The osteocyte as a therapeutic target in the treatment of osteoporosis

Abstract

Osteoporosis is characterized by a low bone-mineral density associated with skeletal fractures. The decrease in bone-mineral density is the consequence of an unbalanced bone-remodeling process, with higher bone resorption than bone formation. The orchestration of the bone-remodeling process is under the control of the most abundant cell in bone, the osteocyte. Functioning as an endocrine cell, osteocytes are also a source of soluble factors that not only target cells on the bone surface, but also target distant organs. Therefore, any drugs targeting the osteocyte functions and signaling pathways will have a major impact on the bone-remodeling process. This review discusses potential advances in drug therapy for osteoporosis, including novel osteocyte-related antiresorptive and anabolic agents that may become available in the coming years.

Keywords

drug therapy osteocyte osteoporosis

Introduction

The mammalian skeleton is a greatly active tissue that undergoes continuous remodeling throughout childhood and adult life. Bone remodeling is needed for microfracture consolidation, and skeleton adaptation to mechanical use, and also for calcium homeostasis [Dallas et al. 2013]. This bone remodeling implicates the coupling of osteoclastic bone resorption and osteoblastic bone formation. Common diseases, such as osteoporosis, multiple myeloma, Paget’s disease, and other bone-metastasized cancers, are characterized by imbalances between the formation and resorption processes [Devogelaer, 2000; Daci et al. 2002; Shankar et al. 2013]. As these pathologies contribute to increase morbidity and mortality worldwide, there is great concern towards improving our understanding of the processes that regulate bone remodeling [Shoback, 2007]. Osteoporosis, which occurs mainly in postmenopausal women, is characterized by excessive bone resorption compared with the formation of new bone. Osteoporosis is thus characterized by a loss of bone strength, a decrease in bone mass, and a worsening in bone quality, leading to an increased risk of fracture [Mosley, 2000].

In addition to the well-known behavior of mature osteoblasts and osteoclasts, and their respective precursor cells, on the bone-remodeling process, there is increasing evidence that osteocytes play important roles in detecting imperfections or microfractures and initiating a targeted bone remodeling [Verborgt et al. 2000; Kogianni and Noble, 2007; Heino et al. 2009]. Osteocytes originate from mesenchymal stem cells through osteoblast lineage differentiation, with only 10–20% of osteoblasts differentiating into osteocytes [Aubin and Turksen, 1996]. During this differentiation, osteocytes become embedded in the bone matrix during the modeling and/or remodeling processes where the bone matrix is synthesized [Rochefort et al. 2010]. Osteocytes remain active in the bone-remodeling process by maintaining connections to the bone surface, to osteoblasts and osteoclasts, and to other osteocytes through an extensive canalicular network. Osteocytes are able to release nitric oxide, prostaglandin E₂, and adenosine-triphosphate that activate bone formation, and sclerostin, Dickkopf-related protein 1 (DKK1), and frizzled-related protein 1 that inhibit bone formation. They are also able to release the receptor activator of the nuclear factor kappa-B ligand (RANKL) to support osteoclastogenesis, but also to secrete the bone-formation inhibitor sclerostin [Winkler et al. 2003; Van Bezooijen et al. 2004; Mulcahy et al. 2011; Nakashima et al. 2011; Moustafa et al. 2012]. Therefore, any drugs that target the remodeling cycle by affecting osteoblasts, osteoclasts, and osteocytes, and/or molecules that control the signaling pathways, will have a major impact on the targeted bone remodeling [Killock, 2011; Moriishi et al. 2012].

The cellular and molecular basis of osteoporosis

Osteoporosis is a systemic skeletal disorder characterized by low bone mass and micro-architectural deterioration of bone tissue with a consequent compromised bone strength and increased susceptibility to fracture. According to the clinical definition of osteoporosis proposed by the World Health Organization, a patient is osteoporotic when the dual-energy X-ray absorptiometry measurement of bone-mineral density is 2.5 standard deviations below the typical peak bone mass of young healthy women [Blake and Fogelman, 2007]. Osteoporosis occurs in both genders, at all ages, and can be separated into three types: (a) primary osteoporosis in which no underlying cause can be clearly identified, but often follows menopause in women and occurs later in life in men; (b) secondary osteoporosis in which the underlying cause is known (e.g. hyperparathyroidism, hypophosphatasia, diabetes types 1 and 2, alcoholism, glucocorticosteroid use, etc.); (c) more rare forms of the disease, such as juvenile, pregnancy-related, and postpartum osteoporosis [Taxel and Kenny, 2000; Schnatz et al. 2010; Cook et al. 2013].

