to restore mechanical stability and promote host-mediated regeneration. The use of bone allografts is well-established in many bone regenerative procedures, but is associated with low rates of ingrowth due to pre-therapeutic graft processing. Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen (O 2 ) and nitrogen (N 2 ) species, is suggested to be advantageous in biomedical implant processing. CPP is a promising tool in allograft processing for improving surface characteristics of bone allografts towards enhanced cellularization and osteoconduction. However, a preclinical assessment regarding the feasibility of pre-therapeutic processing of allogeneic bone grafts with CPP has not yet been performed. Thus, this pilot study aimed to analyze the bone morphology of CPP processed allografts using synchrotron radiation-based microcomputed tomography (SR-µCT) and to analyze the effects of CPP processing on human bone cell viability and function. The analyzes, including co-registration of pre- and post-treatment SR-µCT scans, revealed that the main bone morphological properties (total volume, mineralized volume, surface area, and porosity) remained unaffected by CPP treatment if compared to allografts not treated with CPP. Varying effects on cellular metabolic activity and alkaline phosphatase activity were found in response to different gas mixtures and treatment durations employed for CPP application. It was found that 3 min CPP treatment using a He + 0.1% N 2 gas mixture led to the most favourable outcome regarding a significant increase in bone cell viability and alkaline phosphatase activity. This study highlights the promising potential of pre-therapeuthic bone allograft processing by CPP prior to intraoperative application and emphasizes the need for gas source and treatment time optimization for specific applications., Competing Interests: Authors EmB and BH were employed by Xploraytion GmbH. Author MP was employed by Cells + Tissuebank Austria Gemeinnützige GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fischer, Bortel, Schoon, Behnke, Hesse, Weitkamp, Bekeschus, Pichler, Wassilew and Schulze.)

Background: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used in regenerative medicine and related research involving immunomodulatory, anti-inflammatory, anti-fibrotic and regenerative functions. Isolation of BM-MSCs from samples obtained during total hip arthroplasty (THA) is routinely possible. Advanced age and comorbidities of the majority of patients undergoing THA limit their applicability. Our study aimed to evaluate the potential of bone marrow obtained during periacetabular osteotomy (PAO) as a novel source of BM-MSCs from young donors by analyzing cell yield and cell characteristics., Methods: Bone samples were obtained from the anterior Os ilium or superior Os pubis during PAO and from the femoral cavity during primary THA. Isolation of bone marrow-derived mononuclear cells (BM-MNCs) was performed by density gradient centrifugation. The samples from PAO and THA patients were compared in terms of BM-MSC yield, colony formation and the proportion of BM-MSCs within the BM-MNC population using flow cytometry analysis. The cells were characterized based on the expression of BM-MSC-specific surface markers. The functionality of the cells was compared by quantifying post-thaw viability, metabolic activity, proliferation capacity, senescence-associated beta galactosidase (SA-β-gal) expression, trilineage differentiation potential and major secretome proteins., Results: Isolation of BM-MNCs was possible in a reliable and reproducible manner when using bone from PAO containing more than 0.24 g bone marrow. PAO patients were younger than patients of the THA group. Bone obtained during PAO contained less bone marrow and led to a lower BM-MSC number after the first cell culture passage compared to BM-MSCs obtained during THA. BM-MSCs from PAO samples are characterized by a higher proliferation capacity. This results in a higher yield in cell culture passage two, when normalized to the sample weight. BM-MSCs from PAO patients showed increased secretion of TGF-β1, TIMP2, and VEGF upon osteogenic differentiation. BM-MSCs from PAO and THA patients revealed similar results regarding the onset of SA-β-gal expression and trilineage differentiation capacity., Conclusions: We suggest that bone obtained during PAO is a promising novel source for BM-MSCs from young donors. Limited absolute cell yield due to low sample weight must be considered in early cell culture passages and might be critical for the range of clinical applications possible for BM-MSCs from this source. The higher proliferation capacity and increased growth factor secretion of BM-MSCs from young donors may be beneficial for future regenerative cell therapies, in vitro models, and tissue engineering., (© 2023. The Author(s).)

