the neurobiology of skeletal...

The neurobiology of skeletal pain

Patrick W. MantyhDepartment of Pharmacology and Arizona Cancer Center, University of Arizona, Tucson, AZ 85716, USA

Keywords: age, bone, cartilage, CRPS, marrow, nociceptor

Abstract

Disorders of the skeleton are one of the most common causes of chronic pain and long-term physical disability in the world. Chronicskeletal pain is caused by a remarkably diverse group of conditions including trauma-induced fracture, osteoarthritis, osteoporosis,low back pain, orthopedic procedures, celiac disease, sickle cell disease and bone cancer. While these disorders are diverse, whatthey share in common is that when chronic skeletal pain occurs in these disorders, there are currently few therapies that can fullycontrol the pain without significant unwanted side effects. In this review we focus on recent advances in our knowledge concerningthe unique population of primary afferent sensory nerve fibers that innervate the skeleton, the nociceptive and neuropathic mecha-nisms that are involved in driving skeletal pain, and the neurochemical and structural changes that can occur in sensory and sym-pathetic nerve fibers and the CNS in chronic skeletal pain. We also discuss therapies targeting nerve growth factor or sclerostin fortreating skeletal pain. These therapies have provided unique insight into the factors that drive skeletal pain and the structuraldecline that occurs in the aging skeleton. We conclude by discussing how these advances have changed our understanding andpotentially the therapeutic options for treating and/or preventing chronic pain in the injured, diseased and aged skeleton.

Introduction

Painful skeletal conditions are prevalent and their impact is perva-sive in both the developing and developed world (Lubeck, 2003;Woolf & Pfleger, 2003; Brooks, 2006; Kidd, 2006). A major reasonskeletal pain occurs in such a diverse group of disorders (Fig. 1) isthat the skeleton is required for structural support, movement, pro-tection of the internal organs, mineral and growth factor storage andrelease, and the birth and maturation of blood cells. Importantly, ifskeletal pain is not adequately controlled it oftentimes has secondaryeffects including loss of bone and muscle mass, cardiovascular func-tion and cognitive health, all of which can significantly diminish thepatient’s functional status and quality of life.Skeletal pain tends to increase with age as the mass, quality and

strength of the human skeleton peaks at 25–30 years of age in bothmales and females (Heaney et al., 2000; Seeman, 2002) and thendeclines thereafter (Fig. 2A and B). As the lifespan of individuals inboth the developing and developed world continues to increase (inmany countries it is now >80 years old) and with the rise of lifestylefactors such as obesity and reduction in daily physical activity, bothof which reduce skeletal health, the burden that skeletal pain willexact on individuals and society is expected to increase markedly inthe coming decades (Peltonen et al., 2003; Stovitz et al., 2008).In this review, we discuss the neurobiology of skeletal pain in

terms of the unique repertoire of sensory nerve fibers that innervatethe bone and joint, the sensory innervation in different compart-ments of the skeleton, the remarkable neurochemical and morpho-logical plasticity that can occur in sensory nerves and their axons

that innervate the skeleton following injury or disease, and howmany forms of chronic skeletal pain appear to have both a nocicep-tive and neuropathic component. We conclude by discussing the rolethe central nervous system plays in maintaining and amplifyingchronic skeletal pain and how current and emerging therapies haveprovided insight and potential therapeutic targets for treating/preventing chronic skeletal pain.

The types of sensory nerve fibers that innervate theskeleton

Previous studies have thoroughly and elegantly described thesensory nerve fiber innervation of the mammalian skin (Peters et al.,2002; Funfschilling et al., 2004; Zylka et al., 2005; Pareet al., 2007; Kwan et al., 2009; Albrecht & Rice, 2010; Kalliomakiet al., 2011). These studies have shown that both human and rodentskin are innervated by a rich variety of sensory nerve fibers. Theseinclude the thickly myelinated sensory nerve fibers (type II orA-beta), thinly myelinated sensory nerve fibers (type III or A-delta)and both major classes of unmyelinated nerve fibers (type IV orC-fibers): peptide-rich nerve fibers that express tropomyosin receptorkinase A (TrkA) and calcitonin gene-related peptide (CGRP) as wellas other neuropeptides, and the TrkA� , peptide-poor isolectin B4nerve fibers which generally do not express CGRP (Karanth et al.,1991; Schulze et al., 1997; Zylka et al., 2005; Albrecht et al.,2006; Taylor et al., 2009). Each type of sensory nerve fiber thatinnervates the skin appears to have a unique conduction velocity,neurotransmitter and receptor expression profile, pattern of innerva-tion, and function (Roosterman et al., 2006; Campero et al., 2009;Kwan et al., 2009; Smith & Lewin, 2009). Thickly myelinatedA-beta fibers detect and signal non-noxious stimuli such as fine

Correspondence: P. W. Mantyh, as above.E-mail: [email protected]

Received 6 September 2013, revised 19 November 2013, accepted 25 November 2013

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

European Journal of Neuroscience, Vol. 39, pp. 508–519, 2014 doi:10.1111/ejn.12462

European Journal of Neuroscience

touch, light pressure, and brushing of the skin, do not express neu-ropeptide neurotransmitters, and have conduction velocities >30 m/s.The thinly myelinated A-delta fibers are primarily involved in therapid detection and signaling of nociceptive stimuli such as pinprick and noxious mechanical stimuli, such as pressure and mechan-ical distortion, express neuropeptides such as CGRP, and have con-duction velocities between 2 and 30 m/s. Lastly, the largelyunmyelinated C-fibers (both the peptide-rich and peptide-poor popu-lations) have a slower conduction velocity (<2 m/s) than theA-fibers and are involved in detecting and signaling noxious heat,and mechanical and chemical stimuli.In marked contrast to the skin, the adult skeleton (i.e. the bone

and joint) is innervated largely by thinly myelinated, TrkA+ sensorynerve fibers (A-delta) and the peptide-rich CGRP, TrkA+ sensorynerve fibers (Fig 3), and receive little if any innervation by the lar-ger more rapidly conducting A-beta fibers or the TrkA�, unmyeli-nated peptide-poor C-fibers (Zylka et al., 2005; Jimenez-Andradeet al., 2010). Recent work has shown that this difference in thepopulation of sensory nerve fibers that innervates the adult skin vs.skeleton begins during perinatal and postnatal development. Thus,several transcription factors including Tlx3 and Runx1 establish theunique phenotype and cohort of sensory nerve fibers that innervatethe skin vs. skeleton (Liu & Ma, 2011; Lopes et al., 2012). Func-tionally, the lack of innervation of bone and joint by A-beta sen-sory nerve fibers is probably related to the fact that fine touch, lightpressure and brushing is not required in bone and joint as it is gen-erally a deep structure so that most sensory nerve fibers that inner-

vate the bone and joint are silent nociceptors, i.e. nerve fibers thatare only activated by injury or damage to the bone or joint. Whybone does not receive significant innervation by the TrkA�, pep-tide-poor C-fibers (which do richly innervate the skin) is not com-pletely clear, although it clearly suggests there is less nociceptor‘redundancy’ in bone and joint than in skin (Fig. 3A and B).The above immunohistochemical data concerning the innervation

of the bone and articular cartilage are in close agreement with previ-ous electron microscopy and electrophysiological studies that haveexamined the density and types of nerve fibers that innervate thebone and joint in animals and humans. For example, studies of theperiosteum of the cat humerus (Ivanusic et al., 2006) and the bonemarrow of the dog tibia (Seike, 1976) demonstrated that these bonesare innervated primarily by unmyelinated and thinly myelinatednerve fibers, with few, if any, thickly myelinated nerve fibers. Simi-larly, electrophysiological recordings performed on nerve fibersentering the nutrient foramen of the humerus of the cat demonstratedthat the conduction of nerve fibers separates into two categories,those with conduction velocities of <2 m/s (presumably C-fibers)and those with conduction velocities between 2 and 30 m/s (presum-ably A-delta fibers), with no nerve fibers having conduction veloci-ties >30 m/s, which would correspond to A-beta fibers (Mahnset al., 2006). Additionally, studies using either transgenic animals orretrograde tracers that were applied to different compartments of theskeleton also demonstrated the TrkA�, peptide-poor population ofC-fibers, that richly innervate the skin, does not appear to innervatehuman (Ozawa et al., 2006) and rat intervertebral discs (Ozawaet al., 2003; Aoki et al., 2005; Ohtori et al., 2007), rat hip (Nakaj-ima et al., 2008), rat wrist joint (Kuniyoshi et al., 2007), rat knee,humerus and bones of the skull (Kruger et al., 1989), or the mousefemur (Zylka et al., 2005; Jimenez-Andrade et al., 2010).It should be noted that, while much of the recent work on the

sensory and sympathetic innervation of bone has been done on thelong bones of the body, several studies examining the bones ofthe skull and temporomandibular joint have found that, in general,bones that comprise the skull are generally innervated by the samesubtypes and have an organisation similar to that of the long bonesof the body (Kruger et al., 1989; Silverman & Kruger, 1989; Hill &Elde, 1991; Hill et al., 1991; Takeuchi & Toda, 2003; Zylka et al.,2005; Kosaras et al., 2009). For example, in studies performed bythe same authors on the same animals, it was noted the types, pat-terns and organisation of sensory and sympathetic nerve fibers thatinnervate the rat calvaria and mandible were very similar to thosethat innervate a long bone such as the rat tibia (Hill & Elde, 1991;Hill et al., 1991).