Osteoporosis is associated with typical fractures (e.g. lumbar spine, femoral neck or distal radius, vertebral fractures, and any fracture resulting from a low trauma in the elderly) that are associated with an increase in morbidity and mortality [Lindsay, 1996; Garnero, 2008; Hopkins et al. 2013]. Therefore, the goal of osteoporosis therapies is to prevent these fractures by inhibiting bone resorption and/or by stimulating bone formation [Sun et al. 2013]. While antiresorptive drugs lower bone turnover [Lewiecki, 2013], anabolic therapies increase bone modeling and/or remodeling osteoblastic activity [Khan and Khan, 2006].

Osteoporosis treatments

The most widely prescribed and first-line drugs for bone diseases are the bisphosphonates, such as alendronate, risedronate, ibandronate, or zoledronic acid [Fleisch, 2002]. These molecules are generally considered to be safe drugs, with the clinical benefits surpassing the risks associated with treatment. Due to their widespread usage in many patients suffering from different diseases, several adverse effects have been reported, including nausea, abdominal pain, ocular inflammation, difficulty in swallowing, and the risk of an inflamed esophagus or esophageal ulcers. However, the relationship between the drug and adverse events is often difficult to establish because clear correlations are often missing due to comedication or comorbidities. One of the most severe adverse events of bisphosphonate treatment is jawbone osteonecrosis, defined as an exposed area of bone in the maxillofacial region after a tooth extraction in which a section of jawbone persists for at least 8 weeks [Khosla et al. 2007; Rizzoli et al. 2008], dies, and deteriorates [Aspenberg, 2006]. Incidence of this rare side effect ranges from 1 per 10,000–110,000 patient years to approximately 10% in patients with myeloma [Khosla et al. 2007; Rizzoli et al. 2008]. The occurrence of jawbone osteonecrosis is often connected to underlying dental problems. In addition, bisphosphonate treatment has been linked to stress and atypical fractures of the femoral shaft, but data are not always significant [Schilcher et al. 2011]. These stress fractures seem to occur through inhibition of bone remodeling. When a patient is undergoing long-term bisphosphonate treatment, bone microdamages are not always repaired, and thus accumulate, and eventually lead to fractures occurring on compact bones at sites of high tensional stress.

Hormone-replacement therapies are also an established approach to the treatment and prevention of osteoporosis, with significant improvement in bone-mineral density, and reduction in hip and vertebral fracture. Estrogen started soon after menopause helps to maintain bone density. However, estrogen therapy in women may increase blood clots, and risk of endometrial cancer, breast cancer, and possibly heart disease [Maclean et al. 2008]. Treatment mimicking estrogen, such as raloxifene, has significant beneficial effects on bone density in postmenopausal women, without some of the side effects associated with estrogen, such as breast cancer. Osteoporosis in men may be related to a gradual age-related decrease in testosterone levels that may be treated by testosterone-replacement therapy, which has a lower impact on bone density than direct osteoporosis medications [Maclean et al. 2008].

In addition to these treatments, other bone anabolic pathways can be targeted, such as the powerful parathyroid hormone (PTH) analogous teriparatide that stimulates new bone growth and is more dependent on increasing the activation frequency, or the monoclonal antibody denosumab that binds to RANKL, a protein involved in the formation, function, and survival of bone-resorption osteoclasts. New treatments under development are aimed at targeting sclerostin and the canonical wingless-int (Wnt) signaling, which is more dependent on increasing bone modeling [Bringhurst, 2002; Deal, 2009; Lim and Clarke, 2012].

