THE ANATOMICAL RECORD 293:642–650 (2010)

Masticatory Loading, Function, andPlasticity: A Microanatomical Analysis ofMammalian Circumorbital Soft-Tissue

StructuresELDIN JASAREVIC, JIE NING, ASHLEY N. DANIEL, RACHEL A. MENEGAZ,JEFFREY J. JOHNSON, M. SHARON STACK, AND MATTHEW J. RAVOSA*

Department of Pathology and Anatomical Sciences, University of Missouri School ofMedicine, Columbia, Missouri

ABSTRACTIn contrast to experimental evidence regarding the postorbital bar,

postorbital septum, and browridge, there is exceedingly little evidenceregarding the load-bearing nature of soft-tissue structures of the mamma-lian circumorbital region. This hinders our understanding of pronouncedtransformations during primate origins, in which euprimates evolved apostorbital bar from an ancestor with the primitive mammalian conditionwhere only soft tissues spanned the lateral orbital margin between fron-tal bone and zygomatic arch. To address this significant gap, we investi-gated the postorbital microanatomy of rabbits subjected to long-termvariation in diet-induced masticatory stresses. Rabbits exhibit a mastica-tory complex and feeding behaviors similar to primates, yet retain a moreprimitive mammalian circumorbital region. Three cohorts were obtainedas weanlings and raised on different diets until adult. Following euthana-sia, postorbital soft tissues were dissected away, fixed, and decalcified.These soft tissues were divided into inferior, intermediate, and superiorunits and then dehydrated, embedded, and sectioned. H&E staining wasused to characterize overall architecture. Collagen orientation and com-plexity were evaluated via picrosirius-red staining. Safranin-O identifiedproteoglycan content with additional immunostaining performed to assessType-II collagen expression. Surprisingly, the ligament along the lateralorbital wall was composed of elastic fibrocartilage. A more degraded orga-nization of collagen fibers in this postorbital fibrocartilage is correlatedwith increased masticatory forces due to a more fracture-resistant diet.Furthermore, the lack of marked changes in the extracellular compositionof the lateral orbital wall related to tissue viscoelasticity suggests it isunlikely that long-term exposure to elevated masticatory stresses under-lies the development of a bony postorbital bar. Anat Rec, 293:642–650,2010. VVC 2010 Wiley-Liss, Inc.

Keywords: circumorbital morphology; histology; immuno-histochemistry; adaptive plasticity; ontogeny;masticatory stresses/loads; dietary properties; rabbit

Grant sponsor: NSF and Department of Pathology andAnatomical Sciences (School of Medicine, University ofMissouri); Grant number: BCS-0924592.

*Correspondence to: Matthew J. Ravosa, Department of Pa-thology and Anatomical Sciences, University of Missouri Schoolof Medicine, M303 Medical Sciences Building, 1 Hospital Drive

DC055.07, Columbia, MO 65212. Fax: 573-884-4612.E-mail: ravosam@missouri.edu

Received 7 January 2010; Accepted 11 January 2010

DOI 10.1002/ar.21135Published online in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

The mammalian circumorbital region is characterizedby considerable morphological variation. The primitivecondition for mammals is to possess a ligament runningalong the lateral aspect of the orbit between the frontalbone and zygomatic arch. In primates, all taxa exhibit apostorbital bar with haplorhines exhibiting the derivedcondition of postorbital septum. Although a postorbitalbar has evolved in multiple clades (e.g., primates, her-pestids, felids, pteropodids, and artiodactyls), the postor-bital septum is unique among mammals (Rosenberger,1986; Cartmill, 1970, 1972, 1980, 1992; Ross, 1995; Rossand Hylander, 1996; Ravosa et al., 2000a,b, 2006; Mene-gaz and Kirk, 2009).

