Deletion of Cx43 from osteocytes results in defective bone material properties but does not decrease extrinsic strength in cortical bone

Abstract

Deletion of connexin (Cx) 43 from osteoblasts and osteocytes (OCN-Cre;Cx43(fl/-) mice) or from osteocytes only (DMP1-8kb-Cre;Cx43(fl/fl) mice) results in increased cortical, but not cancellous, osteocyte apoptosis and widening of the femoral midshaft without changes in cortical thickness. Despite the consequent larger moment of inertia, stiffness and ultimate load, measures of mechanical strength assessed by three-point bending, are not higher in either model of Cx43 deficiency due to reduced Young's modulus, a measure of the stiffness of the material per unit of area. In OCN-Cre;Cx43(fl/-) mice, this was accompanied by a reduced ratio of nonreducible/reducible collagen cross-links as assessed by Fourier transformed infrared imaging (FTIRI) in the femoral diaphysis. On the other hand, DMP1-8kb-Cre;Cx43(fl/fl) mice did not show a significant reduction in collagen maturation in the same skeletal site, but a small decrease in mineralization was detected by FTIRI. Remarkably, both osteoblastic and osteocytic cells lacking Cx43 expressed lower mRNA levels of lysyl oxidase, a crucial enzyme involved in collagen maturation. These findings suggest that Cx43 expression in osteoblasts is involved in maintaining the quality of the bone matrix in cortical bone through the maturation of collagen cross-links. Osteocytic Cx43 expression is important also to maintain the stiffness of the bone material, where Cx43 deficiency results in local reduction in mineralization, possibly due to osteocyte apoptosis.

Figure 1. OCN-Cre;Cx43^fl/− and DMP1-8kb-Cre;Cx43^fl/fl mice have decreased material properties in the femur

Mechanical properties of the femur from Cx43^fl/− and OCN-Cre;Cx43^fl/− mice (A) and Cx43^fl/fl and DMP1-8kb-Cre;Cx43^fl/fl mice (B) were determined by 3-point bending. Bars are mean ± SD. *, indicates significant pairwise differences at p<0.05, n = 6-11.

Figure 2. Deletion of Cx43 from osteoblasts and osteocytes results in reduced maturation of collagen cross-links in the femoral midshaft

Bone mineralization, crystallinity and collagen cross-linking were assessed in the femora by FTIRI in (A) cortical compartment of the diaphysis of Cx43^fl/− and OCN-Cre;Cx43^fl/− mice and (B) cancellous compartment of distal femora and cortical compartment of the diaphysis of Cx43^fl/fl and DMP1-8kb-Cre;Cx43^fl/fl mice. Ct: cortical, Cn: cancellous. Bars are mean ± SD. *, p<0.05 by independent Mann Whitney u-tests, n= 3. (C) Bone material density was measured by μCT in the femoral midshaft. Bars are mean ± SD, n=7-10.

Figure 3. Deletion of Cx43 from osteoblasts and osteocytes does not affect vertebral bone

(A) Mechanical properties of the lumbar vertebra L5 from Cx43^fl/− and OCN-Cre;Cx43^fl/− mice were determined by vertebral compression. Bars are mean ± SD. n = 6-11. (B) Bone mineralization, crystallinity and collagen cross-linking were assessed by FTIRI in the cancellous and cortical compartments of lumbar vertebra L5. Bars are mean ± SD, n= 3.

Figure 4. Deletion of Cx43 results in decreased expression of Lox

(A) mRNA levels of the indicated genes in calvaria cells isolated from the progeny of DMP-GFP;DMP1-8kb-Cre separated by FACS in GFP-negative and GFP-positive fractions. Mrsp2 was used as housekeeping gene. OB: osteoblasts; OT: osteocytes. (B) mRNA levels of the indicated genes in RNA purified from tibia diaphysis from wild type C57BL/6 mice. Mrsp2 was used as housekeeping gene. Bars are mean ± SD. *, indicates significant differences at p<0.05 vs Lox levels, n = 6. (C) Expression levels of Cx43, Lox, and Loxl-1 in primary calvaria cells from Cx43^fl/− and OCN-Cre;Cx43^fl/− mice and Cx43^fl/fl and DMP1-8kb-Cre;Cx43^fl/fl mice after 7 days in culture with ascorbic acid to induce differentiation into the osteogenic lineage. Data are expressed as relative to Mrsp2. Bars are mean ± SD. n.s. indicates not significant. (D) Expression levels of Cx43, Lox, and Loxl-1 in osteoblastic OB-6 cells and osteocytic MLO-Y4 cells with (scr shRNA) and without Cx43 (Cx43 shRNA). Data are expressed as relative to Mrsp2. Bars are mean ± SD. *, indicates significant differences at p<0.05, n = 3.