Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

Abstract

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load-bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β-glycyrrhetinic acid, we also demonstrated that this osteocyte-related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial-derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain-related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.

Keywords: coculture; gap junctions; mechanical strain; osteoblasts; osteocytes.

Figure 1

Experimental strategy used. Eighteen‐day‐old Alizarin Red S– and Alcian Blue GX–stained chick embryo depicting the harvest sites for both tibiotarsal and calvarial primary osteoblasts and osteocytes. Primary osteoblasts from both tibiotarsal long bone (LOBs) and calvariae (COBs) were either cultured alone or cocultured with osteocytes. The latter were also derived from both bone sites and were cocultured in either “homotypic” (LOCs + LOBs or COCs + COBs) or “heterotypic” (LOCs + COBs or COCs + LOBs) conditions

Figure 2

Calvarial and tibiotarsal osteocytes retain a morphologically characteristic phenotype. Ob7.3(5)⁺ LOCs and COCs populations (A and B) show the distinct stellate shape of the osteocytic phenotype. Immunocytochemical Ob7.3(5) antibody labelling of bone cells was assessed to ascertain population purity, with negative labelling in the fibroblast‐depleted Ob7.3(5)⁻ population (C) and positive staining in the Ob7.3(5)⁺ population (D). E, Proliferation potential of Ob7.3(5)⁺ and fibroblast‐depleted Ob7.3(5)⁻ populations was assessed by 18‐h pulse treatment with [³H]‐thymidine and incorporation. Both LOCs and COCs were shown to display negligible rates of proliferation. Conversely, LOBs and COBs proliferate avidly; disparity of proliferation allows for the study of osteoblast proliferation in mixed osteocyte‐osteoblast cultures. F, Homotypic cultures of Ob7.3(5)⁺ and fibroblast‐depleted Ob7.3(5)⁻ cells were pulse treated with [³H]‐thymidine. The presence of LOCs in the LOB cultures resulted in an increase of proliferation. Conversely, the presence of COCs in COB culture had no effect on COB proliferation rate. Data are presented to show incorporation of ³H‐thymidine over an 18‐h period, and all 6 wells of a 6‐well plate were used for each variable culture condition (n = 4 experiments in total). The asterisk denotes significance vs osteoblast monocultures (P < .05)

Figure 3

Long bone osteocytes enhance calvarial osteoblast proliferation. To assess whether COBs are unable to respond to osteocyte‐derived proliferative stimuli, COBs were maintained in heterotypic cultures with LOCs. LOCs induced proliferation of COBs, whilst COCs were unable to influence the proliferation of LOBs. This suggests the potential for fundamental differences signalling methods/signals derived from LOCs and COCs. Data are presented to show incorporation of ³H‐thymidine over an 18‐h period and all 6 wells of a 6‐well plate were used for each variable culture condition (n = 3 experiments in total). The asterisk denotes significance vs osteoblast monocultures (P < .05)

Figure 4

Proliferation in mechanically strained, not static, cultures is regulated by gap junctions. A, Mechanical strain increased LOBs proliferation in the absence of gap junction blocker. Somewhat surprisingly, the addition of LOCs in homotypic cultures resulted in a reduction in the proliferation rate following a period of mechanical straining. This effect was dependant on functional gap junctions as this was inhibited by the presence of the gap junction blocker, β‐glycyrrhetinic acid (β‐GA). B, Histogram showing the difference in osteocyte‐derived [³H]‐thymidine incorporation in data presented in A. These experiments identify differential methods of communication between osteocytes and osteoblasts in strained and nonstrained conditions. Data are presented to show incorporation of ³H‐thymidine over an 18‐h period, and 3 cell straining strips were used for each variable culture condition (n = 3 experiments in total; the asterisk denotes significance)

Figure 5

Schema depicting osteocyte‐derived signalling on osteoblasts. The results suggest that the proproliferative influence of osteocytes upon osteoblasts is reversed by the application of strain and that only this reversal is gap junction mediated