Studies were performed evaluating the role of Smad3, a transcription factor mediating canonical TGF-β signaling, on scarring and adhesion formation using an established flexor digitorum longus (FDL) tendon repair model. In unoperated animals the metatarsophalangeal (MTP) range of motion (ROM) was similar in Smad3−/− and wild type (WT) mice while the basal tensile strength of Smad3−/− tendons was significantly (39%) lower than in WT controls. At 14 and 21 days following repair Smad3−/− MTP ROM reached approximately 50% of the level of the basal level and was twice that observed in WT tendon repairs, consistent with reduced adhesion formation. Smad3−/− and WT maximal tensile repair strength on post-operative day 14 was similar. However, Smad3−/− tendon repairs maximal tensile strength on day 21 was 42% lower than observed in matched WT mice, mimicking the relative decrease in strength observed in Smad3−/− FDL tendons under basal conditions. Histology showed reduced "healing callus" in Smad3−/− tendons while quantitative PCR, in situ hybridization, and immunohistochemistry showed decreased col3a1 and col1a1 and increased MMP9 gene and protein expression in repaired Smad3−/− tendons. Thus, Smad3−/− mice have reduced collagen and increased MMP9 gene and protein expression and decreased scarring following tendon FDL tendon repair.

Objective. To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor ␤ (TGF␤) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation.Methods. Joint disease in Smad3-knockout (Smad3 Ϫ/Ϫ ) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNAbinding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3 Ϫ/Ϫ mice. Osteoarthritis (OA) is a chronic joint disease characterized by progressive degenerative changes in the composition, structure, and function of articular tissues. OA primarily affects articular cartilage, although pathologic changes are also observed in the synovial mem- Results. In Smad3

As dominant regulators of osteoclastogenesis and bone resorption, receptor activator of NFKB (RANK), receptor activator of NFKB ligand, and OPG have been identified as ideal drug targets for the treatment of metabolic bone disease. One concern regarding the therapeutic use of RANK signaling inhibitors is their effect on fracture healing. Therefore we tested if uncoupling and osteoclast depletion via RANK blockade affects callus formation, maturation and matrix remodeling, as well as union rates in a mouse tibia fracture model. Low dose (1 mg/kg i.p.) RANK:Fc therapy had no effect on callus formation, matrix maturation and remodeling, and resulted in 100'%1 bony union by day 28. High dose RANK:Fc treatment (10 mgikg i.p.) effectively eliminated osteoclasts at the fracture site on day 14, with no significant effects on fracture healing. When therapy was discontinued, normal numbers of osteoclasts were observed at the fracture site by day 28. However. continuous therapy resulted in a large osteopetrotic callus consisting of both mineralized and unmineralized matrix that was void of osteoclasts, but bony union was unaffected at day 28. We also evaluated this process in the complete absence of RANK signaling using RANK -/-mice. These animals exhibited significant radiographic and histologic evidence of callus formation, indicating that RANK signaling is not required for fracture callus formation. However, only 33% of RANK 4-animals formed bony unions compared to 100'%1 of the osteopetrotic control mice. This defect was most likely a result of decreased blood flow, as evidenced by fewer blood vessels in the RANK 4-animals.Together, these data imply that osteoclast depletion via inhibition of RANK signaling is a viable option for the treatment of pathological bone loss since no adverse effects on fracture healing are observed when therapy is discontinued.

Periprosthetic osteolysis is the most common cause of aseptic loosening in total joint arthroplasty. The role of inflammatory mediators such as prostaglandin E2 (PGE2) and osteoclast promoting factors including RANKL in the pathogenesis of osteolysis has been well characterized. However, the PGE2 receptor (EP1, EP2, or EP4), and cell type in which it is expressed, which is responsible for PGE2 induction of RANKL during wear debris-induced osteolysis, has yet to be elucidated. To address this, we used mice genetically deficient in these EP receptors to assess PGE2 and wear debris responses in vitro and in vivo. Wear debris-induced osteolysis and RANKL expression were observed at similar levels in WT, EP1 2/2 , and EP2 2/2 mice, indicating that these receptors do not mediate PGE2 signals in this process. A conditional knockout approach was used to eliminate EP4 expression in FSP1 + fibroblasts that are the predominant source of RANKL. In the absence of EP4, fibroblasts do not express RANKL after stimulation with particles or PGE2, nor do they exhibit high levels of osteoclasts and osteolysis. These results show that periprosthetic fibroblasts are important mediators of osteolysis through the expression of RANKL, which is induced after PGE2 signaling through the EP4 receptor.

