Age-related inflammation triggers skeletal stem/progenitor cell dysfunctionAnne Marie Josephsona,b, Vivian Bradaschia-Correaa, Sooyeon Leea, Kevin Leclerca, Karan S. Patela, Emma MuinosLopeza, Hannah P. Litwaa, Shane S. Neibarta, Manasa Kadiyalaa, Madeleine Z. Wonga, Matthew M. Mizrahia,Nury L. Yima, Austin J. Rammea, Kenneth A. Egola, and Philipp Leuchta,b,1

aDepartment of Orthopedic Surgery, New York University School of Medicine, New York, NY 10003; and bDepartment of Cell Biology, New York UniversitySchool of Medicine, New York, NY 10016

Edited by Helen M. Blau, Stanford University, Stanford, CA, and approved February 25, 2019 (received for review June 21, 2018)

Aging is associated with impaired tissue regeneration. Stem cellnumber and function have been identified as potential culprits. Wefirst demonstrate a direct correlation between stem cell number andtime to bone fracture union in a human patient cohort. We thendevised an animal model recapitulating this age-associated decline inbone healing and identified increased cellular senescence caused by asystemic and local proinflammatory environment as the major contrib-utor to the decline in skeletal stem/progenitor cell (SSPC) number andfunction. Decoupling age-associated systemic inflammation from chro-nological aging by using transgenic Nfkb1KO mice, we determinedthat the elevated inflammatory environment, and not chronologicalage, was responsible for the decrease in SSPC number and function.By using a pharmacological approach inhibiting NF-κB activation, wedemonstrate a functional rejuvenation of aged SSPCs with decreasedsenescence, increased SSPC number, and increased osteogenic function.Unbiased, whole-genome RNA sequencing confirmed the reversal ofthe aging phenotype. Finally, in an ectopic model of bone healing, wedemonstrate a functional restoration of regenerative potential in agedSSPCs. These data identify aging-associated inflammation as the causeof SSPC dysfunction and provide mechanistic insights into its reversal.

regeneration | skeletal stem cell | senescence | inflammation |bone healing

All tissues are affected by aging, but diseases that weaken theskeleton constitute the most prevalent chronic impairment

in the United States (1). Although skeletal diseases and conditionsare seldom fatal, they can significantly compromise function anddiminish quality of life. Perhaps most importantly, age-relatedchanges in skeletal health may be traced back to the skeletalstem cell. Like other stem-cell pools, skeletal stem/progenitors areimpacted by aging. For example, skeletal stem cells from peopleolder than age 65 y, even if they are healthy, make less bone thanstem cells from younger individuals, irrespective of sex (2). Insteadof becoming bone-producing osteoblasts, skeletal stem cells fromolder people differentiate into fat-producing adipocytes (3), andthis may partly explain why bone-forming ability declines withincreasing age (3, 4).Chronic inflammation in the elderly (“inflamm-aging”) is thought

to be a major contributor to the decline in the regenerative capacityof the skeleton (5). In contrast to a well-balanced inflammatory re-sponse after trauma, which is crucial for successful bone repair (6),chronic unbalanced elevation of proinflammatory cytokines inhibitsregeneration in a variety of other tissues (7). Its effect on the skeletalstem/progenitor cell (SSPC) is yet unknown. To address thisknowledge gap, we hypothesized that chronic inflammation me-diated by NF-κB activation—irrespective of age—contributes to adeterioration of the regenerative function of the stem-cell pool byinducing cellular senescence and decreasing SSPC number andfunction. Our data provide convincing evidence that pharmacologicinhibition of NF-κB activation leads to a functional rejuvenation ofthe SSPC pool, resulting in bone regeneration equal to that seen inyoung animals.