Background: Minced cartilage implantation (MCI) has seen a renaissance in recent years. In this evolved technique, human articular cartilage is harvested with an arthroscopic shaver, augmented with platelet-rich plasma (PRP), and implanted with autologous thrombin. This modified technique combines the possibility of cell-based surgical cartilage repair with a minimally invasive autologous 1-step procedure. However, evidence on cell survival and preserved function after shaver-based mincing and PRP supplementation is limited., Purpose: To evaluate the effects of arthroscopic shaver mincing and augmentation with PRP on human cartilage tissue., Study Design: Controlled laboratory study., Methods: Standardized samples were taken from 12 donors during autologous MCI. A comparison of cell outgrowth, cell viability, proliferation capacity, and ability to produce extracellular matrix-specific proteoglycans after chondrogenic redifferentiation was made between cartilage taken by curettage from the border of the cartilage defect, cartilage tissue minced by an arthroscopic shaver, and cartilage tissue minced by an arthroscopic shaver that was additionally augmented with autologous PRP., Results: There was no difference between all 3 groups in terms of cell outgrowth or proliferation capacity. Metabolic activity relative to the cell number of chondrocytes isolated from shaver-minced cartilage was higher compared with chondrocytes isolated from cartilage that was derived by curettage or shaver-minced cartilage that was augmented with PRP. After chondrogenic stimulation, the normalized proteoglycan content was higher in spheroids of cells derived from shaver-minced cartilage augmented with PRP than in spheroids of cells derived from curettage. A high correlation of cell outgrowth, proliferation capacity, and viability between isolated cells from all 3 groups taken from an individual donor was observed., Conclusion: Chondrocytes isolated from human cartilage tissue that was harvested and minced with an arthroscopic shaver remained viable and proliferative. The augmentation of shaver-minced cartilage with PRP led to the enhanced proteoglycan production of chondrogenic spheroids in vitro, pointing toward the development of a cartilage-specific extracellular matrix. This in vitro study yields promising results regarding the use of an arthroscopic shaver and augmentation with PRP in the context of MCI., Clinical Relevance: Knowledge that shaver mincing and augmentation with PRP are feasible for processing articular cartilage during MCI is highly relevant for surgical cartilage repair.

The study of prostate cancer in vitro relies on established cell lines that lack important physiological characteristics, such as proper polarization and expression of relevant biomarkers. Microphysiological systems (MPS) can replicate cancer microenvironments and lead to cellular phenotypic changes that better represent organ physiology in vitro. In this study, we developed an MPS model comprising conventional prostate cancer cells to evaluate their activity under dynamic culture conditions. Androgen-sensitive (LNCaP) and androgen-insensitive (PC3) cells were grown in conventional and 3D cultures, both static and dynamic. Cell morphology, the secretion of prostate-specific antigen, and the expression of key prostate markers and microRNAs were analyzed. LNCaP formed spheroids in 3D and MPS cultures, with morphological changes supported by the upregulation of cytokeratins and adhesion proteins. LNCaP also maintained a constant prostate-specific antigen secretion in MPS. PC3 cells did not develop complex structures in 3D and MPS cultures. PSA expression at the gene level was downregulated in LNCaP-MPS and considerably upregulated in PC3-MPS. MicroRNA expression was altered by the 3D static and dynamic culture, both intra- and extracellularly. MicroRNAs associated with prostate cancer progression were mostly upregulated in LNCaP-MPS. Overall dynamic cell culture substantially altered the morphology and expression of LNCaP cells, arguably augmenting their prostate cancer phenotype. This novel approach demonstrates that microRNA expression in prostate cancer cells is sensitive to external stimuli and that MPS can effectively promote important physiological changes in conventional prostate cancer models., (© 2023. The Author(s).)

Cold physical plasma (CPP) technology is of high promise for various medical applications. The interplay of specific components of physical plasma with living cells, tissues and organs on a structural and functional level is of paramount interest with the aim to induce therapeutic effects in a controlled and replicable fashion. In contrast to other medical disciplines such as dermatology and oromaxillofacial surgery, research reports on CPP application in orthopaedics are scarce. The present implementation of CPP in orthopaedics involves surface modifications of orthopaedic materials and biomaterials to optimize osseointegration. In addition, the influence of CPP on musculoskeletal cells and tissues is a focus of research, including possible adverse reactions and side effects. Its bactericidal aspects make CPP an attractive supplement to current treatment regimens in case of microbial inflammations such as periprosthetic joint infections. Attributed anticancerogenic and pro-apoptotic effects underline the clinical relevance of CPP as an additive in treating malignant bone lesions. The present review outlines ongoing research in orthopaedics involving CPP; it distinguishes considerations for safe application and the need for more evidence-based research to facilitate robust clinical implementation.

Although Ewing's sarcoma (ES) is a rare, but very aggressive tumor disease affecting the musculoskeletal system, especially in children, it is very aggressive and difficult to treat. Although medical advances and the establishment of chemotherapy represent a turning point in the treatment of ES, resistance to chemotherapy, and its side effects, continue to be problems. New treatment methods such as the application of cold physical plasma (CPP) are considered potential supporting tools since CPP is an exogenous source of reactive oxygen and nitrogen species, which have similar mechanisms of action in the tumor cells as chemotherapy. This study aims to investigate the synergistic effects of CPP and commonly used cytostatic chemotherapeutics on ES cells. The chemotherapy drugs doxorubicin and vincristine, the most commonly used in the treatment of ES, were applied to two different ES cell lines (RD-ES and A673) and their IC 20 and IC 50 were determined. In addition, individual chemotherapeutics in combination with CPP were applied to the ES cells and the effects on cell growth, cell viability, and apoptosis processes were examined. A single CPP treatment resulted in the dose-dependent growth inhibition of ES cells. The combination of different cytostatics and CPP led to significant growth inhibition, a reduction in cell viability, and higher rates of apoptosis compared to cells not additionally exposed to CPP. The combination of CPP treatment and the application of cytostatic drugs to ES cells showed promising results, significantly enhancing the cytotoxic effects of chemotherapeutic agents. These preclinical in vitro data indicate that the use of CPP can enhance the efficacy of common cytostatic chemotherapeutics, and thus support the translation of CPP as an anti-tumor therapy in clinical routine.

