Enhanced Periosteal and Endocortical Responses to Axial Tibial Compression Loading in Conditional Connexin43 Deficient Mice

<div><p>The gap junction protein, connexin43 (Cx43) is involved in mechanotransduction in bone. Recent studies using in vivo models of conditional Cx43 gene (<em>Gja1</em>) deletion in the osteogenic linage have generated inconsistent results, with <em>Gja1</em> ablation resulting in either attenuated or enhanced response to mechanical load, depending upon the skeletal site examined or the type of load applied. To gain further insights on Cx43 and mechanotransduction, we examined bone formation response at both endocortical and periosteal surfaces in 2-month-old mice with conditional <em>Gja1</em> ablation driven by the <em>Dermo1</em> promoter (cKO). Relative to wild type (WT) littermates, it requires a larger amount of compressive force to generate the same periosteal strain in cKO mice. Importantly, cKO mice activate periosteal bone formation at a lower strain level than do WT mice, suggesting an increased sensitivity to mechanical load in Cx43 deficiency. Consistently, trabecular bone mass also increases in mutant mice upon load, while it decreases in WT. On the other hand, bone formation actually decreases on the endocortical surface in WT mice upon application of axial mechanical load, and this response is also accentuated in cKO mice. These changes are associated with increase of <em>Cox-2</em> in both genotypes and further decrease of <em>Sost</em> mRNA in cKO relative to WT bones. Thus, the response of bone forming cells to mechanical load differs between trabecular and cortical components, and remarkably between endocortical and periosteal envelopes. Cx43 deficiency enhances both the periosteal and endocortical response to mechanical load applied as axial compression in growing mice.</p> </div

Effect of axial tibial loading on periosteal and endocortical bone formation in wild type (WT) and <i>Gja1</i> conditional knockout (cKO) mice.

<p>The right tibia was subjected to forces generating 1200 µε or 1900 µε in WT, or 1200 µε in cKO, as noted, while the left was used as non-loaded control. (A) Double calcein/alizarin red labels on cortical sections of tibiae from the different groups, showing both periosteal (Ps) and endocortical (Ec) surfaces. (B) Periosteal mineralizing surfaces per bone surface (Ps.MS/BS) and (C) periosteal bone formation rate per bone surface (Ps.BFR/BS). (D) Endocortical MS/BS (Ec.MS/BS), and (E) endocortical BFR/BS (Ec.BFR/BS). * p<0.05 vs respective Control; # p<0.05 vs WT-1200 Loaded; ANOVA.</p

Relationship between applied force and strain measured at the bone surface in (A) wild type and (B) <i>Gja1</i> conditional knockout (cKO) mice.

<p>Regression equations are given for each genotype, and the slopes of the Regression lines were compared using the test for Parallelism; p<0.05.</p

Effect of axial tibial loading on cortical parameters in wild type (WT) and <i>Gja1</i> conditional knockout (cKO) mice.

<p>The right tibia was subjected to forces generating 1200 µε or 1900 µε in WT, or 1200 µε in cKO, as noted, while the left was used as non-loaded control. (A) Cortical bone volume (Ct.BV). (B) Marrow area (Ma.Ar). (C) Total tissue area (Tt.Ar). Data are expressed as absolute difference between post- and pre-load for each parameter. *p<0.05 vs. respective control; two-tailed t-test.</p

Effect of axial tibial loading on gene expression in vivo.

<p>Mice were sacrificed 2 hours after load (120 cycles, 10 sec interval between cycles), and mRNA extracted from whole bone extracts before qPCR analyses for the genes of interest. a: p<0.05 vs WT control; b: p<0.05 vs WT Loaded and c: p<0.05 vs cKO Control; one-way ANOVA.</p