A new bone substitute developed from 3D-prints of polylactide (PLA) loaded with collagen I : an in vitro study

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering

The influence of bone substitute materials on the bone volume after maxillary sinus augmentation: a microcomputerized tomography study

Objectives: This study aims to evaluate the effect of adding bone substitute materials (BSM) to particulated autogenous bone (PAB) on the volume fraction (Vf) of newly formed bone after maxillary sinus augmentation. Materials and methods: Thirty healthy patients undergoing maxillary sinus augmentation were included. PAB (N = 10), mixtures of PAB and beta-tricalciumphosphate (PAB/β-TCP) (N = 10), as well as PAB and β-TCP and hydroxyapatite (PAB/HA/β-TCP) (N = 10) were randomly used for sinus augmentation. A sample of the graft material was maintained from each patient at time of maxillary sinus augmentation, and Vfs of the PAB and/or BSM in the samples were determined by means of microcomputerized tomography (μ-CT). Five months later, samples of the grafted areas were harvested during implantation using a trephine bur. μ-CT analysis of these samples was performed, and the Vf of bone and BSM were compared with the data obtained 5months earlier from the original material. Results: The mean Vf of the bone showed a statistically significant increase (p < 0.05) in all groups after a healing period of 5months without statistically significant difference between the groups. Conclusions: With regard to the increase of bone volume, it is not relevant if PAB is used alone or combined with β-TCP or HA/β-TCP. Clinical relevance: The amount of PAB and associated donor site morbidity may be reduced by adding BSM for maxillary sinus augmentatio

Evaluation of bone sialoprotein coating of three-dimensional printed calcium phosphate scaffolds in a calvarial defect model in mice

The bioactive coating of calcium phosphate cement (CPC) is a promising approach to enhance the bone-healing properties of bone substitutes. The purpose of this study was to evaluate whether coating CPCs with bone sialoprotein (BSP) results in increased bone formation. Forty-five female C57BL/6NRj mice with an average age of six weeks were divided into three groups. Either a BSP-coated or an uncoated three-dimensional plotted scaffold was implanted into a drilled 2.7-mm diameter calvarial defect, or the defect was left empty (control group; no CPC). Histological analyses revealed that BSP-coated scaffolds were better integrated into the local bone stock eight weeks after implantation. Bone volume/total volume (BV/TV) ratios and bone thickness at the bone-implant contact were analyzed via micro computed tomography (µCT) after eight weeks. BSP-coated scaffolds and uncoated CPC scaffolds increased bone thickness in comparison to the control (CPC + BSP: 691.1 ± 253.5 µm, CPC: 603.1 ± 164.4 µm, no CPC: 261.7 ± 37.8 µm, p < 0.01). Accordingly, BV/TV was enhanced in both scaffold groups (CPC + BSP: 1.3 ± 0.5%, CPC: 0.9 ± 0.5%, no CPC: 0.2 ± 0.3%, p < 0.01). The BSP coating showed a tendency towards an increased bone thickness (p = 0.18) and BV/TV (p = 0.18) in comparison to uncoated CPC scaffolds. However, a significant increase in bone formation through BSP coating was not found

"Sind wir so unwichtig?": Fußballfans zwischen Tradition und Kommerz

Die Verfasser geben einen breit angelegten Überblick über die Entwicklung des Fußballsports und vor allem über seine Kommerzialisierung seit Beginn des 20. Jahrhunderts. Die Voraussetzungen für die beschleunigte Kommerzialisierung wurden in den 1960er Jahren gelegt. Eine kurzfristige Delle im Zuschauerinteressen in den 1980er Jahren wurde zu Beginn der 1990er durch den "Verkauf des Fußballs an das Fernsehen" überwunden, mit dem dem Fußball neue, "erlebnisorientierte" Zuschauerschichten erschlossen wurden. Für die heutigen Zuschauer ist die Differenzierung in "traditionelle" und "neue" Fans typisch. Im Sinne der "Eventisierung" des Fußballs werden die traditionellen Fans mit ihrer Farbenpracht und ihren Gesängen gebraucht, um den "Event" für die "neuen" Fans perfekt zu machen. Einstellungen "traditioneller" Fans werden anhand von Aussagen aus qualitativen Interviews illustriert. (ICE