The success of hip arthroplasty is based on modern materials in addition to the continuous development of surgical techniques and clinical experience gained over six decades. The biocompatible implant materials used in hip arthroplasty can be textured or coated with biomimetic surfaces to ensure durable component ingrowth and moderate host response. Material integrity plays a critical role in the durability of the stable interface between implant components and periprosthetic tissues. Inflammation at the interfaces due to the release of degradation products from the implant materials is one of the causes of hip arthroplasty failure. This review summarizes the implant materials currently used in hip arthroplasty, their preclinical testing and the postoperative neogenesis of periprosthetic tissues, and the interactions of periprosthetic bone and the implant materials at the periprosthetic interfaces., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: The use of cold atmospheric plasma (CAP) in oncology has been intensively investigated over the past 15 years as it inhibits the growth of many tumor cells. It is known that reactive oxidative species (ROS) produced in CAP are responsible for this effect. However, to translate the use of CAP into medical practice, it is essential to know how CAP treatment affects non-malignant cells. Thus, the current in vitro study deals with the effect of CAP on human bone cancer cells and human osteoblasts. Here, identical CAP treatment regimens were applied to the malignant and non-malignant bone cells and their impact was compared., Methods: Two different human bone cancer cell types, U2-OS (osteosarcoma) and A673 (Ewing's sarcoma), and non-malignant primary osteoblasts (HOB) were used. The CAP treatment was performed with the clinically approved kINPen MED. After CAP treatment, growth kinetics and a viability assay were performed. For detecting apoptosis, a caspase-3/7 assay and a TUNEL assay were used. Accumulated ROS was measured in cell culture medium and intracellular. To investigate the influence of CAP on cell motility, a scratch assay was carried out., Results: The CAP treatment showed strong inhibition of cell growth and viability in bone cancer cells. Apoptotic processes were enhanced in the malignant cells. Osteoblasts showed a higher potential for ROS resistance in comparison to malignant cells. There was no difference in cell motility between benign and malignant cells following CAP treatment., Conclusions: Osteoblasts show better tolerance to CAP treatment, indicated by less affected viability compared to CAP-treated bone cancer cells. This points toward the selective effect of CAP on sarcoma cells and represents a further step toward the clinical application of CAP.

Bone generally displays a high intrinsic capacity to regenerate. Nonetheless, large osseous defects sometimes fail to heal. The treatment of such large segmental defects still represents a considerable clinical challenge. The regeneration of large bone defects often proves difficult, since it relies on the formation of large amounts of bone within an environment impedimental to osteogenesis, characterized by soft tissue damage and hampered vascularization. Consequently, research efforts have concentrated on tissue engineering and regenerative medical strategies to resolve this multifaceted challenge. In this review, we summarize, critically evaluate, and discuss present approaches in light of their clinical relevance; we also present future advanced techniques for bone tissue engineering, outlining the steps to realize for their translation from bench to bedside. The discussion includes the physiology of bone healing, requirements and properties of natural and synthetic biomaterials for bone reconstruction, their use in conjunction with cellular components and suitable growth factors, and strategies to improve vascularization and the translation of these regenerative concepts to in vivo applications. We conclude that the ideal all-purpose material for scaffold-guided bone regeneration is currently not available. It seems that a variety of different solutions will be employed, according to the clinical treatment necessary.

Background: In 2020, more than 14,000 aseptic revision procedures for total hip arthroplasty (THA) were registered in Germany. Patient expectations of revision hip arthroplasty are not substantially different from expectations of primary hip replacement., Outcome: However, revision surgery is associated with increased complication rates and a higher proportion of dissatisfied patients. In particular, poorer postoperative function and mobility as well as increased pain levels following revision THA have been described compared to the outcome after primary THA. Quality of life and return-to-work can also be impaired., Survival Rate: Implant survival is influenced by age, BMI, and comorbidities of the patients, but also by the size and complexity of bone defects, the extent of periprosthetic soft tissue compromise and the choice of revision implant(s). In addition, the number of previous revision surgeries inversely correlates with the survival rates. Previous revisions have been shown to be associated with increased risks of aseptic loosening, instability and periprosthetic infection., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests which may be considered as potential competing interests. Wendel Wohlleben is an employee of a company producing and marketing Nanomaterials., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 ( HSD11B1 ) to modulate the endogenous glucocorticoid conversion in SCP-1 cells - a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1 . Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL , respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kragl, Schoon, Tzvetkova, Wenzel, Blaschke, Böcker, Siggelkow and Tzvetkov.)