ResultsSkeletal Stem Cell Frequency Decreases with Aging. To investigate aclinically relevant age-associated effect on skeletal stem cell fre-quency and function, we first examined iliac crest bone graft(ICBG) samples from 36 patients (20 male, 16 female) with agesranging from 24 to 89 y who underwent operative fixation of anupper- or lower-extremity fracture. FACS with CD271 as a humanskeletal stem cell marker (8–11) revealed that SSPC frequencysignificantly declined with increasing age (Fig. 1 A and B). It isgenerally well accepted among orthopedic surgeons that fractures inelderly subjects heal more slowly and less reliably, and therefore weasked whether SSPC frequency correlates with time to bony union.We prospectively evaluated clinical and radiographic fracture unionin this cohort and discovered that a lower SSPC number was as-sociated with longer time to fracture union (Fig. 1C). To identify themechanism involved in this decline in SSPC number and function,we chose a mouse model to further investigate the process ofskeletal stem cell aging.

Aging Impairs Bone Regeneration. To evaluate the extent to whichthe process of aging affects bone healing, we first employed astandardized tibial monocortical defect model in young (12-wk-old) and middle-aged (52-wk-old) male C57BL/6 mice. We an-alyzed bone healing by using histology, histomorphometry, andmicro-CT (μCT). Two weeks after surgery, the injury sites wereanalyzed by histology. Whereas injuries in the young animalsshowed abundant woven bone within the defect site (Fig. 2 A and

Significance

As we age, our capacity for tissue repair and regeneration inresponse to injury declines. Accordingly, bone repair is delayedand impaired in older patients. At the cornerstone of bonehealing is the skeletal stem/progenitor cell (SSPC), whose func-tion and number diminishes with age. However, the mechanismsdriving this decline remain unclear. Here, we identify age-associated inflammation (“inflamm-aging”) as the main culprit ofSSPC dysfunction and provide support for a central role of NF-κBas a mediator of inflamm-aging. Our results show that modifica-tion of the inflammatory environment may be a translational ap-proach to functionally rejuvenate the aged SSPC, therebyimproving the regenerative capacity of the aged skeleton.

Author contributions: A.M.J., V.B.-C., S.L., K.L., and P.L. designed research; A.M.J., V.B.-C.,S.L., K.L., K.S.P., E.M.L., H.P.L., S.S.N., M.K., M.Z.W., M.M.M., N.L.Y., A.J.R., K.A.E., and P.L.performed research; A.M.J., V.B.-C., S.L., K.L., K.S.P., E.M.L., H.P.L., and P.L. analyzed data;and A.M.J. and P.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: philipp.leucht@nyulangone.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810692116/-/DCSupplemental.

Published online March 20, 2019.

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C), the injuries in the middle-aged animals exhibited a smallerarea of woven bone, with bone formation predominantly be-tween the cortical edges (Fig. 2 B and D). μCT imaging and 3Drendering confirmed this finding (Fig. 2 E and F). Histo-morphometry using μCT demonstrated a smaller callus volume[bone volume/total volume (BV/TV)], trabecular number (Tb.N), and trabecular thickness (Tb.Th) and increased trabecularspacing (Tb.Sp; Fig. 2G). This first experiment demonstratedthat 52-wk-old WT mice exhibit a phenotype of age-related im-paired bone regeneration. Thus, we elected to use this age groupfor the subsequent experiments aimed at understanding the ef-fect of aging on bone healing and SSPC function.

Aging Leads to a Decrease in SSPC Number. The key ingredient tosuccessful bone regeneration is the SSPC. To determine whethera decline in SSPC number is responsible for the impaired re-generative capacity of the aging skeleton, as seen in our humancohort, we used FACS with the inclusive SSPC marker LepR(12). CD45−CD31−Ter-119−LepR+ cells (LepR+ cells) comprisea heterogeneous mix of Sca-1+, PDGFRα+, CD51+, and CD105+

SSPCs (SI Appendix, Fig. S1), and Morrison and coworkers (12)demonstrated that LepR+ cells make up 0.3% of bone marrowcells; they differentiate into bone, cartilage, and fat in vivo and invitro and, most importantly, give rise to bone postnatally and inresponse to injury. Bones from middle-aged mice containedsignificantly fewer LepR+ cells compared with bones from youngmice (Fig. 3 A and B), and cfu assays confirmed this finding(Fig. 3C).

Circulating Systemic Factors Lead to Skeletal Stem Cell Aging. Hav-ing now established that SSPC frequency declines in mice simi-larly to our observation in humans, we next sought to identify thecause for this decline in stem cell number. Cell senescence, anirreversible arrest in cell division, has been associated with stemcell attrition in a multitude of other aged tissues (reviewed in ref.13). Cell senescence is accompanied by a senescence-associatedsecretory phenotype (SASP), a local proinflammatory microen-vironment, which acts on surrounding cells and inhibits theirproliferation and cellular function (14). This paracrine effect ofthe SASP then induces senescence in cells within the immediate

vicinity, commencing a vicious cycle that results in a functionaldecline of the entire tissue and organ (14, 15). We hypothesizedthat serum from middle-aged mice contains proinflammatorySASP factors and that this cytokine milieu leads to a functionaldecline of the skeletal stem cell. SSPCs from young (12-wk-old)mice were exposed to sera from middle-aged (52-wk-old) mice invitro (Fig. 4A). Compared with the homochronic control group(young serum/young cells), which demonstrated a linear increasein cell proliferation over a 7-d time course, the heterochronicgroup (middle-aged serum/young cells) exhibited a functional

Fig. 1. Skeletal stem/progenitor cell frequency declines in the aging pa-tient. (A) FACS analysis of ICBG samples from 36 patients (20 male and16 female) of varying ages revealed a significant (P < 0.05) negative corre-lation between age and SSPC number. (B) SSPC frequency is significantlydecreased in patients older than 50 y of age (P < 0.05). (C) SSPC number isnegatively correlated with time to bony union (P < 0.05). Green dots identifyfractures that healed clinically and radiographically within 6 mo. Red dotsmark patients with fracture union after 6 mo.

Fig. 2. Aging results in impaired bone healing. (A and B) Histological sec-tions of tibial monocortical defects 14 d after injury in 12-wk-old and 52-wk-old WT mice stained with Movat’s pentachrome. (C and D) Aniline bluestaining depicting bone matrix deposition within the cortical defect. (E andF) Three-dimensional μCT reconstructions with surface rendering of bonyregenerate (red) showing smaller callus size in the defect site of middle-agedanimals. (G) Analysis of μCT data showing BV/TV (n = 6, P < 0.001), Tb.N (n =6, P < 0.001), Tb.Th (n = 6, P < 0.05), and Tb.Sp (n = 6, P < 0.001) at post-operative day (POD) 7 and POD 14 in young and middle-aged mice. bm,bone marrow; c, cortical bone; is, injury site.

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arrest in cell division (Fig. 4B), suggesting the presence of an“aging” factor within the serum. In line with this assumption,senescence-associated β-gal (SA-β-gal) staining revealed a sig-nificant increase in SSPC senescence after heterochronic serumtreatment (Fig. 4C). Next, we sought to identify whether theSSPCs treated with middle-aged serum started to express SASPfactors themselves. Quantitative RT-PCR for SASP markersdemonstrated that, relative to the homochronic control, in-terleukin-1α (Il1a), tumor necrosis factor-α (Tnfa), and nuclearfactor kappa-light-chain-enhancer of activated B cells p65 (Rela)significantly increased, consistent with a SASP (Fig. 4D). Thus,aging is associated with a systemic cytokine milieu that directlyleads to an arrest of cell division, induces cell senescence, andresults in expression of SASP factors. We confirmed these invitro finding by using an SA-β-gal assay (16) on SSPCs freshlyharvested from young and middle-aged mice, and we discoveredthat middle-aged SSPCs were four times more likely to be se-nescent compared with young SSPCs (Fig. 4 E–G). qRT-PCR ofthe whole bone marrow confirmed an increase in the senescencemarkers Cdkn2a (p16) and Cdkn1a (p21) (17–19) (Fig. 4H).In response to the heterochronic serum treatment, we observed

an increase in Il1a and Tnfa expression in the young SSPCs (Fig.4D). These two proinflammatory cytokines lead to activation ofNF-κB, a key mediator of inflammation (20). Therefore, we sur-veyed young and middle-aged SSPCs for NF-κB activation. Be-cause phosphorylated NF-κBp65 (p-p65) is a prerequisite fornuclear localization and thus NF-κB activation, we first examinedNF-κB activation by using Western blot for p-p65. We observed anincreased p-p65/p65 ratio in middle-aged SSPCs (Fig. 4I). Wethen confirmed increased nuclear localization of NF-κBp65 byusing immunofluorescence and detected a twofold increase inmiddle-aged SSPCs (Fig. 4 J and K). Together, these data confirmactivation of NF-κB as a key inflammatory mediator in middle-aged SSPCs.

These data strongly suggest that the age-associated decline inSSPC frequency is caused by a systemic proinflammatory envi-ronment mediated through NF-κB and likely leading to in-creased cellular senescence.

NF-κB–Mediated Inflammation Induces SASP in Young Skeletal Stem/Progenitor Cells. The age-associated cytokine profile leads to asystemic proinflammatory environment, inducing and potentiat-ing NF-κB activation (Fig. 4) (13), and we hypothesize that thisinflammatory milieu is responsible for the decrease in SSPCnumber and function. NF-κB is the fundamental transcriptionalregulator of inflammation and controls the expression of genesencoding for proinflammatory cytokines, chemokines, and ad-hesion molecules (20). Proinflammatory stress and cell senes-cence activate Nfkb expression (20). We postulate that, withaging, SSPC frequency and function declines, and that this de-crease in SSPC number and function is caused by an increasedinflammatory microenvironment. To experimentally separateinflammation from aging, we used the Nfkb1−/− mouse model,which has served as a model organism for low-level chronic in-flammation in a plethora of studies involving liver regeneration(21), memory loss (22), and stress response (23). Deletion of NF-κB1 results in the activation of the NF-κB, which in turn leads toincreased senescence and accelerated aging. Nfkb1−/− mice lackthe expression of the p105 and p50 NF-κB protein. The lack ofthese two NF-κB subunits results in the inability to form p50:p50 homodimers (repressor of proinflammatory gene expression)while still being able to generate RelA-containing NF-κB dimers(activators of proinflammatory gene expression), which results inan enhanced response to inflammatory stimuli (24, 25). Young,30-wk-old Nfkb1−/− mice housed in a pathogen-free environmentexhibit hallmarks of premature aging with ataxia, kyphosis,sarcopenia, cardiac hypertrophy, and many other age-associatedconditions that are related to an activated chronic inflammatory state(21), thus offering a valuable model organism to study re-generation in a model of low-grade inflammation in the absenceof chronological aging.First, we had to confirm that, in fact, 30-wk-old Nfkb1−/− mice

exhibit a proinflammatory cytokine profile similar to the oneobserved in middle-aged WT mice. qRT-PCR confirmed theexpression of a SASP-like phenotype with up-regulation of Rela(NF-κBp65), Cyclooxygenase 2 (Cox2), Il6, Il10, Tnfa, Il1b, andCdkn2a (p16) in bone marrow stromal and bone-lining cells (Fig.5A). FACS analysis of 30-wk-old, 52-wk-old Nfkb1−/−, and age-matched WT mice demonstrated fewer LepR+ SSPCs in theNfkb1−/− mice (Fig. 5B and SI Appendix, Fig. S2A), further con-firming that inflammation, not aging, is driving the decline in SSPCnumber. We next analyzed the expression of SASP factors withinfreshly isolated LepR-positive SSPCs and found significantly higherexpression levels of Il1b, Il6, and Rela in the SSPC population of 30-wk-old Nfkb1−/− mice compared with age-matched WT mice (SIAppendix, Fig. S2B). Gene-expression analysis of the microenvi-ronment, here captured in Q1/2 (CD31, CD45, and Ter-119–posi-tive cells), revealed no changes in comparison with a similar cellpopulation in age-matched WT mice (SI Appendix, Fig. S2B). Wethen further investigated the microenvironment of Nfkb1−/− miceand separated the myeloid and lymphoid compartments by usingwell-accepted surface markers. Within the lymphoid compartment,Il1b and Rela were down-regulated and Tnfa was up-regulated inNfkb1−/− mice, whereas there were no significant differences forthese cytokines in the myeloid compartment (SI Appendix, Fig. S2).These data suggest a shared contribution of the bone marrow andthe SSPC compartment to the proinflammatory milieu to which theSSPCs then respond with increased SASP expression (SI Appendix,Fig. S2). Next, we sought to test whether this proinflammatoryenvironment in the Nfkb1−/− mice inhibits cell division, similar towhat we had observed in the middle-aged WT mice. We performeda cell proliferation assay and detected an absence of cell

