mouse models of telomere dysfunction phenocopy skeletal ... · features of human age-related...

© 2014. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2014) 7, 583-592 doi:10.1242/dmm.014928

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ABSTRACTA major medical challenge in the elderly is osteoporosis and the highrisk of fracture. Telomere dysfunction is a cause of cellular senescenceand telomere shortening, which occurs with age in cells from mosthuman tissues, including bone. Telomere defects contribute to thepathogenesis of two progeroid disorders characterized by prematureosteoporosis, Werner syndrome and dyskeratosis congenital. It ishypothesized that telomere shortening contributes to bone aging. Weevaluated the skeletal phenotypes of mice with disrupted telomeremaintenance mechanisms as models for human bone aging, includingmutants in Werner helicase (Wrn−/−), telomerase (Terc−/−) andWrn−/−Terc−/− double mutants. Compared with young wild-type (WT)mice, micro-computerized tomography analysis revealed that youngTerc−/− and Wrn−/−Terc−/− mice have decreased trabecular bone volume,trabecular number and trabecular thickness, as well as increasedtrabecular spacing. In cortical bone, young Terc−/− and Wrn−/−Terc−/−

mice have increased cortical thinning, and increased porosity relative toage-matched WT mice. These trabecular and cortical changes wereaccelerated with age in Terc−/− and Wrn−/−Terc−/− mice compared witholder WT mice. Histological quantification of osteoblasts in aged miceshowed a similar number of osteoblasts in all genotypes; however,significant decreases in osteoid, mineralization surface, mineralapposition rate and bone formation rate in older Terc−/− andWrn−/−Terc−/− bone suggest that osteoblast dysfunction is a prominentfeature of precocious aging in these mice. Except in the Wrn−/− singlemutant, osteoclast number did not increase in any genotype. Significantalterations in mechanical parameters (structure model index, degree ofanistrophy and moment of inertia) of the Terc−/− and Wrn−/−Terc−/−

femurs compared with WT mice were also observed. YoungWrn−/−Terc−/− mice had a statistically significant increase in bone-marrowfat content compared with young WT mice, which remained elevated inaged double mutants. Taken together, our results suggest that Terc−/−

and Wrn−/−Terc−/− mutants recapitulate the human bone agingphenotype and are useful models for studying age-related osteoporosis.

KEY WORDS: Aging, Bone histomorphometry, Osteoporosis

RESEARCH ARTICLE

1Department of Medicine, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA 19104, USA. 2Department of Pathology andLaboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.3Department of Clinical Studies-New Bolton Center and Animal Biology, School ofVeterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.4Department of Biostatistics and Epidemiology, Perelman School Of Medicine,University of Pennsylvania, Philadelphia, PA 19104, USA. 5Department ofOrthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania,Philadelphia, PA 19104, USA.

*Author for correspondence ([email protected])

This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricteduse, distribution and reproduction in any medium provided that the original work is properlyattributed.

Received 15 November 2013; Accepted 8 March 2014

INTRODUCTIONHuman senile osteoporosis is generally characterized by low bonemass and altered microarchitectural features, primarily attributed toosteoblast dysfunction, which results in a substantially increased riskof fracture and subsequent disability (Kassem and Marie, 2011;Marie and Kassem, 2011a; Marie and Kassem, 2011b). This differsfrom well-studied postmenopausal osteoporosis, in which significantbone loss is attributed to increased osteoclast resorption concomitantwith the loss of estrogen in women (Aaron et al., 1987; Rehman etal., 1994; Russo et al., 2003; Mullender et al., 2005; Pernow et al.,2009). One of the limitations to more intensive investigations ofsenile osteoporosis is the lack of physiologically relevant models ofaging bone. Although the mechanisms of senile osteoporosis remainto be elucidated, some mechanistic clues have come from geneticconditions that potentiate telomere dysfunction. Two conditions ofparticular interest include dyskeratosis congenita (DC; incidence ~1in 1 million) and Werner syndrome (WS; incidence 1 in 20,000-200,000), both of which are characterized by a complex phenotypeincluding premature osteoporosis. In DC, osteoporosis is anaccelerated form of that seen with physiological aging, whereas theosteoporosis of WS affects the limbs as well as the axial skeleton(Hofer et al., 2005; Mason et al., 2005). One of the most commonforms of DC is caused by a mutation in TERC (the RNA templatecomponent of telomerase), which results in reduced telomeraseactivity. The lack of telomerase activity leads to prematurelyshortened telomeres, telomere uncapping and the association oftelomeric ends with DNA-damage proteins. Taken together, thesefindings suggest that dysfunctional telomeres likely are a majorcontributing factor to the pathology of DC, including osteoporosis(Mason et al., 2005).

WS is most commonly due to a loss-of-function mutation inWRN, which encodes a DNA helicase of the RecQ family andfunctions in the recombinational repair of stalled replication forksor double-strand breaks (Ozgenc and Loeb, 2005). Additionally,WRN is thought to play a role in telomere maintenance (Wyllie etal., 2000; Johnson et al., 2001; Opresko et al., 2002; Crabbe et al.,2004), which might be particularly important in situations whentelomeres become dysfunctional, such as cellular aging.

