J. Comp. Path. 2013, Vol. 149, 495e502 Available online at www.sciencedirect.com

www.elsevier.com/locate/jcpa

DISEASE IN WILDLIFE OR EXOTIC SPECIES

Characterization of Degenerative Changes in theTemporomandibular Joint of the Bengal Tiger

(Panthera tigris tigris) and SiberianTiger (Pantheratigris altaica)

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M. K. Murphy*, B. Arzi*,†, N. Vapniarsky-Arzi‡ and K. A. Athanasiou*,x

*Department of Biomedical Engineering, †Department of Surgical and Radiological Sciences, School of Veterinary Medicine,‡Department of Pathology, Microbiology and Immunology, William R. Pritchard Veterinary Medical Teaching Hospitaland

xDepartment of Orthopaedic Surgery, University of California Davis, Davis, CA, USA

resp

1-99

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Summary

The articulation of the temporomandibular joint (TMJ) is composed of the temporal bone dorsally, themandibular condyle ventrally and a fibrous articular disc. The TMJ disc plays an essential role in distributingload between the two articular surfaces. Degeneration of the disc in the presence of joint pathology has beenshown in man; however, TMJ pathology has not been documented previously in tigers (Panthera tigris). Themandibular condyle and TMJ disc of a Bengal tiger (P. tigris tigris) and a Siberian tiger (P. tigris altaica)were evaluated grossly and the TMJ disc was characterized biochemically andmechanically. Characterizationof the TMJ disc verified region- and direction-dependent biochemical and mechanical properties, reflective ofthe functional demands on the joint. Degenerative joint disease was observed in both cases and this was moresevere in the Siberian tiger. Simultaneous evaluation of joint pathology, biochemical composition andmechan-ical properties of the TMJ disc revealed a loss in functional properties (tensile anisotropy) of the disc as jointpathology advanced from moderate to severe. TMJ degeneration may compromise the ability of the animal toeat and thrive and may be a factor contributing to the endangered status of these species.

� 2013 Elsevier Ltd. All rights reserved.

Keywords: arthritis; biomechanical properties; degenerative joint disease; histopathology

Introduction

The temporomandibular joint (TMJ) is a bilateral di-arthrodial joint with an essential role in mastication,as well as in communication and survival. Its articu-lation is composed of the squamous temporal bonedorsally, the mandibular condyle ventrally and thearticular disc, which divides the joint space into dorsaland ventral components. The anatomy of the joint isvariable in mammals and is closely related to cranialmorphology, masticatory patterns and dietary re-quirements (Nanci, 2003). As such, there is a closerelationship between structure and function of thejoint. This relationship is demonstrated by region-

ondence to: K. A. Athanasiou (e-mail: athanasiou@ucdavis.edu).

75/$ - see front matter

x.doi.org/10.1016/j.jcpa.2013.05.003

and direction-dependent biochemical content andmechanical properties in the components of the joint(Detamore and Athanasiou, 2003; Allen andAthanasiou, 2006; Kalpakci et al., 2011).

The structure of the TMJ disc is generally consis-tent across mammalian species and is closely relatedto the function of the disc. The disc is composed of fi-brocartilaginous tissue that functions to distributeload between the dorsal and ventral bony joint com-ponents during joint motion. The disc is composedprimarily of type I collagen with minimal glycosami-noglycan (GAG) content and low cellularity (Nakanoand Scott, 1989; Detamore et al., 2005; Scapino et al.,2006). However, regional variability between speciesis related to the functional demands (Kalpakci et al.,2011). Independent of species, collagen fibres

� 2013 Elsevier Ltd. All rights reserved.

496 M.K. Murphy et al.

demonstrate rostrocaudal alignment in the central re-gion of the disc and ring-like organization around theperiphery of the disc (Detamore and Athanasiou,2003; Scapino et al., 2006; Arzi et al., 2012). Mechan-ically, the disc demonstrates superior tensile proper-ties in its central region, when tested rostrocaudally,compared with mediolaterally. The fibrocartilagi-nous disc of the TMJ plays a key role in distributingload throughout joint motion, which may be compro-mised in the presence of pathology.