Particles released from cobalt-chromium-molybdenum (CoCrMo) alloys are considered common elicitors of chronic inflammatory adverse effects. There is a lack of data demonstrating particle numbers, size distribution and elemental composition of bone marrow resident particles which would allow for implementation of clinically relevant test strategies in bone marrow models at different degrees of exposure. The aim of this study was to investigate metal particle exposure in human periprosthetic bone marrow of three types of arthroplasty implants. Periprosthetic bone marrow sections from eight patients exposed to CoCrMo particles were analyzed via spatially resolved and synchrotron-based nanoscopic X-ray fluorescence imaging. These analyses revealed lognormal particle size distribution patterns predominantly towards the nanoscale. Analyses of particle numbers and normalization to bone marrow volume and bone marrow cell number indicated particle concentrations of up to 1 × 10 11 particles/ml bone marrow or 2 × 10 4 particles/bone marrow cell, respectively. Analyses of elemental ratios of CoCrMo particles showed that particularly the particles' Co content depends on particle size. The obtained data point towards Co release from arthroprosthetic particles in the course of dealloying and degradation processes of larger particles within periprosthetic bone marrow. This is the first study providing data based on metal particle analyses to be used for future in vitro and in vivo studies of possible toxic effects in human bone marrow following exposure to arthroprosthetic CoCrMo particles of different concentration, size, and elemental composition. Graphical abstract., (© 2022. The Author(s).)

Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen and nitrogen species, is suggested to provide advantages in regenerative medicine. Intraoperative CPP therapy targeting pathologies related to diminished bone quality could be promising in orthopedic surgery. Assessment of a clinically approved plasma jet regarding cellular effects on primary bone marrow mesenchymal stromal cells (hBM-MSCs) from relevant arthroplasty patient cohorts is needed to establish CPP-based therapeutic approaches for bone regeneration. Thus, the aim of this study was to derive biocompatible doses of CPP and subsequent evaluation of human primary hBM-MSCs' osteogenic and immunomodulatory potential. Metabolic activity and cell proliferation were affected in a treatment-time-dependent manner. Morphometric high content imaging analyses revealed a decline in mitochondria and nuclei content and increased cytoskeletal compactness following CPP exposure. Employing a nontoxic exposure regime, investigation on osteogenic differentiation did not enhance osteogenic capacity of hBM-MSCs. Multiplex analysis of major hBM-MSC cytokines, chemokines and growth factors revealed an anti-inflammatory, promatrix-assembling and osteoclast-regulating secretion profile following CPP treatment and osteogenic stimulus. This study can be noted as the first in vitro study addressing the influence of CPP on hBM-MSCs from individual donors of an arthroplasty clientele.

The histopathological examination of the periprosthetic soft tissue and bone has contributed to the identification and description of the morphological features of adverse local tissue reactions (ALTR)/adverse reactions to metallic debris (ARMD). The need of a uniform vocabulary for all disciplines involved in the diagnosis and management of ALTR/ARMD and of clarification of the parameters used in the semi-quantitative scoring systems for their classification has been considered a pre-requisite for a meaningful interdisciplinary evaluation.This review of key terms used for ALTR/ARMD has resulted in the following outcomes: (a) pseudotumor is a descriptive term for ALTR/ARMD, classifiable in two main types according to its cellular composition defining its clinical course; (b) the substitution of the term metallosis with presence of metallic wear debris, since it cannot be used as a category of implant failure or histological diagnosis; (c) the term aseptic lymphocytic-dominated vasculitis- associated lesion (ALVAL) should be replaced due to the absence of a vasculitis with ALLTR/ALRMD for lymphocytic-predominant and AMLTR/AMRMD for macrophage-predominant reaction.This review of the histopathological classifications of ALTR/ARMD has resulted in the following outcomes: (a) distinction between cell death and tissue necrosis; (b) the association of corrosion metallic debris with adverse local lymphocytic reaction and tissue necrosis; (c) the importance of cell and particle debris for the viscosity and density of the lubricating synovial fluid; (d) a consensus classification of lymphocytic infiltrate in soft tissue and bone marrow; (e) evaluation of the macrophage infiltrate in soft tissues and bone marrow; (f) classification of macrophage induced osteolysis/aseptic loosening as a delayed type of ALTR/ARMD; (g) macrophage motility and migration as possible driving factor for osteolysis; (h) usefulness of the histopathological examination for the natural history of the adverse reactions, radiological correlation, post-marketing surveillance, and implant registries.The review of key terms used for the description and histopathological classification of ALTR/ARMD has resulted in a comprehensive, new standard for all disciplines involved in their diagnosis, clinical management, and long-term clinical follow-up. Cite this article: EFORT Open Rev 2021;6:399-419. DOI: 10.1302/2058-5241.6.210013., Competing Interests: ICMJE Conflict of interest statement: DL reports consultancy and expert testimony, acting as witness for plaintiff in metal-on-metal hip litigation for PXD Ltd trading as ExplantLab, and a filed patent to identify risk of ARMD using genetic algorithm with PXD Ltd, all outside the submitted work. The other authors declare no conflict of interest relevant to this work., (© 2021 The author(s).)

