Session I - Basic Science
Do All Bones Heal Through the Same Pathways?
Theodore Miclau, III, MD1; Randall Nacamuli, MD2; Chuanyong Lu, MD1; Zachary Thompson, BS1; Michael Longaker, MD2; Jill A. Helms, DDS, PhD1;
Purpose: Although the process of adult fracture repair in the cranial skeleton clinically resembles that of the appendicular skeleton, the mechanisms that direct cranial skeletal repair are less well understood. One distinct difference between these two skeletal tissues is that the cells that initially form their elements originate from distinct sources. During development, the appendicular skeleton arises from the mesoderm through endochondral ossification, whereas the cranial skeleton is derived from paraxial mesoderm and the cranial neural crest through endochondral or intramembranous ossification or both, depending on the element. Because of the differences in embryonic origin, one hypothesis is that the cellular and molecular mechanisms that govern skeletal repair in bone derived from the cranial neural crest are different from those that regulate healing in bone originating from the mesoderm. This study compared the process of fracture repair in the mandible, which is derived from the neural crest, with that of the tibia, which is derived from the mesoderm.
Methods: All protocols were approved by the Institutional Committee on Animal Research.
Mandible fractures: Twenty-four adult mice between the ages of 10 and 12 weeks were anesthetized. An incision was made over the inferior portion of the right posterior mandible, and the masseter muscle was divided along its length and elevated. The posterior aspect of the mandible was exposed, and a high-speed dental drill was fitted with a 0.2-mm diamond disc that was used to create a complete transverse osteotomy just proximal to the third molar. The soft tissues and skin were closed with nonabsorbable suture. After surgery, mice were given a ground diet and monitored.
Tibia fractures: Thirty-five adult mice were anesthetized as described above, and a closed transverse fracture was created by three-point bending and confirmed radiographically. After surgery, mice were allowed to ambulate freely.
Molecular and cellular analyses: The mice were allowed to heal and were sacrificed at time points corresponding with the inflammatory (3 and 5 days), soft callus (7 and 10 days), hard callus (14 days), and remodeling (21 and 28 days) phases of healing. The skin was removed and the tissue was harvested and prepared for molecular and cellular analyses. Tissues were prepared for histologic examination and in situ hybridization. 35S-labelled sense and antisense UTP-labeled riboprobes were synthesized from plasmids corresponding to osteocalcin (oc), collagen type II (col2) and collagen type X (col10). The histologic staining (Safranin-O and Hall Brunt Quadruple) was performed by following standard protocols.
Quantification of cartilage in callus: Histomorphometric measurements of sections through the entire mandible and tibia calluses were used to determine cartilage volume at 10 days (N = 5), 14 days (N = 5), and 21 days (N = 4) after fracture. The values for average percentage of cartilage volume per callus volume for the fractures were compared at these time points by using a t-test.
Results: Mandible and tibia fracture healing: All mandibular and tibial fractures were significantly displaced, indicating instability. At days 7 and 10, or the soft callus phase of fracture repair, the mandible fractures exhibited new bone formation with no cartilage present at the site of injury by histologic staining. These histologic observations are confirmed by the in situ hybridization data that shows oc-expressing cells (indicating osteogenesis) along the periosteal and endosteal surfaces of the bone, and a lack of col2-expressing cells (present with chondrogenesis) at the fracture site. In contrast, the nonstabilized tibia fracture calluses demonstrated abundant cartilage formation at the fracture site, in addition to the periosteal and endosteal surfaces of the bone ends. By day 14, the hard callus phase of healing, the mandibular fractures demonstrated abundant appositional bone formation with limited cartilage staining at the fracture site. In situ hybridization data indicated that these areas of cartilage had a subset of cells that expressed col2, indicating the presence of immature chondrocytes. At this time point, nonstabilized tibia calluses were composed mostly of hypertrophic cartilage, which was being degraded and replaced by bone. Although the amount of cartilage was diminished relative to tibia fractures at days 7 and 10, there was still significantly more cartilage relative to the mandibular fractures. At day 21, in both mandible and tibia fractures, regenerated bone bridged the fracture segments, and there was little cartilage detectable. At day 28, both mandibular and tibia fractures exhibited bridging bone with no remaining cartilage.
Quantification of cartilage in callus: The data from the histomorphometric analyses supported the above observations. At day 10 after fracture, there was little (1/5) or no (4/5) cartilage present in the calluses (% volume of cartilage/volume of callus = 0.1 ± 0.4%). In contrast, the tibia fractures exhibited significantly large relative volumes of callus (21.5 ± 5.9%; P <0.0001). At day 14, there was still a significantly large difference in the amount of cartilage present between the mandible fractures (1.3 ± 0.8%) and the tibia fractures (13.7 ± 4.7%; P <0.0002). At day 21, both the mandible fractures and the tibia fractures showed minimal cartilage formation (1.5 ± 0.8% and 0.6 ± 0.8%, respectively; P <0.1).
Conclusions: The results of this study indicated that there are distinct differences between fracture repair in the nonstabilized murine mandible and in the tibia. These observations support the possibility that bones may heal through different pathways, depending on the embryonic origin of the injured skeletal tissue. These findings may have important implications for developing clinical therapies to accelerate repair. Future studies will further characterize the cellular and molecular mechanisms that account for these differences.