The aetiology of TMJ pathology remains elusive;however, efforts have beenmade to characterize natu-rally occurring degenerative change in the TMJ ofman and companion animals (Dworkin et al., 1990).Up to 25% of the human population demonstratessymptoms of TMJ dysfunction (Solberg et al., 1979).Notably, 70% of human patients with TMJ disordersdemonstrate disc displacement and evidence of discdegeneration and over 50% demonstrate symptomsof osteoarthritis (Farrar and McCarty, 1979;Bertram et al., 2001). Histologically, displaced discshave been shown to have abnormal collagen fibrearrangement, with fragmentation of fibrils, increasedvasculature and cellular proliferation (Leonardi et al.,2007). Disorders of the TMJ have recently been char-acterized in domestic dogs and cats via computed to-mography (Arzi et al., 2013). In that study,osteoarthritis was found to be the most commonTMJ disorder in dogs and the second most commondisorder in cats. Simultaneous characterization ofthe gross and histological properties of the disc,together with its biochemical content and organiza-tion and mechanical properties, may elucidate func-tional changes occurring with pathology of theTMJ. As TMJ pathology remains poorly understood,an effort to characterize degenerative changes in thetiger population may aid in conservation efforts to-wards the endangered status of the species.

The TMJ disc has not been characterized previ-ously in an exclusively carnivorous mammal in healthor disease. Additionally, TMJ pathology has not yetbeen characterized in the tiger. The present report de-scribes degenerative TMJ disease in two ageing tigers(Panthera tigris altaica and P. tigris tigris).

Materials and Methods

Specimens

The TMJs were isolated from a 16-year-old neuteredfemale Bengal tiger (P. tigris tigris) and a 17-year-oldneutered male Siberian tiger (P. tigris altaica). Both ti-gers were humanely destroyed for reasons not relatedto TMJ arthritis. Each TMJ disc was dissected fromthe condylar process and temporal bone. Images

were taken of the temporal fossa, mandibular condyleand articular disc (Fig. 1). Samples were taken fromrepresentative lesions and normal regions of thecondylar process and stored in 10% neutral bufferedformalin. Bone samples were decalcified with 10%formic acid, prior to embedding in paraffin wax forsectioning.

Periodic irrigation with phosphate buffered saline(PBS) prevented the excised disc specimen from dehy-drating. One disc from each tiger was processed forhistology and biochemical analysis and for mechani-cal characterization. Immediately after dissection,samples for histology and biochemistry were collectedfrom the caudal (C), rostral (R), medial (M), lateral(L) and central (Cent.) regions (Fig. 2). The remain-ing disc tissue was wrapped in gauze soaked with PBScontaining protease inhibitors (10 mMN-ethylmalei-mide and 1 mM phenylmethylsulphonyl fluoride;Sigma, St Louis, Missouri, USA) and frozen at�20�C until mechanical testing.

Histology

Serial sections (5 mm)were stained with haematoxylinand eosin (HE), picrosirius red for collagen contentand organization, and safranin-O for GAG content.Sections stained with picrosirius red were additionallyimaged under polarized light to visualize collagen or-ganization.

Biochemistry

The mass of the samples was measured before and af-ter a 48 h period of lyophilization. Samples were di-gested in 125 mg/ml papain (Sigma) in phosphatebuffer (pH 6.5) containing 2 mM N-acetyl cysteine(Sigma) and 2 mM EDTA for 18 h at 60�C. Afterdigestion, no residual tissue remained. GAG contentwas measured with Blyscan GAG assay (Bicolor,Westbury, New York, USA), based on 1, 9-dimethyl-methyl blue binding. Total collagen was quantifiedafter hydrolyzing samples with 2 N NaOH for20 min at 110�C using a chloramine-T hydroxypro-line assay with Sircol� collagen standards (Bicolor).Cellularity was determined using theQuant-iT Picro-green dsDNA Assay Kit (Invitrogen, Carlsbad, Cali-fornia, USA).

Mechanical Testing

Tensile testing was performed in the intermediatezone of the discs in the rostrocaudal and mediolateraldirections (Fig. 2b). Specimens were tested accordingto the American Society for Testing and Materials(ASTM) D3039 standard test method (ASTM,2008). Uniform specimens were collected using a

Fig. 1. TMJ gross morphology. Mandibular condyle, mandibular fossa and articular disc from the right TMJ of a Bengal and a Siberiantiger. Arrows indicate regions of degeneration. Bar, 1 cm.

