Impact of Metal Ions on Cellular Functions: A Focus on Mesenchymal Stem/Stromal Cell Differentiation.

Metals play a crucial role in the human body, especially as ions in metalloproteins. Essential metals, such as calcium, iron, and zinc are crucial for various physiological functions, but their interactions within biological networks are complex and not fully understood. Mesenchymal stem/stromal cells (MSCs) are essential for tissue regeneration due to their ability to differentiate into various cell types. This review article addresses the effects of physiological and unphysiological, but not directly toxic, metal ion concentrations, particularly concerning MSCs. Overloading or unbalancing of metal ion concentrations can significantly impair the function and differentiation capacity of MSCs. In addition, excessive or unbalanced metal ion concentrations can lead to oxidative stress, which can affect viability or inflammation. Data on the effects of metal ions on MSC differentiation are limited and often contradictory. Future research should, therefore, aim to clarify the mechanisms by which metal ions affect MSC differentiation, focusing on aspects such as metal ion interactions, ion concentrations, exposure duration, and other environmental conditions. Understanding these interactions could ultimately improve the design of biomaterials and implants to promote MSC-mediated tissue regeneration. It could also lead to the development of innovative therapeutic strategies in regenerative medicine.

Novel integrated workflow allows production and in-depth quality assessment of multifactorial reprogrammed skeletal muscle cells from human stem cells.

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach.

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.

BMP-2 Long-Term Stimulation of Human Pre-Osteoblasts Induces Osteogenic Differentiation and Promotes Transdifferentiation and Bone Remodeling Processes.

Bone morphogenic protein (BMP-) 2 plays an important role in the regeneration of bone defects by promoting osteogenic differentiation. However, several animal studies have reported adverse side effects of BMP-2, including osteoclast activation, induction of peroxisome proliferator- activated receptor gamma (PPARG)expression, and inflammation. High BMP-2 concentrations are thought to be responsible for these side effects. For this reason, primary pre-osteoblasts were exposed to lower BMP-2 concentrations (1 and 2 µg/mL). Long-term exposure (up to 28 days) was performed to investigate whether this stimulation protocol may promote osteogenic differentiation without causing the side effects mentioned above. The results showed that BMP-2 treatment for 14 or 28 days resulted in increased osteogenesis, through an increase in runt-related transcription factor 2, osterix, alkaline phosphatase, and integrin-binding sialoprotein expression. However, an increase in tumor necrosis factor alpha and receptor activator of nuclear factor kappa-Β ligand protein levels was observed after BMP-2 exposure, indicating also an increased potential for osteoclast activation by osteoblasts. Additionally, morphological changes like intracellular, filled vacuoles could be detected. Enhanced PPARG and perilipin 1 mRNA transcripts and lipid droplets indicated an induced adipogenic differentiation. Overall, the data demonstrate that long-term BMP-2 exposure promotes not only osteogenic differentiation but also adipogenesis and regulates mediators involved in osteoclast activation in vitro.

Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro.

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.

Devitalizing Effect of High Hydrostatic Pressure on Human Cells-Influence on Cell Death in Osteoblasts and Chondrocytes.

Chemical and physical processing of allografts is associated with a significant reduction in biomechanics. Therefore, treatment of tissue with high hydrostatic pressure (HHP) offers the possibility to devitalize tissue gently without changing biomechanical properties. To obtain an initial assessment of the effectiveness of HHP treatment, human osteoblasts and chondrocytes were treated with different HHPs (100-150 MPa, 250-300 MPa, 450-500 MPa). Devitalization efficiency was determined by analyzing the metabolic activity via WST-1(water-soluble tetrazolium salt) assay. The type of cell death was detected with an apoptosis/necrosis ELISA (enzyme-linked immune sorbent assay) and flow cytometry. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were carried out to detect the degree of cell destruction. After HHP treatment, the metabolic activities of both cell types decreased, whereas HHP of 250 MPa and higher resulted in metabolic inactivation. Further, the highest HHP range induced mostly necrosis while the lower HHP ranges induced apoptosis and necrosis equally. FESEM and TEM analyses of treated osteoblasts revealed pressure-dependent cell damage. In the present study, it could be proven that a pressure range of 250-300 MPa can be used for cell devitalization. However, in order to treat bone and cartilage tissue gently with HHP, the results of our cell experiments must be verified for tissue samples in future studies.