In mice lacking telomerase and with shortened telomeres, Wrn−/−

mutation results in an acceleration of defects seen in Terc−/− mutantsand also the appearance of some pathologies typical of WS (e.g.osteoporosis) (Chang et al., 2004; Du et al., 2004). Previously, weand others found that deficiency in telomerase, alone or incombination with deficiency in the Werner helicase, leads to anaccelerated low-bone-mass phenotype (Chang et al., 2004; Du et al.,2004; Pignolo et al., 2008; Saeed et al., 2011). Declines inmesenchymal progenitor cell number and in osteoblastdifferentiation are the major cellular mechanisms associated withpremature bone loss (Pignolo et al., 2008; Wang et al., 2012). Invitro, osteoclast differentiation and function in Wrn−/−, Terc−/− and

Mouse models of telomere dysfunction phenocopy skeletalchanges found in human age-related osteoporosisTracy A. Brennan1, Kevin P. Egan1, Carter M. Lindborg1, Qijun Chen2, Mariya T. Sweetwyne3, Kurt D. Hankenson3, Sharon X. Xie4, Frederick B. Johnson2 and Robert J. Pignolo1,5,*

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Wrn−/−Terc−/− mutants are comparable to those in age-matched wild-type (WT) mice (Pignolo et al., 2008; Wang et al., 2012).

Here, we test the hypothesis that deficiencies in Terc−/− and/orWrn−/−Terc−/− mutant mice reflect many of the physiologicalfeatures of human age-related osteoporosis, including decreasedbone volume, osteoblast number and osteoblast function, as well asincreased porosity and marrow adiposity. We present evidence thatsupports the use of Terc−/− and Wrn−/−Terc−/− mice as relevantmodels for studying human age-related osteoporosis and suggeststhat telomere-based mechanisms are important in the aging of bonetissue.

RESULTSTelomere-based aging models show accelerated trabecularand cortical bone lossA hallmark of human senile osteoporosis is decreased bone volume,primarily attributed to decreased trabecular bone, which can also beaccompanied by decreased cortical bone volume (Parfitt, 1984;Kleerekoper et al., 1985; Rehman et al., 1994; Mullender et al.,2005; Pernow et al., 2009). High-resolution micro-computerizedtomography (μCT) was used for analysis of the distal femur fromyoung and aged WT, Wrn−/−, Terc−/− and Wrn−/−Terc−/− mice.Fig. 1A shows representative images of the trabecular region of

interest (ROI) for each of the genotypes in the young and agedgroups. Bone volume declined with age across the genotypes (age,P<0.0001; genotype, P<0.0001; interaction, P=0.0012; Fig. 1B,C).At 3 months of age, Terc−/− and Wrn−/−Terc−/− femurs hadstatistically significant decreases in bone volume (41.7% and 47.6%,respectively) compared with the age-matched WT (Fig. 1B). Thiscomparative deficit became more pronounced with age, as reflectedin Fig. 1C. In the aged group, Wrn−/−, Terc−/− and Wrn−/−Terc−/−

mice had statistically significant decreases in bone volume (46.4%,84.9% and 69.8%, respectively) compared with aged WT. Thedecreases in bone volume in young (Fig. 1B) and aged (Fig. 1C)Terc−/− and Wrn−/−Terc−/− were accompanied by significantlydecreased trabecular number (age, P<0.0001; genotype, P<0.0001;interaction, P=0.0027; Fig. 1D,E) and trabecular thickness (age,P=0.0248; genotype, P<0.0001; interaction, P=0.0498; Fig. 1F,G),with a commensurate increase in trabecular separation {age,P<0.0001; genotype, P<0.0001; interaction, P=0.1941 [non-significant (ns)]; Fig. 1H,I}.

Moreover, when the bone volume of young Wrn−/−Terc−/− wascompared with WT aged mice, Wrn−/−Terc−/− (P=0.01) bone volumewas significantly decreased. This accelerated bone loss in youngWrn−/−Terc−/− was accompanied by trends toward both decreasedtrabecular number and trabecular separation as well as a significantdecrease in trabecular thickness (P<0.0001).

Human senile osteoporosis can be accompanied by thinning of thecortical shell and increased cortical porosity causing an increasedrisk of fracture (Feik et al., 1997; Bousson et al., 2001; Ostertag etal., 2009; Nishiyama et al., 2010; Zebaze et al., 2010). Cortical bonewas analyzed for both young and aged mice at the femoral midshaftusing μCT. Fig. 2A shows representative images of the cortical ROI.The cortical bone area decreased significantly with age andgenotype [age, P<0.0001; genotype, P<0.001; interaction, P=0.067(ns)]. Unlike the trabecular changes, in young mice only Terc−/−

