Session V - Fracture Healing

The Effect of Fracture Stability on the Expression of Angiogenic Factors During Fracture Repair

Theodore Miclau, MD; Zachary Thompson, BS; Christian Ogilvie, MD; Diane Hu, PhD; Zena Werb, PhD; Jill A. Helms, PhD, DDS; University of California, San Francisco, San Francisco, CA

Introduction: Throughout life, skeletal tissues exhibit a remarkable ability to adapt to changes in the mechanical environment. Growing evidence indicates that epigenetic factors, such as mechanical forces, are critical regulators of chondrogenesis and osteogenesis during healing. However, the molecular and cellular mechanisms that influence how mesenchymal cells sense different mechanical environments and differentiate into chondrocytes or osteoblasts is largely unknown. One possible explanation is that excessive motion delays angiogenesis, therefore retarding chondrocyte maturation and hypertrophy and resulting in a persistence of cartilage. Conversely, a relatively stable environment allows for vascular ingrowth and accelerates the differentiation of mesenchymal cells into chondrocytes and their subsequent replacement by bone. Our hypothesis is that fracture stability influences the molecular program of the mesenchymal cells at the site of injury, particularly the expression of genes which regulate angiogenesis. The purpose of this study was to analyze the expression of angiogenic genes that are implicated in bone formation during the various stages of stabilized and non-stabilized murine tibial fracture repair.

Methods: All procedures were approved by the University Committee on Animal Research. Forty wild-type mice were divided equally into stabilized and non-stabilized groups. Closed unstable transverse mid-diaphyseal fractures of the tibia were created in 3-point bending, or the fractures were stabilized by placement of a circular ring fixator. Radiographs confirmed the extent of fracture or the position of bone segments. Mice were sacrificed by cervical dislocation at multiple time points corresponding with the 4 stages of fracture repair (inflammatory, soft callus, hard callus, and remodeling). The extent of bone healing and of revascularization were assessed at multiple time points. The fracture callus tissues were prepared for standard histological and molecular analyses, including in-situ hybridization (corresponding to the angiogenic factors VEGF and Ang1, Flt-1 and Flk-1 [VEGF receptors], matrix metalloproteinase 13 [mmp13], indian hedgehog [ihh, cartilage], collagen type II [Col2, cartilage], collagen type X [Col10, hypertrophic cartilage], bone morphogenic protein 6 [bmp6, cartilage], and osteocalcin [bone]).

Results: Non-stabilized fractures healed with increased cartilage formation, whereas the stabilized fractures healed with no, or minimal cartilage. In the non-stabilized fracture callus 3 days after injury, the site was populated predominantly by fibroblast-like cells that weakly expressed collagen type II (col2). By 5 days post-fracture, abundant cartilage was evident at the site of injury, which correlated with the abundant expression of col2, ihh and bmp6. Within 7 days of fracture, the cartilage callus had begun to mature. Ihh and bmp6 were strongly expressed by pre-hypertrophic chondrocytes within the callus, along with angiogenic markers such as VEGF and ang1, the VEGF receptors (flt-1 and flk-1), and mmp13. Bone was also apparent at this time with osteocalcin (oc) expression. Within 28 days of fracture, the callus had completely ossified, and these gene transcripts were no longer detected. Angiogenic factors were detected throughout the bony callus at this stage. Stabilization of the bone fragments substantially decreased the overall amount of cartilage that formed in the callus. In contrast to the non-stabilized fracture calluses, cartilage associated genes, including ihh, col2, and col10 were detected later in the healing process and more transiently. The expression of angiogenic genes was detected early in the healing process and more transiently as well.

Discussion and Conclusion: The results of this study suggest that stabilizing the fracture calluses clearly influences the molecular program of the mesenchymal cells at the site of injury, including those that regulate angiogenesis. In addition, mesenchymal cells in the callus appear to commit to a chondrogenic or osteogenic fate during the initial stages of healing. Mechanical forces may influence mesenchymal cell differentiation by either facilitating or preventing vascular ingrowth into a fracture site. This hypothesis is predicated on the idea that mesenchymal cell fate decisions would be influenced by factors delivered via an intact vasculature, and by new blood vessels providing a source for osteoblasts to the wound site. Further study is necessary to determine the role of mechanical stability in the regulation of angiogenesis throughout fracture repair.