Generally, there are two types of functional models forthe development of bony circumorbital features. The firstproposes that bony circumorbital structures provideinsulation to the lateral orbital wall or postorbitalregion, an explanation derived from the Visual PredationHypothesis (VPH). The VPH posits that the evolution ofa postorbital bar in basal primates with large, conver-gent, and frontated orbits acts to stabilize the postorbitalmargins against ocular distortions that may occur dur-ing routine chewing or biting (Cartmill, 1970, 1972,1974, 1992; Ravosa et al., 2000b, 2006). A similar func-tional argument has been extended to the haplorhinepostorbital septum, where orbital closure is posited toinsulate the orbital contents from the contractions of theanterior temporalis muscle (Cartmill, 1980; Ross, 1995).

Alternatively, others have argued that bony circumor-bital structures are designed to resist masticatorystresses (e.g., Greaves, 1985; Rosenberger, 1986; Tatter-sall, 1995; Wolpoff, 1996; Bookstein et al., 1999; Cox,2008). For instance, Greaves (1985, 1991, 1995) proposedthat the mammalian postorbital bar functions to resisttorsion of the facial skull relative to the braincase dur-ing unilateral mastication. According to this model, tor-sion about the long axis of the skull results in thecompression of the working-side postorbital bar and ten-sion along the balancing-side postorbital bar. A similarargument has been posited regarding the function of thehaplorhine postorbital septum (Rosenberger, 1986).

Subsequent studies have used strain gauges to mea-sure in vivo stress and strain distribution in various cra-niofacial regions during normal chewing and biting.Work in the two major groups of living primates, haplor-hines, and strepsirhines demonstrates that the primatecircumorbital region exhibits much lower peak strainsduring biting and chewing than along the mandible andmidface (Hylander et al., 1991a,b; Ross and Hylander,1996; Ravosa et al., 2000a,b,c, 2006). Thus, in contrastto the inferior craniofacial region, there is no evidencethat the development of bony circumorbital structuressuch as the browridge, postorbital bar, and postorbitalseptum are adaptations to resist masticatory loads dur-ing routine feeding behaviors.

Recent experimental research in pigs, which retainthe primitive mammalian condition of a postorbital liga-ment, provides additional insight into the function of thecircumorbital region (Lemme et al., 2005). Using trans-ducers implanted along the lateral orbital wall, the post-orbital ligament in pigs was found to exhibit averageelongations of about 1% on both the working and balanc-ing sides during alert mastication. This elongationappears differentially related to the activity of the work-ing-side masseter muscle (Lemme et al., 2005). As a 5%

elongation results in the failure of tendon, which isanother highly collagenous connective tissue but lesselastic than ligament (cf., Wainwright et al., 1976), it ispossible that the postorbital ligament exhibits safetyfactors comparable to some masticatory elements (cf.,Hylander et al., 1991a,b; Hylander and Johnson, 1997;Ravosa et al., 2000a,b,c, 2010a).

This novel information helps to frame an outstandingissue regarding the evolution of the mammalian circum-orbital region. Currently, it is unknown if the transfor-mation from the primate ancestral soft-tissue conditionto a bony postorbital bar in basal primates is related tovariation in masticatory stresses, perhaps of a dietarynature, or other unrelated factors (Ravosa et al.,2000a,b,c, 2006). The absence of such information hin-ders a complete understanding of morphological varia-tion characterizing primate and mammalian phylogeny.As described earlier, the experimental data for primateswith a postorbital bar and a postorbital septum indicatethat cranial changes during the evolution of the haplor-hine postorbital septum are unrelated to shifts in diet-related masticatory stresses. On the other hand, prelimi-nary analyses of circumorbital soft tissues in pigs do notnegate the possibility that the postorbital ligament mayexperience significant loads, with the evolutionary trans-formation (versus current function) of the postorbital barpotentially related to variation in masticatory stresses.

