Cigarette smoke inhibits osteogenic differentiation and proliferation of human osteoprogenitor cells in monolayer and three-dimensional collagen gel culture.

Cigarette smoke is a risk factor not only for emphysema but also for other disorders characterized by deficient tissue repair, including osteoporosis. We hypothesized, therefore, that smoke might directly impair bone cell repair processes. To evaluate this, bone marrow osteoprogenitor cells were isolated from normal subjects and cultured in monolayer and in three-dimensional type I collagen gel culture. Human osteoprogenitor cells could be induced to differentiate toward osteoblast-like cells in both culture conditions by osteogenic supplements. Under both culture conditions, cigarette smoke extract (CSE) inhibited the proliferation of osteoprogenitor cells in a concentration-dependent manner. CSE also inhibited differentiation of osteoprogenitor cells toward osteoblast-like cells as assayed by alkaline phosphatase activity and calcium incorporation into cell layer. Cells in monolayer culture were more sensitive to the effect of smoke than cells in three-dimensional gel culture. Similar results were obtained with osteoblast-like cells derived from osteosarcomas. This study, therefore, demonstrates that cigarette smoke may affect bone progenitor cells directly and in this manner may contribute to the development of osteoporosis.

Grain dusts and grain plant components vary in their ability to recruit neutrophils.

Occupational exposure to grain dusts can cause bronchitis, particularly to grain sorghum dust. Bronchitis is associated with the presence of increased numbers of neutrophils. To determine how grain dusts could cause neutrophil recruitment to the airways, extract of whole-grain sorghum, corn, and soybean dusts and of pulverized components of these plants were made with Hanks balanced salt solution (HBSS) and used in direct neutrophil chemotaxis experiments. The glume of the grain sorghum plant, the structure holding the seeds in place, caused the migration of the greatest number of neutrophils compared to HBSS [132 +/- 7 vs. 60 cells/high-power field (hpf) +/- 2 SEM, p < .001], followed by whole-grain sorghum dust (121 +/- 5 cells/hpf). Next, bovine bronchial epithelial cells (BBECs) were obtained from fresh lungs and grown to near confluence before challenge with a 10% solution of grain dust and grain plant extracts. The grain sorghum glume and whole-grain sorghum dusts caused release of the greatest amount of neutrophil chemotactic activity (NCA) from BBECs compared to the medium M199 negative control (141 +/- 6 and 153 +/- 7, respectively, vs. 64 cells/hpf +/- 3 SEM, p < .001). The ability to cause neutrophils to migrate by direct and indirect means did not correlate with levels in the grain dusts of endotoxin, which is known to cause release of NCA from bronchial epithelial cells. Therefore, this article describes additional mechanisms by which grain dusts can cause pulmonary inflammation and that are independent of endotoxin levels.

Serum-free culture of fractionated bovine bronchial epithelial cells.

Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factor beta 1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1 microM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.