High concentrations of dexamethasone suppresses the proliferation but not the differentiation of further maturation of human osteoblast precursor in vitro: relevance to glucocorticoid-induced osteoporosis

Objective. The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). Methods. Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 µm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. Results. At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the 2 and 4, but not ß1, integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. Conclusions. A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

Characterization of the 5' upstream regulatory region of the human myostatin gene: regulation of myostatin gene transcription by dexamethasone in vitro

We cloned and characterized a 3.3-kb fragment containing the 5'-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-kappaB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C(2)C(12) and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

This paper is novel andreports on the in vitro establishment of 3-D cultures of human osteoblasts. These were evaluated for protein markers of bone cells. Sequentially alkaline phosphatase, calcium incorporation for matrix mineralisation and then finally osteocalcin expression were detected in cultures. The extracellular matrix was composed of type 1 collagen and as it mineralised, needle shaped crystals were often associated with matrix vesicles initiating mineralisation. In vivo implantation in nude mice showed progression of mineralisation from the inner region outward with peripheral cells in a non-mineralised matrix. Host vessels invaded the implanted cell area. The research has relevance to musculoskeletal tissue engineering.

The Effects of Delta–9–Tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro.

Objective: to investigate the effects of tetrahydrocannabinol; THC on human sperm function in vitro. Design: laboratory analysis of sperm motility with and without exposure to THC using computer assisted semen analysis (CASA) and acrosome reaction by fluoroscein isothiocyanate labelled peanut agglutinin (FITC-PNA) staining. Setting: An ART unit in a tertiary medical centre. Patients: semen was obtained from 78 men attending the Regional Fertility Centre, Belfast. Interventions: Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with, or without (controls), tetrahydrocannabinol (THC) at concentrations equivalent to therapeutic (0.032�?�¯?�?�­M) and recreational (4.8 and 0.32�?�¯?�?�­M) plasma levels, at 37�?�¯?�?�°C for 3 hours. Main outcome measures: Sperm motility, spontaneous and induced acrosome reactions Results: There was a dose-dependent decrease in percentage progressive motility (-21% at 4.8�??�?�µM, p0.05) in the 90% fraction. The 45% fraction showed a greater decrease in percentage progressive motility (-56% at 4.8�??�?�µM, p=0.011; -23% at 0.32�??�?�µM, p= 0.039; and -28% at 0.032�??�?�µM, p=0.004). A decrease in the straight line velocity; VSL (-10%) and the average path velocity; VAP (-10%) were also observed in the 90% fraction. A significant inhibition (-15% at 4.8�??�?�µM, p=0.04) in spontaneous acrosome reaction was observed in the 90% fraction. The 45% fraction showed a more marked inhibition [-35% (p

The human micro-vascular enclothelial cells in vitro interaction with atomic-nitrogen-doped diamond-like carbon thin films

This paper reports the initial response of atomic nitrogen doped diamond like carbon (DLC) to endothelial cells in vitro. The introduction of nitrogen atoms/molecules to the diamond like carbon structures leads to an atomic structural change favorable to the attachment of human micro-vascular enclothelial cells. Whilst the semi-conductivity induced by nitrogen in DLC is thought to play a part, the increase in the inion-bonded N atoms and N-2 molecules in the atomic doped species (with the exclusion of the charged species) seems to contribute to the improved attachment of human microvascular endothelial cells. The increased endothelial attachment is associated with a lower work function and slightly higher water contact angle in the atomic doped films, where the heavy charged particles are excluded. The films used in the study were synthesized by the RF PECVD technique followed by post deposition doping with nitrogen, and afterwards the films were characterized by XPS, Raman spectroscopy, SIMS and Kelvin probe. The water contact angles were measured, and the counts of the adherent endothelial cells on the samples were carried out. This study is relevant and contributory to improving biocompatibility of surgical implants and prostheses.

Aminolaevulinic acid diffusion characteristics in 'in vitro' normal human skin and actinic keratosis: implications for topical photodynamic therapy

Background: The response rate of aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in certain subtypes of actinic keratosis (AK), such as hypertrophic and hyperkeratotic lesions, is variable, an effect attributable to a supposed lack of ALA penetration. A detailed and depth-related profile of spatial ALA permeation in AK following drug administration would lead to a greater understanding of concentrations achievable before protoporphyrin IX biosynthesis and subsequent PDT.

In vivo and in vitro function of human UDP-galactose 4 '-epimerase variants

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4'-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coil resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three alpha-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia. (C) 2011 Elsevier Masson SAS. All rights reserved.

The effects of hexetidine (Oraldene (TM)) on the adherence of Candida albicans to human buccal epithelial cells in vitro and ex vivo and on in vitro morphogenesis

Purpose. This study reports the effects of hexetidine (Oraldene(TM)) on two virulence attributes of Candida albicans, namely, in vitro and ex vivo adherence of yeast cells to buccal epithelial cells (BEG) and in vitro morphogenesis.

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P <0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P <0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.