Clinical and Imaging Characteristics in Patients with SARS-CoV-2 Infection and Acute Intracranial Hemorrhage

Background and purpose: Intracranial hemorrhage has been observed in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19), but the clinical, imaging, and pathophysiological features of intracranial bleeding during COVID-19 infection remain poorly characterized. This study describes clinical and imaging characteristics of patients with COVID-19 infection who presented with intracranial bleeding in a European multicenter cohort. Methods: This is a multicenter retrospective, observational case series including 18 consecutive patients with COVID-19 infection and intracranial hemorrhage. Data were collected from February to May 2020 at five designated European special care centers for COVID-19. The diagnosis of COVID-19 was based on laboratory-confirmed diagnosis of SARS-CoV-2. Intracranial bleeding was diagnosed on computed tomography (CT) of the brain within one month of the date of COVID-19 diagnosis. The clinical, laboratory, radiologic, and pathologic findings, therapy and outcomes in COVID-19 patients presenting with intracranial bleeding were analyzed. Results: Eighteen patients had evidence of acute intracranial bleeding within 11 days (IQR 9-29) of admission. Six patients had parenchymal hemorrhage (33.3%), 11 had subarachnoid hemorrhage (SAH) (61.1%), and one patient had subdural hemorrhage (5.6%). Three patients presented with intraventricular hemorrhage (IVH) (16.7%). Conclusion: This study represents the largest case series of patients with intracranial hemorrhage diagnosed with COVID-19 based on key European countries with geospatial hotspots of SARS-CoV-2. Isolated SAH along the convexity may be a predominant bleeding manifestation and may occur in a late temporal course of severe COVID-19

Modulation of Osteogenic Cell Morphology by ECM Ligands and Enamel Matrix Derivative

Preduvjet za uspješnu regeneraciju parodontnog tkiva jest odgovarajuća aktivacija populacije za to odgovornih stanica, poput osteoblasta. U tom su slučaju stanična adhezija i sazrijevanje usko povezani s morfologijom stanica i f-aktinskom organizacijom citoskeleta. Mogućnost pojačavanja parodontalnog cijeljenja pomoću pojedinih komponenti izvanstaničnog matriksa (ECM) i derivata caklinskog matriksa dobro je dokumentirana u literaturi. Svrha rada: Svrha ovog istraživanja bila je testirati učinak ECM-bjelančevina, kolagena tipa I, laminina-1 te komercijalnog proizvoda EMD-a na morfološku i citoskeletnu organizaciju osteogenih stanica. Ispitanici i postupci: Tijekom promatranja analizirano je ukupno 2450 osteogenih stanica iz pet različitih staničnih linija (četiri primarnih i jedne komercijalne) kultiviranih na pojedinim supstratima, a analizirali su ih tri neovisna promatrača. Nakon bojenja za f-aktin staničnog citoskeleta i automatizirane CLSM-vizualizacije, stanice su podijeljene u tri skupine ovisno o njihovim morfološkim svojstvima (nezrele, prijelazne i zrele). Osim deskriptivne analize obavljena je bila i multivarijantna logička regresija radi identificiranja odgovarajućih parametara koji utječu na staničnu morfologiju i organizaciju citoskeleta. Rabljeni pojedinačni ligandi kolagena i laminina te posebice EMD poticali su stvaranje zrelog osteogenog fenotipa, premda su bile uočene i određene razlike među korištenim staničnim linijama. Rezultati: Analiza morfologije i citoskeleta pouzdan je način skupljanja prvih podataka o biokompatibilnosti i bioaktivaciji stanica na različitim supstratima. Naši rezultati upućuju na mogući potencijal istraživanih liganada u pojačavanju osteogenoga staničnog pričvrstka i sazrijevanju te time i pomaganju cijeljenja parodontnog tkiva.The precondition for successful periodontal regeneration is adequate activation of relevant cell populations like osteogenic cells. Here, cell adhesion and maturation are closely associated with cell morphology and f-actin cytoskeletal organisation. The potential of solitaire extracellular matrix (ECM) components as well as enamel matrix derivative (EMD) to enhance periodontal healing is well documented. Objective: The aim of the study was to test the impact of the ECM proteins collagen type 1 and laminin-1 as well as commercially available EMD on osteogenic cell morphology and cytoskeletal organisation. Material and methods: In an observational study, a total of 2450 osteogenic cells of 5 different cell lines (4 primary ones and 1 commercial one) cultivated on the respective substrates were analysed by 3 independent observers. After staining for the f-actin cytoskeleton and automated CLSM visualisation, cells were assigned to 3 different categories depending on morphological cell attributes (immature vs. intermediate vs. mature). Besides descriptive analysis, a multivariate logistic regression was performed to identify relevant influence parameters on cell morphology and cytoskeletal organisation. Results: The applied solitaire ligands collagen and laminin and especially EMD promoted a mature osteogenic phenotype. Nevertheless, considerable differences between the investigated cell lines could be identified as well. Analysis of cell morphology and cytoskeletal organisation offers a reliable method of acquiring the first hints of biocompatibility and bio-activation on different substrates. Conclusion: Our results highlight the potential of the investigated ligands to support periodontal regeneration by enhancing osteogenic cell attachment and maturation