Temporomandibular Joint Degeneration in Tigers 497

rectangular cutter with a width of 2.0 mm and alength of 8.0 mm representing a length to width ratioof 4:1. The initial gauge length was considered to bethe grip-to-grip length, set to 4.0 mm, representinga gauge length to gauge width of 2:1. Tensile testingwas conducted using an Instron 5565 (Instron, Nor-wood,Massachusetts, USA). Samples were elongatedat a rate of 1% strain per second (based on the initialgauge/grip-to-grip length of 4.0 mm). Stressestraincurves were generated from the load-displacementdata. The load, elongation and specimen geometrydata were loaded into Matlab (MathWorks, Natick,Massachusetts, USA) and analyzed with a customprogram that plots load normalized to cross-sectional area and strain. User inputs identified thelinear portion of the curve to determine Young’smodulus (EY); ultimate tensile strength (UTS) isthe maximum stress experienced by the tissue.

Fig. 2. Tiger TMJ disc schematic. The TMJ disc was processed forbiochemical, histological and mechanical analysis. (a)Biochemical and histological specimens obtained in fivedisc regions: caudal (C), rostral (R), lateral (L), medial(M) and central (Cent.). (b) Tensile testing was performedthrough the central region in two axes: mediolateral(MeL) and rostrocaudal (ReC).

Results

Gross Morphology and Histology

The TMJs of both tigers were confirmed to havemod-erate to severe degenerative joint disease based ongross and histopathological observations of themandibular condyles and gross observations of themandibular fossa of the squamous temporal bones(Fig. 1). Grossly, areas of cartilage ulceration anderosion, as well as subchondral bone exposure, werenoted in several places. Histologically, the fibrocarti-lage that covers the articulating surfaces of the TMJwas uneven and the osteochondral border was ill-defined (Fig. 3). The subchondral bone containedmultiple cavities filled with dense to loose fibrous tis-sue. The subchondral bone cavities had irregularmargins and occasionally contained osteoclasts withinresorption lacunae. In addition, the subchondralbone was dense, with minimal trabecular bone indi-cating subchondral bone sclerosis. The surface of thearticular zone was ragged and fibrillated. In all re-gions of the TMJ discs, polarized light microscopydemonstrated reduced collagen birefringence in P. ti-

gris altaica compared with P. tigris tigris (Fig. 4).In the rostral aspect of the disc in both tigers most

collagen fibres stained bright pink and about 30%stained pale pink. In the bright pink areas, thecollagen fibres showed parallel arrangement withareas of delicate waviness. In the areas of palecollagen, the fibrillar structure was ill-defined andthe direction of the bundles was irregular. Multifo-cally embedded within the pale areas were clusters

Fig. 3. Histological characterization of mandibular condyle. Histological analysis of themandibular condyle was performed from a repre-sentative lesion and a grossly normal region for each tiger. Both tigers demonstrate regions of subchondral bone pathology and ill-defined osteochondral borders. HE. Bar, 50 mm.

498 M.K. Murphy et al.

of two to five chondrocytes. These areas containedmultiple clefts and were frayed. In the P. tigris altaicasample, there were multiple small-calibre arteries andlarger-calibre congested veins rimmed by thin cuffs ofpale and hypercellular collagen.

The caudal sections of the discs had linear areas ofpale collagen dissected through the centre of the sec-tion by bright pink collagen. Thin streams of palecollagen branched off the main stem and dissectedirregularly throughout the areas of bright pinkcollagen. In the P. tigris altaica sample, there were

Fig. 4. Histological characterization of TMJ disc. As evidenced by Hscopy, the collagen organization was significantly reduced in t

additional pale collagen bundles and small aggregatesof one to three chondrocytes (chondroid change) andmultiple clefts between the collagen bundles.

In the central zone, there were linear areas of palecollagen alternating with areas of bright pink collagen.The fibrillar structure of the pale areas was ill-definedand cellularity was markedly higher in comparisonwith bright pink areas. In the P. tigris altaica specimen,the fibres were separated from each other by clearlinear clefts. The separated fibres were arrangedhaphazardly and occasionally curled. Adjacent to this

E staining and picrosirius red staining with polarized light micro-he specimen from the Siberian tiger. Bar, 100 mm.

