Impaired fracture healing is associated with callus chondro-osseous junction abnormalities in periostin-null and osteopontin-null mice

Periostin and osteopontin are matricellular proteins abundantly expressed in bone fracture callus. Null mutation of either the periostin (<i>Postn</i>) gene or the osteopontin (<i>Spp1</i>) gene can impair bone fracture healing. However, the cell and molecular pathways affected by loss of POSTN or SPP1 which lead to impaired fracture healing are not well understood. To identify potential pathways, a detailed radiological, histological, and immunohistochemical analysis of femur fracture healing in <i>Postn</i>-null (<i>Postn</i>KO), <i>Spp1</i>-null (<i>Spp1</i>KO), and normal (WT) mice was performed. Apparent changes in specific protein levels identified by immunohistochemistry were confirmed by mRNA quantitation. Comparisons between the <i>Postn</i>KO and <i>Spp1</i>KO fracture calluses were confounded by interactions between the two genes; loss of <i>Postn</i> reduced <i>Spp1</i> expression and loss of <i>Spp1</i> reduced <i>Postn</i> expression. Consequently, alterations in fracture healing between mice heterozygous for the <i>Postn</i>-null allele (<i>Postn</i>HET) as well as the <i>Postn</i>KO and <i>Spp1</i>KO mice were similar. Calluses from <i>Postn</i>HET, <i>Postn</i>KO, and <i>Spp1</i>KO mice all had dysmorphic chondro-osseous junctions and reduced numbers of osteoclasts. The dysmorphic chondro-osseous junctions in the <i>Postn</i>HET, <i>Postn</i>KO, and <i>Spp1</i>KO calluses were associated with reduced numbers of MMP-13 expressing hypertrophic chondrocytes, consistent with delayed cartilage resolution. Unlike collagen X expressing callus chondrocytes, chondrocytes only expressed MMP-13 when localized to the chondro-osseous junction or after traversing the chondro-osseous junction. Cyclooxygenase-2 (COX-2) expression also appeared to be reduced in osteoclasts from the <i>Postn</i>HET, <i>Postn</i>KO, and <i>Spp1</i>KO calluses, including in those osteoclasts localized at the chondro-osseous junction. The results indicate that POSTN and SPP1 are necessary for normal chondro-osseous junction formation and that signaling from the chondro-osseous junction, possibly from COX-2 expressing osteoclasts, regulates callus vasculogenesis and chondrocyte hypertrophy necessary for endochondral ossification during fracture healing.