In vitro evaluation of various bioabsorbable and nonresorbable barrier membranes for guided tissue regeneration

<p>Abstract</p> <p>Background</p> <p>Different types of bioabsorbable and nonresorbable membranes have been widely used for guided tissue regeneration (GTR) with its ultimate goal of regenerating lost periodontal structures. The purpose of the present study was to evaluate the biological effects of various bioabsorbable and nonresorbable membranes in cultures of primary human gingival fibroblasts (HGF), periodontal ligament fibroblasts (PDLF) and human osteoblast-like (HOB) cells <it>in vitro</it>.</p> <p>Methods</p> <p>Three commercially available collagen membranes [TutoDent^®(TD), Resodont^®(RD) and BioGide^®(BG)] as well as three nonresorbable polytetrafluoroethylene (PTFE) membranes [ACE (AC), Cytoplast^®(CT) and TefGen-FD^®(TG)] were tested. Cells plated on culture dishes (CD) served as positive controls. The effect of the barrier membranes on HGF, PDLF as well as HOB cells was assessed by the Alamar Blue fluorometric proliferation assay after 1, 2.5, 4, 24 and 48 h time periods. The structural and morphological properties of the membranes were evaluated by scanning electron microscopy (SEM).</p> <p>Results</p> <p>The results showed that of the six barriers tested, TD and RD demonstrated the highest rate of HGF proliferation at both earlier (1 h) and later (48 h) time periods (<it>P </it>< 0.001) compared to all other tested barriers and CD. Similarly, TD, RD and BG had significantly higher numbers of cells at all time periods when compared with the positive control in PDLF culture (<it>P </it>≤ 0.001). In HOB cell culture, the highest rate of cell proliferation was also calculated for TD at all time periods (<it>P </it>< 0.001). SEM observations demonstrated a microporous structure of all collagen membranes, with a compact top surface and a porous bottom surface, whereas the nonresorbable PTFE membranes demonstrated a homogenous structure with a symmetric dense skin layer.</p> <p>Conclusion</p> <p>Results from the present study suggested that GTR membrane materials, per se, may influence cell proliferation in the process of periodontal tissue/bone regeneration. Among the six membranes examined, the bioabsorbable membranes demonstrated to be more suitable to stimulate cellular proliferation compared to nonresorbable PTFE membranes.</p

Supplementary Appendix. All-trans retinoic acid works synergistically with the γ- secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells

Supplement Figure 1: ATRA treatment does not affect the viability of myeloma cell lines. MM.1S, OPM-2 and NCI-H929 cells were treated with ATRA for up to 72 hours. Cell viability was measured by flow cytometry and 7AAD staining (n=6). Bar diagrams show mean values +SD.Supplement Figure 2: ATRA plus crenigacestat treatment enhance BCMA expression on myeloma cell lines. Bar diagram shows BCMA expression on OPM-2 cells (n=3) after treatment with 100 nM ATRA and/or 10 nM GSI crenigacestat for 72 hours. Bar diagram shows mean values +SD. P-values between indicated groups were calculated using unpaired t-test. *p<0.05, **p<0.01.Supplement Figure 3: ATRA treatment leads to increased BCMA transcripts in OPM-2 myeloma cells. BCMA RNA levels in OPM-2 were analyzed by quantitative reverse transcription PCR (qRT-PCR) assay after incubation with increasing doses of ATRA for 48 hours (n=3). Bar diagram shows mean values +SD. P-values between indicated groups were calculated using unpaired t-test. *p<0.05.Supplement Figure 4: ATRA treatment leads to enhanced BCMA expression on primary myeloma cells. Representative flow cytometric analysis of BCMA expression on primary myeloma cells that had been cultured in the absence or presence of ATRA at different concentrations for 72 hours. 7-AAD was used to exclude dead cells from analysis.Supplement Figure 5: ATRA treatment does not impair viability of primary myeloma cells. Viability of primary myeloma cells with or without 72 hours of ATRA treatment was analyzed by flow cytometry and 7-AAD staining (n=5 biological replicates). Bar diagram shows mean values +SD.Supplement Figure 6: sBCMA does not impair BCMA CAR T cell functionality. CD8+ BCMA-CAR T-cells were co-cultured with MM.1S target cells in absence or presence of 150 ng/ml of soluble BCMA. After 4 hours, cytotoxicity was evaluated by bioluminescence- based assay. Diagram shows mean values +/-SD.Supplement Figure 7: ATRA treatment does not increase shedding of sBCMA. sBCMA concentration in the supernatant of OPM-2 and NCI-H929 after incubation with increasing doses of ATRA was analyzed by ELISA. Cell lines were cultured at 1x106/well (n=3 technical replicates). Bar diagrams show mean values +SD, P-values between indicated groups were calculated using 2way ANOVA. n.s. = not significant, *p<0.05, **p<0.01.Supplement Figure 8: BCMA-CAR T-cells confer enhanced cytotoxicity against ATRA plus crenigacestat-treated OPM-2 cells in vitro. OPM-2 cells were incubated with 100 nM ATRA and/or 10 nM GSI for 72 hours or were left untreated. Cytolytic activity of CD8+ BCMA- CAR T-cells was determined in a bioluminescence-based assay after 4h of co-incubation with target cells. Assay was performed in triplicate wells with 5,000 target cells per well. Data are presented as mean values +SD (n=4 biological replicates). P-values between indicated groups were calculated using unpaired t-test. n.s. = not significant, *p<0.05.Supplement Figure 9: Patient-derived BCMA-CAR T-cells confer enhanced cytotoxicity against ATRA-treated MM.1S cells. MM.1S cells were incubated with 50 nM ATRA for 72 hours or were left untreated. Cytolytic activity of MM patient-derived CD8+ BCMA-CAR T-cells was determined in a bioluminescence-based assay after 4h of co-incubation with target cells. Data are presented as mean values +SD of triplicate wells. P-values between indicated groups were calculated using unpaired t-test. *p<0.05, **p<0.01.Peer reviewe

Predation Danger Can Explain Changes in Timing of Migration: The Case of the Barnacle Goose

Understanding stopover decisions of long-distance migratory birds is crucial for conservation and management of these species along their migratory flyway. Recently, an increasing number of Barnacle geese breeding in the Russian Arctic have delayed their departure from their wintering site in the Netherlands by approximately one month and have reduced their staging duration at stopover sites in the Baltic accordingly. Consequently, this extended stay increases agricultural damage in the Netherlands. Using a dynamic state variable approach we explored three hypotheses about the underlying causes of these changes in migratory behavior, possibly related to changes in (i) onset of spring, (ii) potential intake rates and (iii) predation danger at wintering and stopover sites. Our simulations showed that the observed advance in onset of spring contradicts the observed delay of departure, whereas both increased predation danger and decreased intake rates in the Baltic can explain the delay. Decreased intake rates are expected as a result of increased competition for food in the growing Barnacle goose population. However, the effect of predation danger in the model was particularly strong, and we hypothesize that Barnacle geese avoid Baltic stopover sites as a response to the rapidly increasing number of avian predators in the area. Therefore, danger should be considered as an important factor influencing Barnacle goose migratory behavior, and receive more attention in empirical studies