Hox11 Function Is Required for Region-Specific Fracture Repair

Abstract

The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα+/CD51+/LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss-of-function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton. © 2017 American Society for Bone and Mineral Research.

Keywords: ENDOCHONDRAL OSSIFICATION-CARTILAGE; HOX GENES; MESENCHYMAL STROMAL/STEM CELLS; MOLECULAR PATHWAYS-DEVELOPMENT; SKELETAL INJURY/FRACTURE HEALING.

Figure 1. Hox11 is expressed throughout fracture injury in the limb zeugopod

Limb schematic depicts Hoxa11eGFP regional expression (green) and the fracture callus in the zeugopod region (tibia). Hox11 expression is shown using a GFP primary antibody and developing with alkaline phosphatase. (A) Hox11 is expressed at low levels in the hematoma. (B) Hox11 expression expands at 1.5 WPF including the intramedullary space (*) and in the expanded periosteum surround the callus (arrows). (C) Hox11 is expressed near woven bone surfaces in the hard callus at 3 WPF.

Figure 2. Loss of Hox11 function results in defects following fracture injury

(A-D) X-rays (top panels) and cross-sectional views of mCT (lower panels) in control and in Hox11 mutant animals. (A) At 1.5WPF, x-ray and mCT images are comparable between groups. (B) At 3WPF, controls are bridged and mutants are not. (C-D) At 6WPF and 12WPF, most mutants are now bridged, but exhibit delayed remodeling compared to controls. (E) Cross-sectional views of mCT at 21WPF persistent and incomplete bone remodeling in mutant animals. (F) 100% of control animals demonstrate union fractures compared to 71% of Hox11 mutant animals. Statistical analysis carried out by two-tailed Fisher Exact test and by two-tailed Chi Square test; * = p<0.05. (G) mCT analysis shows statistically significant maintenance of callus volume at late stages in mutants. Statistical analysis carried out by Students' T-test; * = p<0.05. WPF; weeks post-fracture.

Figure 3. Intramembranous ossification and vascularization are unchanged in Hox11 mutant fractures

(A) mCT analysis of outer regions of callus show comparable bone formation in regions of intramembranous ossification. (B) Osterix and PECAM-stained sections show bone formation and vascularization in regions of intramembranous ossification. cb; cortical bone. (C-D) PECAM-staining in controls and Hox11 mutants shows comparable vascularization in the early callus (1.5WPF). cb; cortical bone.

Figure 4. Chondroctye differentiation and endochondral ossification is disrupted in the Hox11 mutant callus

(A-B) Histomorphometric quantifications of the mesenchymal, woven bone and cartilage areas from Safranin O/Fast Green-stained sections at 1.5, 3, and 6 weeks post-fracture (WPF). Cartilage was designated by Safranin O. Woven bone and mesenchyme were designated visually; the latter refers to non-woven bone, non-safranin O-positive area. Abundant cartilage formation is visualized in center regions of control calluses; mesenchyme is maintained in similar regions of mutant calluses. Statistical analysis carried out by Students' T-test; * = p<0.05. (C) Sox9, Collagen2a1 and Collagen 10a1-stained sections at 1.5WPF show chondrocyte differentiation in control and mutant calluses. (D) Von Kossa-stained sections show unbridged callus at 3WPF in mutant fractures and undifferentiated mesenchyme at the center of the callus. wb; woven bone.

Figure 5. Osteoclasts are present, express markers of resorption in the Hox11 mutant callus

(A) TRAP-stained callus sections from control and Hox11 mutant animals at 3 and 6 WPF show TRAP+ osteoclasts in calluses. (B) Histomorphometric quantification of osteoclasts per bone surface (%) and number of osteoclasts per 1mm of bone surface is comparable in controls and mutants. (C) Cathepsin K-stained callus sections from control and mutant animals show positive staining in controls and mutants. Safranin O/Fast Green staining on the same sections shows the overlap of CathepsinK with woven bone areas. (D) Images of large, detached osteoclasts in the Hox11 mutant callus compared to control osteoclasts that are flat and attached to the bone surface.

Figure 6. Bone matrix organization is disrupted due to the loss of Hox11 function

(A-B) Raman spectroscopy of woven bone callus and cortical bone outside the callus from controls and Hox11 mutants at 3 WPF. Parameters measured include mineral crystalinity (A) and mineral to matrix [Pro+Hyp] ratio (B). (C-D) Picrosirius red-stained sections with brightfield (top panels) or polarized light microscopy (bottom panels) for woven bone in fracture callus (C) and cortical bone out the callus (D). (E) Sclerostin-stained cortical bone shows disorganized osteocytes in Hox11 mutants.