showed a decrease (4.0%) in cortical area compared with age-matched WT (Fig. 2A,B). However, significant decreases in corticalarea were seen in aged Terc−/− and Wrn−/−Terc−/− mice comparedwith older WT mice (28.2% and 29.7%, respectively; Fig. 2C). Inaddition to changes in cortical area, cortical thickness significantlydecreased (age, P=0.0003; genotype, P<0.0001; interaction,P=0.0037). The cortical thickness of Terc−/− and Wrn−/−Terc−/−

mutants was significantly decreased in both young and aged groupscompared with their respective WT counterparts (Fig. 2D,E). Theeffects of accelerated aging in the young Terc−/− and Wrn−/−Terc−/−

femurs were further highlighted by their statistically significantdecrease in cortical thickness compared with aged WT femurs(P=0.0021 and P=0.0001, respectively). Although the corticalthickness decreased with age for Terc−/− and Wrn−/−Terc−/− femurs,there were no statistically significant differences in the endosteal[age, P=0.0012; genotype, P=0.7552 (ns); interaction, P=0.6157(ns)] or periosteal [age, P=0.0302; genotype, P=0.1594 (ns);interaction, P=0.2842 (ns)] surfaces between these genotypescompared with young or aged WT. Average animal weight withinyoung (28.5±1.4 g, WT; 28.2±0.6 g, Wrn−/−; 25.6±1.3 g, Terc−/−;24.4±1.4 g, Wrn−/−Terc−/−) and older (32.8±0.8 g, WT; 32.2±0.7 g,Wrn−/−; 29.0±2.0 g, Terc−/−; 30.2±2.0 g, Wrn−/−Terc−/−) groups werenot statistically significant compared with WT.

Cortical porosity increased with age and genotype (age,P=0.0038; genotype, P<0.0001; interaction, P=0.0022). Relative tothe WT mice, porosity increased in Terc−/− and Wrn−/−Terc−/−

mutants, in both young (27.1% and 19.0%, respectively) and aged(49.3% and 60.1%, respectively) groups (Fig. 2F,G). Whencompared with aged WT femurs, porosity in the cortical bone of

RESEARCH ARTICLE Disease Models & Mechanisms (2014) doi:10.1242/dmm.014928

TRANSLATIONAL IMPACTClinical issueHuman age-related osteoporosis poses a major problem to both elderlymen and women. The low bone mass and altered microarchitecturalfeatures of the condition, which are mainly attributed to osteoblastdysfunction, increase the risk of bone fracture. It is predicted that theannual number of hip fractures worldwide due to osteoporosis will risethreefold from the 1990 figure of 1.7 million to 6.3 million by 2050.Therefore, it is crucial to develop novel therapies to help treat thiscondition. Although postmenopausal osteoporosis, which is attributed toincreased osteoclast activity (osteoclasts destroy bone whereasosteoblasts build bone; normal bone is constantly remodeled by thesetwo cell types), has been well studied, far less is known about themechanisms of age-related osteoporosis. This is in part due to a lack ofphysiologically relevant models of this type of osteoporosis.

ResultsHere, the authors investigate the premature osteoporosis that occurs inmouse models based on two human genetic progeroid (premature aging)conditions, Werner syndrome and dyskeratosis congenita. Telomeredefects contribute to both of these disorders, and telomere shorteningoccurs with age in cells from most human tissues, including bone. It ispossible, therefore, that telomere shortening contributes to bone aging.To test this hypothesis and to evaluate the physiological similarity ofosteoporosis in Terc–/– mice (telomerase mutants; a model fordyskeratosis congenita), Wrn–/– mice (a model for Werner’s syndrome),Terc–/–Wrn–/– double-mutant mice and the human condition, the authorsevaluate the skeletal phenotypes of the mutant mice. They show thatthese models reflect the major features of senile bone loss, includingdecreased trabecular and cortical bone volume, increased corticalporosity, and impairment of osteoblast function as a prominent cause ofaccelerated bone aging.

Implications and future directionsThese findings suggest that Terc–/– and Wrn–/–Terc–/– mice recapitulatethe bone changes that occur in aging human bones and establish thesemice as appropriate models for studying senile osteoporosis. Moreover,they support the hypothesis that telomere-based mechanisms areimportant in bone aging. The models described will be useful for theidentification and dissection of signaling pathways that are altered inresponse to telomere-based bone aging. Moreover, further studiesincorporating these models should aid in the design and preclinicalevaluation of potential therapeutic treatments for age-related bone loss.

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either young Terc−/− or young Wrn−/−Terc−/− mice was significantlyincreased (P=0.0008 and P=0.0024, respectively).

Taken together, our data show that the accelerated bone aging inTerc−/− and Wrn−/−Terc−/− mouse models not only displayscharacteristic features of human senile osteoporosis, as examined atthe structural level by μCT, but that these characteristics presentearlier and in general are more pronounced than in older WT mice.