To fill this significant gap in our understanding of theevolutionary morphology of the mammalian circumorbi-tal region, this experimental study examines the long-term effects of diet-induced variation in mechanical load-ing during mastication on the microanatomy of postorbi-tal soft tissues in growing rabbits. The New Zealanddomestic white rabbit (Oryctolagus cuniculus) is a par-ticularly unique experimental model, in that it exhibitsthe primitive mammalian condition, where the lateralorbital wall consists of soft tissues and a number of im-portant similarities in the form and function of the mas-ticatory apparatus shared with primates. For instance,considerable in vivo data exist for rabbits regarding jaw-adductor muscle activity, jaw-kinematic and jaw-loadingpatterns, masticatory function during ontogeny, intra-cortical remodeling, and diet-related feeding behaviors(Weijs and de Jongh, 1977; Weijs and Dantuma, 1981;Weijs et al., 1987, 1989; Langenbach et al., 1991, 1992,2001; Hirano et al., 2000; Langenbach and van Eijden,2001; Taylor et al., 2006; Menegaz et al., 2009, 2010;Ravosa et al., 2007, 2008a,b, 2010b). Similar to a num-ber of mammalian taxa including primates, rabbits dem-onstrate postnatal plasticity of masticatory soft and hardtissues in response to diet-induced variation in loadingpatterns (Beecher and Corruccini, 1981; Bouvier andHylander, 1981, 1982, 1984, 1996a,b; Beecher et al.,1983; Kiliaridis et al., 1985; Bouvier, 1987, 1988;Yamada and Kimmel, 1991; Ravosa et al., 2007, 2008a,b;Menegaz et al., 2009, 2010). Like anthropoids, rabbitshave a deep face and a high jaw joint capable of bothrotation and translation (Weijs and Dantuma, 1981;Crompton et al., 2006). Furthermore, both anthropoidsand rabbits show delayed activity of the balancing-sidedeep masseter muscle underlying transverse mandibularmovements during unilateral mastication (Hylanderet al., 1987, 2000; Weijs et al., 1989).

Although functional changes due to masticatory load-ing are documented for bony elements in the craniofacial

CIRCUMORBITAL LOADING, FUNCTION, AND PLASTICITY 643

skeleton (e.g., mandible; Ravosa et al., 2007), little isknown regarding the plasticity responses of postorbitalsoft tissues to variation in masticatory stresses.Although dietary shifts have not been posited for pri-mate origins (cf., Cartmill, 1992), the derivation of apostorbital bar and postorbital septum led us to test fora circumorbital response to variation in loading condi-tions. Clarifying the long term, natural influence ofaltered masticatory stresses on postorbital soft tissues isfundamental for interpreting the behavioral and ecologi-cal correlates of morphological variation in extant andextinct mammals as well as for understanding the bio-mechanics and function of routinely loaded cranialstructures.

Here, we extend what is currently known about hardtissue biomechanics in order to test the hypothesis thatvariation in masticatory stress produced by differencesin dietary properties is correlated with variation in post-orbital soft-tissue morphology. In the facial skull andfeeding apparatus of the same animal model, increasedmasticatory loading has been observed to result in largerjaw-joint proportions, greater cortical bone thickness,and elevated hard-tissue biomineralization (Ravosaet al., 2007, 2008a,b; Menegaz et al., 2009, 2010). If mas-ticatory stress plays a role in the growth and form of thecircumorbital region, similar adaptive responses shouldbe observed in the soft-tissue structures of the lateral or-bital wall. Thus, plasticity studies of this kind can beused to inform our notions regarding function, biome-chanics, and adaptation (sensu Gotthard and Nylin,1995). On the one hand, differences in postorbital soft-tissue structures between dietary loading cohorts wouldsuggest that this circumorbital structure is influencedby variation in chewing stresses, thus contrasting withdata for bony elements of the circumorbital region(Hylander et al., 1991a,b; Ross and Hylander, 1996;Ravosa et al., 2000a,b,c, 2006). On the other hand, alack of differences in postorbital soft-tissue structuresbetween dietary loading cohorts would contrast with dif-ferences observed for masticatory structures (Ravosaet al., 2007; Menegaz et al., 2009, 2010), and insteadsuggest that postorbital soft tissues are unaffected byvariation in chewing stresses much like bony circumorbi-tal structures (Ravosa et al., 2000c).

