The dysregulation of the Wnt/ß-catenin signaling pathway during chondro- and osteogenesis is associated with the development of osteoarthritis and hyperostosis in STR/ort mice

Der Wnt/ß-Catenin-Signalweg steuert die embryonale Skelettentwicklung und die Remodellierung der Knochenmasse. Vor diesem Hintergrund wurde untersucht, ob eine Fehlregulierung des Signalwegs 1) mit der beim STR/ort-Mausstamm beschriebenen Arthrose und 2) mit dem hier neu charakterisierten Knochenmassedefekt des Mausstamms assoziiert ist. Für Sfrp1, ein Antagonist des Signalwegs, wurden DNA-Sequenzabweichungen und eine differentielle Expression auf RNA- und Proteinebene während der Knorpel- und Knochenentwicklung identifiziert. Hieraus resultiert eine verstärkte Aktivierung des Wnt-Signalwegs.The Wnt/ß-catenin pathway regulates the embryonic skeletal development and the remodelling of the bone mass. In this context we wanted to know if a dysregulation of the signaling pathway 1) could be associated with osteoarthritis in STR/ort mice and 2) could be associated with the here newly characterized bone mass defect in these mice. For Sfrp1, an antagonist of the Wnt signaling pathway, we identified DNA sequence variations and a differential expression at the RNA- and protein level during cartilage and bone development. This results in an increased activation of the Wnt signaling pathway

Transient supplementation of growth factor TGF-β1 effectively initiates chondrogenic redifferentiation of human chondrocytes

Cartilage tissue is avascular with less regeneration potential and therefore, cartilage regeneration is still a major challenge for therapeutic approaches. Commonly used treatment strategies involve the transplantation of autologous chondrocytes into the defect. Before that, it is required to increase the cell number in vitro resulting in unwanted chondrocyte dedifferentiation. This could impair subsequent tissue regeneration. Both growth factors TGF-ß1 and IGF-1 are used as strong inducer of chondrogenic redifferentiation, however, a controlled application of TGF-ß1 is essential to avoid adverse effects. Therefore, in the present study, we investigated the time-dependent influence of TGF-ß1 administration on chondrocyte redifferentiation

Surface Modifications of Dental Ceramic Implants with Different Glass Solder Matrices: In Vitro Analyses with Human Primary Osteoblasts and Epithelial Cells

Ceramic materials show excellent esthetic behavior, along with an absence of hypersensitivity, making them a possible alternative implant material in dental surgery. However, their surface properties enable only limited osseointegration compared to titanium implants. Within this study, a novel surface coating technique for enhanced osseointegration was investigated biologically and mechanically. Specimens of tetragonal zirconia polycrystal (TZP) and aluminum toughened zirconia (ATZ) were modified with glass solder matrices in two configurations which mainly consisted of SiO2, Al2O3, K2O, and Na2O. The influence on human osteoblastic and epithelial cell viability was examined by means of a WST-1 assay as well as live/dead staining. A C1CP-ELISA was carried out to verify procollagen type I production. Uncoated/sandblasted ceramic specimens and sandblasted titanium surfaces were investigated as a reference. Furthermore, mechanical investigations of bilaterally coated pellets were conducted with respect to surface roughness and adhesive strength of the different coatings. These tests could demonstrate a mechanically stable implant coating with glass solder matrices. The coated ceramic specimens show enhanced osteoblastic and partly epithelial viability and matrix production compared to the titanium control. Hence, the new glass solder matrix coating could improve bone cell growth as a prerequisite for enhanced osseointegration of ceramic implants

Co-Culture of S. epidermidis and Human Osteoblasts on Implant Surfaces: An Advanced In Vitro Model for Implant-Associated Infections.

OBJECTIVES:Total joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings. MATERIALS AND METHODS:For initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R®, and PALACOS R® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces. RESULTS:After 2 days, we observed approximately 104 CFU/ml biofilm-bound S. epidermidis (103 CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 105 CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB. CONCLUSION:We developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments

Influence of Different Three-Dimensional Open Porous Titanium Scaffold Designs on Human Osteoblasts Behavior in Static and Dynamic Cell Investigations

In the treatment of osseous defects micro-structured three-dimensional materials for bone replacement serve as leading structure for cell migration, proliferation and bone formation. The scaffold design and culture conditions are crucial for the limited diffusion distance of nutrients and oxygen. In static culture, decreased cell activity and irregular distribution occur within the scaffold. Dynamic conditions entail physical stimulation and constant medium perfusion imitating physiological nutrient supply and metabolite disposal. Therefore, we investigated the influence of different scaffold configurations and cultivation methods on human osteoblasts. Cells were seeded on three-dimensional porous Ti-6Al-4V scaffolds manufactured with selective laser melting (SLM) or electron beam melting (EBM) varying in porosity, pore size and basic structure (cubic, diagonal, pyramidal) and cultured under static and dynamic conditions. Cell viability, migration and matrix production were examined via mitochondrial activity assay, fluorescence staining and ELISA. All scaffolds showed an increasing cell activity and matrix production under static conditions over time. Expectations about the dynamic culture were only partially fulfilled, since it enabled proliferation alike the static one and enhanced cell migration. Overall, the SLM manufactured scaffold with the highest porosity, small pore size and pyramidal basic structure proved to be the most suitable structure for cell proliferation and migration

Nanoparticle internalization verified by Prussian Blue staining.

<p>After incubation of ASC with BNF starch nanoparticles (10/25/50 µg Fe/ml) and nanomag-D-spio nanoparticles (25/50/100 µg Fe/ml), iron oxide of internalized particles was visualized by Prussian Blue staining (Zeiss Axiovert 40 CFL, Carl Zeiss Microscopy GmbH, Jena, Germany; scale bars  = 50 µm).</p

Viability/Cytotoxicity test after nanoparticle labeling.

<p>After treatment of ASC with BNF starch nanoparticles (10/25/50 µg Fe/ml) and nanomag-D-spio nanoparticles, a viability/cytotoxicity assay was performed up to 14 days following labeling. Viable cells and nuclei of apoptotic cells were stained with calcein AM (green) and ethidium homodimer (red), respectively. Cells were counterstained with Hoechst 33342 (blue). No cytotoxic effects were detected due to nanoparticle labeling (Axio Observer, Carl Zeiss Microscopy GmbH, Jena, Germany; scale bars  = 50 µm).</p

Effect of labeling on adipogenic differentiation of ASC.

<p>After cell labeling with (A) BNF starch nanoparticles and (B) nanomag-D-spio nanoparticles, adipogenic differentiation conditions were provided for 21 days and lipid droplet deposition was measured. BNF starch labeling of ASC resulted in a dose-dependent reduction of adipogenic differentiation potential (n = 4; boxplots, Mann-Whitney U-test; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Proliferation of nanoparticle-labeled ASC.

<p>Cells were treated with (A) BNF starch nanoparticles and (B) nanomag-D-spio nanoparticles and cell numbers were determined up to 10 days after labeling. Cell treatment with both nanoparticle types resulted generally in a higher proliferation rate compared to control cells (n = 4; median, error bars represent 25^th and 75^th percentiles).</p