The osteocyte within bone tissue

Osteocytes, the most abundant cells in bone, represent 90–95% of all cells in the adult skeleton [Rochefort et al. 2010]. They are able to live for several decades within the bone matrix, whereas osteoblasts and osteoclasts have a lifespan of only a few days or weeks [Bonewald, 2011]. Osteocytes represent the terminal differentiation of osteoblasts [Klein-Nulend et al. 2003]. These cells send out long dendritic processes (the dendrites) through fine channels within the bone matrix (the canaliculi), thus forming a large dendritic network connecting these cells with each other and with osteoblasts, lining cells, and osteoclasts [Rosser and Bonewald, 2012]. Osteocytes and their dendritic processes are bathed in an interstitial fluid (the bone fluid flow) [Bivi et al. 2012]. The osteocytic dendrites are particularly important in the mechanical sensitivity of these cells [Burra et al. 2010], as well as the mechanical signals recorded by the cilia [Hoey et al. 2011; Uzbekov et al. 2012]. More sensitive than osteoblasts, osteocytes are able to respond to mechanical stimulation, particularly shear stress forces [Klein-Nulend et al. 2002], by secreting several molecules, including insulin-like growth factors, osteocalcin, sclerostin, c-fos, prostanoids, and nitric oxide [Uzbekov et al. 2012; Dallas et al. 2013]. Among all the functions assigned to this cell (see an overview in Figure 1) [Dallas et al. 2013]), osteocyte mechanoreception may stimulate the Wnt-signaling pathway as a negative regulator of sclerostin secretion, itself acting as a negative regulator of bone formation [Ozcivici et al. 2010; Post et al. 2013].

Figure 1.

Functions assigned to osteocytes.

PTH-related therapies

The mechanism of action of the recombinant human PTH drug is still under investigation, but it probably affects multiple signaling pathways and alters the biological activity of several bone cells, including osteoblasts, lining cells, osteoclasts, and osteocytes [O’Brien et al. 2008: Bellido et al. 2013]. The PTH stimulates bone formation by increasing the number of osteoblasts [Wang et al. 2007]. The PTH effects are mediated by a G-protein-coupled receptor, the PTH receptor 1 [Maeda et al. 2013; Van Der Lee et al. 2013]. Different recombinant peptides, mimicking this PTH receptor 1, have different anabolic effects. Thus, the cyclic amino-terminal fragment may have a more anabolic profile than the PTH_1–34 or PTH_1–84 fragments [Fraher et al. 1999; Whitfield, 2006; Henriksen et al. 2013]. Selected amino-acid substitutions at various positions in the PTH_1–28 fragment have also revealed an increased activity of this recombinant hormone [Yang et al. 2007].

The release regulation of PTH, and the related regulation of calcium homeostasis, is under the influence of the calcium-sensing receptor, a G-protein-coupled, seven-pass transmembrane molecule present in the parathyroid gland, the kidney, and in osteoblasts and osteocytes [Brown, 2007; Fromigue et al. 2009; Xue et al. 2012]. Allosteric modulators of this calcium-sensing receptor can affect the secretion of PTH [Trivedi et al. 2008; Riccardi, 2012]. Positive calcium-sensing receptor agonists, called calcimimetics, such as cinacalcet, can reduce PTH secretion in patients with hyperparathyroidism and renal disease [Li et al. 2013; Tsuruta et al. 2013], whereas negative antagonists of this receptor, called calcilytics, can inhibit the receptor function thus inducing the release of a PTH pulse [Cabal et al. 2013]. Therefore, these molecules may represent new targets in the treatment of osteoporosis [Fraser et al. 2004; Nemeth, 2004; John et al. 2011]. However, to be useful as anabolic agents, calcilytic agents must induce the release of sufficient PTH to be anabolic, they must have a short half-life since sustained activation would result in prolonged PTH secretion and a catabolic state (hyperparathyroidism), and they should not deplete the parathyroid gland, and not result in hyperplasia [Avlani et al. 2013]. Recently, a calcilytic agent, called ronacaleret, has shown a strong PTH response, with a short half-life, and an increase in both cortical and trabecular bone formation in rodents [Balan et al. 2009; Atchison et al. 2011]. However, a recent clinical trial involving ronacaleret in humans showed a small, nondose-dependent increase in bone-mineral density in the lumbar spine at 6 months [Fitzpatrick et al. 2012].

Recent studies demonstrated that some actions of PTH on the skeleton are mediated by direct effects on osteocytes [Bellido et al. 2013]. PTH thus down-regulates the expression of the SOST gene, encoding the potent inhibitor of bone formation sclerostin expressed in osteocytes (see below) [Bellido et al. 2005; Keller and Kneissel, 2005]. Furthermore, PTH increases the expression of fibroblast growth factor 23, a hormone expressed in osteocytes (and osteoblasts) that regulates phosphate reabsorption in kidney and contributes to mineral homeostasis [Lavi-Moshayoff et al. 2010; Bellido et al. 2013].

