A bioactive hybrid three-dimensional tissue-engineering construct for cartilage repair

The aim was to develop a hybrid three-dimensional-tissue engineering construct for chondrogenesis. The hypothesis was that they support chondrogenesis. A biodegradable, highly porous polycaprolactone-grate was produced by solid freeform fabrication. The polycaprolactone support was coated with a chitosan/polyethylene oxide nanofibre sheet produced by electrospinning. Transforming growth factor-3-induced chondrogenesis was followed using the following markers: sex determining region Y/-box 9, runt-related transcription factor 2 and collagen II and X in quantitative real-time polymerase chain reaction, histology and immunostaining. A polycaprolactone-grate and an optimized chitosan/polyethylene oxide nanofibre sheet supported cellular aggregation, chondrogenesis and matrix formation. In tissue engineering constructs, the sheets were seeded first with mesenchymal stem cells and then piled up according to the lasagne principle. The advantages of such a construct are (1) the cells do not need to migrate to the tissue engineering construct and therefore pore size and interconnectivity problems are omitted and (2) the cell-tight nanofibre sheet and collagen-fibre network mimic a cell culture platform for mesenchymal stem cells/chondrocytes (preventing escape) and hinders in-growth of fibroblasts and fibrous scarring (preventing capture). This allows time for the slowly progressing, multiphase true cartilage regeneration.Peer reviewe

Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum

Abstract Introduction Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally. Methods We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified. Results Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP. Conclusions Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option

Identification of histamine receptor subtypes in skeletal myogenesis

investigated in mouse skeletal myogenesis. Therefore, the present study aimed to investigate the HR‑subtypes in skeletal myogenesis. The myogenesis of C2C12 skeletal myoblasts was evaluated using desmin, myogenin and myosin heavy chain (Myh) as early, intermediate and late differentiation markers, respectively. Reverse transcription‑quantitative polymerase chain reaction and immunostaining were performed and the messenger RNA (mRNA) expression levels of the HR‑subtypes and markers were determined. H1R mRNA was found to be highly expressed in myoblasts at day 0; however, the expression levels were reduced as differentiation progressed. By contrast, H2R mRNA expression remained constant, while H3R mRNA expression increased by 28‑, 103‑ and 198‑fold at days 2, 4 and 6 compared with the baseline level (day 0), respectively. In addition, Myh expression increased by 7,718‑, 94,487‑ and 286,288‑fold on days 2, 4 and 6 compared with the baseline expression level (day 0). Weak positive staining of the cells for H3R protein was observed on day 2, whereas highly positive staining was observed on days 4 and 6. HR expression during myogenesis was, in part, regulated by the stage of differentiation. These results along with previous findings indicated possible involvement of H1R in the regulation of progenitor cell mitogenesis and of H2R in the relaxation of acetylcholine‑stimulated contraction of muscle cells, following the activation of professional histamine‑producing cells, including mast cells. By contrast, H3R may participate in the regulation of specialized myocyte functions, potentially by maintaining the relaxed state under the influence of constitutive H3R activity and low histamine concentrations, locally produced/released by non‑professional histamine‑producing cells

Cholecystokinin 2 receptor-deficient mice display altered function of brain dopaminergic system

Rationale: Cholecystokinin (CCK) has been shown to coexist and interact with dopamine in the regulation of behaviour. Two different CCK receptors (CCK1 and CCK2) have an opposite influence on the activity of dopamine neurons. Stimulation of CCK2 receptors decreases the release of dopamine and that receptor could mediate the neuroleptic-like effect of CCK. Objective: To investigate the activity of the dopaminergic system in pharmacological experiments on CCK2 receptor (CCK2R)-deficient mice. Methods: We used age- and sex-matched littermates in all our experiments. To evaluate the behavioural differences, we performed the rotarod test and measured the locomotor activity of animals using computer-connected photoelectric motility boxes. Amphetamine and apomorphine, two dopaminergic drugs with different pharmacodynamic properties, were used to influence the activity of the dopaminergic system in the brain. Neurochemical differences related to the different genotype were analysed by means of high-performance liquid chromatography and radioligand binding studies. Results: Motor co-ordination was significantly impaired in the rotarod test of CCK2R receptor-deficient mice. Moreover, the locomotor activity of heterozygous (+/–) and homozygous (–/–) CCK2R receptor-deficient mice was somewhat reduced. A low dose of apomorphine (0.1 mg/kg), an unselective agonist of dopamine receptors, suppressed locomotor activity significantly more in homozygous (–/–) and heterozygous (+/–) mutant mice than in their wild-type (+/+) littermates. Amphetamine (3–6 mg/kg), increasing release of dopamine from the presynaptic terminals, caused a dose-dependent motor stimulation in wild-type (+/+) mice. In heterozygous (+/–) and homozygous (–/–) mice, a lower dose of amphetamine (3 mg/kg) did not alter the locomotor activity, whereas the higher dose of (6 mg/kg) induced a significantly stronger increase in locomotor activity in homozygous (–/–) mice than in their heterozygous (+/–) and wild-type (+/+) littermates. Despite the changes in the action of apomorphine and amphetamine in homozygous (–/–) mice, we did not find any significant differences in the concentration of dopamine and their metabolites in the striatum or cortex. However, the density of dopamine D2 receptors was significantly increased in the striatum of homozygous (–/–) animals compared with wild-type (+/+) mice. Conclusions: The targeted mutation of the CCK2 receptor gene induced gene dose-dependent changes in the activity of the dopaminergic system. The sensitivity of presynaptic dopamine receptors was increased in heterozygous (+/–) and homozygous (–/–) animals, whereas the increase in sensitivity of postsynaptic dopamine receptors was apparent only in homozygous (–/–) mice. Targeted mutagenesis Wild-type Heterozygous Homozygous Dopamine Serotonin Receptor Cholecystokinin Cholecystokinin2 receptor Motor co-ordination Rotarod test Motility boxes Locomotor activit

Support Mechanisms and Value Creation in Multi-Stakeholder Networks for Digital Innovation in Education: A Cross-Country Study

This study investigates the impact of support mechanisms on enhancing the participation of network members in diverse multi-stakeholder networks with a primary focus on digital innovation in education. The study was conducted in collaboration with national networks from partner countries of a H2020 project, which aims to accelerate digital innovation in and across schools. Twenty-six international stakeholders from various backgrounds, including teachers, school leaders, mentors, educational technologists, policy makers, and a researcher were interviewed (n = 14) or surveyed (n = 12). Using Wenger et al.’s [1] value creation framework, the study identified participants’ perceptions of various activities, interactions, and structures that enable network members to generate value. The results indicated overarching values for members to be associated with capacity building opportunities, joint projects, opportunities for expanding impact through recognized expertise, access to personalized support and guidance, and knowledge sharing platforms

Bone Regeneration and stem cells.

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed