After bone injury, developmental processes such as endochondral and intramembranous ossification are recapitulated as the skeleton regenerates. In contrast to development, skeletal healing involves inflammation. During the early stages of healing a variety of inflammatory cells infiltrate the injured site, debride the wound, and stimulate the repair process. Little is known about the inflammatory process during bone repair. In this work, we examined the effect of a pro-inflammatory cytokine, Interleukin-1 beta (IL-1b), on osteoblast and stem cell differentiation and on intramembranous and endochondral ossification, because IL-1b exerts effects on skeletal homeostasis and is upregulated in response to fracture. We determined that IL-1b stimulated proliferation of osteoblasts and production of mineralized bone matrix, but suppressed proliferation and inhibited differentiation of bone marrow derived MSCs. We next performed loss-and gain-offunction experiments to determine if altering IL-1b signaling affects fracture healing. We did not detect any differences in callus, cartilage, and bone matrix production during healing of nonstabilized or stabilized fractures in mice that lacked the IL-1b receptor compared to wildtype animals. We observed subtle alterations in the healing process after administering IL-1b during the early phases of repair. At day 10 after injury, the ratio of cartilage to callus was increased, and by day 14, the proportion of cartilage to total callus and to bone was reduced. These changes could reflect a slight acceleration of endochondral ossification, or direct effects on cartilage and bone formation.

Age affects fracture repair; however, the underlying mechanisms are not well understood. The goal of this study was to assess the effects that age has on vascularization during fracture healing. Tibial fractures were created in juvenile (4-week-old), middle-aged (6-month-old), and elderly (18-month-old) mice. The length density and surface density of blood vessels within fracture calluses were analyzed using stereology at 7 days after fracture. The expression of molecules that regulate vascular invasion of the fracture callus was also compared among the three age groups by immunohistochemistry and in situ hybridization. At 7 days after fracture, juvenile mice had a higher surface density of blood vessels compared to the middle-aged and elderly. Hypoxia-inducible factor-1a protein and transcripts of vascular endothelial growth factor were detected at 3 days postinjury in juvenile but not middle-aged and elderly mice. Stronger Mmp-9 and -13 expression was detected in fracture calluses at day 7 in the juvenile compared to the middle-aged and elderly mice. At 21 days postfracture, expression of both Mmps was more robust in the elderly than juvenile and middle-aged animals. These data indicate that age affects vascularization during fracture repair, and the changes we observed are directly correlated with altered expression of biochemical factors that regulate the process of angiogenesis. However, whether the increased vascularization is the cause or result of accelerated bone repair in juvenile animals remains unknown. Nonetheless, our results indicate that enhancing vascularization during fracture repair in the elderly may provide unique therapeutic opportunities. ß

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Integrins are heterodimeric transmembrane proteins that mediate cell-matrix interactions and modulate cell behavior. Beta3 subunit is a component of aIIb3 and aVb3 integrins. In this study, we first determined that b3 transcripts are expressed by cells within fracture calluses at 7 and 10 days after injury in a mouse model. We then analyzed fracture healing in mice deficient of b3 integrin with molecular, histomorphometric, and biomechanical techniques. We found that lack of b3 integrin results in an extended bleeding time and leads to more bone formation and accelerated cartilage maturation at 7 days after injury. However, b3 deficiency does not appear to affect later fracture healing. At days 14 and 21, histological appearance or biomechanical properties of fracture calluses are similar between wild type and mutant mice. We also found that altered fracture healing in b3-null mice is not associated with accelerated angiogenesis, because no significant difference of length density and surface density of blood vessels in fracture limbs was detected at 3 days after injury between wild type and b3-null mice. In conclusion, our findings demonstrate that b3 integrin plays an important role during early fracture healing. Further research is required to determine the underlying mechanisms. ß