Axis inhibition proteins 1 and 2 (Axin1 and Axin2) are scaffolding proteins that modulate at least two signaling pathways that are crucial in skeletogenesis: the Wnt/b-catenin and TGF-b signaling pathways. To determine whether Axin2 is important in skeletogenesis, we examined the skeletal phenotype of Axin2-null mice in a wild-type or Axin1 þ/À background. Animals with disrupted Axin2 expression displayed a runt phenotype when compared to heterozygous littermates. Whole-mount and tissue b-galactosidase staining of Axin2 LacZ/LacZ mice revealed that Axin2 is expressed in cartilage tissue, and histological sections from knockout animals showed shorter hypertrophic zones in the growth plate. Primary chondrocytes were isolated from Axin2-null and wild-type mice, cultured, and assayed for type X collagen gene expression. While type II collagen levels were depressed in cells from Axin2-deficient animals, type X collagen gene expression was enhanced. There was no difference in BrdU incorporation between null and heterozygous mice, suggesting that loss of Axin2 does not alter chondrocyte proliferation. Taken together, these findings reveal that disruption of Axin2 expression results in accelerated chondrocyte maturation. In the presence of a heterozygous deficiency of Axin1, Axin2 was also shown to play a critical role in craniofacial and axial skeleton development. ß

As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E2 (PGE2) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro–computed tomographic (µCT), and biomechanical measures. EP1−/− mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1−/− mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)–positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1−/− mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE2 receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing. © 2011 American Society for Bone and Mineral Research.

The attenuation of an in vitro inflammatory response in RAW 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) endotoxin was tested using sol-gel-derived bioactive glasses. Three general types of sol-gel-derived samples were evaluated: 58S, zinc-containing glasses, and copper-containing glasses. Distinct experimental procedures were used to test the potential of bioactive glasses to attenuate the inflammatory response in three situations: (1) therapeutically following LPS stimulation, (2) prophylactically before LPS stimulation of macrophages, and (3) indirectly via the glass dissolution products after stimulation with LPS. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to monitor the concentration of tumor necrosis factor-alpha (TNF-alpha) secreted by macrophage cells. The strongest reduction in TNF-alpha concentration was observed when macrophage cells were first incubated with bioactive glass powder and then stimulated with LPS. This suggests a possible prophylactic application of these bioactive glasses for the prevention of inflammation. The 58S glass was capable of reducing the expression of TNF-alpha by macrophages, although the zinc- and copper-containing were more effective at suppressing the inflammatory response. The additional benefit of using zinc- and copper-doped bioactive glasses may be explained by the direct interactions of zinc and copper ions in key regulatory pathways for the inflammation response.

The goal of this study was to define the anti-osteoclastogenic and/or anti-inflammatory role of IL-6 in inflammatory bone resorption using in vivo and in vitro methods. To this end, titanium particles were placed on murine calvaria, and bone resorption and osteoclast formation quantified in wildtype and IL-6 -/-mice. In this model, calvarial bone loss and osteoclast formation were increased in titanium-treated IL-6 -/-mice. Although basal numbers of splenic osteoclast precursors (OCP) were similar, IL-6 -/-mice treated with particles in vivo had increased splenic OCP suggesting an enhanced systemic inflammatory response. In vitro osteoclastogenesis was measured using splenic (OCP) at various stages of maturation, including splenocytes from WT, IL-6 -/-and TNFα transgenic mice. ELISA was used to measure TNFα production. IL-6 inhibited osteoclastogenesis in early OCP obtained from wild-type and IL-6 -/-spleens. Pre-treatment of OCP with M-CSF for three days increased the CD11b high /c-Fms+ cell population, resulting in an intermediate staged OCP. Osteoclastogenesis was unaffected by IL-6 in M-CSF pre-treated and TNFα transgenic derived OCP. IL-6 -/-splenocytes secreted greater concentrations of TNFα in response to titanium particles than WT; addition of exogenous IL-6 to these cultures decreased TNFα expression while anti-IL-6 antibody increased TNFα. While IL-6 lacks effects on intermediate staged precursors, the dominant in vivo effects of IL-6 appear to be related to strong suppression of early OCP differentiation and an anti-inflammatory effect targeting TNFα. Thus, the absence of IL-6 results in increased inflammatory bone loss.