Osteoblast dysfunction is the primary cellular mechanismfor osteoporosis in telomere-based bone agingThe decrease in bone volume seen in human senile osteoporosis isgenerally attributed to decreased bone formation due to osteoblastdysfunction, in contrast to menopausal osteoporosis in whichincreased osteoclast resorption seems to be the predominantmechanism (Rehman et al., 1994; Clarke et al., 1996; Pernow et al.,2009). Therefore, to assess osteoblast function, calcein doublelabeling was used to quantify dynamic parameters of bone formation

in young and aged mice (Fig. 3A). Although the differencesobserved in mineralized surface [mineralized surface (MS)/bonesurface (BS), %] [age, P=0.0861 (ns); genotype, P=0.0495;interaction, P=0.0091; Fig. 3B,C] and bone formation rate (BFR;%/year) [age, P=0.1213 (ns); genotype, P=0.0061; interaction,P=0.0137; Fig. 3F,G] were related to telomere-based defects alone,decreases in mineral apposition rate (MAR; μm/day) wereinfluenced by both age and genotype (age, P=0.0074; genotype,P=0.0004; interaction, P=0.0031; Fig. 3D,E). Young Wrn−/−, Terc−/−

and Wrn−/−Terc−/− mice showed no statistically significantdifferences in MS, MAR or BFR compared with young WT mice(Fig. 3B,D,F). However, MS, MAR and BFR significantly anddramatically declined in aged Terc−/− and Wrn−/−Terc−/− micecompared with WT mice that were on average more than 30% older(Fig. 3C,E,G).

Concomitant with decreased kinetic parameters of boneformation, osteoid significantly decreased in the aged Terc−/− and

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Fig. 1. Telomere-based changes in Terc−/− and Wrn−/−Terc−/−trabecular bone parameters. (A) Three-dimensionalrepresentation of the trabecular ROI in the distal femurs. Distalfemurs from young and aged male mice were evaluated formicroarchitectural changes including (B,C) bone volume/totalvolume (BV/TV; %), (D,E) trabecular number (Tb. N; /mm), (F,G)trabecular thickness (Tb. Th; mm) and (H,I) trabecularseparation (Tb. Sp.; mm). Data represent means ± s.e.m.Statistical significance is *P<0.05, **P<0.005, ***P<0.0001 and****P<0.0001 compared with WT in each age group.

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Wrn−/−Terc−/− femurs compared with WT (age, P<0.0001; genotype,P=0.0008; interaction, P=0.0350; Fig. 4A,C,D). At the cellular level,osteoblast numbers decreased significantly with age and across allgenotypes [age, P<0.0001; genotype, P=0.0700 (ns); interaction,P=0.9236 (ns); Fig. 4E]. Importantly, only the Wrn−/− single mutanthad a statistically significant increase in osteoclast number with age[age, P=0.1764 (ns); genotype, P<0.0001; interaction, P=0.0150;Fig. 4B,F]. Thus, taken together with their profoundly decreasedcapacity for osteoid production, mineralization and bone formation,this strongly suggests that, like in human senile bone loss, osteoblastdysfunction is the primary mechanism for osteoporosis in Terc−/−

and Wrn−/−Terc−/− mutants.Osteoblast dysfunction in the Terc−/− and Wrn−/−Terc−/− mutants

was accompanied by alterations in mechanical properties. YoungTerc−/− and Wrn−/−Terc−/− mutant femurs had a statisticallysignificant increase in structure model index (SMI), which continuedto increase with age [age, P<0.0001; genotype, P<0.0001;interaction, P=0.2378 (ns); Fig. 5A,D] and indicating a more rod-like trabecular structure. The degree of anisotropy (DA) wasstatistically decreased in young Wrn−/− and Wrn−/−Terc−/− mutants[age, P=0.0004; genotype, P=0.0078; interaction, P=0.7226 (ns);Fig. 5B,E]. The moment of inertia (MOI) was statistically decreasedin young Terc−/− femurs and, in the aged group, the MOIsignificantly decreased in both Terc−/− and Wrn−/−Terc−/− mutants

[interaction, P=0.1002 (ns); genotype, P<0.0001; age, P=0.1898(ns), Fig. 5C,F]. These changes in mechanical parameters areconsistent with those seen in aging humans (Bono and Einhorn,2003; Russo et al., 2003; Jiang et al., 2005).

High bone-marrow adiposity in Terc–/– and Wrn–/–Terc–/–

mutantsAdiposity increases with human bone aging and more severely withosteoporosis (Justesen et al., 2001; Rosen and Bouxsein, 2006). Ouranalysis of adiposity showed no relationship with aging and/orgenotype on percent adipose tissue [age, P=0.1027 (ns); genotype,P=0.3390 (ns); interaction, P=0.5074 (ns)]. However, an analysis ofadipocyte volume in young Wrn−/−Terc−/− mice compared withyoung WT animals demonstrated that the former have a statisticallysignificant increase in bone-marrow fat content (P<0.05), whichremained elevated in aged double mutants (Fig. 6). In addition, agedTerc−/− and Wrn−/−Terc−/− mice had bone-marrow fat content similarto much older WT mice.

