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. 2013 Jan;31(1):147-54.
doi: 10.1002/jor.22178. Epub 2012 Jun 20.

Osteoblast and osteocyte-specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing

Alayna E Loiselle  1 , Emmanuel M Paul, Gregory S Lewis, Henry J Donahue
Affiliations

Osteoblast and osteocyte-specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing

Alayna E Loiselle et al. J Orthop Res. 2013 Jan.

Abstract

Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte-specific loss of Cx43 display decreased gap junctional intercellular communication (GJIC), bone density, and cortical thickness. To determine the role of Cx43 in fracture healing, a closed femur fracture was induced in Osteocalcin-Cre+; Cx43(flox/flox) (Cx43cKO) and Cre-; Cx43(flox/flox) (WT) mice. We tested the hypothesis that loss of Cx43 results in decreased bone formation and impaired healing following fracture. Here, we show that osteoblast and osteocyte-specific deletion of Cx43 results in decreased bone formation, bone remodeling, and mechanical properties during fracture healing. Cx43cKO mice display decreased bone volume, total volume, and fewer TRAP+ osteoclasts. Furthermore, loss of Cx43 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post-fracture, compared to WT mice. These studies identify a novel role for the gap junction protein Cx43 during fracture healing, suggesting that loss of Cx43 can result in both decreased bone formation and bone resorption. Therefore, enhancing Cx43 expression or GJIC may provide a novel means to enhance bone formation during fracture healing.

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Figures

Figure 1
Figure 1
Immunohistochemical staining of Cx43 at [A] 10, [B] 14, [C] 21, [D] 28, [E] 35 days post-fracture. Cx43 expression occurs in osteoblasts lining new bone formation (white arrows [A-D]), and in osteocytes (yellow arrows [C]), in chondrocytes of the callus (yellow arrow [A]), and in marrow cells (yellow arrow [D]). A lack of Cx43 staining is observed in osteocytes/ osteoblasts at 35 days post-fracture (blue arrow [E]). [F] Non-immune IgG control shows a lack of Cx43 expression. 20X magnification. Scale bars represent 100 microns.
Figure 2
Figure 2
Histology and histomorphometry of WT [A, C, E, G, I], and Cx43cKO [B, D, F, H, J] fractures at [A&B] 10, [C&D] 14, [E&F] 21, [G&H] 28, and [I&J] 35 days post-fracture respectively. Sections were stained with Alcian Blue Hematoxylin/ Orange G. Cx43cKO fractures have larger areas of Alcian blue stained cartilage at 14 days [D&K]. Increased areas of new woven bone formation are apparent in the WT callus at 21 and 28 days (blue arrows, [E, G]). Cortical bone is not resorbed in Cx43cKO fractures (black arrows, [F, H]). 4x magnification. Scale bars represent 400 microns. [K&L] Histomorphometry of [K] cartilage, and [L] mineralized tissue as a proportion of total callus area from 10-35 days post-fracture. Data are presented as ± SEM, n=3/group, *=p<0.05.
Figure 3
Figure 3
[A-D] Representative images of TRAP staining of osteoclasts in WT [A, C] and Cx43cKO [B, D] fractures at 14 [A, B] and 21 [C, D] days post-fracture. [E] Quantification of TRAP+ osteoclasts. Data are presented ± SEM, n=3, *p<0.05. TRAP+ osteoclasts were counted for three sections/specimen, with three specimens/group/time-point. Scale bars represent 100 microns.
Figure 4
Figure 4
[A-H] Representative three-dimensional reconstructions of microCT scans of WT [A-D] and Cx43cKO [E-H] fractures at [A, E] 14, [B, F] 21, [C, G] 28, and [D, H] 35 days post-fracture. [I-L]: Quantification of callus mineralization during fracture healing in WT (white bars) and Cx43cKO (black bars). [I] Total callus bone volume (BV), [J] Total callus volume (TV), [K] BV/TV, [L] Low-density bone volume. Data are presented ±SEM, N=8-12/ group, *=p<0.05.
Figure 5
Figure 5
[A] Ultimate Torque (Tult) at failure of WT (white bars) and Cx43cKO fractures (black bars) from 14-35 days post-fracture. [B] Torsional Rigidity. Data are presented ±SEM, N=8-12/ group, *=p<0.05.
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