Alternating Electric Fields Modify the Function of Human Osteoblasts Growing on and in the Surroundings of Titanium Electrodes.

Endogenous electric fields created in bone tissue as a response to mechanical loading are known to influence the activity and differentiation of bone and precursor cells. Thus, electrical stimulation offers an adjunct therapy option for the promotion of bone regeneration. Understanding the influence of electric fields on bone cell function and the identification of suitable electrical stimulation parameters are crucial for the clinical success of stimulation therapy. Therefore, we investigated the impact of alternating electric fields on human osteoblasts that were seeded on titanium electrodes, which delivered the electrical stimulation. Moreover, osteoblasts were seeded on collagen-coated coverslips near the electrodes, representing the bone stock surrounding the implant. Next, 0.2 V, 1.4 V, or 2.8 V were applied to the in vitro system with 20 Hz frequency. After one, three, and seven days, the osteoblast morphology and expression of osteogenic genes were analysed. The actin organisation, as well as the proliferation, were not affected by the electrical stimulation. Changes in the gene expression and protein accumulation after electrical stimulation were voltage-dependent. After three days, the osteogenic gene expression and alkaline phosphatase activity were up to 2.35-fold higher following the electrical stimulation with 0.2 V and 1.4 V on electrodes and coverslips compared to controls. Furthermore, collagen type I mRNA, as well as the amount of the C-terminal propeptide of collagen type I were increased after the stimulation with 0.2 V and 1.4 V, while the higher electrical stimulation with 2.8 V led to decreased levels, especially on the electrodes.

Label-Free Monitoring of Uptake and Toxicity of Endoprosthetic Wear Particles in Human Cell Cultures.

The evaluation of the biological effects of endoprosthetic wear particles on cells in vitro relies on a variety of test assays. However, most of these methods are susceptible to particle-induced interferences; therefore, label-free testing approaches emerge as more reliable alternatives. In this study, impedance-based real-time monitoring of cellular viability and metabolic activity were performed following exposure to metallic and ceramic wear particles. Moreover, label-free imaging of particle-exposed cells was done by high-resolution darkfield microscopy (HR-ODM) and field emission scanning electron microscopy (FESEM). The isolated human fibroblasts were exposed to CoCr28Mo6 and alumina matrix composite (AMC) ceramic particles. HR-ODM and FESEM revealed ingested particles. For impedance measurements, cells were seeded on gold-plated microelectrodes. Cellular behavior was monitored over a period of 48 h. CoCr28Mo6 and AMC particle exposure affected cell viability in a concentration-dependent manner, i.e., 0.01 mg/mL particle solutions led to small changes in cell viability, while 0.05 mg/mL resulted in a significant reduction of viability. The effects were more pronounced after exposure to CoCr28Mo6 particles. The results were in line with light and darkfield microcopy observations indicating that the chosen methods are valuable tools to assess cytotoxicity and cellular behavior following exposure to endoprosthetic wear particles.

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro.

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.

Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts.

Introduction: The release of mature interleukin (IL-) 1ß from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals. Methods: Temporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data. Results: Exposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1ß was unaffected after contact with CoCr particles. Discussion: Although CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts.

Advanced Ti-Nb-Ta Alloys for Bone Implants with Improved Functionality.