MATERIALS AND METHODSSample

The sample consisted of 30 genetically similar NewZealand domestic white rabbits (O. cuniculus) that were

obtained as weanlings (4 weeks old) and raised for 26weeks until attaining adult status at 30 weeks old(Sorensen et al., 1968; Yardin, 1974). In accordance withan ACUC-approved protocol, three dietary cohorts of 10rabbits each were established to induce postweaningvariation in jaw-muscle forces and masticatory loads(cf., Ravosa et al., 2007, 2010b; Menegaz et al., 2009,2010). Weaning was chosen as the starting point for die-tary manipulation because plasticity decreases with age(Hinton and McNamara, 1984; Meyer, 1987; Bouvier,1988; Rubin et al., 1992; Ravosa et al., 2008b), becausethis approximates shifts in masticatory function in thewild (cf., Ravosa et al., 2007, 2008a,b), and because itminimizes the confounding influence of diets other thanthose used in our protocol.

Cohorts were separated into an ‘‘under-use’’ cohort fedonly powdered pellets (n ¼ 10), a ‘‘control’’ cohort fedintact Harlan TekLad rabbit pellets (n ¼ 10), and an‘‘over-use’’ cohort fed whole rabbit pellets supplementeddaily with two 2.5-cm cube hay blocks (n ¼ 10). Table 1summarizes the dietary mechanical properties of the ex-perimental food. In rabbits and other mammals, frac-ture-resistant foods such as pellets and hay requireabsolutely larger jaw-muscle activity and greater cyclicalloading during food processing, which in turn increasesmasticatory stresses (Weijs and de Jongh, 1977; Bouvierand Hylander, 1981; Weijs and Dantuma, 1981; Weijset al., 1987, 1989; Hylander et al., 1992, 1998, 2000;Ravosa et al., 2007, 2008a,b). The inclusion of pellets inthe diet of all rabbits facilitated adequate nutrition fornormal postweaning growth.

Histology and Immunohistochemistry

Once euthanized at 30 weeks, rabbit crania were fixedin 10% buffered formalin. The eyes were enucleated byremoving the superficial connective tissues then trans-ecting the optic nerve. The postorbital ligament spansthe bony gap along the lateral orbital margin betweenthe temporal process of the frontal bone and the frontalprocess of the temporal (squamosal) bone (see Fig. 1).Once enucleated, surrounding connective tissues werefurther removed to expose lateral orbital wall tissues.Only right-sided postorbital soft tissues were dissectedaway for histology. These were fixed in 10% buffered for-malin for 48 hr, followed by a 24-hr decalcification periodusing Cal-Ex II (Fisher Scientific). Decalcified soft tis-sues from each specimen were segmented into inferior,intermediate, and superior sections (see Fig. 1) and thendehydrated and paraffin embedded at the University of

TABLE 1. Dietary mechanical properties of rabbit experimental foods measured with a portable foodtester

Food items(sample size)

Young’s modulus:E in MPa (mean, range)

Toughness:R in Jm�2 (mean, range)

Hardness:H in MPa (mean, range)

Pellets (n ¼ 10) 29.2 (17.0–41.0) – 11.8 (6.3–19.9)Wet hay (n ¼ 15) 277.8 (124.9–451.0) 1759.2 (643.6–3251.9) –Dry hay (n ¼ 15) 3335.6 (1476.8–6711.4) 2759.8 (434.0–6625.5) –

Ground pellets require minimal oral preparation, which reduces the amount of cyclical loading during unilateral mastica-tion (under-use). The control diet consisted of intact pellets. Hay requires greater forces to process, which, in addition tothe greater processing required for intact pellets, results in increased peak loads and increased cyclical loading duringmolar biting and chewing (over-use). The mechanical properties of wet hay model the exposure of hay to saliva. Missingvalues are due to the inability to perform that particular test on the item (due to its size, shape, or properties).

644 JASAREVIC ET AL.

Missouri Veterinary Medical Diagnostics Laboratory(VMDL). Data for superior and inferior regions are notpresented here, because the inferior and superior tissuesegments were largely uniform in microanatomy regard-less of loading regime. Thus, only data for the intermedi-ate region of the postorbital soft tissues are presentedand discussed herein.