Loosening of orthodontic and orthopedic implants is a critical and common clinical problem. To minimize the numbers of revision surgeries due to peri-implant inflammation or insufficient osseointegration, developments of new implant manufacturing strategies are indicated. Ultrafast laser surface texturing is a promising contact-free technology to modify the physicochemical properties of surfaces toward an anti-infectious functionalization. This work aims to texture Ti6Al4V surfaces with ultraviolet (UV) and green (GR) radiation for the manufacturing of laser-induced periodic surface structures (LIPSS). The assessment of these surface modifications addresses key aspects of topography, morphology and chemical composition. Human primary mesenchymal stromal cells (hMSCs) were cultured on laser-textured and polished Ti6Al4V to characterize the surfaces in terms of their in vitro biocompatibility, cytotoxicity, and metal release. The outcomes of the in vitro experiment show the successful culture of hMSCs on textured Ti6Al4V surfaces developed within this work. Cells cultured on LIPSS surfaces were not compromised in terms of their viability if compared to polished surfaces. Yet, the hMSC culture on UV-LIPSS show significantly lower lactate dehydrogenase and titanium release into the supernatant compared to polished. Thus, the presented surface modification can be a promising approach for future applications in orthodontics and orthopedics.

In recent years, increasing concern has been raised regarding potential systemic toxicity of metals released from arthroplasty implants. A lack of valid metal thresholds for human (organ) toxicity and the prospect of multi-decade survival of modern hip and knee replacements pose special challenges. Indeed, evidence of systemic effects of metals released from such implants is largely missing. Systemic cobalt exposure has repeatedly been associated with cardiotoxic and neurotoxic effects, and also with thyroid dysfunction. The toxic potential of chromium is considered less pronounced. Yet, in arthroplasty there is usually a co-exposure to chromium and cobalt which complicates evaluation of element-specific effects. Toxicity of titanium dioxide nanoparticles has been subject to debate among international regulatory authorities. Their wide use in a variety of products in everyday life, such as toothpaste, cosmetics and food colorants, hampers the assessment of an arthroplasty-induced systemic titanium exposure. To date there is no clear evidence for systemic complications due to titanium dioxide released from arthroplasty implants. Release of further metals such as tantalum, niobium, nickel, vanadium and zirconium from hip and knee replacement implants has been described occasionally, but systemic effects of respective long-term exposure scenarios are unknown. Generally, the characterization of all released metals regarding their chemical and physical specifications is critical for the evaluation of potential systemic risks. Systematic studies investigating the accumulation of metals relevant in arthroplasty in different organs/organ systems and the biological consequences of such accumulations are urgently needed., Competing Interests: The authors gratefully acknowledge financial support of research projects by the ENDO-Verein e. V. (Hamburg, Germany), the Deutsche Arthrose-Hilfe e. V. (P327-A844-AE-EP1), the Einstein Foundation Berlin through the Einstein Center for Regenerative Therapies (EZ-2016-289), the Berlin-Brandenburg Center for Regenerative Therapies and the Berlin-Brandenburg School for Regenerative Therapies (GSC 203). The authors further acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité – Universitätsmedizin Berlin. The authors received the AE basic science research award 2017 by the Deutsche Gesellschaft fuer Endoprothetik e. V. (AE) and the Stiftung Endoprothetik e. V. In 2017, Anastasia Rakow received a “quality and safety in arthroplasty” scholarship of the Deutsche Gesellschaft fuer Orthopaedie und Unfallchirurgie (DGOU). The above funders had no role in design, data collection and analysis, decision to publish, or preparation of this manuscript./Die Autoren erklären, dass sie innerhalb der vergangenen 3 Jahre Forschungsunterstützung von dem ENDO-Verein e. V. (Hamburg, Deutschland), der Deutschen Arthrose-Hilfe e. V (P327-A844-AE-EP1), der Einstein Stiftung Berlin bzw. dem Einstein Center for Regenerative Therapies (EZ-2016-289) und dem Berlin-Brandenburger Centrum für Regenerative Therapien und der Berlin-Brandenburger Schule für Regenerative Therapien (GSC 203) erhalten haben. Sie erklären ferner, dass sie finanzielle Unterstützung für Open Access Publikationen von der Deutschen Forschungsgemeinschaft DFG und dem Open Access Publication Fund der Charité – Universitätsmedizin Berlin erhalten haben. Die Autoren erhielten den AE-Preis 2017 auf dem Gebiet der Grundlagenforschung von der Deutschen Gesellschaft für Endoprothetik e. V. (AE) und der Stiftung Endoprothetik e. V.; Anastasia Rakow erhielt 2017 ein Stipendium („Qualität und Sicherheit in der Endoprothetik“) der Deutschen Gesellschaft für Orthopädie und Unfallchirurgie (DGOU). Konzeption, Inhalt und Veröffentlichung der vorliegenden Arbeit sowie Akquise und Analysen hier erwähnter Daten blieben von genannten Institutionen/Organisationen/Stiftungen/Vereinen/Fachgesellschaften unbeeinflusst., (Thieme. All rights reserved.)