The organisation of sensory nerve fibers that innervatethe skeleton

Although bone and joint (the two tissues which comprise the skele-ton) appear to be innervated by the same sub-populations of noci-ceptive nerve fibers (A-delta and peptide-rich C-fibers), the density,pattern and morphology of nerve fibers in the two compartments ofbone and joint are strikingly different (Mach et al., 2002; Kuniyoshiet al., 2007; Casta~neda-Corral et al., 2011; Aso et al., 2013). Theperiosteum, which is the thin fibrous sheath that covers the entiresurface of bone with the exception of the articular surface of thejoint, receives the most dense sensory innervation (i.e. greatest num-ber of sensory nerve fibers per unit area) of any compartment of theskeleton. The periosteum is composed of a thin outer ‘fibrous layer’and an inner bone lining ‘cambium layer’ which contains theprogenitor cells that are intimately involved and required for bone

Fig. 1. List of human disorders that are frequently accompanied by skeletalpain. A major reason that skeletal pain occurs in such a diverse group of dis-orders is that the skeleton participates in a variety of functions includingstructural support, ambulation, protection of internal organs, storage andrelease of minerals and growth factors, and the birth and maturation of bloodcells. As the skeleton is composed of tissues with very distinct microenviron-ments, (such as articular cartilage, periosteum, mineralised bone, and bonemarrow) injury, aging or disease in any of these compartments can result inskeletal pain. For an extensive list of diseases that are frequently accompa-nied by skeletal pain, see http://www.rightdiagnosis.com/symptoms/bone_-pain/common.htm; in children, http://www.nof.org/articles/5; and for a list ofrare (orphan) bone diseases that may be accompanied by skeletal pain, http://www.usbji.org/projects/RBDPN_op.cfm?dirID=252.

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.European Journal of Neuroscience, 39, 508–519

Neurobiology of skeletal pain 509

remodeling and fracture repair in adult bones (Hutmacher & Sittin-ger, 2003; Zhang et al., 2005; Bielby et al., 2007). In the perios-teum, the A-delta and C-sensory nerve fibers are arranged in afishnet-like pattern, which appears to be designed to act as a ‘neuralnet’ to detect mechanical injury or distortion of the underlying corti-cal bone (Martin et al., 2007). Thus, the initial sharp pain feltupon a kick to the shin bone (i.e. the tibia) or a fracture of anybone, is probably detected by mechanotransducers expressed by theperiosteal A-delta and C-sensory fibers. Mechanotransducers are ionchannels that detect mechanical stimuli such as stretching and pres-sure, although the exact set of mechanaotranducers that areexpressed in each subset of mammalian sensory nerve fibers has yetto be fully elucidated (Delmas et al., 2011). Following bone frac-ture, movement before effective stabilisation of the fractured bonecan be remarkably painful. This pain is most likely attributable tothe normally silent mechanosensitive nerve fibers in the periosteum

that are now sensitised, discharging, and signaling the pain. Whenthe bone is stabilised with either an external cast or internal rod, sig-nificant relief of this sharp, stabbing and arresting pain occurs. Incontrast, the dull aching pain following injury, bruising, or stabilisa-tion of the fractured bone is probably driven by the unmyelinatedC-fibers present in the periosteum, marrow, and mineralised bone(Martin et al., 2007; Jimenez-Andrade et al., 2009).The cortical bone and bone marrow are also innervated by the

same population of A-delta and C-sensory nerve fibers that innervatethe periosteum, although the relative density of sensory nerve fibersper unit area is markedly lower (Fig. 3A and B). The relative densi-ties of A-delta and C-sensory nerve fibers in the periosteum, marrowand cortical bone are 100 : 2 : 0.1, respectively (Casta~neda-Corralet al., 2011). In the cortical bone, both A-delta and C-fiberstypically colocalise with blood vessels that run through the Haveri-sian and Volkmann canals although it is clear that the majority of

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Fig. 2. Changes in the skeleton and sclerostin levels with normal aging. (A) Human bone mass usually peaks at 25–30 years of age in both men and womenand then generally declines thereafter, (B) resulting in stereotypic changes in posture and height. (C) One factor that appears to participate in driving age-relatedbone loss is sclerostin (here measured in human plasma) which inhibits bone formation and increases linearly with age (Figure reprinted with permission fromArdawi et al., 2011). (D) The major cell type that expresses sclerostin in the adult is the osteocyte in bone and mechanical loading of the bone reduces theexpression and release of sclerostin. Sclerostin appears to exert its inhibitory effect by blocking bone progenitor cells and osteoblast function. (E) Administrationof an antibody that sequesters sclerostin (Scl-Ab) has been shown to accelerate fracture healing in the primate tibia (reprinted with permission from Ominskyet al., 2011). (F) Administration of Scl-Ab to normal rats also shows a dose-related effect (as analysed by lCT) in stimulating bone formation in the femoralneck of the normal femur, distal head of the femur and the L5 vertebrae (Figure reprinted with permission from Li et al., 2010).


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blood vessels in Haversian or Volkmann canal in the mineralisedbone are not innervated by sensory nerve fibers, which may be onereason why bone microfractures may not at least initially be per-ceived as painful. Likewise, the bone marrow is innervated byA-delta and C-sensory nerve fibers and, in most cases, these nervefibers are associated with blood vessels.In sharp contrast to periosteum, cortical bone and bone marrow,

the normal articular cartilage of the joint (including the knee andtemporomandibular joint) appears to completely lack any detectableinnervation by sensory nerve fibers or vascularisation by blood ves-sels (Archer et al., 2012), although the neighboring synovial mem-brane and the subchondral bone (i.e. bone immediately below thearticular cartilage) in the normal joint is innervated by sensory nervefibers and receives a significant vascular supply (Donaldson, 2009;Kelly et al., 2012) (Fig. 3A). In light of this lack of sensory nervefibers in normal articular cartilage, the initial source of pain follow-ing acute injury of the joint in osteoarthritis (OA) (Campero et al.,2009) or temporomandibular joint disorder (TMD) must arise fromadjacent structures such as the bone, ligaments, synovium andmuscle. However, as discussed below, nerve fibers may undergosprouting and begin innervating the damaged articular cartilage fol-lowing injury, inflammation or aging, and thus serve as a generatorof chronic knee or TMD pain.

Sympathetic nerve fibers and skeleton pain

While primary afferent sensory nerve fibers are clearly involved indriving skeletal pain, the skeleton also receives a significant innerva-

tion by both adrenergic and cholinergic sympathetic nerve fibers.Previous studies have shown that sympathetic nerve fibers in bonecan regulate bone destruction, bone formation, vasodilation, vaso-constriction, macrophage infiltration and bone progenitor cell func-tion. As such, they may play a significant role in health and diseaseprogression in both cartilage (i.e. rheumatoid arthritis) and bone (i.e.osteoporosis) and thus via modulating disease progression play asignificant role in driving skeletal pain (Asmus et al., 2000; Elenkovet al., 2000; Bataille et al., 2012; Eimar et al., 2013).It has also been shown that, following injury to the skeleton, sym-

pathetic nerve fibers can modulate sensory nerve fiber function andthis pathological interaction between sensory and sympathetic nervefibers may play a role in OA and complex regional pain syndrome(Pepper et al., 2013). For example, in many cases of chronic skele-tal pain, when significant sprouting of TrkA+ sensory nerve fibersoccurs this is also accompanied by nearby sprouting on TrkA+, tyro-sine hydroxylase expressing sympathetic nerve fibers (Ghilardiet al., 2011). For example, in a rodent model of knee OA it wasshown that sympathetic nerve sprouting occurred in the inflamedjoint and that pharmacological suppression of sympathetic fiberfunction significantly decreased the OA pain-related behaviors (Lon-go et al., 2013). Previous data has suggested that noradrenalinereleased from these newly sprouted sympathetic fibers can bind toadrenergic receptors expressed by nearby sensory nerve fibers, thuscontributing to pain-related behavior associated with arthritis. Inter-estingly, noradrenaline injected into normal peripheral tissuesdoes not produce pain. However, in injured or inflamed tissues ithas varying effects, including aggravation of pain in neuropathy

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Fig. 3. The types of sensory nerve fibers that innervate the skeleton, a list of stromal cells and algogenic substances that can be released from these cells.(A) Schematic diagram illustrating the general organisation and pattern of sensory innervation of the skeleton. The types of sensory neurons that innervate thebone are unmyelinated C fibers and thinly myelinated A-delta fibers. The relative density of A-delta and C sensory fibers (nerve fibers per unit area) is greatestin the periosteum, followed by the bone marrow and cortical bone, with a ratio of 100 : 2 : 0.1, respectively. (B) Primary afferent neurons innervating the skele-ton have their cell bodies in the dorsal root ganglia (DRG) and project to the spinal cord. The great majority (>80%) of sensory nerve fibers that innervate thebone and articular cartilage express TrkA (which is the cognate receptor for NGF), whereas < 30% of the nerve fibers that innervate the skin express TrkA. (C)Bone contains a remarkably diverse population of stromal, myeloid and other cells which can release a wide variety of factors (some of which are listed in thearrow) that can activate and sensitise the primary afferent nerve fibers that innervate the bone.



presumably due to injury-induced expression of novel noradrenergicreceptors by nociceptors, sprouting of sympathetic nerve fibers, andpronociceptive changes in the ion channel properties on primaryafferent nociceptors (Pertovaara, 2013). Developing a better under-standing of the normal and pathological interactions of sensory andsympathetic nerve fibers in the skeleton may help understand howthe nerve fibers may be involved in modulating both skeletal painand disease progression.

Clinically relevant models of skeletal pain

Recent research attempting to define the mechanisms that drive skel-etal pain have significantly benefited from the development of newpreclinical models of skeletal pain (Honore & Mantyh, 2000; Hal-vorson et al., 2005; Ballas et al., 2012; Pepper et al., 2013; Vincentet al., 2013). The development and usefulness of these models ispredicated on three insights.First, as noted above, the skeleton is innervated by a very differ-

ent repertoire of sensory nerve fibers from the skin and thus modelsof skin pain and skeletal pain cannot serve as surrogates of eachother.Second, even when modeling skeletal pain, it is very useful to

attempt to have the model closely mirror a specific type of humanskeletal pain, as the various microenvironments in the skeleton (mar-row, mineralised bone, articular cartilage and periosteum) involvedin driving the pain can be remarkably heterogeneous (Ballas et al.,2012; Mantyh, 2013). Thus, while there may be similarities in the

mechanisms that drive skeletal pain in different diseases, the mecha-nisms that drive sickle cell pain due to a vascular occlusion episodein the marrow may be quite different from the pain followingmechanical injury to articular cartilage or periosteum.Third, models of skeletal pain that closely mirror the underlying

skeletal disease process have the advantage of being able to simulta-neously determine not only whether the therapy is an effective anal-gesic but whether it also has an impact the disease process itself.Examples of therapies having an analgesic and disease modifyingeffect in skeletal disorders are bisphosphonates and Denosumab inbone cancer, NSAIDS and COX-2 inhibitors in bone fracture, andtumor necrosis factor (TNF)-alpha in rheumatoid arthritis (Gaston &Simpson, 2007; Brown-Glaberman & Stopeck, 2013; Sakthiswary &Das, 2013).