To provide a comparison between postorbital soft tis-sues and a cranial structure known to be influenced bymasticatory stresses (Ravosa et al., 2007), the left-sidedtemporomandibular joint (TMJ) from each of the abovespecimens was fixed, decalcified, embedded, and sec-tioned. Coronal sections (6 lm) from the intermediatejoint region were used for histological and immunohisto-chemical analyses of extracellular matrix (ECM) compo-sition and properties of TMJ articular cartilage. Suchassays were performed following similar analyses ofpostorbital soft tissues.

Several histological and immunohistochemical stainswere used to analyze changes in the protein composi-tion of the postorbital ligament as a function of die-tary properties (Kiernan, 1999; Ravosa et al., 2007,2008a,b). H&E staining was used to identify generalsoft-tissue architecture. Collagen orientation and inten-sity were defined through picrosirius-red staining(Junqueira et al., 1979). Safranin-O/fast green immu-nohistochemistry (Kiernan, 1999) identified proteogly-can content in the ECM of the soft tissue, and thusvariation in tissue viscoelasticity. Immunostaining(Kiernan, 1999) was used to identify variation inType-II collagen expression among loading cohorts,which in turn tracks variation in tissue viscoelasticity.The latter two staining protocols were similarlyapplied to TMJ articular cartilage for comparison withthe postorbital ligament.

RESULTS

H&E highlights general variation in tissue organiza-tion among loading cohorts and postorbital soft-tissue sec-tions (Fig. 2). H&E staining shows that the postorbitalligament is composed of elastic fibrocartilage, rather thandense connective tissue. Identifying postorbital soft-tissuetype requires consideration of cell type. Dense connectivetissues are composed of ligament with fibroblasts at thematrix interface, whereas a fibrocartilaginous ligamentcontains chondrocytes. The inset in Fig. 2 illustrates mor-phology typical of fibrocartilage, which is commonlydescribed as dense fibrous tissue arranged in bundleswith chondrocytes embedded among the bundles. Fibro-cartilage appears designed to resist compressive loadsand to protect blood vessels from compression (Benjaminand Ralphs, 1998). Similar to other connective tissues,fibrocartilage in the postorbital region may be a precursorto osteogenesis and ossification (Carter, 1987). Indeed, theintermediate section of ‘‘over-use’’ cohort shows increaseddisorganization and degradation, which may be indicativeof tissue turnover and osteogenesis. However, tissuesstained for Type-II collagen expression (see later) gave lit-tle indication of being cleaved, which suggests that osteo-genesis in this region may be unlikely.

The postorbital ligament in ‘‘under-use,’’ control, and‘‘over-use’’ cohorts indicate low levels of safranin-Ostaining for proteoglycans, which suggests a lower visco-elastic capacity of these soft tissues (Fig. 3). Further, asthere is little variation across dietary groups, there is aminimal postorbital response to masticatory loading, andthus minor variation in tissue viscoelasticity across die-tary cohorts. These findings are in marked contrast tofindings for soft tissues in the rabbit masticatory com-plex (Fig. 4). In particular, the TMJ shows considerable

Fig. 1. Schematic representation of the location of the three rabbit postorbital sections. The postorbitalligament spans the bony gap along the lateral orbital margin between the temporal process of the frontalbone and the frontal process of the temporal (squamosal) bone. The two red dotted lines represent theorientation of the postorbital ligament.

CIRCUMORBITAL LOADING, FUNCTION, AND PLASTICITY 645

variation across dietary cohorts, with ‘‘under-use,’’ con-trol and then ‘‘over-use’’ groups exhibiting progressivelylower levels of safranin-O staining, indicative ofdecreased articular cartilage viscoelasticity (see alsoRavosa et al., 2007).