Temporomandibular Joint Degeneration in Tigers 499

area there were regions of increased cellularity, slightlylighter collagen staining and few slit-like clear spaces.

The fibres in the lateral regions of the discs wereuniformly bright pink with minimal cellularity. Thefibres running parallel to each other formed thickbundles that regularly and orthogonally intersectedother similarly arranged bundles. In the P. tigris alta-ica sample, the fibres contained alternating pale andbright pink collagen bundles. The cellularity of thepale areas was generally higher and fibre arrange-ment was poorly defined. One pale area containedmyxomatous matrix that was cavitated by up to 10round empty lacunae (i.e. myxomatous change ratherthan chondroid change).

The medial aspects of the discs contained mostlypale collagen fibres alternating with areas of brightpink collagen fibres. The fibrillar structure was notapparent in the pale fibres and their arrangementwas haphazard with occasional whirling and orthog-onal intersection. In the P. tigris altaica specimen,there were multiple small-calibre arteries andlarger-calibre congested veins.

Picrosirius red and safranin-O staining confirmedthe disc to be fibrocartilaginous, showing intensecollagen staining and minimal GAG staining acrossregions (data not shown). Both tigers demonstrateddegeneration grossly and histologically in the bonyjoint components and the articular disc. Histological-ly, degeneration was more advanced in the TMJ ofthe P. tigris altaica (Fig. 4).

Dental Disease

Both tigers exhibited severe lesions in the dentitionand dental fractures with pulp exposure (Fig. 5).The specimen from P. tigris tigris demonstrated acrown fracture of the right maxillary canine toothand the specimen from P. tigris altaica exhibited severeabrasion at the maxillary and mandibular canine

Fig. 5. Lesions in the dentition of the Bengal tiger and Siberiantiger. (a) The Bengal tiger demonstrated a complicatedcrown fracture of the right maxillary canine tooth withperiapical lesion and fenestration through the mucosa.(b) The Siberian tiger demonstrated severe abrasion ofthe maxillary (white arrows) and mandibular (black ar-rows) canine teeth with pulp exposure. The dental lesionswere indicative of chronic abnormal chewing behaviour.

teeth with pulp exposure. Although complete perti-nent history with regards to chewing behaviour wasnot available, the dental lesions found were indicativeof severe, chronic paranormal chewing behaviour.

Mechanical Properties

The mechanical properties of the TMJ discs werecharacterized (Fig. 6). The sample from P. tigris tigris

demonstrated greater tensile stiffness and strength inthe rostrocaudal direction compared with the medio-lateral direction. The disc demonstrated stiffness of29.50 MPa and strength of 9.77 MPa rostrocaudallyand stiffness of 10.64 MPa and strength of 3.22 MPamediolaterally. The specimen from P. tigris altaica

did not demonstrate superior tensile properties inthe rostrocaudal direction, compared with the medio-lateral direction. This disc demonstrated stiffness of7.48 MPa and strength of 2.93 MPa rostrocaudallyand stiffness of 11.97 MPa and strength of 4.02 MPamediolaterally. Thus, the disc demonstrated aniso-tropic tensile properties in P. tigris tigris, whereas ten-sile anisotropy was not present in P. tigris altaica

Biochemical Content

The biochemical content varied between regions ofthe disc (Table 1). Hydration varied regionally andwas greatest in both tigers in the medial and lateral

Fig. 6. Tensile properties of the TMJ disc. Biomechanical proper-ties of the TMJ discs were tested in uniaxial tension, rostro-caudally and mediolaterally (Fig. 1). The specimen fromthe Bengal tiger demonstrated tensile anisotropy whilethat from the Siberian tiger demonstrated a loss of anisot-ropy in mechanical properties.

Table 1

Biochemical properties of the tiger TMJ disc

Region Hydration (%) DNA/DW (%) sGAG/DW (%) Collagen/DW (%)

Bengal tiger Central 72.21 0.027 0.99 62.67

Caudal 69.91 0.088 0.59 72.70Rostral 75.73 0.092 0.45 68.29

Lateral 76.38 0.042 1.07 67.24

Medial 79.92 0.091 0.35 71.26

Siberian tiger Central 68.98 0.043 0.96 71.36Caudal 73.16 0.053 1.42 61.00

Rostral 72.71 0.091 0.37 69.27

Lateral 77.65 0.090 0.82 70.10Medial 73.50 0.049 0.82 63.50

DNA, sGAG and collagen content reported per tissue dry weight.

