(KJJ) S U M M A R Y Rat and mouse femur and tibia fracture calluses were collected over various time increments of healing. Serial sections were produced at spatial segments across the fracture callus. Standard histological methods and in situ hybridization to col1a1 and col2a1 mRNAs were used to define areas of cartilage and bone formation as well as tissue areas undergoing remodeling. Computer-assisted reconstructions of histological sections were used to generate three-dimensional images of the spatial morphogenesis of the fracture calluses. Endochondral bone formation occurred in an asymmetrical manner in both the femur and tibia, with cartilage tissues seen primarily proximal or distal to the fractures in the respective calluses of these bones. Remodeling of the calcified cartilage proceeded from the edges of the callus inward toward the fracture producing an inner-supporting trabecular structure over which a thin outer cortical shell forms. These data suggest that the specific developmental mechanisms that control the asymmetrical pattern of endochondral bone formation in fracture healing recapitulated the original asymmetry of development of a given bone because femur and tibia grow predominantly from their respective distal and proximal physis. These data further show that remodeling of the calcified cartilage produces a trabecular bone structure unique to fracture healing that provides the rapid regain in weight-bearing capacity to the injured bone.

ABSTRACT:Introduction: Distraction osteogenesis (DO) is characterized by the induction of highly vascularized new bone formation through an intramembranous process largely devoid of the formation of cartilage. Materials and Methods: To test the hypothesis that DO is strictly dependent on vascualrization, we inhibited vascular endothelial growth factor (VEGF) activity by antibody blockade of both receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1) or only VEGFR2 (Flk-1) in a previously developed murine tibia DO model. During normal DO, VEGFR1 (Flt-1), VEGFR2 (Flk-1), VEGFR3 (Flt4) and all four VEGF ligand (A, B, C, and D) mRNAs are induced. Results: The expression of mRNA for the receptors generally paralleled those of the ligands during the period of active distraction. Bone formation, as assessed by CT, showed a significant decrease with the double antibody treatment and a smaller decrease with single antibody treatment. Vessel volume, number, and connectivity showed progressive and significant inhibition in all of these of parameters between the single and double antibody blockade. Molecular analysis showed significant inhibition in skeletal cell development with the single and double antibody blockade of both VEGFR1 and 2. Interestingly, the single antibody treatment led to selective early development of chondrogenesis, whereas the double antibody treatment led to a failure of both osteogenesis and chondrogenesis. Conclusions: Both VEGFR1 and VEGFR2 are functionally essential in blood vessel and bone formation during DO and are needed to promote osteogenic over chondrogenic lineage progression.

These studies examined how genetic differences that regulate architectural and bone material properties would be expressed during fracture healing and determine whether any of these features would affect rates of healing as defined by regain of strength. Controlled fractures were generated in three inbred strains of mice: A/J, C57Bl/6J (B6), and C3H/HeJ (C3H). Both the A/J and B6 strains showed faster healing than the C3H strain based on regains in strength and stiffness. Strain-specific architectural features such as moment of inertia, cross-sectional area, and cortical thickness were all recapitulated during the development of the callus tissues. None of these traits were directly relatable to rates of fracture healing. However, rates of healing were related to variations in the temporal patterns of chondrogenic and osteogenic lineage development. The B6 strain expressed the highest percentage of cartilage gene products and had the longest period of chondrocyte maturation and hypertrophy. The slowest healing strain (C3H) had the shortest period of chondrogenic development and earliest initiation of osteogenic development. Although the A/J strain showed an almost identical pattern of chondrogenic development as the C3H strain, A/J initiated osteogenic development several days later than C3H during fracture healing. Long bone growth plates at 28 days after birth showed similar strain-specific variation in cartilage tissue development as seen in fracture healing. Thus, the B6 strain had the largest growth plate heights, cell numbers per column, and the largest cell size, whereas the C3H columns were the shortest, had the smallest number of cells per column, and showed the smallest cell sizes. These results show that (1) different strains of mice express variations of skeletal stem cell lineage differentiation and (2) that these variations affect the rate of fracture healing.