J. Edward Puzas

Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair

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.

The role of Axin2 in calvarial morphogenesis and craniosynostosis

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of β-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2 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.

BMP-6 Is an Autocrine Stimulator of Chondrocyte Differentiation

While parathyroid hormone‐related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13‐day embryos, and the role of bone morphogenetic protein‐6 (BMP‐6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP‐6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10−7 M) at the time of plating completely abolished BMP‐6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP‐6 and type X collagen mRNA, with BMP‐6 again preceding type X collagen expression. The addition of exogenous BMP‐6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP‐6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP‐6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP‐6, it indirectly regulates Ihh expression through BMP‐6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP‐6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP‐6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP‐6.

In Vivo RANK Signaling Blockade Using the Receptor Activator of NF‐κB:Fc Effectively Prevents and Ameliorates Wear Debris‐Induced Osteolysis via Osteoclast Depletion Without Inhibiting Osteogenesis

Prosthesis failure due to wear debris‐induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF‐κB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)‐induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti‐treated RANK−/− mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti‐treated were still depleted for multinucleated tartrate‐resistant acid phosphatase‐positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris‐induced osteolysis without jeopardizing osteogenesis.

Reduced COX-2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX‐2), the inducible regulator of prostaglandin E2 (PGE2) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX‐2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX‐2 is expressed primarily in early cartilage precursors that co‐express col‐2. COX‐2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX‐2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX‐2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging.

Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysis

Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone‐implant interface, which is caused by wear debris‐induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the efficacy of stably transfected fibroblast‐like synoviocytes (FLS) expressing OPG in preventing wear debris‐induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/106 cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly, implantation of 107 FLS–OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS‐LacZ control cells, which persisted for 3 days as determined by X‐gal staining, together with the Ti particles, had a 6‐fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS‐OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls.

PTHrP Modulates Chondrocyte Differentiation through AP-1 and CREB Signaling

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor β. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.

Heterotopic ossification: Clinical and cellular aspects

Heterotopic ossification is the formation of mature trabecular bone in sites where it is not normally present. This differs from dystrophic calcification, or calciphylaxis, which is the acellular formation of amorphous calcium phosphate deposits. Heterotopic ossification has been described in the literature as para-osteo anthropathy [l , 2], myositis ossificans [3,4], peri-articular new bone formation [5], periarticular ectopic ossification [6], neurogenic osteoma [2], neurogenic ossifying f ibromyopathy [1], and heterotopic calcification [7]. The terms heterotopic bone formation and heterotopic ossification are preferred, however, as they are both accurate and descriptive. This syndrome has been associated with a number of conditions, and is seen most commonly following hip surgery, neurologic injury, or severe burns. Many etiologic factors have been proposed to explain this phenomenon, including allergic hypersensitivity, venous abnormality, neural factors, and local trauma. This review focuses on the clinical and histologic features of heterotopic bone, and the potential role of local growth factors and mitogens in its development. In addition, the more common etiologies and treatment options are discussed.

ALK2 Functions as a BMP Type I Receptor and Induces Indian Hedgehog in Chondrocytes During Skeletal Development

Growth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptor ALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type I BMP receptors results in maturation‐independent expression of Ihh in chondrocytes in vitro and in vivo. The findings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation.

COX-1 and COX-2 expression in osteoid osteomas

Osteoid osteoma is a benign bone forming neoplasm that is characterized by its small size (less than 2 cm), self‐limited growth, and the tendency to cause extensive reactive changes in the adjacent tissue. The lesion classically presents with severe pain at night that is dramatically relieved by NSAIDs. The tumor has been shown to express very high levels of prostaglandins, particularly PGE2 and PG12. The high local levels of these prostaglandins are presumed to be the cause of the intense pain seen in patients with this lesion. One generally accepted form of treatment is the prolonged use of NSAIDs. Since the cyclooxygenases are thought to be the source of these prostaglandins, and the central target of NSAIDs, we evaluated the expression of cyclooxygenase‐1 (COX‐1) and cyclooxygenase‐2 (COX‐2) in osteoid osteoma tissues from patients following surgery. In the 12 specimens examined we found that the tumor osteoblasts had strong immunohistochemical staining for COX‐2, while the staining in the surrounding host osteoblasts in the reactive bone was scant. Significant COX‐1 staining was also detected in both tumor and host osteoblasts. For comparison we examined the COX expression in human fracture callus, fibrous dysplasia, osteoblastoma, osteofibrous dysplasia, and myositis ossificans. With the exception of fracture callus, very limited amounts of COX‐2 could be detected in these tissues. Taken together, we conclude that the increased production of prostaglandins by osteoid osteomas implicates that COX‐2 is one of the mediators of this condition. These findings suggest that the newly selective COX‐2 inhibitors could be used to more safely treat osteoid osteomas.