500 M.K. Murphy et al.

regions. Water content ranged from 68.98 to 79.92%.The discs generally demonstrated low cellularity and<0.1% DNA by dry weight. Cellularity was greatestin samples from both tigers taken from the rostral re-gion. The sulphated GAG (sGAG) content was alsovery low in the TMJ discs, on average <1% by dryweight. The discs were composed primarily ofcollagen with collagen content ranging from 61.27to 71.26%by dry weight. Collagen content was great-est in the specimen from P. tigris tigris in the caudal re-gion, while in the sample from P. tigris altaica it waslowest in the caudal region. Biochemical content var-ied regionally and overall demonstrated a fibrousarticular disc with low cellularity.

Discussion

The aetiology of TMJ disease remains poorly under-stood. Efforts have been made to characterize thestructure and function of the TMJ disc across species(Detamore and Athanasiou, 2003; Detamore et al.,2005; Allen and Athanasiou, 2006; Singh andDetamore, 2008, 2009; Kalpakci et al., 2011); howev-er, the disc has not been characterized previously incarnivorous animals. In the present study, the TMJwas characterized histologically, biochemically andmechanically in two subspecies of tiger. Both tigersdemonstrated degenerative joint disease in both thebony and soft tissue joint components. The discs re-flected marked change in matrix structure, as wellas reduced mechanical functionality. In the case of se-vere degeneration, present in the specimen from theSiberian tiger, histological, biochemical and mechan-ical properties reflected a loss in the functionality ofthe TMJ disc. With all six subspecies of tiger thatremain in the wild being endangered, elucidatingthe pathology of TMJ disease may aid in conservationefforts and may be translatable to human health.

The essential functions of the TMJ may becomecompromised in the presence of degenerative joint

disease. The function of this joint is closely relatedto dietary requirements and cranial morphology(Nanci, 2003). The tiger has a dorsally convexmandibular condyle that fits into the ventrallyconcave mandibular fossa of the squamous bone.This morphology limits the joint to the exclusivelyhinge motion necessary for opening and closing themouth (Perrin et al., 2002). A similar morphologyhas been previously described in domestic cats. Thedomestic feline TMJ morphology is restrictive, suchthat independent movement of the mandible is mini-mal, with the exception of hinge movements (Caporn,1995; Lantz andVerstraete, 2012). However, this mo-tion may be compromised in the presence of jointdegeneration. It was hypothesized that the tigerTMJ disc would reflect rostrocaudal fibre alignment,supporting the distribution of load through hingingmotion. Furthermore, it was hypothesized that suchfibre alignment would be associated with increasedtensile properties in the rostrocaudal direction,compared with the mediolateral direction, reflectingmechanical anisotropy. The anticipated rostrocaudalfibre alignment and mechanical anisotropy wasconfirmed in the specimen from the Bengal tiger.However, moderate degenerative joint disease wasconfirmed grossly and histologically. In contrast, thespecimen from the Siberian tiger demonstratedmore advanced degenerative joint disease, confirmedgrossly and histologically. In this case, compromise infibre organization and structure and complete loss ofmechanical anisotropy was observed. Degenerativejoint disease may compromise essential functions ofthe TMJ, which normally facilitates the hinging mo-tion of the mandible employed by carnivores.

Severe dental abrasions and tooth fractures,observed in both cases presented here, may be indic-ative of abnormal chewing behaviour. Additionally,the dental lesions were observed concomitantlywith TMJ degeneration. An association betweenabnormal chewing habits and TMJ disease symptoms

Temporomandibular Joint Degeneration in Tigers 501

has been demonstrated in man (Alamoudi, 2001;Magnusson et al., 2005). Excessive or sustained phys-ical stress may exceed the normal adaptive capacity ofTMJ articular structures (Arnett et al., 1996; Tanakaet al., 2008;Machon et al., 2011). Such behaviour mayresult in initiation and progression of TMJ degenera-tive joint disease.