Fig. 3. SSPC frequency declines with aging. (A) Representative FACS plots ofyoung and middle-aged skeletal elements showing decrease in LepR+ SSPCswith aging. (B) Summary plot of FACS data demonstrating decrease in SSPCfrequency in 52-wk-old (wo) mice (n = 5, P < 0.01). (C) cfu assay of young andmiddle-aged bone marrow showing representative colony staining andgraphical depiction of quantification (n = 3, P < 0.0001).

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proliferation in SSPCs from 30-wk-old Nfkb1−/− mice, whereasage-matched WT SSPCs exhibited a linear increase in cell pro-liferation (Fig. 5C). An absence of cell proliferation can be at-tributed to apoptosis, senescence, quiescence, and prematuredifferentiation. We used CellTrace to identify cell-cycle activityof WT and Nfkb1−/− cells and demonstrated that Nfkb1−/− cellsare low-cycling as they are preferentially found in generation 1(SI Appendix, Fig. S3 A and B). We then further analyzed thecells within generation 1 and showed that fewer than 1% of cellswere apoptotic, without difference between the two groups (SIAppendix, Fig. S3C). Last, premature differentiation was ruledout by using cell-surface markers characteristic for stem cells.We observed no difference in PDGFRα+ Sca-1+ cell numberbetween the two cell populations (SI Appendix, Fig. S3C).Thus far, we have shown that the proinflammatory environment

in Nfkb1−/− mice leads to a decrease in SSPC number, an increasein cell senescence, and a decrease in cell division. Next, we in-vestigated the trilineage potential of Nfkb1−/− SSPCs. First, weexposed 30-wk-old SSPCs to osteogenic and growth media (GM)and assessed mineral deposition. Whereas WT cells formed con-fluent Alizarin red-positive mineral, Nfkb1−/− cells deposited onlysmall islands of Alizarin red-positive bone matrix when subjectedto osteogenic differentiation media (Fig. 5D). Quantification

revealed diminished mineral deposition and alkaline phosphataseactivity in Nfkb1−/− cells (Fig. 5E). This decrease in osteogenesis ofthe Nfkb1−/− cells was confirmed by qRT-PCR for Runx2, osterix(Osx), and alkaline phosphatase (Alk Phos; Fig. 5F). Second, weanalyzed chondrogenic and adipogenic differentiation in Nfkb1−/−

cells. Chondrogenesis, assessed by using micromass cultures,revealed smaller and disorganized micromasses without the char-acteristic hypertrophic center surrounded by less differentiatedchondrocytes in the periphery, as seen in the WT micromasses (SIAppendix, Fig. S4A). Adipogenic differentiation revealed an in-crease in Oil Red O-positive cells. In addition, we observed agreater number of adipocytes in the tibial bone marrow ofNfkb1−/− mice (SI Appendix, Fig. S4B). Finally, to identify whetherthe Nfkb1−/− phenotype is truly related to the proinflammatoryenvironment, we treated young WT SSPCs with serum from youngNfkb1−/− mice. Serum treatment resulted in a uniform increase inproinflammatory cytokine expression in the young cells (SI Ap-pendix, Fig. S5). The previously described heterochronic serumexperiments in WT mice (Fig. 4) demonstrated an arrest in pro-liferation in response to treatment with serum from middle-agedserum (Fig. 4B). We sought to determine whether this same effecton proliferation can be observed when cells were treated withserum from Nfkb1−/− mice. We used proliferating cell nuclear

