Effects of genetic variability on fracture healing: a temporal study of gene expression and callus phenotype

Abstract

Bones have a large intrinsic capacity for repair and regeneration following an injury, however, an estimated 5-10% of nearly 8 million fractures that occur every year in the United States lead to nonunions. The process of bone regeneration is a complex trait that brings together different complements of molecular and cellular interactions to carry out its necessary mechanical functions. These interactions may be attributable to the effects of genetic variations that contribute to differences in bone morphology and fracture healing. This study was undertaken to determine how genetic variability that controls phenotypic qualities of bone affect rates and patterns of fracture healing. Three inbred strains of mice (A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3)) with known structural and biomechanical differences resulting from fetal bone development were examined. Transverse fractures were generated in the femur and healing traits were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), micro-computed tomography (μCT), biomechanical torsional testing, and cartilage contrast-enhanced micro-computed tomography (CECT). The temporal analysis of gene expression revealed that B6 had the longest duration of chondrocyte maturation and the greatest relative expression of osteogenic genes relative to either C3 or AJ. While AJ and C3 exhibited similar patterns of chondrogenesis, AJ initiated osteogenesis earlier than C3. These results suggest that compared to either AJ or B6, the C3 strain exhibited the least temporal coordination between the chondrogenic and osteogenic stages. Consistent with the relative patterns of RNA expression, μCT evaluations at day 21 post fracture showed that B6 had higher callus mineralization than AJ and C3. μCT, cartilage CECT, and biomechanical testing revealed less tissue mineralization and more cartilage near the fracture gap, which indicated a less developed bony bridge in C3 calluses at day 21 post fracture. The lack of large amounts of cartilage in calluses of all strains by day 21 indicated that all strains had initiated osteogenesis by this time. Taken together, these results showed that mice with different genetic backgrounds express different patterns of mobilization and renewal of skeletal stem cells with differing temporal progressions of chondrogenic and osteogenic differentiation. These data further show that these variations affect the phenotypic properties of fracture calluses during the process of fracture healing.