DISCUSSIONSenile osteoporosis results in a substantially increased risk offracture and subsequent disability (Raisz and Rodan, 2003).Although postmenopausal osteoporosis has been well studied, farless is known about the mechanisms of age-related osteoporosis


Fig. 2. Telomere-based changes in Terc−/− and Wrn−/−Terc−/−cortical parameters. (A) Three-dimensional representation ofthe cortical ROI in the femur. The femoral midshaft from youngand aged male mice were evaluated for cortical bone changesincluding (B,C) cortical area/total area (Ct. Ar/Tt. Ar; %), (D,E) cortical thickness (Ct. Th; mm) and (F,G) total pore volume/bone volume (porosity; %). Data represent means ± s.e.m.Statistical significance is *P<0.05, **P<0.005, ***P<0.0025,****P<0.0001, compared with WT in each age group.

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affecting both men and women. In this study, we assessed telomere-based aging mouse models according to key characteristics ofhuman senile bone loss, both to evaluate their physiologicalsimilarities to the human condition as well as to substantiate theimportance of telomere dysfunction as a mechanism for skeletalaging. Although previous data suggest that telomerase-based modelsof accelerated aging have osteoporosis, our current study shows thatthese models reflect the major features of senile bone loss, and thatimpairment of osteoblast function is a prominent aspect ofaccelerated bone aging. Inadequate osteoblast-mediatedmineralization during bone remodeling is likely to be responsible forosteopenia in mouse models of physiologic as well as prematureaging (Jilka, 2013).

Bone loss in both trabecular and cortical locations is a hallmarkof age-related osteoporosis. Similar to human senile osteoporosis,Terc−/− and Wrn−/−Terc−/− mice had decreased trabecular bonevolume and trabecular number (Table 1). A significant decrease inTerc−/− trabecular bone has also been reported (Saeed et al., 2011)from histological analysis in the spinal region (L5 vertebra).Although trabecular thickness also decreased with age in ourmodels, no consensus has been reached for humans, where reportshave shown a decline in trabecular thickness (Parfitt et al., 1983;Birkenhäger-Frenkel et al., 1988), a decline in men only(Wakamatsu and Sissons, 1969; Aaron et al., 1987; Mellish et al.,1989), and a decline in both men and women (Rehman et al., 1994).These discrepancies could be attributed to human variation;however, when looking at severely osteoporotic male bone samples,Pernow et al. showed that trabecular thickness was significantlydecreased (Pernow et al., 2009).

Cortical bone loss in human senile osteoporosis is structurallyidentified by cortical thinning and increased porosity (Feik et al.,1997; Ostertag et al., 2009; Nishiyama et al., 2010; Zebaze et al.,2010; Macdonald et al., 2011). μCT analysis of Terc−/− andWrn−/−Terc−/− mice showed that they recapitulate this phenotype byhaving both a significant decrease in cortical thickness and asignificant increase in porosity, which together contributed to thesignificant loss of cortical bone volume compared with WT mice(Table 1).

Age-related bone loss in humans is attributed to osteoblastdysfunction in the presence of normal osteoclast activity (Marie andKassem, 2011b). Although the number of osteoblasts present in theaged mice was comparable across genotypes, parameters of boneformation (including osteoid, mineralization, MAR and BFR) weresignificantly decreased in both Terc−/− and Wrn−/−Terc−/− micecompared with the WT counterparts (Table 1). Taken together, ourdata suggest that osteoblasts present in the Terc−/− and Wrn−/−Terc−/−

mice are dysfunctional. This is supported by similar data reportedfor the analysis of tibiae in Terc−/− mice (Saeed et al., 2011). Inaddition, our previous work using Terc−/− and Wrn−/−Terc−/− micedemonstrated dysfunctional osteoblast differentiation in vitro,independent of cell proliferation (Pignolo et al., 2008; Wang et al.,2012). The contribution of osteoblast dysfunction to theseosteoporotic phenotypes is further highlighted by the fact thatTRAP+ osteoclasts in Terc−/− and Wrn−/−Terc−/− bones remainedrelatively unchanged in the young versus older groups. This issupported by our previous work that demonstrated that osteoclastactivity was similar in WT, Terc−/− and Wrn−/−Terc−/− osteoclasts invitro (Wang et al., 2012). Thus, telomere-based osteoporosis is

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Fig. 3. Decreased kinetic parameters of bone formation inaged Terc−/− and Wrn−/−Terc−/− mice. (A) Representativefluorescently labeled sections of metaphyseal distal femurs foryoung and aged WT, Wrn−/−, Terc−/− and Wrn−/−Terc−/− animals.(B,C) Mineralizing surface/bone surface (MS/BS; %), (D,E)mineral apposition rate (MAR; μm/day) and (F,G) bone formationrate (BFR; %/yr) are shown. Data represent means ± s.e.m.Statistical significance is *P<0.05, **P<0.01 and ***P<0.0025,compared with WT in each age group (n=5 per group).

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primarily due to age-related osteoblast dysfunction in the context ofunchanged osteoclast function.

