Characterisation of human co-culture systems for applications in bone tissue engineering

For the successful integration of bone tissue engineering constructs into patients, an adequate supply with oxygen and nutrients is critical. Therefore, prevascularisation of bone tissue engineering constructs is desirable for bone formation, remodelling and regeneration. Co-culture systems, consisting of human endothelial cells and primary osteoblasts (pOB) as well as osteosarcoma cell lines, represent a promising method for studying the mechanisms involved in the vascularisation of constructs in bone tissue en- gineering and could provide new insights into the molecular and cellular mechanisms that control essential processes during angiogenesis. The present study demonstrated the im- portant components of co-culture systems with a focus on bone tissue replacement and the angiogenic effects of pOB and osteosarcoma cell lines on human endothelial cells. Furthermore, the studies emphasised an overall approach for analysis of signal molecules that are involved in the angiogenic activation of human endothelial cells by the regulation of VEGF-related pathways at the transcriptional and translational levels. The osteosarcoma cell lines Cal-72, MG-63 and SaOS-2, as well as pOB from several donors, differed in their angiogenesis-inducing potential in 2-D and 3-D co-culture systems. SaOS-2 cells appeared to have a high osteogenic differentiation level with no detectable angiogenesis-inducing potential in co-culture with human endothelial cells. The angiogenic potential of the osteoblast-like cells is mainly correlated with the upregulation of essential angiogenic growth factors, such as VEGF, bFGF and HGF and the downregulation of the angiogenesis inhibitor, endostatin. However, other factors involved in angiogenic regulation were found to differ between SaOS-2 cells, compared to Cal-72 and MG-63. The present study focuses on VEGF pathway-effecting genes as key players in the regulation of angiogenesis. The levels of VEGF and VEGF-effecting genes, such as TGF-α and TIMP-2 are down-regulated in SaOS-2 cells. In contrast, direct regulators of VEGF, such as IL6, IL8 and TNF are strongly upregulated, which indicates disruptions in growth factor regulating pathways in SaOS-2 cells. Potential pathways, which could be involved include MEK, PI3K, MAPK, STAT3, AKT or ERK. Additional treatment of co-cultures with single growth factors did not accelerate or improve the angiogenesis-inducing potential of SaOS-2 cells. Knowledge of the detailed molecular mechanisms involved in angiogenesis control will hopefully allow improved approaches to be developed for prevascularisation of bone tissue engineering constructs.

Wound stimulation by growth-arrested human keratinocytes and fibroblasts: HP802-247, a new-generation allogeneic tissue engineering product

HP802-247 is a new-generation, allogeneic tissue engineering product consisting of growth-arrested, human keratinocytes (K) and fibroblasts (F) delivered in a fibrin matrix by a spray device.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Engineering actin-resistant human DNase I for treatment of cystic fibrosis.

Human deoxyribonuclease I (DNase I), an enzyme recently approved for treatment of cystic fibrosis (CF), has been engineered to create two classes of mutants: actin-resistant variants, which still catalyze DNA hydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but still bind G-actin. Actin-resistant variants with the least affinity for actin, as measured by an actin binding ELISA and actin inhibition of [33P] DNA hydrolysis, resulted from the introduction of charged, aliphatic, or aromatic residues at Ala-114 or charged residues on the central hydrophobic actin binding interface at Tyr-65 or Val-67. In CF sputum, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelasticity as determined in sputum compaction assays. The reduced viscoelasticity correlated with reduced DNA length as measured by pulsed-field gel electrophoresis. In contrast, the active site variants H252A or H134A had no effect on altering either viscoelasticity or DNA length in CF sputum. The data from both the active site and actin-resistant variants demonstrate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin). The increased potency of the actin-resistant variants indicates that G-actin is a significant inhibitor of DNase I in CF sputum. These results further suggest that actin-resistant DNase I variants may have improved efficacy in CF patients.

Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

Kinetochores assemble on distinct 'centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4-K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.

Profiles, mechanical engineering : human resources and funding.

"NSF 87-309".

Profiles, civil engineering : human resources and funding /

"November 1989"--p. iii.

Human genetic engineering [microform] : hearings before the Subcommittee on Investigations and Oversight of the Committee on Science and Technology, U.S. House of Representatives, Ninety-seventh Congress, second session, November 16, 17, 18, 1982.

"No. 170."

Gravity spun polycaprolactone fibers for applications in vascular tissue engineering:proliferation and function of human vascular endothelial cells

Poly(ε-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under low-shear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 × 104 cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering. © Mary Ann Liebert, Inc.