Nociceptive component of skeletal pain

Until recently, the major factors thought to drive acute and chronicskeletal pain were that following overuse, injury, inflammation, dis-ease or aging of the bone or cartilage, the sensory nerve fibers thatinnervate the skeleton were activated and/or sensitised by eitherdirect mechanical injury or the release of algogenic substances fromthe bone or joint (Fig. 3C).A good example of mechanical distortion of sensory nerve fibers

inducing significant skeletal pain is the immediate pain that is per-ceived following bone fracture. Initial fracture of the bone is thoughtto be detected by the mechanotransducers expressed by nociceptors

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Fig. 4. The efficacy of anti-NGF in blocking skeletal pain in pre-clinical and human models. (A) Radiograph of the normal mouse bone with intramedullarypin, and following a closed femoral fracture that generates fracture pain. (B) Sustained anti-NGF therapy (commenced immediately after fracture) attenuatedfracture-induced skeletal pain-related guarding behaviors by 40–50%. Figure reprinted with permission from Koewler et al. (2007). (C) Radiograph of thenormal and osteoarthritic human knee. (D) The assessment of the patient’s knee pain while walking showed a significant reduction (40–50%) with human anti-NGF therapy (Tanezumab). A decrease in the change from baseline indicates an improvement (i.e., less pain). Figure reprinted with permission from Lane et al.(2010).


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that densely innervate the periosteum, with this initial sharp,stabbing and arresting pain being transmitted by the thinly myelin-ated A-delta nerve fibers. Upon stabilisation of the fracture, the ini-tial sharp, stabbing pain usually subsides and is replaced by a lesserdull, aching pain that is most likely conveyed by the C-fibers in theperiosteum, bone and marrow. This dull aching pain is probably dueto activation and sensitisation of both the A-delta and C-fibers byalgogenic factors released by the fractured bone cells and the invad-ing inflammatory and immune cells. These algogenic factors includebradykinin, colony stimulating factors, nerve growth factor (NGF),prostaglandin E2, serotonin, protease-activated receptor 2, and TNF-alpha (Fig. 3C) (Inglis et al., 2005; Schweizerhof et al., 2009; Gold& Gebhart, 2010; Kurejova et al., 2010; Ye et al., 2011; Lam et al.,2012). If mechanical distortion of the periosteum of the fracturedbone recurs, the now sensitised A-delta and C-fibers avidly dis-charge, and the sharp, stabbing pain immediately returns.A similar duality of sharp stabbing pain, and a lesser dull aching

pain, appears to occur following injury to the articular cartilage inOA or TMD. With either trauma or degeneration of the articular car-tilage of the joint, A-delta and C-fibers in the synovium and sub-chondral bone are sensitised by algogenic substances so thatnormally non-noxious loading and movement of the joint are per-ceived as noxious stimuli. As the normal articular cartilage com-pletely lacks any detectable innervation by sensory nerve fibers, theexact location(s) of nerve fibers that drive OA pain are as yetunknown. Thus, while OA of TMD pain may arise from directmechanical stimulation of sensitised nerve fibers that are present inthe adjacent bone (Kidd, 2006; Hunter et al., 2008), if this were theonly mechanism driving arthritic joint pain, a clear correlation

should appear between joint deterioration or destruction and jointpain. However, in both trauma and age-related OA and TMD, thereis a poor correlation between the extent of joint destruction and thefrequency and severity of joint pain (Felson et al., 1987; Hannanet al., 2000; Bedson & Croft, 2008).Decades of research have demonstrated that NGF directly acti-

vates and sensitises TrkA+ sensory neurons and that NGF activationof TrkA could play a key role in the sensitisation of TrkA+ nocicep-tors to mechanical, chemical and thermal stimuli (Levi-Montalcini,1987; Lewin & Mendell, 1993; Petruska & Mendell, 2004). It hadbeen shown that NGF induces a rapid phosphorylation and sensitisa-tion of transient receptor potential cation channel subfamily V mem-ber 1 (TRPV1) channel (which detects acid and heat), as well asnociceptor mechanotransducers (Mendell et al., 1999; Zhang et al.,2005). Additionally, retrograde transport of the NGF–TrkA complexto the neuronal cell body of nociceptors induces increased synthesisof the neurotransmitters substance P and CGRP, and receptors(bradykinin), channels (P2X3, TRPV1, ASIC-3 and sodium chan-nels), transcription factors (ATF-3) and structural molecules (neuro-filaments and the sodium channel-anchoring molecule p11) (Julius& Basbaum, 2001; Patapoutian & Reichardt, 2001). However,sequestration of NGF showed little efficacy in a rat skin incisionmodel of pain, suggesting that targeting of NGF–TrkA may not beefficacious in relieving chronic pain in skin (Zahn et al., 2004; Ba-nik et al., 2005).Although anti-NGF did not appear to have a major effect on

reducing incisional skin pain, a major question was that, given thatmost sensory nerve fibers that innervated the skeleton were TrkA+,could blockage of NFG–TrkA significantly reduce severe skeletal

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Fig. 5. Ectopic sprouting of primary afferent sensory nerve fibers occurs in a variety of skeletal pain conditions. (A) Confocal images of sections of (A) thenormal and (B) the inflamed knee joint that have been immunostained for DAPI, which stains nuclei, and growth-associated protein (GAP-43), which stainssprouting nerve fibers. Twenty-eight days after the initial injection of Complete Freund’s Adjuvant (CFA) into the rat knee-joint, a significant number of GAP-43+ nerve fibers in the synovial knee joint had sprouted and had a disorganised appearance, as compared with vehicle-injected mice. Reprinted with permissionfrom Jimenez-Andrade & Mantyh (2012). Confocal images of periosteal whole mounts of (C) normal and (D) sarcoma tumor-bearing bone immunostained forcalcitonin gene-related peptide (CGRP+) and green fluorescent protein (GFP)-labeled sarcoma cells. As tumor cells invade the periosteum of the bone, ectopicsprouting of CGRP+ sensory fibers occurs and neuroma-like structures form. Reprinted with permission from Mantyh et al. (2010). Confocal images of bonemarrow of (E) normal and (F) prostate tumor-bearing bone marrow, immunostained for DAPI, CGRP and GFP-expressing prostate cancer cells. Note that in thenormal marrow, CGRP+ nerve fibers appear as single nerve fibers with a highly linear morphology. As GFP+ prostate tumor cells proliferate and form tumorcolonies, the CGRP+ sensory nerve fibers undergo marked sprouting which produces a highly branched, disorganised and dense meshwork of sensory nervefibers that is never observed in normal marrow. Reprinted with permission from Jimenez-Andrade et al. (2010).



pain?. To test this hypothesis, blockade of NGF–TrkA was tested inthree separate mouse models of bone cancer pain using sarcoma,prostate and breast cancer cell implantation, all of which generated asignificant skeletal pain that was accompanied by bone remodelingsimilar to that seen in human patients with bone cancer. Administra-tion of anti-NGF attenuated skeletal pain by 50% in all three mousemodels of bone cancer pain, regardless of whether the injected can-cer cells expressed NGF (Mantyh et al., 2010; Jimenez-Andradeet al., 2011). These data demonstrated that NGF appeared to play asignificant role in driving severe bone cancer pain, and that therelief of bone cancer pain did not depend on the type of tumor orwhether the cancer cells expressed NGF (Halvorson et al., 2005).These data suggested not only that blockade of NGF–TrkA couldsignificantly attenuate skeletal pain but that blockade of NGF–TrkAmight also relieve non-malignant skeletal pain as the major sourceof NGF was not from the cancer cells themselves but from bonestromal, immune and/or inflammatory cells that are present in bothnon-malignant and malignant skeletal conditions.The fact that the efficacy of anti-NGF therapy was not limited to

malignant mouse models was also demonstrated in rodent models ofknee arthritis pain in the rat (Albrecht et al., 2006; Ashraf et al.,2013) and mouse (Ghilardi et al., 2012), bone fracture pain in themouse (Jimenez-Andrade et al., 2007; Koewler et al., 2007; Ghi-lardi et al., 2011) and the rat (Sabsovich et al., 2008). In most ofthese studies similar efficacy (an ~50% reduction in pain-relatedbehaviors) was observed as in the malignant models of skeletal pain(Fig. 4). Subsequent human clinical trials demonstrated that anti-NGF therapy could relieve OA pain by 50% and human low backpain by 40% (Fig. 4) whereas in human visceral pain (interstitialcystitis) and human neuropathic pain (sciatica), anti-NGF showedless efficacy (Nickel et al., 2012).

Neuropathic component of skeletal pain

While most preclinical and clinical scientists who study skeletal painwould agree that there is a significant nociceptive component inmost chronic skeletal pains (OA, TMD, bone cancer, bone fractureand sickle cell), recent data has suggested there may also be a neu-ropathic component in many types of skeletal pain. For example, inthe sarcoma, prostate and breast mouse models of bone cancer pain,as tumor cells invade the normal tissue the tumor appears to firstcome into contact then injure and destroy the very distal processesof sensory fibers. This tumor-induced injury and destruction of thedistal ends of the sensory nerve fibers that innervate the skeleton isaccompanied by an increase in ongoing and movement-evoked painbehaviors. This data, along with data suggesting sensory nerve fibersare injured in OA and low back pain, suggests a component ofmalignant and non-malignant skeletal pain can be neuropathic in ori-gin (Ohtori et al., 2012; Schaible, 2012). This, as well as the factthat several skeletal pains can be attenuated by gabapentin (Rosen-berg et al., 1997; Wetzel & Connelly, 1997), which is approved forthe treatment of neuropathic pain, suggests that injury to sensorynerve fibers may play a role in several types of skeletal pain (Dono-van-Rodriguez et al., 2005; Kurejova et al., 2010).Another intriguing, but largely unexplored, mechanism by which

skeletal pain may be generated is not by injury, but rather by anactive and pathological sprouting and neuroma formation by sensoryand sympathetic nerve fibers that innervate the skeleton. To explorethis possibility, active nerve sprouting and formation of neuroma-like structures was examined using mouse models of sarcoma, breastand prostate bone cancer cells growing in the bone marrow. Usingthese models it was noted that there was a remarkable and dramatic

sprouting of sensory and sympathetic nerve fibers, and that thesenewly sprouted nerve fibers (which can be observed in the perios-teum, mineralised bond and marrow) have a unique morphology,organisation and high density that is never observed in the normalbone (Fig. 5). What is impressive is how exuberant this sproutingcan be: in the bone cancer prostate model (where the prostate cancercells do not express NGF) the number of nerve fibers per unit areaincreased to 10–709 what is observed in the normal bone marrow.This sprouting appears to require NGF, as sustained administrationof anti-NGF or a Pan-Trk inhibitor largely blocked the pathologicalsprouting of sensory nerve fibers and the formation of neuroma-likestructures, and significantly inhibited the generation of pain (Mantyhet al., 2010; Ghilardi et al., 2012).The above studies suggest ectopic sprouting of nerve fibers may

occur in several types of bone cancer pain, whereas previous stud-ies have reported that ectopic sprouting of sensory nerve fibers alsois observed in several types of non-malignant skeletal pain. Forexample, in human chronic vertebral discogenic pain it has beenshown that there is sprouting of CGRP sensory nerves fibers intonormally aneural and avascular areas of the human intervertebraldisc (Freemont et al., 2002), also sprouting of CGRP nerve fibersinto the callus that forms around the bone fracture in rat, and in thesynovoium of arthritic joints of humans and animals (Buma et al.,1992; Wu et al., 2002; Suri et al., 2007). Similar to what isobserved in models of bone cancer, it has been suggested that thesprouting of sensory nerve fibers in non-malignant skeletal pain isdriven by NGF released by endogenous stromal, immune and/orinflammatory cells (Ehrhard et al., 1993; Skaper et al., 2001; Articoet al., 2008).Given the above observations on ectopic sprouting in the injured

or diseased skeleton, a major question is to what extent does thispathological sprouting drive skeletal pain? Previous studies in ani-mals and humans have demonstrated that inappropriate sproutingand/or neuroma formation in non-skeletal conditions is often isaccompanied by a remarkable change in the phenotype and hyper-sensitivity to normally non-noxious stimuli (Kryger et al., 2001).Given the exuberant nature of this observed sprouting in the injuredskeleton, and that in adult humans newly sprouted sensory nervefibers (such as that which follows nerve transection of injury) fre-quently exhibit both a spontaneous and movement-evoked ectopicdischarges, it will be important to further define the role that ectopicnerve sprouting plays in the generation and maintenance of achronic skeletal pain.

Central sensitisation and skeletal pain

Studies in both animals and humans have demonstrated that, follow-ing injury to the skin, muscle and joint, neurons responsible for painprocessing in the spinal cord and brain can also undergo sensitisa-tion (i.e. ‘central sensitisation’) that amplifies the perception andseverity of pain (Woolf, 1983; Woolf & Salter, 2000). Central sensi-tisation is thought to occur when the chemical, electrophysiologicaland pharmacological systems that transmit and modulate pain arealtered in the spinal cord and higher centers of the brain. Thesechanges cause an exaggerated perception of painful stimuli so thatnormally mild painful stimuli are perceived as highly painful (hyper-algesia) and normal non-painful stimuli such as normal loading oruse of joint or bone is now perceived as a painful event (allodynia).Additionally, central sensitisation also contributes to the phenome-non of referred pain, where areas adjacent to the initial injurybecome hypersensitive. A common example of this is in whiplashinjuries to the neck: following injury to one cervical vertebra,


514 P. W. Mantyh

nearby areas such as the shoulder, arm and back also become hyper-sensitive (Graven-Nielsen & Arendt-Nielsen, 2010).Currently, we know remarkably little as to the specific mecha-

nisms that drive central sensitisation in chronic skeletal pain.Changes that have been described include upregulation of dynor-phin, CGRP and substance P in the spinal cord, astrocyte hypertro-phy and microglial activation (Voscopoulos & Lema, 2010). Otherreports have demonstrated that increases in skeletal pain-relatedbehaviors are accompanied by increased expression of NR2B (anNMDA receptor subunit), and interleukin-1b (released from glialcells) and enhanced phosphorylation of NMDA receptor NR-1subunits (Ren & Dubner, 2008). Understanding the specific cascadeof changes that generate and maintain central sensitisation wouldsignificantly expand our ability to design targeted therapies to treatthe central sensitisation that occurs following many types of skeletalpain. These neurochemical and electrophysiological changes appearto occur in the ascending and descending pain pathways of thespinal cord, brainstem, thalamus and cerebral cortex, and all of thesechanges may contribute to the amplified perception of skeletal pain(Basbaum & Fields, 1978; Gebhart, 2004; Robinson et al., 2004;Hunt, 2009; Latremoliere & Woolf, 2009; Woolf, 2011; Yoshidaet al., 2013).While we do not yet know the specific mechanisms that generate

central sensitisation, what we do know is that injury to the skeletonseems to be much more effective at inducing central sensitisation ascompared to injury to skin or muscle (Woolf & Wall, 1986). Forexample as noted by Woolf and Wall ‘…a twisted ankle invokesrelatively little destruction of tissue and elicits an abrupt localisedstabbing pain that dies down quickly but is followed by a prolongedperiod of spreading, poorly localised deep pain, and tenderness thataffects reflexes and gait. In contrast, localised skin damage producesan acute burst of pain that gradually dies down over minutes but isassociated with a spatially restricted response of flair, wheal and sur-rounding tenderness’ (Woolf & Wall, 1986). These authors alsonoted that small skin lesions produce comparatively less widespreadand prolonged disturbances to sensation and reflex patterns thanskeletal injuries. They then postulated that the varied pattern ofpost-injury pain hypersensitivities resulting from injury to differenttissues may be the consequence of the activation of distinct primaryafferent neurons with differing central actions, or that the uniquesubtypes of sensory neurons that innervate the skeleton might beuniquely effective at inducing central sensitisation in the spinal cordand higher centers of the brain.

Anabolic therapies and skeletal pain

Analgesics with increased efficacy and fewer side effects are clearlyneeded to improve control of skeletal pain. However, another com-plimentary approach would be therapies that induce bone or carti-lage formation and/or promote more rapid healing following injury,disease or aging of the skeleton. Currently, injury and age-relatedloss of bone injury to the skeleton, and the frequently slow healingof these injures that comes with aging, have enormous consequencesin terms of recovery rates and the functional status of the affectedindividual (Wang & Seeman, 2008; Ominsky et al., 2010; Woolfet al., 2012) . Thus, most fractures in people over age 50 are theresult of age-related bone loss and the resulting fragility of bone,and these fractures result in high rates of hospitalisation, and fre-quently a permanent decrease in function status, and mortality(Woolf & Pfleger, 2003).Currently, there are two main classes of drugs available to treat

age-related bone loss. Antiresorptives (e.g., bisphosphonates,

denosumab) which work by slowing bone loss by inhibiting theactivity of osteoclasts. However, with long-term use there is adecrease in the rate of bone formation because the cyclic activityof osteoclasts and osteoblasts is disrupted. The disturbance slowsbone remodeling (Baron & Hesse, 2012) and leads to a state oflow bone turnover, which over time can cause the accumulationof microcracks and compromise the strength and integrity of thebone.The other relevant class of drugs is osteoanabolic: true bone-

building agents. The first osteoanabolic to be put into clinical usewas intermittent parathyroid hormone (PTH), which exerts its effectsby preferentially stimulating osteoblasts over osteoclasts. However,bone density seems to plateau after 18–24 months of PTH therapy,and the treatment may also increase the risk of osteosarcoma (Baron& Hesse, 2012). Bone morphogenic proteins represent another ana-bolic option, though these are limited by their high cost (Lane et al.,2010) and the difficulties of administration (Albrecht et al., 2006;Lane & Silverman, 2010). Thus, the niche for a safe and effectiveosteoanabolic drug to treat age-related bone loss remains largelyunfilled.Several new therapeutic targets for building bone have profoundly

changed our understanding as to why age-related bone loss occurs.Two inhibitory proteins, known as sclerostin (Burgers & Williams,2013) and Dickkopf-1 (DKK1), have been identified and both pro-teins interact with the Wnt coreceptors LRP5/6 to inhibit the canoni-cal Wnt/beta-catenin signaling pathway, causing a decrease in boneformation (Moester et al., 2010; Palaniswamy et al., 2010; Pasztyet al., 2010; Lewiecki, 2011; Costa & Bilezikian, 2012; Ke et al.,2012; Lim & Clarke, 2012; Ohlsson, 2013). DKK1 appears to havea greater role in developing animals than in adults (Ke et al., 2012).In contrast, sclerostin which is a small (24 Da) secreted glycoproteinthat is expressed by osteocytes in bone (Moester et al., 2010; Costa& Bilezikian, 2012) appears to play a greater role in the adult thanin the the developing skeleton. In adults, the expression and releaseof sclerostin by osteocytes is modulated by local mechanical loadingof the bone (Costa & Bilezikian, 2012) (Fig. 2D). Aside from itsnormal physiological role, sclerostin has been directly implicated asa factor in skeletal disease, as studies in humans have shown a rela-tively linear increase in plasma levels of sclerostin with age (Mod-der et al., 2011) (Fig. 2C) and older women with high expressionof sclerostin have greater risk of hip fracture (Albrecht et al., 2006).An antibody that sequesters sclerostin has been developed andadministration of this therapy increased bone growth and acceleratedfracture healing in preclinical trials in osteoporotic rats and monkeys(Li et al., 2009; Ominsky et al., 2010) (Fig. 2E). In a Phase Istudy, a single dose of anti-sclerostin antibody increased bone den-sity in the hip and spine in healthy men and postmenopausal women(Padhi et al., 2011)(Fig. 2F). The administration of the drug for1 year was well tolerated, and in a Phase II trial, for osteoporoticwomen, it increased bone density (Reid, 2012; Burgers & Williams,2013).In many older patients with bone fractures due to low-impact

falls, appropriate healing of the skeleton never occurs and manypatients develop chronic pain at the site of non-healed injury. Giventhe exuberant sensory nerve sprouting that has been shown follow-ing other skeletal injury and diseases, one possibility is that ectopicsensory nerve sprouting occurs at the non-healed fracture site andthis then drives chronic skeletal pain. However, if ectopic nervesprouting and skeletal pain could be attenuated by blockade ofNGF–TrkA, and fracture healing accelerated by blocking sclerostin,this could potentially transform how we treat fracture healing in theaged or diseased skeleton.



Analgesics and their potential effects on the skeleton

Although it may seem counterintuitive, one question that needs tobe addressed when considering therapies to relieve skeletal pain ishow much relief of skeletal pain is desirable? Obviously, one wouldprobably want to eliminate all skeletal pain due to bone cancer, leu-kemia, fibrous dysplasia, Gaucher’s disease and sickle-cell disease,as these skeletal pains appear to serve little or no protective purpose.However, in patients with skeletal pain due to OA, TMD, bone frac-ture or aging, eliminating all skeletal pain could potentially lead toinappropriate loading and/or overuse of the skeleton that could drivemore rapid deterioration of the diseased, injured or aged skeleton.For example, this issue as to the extent of relief of skeletal painoccurred in a phase III clinical trial with anti-NGF therapy inpatients with knee and hip OA. Here it was noted that a small sub-set of patients being treated simultaneously with anti-NGF and anNSAID developed rapidly progressing OA although it was unclearwhether this deterioration was due to patients overusing theirarthritic joints or a direct effect of anti-NGF and NSAIDS on theskeleton itself (Lane & Silverman, 2010).A second major issue is whether any analgesics used to treat skel-

etal pain may have direct negative effects on skeletal health and/orhealing of the injured skeleton. Here the answer is probably yes, asthe administration of Ibuprofen or COX-2 inhibitors have beenshown to markedly slow down fracture healing in rodent models ofbone fracture (O’Connor et al., 2009; Barry, 2010). While themechanism(s) by which these side effects occurs is not completelyclear, prostaglandins do appear to play a significant role in Wntsignaling (Genetos et al., 2011), a key pathway required for boneformation in both the normal and fractured bone. Thus, inhibition ofprostaglandin synthesis might be expected to inhibit bone formation.Which and how much NSAIDS and COX-2 inhibitors actuallyreduce normal bone formation and fracture healing in humansremains to be determined (Su & O’Connor, 2013). However, whatis clear is that it is probably wise to include bone formation andskeletal healing as endpoints in assessing novel skeletal analgesicsin both preclinical studies and human clinical trials.Lastly, if reducing skeletal pain by any mechanism has the poten-

tial to induce overuse of the injured, diseased or aged skeleton bythe patient, is there an approach that ameliorates or prevents poten-tial injury do to overuse? Obviously, more effective monitoring andfeedback to the patient concerning desired activity and movementcould prevent overuse. However, another possibility would be tosimultaneously block NGF to attenuate skeletal pain and block scle-rostin to promote bone formation and fracture healing. Whether thiscombined therapy would simultaneously reduce skeletal pain,increase bone formation and healing, and improve the patient’s func-tional status and ability to participate in physical therapy has not yetbeen determined.

Future perspectives

Although skeletal pain is one of the most common causes of chronicpain and long-term physical disability, we currently have relativelyfew pharmacological therapies that can fully manage the pain, stim-ulate skeletal repair following injury, or reverse the bone and carti-lage loss that occurs with aging. However, in the last decadesignificant progress has been made in developing more clinically rel-evant models of skeletal pain, understanding some of the mecha-nisms that drive skeletal pain, and identifying key moleculesinvolved in the regulation of skeletal repair and the decline in theskeleton that occurs with aging.

In terms of skeletal pain, we now know that both the bone andjoint are innervated by a limited repertoire of sensory nerve fibersand that many forms of skeletal pain probably have both a noci-ceptive and a neuropathic component. Researchers have identifiedthe restricted repertoire of primary afferent sensory nerve fibersthat innervate the skeleton, the majority of which express TrkAand thus respond to NGF released by bone stromal cells followinginjury or in disease. Sensory nerve fibers in an injured or diseasedskeleton also display a remarkable neurochemical and morphologi-cal plasticity by upregulating neurotransmitters and receptors andundergoing a level of nerve sprouting that is never observed inthe normal skeleton. In agreement with these observations, humanclinical trials have demonstrated that sequestration of NGF resultsin significant attenuation of OA and low back pain.Progress has also been made in beginning to understand the

molecules that contribute to the deterioration and lack of effectiveskeletal repair that frequently occur with aging. For example, scle-rostin has been identified as a protein that is expressed andreleased by osteocytes when the bone is mechanically unloaded,inhibits bone formation, and whose levels increase dramaticallywith age. As the incidence of low-trauma fractures increases andthe rate of rapid and effective bone fracture healing decreases withage, inhibiting sclerostin may reduce the likelihood of fragilityfractures, stimulate more rapid and effective fracture healing, andthereby reduce the incidence and duration of skeletal pain that fre-quently accompanies failed healing of bone fractures. Indeed, datafrom preclinical and human studies suggests that inhibition ofendogenous sclerostin can both build bone and promote fracturehealing in both young and aged bone (Lane & Silverman, 2010;Moester et al., 2010).Whether therapies targeting the NGF or sclerostin will ultimately

receive approval for broad use in humans will depend on theirsafety and side-effect profile. However, what is clear is that NGFplays a major role in driving skeletal pain and sclerostin plays amajor role in driving the age-related decline in normal bone remod-eling and fracture healing. Increasing our understanding of themechanisms that drive skeletal pain and defining the factors thatcontrol bone remodeling in the young and old have the potential tofundamentally transform our understanding and ability to preventand/or treat skeletal pain due to injury, disease, and aging.

Acknowledgements

This work was supported by the National Institutes of Health grants(NS023970, CA154550, CA157449). P.W.M. received anti-NGF as aresearch tool and gift from Rinat Laboratories, Pfizer Inc. (South San Fran-cisco, CA, USA) and has served as a consultant and received research grantsfrom Abbott (Abbott Park, IL, USA), Adolor (Exton, PA, USA), Array Bio-pharma (Boulder, CO, USA), Johnson and Johnson (New Brunswick, NJ,USA), Pfizer (New York, NY, USA), Plexxikon (Berkeley, CA, USA) andRoche (South San Francisco, CA, USA). The author thanks Stephane Char-tier, Michelle Fealk, Lisa Majuta, Gwen McCaffrey and Michelle L. Thomp-son for editing and reviewing the manuscript.

Abbreviations

CGRP, calcitonin gene-related peptide; NGF, nerve growth factor; OA,osteoarthritis; TMD, temporomandibular joint disorder; TrkA, tropomyosinreceptor kinase A.

References

Albrecht, P.J. & Rice, F.L. (2010) Role of small-fiber afferents in pain mech-anisms with implications on diagnosis and treatment. Curr. Pain HeadacheR., 14, 179–188.


516 P. W. Mantyh

Albrecht, P.J., Hines, S., Eisenberg, E., Pud, D., Finlay, D.R., Connolly,M.K., Pare, M., Davar, G. & Rice, F.L. (2006) Pathologic alterations ofcutaneous innervation and vasculature in affected limbs from patients withcomplex regional pain syndrome. Pain, 120, 244–266.

Aoki, Y., Ohtori, S., Takahashi, K., Ino, H., Douya, H., Ozawa, T., Saito, T.& Moriya, H. (2005) Expression and co-expression of VR1, CGRP, andIB4-binding glycoprotein in dorsal root ganglion neurons in rats: differ-ences between the disc afferents and the cutaneous afferents. Spine, 30,1496–1500.

Archer, C.W., Williams, R., Nelson, L. & Khan, I.M. (2012) Articular carti-lage-derived stem cells: identification, characterisation and their role inspontaneous repair. Rheumatol. Curr. Res., S3, 005.

Artico, M., Bronzetti, E., Felici, L.M., Alicino, V., Ionta, B., Bronzetti, B.,Magliulo, G., Grande, C., Zamai, L., Pasquantonio, G. & De Vincentiis,M. (2008) Neurotrophins and their receptors in human lingual tonsil: animmunohistochemical analysis. Oncol. Rep., 20, 1201–1206.

Ashraf, S., Mapp, P.I., Burston, J., Bennett, A.J., Chapman, V. & Walsh, D.A.(2013) Augmented pain behavioural responses to intra-articular injection ofnerve growth factor in two animal models of osteoarthritis. Ann. Rheum.Dis., doi: 10.1136/annrheumdis-2013-203416. [Epub ahead of print].

Asmus, S.E., Parsons, S. & Landis, S.C. (2000) Developmental changes inthe transmitter properties of sympathetic neurons that innervate the perios-teum. J. Neurosci., 20, 1495–1504.

Aso, K., Ikeuchi, M., Izumi, M., Sugimura, N., Kato, T., Ushida, T. & Tani,T. (2013) Nociceptive phenotype of dorsal root ganglia neurons innervat-ing the subchondral bone in rat knee joints. Eur. J. Pain., doi: 10.1002/j.1532-2149.2013.00360.x. [Epub ahead of print].

Ballas, S.K., Gupta, K. & Adams-Graves, P. (2012) Sickle cell pain: a criti-cal reappraisal. Blood, 120, 3647–3656.

Banik, R.K., Subieta, A.R., Wu, C. & Brennan, T.J. (2005) Increased nervegrowth factor after rat plantar incision contributes to guarding behaviorand heat hyperalgesia. Pain, 117, 68–76.

Baron, R. & Hesse, E. (2012) Update on bone anabolics in osteoporosistreatment: rationale, current status, and perspectives. J. Clin. Endocr.Metab., 97, 311–325.

Barry, S. (2010) Non-steroidal anti-inflammatory drugs inhibit bone healing:a review. Vet. Comp. Orthopaed., 23, 385–392.

Basbaum, A.I. & Fields, H.L. (1978) Endogenous pain control mechanisms:review and hypothesis. Ann. Neurol., 4, 451–462.

Bataille, C., Mauprivez, C., Hay, E., Baroukh, B., Brun, A., Chaussain, C.,Marie, P.J., Saffar, J.L. & Cherruau, M. (2012) Different sympathetic path-ways control the metabolism of distinct bone envelopes. Bone, 50, 1162–1172.

Bedson, J. & Croft, P.R. (2008) The discordance between clinical and radio-graphic knee osteoarthritis: a systematic search and summary of the litera-ture. BMC Musculoskel. Dis., 9, 116.