Osteoblast dysfunction coincides with deleterious changes inmechanical properties of bone. The Terc−/− and Wrn−/−Terc−/−

trabecular bone had statistically increased SMIs in both young andaged groups, consistent with changes seen in aging bone thatdemonstrate a transition from a more plate-like trabecular structure toa more rod-like structure typical of osteoporotic bone (Ding and Hvid,2000; Chen et al., 2013). Although the DA varies with skeletal site,with human bone aging there is initially an increase in anisotropyfollowed by a period of trabecular perforation and an eventualdecrease in anisotropy (Jiang et al., 2005). In young Wrn−/− andWrn−/−Terc−/− mutants, the DA is decreased and this level ismaintained with aging in these genotypes. Furthermore, the MOI was

significantly decreased in Terc−/− and Wrn−/−Terc−/− cortical bone.This decrease in MOI is characteristic of osteoporotic bone that ismore easily fractured (Bono and Einhorn, 2003; Russo et al., 2003).

In bone, there seems to be a reciprocal relationship between thenumber of osteoblasts and adipocytes, with increased bone-marrowadiposity manifesting as another prominent feature of human senileosteoporosis (Justesen et al., 2001; Rosen and Bouxsein, 2006).Aged WT bone marrow had a significant increase in adiposity. By3 months of age, however, Wrn−/−Terc−/− mice already had asignificantly increased volume of bone-marrow adipose tissue, andyoung Terc−/− mice displayed a trend toward increased bone-marrowadiposity compared with WT counterparts.

Age-related osteoporosis is a complex condition that requiresphysiologically relevant models. An early model of senile bone loss


Fig. 4. Declines in osteoblast number and osteoid, but not osteoclast number, in aging bone.(A,B) Representative sections of metaphyseal distal femursfrom young and aged WT, Wrn−/−, Terc−/− and Wrn−/−Terc−/−

mice stained with Goldner’s trichrome (A; arrowheadsindicate osteoid; arrows indicate osteoblasts) and tartrate-resistant acid phosphatase (TRAP; B). (C,D) Decreasedosteoid is present in young Terc−/− and older Terc−/− andWrn−/−Terc−/− mice compared with WT. Data representmeans ± s.e.m. Statistical significance is *P<0.05 and**P<0.01, compared with WT in each age group (n=5 pergroup). (E) Decreased osteoblast number per mm bonesurface (Ob. N/BS) occurs in wild-type and mutantgenotypes with age. (F) Unchanged number of osteoclastsper mm bone surface (OCL N/BS) is seen with aging in WT,Terc−/− and Wrn−/−Terc−/− mice (n≥5 samples).(E,F) Statistical significance is *P<0.05 and **P<0.01,compared with young genotype-matched group (n=5 pergroup).

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involved using mouse strains that had a low peak bone mass,including C57BL/6J, that would develop osteoporosis naturally withage (Matsushita et al., 1986; Perkins et al., 1994; Bergman et al.,1996; Takeda et al., 1997b; Takeda et al., 1997a; Takeda et al.,1997c; Cao et al., 2003; Ferguson et al., 2003). However, the longtime over which these strains develop characteristic features of boneaging make these models less appealing. To obviate this problem,strategies were implemented to mimic bone aging throughovariectomy, orchiectomy or bone-marrow ablation (Wink and Felts,1980; Kalu, 1984; Liang et al., 1992; Mosekilde et al., 1994; Zhanget al., 1998). Although these approaches will cause an osteoporoticphenotype, they are better models for secondary causes of bone loss(e.g. sex-hormone deficiency) rather than primary age-relatedosteoporosis. Advantages and limitations of mouse models for thestudy of bone aging, and their applicability to the human condition,have been comprehensively reviewed (Jilka, 2013).

The use of genetically modified animals has enabled thedevelopment of osteoporotic mouse models (Kawaguchi et al., 1999;Vogel et al., 1999; Li et al., 2000; Bonyadi et al., 2003; Ito et al.,2003; Sun et al., 2004; Rasheed et al., 2006), but raises the questionof how to qualify genetic changes as relevant to age-related boneloss specifically. The gene(s) modified in a proposed model of senileosteoporosis should (a) effect osteoblast differentiation and/orfunction; (b) accelerate the processes naturally impacted by aging,preferably on both the cellular and organismal level; and (c) resultin a bone phenotype that recapitulates aspects of age-related boneloss in humans. Previous research has validated the influence of theWrn and Terc genes on aging (Epstein et al., 1966; Chang et al.,2004; Mason et al., 2005; Ozgenc and Loeb, 2005). The results ofthe present study demonstrate that the Terc−/− and Wrn−/−Terc−/−

mutations in mice cause a bone phenotype very similar to that seenin age-related bone loss in humans (Table 1), including osteoblastdysfunction, and thus are appropriate models for studying senileosteoporosis.