The additive manufacturing of titanium-niobium-tantalum alloys with nominal chemical compositions Ti-xNb-6Ta (x = 20, 27, 35) by means of laser beam powder bed fusion is reported, and their potential as implant materials is elaborated by mechanical and biological characterization. The properties of dense specimens manufactured in different build orientations and of open porous Ti-20Nb-6Ta specimens are evaluated. Compression tests indicate that strength and elasticity are influenced by the chemical composition and build orientation. The minimum elasticity is always observed in the 90° orientation. It is lowest for Ti-20Nb-6Ta (43.2 ± 2.7 GPa) and can be further reduced to 8.1 ± 1.0 GPa for open porous specimens (p < 0.001). Furthermore, human osteoblasts are cultivated for 7 and 14 days on as-printed specimens and their biological response is compared to that of Ti-6Al-4V. Build orientation and cultivation time significantly affect the gene expression profile of osteogenic differentiation markers. Incomplete cell spreading is observed in specimens manufactured in 0° build orientation, whereas widely stretched cells are observed in 90° build orientation, i.e., parallel to the build direction. Compared to Ti-6Al-4V, Ti-Nb-Ta specimens promote improved osteogenesis and reduce the induction of inflammation. Accordingly, Ti-xNb-6Ta alloys have favorable mechanical and biological properties with great potential for application in orthopedic implants.

Systematic enhancement of microbial decontamination efficiency in bone graft processing by means of high hydrostatic pressure using Escherichia coli as a model organism.

To obtain bone allografts that are safe for transplantation, several processing steps for decellularization and decontamination have to be applied. Currently available processing methods, although well-established, may interfere with the biomechanical properties of the bone. High hydrostatic pressure (HHP) is known to devitalize tissues effectively while leaving the extracellular matrix intact. However, little is known about the inactivation of the contaminating microorganisms by HHP. This study aims to investigate the ability of high-pressure decontamination and to establish a treatment protocol that is able to successfully inactivate microorganisms with the final goal to sterilize bone specimens. Using Escherichia coli (E. coli) as a model organism, HHP treatment parameters like temperature and duration, pressurization medium, and the number of treatment cycles were systematically adjusted to maximize the efficiency of inactivating logarithmic and stationary phase bacteria. Towards that we quantified colony-forming units (cfu) after treatment and investigated morphological changes via Field Emission Scanning Electron Microscopy (FESEM). Additionally, we tested the decontamination efficiency of HHP in bovine cancellous bone blocks that were contaminated with bacteria. Finally, two further model organisms were evaluated, namely Pseudomonas fluorescens as a Gram-negative microorganism and Micrococcus luteus as a Gram-positive representative. A HHP protocol, using 350 MPa, was able to sterilize a suspension of stationary phase E. coli, leading to a logarithmic reduction factor (log RF) of at least -7.99 (±0.43). The decontamination of bone blocks was less successful, indicating a protective effect of the surrounding tissue. Sterilization of 100% of the samples was achieved when a protocol optimized in terms of treatment temperature, duration, pressurization medium, and number and/or interval of cycles, respectively, was applied to bone blocks artificially contaminated with a suspension containing 104 cfu/mL. Hence, we here successfully established protocols for inactivating Gram-negative model microorganisms by HHP of up to 350 MPa, while pressure levels of 600 MPa were needed to inactivate the Gram-positive model organism. Thus, this study provides a basis for further investigations on different pathogenic bacteria that could enable the use of HHP in the decontamination of bone grafts intended for transplantation.

In vivo endocultivation of CAD/CAM hybrid scaffolds in the omentum majus in miniature pigs.

PURPOSE: Correction of bony mandibular defects is a challenge in oral and maxillofacial surgery due to aesthetic and functional requirements. This study investigated the potential of a novel hybrid scaffold for bone regeneration and degradation assessment of the ceramic within the omentum majus over 6 months and the extent to which rhBMP-2 as a growth factor, alone or combined with a hydrogel, affects regeneration. MATERIALS AND METHODS: In this animal study, 10 Göttingen minipigs each had one scaffold implanted in the greater omentum. Five animals had scaffolds loaded with a collagen hydrogel and rhBMP-2, and the other five animals (control group) had scaffolds loaded with rhBMP-2 only. Fluorochrome injections and computed tomography (CT) were performed regularly. After 6 months, the animals were euthanized, and samples were collected for microCT and histological evaluations. RESULTS: Fluorescent and light microscopic and a CT morphological density evaluation showed continuous bone growth until week 16 in both groups. Regarding the ratio of bone attachment to the Zr02 support struts, the rhBMP-2 loaded collagen hydrogel group showed with 63% a significantly higher attachment (p > 0.001) than the rhBMP-2 control group (49%). CONCLUSION: In this study, bone growth was induced in all omentum majus specimens until post-operative week 16. Furthermore, hydrogel and rhBMP-2 together resulted in better bone-scaffold integration than rhBMP-2 alone. Further studies should investigate whether implantation of the scaffolds in the jaw after an appropriate period of bone regeneration leads to a stable situation and the desired results.

