Regis J. O'Keefe

Atypical Subtrochanteric and Diaphyseal Femoral Fractures: Report of a Task Force of the American Society for Bone and Mineral Research

Bisphosphonates (BPs) and denosumab reduce the risk of spine and nonspine fractures. Atypical femur fractures (AFFs) located in the subtrochanteric region and diaphysis of the femur have been reported in patients taking BPs and in patients on denosumab, but they also occur in patients with no exposure to these drugs. In this report, we review studies on the epidemiology, pathogenesis, and medical management of AFFs, published since 2010. This newer evidence suggests that AFFs are stress or insufficiency fractures. The original case definition was revised to highlight radiographic features that distinguish AFFs from ordinary osteoporotic femoral diaphyseal fractures and to provide guidance on the importance of their transverse orientation. The requirement that fractures be noncomminuted was relaxed to include minimal comminution. The periosteal stress reaction at the fracture site was changed from a minor to a major feature. The association with specific diseases and drug exposures was removed from the minor features, because it was considered that these associations should be sought rather than be included in the case definition. Studies with radiographic review consistently report significant associations between AFFs and BP use, although the strength of associations and magnitude of effect vary. Although the relative risk of patients with AFFs taking BPs is high, the absolute risk of AFFs in patients on BPs is low, ranging from 3.2 to 50 cases per 100,000 person‐years. However, long‐term use may be associated with higher risk (∼100 per 100,000 person‐years). BPs localize in areas that are developing stress fractures; suppression of targeted intracortical remodeling at the site of an AFF could impair the processes by which stress fractures normally heal. When BPs are stopped, risk of an AFF may decline. Lower limb geometry and Asian ethnicity may contribute to the risk of AFFs. There is inconsistent evidence that teriparatide may advance healing of AFFs.

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.

Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy

Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL- and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts.

Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP‐2‐producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene‐enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.

The Biology of the Growth Plate

• Chondrocytes in the growth plate undergo a complex series of molecular, biochemical, and morphological changes during the process of differentiation. • Longitudinal growth depends on both proliferation and hypertrophy of chondrocytes in the growth plate. • Integrated growth factor signaling pathways control the key transition between proliferation and hypertrophy that is required for completion of the maturation process. • Terminal chondrocyte maturation is associated with programmed cell death and the secretion of matrix and other factors that promote matrix calcification and vascular invasion. • Systemic hormones interact with local growth factors and signaling pathways to influence the rate of growth and lead to closure of the growth plate. In order for paired human limbs to reach the same adult length and proportions, the longitudinal growth of the skeleton must be tightly regulated. The regulation of longitudinal growth at the growth plate occurs generally through the intimate interaction of circulating systemic hormones and locally produced peptide growth factors, the net result of which is to trigger changes in gene expression by growth plate chondrocytes. These molecular events lead to precisely orchestrated alterations in chondrocyte size, extracellular matrix components, secreted enzymes and growth factors, and receptor expression. The culmination of these events is calcification of the matrix, chondrocyte apoptosis, and completion of endochondral bone formation. This review will highlight some of the advances in genetics and cell biology in the past decade that have begun to illuminate some of the important regulatory mechanisms governing the biology of the growth plate. The growth plate can be divided into a series of anatomic zones that distinguish unique morphological and biochemical stages during the process of chondrocyte differentiation. In the resting zone, the ratio of extracellular matrix to cell volume is high and the cells are in a relatively quiescent state. In the proliferating …

RANK Signaling Is Not Required for TNFα‐Mediated Increase in CD11bhi Osteoclast Precursors but Is Essential for Mature Osteoclast Formation in TNFα‐Mediated Inflammatory Arthritis

To address the controversy of whether TNFα can compensate for RANKL in osteoclastogenesis in vivo, we used a TNFα‐induced animal model of inflammatory arthritis and blocked RANKL/RANK signaling. TNFα increased osteoclast precursors available for RANK‐dependent osteoclastogenesis. RANK signaling is not required for the TNFα‐stimulated increase in CD11bhi osteoclast precursors but is essential for mature osteoclast formation.

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.

Efficacy of etanercept for wear debris-induced osteolysis.

A major limitation of total joint arthroplasty is that up to 20% of patients require revision surgery to correct prosthetic loosening. Aseptic loosening is believed to result from the phagocytosis of wear debris particles by macrophages, which secrete proinflammatory cytokines that stimulate osteolysis. Tumor necrosis factor α (TNF‐α) has been shown to be one of the prominent cytokines in this cascade and to be involved critically in the generation of particle‐induced osteolysis. Etanercept is a soluble inhibitor of TNF‐α, which is widely used for the treatment of rheumatoid arthritis. Here, we show this agents ability to prevent wear debris‐induced osteolysis. In vitro we show that Etanercept can inhibit directly osteoclastic bone resorption in a bone wafer pit assay, as well as cytokine production from titanium (Ti)‐stimulated macrophages. Using a quantitative in vivo model of wear debris‐induced osteolysis, we show that Etanercept prevents bone resorption and osteoclastogenesis. In mice treated with Etanercept at the time of osteolysis induction, bone resorption and osteoclast numbers were reduced to background levels in both normal and human TNF‐α (hTNF‐α) transgenic mice. In an effort to evaluate its effect on established osteolysis, Etanercept was administered 5 days after Ti implantation, and we observed that further osteolysis was prevented. These data support the concept that TNF‐α is involved critically in osteoclastogenesis and bone resorption during periprosthetic osteolysis and suggest that soluble TNF‐α inhibitors may be useful as therapeutic agents for the treatment of prosthetic loosening in humans.

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.