Bielby, R., Jones, E. & McGonagle, D. (2007) The role of mesenchymalstem cells in maintenance and repair of bone. Injury, 38(Suppl 1), S26–S32.

Brooks, P.M. (2006) The burden of musculoskeletal disease–a global per-spective. Clin. Rheumatol., 25, 778–781.

Brown-Glaberman, U. & Stopeck, A.T. (2013) Impact of denosumab onbone mass in cancer patients. Clin. Pharmacol., 5, 117–129.

Buma, P., Verschuren, C., Versleyen, D., Van der Kraan, P. & Oestreicher,A.B. (1992) Calcitonin gene-related peptide, substance P and GAP-43/B-50 immunoreactivity in the normal and arthrotic knee joint of the mouse.Histochemistry, 98, 327–339.

Burgers, T.A. & Williams, B.O. (2013) Regulation of Wnt/beta-cateninsignaling within and from osteocytes. Bone, 54, 244–249.

Campero, M., Baumann, T.K., Bostock, H. & Ochoa, J.L. (2009) Humancutaneous C fibres activated by cooling, heating and menthol. J. Physiol.,587, 5633–5652.

Casta~neda-Corral, G., Jimenez-Andrade, J.M., Bloom, A.P., Taylor, R.N.,Mantyh, W.G., Kaczmarska, M.J., Ghilardi, J.R. & Mantyh, P.W. (2011)The majority of myelinated and unmyelinated sensory nerve fibers thatinnervate bone express the tropomyosin receptor kinase A. Neuroscience,178, 196–207.

Costa, A.G. & Bilezikian, J.P. (2012) Sclerostin: therapeutic horizons basedupon its actions. Curr. Osteoporos Rep., 10, 64–72.

Delmas, P., Hao, J. & Rodat-Despoix, L. (2011) Molecular mechanisms ofmechanotransduction in mammalian sensory neurons. Nat. Rev. Neurosci.,12, 139–153.

Donaldson, L.F. (2009) Neurogenic mechanisms in arthritis. In Gabor,J. (Ed.), Neurogenic Inflammation in Health and Disease. Elsevier,Oxford, pp. 211–241.

Donovan-Rodriguez, T., Dickenson, A.H. & Urch, C.E. (2005) Gabapentinnormalizes spinal neuronal responses that correlate with behavior in a ratmodel of cancer-induced bone pain. Anesthesiology, 102, 132–140.

Ehrhard, P.B., Erb, P., Graumann, U. & Otten, U. (1993) Expression ofnerve growth factor and nerve growth factor receptor tyrosine kinase Trkin activated CD4-positive T-cell clones. Proc. Natl. Acad. Sci. USA, 90,10984–10988.

Eimar, H., Tamimi, I., Murshed, M. & Tamimi, F. (2013) Cholinergic regu-lation of bone. J. Musculoskelet. Neuronal Interact., 13, 124–132.

Elenkov, I.J., Wilder, R.L., Chrousos, G.P. & Vizi, E.S. (2000) The sympa-thetic nerve–an integrative interface between two supersystems: the brainand the immune system. Pharmacol. Rev., 52, 595–638.

Felson, D.T., Naimark, A., Anderson, J., Kazis, L., Castelli, W. & Meenan,R.F. (1987) The prevalence of knee osteoarthritis in the elderly. TheFramingham Osteoarthritis Study. Arthritis Rheum., 30, 914–918.

Freemont, A.J., Watkins, A., Le Maitre, C., Baird, P., Jeziorska, M., Knight,M.T., Ross, E.R., O’Brien, J.P. & Hoyland, J.A. (2002) Nerve growth fac-tor expression and innervation of the painful intervertebral disc. J. Pathol.,197, 286–292.

Funfschilling, U., Ng, Y.G., Zang, K., Miyazaki, J., Reichardt, L.F. & Rice,F.L. (2004) TrkC kinase expression in distinct subsets of cutaneoustrigeminal innervation and nonneuronal cells. J. Comp. Neurol., 480,392–414.

Gaston, M.S. & Simpson, A.H.R.W. (2007) Inhibition of fracture healing. J.Bone Joint Surg. Br., 89B, 1553–1560.

Gebhart, G.F. (2004) Descending modulation of pain. Neurosci. Biobehav.R., 27, 729–737.

Genetos, D.C., Yellowley, C.E. & Loots, G.G. (2011) Prostaglandin E2 sig-nals through PTGER2 to regulate sclerostin expression. PLoS ONE, 6,e17772.

Ghilardi, J.R., Freeman, K.T., Jimenez-Andrade, J.M., Mantyh, W.G.,Bloom, A.P., Bouhana, K.S., Trollinger, D., Winkler, J., Lee, P., Andrews,S.W., Kuskowski, M.A. & Mantyh, P.W. (2011) Sustained blockade ofneurotrophin receptors TrkA, TrkB and TrkC reduces non-malignant skele-tal pain but not the maintenance of sensory and sympathetic nerve fibers.Bone, 48, 389–398.

Ghilardi, J.R., Freeman, K.T., Jimenez-Andrade, J.M., Coughlin, K.A.,Kaczmarska, M.J., Castaneda-Corral, G., Bloom, A.P., Kuskowski, M.A.& Mantyh, P.W. (2012) Neuroplasticity of sensory and sympathetic nervefibers in a mouse model of a painful arthritic joint. Arthritis Rheum., 64,2223–2232.

Gold, M.S. & Gebhart, G.F. (2010) Nociceptor sensitization in pain patho-genesis. Nat. Med., 16, 1248–1257.

Graven-Nielsen, T. & Arendt-Nielsen, L. (2010) Assessment of mechanismsin localized and widespread musculoskeletal pain. Nat. Rev. Rheumatol., 6,599–606.

Halvorson, K.G., Kubota, K., Sevcik, M.A., Lindsay, T.H., Sotillo, J.E., Ghi-lardi, J.R., Rosol, T.J., Boustany, L., Shelton, D.L. & Mantyh, P.W.(2005) A blocking antibody to nerve growth factor attenuates skeletal paininduced by prostate tumor cells growing in bone. Cancer Res., 65, 9426–9435.

Hannan, M.T., Felson, D.T. & Pincus, T. (2000) Analysis of the discordancebetween radiographic changes and knee pain in osteoarthritis of the knee.J. Rheumatol., 27, 1513–1517.

Heaney, R.P., Abrams, S., Dawson-Hughes, B., Looker, A., Marcus, R.,Matkovic, V. & Weaver, C. (2000) Peak bone mass. Osteoporosis Int., 11,985–1009.

Hill, E.L. & Elde, R. (1991) Distribution of CGRP-, VIP-, D beta H-, SP-,and NPY-immunoreactive nerves in the periosteum of the rat. Cell TissueRes., 264, 469–480.

Hill, E.L., Turner, R. & Elde, R. (1991) Effects of neonatal sympathectomyand capsaicin treatment on bone remodeling in rats. Neuroscience, 44,747–755.

Honore, P. & Mantyh, P.W. (2000) Bone cancer pain: from mechanism tomodel to therapy. Pain Med., 1, 303–309.

Hunt, S.P. (2009) Genes and the dynamics of pain control. Funct. Neurol.,24, 9–15.

Hunter, D.J., McDougall, J.J. & Keefe, F.J. (2008) The symptoms of osteoar-thritis and the genesis of pain. Rheum. Dis. Clin. N. Am., 34, 623–643.

Hutmacher, D.W. & Sittinger, M. (2003) Periosteal cells in bone tissue engi-neering. Tissue Eng., 9(Suppl 1), S45–S64.

Inglis, J.J., Nissim, A., Lees, D.M., Hunt, S.P., Chernajovsky, Y. & Kidd,B.L. (2005) The differential contribution of tumour necrosis factor to ther-mal and mechanical hyperalgesia during chronic inflammation. ArthritisRes. Ther., 7, R807–R816.



Ivanusic, J.J., Mahns, D.A., Sahai, V. & Rowe, M.J. (2006) Absence oflarge-diameter sensory fibres in a nerve to the cat humerus. J. Anat., 208,251–255.

Jimenez-Andrade, J.M., Martin, C.D., Koewler, N.J., Freeman, K.T., Sulli-van, L.J., Halvorson, K.G., Barthold, C.M., Peters, C.M., Buus, R.J.,Ghilardi, J.R., Lewis, J.L., Kuskowski, M.A. & Mantyh, P.W. (2007)Nerve growth factor sequestering therapy attenuates non-malignant skeletalpain following fracture. Pain, 133, 183–196.

Jimenez-Andrade, J.M., Bloom, A.P., Mantyh, W.G., Koewler, N.J., Free-man, K.T., Delong, D., Ghilardi, J.R., Kuskowski, M.A. & Mantyh, P.W.(2009) Capsaicin-sensitive sensory nerve fibers contribute to the generationand maintenance of skeletal fracture pain. Neuroscience, 162, 1244–1254.

Jimenez-Andrade, J.M., Mantyh, W.G., Bloom, A.P., Xu, H., Ferng, A.S.,Dussor, G., Vanderah, T.W. & Mantyh, P.W. (2010) A phenotypicallyrestricted set of primary afferent nerve fibers innervate the bone versusskin: therapeutic opportunity for treating skeletal pain. Bone, 46, 306–313.

Jimenez-Andrade, J.M., Ghilardi, J.R., Castaneda-Corral, G., Kuskowski,M.A. & Mantyh, P.W. (2011) Preventive or late administration of anti-NGF therapy attenuates tumor-induced nerve sprouting, neuroma forma-tion, and cancer pain. Pain, 152, 2564–2574.

Julius, D. & Basbaum, A.I. (2001) Molecular mechanisms of nociception.Nature, 413, 203–210.

Kalliomaki, M., Kieseritzky, J.V., Schmidt, R., Hagglof, B., Karlsten, R.,Sjogren, N., Albrecht, P., Gee, L., Rice, F., Wiig, M., Schmelz, M. &Gordh, T. (2011) Structural and functional differences between neuropathywith and without pain? Exp. Neurol., 231, 199–206.

Karanth, S.S., Springall, D.R., Kuhn, D.M., Levene, M.M. & Polak, J.M.(1991) An immunocytochemical study of cutaneous innervation and thedistribution of neuropeptides and protein gene product 9.5 in man andcommonly employed laboratory animals. Am. J. Anat., 191, 369–383.