High hydrostatic pressure treatment for advanced tissue grafts in reconstructive head and neck surgery.

The increasing importance of regenerative medicine has resulted in a growing need for advanced tissue replacement materials in head and neck surgery. Allo- and xenogenic graft processing is often time-consuming and can deteriorate the extracellular matrix (ECM). High hydrostatic pressure (HHP)-treatment could allow specific devitalization while retaining the essential properties of the ECM. Porcine connective tissue and cartilage were HHP-treated at 100-400 MPa for 10 min. Structural modifications following HHP-exposure were examined using electron microscopy, while devitalization was assessed through metabolism and cell death analyses. Furthermore, ECM alterations and decellularization were evaluated by histology, biomechanical testing, and DNA content analysis. Additionally, the inflammatory potential of HHP-treated tissue was evaluated in vivo using a dorsal skinfold chamber in a mouse model. The devitalization effects of HHP were dose-dependent, with a threshold identified at 200 MPa for fibroblasts and chondrocytes. At this pressure level, HHP induced structural alterations in cells, with a shift toward late-stage apoptosis. HHP-treatment preserved ECM structure and biomechanical properties, but did not remove cell debris from the tissue. This study observed a pressure-dependent increase of markers suggesting the occurrence of immunogenic cell death. In vivo investigations revealed an absence of inflammatory responses to HHP-treated tissue, indicating a favorable biological response to HHP. In conclusion, application of HHP devitalizes fibroblasts and chondrocytes at 200 MPa while retaining the essential properties of the ECM. Prospectively, HHP may simplify the preparation of allo- and xenogenic tissue replacement materials and increase the availability of grafts in head and neck surgery.

Bone regeneration in critical-size defects of the mandible using biomechanically adapted CAD/CAM hybrid scaffolds: An in vivo study in miniature pigs.

The study aimed to analyze bone regeneration in critical-size defects using hybrid scaffolds biomechanically adapted to the specific defect and adding the growth factor rhBMP-2. For this animal study, ten minipigs underwent bilateral defects in the corpus mandibulae and were subsequently treated with novel cylindrical hybrid scaffolds. These scaffolds were designed digitally to suit the biomechanical requirements of the mandibular defect, utilizing finite element analysis. The scaffolds comprised zirconium dioxide-tricalcium phosphate (ZrO2-TCP) support struts and TCP foam ceramics. One scaffold in each animal was loaded with rhBMP-2 (100 µg/cm³), while the other served as an unloaded negative control. Fluorescent dyes were administered every 2 weeks, and computed tomography (CT) scans were conducted every 4 weeks. Euthanasia was performed after 3 months, and samples were collected for examination using micro-CT and histological evaluation of both hard and soft tissue. Intravital CT examinations revealed minor changes in radiographic density from 4 to 12 weeks postoperatively. In the group treated with rhBMP-2, radiographic density shifted from 2513 ± 128 (mean ± SD) to 2606 ± 115 Hounsfield units (HU), while the group without rhBMP-2 showed a change from 2430 ± 131 to 2601 ± 67 HU. Prior to implantation, the radiological density of samples measured 1508 ± 30 mg HA/cm³, whereas post-mortem densities were 1346 ± 71 mg HA/cm³ in the rhBMP-2 group and 1282 ± 91 mg HA/cm³ in the control group (p = 0.045), as indicated by micro-CT measurements. The histological assessment demonstrated successful ossification in all specimens. The newly formed bone area proportion was significantly greater in the rhBMP-2 group (48 ± 10%) compared with the control group without rhBMP-2 (42 ± 9%, p = 0.03). The mean area proportion of remaining TCP foam was 23 ± 8% with rhBMP-2 and 24 ± 10% without rhBMP-2. Successful bone regeneration was accomplished by implanting hybrid scaffolds into critical-size mandibular defects. Loading these scaffolds with rhBMP-2 led to enhanced bone regeneration and a uniform distribution of new bone formation within the hybrid scaffolds. Further studies are required to determine the adaptability of hybrid scaffolds for larger and potentially segmental defects in the maxillofacial region.

