Extensive cell‐cell‐coupling via gap junctions has been suspected to play an essential role for osteoblast development. Here, osteoblast‐like cells (OBL) from connexin(Cx)43 knock out mice were used to explore the role of Cx43 for osteoblast differentiation. Primary cultures of OBL were derived from calvaria of homozygous (Cx43‐/‐) and heterozygous (Cx43+/–) knock out mice and also from wild type controls (Cx43+/+). In Cx43‐/‐ OBL Lucifer Yellow dye coupling was largely abolished demonstrating that small molecules could no longer be transferred among neighboring cells. Cx43‐/‐ OBL grew out very slowly from calvarial fragments. Nevertheless their cell density around explants was increased 3‐fold vs. controls after 3 weeks. Histochemistry showed that in many Cx43‐/‐ OBL there was an increased alkaline phosphatase activity within the cytoplasm and close to the cell membrane. Mineralization was diminished in Cx43‐/‐ cultures. In heterozygous Cx43+/– OBL all aforementioned effects were less pronounced, pointing to a gene‐dosage effect. Data suggest that the loss of Cx43 indirectly impairs the osteoblastic phenotype, e.g. by disturbing cellular functions such as motility and/or secretion. If this holds true, all parameters in the interphase of enosseous implants which lower gap junction expression will also affect bone regeneration.
Intercellular communication of osteoblast-like cells (OBL) is discussed to be a prerequisite for osseointegration of implants. This signaling involves changes of the intracellular calcium concentration ([Ca 2+ ] i ) which can be elicited by mechanical stimuli and are propagated from cell to cell. These [Ca 2+ ] i signals have originally been assumed to pass exclusively through gap junctions. However, substances such as purine nucleotides (ATP, UTP) are also released by osteoblasts upon mechanical stimulation and evoke [Ca 2+ ] i signals in neighboring cells after binding to purine receptors. In this investigation we studied the impact of the major gap junction protein connexin 43 (Cx43) on ATP-mediated calcium signaling of OBL in vitro. We compared OBL derived from calvaria of Cx43 deficient (Cx43-/-, Cx43+/-) and wild type animals (Cx43+/ +). While application of ATP to Cx43+/+ or Cx43+/-OBL evoked a single transient increase of [Ca 2+ ] i , the response of most Cx43-/-OBL was pronounced in that the initial [Ca 2+ ] i peak was larger and followed by 3-10 repetitive calcium waves. With respect to shape and number of peaks different types of responses co-existed in neighboring OBL, possibly pointing to differences in purinoceptor subtypes and/or filling states of intracellular calcium stores. Data show that ATP signaling of osteoblasts is changed in Cx43 deficient OBL, suggesting a compensatory role of purinoceptor mediated [Ca 2+ ] i signaling secondary to an impaired gap junction communication in Cx43-/-OBL.
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