Session I - Combined Session (International Society for Fracture Repair)

The Effect of Early Stability on Fracture Repair

Theodore Miclau, III, MD; Zachary Thompson, BS; Celine Colnot, PhD; Diane Hu, MD; Zena Werb, PhD; Jill Helms, DDS; S. Huang; University of California, San Francisco, San Francisco General Hospital, Department of Orthopaedic Surgery, San Francisco, California, USA

Introduction: Fracture repair is an ideal context for addressing the fundamental questions underlying skeletal tissue regeneration, remineralization, and remodeling. Fractures heal by two mechanisms: endochondral and intramembranous ossification. In both cases, in response to locally increased levels of growth factors and cytokines, mesenchymal cells migrate to the wound site, where they differentiate into chondrocytes or osteoblasts. The mechanical environment influences this cell fate decision; motion at the site of injury is associated with the formation of a cartilage intermediate, whereas rigidly stabilized fractures appear to heal by the direct differentiation of mesenchymal cells into osteoblasts. Precisely how cells perceive and respond to changes in the local mechanical environment is unclear, and when this cell fate commitment is made is unknown.

Purpose: Our goal was to ascertain the time frame in which cells become irreversibly committed to either chondrogenic or osteoblastic cell fates. We compared fracture healing in mice in which the bone segments were left unstable for varying amounts of time, then stabilized and allowed to heal. The extent to which the fractures healed through intramembranous or endochondral ossification was determined by molecular, cellular, and histological analyses.

Methods:

Fracture Technique: All protocols were approved by the Institutional Committee on Animal Research. Adult mice were anesthetized by intraperitoneal injection of Avertin. Closed transverse fractures were created through the left tibial diaphysis by three-point bending and were confirmed by radiography.

Surgical procedure(stabilized fractures): After shaving, the proximal and distal metaphyses of the left tibia were transfixed with use of four 0.25-mm pins oriented perpendicular to the long axis of the tibia, 90° to each other. An external fixation pin was positioned, and after creation of the transverse fracture, the second ring was secured to the distal pins. A third threaded rod was placed through proximal and distal rings to provide further stability at the time of definitive fixation. Mice were allowed to ambulate freely, which typically occurred within 24 hours. Groups: Animals were divided into three groups: mice in which closed fractures were stabilized for the entire healing period; mice in which the fractures were unstable for 24 hours, then stabilized for the remaining healing period; and mice whose bone segments were left unstabilized for 48 hours, then stabilized for the remaining healing period. The mice were allowed to heal for 10 days after fracture.

Molecular and Cellular Analyses: The fracture callus tissues were prepared for standard histological and molecular analyses, including immunohistochemistry (platelet endothelial cell adhesion molecule, PECAM) and in situ hybridization (collagen type II, Col2 cartilage), (collagen type X, col10, hypertrophic cartilage), and (osteocalcin, bone).

Results: Fractures that were stabilized for the entire healing period generated new bone through the direct differentiation of mesenchymal cells into osteoblasts, as evidenced by the presence of osteocalcin, and the absence of collagen type IIa transcripts in mesenchymal cells at the site of injury. Similarly, the majority of fractures that were unstable for 24 hours also healed by intramembranous ossification. Histologic staining and in situ hybridization analyses indicated that mesenchymal cells expressed osteocalcin but not collagen type IIa at multiple time points after fracture. In contrast, most fractures that were left unstable for 48 hours healed through endochondral ossification.

Discussion/Conclusion: These data strongly suggest that the mechanical environment exerts its influence on bone regeneration within the earliest days of fracture healing. The results of this study provide evidence that regulating the mechanical environment during the initial inflammatory phase influences the differentiation of mesenchymal stem cells to an osteogenic or chondrogenic cell fate. Within 48 hours of sustaining an injury, cells at the fracture site have begun to express molecular markers that indicate their ultimate differentiation into osteoblasts or chondrocytes. We are currently exploring the molecular signals that are involved in transducing these mechanical stimuli during the early stages of fracture repair.