Multifunctional Hybrid Material for Endoprosthetic Implants Based on Alumina-Toughened Zirconia Ceramics and Additively Manufactured TiNbTa Alloys.

Aseptic implant loosening after a total joint replacement is partially influenced by material-specific factors when cobalt-chromium alloys are used, including osteolysis induced by wear and corrosion products and stress shielding. Here, we aim to characterize a hybrid material consisting of alumina-toughened zirconia (ATZ) ceramics and additively manufactured Ti-35Nb-6Ta (TiNbTa) alloys, which are joined by a glass solder. The structure of the joint, the static and fatigue shear strength, the influence of accelerated aging, and the cytotoxicity with human osteoblasts are characterized. Furthermore, the biomechanical properties of the functional demonstrators of a femoral component for total knee replacements are evaluated. The TiNbTa-ATZ specimens showed a homogenous joint with statistically distributed micro-pores and a slight accumulation of Al-rich compounds at the glass solder-TiNbTa interface. Shear strengths of 26.4 ± 4.2 MPa and 38.2 ± 14.4 MPa were achieved for the TiNbTa-ATZ and Ti-ATZ specimens, respectively, and they were not significantly affected by the titanium material used, nor by accelerated aging (p = 0.07). All of the specimens survived 107 cycles of shear loading to 10 MPa. Furthermore, the TiNbTa-ATZ did not impair the proliferation and metabolic activity of the human osteoblasts. Functional demonstrators made of TiNbTa-ATZ provided a maximum bearable extension-flexion moment of 40.7 ± 2.2 Nm. The biomechanical and biological properties of TiNbTa-ATZ demonstrate potential applications for endoprosthetic implants.

Isolation of TiNbN wear particles from a coated metal-on-metal bearing: Morphological characterization and in vitro evaluation of cytotoxicity in human osteoblasts.

To improve the wear resistance of articulating metallic joint endoprostheses, the surfaces can be coated with titanium niobium nitride (TiNbN). Under poor tribological conditions or malalignment, wear can occur on these implant surfaces in situ. This study investigated the biological response of human osteoblasts to wear particles generated from TiNbN-coated hip implants. Abrasive particles were generated in a hip simulator according to ISO 14242-1/-2 and extracted with Proteinase K. Particle characteristics were evaluated by electron microscopy and energy dispersive x-ray spectroscopy (EDS), inductively coupled plasma mass spectrometry (ICP-MS) and dynamic light scattering (DLS) measurements. Human osteoblasts were exposed to different particle dilutions (1:20, 1:50, and 1:100), and cell viability and gene expression levels of osteogenic markers and inflammatory mediators were analyzed after 4 and 7 days. Using ICP-MS, EDS, and DLS measurements, ~70% of the particles were identified as TiNbN, ranging from 39 to 94 nm. The particles exhibited a flat and subangular morphology. Exposure to particles did not influence cell viability and osteoblastic differentiation capacity. Protein levels of collagen type 1, osteoprotegerin, and receptor activator of nuclear factor κB ligand were almost unaffected. Moreover, the pro-inflammatory response via interleukins 6 and 8 was minor induced after particle contact. A high number of TiNbN wear particles only slightly affected osteoblasts' differentiation ability and inflammatory response compared to metallic particles. Nevertheless, further studies should investigate the role of these particles in peri-implant bone tissue, especially concerning other cell types.

