OTA 2006 Posters

Scientific Poster #91 Basic Science

Aging Altered Angiogenesis during Fracture Healing
Chuanyong Lu, MD (n); Eric Hansen, MD (n); Anna Sapozhnikova, BA (n); Diane Hu, MD (n); Ralph Marcucio, PhD (n); Theodore Miclau, MD (n);
University of California at San Francisco, San Francisco, California, USA

Purpose: Aging delays healing of fractures by reducing the rate of bone and cartilage formation. However, the underlying mechanism is not well understood. Angiogenesis, the sprouting of new capillaries from existing blood vessels, is crucial for normal fracture healing and is altered in aged animals. We hypothesized that aging may alter angiogenesis, leading to delayed fracture healing. To test this hypothesis, we compared vascularization and expression patterns of genes that are required for angiogenesis during fracture healing among the mice of different ages.

Methods: Nonstabilized tibia fractures were created by 3-point bending in 1-month (juvenile), 6-month (middle-aged), and 18-month (elderly) old mice. Animals were sacrificed at 3, 5, 7, 10, 14, 21, and 28 days postfracture (3-5 animals/time point/age group). The fractured tibia were collected, fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and sectioned. Cartilage and bone were stained with Safranin O/Fast Green (SO/FG) and Trichrome respectively. To visualize blood vessels, immunohistochemistry using an anti-PECAM (platelet endothelial cell adhesion molecule) antibody was performed on one section from the middle of the fracture site in each animal. Vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and MMP-13 transcripts were detected by in situ hybridization.

Results: By 3 days postfracture, the hematoma between fracture ends was not vascularized. New blood vessels were observed in the bone marrow close to the fractured bone ends in juvenile mice. The injured bone marrow of middle-aged and elderly mice appeared to be vascularized to a lesser extent. Blood vessels were present in periosteum and surrounding soft tissue but did not appear to exhibit any significant differences among the three age groups. By 5 days, the fracture calluses were more cellular than that at 3 days postfracture. New blood vessels were observed throughout the calluses with islands of avascular cartilage in mice of all ages. At this time, blood vessel invasion into cartilage, a hallmark of endochondral ossification, was observed in juvenile mice but not in adult and elderly mice. By 10 days after fracture, endochondral ossification was actively occurring in mice of all ages. The process of endochondral ossification was prolonged in the middle-aged and elderly mice compared to juvenile mice. VEGF, MMP-9, and MMP-13 transcripts were expressed at high levels by hypertrophic chondrocytes and inflammatory cells at the ossification front, and were detected earlier in juvenile mice. Expression of these genes was protracted in middle-aged and elderly mice.

Conclusion/Significance: Aging animals exhibit decreased ability to heal skeletal injuries, which could be exacerbated by decreased angiogenic potential. Our results indicate that aging affected the vascularization of fracture calluses and also delayed and protracted VEGF, MMP-9, and MMP-13 expression. These changes correlated with the age-related delays of chondrogenesis and endochondral ossification during fracture healing. Our ongoing research is focusing on determining the mechanistic relationship between angiogenesis and fracture healing, and how alterations in angiogenesis during aging impact fracture repair.

If noted, the author indicates something of value received. The codes are identified as a-research or institutional support; b-miscellaneous funding; c-royalties; d-stock options; e-consultant or employee; n-no conflicts disclosed, and *disclosure not available at time of printing.
· The FDA has not cleared this drug and/or medical device for the use described in this presentation (i.e., the drug or medical device is being discussed for an "off label" use). · · FDA information not available at time of printing.