Metallic implants are frequently used in medicine to support and replace degenerated tissues. Implant loosening due to particle exposure remains a major cause for revision arthroplasty. The exact role of metal debris in sterile peri-implant inflammation is controversial, as it remains unclear whether and how metals chemically alter and potentially accumulate behind an insulating peri-implant membrane, in the adjacent bone and bone marrow (BM). An intensively focused and bright synchrotron X-ray beam allows for spatially resolving the multi-elemental composition of peri-implant tissues from patients undergoing revision surgery. In peri-implant BM, particulate cobalt (Co) is exclusively co-localized with chromium (Cr), non-particulate Cr accumulates in the BM matrix. Particles consisting of Co and Cr contain less Co than bulk alloy, which indicates a pronounced dissolution capacity. Particulate titanium (Ti) is abundant in the BM and analyzed Ti nanoparticles predominantly consist of titanium dioxide in the anatase crystal phase. Co and Cr but not Ti integrate into peri-implant bone trabeculae. The characteristic of Cr to accumulate in the intertrabecular matrix and trabecular bone is reproducible in a human 3D in vitro model. This study illustrates the importance of updating the view on long-term consequences of biomaterial usage and reveals toxicokinetics within highly sensitive organs., Competing Interests: U.M. is shareholder and CEO of TissUse GmbH. B.H. is shareholder and CEO of Xploraytion GmbH. C.P. serves as consultant for Aesculap, CeramTec, Zimmer Biomet, DePuy Synthes, Smith & Nephew, receives royalties from Smith & Nephew, DePuy Synthes, and receives institutional funding and research support from Aesculap. G.N.D. serves as consultant for CeramTec and DePuy Synthes and receives institutional funding and research support from Aesculap, CeramTec, DePuy Synthes, LINK, OHST, Peter Brehm, Smith & Nephew, Zimmer Biomet. These companies did not financially support this study, had no role in study design, sample collection, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2020 The Authors. Published by Wiley‐VCH GmbH.)

Delayed type hypersensitivity (DTH) reactions are considered infrequent complications in arthroplasty, but have been recognized to be associated with devastating morbidity and substantial decrease in quality of life of affected patients. Chronic inflammation of artificial joints and associated loss of peri-implant bone often require revision surgery. Methods for the diagnosis of implant-related DTH are available but infrequently considered to the full extent. Sequential diagnostics based on exclusion of septic complications, local and systemic metal level determination, lymphocyte transformation testing (LTT), and local T cell subset analysis are required for an unequivocal DTH diagnosis. Here, we report on a patient with a history of chronic rheumatoid arthritis and an unfavorable outcome of unilateral knee arthroplasty. This case illustrates pitfalls and difficulties in the course of recurrent inflammation following joint replacement. In the early course, suspicion of low-grade bacterial infection led to three two-stage revisions. Afterwards, the joint was proven to be sterile. However, metal level quantification revealed release of especially cobalt and chromium from the joint, LTT indicated persisting cobalt and nickel sensitization and subset analysis of T cells from the synovium suggested DTH as a root cause for the inflammatory symptoms. This report aims to recommend the depicted diagnostic algorithm as an adequate tool for future DTH detection. Yet, systemic to local subset ratios for effector memory and regulatory T cells should be derived from sufficient patient numbers to establish it as a diagnostic marker. Moreover, future prospects regarding implant-related DTH diagnostics are discussed. Therapeutic options for the portrayed patient are proposed, considering pharmaceutical, cell-therapeutic and surgical aspects. Patients who experience peri-implant inflammation but do not have obvious mechanical or infectious problems remain a diagnostic challenge and are at high risk of being treated inadequately. Since potentially sensitizing materials are regularly used in arthroplasty, it is essential to detect cases of acute DTH-derived inflammation of an artificial joint at early postoperative stages. This would reduce the severity of inflammation-related long-term consequences for affected patients and may avoid unnecessary revision surgery., (Copyright © 2019 Schoon, Ort, Huesker, Geissler and Rakow.)

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo -expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank ( n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics., (Copyright © 2019 Andrzejewska, Catar, Schoon, Qazi, Sass, Jacobi, Blankenstein, Reinke, Krüger, Streitz, Schlickeiser, Richter, Souidi, Beez, Kamhieh-Milz, Krüger, Zemojtel, Jürchott, Strunk, Reinke, Duda, Moll and Geissler.)