IL-6-induced response of human osteoblasts from patients with rheumatoid arthritis after inhibition of the signaling pathway.

Interleukin (IL-) 6 is a critical factor in inflammatory processes of rheumatoid arthritis (RA). This is of high interest as the progression of RA may lead to the implantation of joint endoprostheses, which is associated with a pro-inflammatory increase in IL-6 in the periprosthetic tissue. Biological agents such as sarilumab have been developed to inhibit IL-6-mediated signaling. However, IL-6 signaling blockade should consider the inhibition of inflammatory processes and the regenerative functions of IL-6. This in vitro study investigated whether inhibiting IL-6 receptors can affect the differentiation of osteoblasts isolated from patients with RA. Since wear particles can be generated at the articular surfaces of endoprostheses leading to osteolysis and implant loosening, the potential of sarilumab to inhibit wear particle-induced pro-inflammatory processes should be investigated. Both in monocultures and indirect co-cultures with osteoclast-like cells (OLCs), human osteoblasts were stimulated with 50 ng/mL each of IL-6 + sIL-6R and in combination with sarilumab (250 nM) to characterize cell viability and osteogenic differentiation capacity. Furthermore, the influence of IL-6 + sIL-6R or sarilumab on viability, differentiation, and inflammation was evaluated in osteoblasts exposed to particles. Stimulation with IL-6 + sIL-6R and sarilumab did not affect cell viability. Except for the significant induction of RUNX2 mRNA by IL-6 + sIL-6R and a significant reduction with sarilumab, no effects on cell differentiation and mineralization could be detected. Furthermore, the different stimulations did not affect the osteogenic and osteoclastic differentiation of co-cultured cells. Compared to the osteoblastic monocultures, a decreased release of IL-8 was triggered in the co-culture. Among these, treatment with sarilumab alone resulted in the greatest reduction of IL-8. The co-culture also showed clearly increased OPN concentrations than the respective monocultures, with OPN secretion apparently triggered by the OLCs. Particle exposure demonstrated decreased osteogenic differentiation using different treatment strategies. However, sarilumab administration caused a trend toward a decrease in IL-8 production after stimulation with IL-6 + sIL-6R. The blockade of IL-6 and its pathway have no significant effect on the osteogenic and osteoclastic differentiation of bone cells derived from patients with RA. Nonetheless, observed effects on the reduced IL-8 secretion need further investigation.

Corrosion Products from Metallic Implants Induce ROS and Cell Death in Human Motoneurons In Vitro.

Due to advances in surgical procedures and the biocompatibility of materials used in total joint replacement, more and younger patients are undergoing these procedures. Although state-of-the-art joint replacements can last 20 years or longer, wear and corrosion is still a major risk for implant failure, and patients with these implants are exposed for longer to these corrosive products. It is therefore important to investigate the potential effects on the whole organism. Released nanoparticles and ions derived from commonly used metal implants consist, among others, of cobalt, nickel, and chromium. The effect of these metallic products in the process of osteolysis and aseptic implant loosening has already been studied; however, the systemic effect on other cell types, including neurons, remains elusive. To this end, we used human iPSC-derived motoneurons to investigate the effects of metal ions on human neurons. We treated human motoneurons with ion concentrations regularly found in patients, stained them with MitoSOX and propidium iodide, and analyzed them with fluorescence-assisted cell sorting (FACS). We found that upon treatment human motoneurons suffered from the formation of ROS and subsequently died. These effects were most prominent in motoneurons treated with 500 µM of cobalt or nickel, in which we observed significant cell death, whereas chromium showed fewer ROS and no apparent impairment of motoneurons. Our results show that the wear and corrosive products of metal implants at concentrations readily available in peri-implant tissues induced ROS and subsequently cell death in an iPSC-derived motoneuron cell model. We therefore conclude that monitoring of neuronal impairment is important in patients undergoing total joint replacement.