Calvaria derived osteogenic cells: phenotypic expression in culture.

The osteoblast phenotype is characterized by its ability to (a) synthesize a well defined mineralized collagenous matrix, (b) regulate the remodeling process by synthesizing local hormones (PGE2) and specific molecules (osteocalcin) and enzymes (alkaline phosphatase and collagenase), (c) respond to a variety of hormones (PTH, PGs, vitamin-D metabolites, steroids and growth factors), (d) respond to mechanical stimulation. Most of osteoblast culture systems meet many of the above qualifications though most fail to show the PTH effect on DNA synthesis, (c), and mechanical stimulation (d). Here we show that by using trypsin digestion and serum-containing low calcium medium (0.25 mM), all the above listed osteoblast phenotypic characteristics are demonstrated including their responsiveness to mechanical stimulation and the PTH effect on DNA synthesis.

Rat epiphyseal cells in culture: responsiveness to bone-seeking hormones.

Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJb medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E2 (PGE2) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE2 (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)2D3 increased significantly the [3H]thymidine incorporation into DNA, whereas 1,25(OH)2D3 effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.

The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2.

Mechanical forces applied to cultured bone cells induce the production of cAMP via stimulation of the formation of prostaglandin E2 (PGE2) and its release into the medium, resulting in stimulation of adenylate cyclase. In this paper we show that either the antibiotic gentamycin (100 micrograms/ml) or antiphospholipid antibodies (0.1%) which bind to membrane phospholipids abolish cAMP formation induced by mechanical forces; exogenously added arachidonic acid or PGE2 stimulates cAMP formation, even in the presence of these agents. Addition of exogenous phospholipase A2 (but not phospholipase C) causes an increase in the formation of cAMP in bone cells, a response that is also inhibited by gentamycin or antiphospholipase antibodies. These observations suggest that mechanical forces exert their effect on bone cells via the following chain of events: (1) activation of phospholipase A2, (2) release of arachidonic acid, (3) increased PGE synthesis, (4) augmented cAMP production.

Developmental changes in responsiveness to vitamin D metabolites.

We have demonstrated that epiphyseal chondroblasts contain specific receptors for 24R,25-dihydroxy vitamin D3(24,25(OH)2D3) while diaphyseal osteoblasts contain specific receptors for 1 alpha 25-dihydroxy vitamin D3(1,25(OH)2D3). Both metabolites induce DNA synthesis and creatine kinase (CKBB) activity. We have also found that the responsiveness of rat kidney to these metabolites changes during development. In embryonic and early postnatal stages, the kidney responds to 24,25(OH)2D3, later to both 24,25(OH)2D3 and 1,25(OH)2D3, and the mature kidney only to 1,25(OH)2D3. These responses correlate with changes in the specific receptors present in the kidney. Furthermore, we have compared developmental changes in skeletal (epiphysis, diaphysis and mandibular condyle) and non-skeletal (kidney, cerebellum, cerebrum, liver and pituitary) tissue in both rat (a postnatal developer) and rabbit (a perinatal developer). Epiphyseal or diaphyseal chondroblasts at any stage of development were predominantly responsive to 24,25(OH)2D3, whereas osteoblasts were responsive to 1,25(OH)2D3. In contrast, condylar chondroblasts, kidney, cerebellum and pituitary responded to 24,25(OH)2D3 during early development and subsequently developed responsiveness to 1,25(OH)2D3. Using primary cell cultures from kidneys at different stages of maturation, we showed the same developmental pattern as in vivo. Chronic treatment of the cells with 24,25(OH)2D3, but not 1,25(OH)2D3, caused precocious development of responsiveness to 1,25(OH)2D3 in culture. We suggest that 24,25(OH)2D3 acts as a maturation factor, during early development in kidney, and probably in other tissues, possibly by induction of receptor to 1,25(OH)2D3, accompanied by down-regulation of its own receptor.

Stimulation of skeletal-derived cell cultures by different electric field intensities is cell-specific.

Pulsed electric stimulation, coupled capacitively to different cell cultures of skeletal origin, caused immediate changes in the cellular levels of cyclic AMP and a later enhanced DNA synthesis. Changes both in cyclic AMP level and DNA synthesis were correlated with the strength of the applied electric field. Cultures of calvaria bone cells which contain mainly two cell types, parathyroid hormone responsive cells (osteoblast-like) and prostaglandin E2 responsive cells (fibroblast-like), respond to both low (13 V/cm) and to high (54 V/cm) electric field strength, with no response at intermediate (24 V/cm) field strength. Rat epiphyseal cartilage responded like bone cells both to low and high field intensities, while rat condylar cartilage responded only to the intermediate field strength. Moreover, subcultures of calvaria bone cells, which lost their osteoblastic phenotype expression during subculturing, were responsive only to low field strength. On the other hand, osteoblast-enriched cultures, derived from calvaria bone grown in low calcium, were responsive only to the high field strength. These findings suggest that the response to various electric field intensities is cell-specific and might be used as an additional parameter to characterize cell types. Our study points to the possibility that when exposing a whole organ to an electrical stimulation it is possible to affect specifically only one cell population out of the many cell types existing in the organ.

Mechanical and hormonal stimulation of cell cultures derived from young rat mandible condyle.

Collagenase digestion of young rat condyles released cells which were grown in culture during two weeks. Morphologically, two populations of cells were distinguished, one of which reacted to alkaline phosphatase and resembled chondroblast or osteoblast-like cells. Parathyroid hormone stimulated a 2-fold increase in cellular cyclic AMP, whereas calcitonin had no effect. Physical forces activated cellular cyclic AMP to a 2.5-fold of control levels and increased the incorporation of radioactive thymidine into DNA by 50 per cent. In contrast to cultured bone cells, the response to physical forces was not inhibited by indomethacin in cultured condyle cells. It seems, therefore, that condyle cells are specific in their response to bone-seeking hormones and physical forces.

Biochemical pathways involved in the translation of physical stimulus into biological message.

Studies from our laboratory revealed that direct application of physical strain (PS) to cultured bone cells stimulated synthesis of prostaglandin E2 (PGE2) in a specific population of cells. We found that PGE2 induced the cellular production of cAMP in several bone cell types, whereas the induction of DNA synthesis was limited to osteoblastlike cells. Indirect evidence indicated that PS induced the osteoblastlike cells to synthesize PGE2. Other cell types, for example, chondrocytes, when activated by PS, can be induced to produce cAMP and induce DNA synthesis not mediated by PGE2. We have also found that electric stimulation of different populations of bone cells is specifically induced by a certain intensity of the electric field. It seems that electric stimulus circumvents the PGE2 effect and triggers the adenyl cyclase system in the cell directly. The electric field also induces DNA synthesis not via PGE2 production.