The intermediate aspect of ‘‘under-use,’’ control, and‘‘over-use’’ dietary cohorts exhibit low levels of postorbi-tal soft-tissue immunostaining (Fig. 5). For the mostpart, there is little variation across groups in Type-II col-lagen staining intensity, suggestive of the absence ofmasticatory-related variation in the viscoelasticity ofpostorbital tissues. However, in the ‘‘over-use’’ dietgroup, there is greater expression of Type-II collagenlocalized to sites where collagen fibers appear to bedegraded. As noted earlier for diet-induced patterns ofvariation in proteoglycan content, TMJ articular carti-lage exhibits similar variation in the expression of Type-II collagen (Fig. 4), with ‘‘under-use,’’ control and then‘‘over-use’’ groups having progressively lower levels ofstaining, indicative of decreased TMJ articular cartilageviscoelasticity (see also Ravosa et al., 2007).

Picrosirius-red staining indicates a significantlyaltered collagen fibril organization in the ‘‘over-use’’cohort relative to ‘‘under-use’’ rabbits (Fig. 6). Although‘‘under-use’’ rabbits show parallel bundles of collagenfibers, ‘‘over-use’’ collagen exhibits a disorganized andless complex fiber structure. These data suggest that col-

lagen fiber organization of the postorbital ligament isaltered by masticatory loading.

DISCUSSIONCircumorbital Function

This experimental study evaluated the plasticity andfunction of circumorbital soft-tissue structures duringmasticatory function in a rabbit model of the primitivecondition of the mammalian lateral orbital wall, a condi-tion resembling that for the ancestors of primates(Cartmill, 1992; Ravosa et al., 2000a,b, 2006; Bloch andBoyer, 2002). A series of histological and immunohisto-chemical techniques were used to identify variation inpostorbital soft-tissue architecture, collagen fiber organi-zation, and ECM composition as a function of diet-induced masticatory stresses.

To an extent, previous descriptions of the mammaliancircumorbital region failed to adequately describe thestructure of soft tissues along the postorbital region. Therabbit postorbital region is composed of dense fibrous tis-sue bundles with chondrocytes among the bundles.Fibrocartilage is considered a ‘‘transitional tissue,’’ as itcan differentiate from both dense fibrous connective tis-sue and hyaline cartilage (Benjamin and Ralphs, 1998).Fibrocartilage is generally avascular, and the lack ofblood vessels allows it to resist routine compressive

Fig. 2. Coronal section of intermediate aspect of the adult rabbitpostorbital fibrocartilage stained with H&E. Sections from (A) ‘‘under-use,’’ (B) ‘‘control,’’ and (C) ‘‘over-use’’ rabbit postorbital fibrocartilage.The inset in Fig. 2a shows chondrocytes (indicated by the arrows) sit-

uated within bundles of collagen fibers, which suggests that the post-orbital soft-tissue structure is elastic fibrocartilage not denseconnective tissue.

Fig. 3. Coronal section of intermediate aspect of the adult rabbitpostorbital fibrocartilage stained with Safranin-O. Sections from (A)‘‘under-use,’’ (B) ‘‘control,’’ and (C) ‘‘over-use’’ rabbit postorbital fibro-

cartilage. Unlike the rabbit TMJ tissues, the rabbit postorbital fibrocar-tilage indicates minimal staining for proteoglycans, variation inviscoelasticity and tissue response to altered masticatory stresses.

646 JASAREVIC ET AL.

loads. Consistent with the general microstructure offibrocartilage, rabbit postorbital tissues were character-ized by variation in their internal organization (see ear-lier). The more complex combination of a basket-weavepattern and parallel arrangement of collagen fibrils wasunique to the intermediate aspect of the postorbital liga-ment, independent of cohort. When cohorts are analyzedseparately, the intermediate section of the postorbitalsoft tissues in control and ‘‘under-use’’ rabbits indicates

a similar arrangement, whereas a more irregular fibrilorientation is observed in ‘‘over-use’’ rabbits. This find-ing suggests that long term increases in masticatoryloading results in a degradative response, wherebypostorbital fibrocartilage exhibits decreases in multidir-ectional collagen fiber orientation and subsequent reduc-tions in the ability to resist multidirectional stresses.