MATERIALS AND METHODSAnimalsThe University of Pennsylvania Institutional Animal Care and UseCommittee approved the use of mice described in this study. Mutant micehad the Wrn−/− and Terc−/− alleles backcrossed on the C57BL/6J background(for >11 generations). Fourth-generation (G4) Wrn−/−Terc−/− mice wereproduced as previously described (Pignolo et al., 2008; Wang et al., 2012).WT mice were obtained via standard matings using the C57Bl/6J strain thatwas used for backcrossing the Wrn−/− and Terc−/− alleles. Wrn−/− mutantswere generated by crossing Wrn+/− mutants. Terc−/− and Wrn−/−Terc−/− miceused in experiments were from G4 lineages. Young animals across allgenotypes were used in experiments at 3 months of age. Aged WT andWrn−/− mutant animals were sacrificed at 15 months. Terc−/− andWrn−/−Terc−/− mutants were sacrificed by 15 months or earlier, when theyexhibited signs of significant suffering and impending demise includingsevere lethargy and/or major (>15%) weight loss. Based on our previousexperience, animals exhibiting these moribund features died within 1 week.The average age of the older Terc−/− group was 11 months of age and thatof the older Wrn−/−Terc−/− group 10 months of age.

μCT analysisTrabecular and cortical boneHigh-resolution images of femurs were acquired by using a Scanco VivaCT40 device (Bruettisellen, Switzerland). The femurs were scanned with asource voltage of 55 kV, a source current of 142 μA and an isotropic voxelsize of 10.5 μm. After scanning, three-dimensional image data wasreconstructed and structural indices were calculated using Scanco μCT V6.1software.

The area for trabecular analysis started 57.5 μm proximal from the growthplate at the distal end of the femur and extended proximally 1050 μm towardthe femoral head. The ROI for measurement of trabecular microarchitecturalvariables was defined manually by outlining the bone within theendocortical margins and allowing a few voxels between the ROI and theendocortical margin, as described previously (Bouxsein et al., 2010). Anupper threshold of 1000 Hounsfield units and a lower threshold of 220Hounsfield units were used to delineate each pixel as ‘bone’ or ‘non-bone’.Representative three-dimensional cross-sectional images for each genotypewithin the young and aged groups are shown in gray scale. Trabecular bonevolume per total volume (BV/TV), mean trabecular thickness (Tb. Th),mean trabecular number (Tb. N, and mean trabecular separation (Tb. Sp)were calculated using Scanco μCT V6.1 software.

The cortical ROI was defined as extending 262.5 μm distally and262.5 μm proximally from the midshaft. An upper threshold of 1000Hounsfield units and a lower threshold of 260 Hounsfield units were usedto delineate each pixel as ‘bone’ or ‘non-bone’. Cortical area per total area(Ct. Ar/Tt. Ar), average cortical thickness (Ct. Th), periosteal surface andendosteal surface were calculated using Scanco μCT V6.1 software.

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Fig. 5. Mechanical alterations in Terc−/− and Wrn−/−Terc−/− boneaccompany their osteoporotic phenotype. Trabecular bone analysis ofyoung and aged femurs for (A,D) structure model index (SMI) and (B,E)degree of anisotropy (DA). (C,F) Cortical moment of inertia (MOI; mm4) isdecreased in young Terc−/− and older Terc−/− and Wrn−/−Terc−/− micecompared with WT. Data represent means ± s.e.m. Statistical significance is*P<0.05 and ***P<0.0001 compared with WT in each age group.

Fig. 6. Increase in bone-marrow fat content in Wrn−/−Terc−/− mutants.Adipocyte volume was measured in WT, Wrn−/−, Terc−/− and Wrn−/−Terc−/−

femurs from young and aged animals. Data represent average ± s.e.m.Statistical significance is ****P<0.0001 or *P<0.05 compared with young WT(n=5 per group).

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PorosityHigh-resolution images of femurs were acquired by using a Scanco μCT 35device (Bruettisellen, Switzerland). The femurs were scanned with a sourcevoltage of 55 kV, a source current of 142 μA and an isotropic voxel size of6 μm. Porosity was assessed from the midshaft and extending 600 μmtoward the proximal femur head. An upper threshold of 1000 Hounsfieldunits and a lower threshold of 447 Hounsfield units were used to delineateeach pixel as ‘bone’ or ‘pores’. After scanning, porosity was calculated usingScanco μCT V6.1 software.

Histological analysisTo measure dynamic bone formation parameters, mice were injectedintraperitoneally with calcein (Sigma, St Louis, MO, USA; 30 mg/kg bodyweight) and xylenol orange (Sigma, St Louis, MO, USA; 90 mg/kg bodyweight) on day 9 and day 2 before tissue harvest.

Mouse hind limbs were excised, cleaned of soft tissue and fixed in 3.7%formaldehyde for 72 hours. Isolated bone tissue was dehydrated in gradedalcohols (70 to 100%), cleared in xylene and embedded in methylmethacrylate (80% methyl methacrylate, 20% dibutyl phthalate, 2.5%benzoyl peroxide) by standard methods. Femurs placed in plastic blockswere cut longitudinally to expose trabecular bone using a Polycut-Smotorized microtome (Reichert-Jung, Nossloch, Germany). Consecutive 5-μm sections were collected beginning 25 μm into trabecular bone forhistological analysis. For all animals, except those in the aged Terc−/− andWrn−/−Terc−/− groups, at least nine consecutive sections were used foranalysis; in the latter two groups, at least 18 consecutive sections were used.