Wnt signaling, sclerostin, and DKK1

Wnt proteins are a large family of extracellular (secreted) cysteine-rich glycoproteins that regulate bone remodeling, but are also involved in several other physiopathological situations, including prostate adenocarcinoma [Yu et al. 2011], renal cancer [Banumathy and Cairns, 2010], most sporadic colorectal cancers [Scholer-Dahirel et al. 2011], melanoma [Lucero et al. 2010], breast cancer [Bu et al. 2008], as well as parathyroid carcinoma [Svedlund et al. 2010], and glioma [Liu et al. 2011].

The biological action of Wnt passes through canonical and noncanonical pathways. Canonical Wnt signaling employs extracellular Wnt ligands that bind frizzled and lipoprotein receptor-related protein (LRP) 5/6 coreceptors at the cell surface to transduce a signal that results in the intracellular activation of β-catenin (Figure 2). This canonical Wnt pathway regulates production of the β-catenin transcription factor by inhibiting its phosphorylation, ubiquitination, and degradation. Noncanonical Wnt signaling is defined as Wnt-initiated or frizzled-initiated signaling that is independent of β-catenin transcriptional function [Lee et al. 2010; Baron and Kneissel, 2013]. Noncanonical Wnt pathways are diverse and include Wnt-cGMP/Ca2+ signaling, Wnt-ROR2 signaling, Wnt-RYK signaling, Wnt-mTOR signaling, etc. [Lee et al. 2010; Sassi et al. 2013].

Figure 2.

Wnt pathway, sclerostin, and DKK1; effects of monoclonal antibodies on bone.

The Wnt protein also allows the activation of a protein complex consisting of axin, adenomatous polyposis coli, and glycogen synthase kinase 3 activating an intracellular signal. When the Wnt protein is absent, the membrane receptors frizzled and LRP5/6 are dissociated and glycogen synthase kinase 3 phosphorylates β-catenin, which is then degraded through the ubiquitin/proteasome pathway [Boudin et al. 2013]. When the Wnt protein is present, the frizzled membrane receptors are associated with LRP5/6, the protein complex is disrupted, and the phosphorylation of β-catenin does not occur. Therefore β-catenin accumulates, and is then translocated to the cell nucleus, and binds to transcription factors that can affect the transcription of genes related to bone formation [Lee et al. 2010; Baron and Kneissel, 2013; Boudin et al. 2013].

Elements of the Wnt-signaling pathway are well conserved in evolution and are found in primitive metazoans, such as cnidarians [Lengfeld et al. 2009]. The Wnt antagonist DKK1 is expressed by early invertebrates, such as the Hydra [Guder et al. 2006], whereas the expression of sclerostin is not found until the emergence of bony vertebrates, indicating a more specific role for sclerostin in the development and maintenance of the skeleton and a broader role for DKK1.

In addition to binding LRP5/6, both sclerostin and DKK1 can bind other transmembrane molecules, such as LRP4, also known as multiple epidermal growth factor-like domains 7 (Megf7) to increase their inhibitory activity on the Wnt-signaling pathway (Figure 2) [Choi et al. 2009]. Indeed, Megf4 KO mice exhibit limb abnormalities with polysyndactyly [Simon-Chazottes et al. 2006], whereas loss-of-function mutations of this factor in humans, also known as Cenani–Lenz syndrome, cause syndactyly, kidney malformations, and bone overgrowth [Li et al. 2010b]. This indicates that LRP4 acts as a negative regulator of LRP5/6 signaling by increasing the inhibitory activity of sclerostin on the Wnt pathway, without affecting the activity of DKK1 [Leupin et al. 2011].

DKK1 may also bind to a two-member family of proteins referred to as Kremen-1 and Kremen-2, leading to the removal of LRP5/6 from the cell surface (Figure 2) [Mao et al. 2002]. Invalidation of both Kremen-1 and -2 in mice lead to subtle patterning defects in the forelimb with increased bone formation that were additionally enhanced by the deletion of a single DKK1 allele, indicating the importance of these factors in the regulation of bone mass through modulation of LRP5/6 [Ellwanger et al. 2008; Schulze et al. 2010]. However, the homozygous deletion of either Kremen-1 or -2 lead to normal bone formation and bone mass, suggesting a functional redundancy of Kremen-1 and -2 [Schulze et al. 2010].