Aggrecan, a hydrophilic molecule in fibrocartilage isconfined in the ECM, when the tissue is compressed

Fig. 4. Coronal sections of middle TMJ sites from ‘‘under-use’’(A, D) diet, ‘‘control,’’ (B, E) and ‘‘over-use’’ (C, F) diet adult rabbitsstained with safranin-O (A–C) to identify proteoglycan content and aprimary antibody directed against Type-II collagen (D–F). TMJ articularcartilage of ‘‘over-use’’ (C, F) rabbits exhibits reduced Type-II collagen

and lower proteoglycan content, which is indicative of reduced articu-lar cartilage viscoelasticity. Thus, cartilage in TMJs routinely subjectedto elevated loading due to a more fracture-resistant diet showsreduced ability to resist compressive loads during biting and chewing.

Fig. 5. Coronal section of intermediate aspect of the adult rabbit postorbital fibrocartilage stained forType-II collagen. Sections from (A) ‘‘under-use,’’ (B) ‘‘control,’’ and (C) ‘‘over-use’’ rabbit postorbital fibro-cartilage. Unlike the rabbit TMJ tissues, rabbit postorbital fibrocartilage indicates less variation in tissueviscoelasticity and subtle tissue responses to altered loads.

CIRCUMORBITAL LOADING, FUNCTION, AND PLASTICITY 647

by the basket-weave arrangement of collagen fibrils(Benjamin and Ralphs, 2004). In vivo loading modelsindicated that aggrecan gene expression was both loadand frequency dependent in mouse intervertebral discswith higher-stress cohorts exhibiting short-term eleva-tions in aggrecan gene expression (MacLean et al.,2003). Long-term exposure to elevated compressive loadsdownregulates aggrecan gene expression, which maylead to a myriad of biomechanical effects including celldeath, loss of meshwork arrangement, and subsequentdegeneration of collagen fibers. This would suggest thatthe exposure to long-term loading may decrease tissuecohesion, and thus overall structural integrity, a patterncharacteristic of postorbital cartilage and jaw-joint carti-lage (Ravosa et al., 2007, 2008a,b).

The extent to which such variation in ECM expressionis observed in other mammals with a primitive postorbi-tal region remains to be determined. Unlike inferior cra-niofacial regions such as the symphysis and TMJ(Ravosa et al., 2007, 2008a,b), there was minimal varia-tion across cohorts in proteoglycan expression in the rab-bit postorbital ligament. As tissue viscoelasticity islikewise presumably low across such groups, this sug-gests that postorbital soft tissues are unlikely to bedesigned for resisting significant stresses during chew-ing and biting. Results of Type-II collagen stainingacross dietary cohorts offer further support for thisinterpretation.

The avascular nature of the rabbit postorbital fibro-cartilage prevents the tissue from being able to respond(or adapt) to long-term loading patterns. Conversely,fibrocartilage exposed to low and normal loadingregimes maintains multidirectional collagen fiber orien-tation (e.g., Provenzano and Vanderby, 2006), whichmay demonstrate a nonelastic response in the ‘‘under-use’’ and normal cohorts (Figs. 3 and 4). Picrosirius-redstaining identified variation across rabbit dietarygroups, where the complexity of collagen fiber orienta-tion appears to be inversely related to masticatorystresses. Whether this represents permanent cartilagedegeneration or collagen degradation as a precursor totissue biomineralization is currently unknown,

although preliminary analyses suggest little support forthe latter interpretation.

