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

Sildenafil citrate improves sperm motility but causes a premature acrosome reaction in vitro

Objective: to determine if sildenafil citrate; a cyclic monophosphate specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction. Design: laboratory analysis of sperm motility after exposure to sildenafil citrate using computer assisted semen analysis (CASA) and acrosome reaction by fluoroscein isothiocyanate labelled peanut agglutinin (FITC-PNA) staining. Setting: An ART unit Patients: 57 male patients 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 sildenafil citrate ( 0.67uM) at 37ï?°C for up to 180 minutes. Main outcome measures: both the numbers and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Further, samples revealed that these effects were consistent in the 90% and 45% subpopulations of sperm. In both subpopulations, sildenafil also caused a significant increase in the proportion of acrosome reacted sperm - 22.1% compared with 11.8% in the control group of the good quality fraction (p