Inhibitors of the Wnt pathway can target either frizzled (serum frizzled-related proteins), Wnt (Wnt inhibitory factors), or LRP5/6 (DKK1 and the osteocyte-released sclerostin) (Figure 2) [Rybchyn et al. 2011]. These agonist molecules can prevent Wnt from activating the frizzled LRP5/6 signaling pathway, inducing a decrease in Wnt signaling, and thus a significant bone decrease in bone formation [Glantschnig et al. 2011]. On the other hand, deficiencies in these inhibitors or antibodies targeting these inhibitors induce an increase of Wnt signaling and, therefore, induce an increase in bone formation [Glantschnig et al. 2010].

Complexity of Wnt signaling has increased since the recent description of distinct ligand-binding domains on LRP5/6 receptors that recognize different classes of Wnt proteins and inhibitors [Bourhis et al. 2010; Ettenberg et al. 2010; Gong et al. 2010]. The Wnt1 class, comprising Wnt 1, 2, 6, 7a, 7b, 9a, 9b, and 10b, was reported to bind the first β-sheet of LRP5/6, whereas the Wnt3 class, including Wnt3 and 3a, was shown to bind the third β-sheet of LRP5/6 (Figure 2). DKK1 was revealed to bind indifferently the first and third β-sheets of LRP5/6 [Bourhis et al. 2010], whereas sclerostin bound only the first β-sheet of LRP5/6 (Figure 2) [Ettenberg et al. 2010]. Therefore, DKK1 inhibited both the Wnt1 and Wnt3 classes, and sclerostin inhibited the Wnt1 class and enhanced the Wnt3 class [Ettenberg et al. 2010].

The sclerostin protein is encoded by the human SOST gene [Winkler et al. 2003; Robling et al. 2008; Cohen-Kfir et al. 2011]. Homozygous mutation of loss of expression of the SOST gene is responsible for two rare genetic disorders, sclerosteosis and van Buchem disease, which are associated with general progressive skeletal overgrowth and sclerosis of the axial and appendicular skeleton [Brunkow et al. 2001; Bhadada et al. 2013]. In sclerosteosis, bone formation is stimulated by the absence or decreased synthesis of sclerostin in humans, whereas bone resorption is not (or only mildly) affected [Van Lierop et al. 2011]. Van Buchem disease is a disorder resembling sclerosteosis but distinguished by its less severe phenotype and absence of hand malformations, such as syndactyly [Staehling-Hampton et al. 2002]. The difference between these two disorders might be explained by the fact that the deleted genomic region in van Buchem disease includes no regulatory elements required for sclerostin expression during the embryologic steps of digit formation [Brunkow et al. 2001; Uitterlinden et al. 2004]. Finally, heterozygous mutations in the SOST gene cause a mild increase in bone mass and fewer skeletal complications [Van Lierop et al. 2011].

SOST mRNA is expressed in many tissues during embryogenesis. However, the sclerostin protein is found only postnatally in terminally differentiated cells embedded within a mineralized matrix, including osteocytes, mineralized hypertrophic chondrocytes, and cementocytes [Ohyama et al. 2004]. In humans, SOST mRNA is detectable in tissues such as heart, aorta, liver, odontoblasts, and kidney, whereas sclerostin protein has never been detected in any organs other than bone [Balemans et al. 2001; Moester et al. 2010]. Since the sclerostin protein is almost exclusively produced by osteocytes in adult murine and human bone, antibodies targeting this offer a way to target specifically bone formation [Winkler et al. 2003; Van Bezooijen et al. 2005; Robling et al. 2008]. Sclerostin antibodies thus increase bone formation in osteopenic estrogen-deficient rats [Keller and Kneissel, 2005; Li et al. 2009]. A single subcutaneous dose of a sclerostin antibody in postmenopausal women resulted in an increase in bone density and bone formation markers, without any modification of bone-resorption markers [Papapoulos, 2011; Lewiecki, 2013].