Circumorbital Evolution

What are the implications of the earlier evidenceregarding postorbital soft-tissue plasticity for under-standing circumorbital adaptation in primate and non-primate mammals? Most importantly, there is noevidence that long-term elevations in masticatory stresscan induce the epigenetic transformation of soft tissuesof the lateral orbital wall into a bony postorbital bar.Rather, ECM components related to tissue viscoelasticityshow a greater potential for elevated load-resisting abil-ities and a larger plasticity response in TMJ articularcartilage versus the postorbital ligament. Although die-tary shifts has not been posited for primate origins, thedata presented here have broader implications for mam-mals. In particular, as noted for the postorbital bar andseptum, it is unlikely that circumorbital soft tissuesshow adaptive responses to increases in routine mastica-tory forces. Indeed, contrary to predictions of mastica-tory-stress models of the circumorbital region, elevatedloading results in the degradation of circumorbital soft-tissue structures. Although masticatory elements suchas the mandibular symphysis and TMJ vary dynamicallyin response to functional requirements, the postnatalplasticity of circumorbital soft tissues is much less pro-nounced under the loading regimes tested here. Theseresults provide no support for the supposition that evolu-tion of the postorbital bar in various mammal clades (fel-ids, herpestids, pteropodids, etc.) is due to elevatedmasticatory stresses. Instead, the best evidence to datesuggests that increased orbital convergence and/or or-bital frontation may require a stiff lateral orbital marginto maintain high levels of stereoscopic nocturnal visualacuity during predatory behaviors (Cartmill, 1970, 1972,1974, 1992; Noble et al., 2000; Ravosa et al., 2000b,2006; Menegaz and Kirk, 2009). Obviously, this is notthe final word regarding the evolution of the postorbitalbar as there are many clades in which high levels of vis-ual acuity remain to be implicated as selective pressures

Fig. 6. Coronal section of intermediate aspect of the adult rabbitpostorbital fibrocartilage stained with Picrosirius-red. Sections from (A)‘‘under-use,’’ (B) ‘‘control,’’ and (C) ‘‘over-use’’ rabbit postorbital fibro-cartilage. Picrosirius-red stain indicates significant variation across di-

etary cohorts; complexity of collagen orientation is inversely related tomasticatory loads, which may imply a degradative, not an adaptive,response.

648 JASAREVIC ET AL.

underlying morphological differences in the mammaliancircumorbital region.

CONCLUSIONS

To further address circumorbital function and evolu-tion, we examined the long-term plasticity of circumorbi-tal soft tissues in a rabbit model of the primitivemammalian condition. Masticatory forces related to long-term processing of a tougher or more resistant dietappear to result in a reduced organizational complexity ofcircumorbital soft tissues, particularly collagen fibersindicating a decreased ability of the postorbital ligamentto resist multidirectional loads. However, although diet-related variation in masticatory stress may affect ECMcomposition and structural properties in certain cranio-mandibular elements (i.e., TMJ or symphysis), currentexperimental data provide minimal evidence that circum-orbital tissues are similarly influenced by masticatoryforces. Indeed, the postorbital ligament only maintains anability to resist multidirectional loads when such stressesare moderate-to-low in magnitude, and there are fewindicators that ECM composition confers much tissue vis-coelasticity in any of the treatment groups. Rather, long-term exposure to elevated cyclical mechanical loadingmay be maladaptive, such that the soft- to hard-tissuetransformation in the postorbital region of primatesoccurred due to different selective pressures almost cer-tainly unrelated to mechanical loading.

In sum, an ‘‘osteocentric’’ focus of research on circum-orbital biomechanics has hindered our understanding ofcraniofacial evolution in primates and other mammals.As this is the first study aimed at identifying the micro-anatomical function and plasticity of the postorbitalregion of a species retaining the basal mammalian condi-tion, future investigations would benefit from similarwork on the development of the circumorbital region inother species. Another point for further consideration isthe role of masticatory loading on tissue turnover, partic-ularly addressing whether long term, elevated mastica-tory stresses result in permanent tissue degradation(i.e., degeneration) or if tissue degradation is a precursorto osteogenesis.

ACKNOWLEDGMENTS

J. Organ, T. Smith, V. DeLeon, and Q. Wang arethanked for inviting us to contribute to their specialissue on experimental approaches to morphology. B.Wright kindly performed the analyses of rabbit foodproperties. W. Phillips is thanked for helping with initialphases of this project. We are also indebted to L. Cous-sens for the Picrosirius-red protocol and D. Miller foraccess to and assistance with a polarizing microscope.E.J. and A.N.D. were supported by MU LS UROP Fel-lowships. R.A.M. was supported by MU Life Sciencesand NSF Graduate Research Fellowships. Lastly, thismanuscript benefited from the comments of V. DeLeonand two anonymous reviewers.

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