mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of β-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis. Research article Development and disease Development 1996 modulating the canonical Wnt pathway, was first identified in a mouse mutant strain (Zeng et al., 1997). Substantial evidence has established that Axin1 and its homolog Axin2/conductin/ Axil plays a central role in regulating the stability of β-catenin, which is a crucial event in cellular response to Wnt signaling (Kikuchi, 2000;Miller et al., 1999;Moon et al., 2002;Peifer and Polakis, 2000). Axins serve as scaffold proteins directly associating with several Wnt signaling molecules, including disheveled, the serine/threonine kinase GSK-3, β-catenin, adenomatous polypopsis coli (APC) and the serine/threonine protein phosphatase 2A (PP2A) (Behrens et al., 1998;Fagotto et al., 1999;Hedgepeth et al., 1999;Hsu et al., 1999;Itoh et al., 1998;Julius et al., 2000;Kishida et al., 1998;Sakanaka et al., 1998). In the absence of a Wnt signal, the Axin-dependent complex mediates β-catenin degradation, while Wnt signals perturb formation of this complex (Farr et al., 2000;Li et al., 1999;Smalley et al., 1999;Yanagawa et al., 1995). Therefore, β-catenin is accumulated and binds to LEF/TCF family proteins to activate target genes (Behrens et al., 1996; Brannon et al., 1997;Molenaar et al., 1996). Wnt signaling controls early craniofacial morphogenesis (Parr et al., 1993). Wnt1 and Wnt3a are both expressed in the dorsolateral region of the neural tube that gives rise to CNC (McMahon et al., 1992). Although inactivation of either Wnt1 or Wnt3a gene did not cause defects in craniofacial development (McMahon and Bradley, 1990;Takada et al., 1994), mice in which both the Wnt1 and Wnt3a genes are inactivated showed a marked deficiency in CNC derivatives (Ikeya et al., 1997). Furthermore, downstream components of the Wnt signaling pathway, including Lrp6, APC and β-catenin, have also been implicated in craniofacial development (Brault et al., 2001;Hasegawa et al., 2002;Mitchell et al., 2001). Nevertheless, the importance of the Wnt pathway in intramembranous ossification during mammalian skull formation remains unclear.In this study, we have investigated the involvement of Axin2 in cranial skeletogenesis. Targeted disruption of Axin2 did not cause obvious embryonic abnormalities, although Axin2 is highly expressed in CNC. However, our data demonstrate that Axin2 is required for skull development at early postnatal stages...

Preclinical and clinical studies suggest a possible role for cyclooxygenases in bone repair and create concerns about the use of nonsteroidal antiinflammatory drugs in patients with skeletal injury. We utilized wild-type, COX-1(-/-), and COX-2(-/-) mice to demonstrate that COX-2 plays an essential role in both endochondral and intramembranous bone formation during skeletal repair. The healing of stabilized tibia fractures was significantly delayed in COX-2(-/-) mice compared with COX-1(-/-) and wild-type controls. The histology was characterized by a persistence of undifferentiated mesenchyme and a marked reduction in osteoblastogenesis that resulted in a high incidence of fibrous nonunion in the COX-2(-/-) mice. Similarly, intramembranous bone formation on the calvaria was reduced 60% in COX-2(-/-) mice following in vivo injection of FGF-1 compared with either COX-1(-/-) or wild-type mice. To elucidate the mechanism involved in reduced bone formation, osteoblastogenesis was studied in bone marrow stromal cell cultures obtained from COX-2(-/-) and wild-type mice. Bone nodule formation was reduced 50% in COX-2(-/-) mice. The defect in osteogenesis was completely rescued by addition of prostaglandin E2 (PGE(2)) to the cultures. In the presence of bone morphogenetic protein (BMP-2), bone nodule formation was enhanced to a similar level above that observed with PGE(2) alone in both control and COX-2(-/-) cultures, indicating that BMPs complement COX-2 deficiency and are downstream of prostaglandins. Furthermore, we found that the defect in COX-2(-/-) cultures correlated with significantly reduced levels of cbfa1 and osterix, two genes necessary for bone formation. Addition of PGE(2) rescued this defect, while BMP-2 enhanced cbfa1 and osterix in both COX-2(-/-) and wild-type cultures. Finally, the effects of these agents were additive, indicating that COX-2 is involved in maximal induction of osteogenesis. These results provide a model whereby COX-2 regulates the induction of cbfa1 and osterix to mediate normal skeletal repair.

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Due to irreversible joint destruction caused by the various arthritides, more than 400,000 total joint arthroplasties are performed each year in the United States. As many as 20% of these require revision surgery because of aseptic loosening. The current paradigm to explain aseptic loosening is that wear debris generated from the prosthesis stimulates the release of proinflammatory cytokines (i.e., tumor necrosis factor-alpha and interleukins 1 and 6) following phagocytosis by resident macrophages. These cytokines, in turn, initiate an inflammatory response, with the development of an erosive pannus that stimulates bone resorption by osteoclasts. In support of this model, we have previously shown that human monocytes produce large quantities of tumor necrosis factor-alpha in response to titanium particles in vitro. In the current study, we characterized the role of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in the proinflammatory response to titanium particles in vitro and in vivo. Using the mouse macrophage cell line J774, we showed that these cells produce an amount of tumor necrosis factor-alpha in response to titanium particles similar to that produced by human peripheral blood monocytes. The production of tumor necrosis factor-alpha was preceded by a drop in cellular levels of inhibitory factor-kappaBalpha protein and translocation of p50/p65 nuclear transcription factor-KB to the nucleus 30 minutes after stimulation. Levels of tumor necrosis factor-alpha and inhibitory factor-kappaBalpha mRNA increased 30 minutes after stimulation, consistent with the activation of nuclear transcription factor-kappaB. Interleukin-6 mRNA was first seen 4 hours after the addition of the titanium particles, indicating that the production of this cytokine is secondary to the immediate nuclear transcription factor-kappaB response. To test the relevance of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in response to titanium particles in vivo, we adopted an animal model in which the particles were surgically implanted on the calvaria of mice. The animals displayed a dramatic histological response to the debris, with the formation of fibrous tissue and extensive bone resorption after only 1 week. With use of immunohistochemistry and tartrate-resistant acid phosphatase staining, tumor necrosis factor-alpha and osteoclasts were readily detected at the site of inflammation and bone resorption in the calvaria of the treated mice. By testing mice that genetically over-produce tumor necrosis factor-alpha (hTNFalpha-Tg), those defective in tumor necrosis factor-alpha signaling (TNF-RI-/-), and those that are nuclear transcription factor-kappaB1-deficient (NFkappaB1-/-), we evaluated the importance of tumor necrosis factor-alpha/nuclear transcription factor-kappaB signaling in the biological processes responsible for aseptic loosening. The hTNFalpha-Tg mice had a grossly exaggerated response, the TNF-RI(-/-) mice showed little evidence of inflammation or bone resorption, and the n...

Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p < 0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection. ß