Next to sclerostin, mutations in the gene encoding LRP5/6 inducing a gain of function cause an increase in bone mass [Kim et al. 2007; Hoeppner et al. 2009]. These mutations induce impairment of the binding of DKK1 to frizzled LRP5/6, thus allowing an increase in the Wnt-signaling pathway, and an increase in bone formation [Choi et al. 2009]. Antibodies targeting DKK1 and/or LRP5/6 induce the increase in bone mass, volume, and formation in rodents [Van Dinther et al. 2013]. Therefore, antibodies targeting DKK1 could also be used as anabolic or antiresorptive agents for the treatment of patients with low bone mass [Ahn et al. 2011].

Effects of monoclonal antibodies on bone

The pharmacologic inhibition of the sclerostin protein, using monoclonal antibody, has confirmed efficacy in animal models of bone diseases, including estrogen deficiency-induced bone loss [Li et al. 2009], age-related, or androgen deficiency-induced bone loss [Li et al. 2010a], disuse/immobilization-induced bone loss [Tian et al. 2011], glucocorticoid-induced bone loss [Marenzana et al. 2011], chronic inflammation-induced bone loss [Eddleston et al. 2009], bone loss associated with type 2 diabetes mellitus [Gaudio et al. 2012], as well as in a rodent model of osteogenesis imperfecta [Sinder et al. 2013], and fracture healing [Gamie et al. 2012; Cui et al. 2013]. Effects of sclerostin monoclonal antibody have also been reported in human preclinical models of bone loss, including osteogenesis imperfecta, fracture healing, implant diseases. and other bone disorders (Figure 2) [Eddleston et al. 2009; Li et al. 2009, 2010a]. The use of sclerostin antibody in these conditions has demonstrated a consistent ability to increase bone formation, bone mass, and bone strength [Ke et al. 2012].

A human antisclerostin antibody, known as AMG 785 or romosozumab, is being developed by Amgen (Thousand Oaks, CA, USA) and UCB Inc. (Smyrna, GA, USA), and a phase l randomized, double-blinded, placebo-controlled study using this antibody has been conducted on men and postmenopausal women [Padhi et al. 2011]. In this trial, antibody injection was associated with substantial increases in bone-formation markers and reductions in bone-resorption markers, as well as a dose-dependent increase in bone-mineral density at the lumbar spine and total hip after 3 months. Results from the phase ll trial on postmenopausal osteoporosis have not been published at this time, but a recent press release by Amgen and UCB Inc. reported positive results with the cohort using the monoclonal antisclerostin antibody, including a significant increase in bone-mineral density of the lumbar spine at 12 months compared with placebo. Furthermore, this antisclerostin antibody was positively compared with teriparatide and alendronate. Positive phase ll results of this antibody in patients with postmenopausal osteoporosis have been announced by Amgen and UCB Inc. (NCT00896532, May 2009), whereas phase lll programs on fracture healing, and in patients with postmenopausal osteoporosis are currently ongoing (NCT01631214, May 2012; NCT01796301, February 2013).

In the same manner, the pharmacologic inhibition of DKK1, using monoclonal antibody, has also demonstrated efficacy in animal models of bone diseases, including gonad-intact rodents [Li et al. 2006], rodent models of fracture healing [Li et al. 2011], and implant fixation [Agholme et al. 2011]. However, unlike Scl-Ab, DKK1 demonstrated no efficacy in adult ovariectomized rats [Li et al. 2006], and a modest improvement in ovariectomized rhesus monkeys [Glantschnig et al. 2011; Li et al. 2011]. DKK1 antibody demonstrated efficacy in rodent models of rheumatoid arthritis [Diarra et al. 2007], ankylosing spondylitis [Uderhardt et al. 2010], and multiple myeloma [Fulciniti et al. 2009].

Conclusion

Treatments of low bone mass and osteoporosis have advanced significantly beyond hormone therapy administered at menopause. These advances are mainly the result of increased understanding of the mechanisms underlying osteoblast, osteoclast, and osteocyte biology. Novel anabolic and antiresorptive agents, affecting osteocyte-associated sclerostin, the calcium-sensing receptor, or Wnt signaling, offer promise for the treatment of bone disorders. Additional therapies treating patients with established fractures are needed next to reduce the burden of this other disease.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest statement

The authors declare no conflict of interest in preparing this article.

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