Ke, H.Z., Richards, W.G., Li, X. & Ominsky, M.S. (2012) Sclerostin andDickkopf-1 as therapeutic targets in bone diseases. Endocr. Rev., 33,747–783.

Kelly, S., Dunham, J.P., Murray, F., Read, S., Donaldson, L.F. & Lawson,S.N. (2012) Spontaneous firing in C-fibers and increased mechanical sensi-tivity in A-fibers of knee joint-associated mechanoreceptive primary affer-ent neurones during MIA-induced osteoarthritis in the rat. Osteoarthr.Cartilage, 20, 305–313.

Kidd, B.L. (2006) Osteoarthritis and joint pain. Pain, 123, 6–9.Koewler, N.J., Freeman, K.T., Buus, R.J., Herrera, M.B., Jimenez-Andrade,J.M., Ghilardi, J.R., Peters, C.M., Sullivan, L.J., Kuskowski, M.A., Lewis,J.L. & Mantyh, P.W. (2007) Effects of a monoclonal antibody raisedagainst nerve growth factor on skeletal pain and bone healing after fractureof the C57BL/6J mouse femur. J. Bone Miner. Res., 22, 1732–1742.

Kosaras, B., Jakubowski, M., Kainz, V. & Burstein, R. (2009) Sensory inner-vation of the calvarial bones of the mouse. J. Comp. Neurol., 515,331–348.

Kruger, L., Silverman, J.D., Mantyh, P.W., Sternini, C. & Brecha, N.C.(1989) Peripheral patterns of calcitonin-gene-related peptide generalsomatic sensory innervation: cutaneous and deep terminations. J. Comp.Neurol., 280, 291–302.

Kryger, G.S., Kryger, Z., Zhang, F., Shelton, D.L., Lineaweaver, W.C. &Buncke, H.J. (2001) Nerve growth factor inhibition prevents traumaticneuroma formation in the rat. J. Hand Surg., 26, 635–644.

Kuniyoshi, K., Ohtori, S., Ochiai, N., Murata, R., Matsudo, T., Yamada, T.,Ochiai, S.S., Moriya, H. & Takahashi, K. (2007) Characteristics of sensoryDRG neurons innervating the wrist joint in rats. Eur. J. Pain, 11,323–328.

Kurejova, M., Nattenmuller, U., Hildebrandt, U., Selvaraj, D., Stosser, S. &Kuner, R. (2010) An improved behavioural assay demonstrates that ultra-sound vocalizations constitute a reliable indicator of chronic cancer painand neuropathic pain. Mol. Pain, 6, 18.

Kwan, K.Y., Glazer, J.M., Corey, D.P., Rice, F.L. & Stucky, C.L. (2009)TRPA1 modulates mechanotransduction in cutaneous sensory neurons.J. Neurosci., 29, 4808–4819.

Lam, D.K., Dang, D., Zhang, J., Dolan, J.C. & Schmidt, B.L. (2012) Novelanimal models of acute and chronic cancer pain: a pivotal role for PAR2.J. Neurosci., 32, 14178–14183.

Lane, N.E. & Silverman, S.L. (2010) Anabolic therapies. Curr. OsteoporosRep., 8, 23–27.

Lane, N.E., Schnitzer, T.J., Birbara, C.A., Mokhtarani, M., Shelton, D.L.,Smith, M.D. & Brown, M.T. (2010) Tanezumab for the treatment of painfrom osteoarthritis of the knee. New Engl. J. Med., 363, 1521–1531.

Latremoliere, A. & Woolf, C.J. (2009) Central sensitization: a generator ofpain hypersensitivity by central neural plasticity. J. Pain, 10, 895–926.

Levi-Montalcini, R. (1987) The nerve growth factor 35 years later. Science,237, 1154–1162.

Lewiecki, E.M. (2011) Sclerostin: a novel target for intervention in the treat-ment of osteoporosis. Discov. Med., 12, 263–273.

Lewin, G.R. & Mendell, L.M. (1993) Nerve growth factor and nociception.Trends Neurosci., 16, 353–359.

Li, X., Ominsky, M.S., Warmington, K.S., Morony, S., Gong, J., Cao, J.,Gao, Y., Shalhoub, V., Tipton, B., Haldankar, R., Chen, Q., Winters, A.,Boone, T., Geng, Z., Niu, Q.T., Ke, H.Z., Kostenuik, P.J., Simonet, W.S.,Lacey, D.L. & Paszty, C. (2009) Sclerostin antibody treatment increasesbone formation, bone mass, and bone strength in a rat model of postmeno-pausal osteoporosis. J. Bone Miner. Res., 24, 578–588.

Lim, V. & Clarke, B.L. (2012) New therapeutic targets for osteoporosis:beyond denosumab. Maturitas, 73, 269–272.

Liu, Y. & Ma, Q. (2011) Generation of somatic sensory neuron diversity andimplications on sensory coding. Curr. Opin. Neurobiol., 21, 52–60.

Longo, G., Osikowicz, M. & Ribeiro-da-Silva, A. (2013) Sympathetic fibersprouting in inflamed joints and adjacent skin contributes to pain-relatedbehavior in arthritis. J. Neurosci., 33, 10066–10074.

Lopes, C., Liu, Z., Xu, Y. & Ma, Q. (2012) Tlx3 and Runx1 act in combina-tion to coordinate the development of a cohort of nociceptors, thermocep-tors, and pruriceptors. J. Neurosci., 32, 9706–9715.

Lubeck, D.P. (2003) The costs of musculoskeletal disease: health needsassessment and health economics. Best Pract. Res. Cl. Rh., 17, 529–539.

Mach, D.B., Rogers, S.D., Sabino, M.C., Luger, N.M., Schwei, M.J., Pomo-nis, J.D., Keyser, C.P., Clohisy, D.R., O’Leary, P. & Mantyh, P.W. (2002)Origins of skeletal pain: sensory and sympathetic innervation of the mousefemur. Neuroscience, 113, 115–166.

Mahns, D.A., Ivanusic, J.J., Sahai, V. & Rowe, M.J. (2006) An intact periph-eral nerve preparation for monitoring the activity of single, periosteal affer-ent nerve fibres. J. Neurosci. Meth., 156, 140–144.

Mantyh, P. (2013) Bone cancer pain: causes, consequences, and therapeuticopportunities. Pain, 154S, S54–S62.

Mantyh, W.G., Jimenez-Andrade, J.M., Stake, J.I., Bloom, A.P.,Kaczmarska, M.J., Taylor, R.N., Freeman, K.T., Ghilardi, J.R., Kuskow-ski, M.A. & Mantyh, P.W. (2010) Blockade of nerve sprouting and neu-roma formation markedly attenuates the development of late stage cancerpain. Neuroscience, 171, 588–598.

Martin, C.D., Jimenez-Andrade, J.M., Ghilardi, J.R. & Mantyh, P.W. (2007)Organization of a unique net-like meshwork of CGRP+ sensory fibers inthe mouse periosteum: implications for the generation and maintenance ofbone fracture pain. Neurosci. Lett., 427, 148–152.

Mendell, L.M., Albers, K.M. & Davis, B.M. (1999) Neurotrophins, nocicep-tors, and pain. Microsc. Res. Techniq., 45, 252–261.

Modder, U.I., Hoey, K.A., Amin, S., McCready, L.K., Achenbach, S.J.,Riggs, B.L., Melton, L.J. 3rd. & Khosla, S. (2011) Relation of age, gen-der, and bone mass to circulating sclerostin levels in women and men. J.Bone Miner. Res., 26, 373–379.

Moester, M.J., Papapoulos, S.E., Lowik, C.W. & van Bezooijen, R.L. (2010)Sclerostin: current knowledge and future perspectives. Calcified TissueInt., 87, 99–107.

Nakajima, T., Ohtori, S., Yamamoto, S., Takahashi, K. & Harada, Y. (2008)Differences in innervation and innervated neurons between hip and ingui-nal skin. Clin. Orthop. Relat. R., 466, 2527–2532.

Nickel, J.C., Atkinson, G., Krieger, J.N., Mills, I.W., Pontari, M., Shoskes,D.A. & Crook, T.J. (2012) Preliminary assessment of safety and efficacyin proof-of-concept, randomized clinical trial of tanezumab for chronicprostatitis/chronic pelvic pain syndrome. Urology, 80, 1105–1110.

O’Connor, J.P., Capo, J.T., Tan, V., Cottrell, J.A., Manigrasso, M.B., Bontem-po, N. & Parsons, J.R. (2009) A comparison of the effects of ibuprofen androfecoxib on rabbit fibula osteotomy healing. Acta Orthop., 80, 597–605.

Ohlsson, C. (2013) Bone metabolism in 2012: novel osteoporosis targets.Nat. Rev. Endocrinol., 9, 72–74.

Ohtori, S., Inoue, G., Koshi, T., Ito, T., Yamashita, M., Yamauchi, K.,Suzuki, M., Doya, H., Moriya, H., Takahashi, Y. & Takahashi, K. (2007)Characteristics of sensory dorsal root ganglia neurons innervating the lum-bar vertebral body in rats. J. Pain, 8, 483–488.

Ohtori, S., Orita, S., Yamashita, M., Ishikawa, T., Ito, T., Shigemura, T.,Nishiyama, H., Konno, S., Ohta, H., Takaso, M., Inoue, G., Eguchi, Y.,Ochiai, N., Kishida, S., Kuniyoshi, K., Aoki, Y., Arai, G., Miyagi, M.,Kamoda, H., Suzkuki, M., Nakamura, J., Furuya, T., Kubota, G., Sakuma,Y., Oikawa, Y., Suzuki, M., Sasho, T., Nakagawa, K., Toyone, T. &Takahashi, K. (2012) Existence of a neuropathic pain component inpatients with osteoarthritis of the knee. Yonsei Med. J., 53, 801–805.


518 P. W. Mantyh

Ominsky, M.S., Vlasseros, F., Jolette, J., Smith, S.Y., Stouch, B., Doellgast,G., Gong, J., Gao, Y., Cao, J., Graham, K., Tipton, B., Cai, J., Deshpande,R., Zhou, L., Hale, M.D., Lightwood, D.J., Henry, A.J., Popplewell, A.G.,Moore, A.R., Robinson, M.K., Lacey, D.L., Simonet, W.S. & Paszty, C.(2010) Two doses of sclerostin antibody in cynomolgus monkeys increasesbone formation, bone mineral density, and bone strength. J. Bone Miner.Res., 25, 948–959.