Arthroplasty ranks among the greatest achievements of surgical medicine, with total hip replacement termed "the operation of the century." Despite its wide success, arthroplasty bears risks, such as local reactions to implant derived wear and corrosion products. Prevalence of allergies across Western society increases and along the number of reported hypersensitivity reactions to orthopedic implant materials. In this context the main focus is on delayed hypersensitivity (DTH). This mechanism is mainly attributed to T cells and an overreaction of the adaptive immune system. Arthroplasty implant materials are in direct contact with bone marrow (BM), which is discussed as a secondary lymphoid organ. However, the mechanisms of sensitization toward implant wear remain elusive. Nickel and cobalt ions can form haptens with native peptides to activate immune cell receptors and are therefore common T helper allergens in cutaneous DTH. The rising prevalence of metal-related allergy in the general population and evidence for the immune-modulating function of BM allow for the assumption hypersensitivity reactions could occur in peri-implant BM. There is evidence that pro-inflammatory factors released during DTH reactions enhance osteoclast activity and inhibit osteoblast function, an imbalance characteristic for osteolysis. Even though some mechanisms are understood, hypersensitivity has remained a diagnosis of exclusion. This review aims to summarize current views on the pathomechanism of DTH in arthroplasty with emphasis on BM and discusses recent advances and future directions for basic research and clinical diagnostics., (Copyright © 2019 Ort, Geissler, Rakow and Schoon.)

Vascularization of the fracture site and cell-mediated deposition of the mineralized matrix are crucial determinants for successful bone regeneration after injury. Ceramic biomaterials such as bioactive glasses (BAGs) that release bioactive ions have shown promising results in bone defect regeneration. However, it remains unclear how the dosage and composition of bioactive ions influence the angiogenic and osteogenic behavior of primary human mesenchymal stromal cells (MSCs). Here, we show that exposure to ionic dissolution products from 1393 and 45S5 BAGs can evoke distinct angiogenic and osteogenic responses from primary MSCs in a dose- and composition-dependent manner. Significantly higher concentrations of the pro-angiogenic factors VEGF, HGF, PIGF, angiopoietin, and angiogenin were detected in conditioned media (CM) from MSCs exposed to 45S5, but not 1393, BAGs. Application of this CM to human umbilical vein endothelial cells (HUVECs) resulted in robust 2D tube formation in vitro. Osteogenic differentiation of MSCs was assessed by gene expression analysis and mineralization assays. Low concentrations (0.1% w/v) of 1393 BAGs significantly enhanced the gene expression of RUNX2 and ALP and induced an earlier onset of matrix mineralization compared to all other groups. We further tested whether simultaneous exposure to both BAGs would improve both angiogenic secretion and osteogenic differentiation of MSCs, and did not find evidence to support this hypothesis. Our results provide evidence of BAG composition-dependent enhancement of primary human MSCs' regenerative function, besides also underlining the importance of an in vitro evaluation of the dose-response relationship to translate BAG based approaches into safe and effective clinical therapies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2827-2837, 2018., 2018., (© 2018 Wiley Periodicals, Inc.)

Background: Vascular calcification is enhanced in uraemic chronic haemodialysis patients, likely due to the accumulation of midsize uraemic toxins, such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Here we have assessed the impact of uraemia on vascular smooth muscle cell (VSMC) calcification and examined the role of IL-6 and TNF-α as possible mediators and, most importantly, its underlying signalling pathway in VSMCs., Methods: VSMCs were incubated with samples of uraemic serum obtained from patients treated with haemodialysis for renal failure in the Permeability Enhancement to Reduce Chronic Inflammation-I clinical trial. The VSMCs were assessed for IL-6 gene regulation and promoter activation in response to uraemic serum and TNF-α with reporter assays and electrophoretic mobility shift assay and for osteoblastic transition, cellular calcification and cell viability upon osteogenic differentiation., Results: Uraemic serum contained higher levels of TNF-α and IL-6 compared with serum from healthy individuals. Exposure of VSMCs to uraemic serum or recombinant TNF-α lead to a strong upregulation of IL-6 mRNA expression and protein secretion, which was mediated by activator protein 1 (AP-1)/c-FOS-pathway signalling. Uraemic serum induced osteoblastic transition and calcification of VSMCs could be strongly attenuated by blocking TNF-α, IL-6 or AP-1/c-FOS signalling, which was accompanied by improved cell viability., Conclusion: These results demonstrate that uraemic serum contains higher levels of uraemic toxins TNF-α and IL-6 and that uraemia promotes vascular calcification through a signalling pathway involving TNF-α, IL-6 and the AP-1/c-FOS cytokine-signalling axis. Thus treatment modalities aiming to reduce systemic TNF-α and IL-6 levels in chronic haemodialysis patients should be evaluated in future clinical trials.

