Sutures Possess Strong Regenerative Capacity for Calvarial Bone Injury

Abstract

Repair of calvarial bony defects remains challenging for craniofacial surgeons. Injury experiments on animal calvarial bones are widely used to study healing mechanisms and test tissue engineering approaches. Previously, we identified Gli1+ cells within the calvarial sutures as stem cells supporting calvarial bone turnover and injury repair. In this study, we tested the regenerative capacity of the suture region compared with other areas of calvarial bone. Injuries were made to mouse sagittal sutures or other areas of the calvarial bone at varying distances from the suture. Samples were collected at different time points after injury for evaluation. MicroCT and histological analyses were conducted. EdU incorporation analysis was performed to assay cell proliferation. Gli1-Cre^ERT2;Tdtomato^flox mice were used to trace the fate of Gli1+ stem cells after injury. Calvarial sutures possess much stronger regeneration capability than the nonsuture bony areas of the calvaria. The healing rate of the calvarial bone is inversely proportional to the distance between the suture and injury site: injuries closer to the suture heal faster. After complete removal of the sagittal suture, regeneration and restoration of normal organization occur within 6 weeks. Gli1+ cells within the suture mesenchyme are the cellular source for injury repair and bone regeneration. These results demonstrate that calvarial bone healing is not an evenly distributed event on the calvarial surface. Sutures contain stem cells and are the origin of calvarial bone tissue regeneration. Therefore, current practice in calvarial surgery needs to be reevaluated and modified. These findings also necessitate the design of new approaches for repairing calvarial bony defects.

Keywords: calvarial bone injury and repair; suture stem cells.

FIG. 1.

The suture possesses stronger regenerative capacity than other regions of the calvarial bone. (A) Representation of sites of injuries (blue circles) to the skulls of 6-week-old mice. Three holes, 2 mm in diameter, were drilled in different locations of the parietal bone. (B, C) MicroCT (B) and light microscopic imaging (C) of calvarial bones 1 month after injury. (D) MicroCT image 1 month after injury. Asterisk indicates injury site on the suture and arrows indicate injury sites in the center of the parietal bone. (E) Fast Red staining of the harvested parietal bone 1 month after injury. Boxed regions E-a and E-b are enlarged (in F and G, respectively). Scale bars (A–C), 1 mm. Scale bars (D–G), 200 μm.

FIG. 2.

Time course of suture injury repair. (A–D) MicroCT images of skulls 1–4 weeks after injury with sutures of 6-week-old mice. (E–H) Fast Red staining of sections of skulls 1–4 weeks after injury with sutures of 6-week-old mice. Dotted lines outline the bony surfaces of the calvaria. Scale bars (A–D), 1 mm. Scale bars (E–H), 200 μm.

FIG. 3.

Regeneration of sagittal sutures after complete removal. (A–D) MicroCT analysis of skulls 1, 2, 3, and 6 weeks after removal of a 2 × 5 mm rectangular region, including the entire sagittal suture from 6-week-old mice. Dotted lines outline the approximate original sizes of the injury sites. Scale bars, 1 mm.

FIG. 4.

The healing capacity of an injury site is inversely proportional to its distance from the suture. (A) Representation of injury sites (blue circles) on skulls of 6-week-old mice; (a) on the suture; (b) 0.5 mm from the suture; (c) 1 mm from the suture; and (d) and (d′) 2 mm from the suture. (B, C) MicroCT analysis of calvarial bones 1 month after injury. (D) Quantification of results from experiments is represented (B, C). n = 5. *P < 0.01. Scale bars, 1 mm.

FIG. 5.

The suture responds to injury in a manner distinct from nonsuture regions. (A–D) EdU staining (red) of sutures from mice uninjured (A) and 4 days postinjury (B). Boxed regions (B) are shown enlarged (C, D). Dotted lines outline the bony surfaces of the calvaria. (E) Visualization of Gli1-CE;Tdtomato^flox (GT) mice 2 weeks after induction with tamoxifen at 1 month of age and injury ∼1 mm from the suture. Red staining indicates Gli1+ cells. Experimental procedures are described on the left. Asterisk indicates the migrating Gli1+ cells. (F) β-Gal staining (blue) of sutures of Gli1-LacZ mice 4 weeks after injury to the suture. Experimental procedures are described on the left. Dotted circles outline the approximate injury site. Scale bars (A–D), 200 μm. Scale bars (E, F), 1 mm.

FIG. 6.

Rabbit calvarial sutures possess stronger regenerative capacity than nonsuture regions. MicroCT imaging of calvarial bones 1 month after injury of 2-month-old rabbits. Holes, 4 mm in diameter, were drilled in the parietal bones. One injury was centered on the sagittal suture and the other on the parietal bone. Dotted circles outline the injury locations. Scale bar, 1 mm.