Grossly and histologically, the TMJ discs of both ti-gers demonstrated evidence of degenerative changeand compromise of fibre organization. Both discsdemonstrated regions of haphazard fibre arrange-ment and the appearance of fibre separation. Thespecimen from the Siberian tiger demonstratedmore severe disc degeneration. Fibres were loosely ar-ranged and separated by clear linear clefts. The pooralignment in the regions of this specimen was furtherconfirmed by polarized light microscopy. Histologi-cally, the disc showed increased cellularity whencompared with that from the Bengal tiger andbiochemical analysis confirmed this in the centraland lateral regions. Additionally, the specimen fromthe Bengal tiger demonstrated the presence of small-calibre arteries and large-calibre congested veins inthe rostral and lateral regions, which may be indica-tive of degeneration (Leonardi et al., 2010). Concern-ing degenerative joint disease, the earliest changes inosteoarthritic cartilage are characterized by a diffuseincrease in cellularity, depletion of proteoglycan con-tent (Collins, 1951; Bollet et al., 1963) and loss of fibreorganization (Leonardi et al., 2007). With regard todisc degeneration in man, fibrosis, fibrillation, hyali-nization and perforation have been reported(Wilkes, 1989). Advanced degeneration in the centralregion of the specimen from the Siberian tiger was re-flected biochemically and histologically.

In parallel with the compromise in structure andfunction of the TMJ discs, the bony joint componentsof both tigers demonstrated gross and histologicaldegeneration. Grossly, both the fossa and mandibularcartilage showed areas of ulceration and erosion. His-tologically, the fibrocartilage of the mandibular con-dyles was uneven and eroded in some regions. Thesubchondral bone reflected degeneration with multi-ple cavities. While the aetiology is not understood,degeneration in the bony joint components occurredtogether with degeneration of the TMJ disc. Degener-ation of the TMJ disc was parallelled by articularcartilage and subchondral bone degeneration in thedorsal and ventral bony joint components of bothspecimens.

Mechanical analysis of disc functionality demon-strated compromise of the anisotropic tensile proper-ties when severe joint disease was present. Basedon literature findings across several species and thefunctional demands of tiger masticatory patterns,

mechanical anisotropy was anticipated (Detamoreand Athanasiou, 2003; Kalpakci et al., 2011). Asanticipated, the specimen from the Bengal tigerdemonstrated mechanical anisotropy in the centralregion of the disc; the tensile properties in the rostro-caudal direction were greater than that in the medio-lateral direction. However, this was not observed inthe specimen from the Siberian tiger, which alsoshowed more severe degeneration histologically.The tensile properties of the specimen from the Bengaltiger were similar in magnitude in the two directionstested. Most notably, comparing the rostrocaudaltensile properties of the two tigers, the specimenfrom the Bengal tiger showed 75% less stiffness and70% less strength. Mediolaterally, there was only a10% difference in stiffness. This discrepancy in me-chanical properties parallels the previously describedage-related decrease in tensile properties of articularcartilage, which peak in the third decade(Kempson, 1982). This mechanical compromise hasbeen related to organizational changes in the collagennetwork, including the fibrils becoming more widelyspaced, as observed histologically here, and a decreasein collagen cross linking (not quantified here). Thus,the results of the present study suggest that in the pres-ence of severe degeneration, mechanical anisotropy ofthe TMJ disc is compromised.

A structureefunction relationship was demon-strated in the tiger TMJ disc that was compromisedin the presence of severe degenerative joint disease.Regional variations were observed in biochemicalanalysis thatmay correlate with functional loading re-quirements. Pathology was indicated by the loss offibrillar structure of the disc, ultimately compromisingits mechanical functionality. With all six of the re-maining tiger subspecies listed as endangered, pathol-ogy of the TMJ may play an important role in thesurvival of these animals. Further investigation of theTMJ in healthy carnivorous animals is needed toelucidate regional variations and structureefunctionrelationships associated with normal joint motion.

Acknowledgements

The authors thankDr.M.Mok andDr. S. Kubiski forproviding gross dental images. This material is basedon work supported by the National Science Founda-tion Graduate Research Fellowship under GrantNo. DGE-1148897. Additionally, this work is sup-ported by grants R01DE015038 and R01DE019666from the National Institutes of Health.

Conflict of Interest Statement

The authors have no conflicts of interest to disclose.

502 M.K. Murphy et al.

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½ R

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eceived, February 9th, 2013

ccepted, May 8th, 2013

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