Ozawa, T., Aoki, Y., Ohtori, S., Takahashi, K., Chiba, T., Ino, H. & Moriya,H. (2003) The dorsal portion of the lumbar intervertebral disc is innervatedprimarily by small peptide-containing dorsal root ganglion neurons in rats.Neurosci. Lett., 344, 65–67.

Ozawa, T., Ohtori, S., Inoue, G., Aoki, Y., Moriya, H. & Takahashi, K. (2006)The degenerated lumbar intervertebral disc is innervated primarily by pep-tide-containing sensory nerve fibers in humans. Spine, 31, 2418–2422.

Padhi, D., Jang, G., Stouch, B., Fang, L. & Posvar, E. (2011) Single-dose,placebo-controlled, randomized study of AMG 785, a sclerostin monoclo-nal antibody. J. Bone Miner. Res., 26, 19–26.

Palaniswamy, C., Selvaraj, D.R., Rao, V. & Patel, U. (2010) Newer therapiesfor osteoporosis. Am. J. Ther., 17, 197–200.

Pare, M., Albrecht, P.J., Noto, C.J., Bodkin, N.L., Pittenger, G.L., Schreyer,D.J., Tigno, X.T., Hansen, B.C. & Rice, F.L. (2007) Differential hypertro-phy and atrophy among all types of cutaneous innervation in the glabrousskin of the monkey hand during aging and naturally occurring type 2 dia-betes. J. Comp. Neurol., 501, 543–567.

Paszty, C., Turner, C.H. & Robinson, M.K. (2010) Sclerostin: a gem fromthe genome leads to bone-building antibodies. J. Bone Miner. Res., 25,1897–1904.

Patapoutian, A. & Reichardt, L.F. (2001) Trk receptors: mediators of neuro-trophin action. Curr. Opin. Neurobiol., 11, 272–280.

Peltonen, M., Lindroos, A.K. & Torgerson, J.S. (2003) Musculoskeletal pain inthe obese: a comparison with a general population and long-term changesafter conventional and surgical obesity treatment. Pain, 104, 549–557.

Pepper, A., Li, W., Kingery, W.S., Angst, M.S., Curtin, C.M. & Clark, J.D.(2013) Changes resembling complex regional pain syndrome followingsurgery and immobilization. J. Pain, 14, 516–524.

Pertovaara, A. (2013) The noradrenergic pain regulation system: a potentialtarget for pain therapy. Eur. J. Pharmacol., 716, 2–7.

Peters, E.M., Botchkarev, V.A., Muller-Rover, S., Moll, I., Rice, F.L. &Paus, R. (2002) Developmental timing of hair follicle and dorsal skininnervation in mice. J. Comp. Neurol., 448, 28–52.

Petruska, J.C. & Mendell, L.M. (2004) The many functions of nerve growthfactor: multiple actions on nociceptors. Neurosci. Lett., 361, 168–171.

Reid, I.R. (2012) Osteoporosis treatment at ASBMR 2012. IBMS BoneKEy,9, 245.

Ren, K. & Dubner, R. (2008) Neuron-glia crosstalk gets serious: role in painhypersensitivity. Curr. Opin. Anesthesio., 21, 570–579.

Robinson, D.A., Calejesan, A.A., Wei, F., Gebhart, G.F. & Zhuo, M. (2004)Endogenous facilitation: from molecular mechanisms to persistent pain.Curr. Neurovasc. Res., 1, 11–20.

Roosterman, D., Goerge, T., Schneider, S.W., Bunnett, N.W. & Steinhoff,M. (2006) Neuronal control of skin function: the skin as a neuroimmu-noendocrine organ. Physiol. Rev., 86, 1309–1379.

Rosenberg, J.M., Harrell, C., Ristic, H., Werner, R.A. & de Rosayro, A.M.(1997) The effect of gabapentin on neuropathic pain. Clin. J. Pain, 13,251–255.

Sabsovich, I., Wei, T., Guo, T.Z., Zhao, R., Shi, X., Li, X., Yeomans, D.C.,Klyukinov, M., Kingery, W.S. & Clark, J.D. (2008) Effect of anti-NGFantibodies in a rat tibia fracture model of complex regional pain syndrometype I. Pain, 138, 47–60.

Sakthiswary, R. & Das, S. (2013) The effects of TNFalpha antagonist ther-apy on bone metabolism in rheumatoid arthritis: a systematic review. Curr.Drug Targets, 14, 1552–1557.

Schaible, H.G. (2012) Mechanisms of chronic pain in osteoarthritis. Curr.Rheumatol. Rep., 14, 549–556.

Schulze, E., Witt, M., Fink, T., Hofer, A. & Funk, R.H. (1997) Immunohis-tochemical detection of human skin nerve fibers. Acta Histochem., 99,301–309.

Schweizerhof, M., Stosser, S., Kurejova, M., Njoo, C., Gangadharan, V.,Agarwal, N., Schmelz, M., Bali, K.K., Michalski, C.W., Brugger, S.,

Dickenson, A., Simone, D.A. & Kuner, R. (2009) Hematopoietic colony-stimulating factors mediate tumor-nerve interactions and bone cancer pain.Nat. Med., 15, 802–807.

Seeman, E. (2002) Pathogenesis of bone fragility in women and men. Lancet,359, 1841–1850.

Seike, W. (1976) Electrophysiological and histological studies on the sensibil-ity of the bone marrow nerve terminal. Yonago Acta Med., 20, 192–211.

Silverman, J.D. & Kruger, L. (1989) Calcitonin-gene-related-peptide-immu-noreactive innervation of the rat head with emphasis on specializedsensory structures. J. Comp. Neurol., 280, 303–330.

Skaper, S.D., Pollock, M. & Facci, L. (2001) Mast cells differentially expressand release active high molecular weight neurotrophins. Brain Res. Mol.Brain Res., 97, 177–185.

Smith, E.S. & Lewin, G.R. (2009) Nociceptors: a phylogenetic view. J.Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol., 195,1089–1106.

Stovitz, S.D., Pardee, P.E., Vazquez, G., Duval, S. & Schwimmer, J.B.(2008) Musculoskeletal pain in obese children and adolescents. ActaPaediatr., 97, 489–493.

Su, B. & O’Connor, J.P. (2013) NSAID therapy effects on healing of bone,tendon, and the enthesis. J. Appl. Physiol., 115, 892–899.

Suri, S., Gill, S.E., Massena de Camin, S., Wilson, D., McWilliams, D.F. &Walsh, D.A. (2007) Neurovascular invasion at the osteochondral junctionand in osteophytes in osteoarthritis. Ann. Rheum. Dis., 66, 1423–1428.

Takeuchi, Y. & Toda, K. (2003) Subtypes of nociceptive units in the rat tem-poromandibular joint. Brain Res. Bull., 61, 603–608.

Taylor, A.M., Peleshok, J.C. & Ribeiro-da-Silva, A. (2009) Distribution ofP2X(3)-immunoreactive fibers in hairy and glabrous skin of the rat. J.Comp. Neurol., 514, 555–566.

Vincent, L., Vang, D., Nguyen, J., Gupta, M., Luk, K., Ericson, M.E.,Simone, D.A. & Gupta, K. (2013) Mast cell activation contributes tosickle cell pathobiology and pain in mice. Blood, 122, 1853–1862.

Voscopoulos, C. & Lema, M. (2010) When does acute pain become chronic?Brit. J. Anaesth., 105(Suppl 1), i69–i85.

Wang, Q. & Seeman, E. (2008) Skeletal growth and peak bone strength. BestPract. Res. Cl. En., 22, 687–700.

Wetzel, C.H. & Connelly, J.F. (1997) Use of gabapentin in pain manage-ment. Annals Pharmacother., 31, 1082–1083.

Woolf, C.J. (1983) Evidence for a central component of post-injury painhypersensitivity. Nature, 306, 686–688.

Woolf, C.J. (2011) Central sensitization: implications for the diagnosis andtreatment of pain. Pain, 152, S2–S15.

Woolf, A.D. & Pfleger, B. (2003) Burden of major musculoskeletal condi-tions. B. World Health Organ., 81, 646–656.

Woolf, A.D., Erwin, J. & March, L. (2012) The need to address the burdenof musculoskeletal conditions. Best Pract. Res. Cl. Rh., 26, 183–224.

Woolf, C.J. & Salter, M.W. (2000) Neuronal plasticity: increasing the gainin pain. Science, 288, 1765–1769.

Woolf, C.J. & Wall, P.D. (1986) Relative effectiveness of C primary afferentfibers of different origins in evoking a prolonged facilitation of the flexorreflex in the rat. J. Neurosci., 6, 1433–1442.

Wu, Z., Nagata, K. & Iijima, T. (2002) Involvement of sensory nerves andimmune cells in osteophyte formation in the ankle joint of adjuvantarthritic rats. Histochem. Cell Biol., 118, 213–220.

Ye, Y., Dang, D., Zhang, J., Viet, C.T., Lam, D.K., Dolan, J.C., Gibbs, J.L.& Schmidt, B.L. (2011) Nerve growth factor links oral cancer progression,pain, and cachexia. Mol. Cancer Ther., 10, 1667–1676.

Yoshida, W., Seymour, B., Koltzenburg, M. & Dolan, R.J. (2013) Uncer-tainty increases pain: evidence for a novel mechanism of pain modulationinvolving the periaqueductal gray. J. Neurosci., 33, 5638–5646.

Zahn, P.K., Subieta, A., Park, S.S. & Brennan, T.J. (2004) Effect of block-ade of nerve growth factor and tumor necrosis factor on pain behaviorsafter plantar incision. J. Pain, 5, 157–163.

Zhang, X., Huang, J. & McNaughton, P.A. (2005) NGF rapidly increasesmembrane expression of TRPV1 heat-gated ion channels. EMBO J., 24,4211–4223.

Zylka, M.J., Rice, F.L. & Anderson, D.J. (2005) Topographically distinctepidermal nociceptive circuits revealed by axonal tracers targeted toMrgprd. Neuron, 45, 17–25.



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