Representing human expertise by the OWL web ontology language to support knowledge engineering in decision support systems

Clinical decision support systems (CDSSs) often base their knowledge and advice on human expertise. Knowledge representation needs to be in a format that can be easily understood by human users as well as supporting ongoing knowledge engineering, including evolution and consistency of knowledge. This paper reports on the development of an ontology specification for managing knowledge engineering in a CDSS for assessing and managing risks associated with mental-health problems. The Galatean Risk and Safety Tool, GRiST, represents mental-health expertise in the form of a psychological model of classification. The hierarchical structure was directly represented in the machine using an XML document. Functionality of the model and knowledge management were controlled using attributes in the XML nodes, with an accompanying paper manual for specifying how end-user tools should behave when interfacing with the XML. This paper explains the advantages of using the web-ontology language, OWL, as the specification, details some of the issues and problems encountered in translating the psychological model to OWL, and shows how OWL benefits knowledge engineering. The conclusions are that OWL can have an important role in managing complex knowledge domains for systems based on human expertise without impeding the end-users' understanding of the knowledge base. The generic classification model underpinning GRiST makes it applicable to many decision domains and the accompanying OWL specification facilitates its implementation.

SFPE Engineering Guide to Human Behavior in Fire

Abstract not available

Vibration-induced extra torque during electrically-evoked contractions of the human calf muscles

Background: High-frequency trains of electrical stimulation applied over the lower limb muscles can generate forces higher than would be expected from a peripheral mechanism (i.e. by direct activation of motor axons). This phenomenon is presumably originated within the central nervous system by synaptic input from Ia afferents to motoneurons and is consistent with the development of plateau potentials. The first objective of this work was to investigate if vibration (sinusoidal or random) applied to the Achilles tendon is also able to generate large magnitude extra torques in the triceps surae muscle group. The second objective was to verify if the extra torques that were found were accompanied by increases in motoneuron excitability. Methods: Subjects (n = 6) were seated on a chair and the right foot was strapped to a pedal attached to a torque meter. The isometric ankle torque was measured in response to different patterns of coupled electrical (20-Hz, rectangular 1-ms pulses) and mechanical stimuli (either 100-Hz sinusoid or gaussian white noise) applied to the triceps surae muscle group. In an additional investigation, M(max) and F-waves were elicited at different times before or after the vibratory stimulation. Results: The vibratory bursts could generate substantial self-sustained extra torques, either with or without the background 20-Hz electrical stimulation applied simultaneously with the vibration. The extra torque generation was accompanied by increased motoneuron excitability, since an increase in the peak-to-peak amplitude of soleus F waves was observed. The delivery of electrical stimulation following the vibration was essential to keep the maintained extra torques and increased F-waves. Conclusions: These results show that vibratory stimuli applied with a background electrical stimulation generate considerable force levels (up to about 50% MVC) due to the spinal recruitment of motoneurons. The association of vibration and electrical stimulation could be beneficial for many therapeutic interventions and vibration-based exercise programs. The command for the vibration-induced extra torques presumably activates spinal motoneurons following the size principle, which is a desirable feature for stimulation paradigms.

Shear Stress Induces Nitric Oxide-Mediated Vascular Endothelial Growth Factor Production in Human Adipose Tissue Mesenchymal Stem Cells

It has been demonstrated that human adipose tissue-derived mesenchymal stem cells (hASCs) enhance vascular density in ischemic tissues, suggesting that they can differentiate into vascular cells or release angiogenic factors that may stimulate neoangiogenesis. Moreover, there is evidence that shear stress (SS) may activate proliferation and differentiation of embryonic and endothelial precursor stem cells into endothelial cells (ECs). In this work, we investigated the effect of laminar SS in promoting differentiation of hASCs into ECs. SS (10 dyn/cm(2) up to 96 h), produced by a cone plate system, failed to induce EC markers (CD31, vWF, Flk-1) on hASC assayed by RT-PCR and flow cytometry. In contrast, there was a cumulative production of nitric oxide (determined by Griess Reaction) and vascular endothelial growth factor (VEGF; by ELISA) up to 96 h of SS stimulation ( NO(2)(-) in nmol/10(4) cells: static: 0.20 +/- 0.03; SS: 1.78 +/- 0.38, n = 6; VEGF in pg/10(4) cells: static: 191.31 +/- v35.29; SS: 372.80 +/- 46.74, n = 6, P < 0.05). Interestingly, the VEGF production was abrogated by 5 mM N(G)-L-nitro-arginine methyl ester (L-NAME) treatment (VEGF in pg/10(4) cells: SS: 378.80 +/- 46.74, n = 6; SS + L-NAME: 205.84 +/- 91.66, n = 4, P < 0.05). The results indicate that even though SS failed to induce EC surface markers in hASC under the tested conditions, it stimulated NO-dependent VEGF production.