Dye and electric coupling between osteoblast-like cells in culture

Abstract

Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cells in vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4-5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (pCO2 > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca2+ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.

Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation

“…They play a vital role in skeletal health by acting as mechanosensors that monitor the mechanical environment within bone tissue and signalling to osteoblasts and osteoclasts to remodel the tissue so that bone strength is maintained throughout life (Klein-Nulend et al, 1995; Lanyon, 1993; Jee, 2001). Osteocytes are formed when osteoblasts undergo a dramatic phenotypic transition as they become embedded within newly deposited bone matrix.…”

Dynamic changes of intracellular calcium in osteoblast-like cells: effects on dye coupling

“…Previously, we have shown that antiepileptic drugs, e. g. phenytoin, could reduce the electric coupling between ROB cells within 5 min [5] which could be a first hint that osteomalacia may not only be caused by disturbances of, e. g., the vitamin-D metabolism. In this respect, all effects of implant materials, which are capable of modulating gap junctional coupling either by changes of the local milieu, effects of local strains, etc., our due to metal ions released from implant materials may also change cell functions (proliferation, differentiation, etc.)…”

“…Calvarial fragments derived from newborn rats were explanted onto collagen-coated glass-coverslips as described [5]. Cells were maintained at 35 8C in Hepes (20 mM) buffered minimum essential medium (88 %) which was supplemented with fetal calf serum (10 %), glutamine (2 mM) and penicillin (50 I.U.…”

“…In control saline, the initial ratio (R) was 0. 5 In control saline, intercellular coupling was extensive: the incidence of dye spread was 94 %, i. e. 68 of 72 possible primary neighboring cells received dye from an adjacent cells.…”

“…Since such signals might travel to adjacent cells via gap junctions, these channels allow signal transmission [4]. Parallel intracellular recordings and injections of Lucifer Yellow revealed high electric and dye coupling via gap junctions between rat osteoblast-like (ROB) cells in primary culture [5]. Gap junctions in bone were found to be composed mainly of connexin43 (Cx43) and in less amounts of connex-in45 (Cx45) [6 ± 10].…”