Porous tantalum components are widely used for complex acetabular reconstructions in revision hip arthroplasty. Multiple other metal alloys such as titanium-aluminum-vanadium or cobalt-chromium-molybdenum are principally used in artificial joint setups. We report a case of tantalum component failure being both cause and effect of a multiple metal exposure. Our aims were to assess and to characterize associated particle exposure and biological consequences. Metal level quantification revealed substantial in vivo exposure to particulate and dissociated tantalum, zirconium, chromium, cobalt, molybdenum, titanium, aluminum and vanadium in periprosthetic compartments. Aside from micron-sized particles, nanoparticles of a broad size range and elemental composition were obtained. Histological exams verified a spectrum of necrotic changes in the periprosthetic tissues. In the presented case tantalum release was accompanied by concomitance of particles originating from other utilized metals. We conclude that an overall in vivo exposure assessment is mandatory for realistic appraisal of metal toxicity and associated risks., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In hip arthroplasty the implants' articulating surfaces can be made of a cobalt-chromium-molybdenum (CoCrMo) alloy. The use of these metal-on-metal (MoM) pairings can lead to the release of wear products such as metallic particles and dissociated metal species, raising concerns regarding their safety amongst orthopedic surgeons and the public. MoM-wear particles are reported to be heterogeneous in their physicochemical properties, are capable of inducing adverse effects on a cellular level and are thought to be involved in relevant clinical problems like aseptic osteolysis. Yet, it remains elusive how MoM-wear affects bone forming cells and their progenitors: bone marrow residing mesenchymal stromal cells (MSCs). This study introduces an assessment of the in vivo exposure to particulate and dissociated Co and Cr and evaluates the effects of MoM-wear on MSCs. The exposure to MoM-wear products in vivo and in vitro leads to a decrease in MSCs' osteogenic matrix mineralization and alkaline phosphatase activity on a cellular and systemic level. In conclusion, MoM-wear products are released in the periprosthetic region and elevate bone marrow Co and Cr concentrations towards levels that impair osteogenic differentiation of MSCs. Therefore, the ongoing use of CoCrMo alloys for articulating surfaces in joint replacement implants needs critical reconsideration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells (MSCs) are promising candidates in regenerative cell-therapies. However, optimizing their number and route of delivery remains a critical issue, which can be addressed by monitoring the MSCs' bio-distribution in vivo using super-paramagnetic iron-oxide nanoparticles (SPIONs). In this study, amino-polyvinyl alcohol coated (A-PVA) SPIONs are introduced for cell-labeling and visualization by magnetic resonance imaging (MRI) of human MSCs. Size and surface charge of A-PVA-SPIONs differ depending on their solvent. Under MSC-labeling conditions, A-PVA-SPIONs have a hydrodynamic diameter of 42 ± 2 nm and a negative Zeta potential of 25 ± 5 mV, which enable efficient internalization by MSCs without the need to use transfection agents. Transmission X-ray microscopy localizes A-PVA-SPIONs in intracellular vesicles and as cytosolic single particles. After identifying non-interfering cell-assays and determining the delivered and cellular dose, in addition to the administered dose, A-PVA-SPIONs are found to be non-toxic to MSCs and non-destructive towards their multi-lineage differentiation potential. Surprisingly, MSC migration is increased. In MRI, A-PVA-SPION-labeled MSCs are successfully visualized in vitro and in vivo. In conclusion, A-PVA-SPIONs have no unfavorable influences on MSCs, although it becomes evident how sensitive their functional behavior is towards SPION-labeling. And A-PVA-SPIONs allow MSC-monitoring in vivo., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bone regeneration is influenced by mesenchymal stromal cells (MSCs) and mechanical conditions. How healing outcome and mechanical stability are linked on the cellular level, however, remains elusive. Cyclic-compressive loading of MSCs affects the expression of molecules involved in angiogenesis and matrix assembly, but also reduces the expression of CD73, an ecto-5'-nucleotidase, which plays a crucial role in extracellular adenosine generation. Although, for almost 20 years, CD73 has been a major cell surface marker defining MSCs, little is known about its function in these cells. Therefore, the aim of this study was to determine the putative involvement of CD73 in MSC differentiation after cyclic-compressive loading. After cultivation in appropriate differentiation media, chondrogenic differentiation ability was significantly increased in loaded MSCs, hence following current models. Through treatment with the CD73 inhibitor adenosine 5'-(α, β-methylene) diphosphate, chondrogenic matrix deposition was further increased; in contrast, mineral matrix deposition and expression of osteogenic markers was reduced. One major signal transduction pathway, which is activated via CD73-mediated adenosine, is the adenosine receptor pathway. Thus, the adenosine receptor expression pattern was investigated. MSCs expressed the four known adenosine receptors at the mRNA level. After mechanical stimulation of MSCs, Adora2a was down-regulated. These data point towards a role of CD73 in MSC differentiation possibly via A2AR signalling, which is mutually regulated with CD73. In conclusion, the findings of this study suggest that CD73 is another regulatory factor in osteo-/chondrogenic differentiation of MSCs and may provide a - thus far underestimated - therapeutic target to guide bone regeneration.