Femur sections were deplasticized in xylene and assessed for kineticparameters of bone formation. Selected ROIs (100 μm distal to the growthplate and 50 μm in from the endosteal cortical bone) were visualized usinga Nikon Eclipse 90i microscope. Image capture was performed using NISElements Imaging Software 3.10 Sp2 and a Nikon DS-Fi1 camera using

Nikon 4×/0.2 Plan Apo and 40×/0.95 Plan Apo objectives. The BioquantOsteo II digitizing system (R&M Biometrics, Nashville, TN, USA) wasused for image analysis according to the manufacturer’s instructions.Mineralizing surface (MS/BS %) and MAR (μm/day) were quantified fromROIs in trabecular bone using a Nikon 20×/0.75 Plan Apo objective. BFRwas calculated from mineralizing surface and MAR. Femur tissue sectionswere then stained with Goldner’s Trichrome or TRAP as previouslydescribed (Brennan et al., 2011; Singh et al., 2013). Osteoid (mm),osteoblast number (Ob N/BS, /mm) and osteoclast number (OCL N/BS,/mm) were quantified within the ROI described above (Parfitt et al., 1987)using Nikon 40×/0.95 Plan Apo objectives. Adipocytes (adipocyte vol/tissuevol) were identified morphologically and quantified as previously described(Elbaz et al., 2009) using a Nikon 20×/0.75 Plan Apo objective. Imagecapture was performed as described above.

StatisticsTo determine the effects of genotypes, two-way analysis of variance(ANOVA) was used with the following factors: age (young and aged) andgenotype (WT, Wrn−/−, Terc−/−, Wrn−/−Terc−/−). The Tukey post-hoc methodfor multiple comparisons was used to compare results among young WT,Wrn−/−, Terc−/− and Wrn−/−Terc−/− mice, as well as among aged WT, Wrn−/−,Terc−/− and Wrn−/−Terc−/− mice (within group comparison), and to comparebetween age-grouped genotypes (intergroup comparison). All statisticalanalyses were performed using GraphPad Prism 4.0 software (San Diego,CA, USA). An adjusted P-value of <0.05 was considered significant for allanalyses. All statistical tests are two-sided. Data are represented as mean ±standard error of the mean (s.e.m.).

AcknowledgementsWe thank Dr Sherry Liu (University of Pennsylvania Perelman School of Medicine)for technical support and helpful discussions regarding the μCT work in this paper.


Table 1. Mouse models of telomere dysfunction recapitulate bone changes in human senile osteoporosis Characteristic Human senile osteoporosis WT (15 m) Wrn–/– (15 m) Terc–/– (3-15 m) Wrn–/–Terc–/– (3-15 m)

Trabecular bone volume Decreased (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

–

Trabecular thickness Decreased (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

– –

Trabecular number Decreased (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

–

Trabecular separation Increased (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

Cortical bone volume Decreased (Rehman et al., 1994; Clarke et al., 1996; Bell et al., 1999; Mullender et al., 2005; Macdonald et al., 2011)

– –

Cortical thickness Decreased (Rehman et al., 1994; Clarke et al., 1996; Bell et al., 1999; Mullender et al., 2005; Macdonald et al., 2011)

– –

Porosity Increased (Feik et al., 1997; Bousson et al., 2001; Ostertag et al., 2009; Nishiyama et al., 2010; Zebaze et al., 2010; Macdonald et al., 2011)

– –

Osteoblast number Decreased (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

OB function: MS/BS MAR BFR Osteoid

Decreased (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

– – – –

– – –

Osteoclast number Unchanged (Parfitt, 1984; Kleerekoper et al., 1985; Aaron et al., 1987; Rehman et al., 1994; Mullender et al., 2005; Pernow et al., 2009; Macdonald et al., 2011)

–

–

Adipocytes Increased (Ostertag et al., 2009; Zebaze et al., 2010) – – Arrows represent relative increase or decrease compared to young WT. m, months.

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Competing interestsThe authors declare no competing financial interests.

Author contributionsT.A.B.: collection and/or assembly of data, data analysis and interpretation,manuscript writing and final approval of manuscript; K.P.E. and Q.C.: provision ofstudy materials, final approval of manuscript; C.M.L. and M.T.S.: collection of data,final approval of manuscript; K.D.H.: collection and/or assembly of data, dataanalysis and interpretation, manuscript writing, and final approval of manuscript;S.X.X.: data analysis and interpretation, manuscript writing, and final approval ofmanuscript; F.B.J.: collection and/or assembly of data, provision of study materials,data analysis and interpretation, manuscript writing, and final approval ofmanuscript; R.J.P.: conception and design, financial support, collection and/orassembly of data, data analysis and interpretation, manuscript writing, and finalapproval of manuscript.

FundingThis work is supported by National Institutes of Health/National Institute on Aginggrant R01AG028873 (R.J.P.) and a National Institutes of Health/National Instituteof Arthritis, Musculoskeletal, and Skin Diseases grant P30-AR050950 sub-project(R.J.P.).

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