Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue

The regeneration of cartilage lesions still represents a major challenge. Cartilage has a tissue-specific architecture, complicating recreation by synthetic biomaterials. A novel approach for reconstruction is the use of devitalised cartilage. Treatment with high hydrostatic pressure (HHP) achieves devitalisation while biomechanical properties are remained. Therefore, in the present study, cartilage was devitalised using HHP treatment and the potential for revitalisation with chondrocytes and mesenchymal stem cells (MSCs) was investigated. The devitalisation of cartilage was performed by application of 480 MPa over 10 minutes. Effective cellular inactivation was demonstrated by the trypan blue exclusion test and DNA quantification. Histology and electron microscopy examinations showed undamaged cartilage structure after HHP treatment. For revitalisation chondrocytes and MSCs were cultured on devitalised cartilage without supplementation of chondrogenic growth factors. Both chondrocytes and MSCs significantly increased expression of cartilage- specific genes. ECM stainings showed neocartilage-like structure with positive AZAN staining as well as collagen type II and aggrecan deposition after three weeks of cultivation. Our results showed that HHP treatment caused devitalisation of cartilage tissue. ECM proteins were not influenced, thus, providing a scaffold for chondrogenic differentiation of MSCs and chondrocytes. Therefore, using HHP-treated tissue might be a promising approach for cartilage repair

Should I stay or should I go?:Navigating contradictory temporal logics in the Dutch asylum system

Temporal logics in migration policymaking become evident in migrants' oscillation between waiting and moving. Scholars explored temporal experiences of migrants stuck at borders, in asylum procedures and detention centres, showing how states use time to govern people's (im)mobility. Yet, there is little insight into what happens after migrants gain protection and how migration officials experience problems with time in their work. Drawing on the Netherlands as a case study, we analyse the implications of temporal logics in migration and integration policymaking for migrants moving through the asylum system and for migration officials. Building on temporal governance literature, we show that temporal logics can be ambiguous due to policies separating asylum-seeker reception from refugee integration. Asylum seeker reception is governed by an exclusionary migration policy, resulting in slow periods of waiting, whereas integration policy aims for refugees' fast inclusion leading to accelerated timelines. We challenge the idea of a clear-cut distinction between migration and integration temporalities, showing that differing temporal logics create in-between situations. For example, refugees may be expected to integrate while remaining 'stuck' in reception centres, sending conflicting messages about expectations, and leading to personal, mental, and health consequences. For migration officials, effective implementation is hindered when policy objectives differ significantly

Are small towns a battleground for migration governance?:Negotiating refugee integration in small towns in the Netherlands

A surge in refugee arrivals post-2015 challenged small localities across Europe to respond to ‘refugee integration’ locally. Existing literature focuses little on governance dynamics in small localities, particularly regarding actors’ roles and relations. Based on fieldwork in four Dutch localities, we analyse the involvement of different actor types and explore if/how they develop collaborative relations. We show that small town governments engage in different forms of collaborative governance, from consolidated actor networks and intensive collaboration between local governments and CSOs to fragmented networks with more passive governments. However, we also find conflicts among actors due to power asymmetries and diverging interests and values. We identify several factors shaping interaction patterns, including localities’ size, political orientation, political leadership, residents’ attitudes and municipal decision-making. Despite outsourcing tasks, local governments remain pivotal in integration governance, giving them a crucial role in creating inclusive, participatory spaces, preventing actors’ alienation and designing efficient policy responses

Educating physicians on strong opioids by descriptive versus simulated-experience formats: a randomized controlled trial

Background: Long-term prescriptions of strong opioids for chronic noncancer pain-which are not supported by scientific evidence-suggest miscalibrated risk perceptions among those who prescribe, dispense, and take opioids. Because risk perceptions and behaviors can differ depending on whether people learn about risks through description or experience, we investigated the effects of descriptive versus simulated-experience educative formats on physicians' risk perceptions of strong opioids and their prescription behavior for managing chronic noncancer pain. Methods: Three hundred general practitioners and 300 pain specialists in Germany-enrolled separately in two independent exploratory randomized controlled online trials-were randomly assigned to either a descriptive format (fact box) or a simulated-experience format (interactive simulation). Primary endpoints: Objective risk perception (numerical estimates of opioids' benefits and harms), actual prescriptions of seven therapy options for managing chronic pain. Secondary endpoint: Implementation of intended prescriptions of seven therapy options for managing chronic pain. Results: Both formats improved the proportion of correct numerical estimates of strong opioids' benefits and harms immediately after intervention, with no notable differences between formats. Compared to description, simulated experience led to significantly lower reported actual prescription rates for strong and/or weak opioids, and was more effective at increasing prescription rates for non-drug-based therapies (e.g., means of opioid reduction) from baseline to follow-up for both general practitioners and pain specialists. Simulated experience also resulted in a higher implementation of intended behavior for some drug-based and non-drug-based therapies. Conclusions: The two formats, which recruit different cognitive processes, may serve different risk-communication goals: If the goal is to improve exact risk perception, descriptive and simulated-experience formats are likely to be equally suitable. If, however, the goal is to boost less risky prescription habits, simulated experience may be the better choice

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.publishedVersio

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

IntroductionThe release of mature interleukin (IL-) 1β 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.MethodsTemporal 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.ResultsExposure 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-1β was unaffected after contact with CoCr particles.DiscussionAlthough 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

Biomimetic Calcium Phosphate Coatings for Bioactivation of Titanium Implant Surfaces: Methodological Approach and In Vitro Evaluation of Biocompatibility

Ti6Al4V as a common implant material features good mechanical properties and corrosion resistance. However, untreated, it lacks bioactivity. In contrast, coatings with calcium phosphates (CaP) were shown to improve cell–material interactions in bone tissue engineering. Therefore, this work aimed to investigate how to tailor biomimetic CaP coatings on Ti6Al4V substrates using modified biomimetic calcium phosphate (BCP) coating solutions. Furthermore, the impact of substrate immersion in a 1 M alkaline CaCl2 solution (pH = 10) on subsequent CaP coating formation was examined. CaP coatings were characterized via scanning electron microscopy, x-ray diffraction, energy-dispersive x-ray spectroscopy, and laser-scanning microscope. Biocompatibility of coatings was carried out with primary human osteoblasts analyzing cell morphology, proliferation, collagen type 1, and interleukin 6 and 8 release. Results indicate a successful formation of low crystalline hydroxyapatite (HA) on top of every sample after immersion in each BCP coating solution after 14 days. Furthermore, HA coating promoted cell proliferation and reduced the concentration of interleukins compared to the uncoated surface, assuming increased biocompatibility