Randy N. Rosier

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.

Activation of β-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult β-catenin conditional activation mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐cateninfx(Ex3)/fx(Ex3) mice with Col2a1‐CreERT2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreERT2;β‐cateninfx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.Denosumab is a fully human monoclonal antibody that inhibits bone resorption by neutralizing RANKL, a key mediator of osteoclast formation, function, and survival. This phase 3, multicenter, doubleblind study compared the efficacy and safety of denosumab with alendronate in postmenopausal women with low bone mass. One thousand one hundred eighty-nine postmenopausal women with a T-score <or= -2.0 at the lumbar spine or total hip were randomized 1:1 to receive subcutaneous denosumab injections (60 mg every 6 mo [Q6M]) plus oral placebo weekly (n = 594) or oral alendronate weekly (70 mg) plus subcutaneous placebo injections Q6M (n = 595). Changes in BMD were assessed at the total hip, femoral neck, trochanter, lumbar spine, and one-third radius at 6 and 12 mo and in bone turnover markers at months 1, 3, 6, 9, and 12. Safety was evaluated by monitoring adverse events and laboratory values. At the total hip, denosumab significantly increased BMD compared with alendronate at month 12 (3.5% versus 2.6%; p < 0.0001). Furthermore, significantly greater increases in BMD were observed with denosumab treatment at all measured skeletal sites (12-mo treatment difference: 0.6%, femoral neck; 1.0%, trochanter; 1.1%, lumbar spine; 0.6%, one-third radius; p <or= 0.0002 all sites). Denosumab treatment led to significantly greater reduction of bone turnover markers compared with alendronate therapy. Adverse events and laboratory values were similar for denosumab- and alendronate-treated subjects. Denosumab showed significantly larger gains in BMD and greater reduction in bone turnover markers compared with alendronate. The overall safety profile was similar for both treatments.

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.

Inhibition of β-catenin signaling in articular chondrocytes results in articular cartilage destruction

OBJECTIVE Osteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/beta-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of beta-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit beta-catenin signaling in chondrocytes. METHODS The expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x(L), and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes. RESULTS Expression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICAT-transgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x(L) expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice. CONCLUSION Inhibition of beta-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICAT- transgenic mice.

Articular cartilage biology.

Abstract Articular cartilage is a complex tissue maintained by chondrocytes, which undergo metabolic changes as a result of aging, disease, and injury. These changes may hinder tissue maintenance and repair, resulting in accelerated loss of articular surface and leading to end‐stage arthritis. Researchers are investigating both normal and pathologic cellular and molecular processes as well as the development of chondroprotective agents to improve the metabolic function of articular cartilage. Current research is helping to clarify the mechanisms by which a variety of agents, such as glucosamine, chondroitin sulfate, hyaluronic acid, green tea, glucocorticoids, and nonsteroidal anti‐inflammatory drugs, can modify the symptoms and course of osteoarthritis. Also under investigation are methods of stimulating repair or replacing damaged cartilage, such as matrix metalloproteinase inhibitors, gene therapy, growth factors, cytokine inhibitors, and artificial cartilage substitutes. Tissue engineering, the combining of artificial matrices with cells and growth factors or genes, offers great potential for improving patient care.

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.

The potential role of transforming growth factor beta in fracture healing

As shown in previous studies, the transforming growth factor beta superfamily of growth factors is involved in many aspects of skeletal development and regulation, including fracture repair and bone regeneration. Several studies have shown transforming growth factor beta messenger ribonucleicacid and protein expression in cells comprising fracture callus. In healing fractures in a chick model, differential isoform expression of the transforming growth factor betas was observed by in situ hybridization, with more prominent expression of the transforming growth factor beta 2 and transforming growth factor beta 3 isoforms. Small amounts of transforming growth factor beta 1 were present in early callus and increased in expression later during chondrogenesis and endochondral ossification. These findings resemble those reported in rat and human fracture callus. Transforming growth factor beta 4 expression was not significant in the chick fracture model. Transforming growth factor beta can function as a morphogen when injected subperiosteally, inducing cartilage and bone formation that morphologically resembles many of the events occurring in fracture callus. Exogenous transforming growth factor beta has been used in several critical size defect models of bone regeneration and fracture healing, with most of the studies showing increased bone or callus formation and increased mechanical stability. Numerous variables, including markedly different dose ranges and differing isoforms, dosing regimens, delivery methods, animal models, and various times and endpoint measures for analysis, make it difficult to comparatively assess the effects of transforming growth factor beta on bone healing. Additional study is necessary to satisfactorily determine the role of transforming growth factor beta in normal fracture healing and its potential for use in augmenting this process.

Increased Levels of Tumor Necrosis Factor-α and Interleukin-6 Protein and Messenger RNA in Human Peripheral Blood Monocytes due to Titanium Particles*

Cytokines produced by macrophages in the periprosthetic membranes surrounding joint replacements have been implicated as causal agents in osteolysis and prosthetic loosening. The present study characterizes the response of human peripheral blood monocytes to titanium particles. Monocytes were obtained from volunteers and blood that had been donated to the American Red Cross and were cultured in the presence of titanium particles (one to three micrometers in diameter). There were consistent dose-dependent increases in the production of TNF-&agr; (tumor necrosis factor-&agr;) and IL-6 (interleukin-6) protein, with the greatest stimulation generally observed with a concentration of 6 x 105 to 6 x 106 particles of titanium per milliliter. The level of TNF-&agr; was the greatest (fifty to 1000 times greater than the control level) after eight hours of exposure to titanium particles; the level of IL-6 was two to five times greater than the control level after sixteen hours of exposure. These increases were similar to those observed after stimulation with lipopolysaccharide and depended on de novo synthesis rather than on release from intracellular stores. The production of TNF-&agr; was inhibited in a dose-dependent manner by the translational inhibitor cycloheximide and the transcriptional inhibitor actinomycin D, indicating the requirement for both mRNA (messenger RNA) and protein synthesis for the induction of cytokine synthesis by titanium particles. Although the increase in the levels of cytokine mRNA in response to titanium was rapid (thirty to ninety minutes), the increase in the level of TNF-&agr; mRNA preceded that of IL-6 mRNA. The level of TNF-&agr; mRNA was the greatest at ninety minutes and the level of IL-6 mRNA was the greatest at three hours. After stimulation with titanium particles, the level of TNF-&agr; mRNA was increased as much as fivefold and the level of IL-6 mRNA, as much as twelvefold. CLINICAL RELEVANCE: Awareness of the importance of wear debris particles in cytokine-induced bone resorption has resulted in improvements in the designs of implants and in operative techniques to reduce wear of components. The present study further elucidates the biological mechanisms involved in periprosthetic osteolysis. Titanium-stimulated biosynthesis of the cytokines TNF-&agr; and IL-6, which are both potent stimulators of bone resorption, requires increases in the synthesis of both mRNA and protein by monocytes. An understanding of the complex mechanisms of the induction of cytokine synthesis by particles of wear debris will facilitate the design of pharmacological agents to control periprosthetic bone resorption. These agents, in combination with other efforts to reduce the generation of wear debris, may improve the longevity of orthopaedic implants.

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.