Fig. 4. Systemic cytokines are responsible for aging phenotype. (A) Schematic illustration depicting homochronic (young serum/young cells) and hetero-chronic (serum from middle-aged mice/young cells) in vitro culture conditions. (B) Cell proliferation assay of young SSPCs treated with sera from young ormiddle-aged mice showing an inhibitory effect of middle-aged sera on mitotic activity (n = 5). (C) Young SSPCs subjected to middle-aged serum exhibit morecell senescence at 4 d (n = 3, P < 0.01) and 7 d (n = 3, P < 0.001) as measured by SA-β-gal staining. (D) qRT-PCR shows induction of the expression of senescence-associated genes Il1a, Tnfa, and Rela in cells subjected to sera from middle-aged mice (n = 3, P < 0.05). (E–G) SA-β-gal staining of SSPCs from 12- and 52-wk-oldmice showing increased senescence (arrowheads) in the aging animal. Quantification reveals a significant increase in senescence at 52 wk of age (n = 4, P <0.01). (H) Consistent with the SA-β-gal staining, expression levels of Cdkn2a (p16) and cdkn1a (p21) were elevated in the middle-aged bone compartment (n =7, P < 0.05). (I) Western blot for p-p65 reveals increased NF-κB activation in the middle-aged SSPCs (n = 3, P < 0.05). (J) Immunofluorescence of NF-κBp65 inyoung and middle-aged SSPCs revealed nuclear localization of NF-κBp65 in the middle-aged cells. (K) Quantification of NF-κBp65 (p65) nuclear localizationdemonstrates increased NF-κB activation in 52-wk-old SSPCs (n = 3, P < 0.001).

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antigen (Pcna) gene expression to evaluate cell division in thisexperiment and observed a decrease in Pcna expression in the cellstreated with Nfkb1−/− serum (SI Appendix, Fig. S5). Collectively,these data confirmed that heightened inflammation, and notchronological aging, is responsible for a decrease in number andfunction of SSPCs.

Inflammation-Associated SSPC Decline Is Reversible. Increased cellsenescence (26), predominant adipogenic differentiation of SSPCs(27), and amplified apoptosis (28) are all associated with aging andresult in an increased inflammatory response known as inflamm-aging (29, 30). First, we sought to identify whether the inflamm-aging phenotype is detectable in middle-aged mice on a systemiclevel. We performed multiplex analysis on serum from young andmiddle-aged mice and detected increases of the proinflammatorycytokines IFN-γ, TNF-α, and IL-6 in the aging animals (Fig. 6A).We confirmed this finding by qRT-PCR, which revealed up-regulated expression of the proinflammatory cytokines Rela, Tnfa,Il6, Il1a, and Il1b in SSPCs from middle-aged animals (Fig. 6B).These experiments confirmed that 52-wk-old WT mice exhibit aninflammatory cytokine profile consistent with inflamm-aging. If, infact, chronic low-grade inflammation is responsible for the decreasein SSPC number, increased senescence, and overall decreasedregenerative potential of aged animals, treatment with an antiin-flammatory drug may overcome these negative effects. We there-fore treated 52-wk-old mice with sodium salicylate, a low-gradeantiinflammatory agent proven to inhibit NF-κB pathway activation(31–33), for 8 wk, and then harvested SSPCs and analyzed the ex-pression levels of the aforementioned pro- and antiinflammatorycytokines. Salicylate treatment resulted in a decrease of the proin-flammatory cytokines Rela, Cox2, and Il1b (Fig. 6B). This confirmedthat the treatment protocol successfully repressed aging-induced

chronic inflammation, as the expression level returned to levelsmeasured in young animals.As we postulated that the age-associated elevation of inflammatory

cytokines results in increased NF-κB activation, we wanted to de-termine whether the observed systemic NSAID-induced reduction incytokine levels resulted in decreased NF-κB signaling. We againtreated young SSPCs with serum from young, middle-aged, andmiddle-aged NSAID-treated mice in vitro. This experiment revealedthat serum from middle-aged mice treated with sodium salicylate didnot result in nuclear localization of NF-κBp65, as shown by immu-nofluorescence and quantification (Fig. 6C). We confirmed this ob-servation by using Western blot for p-p65, which was increased in thecells treated with middle-aged serum and returned to juvenile levelswhen treated with serum from NSAID-treated middle-aged mice(Fig. 6D).Previously, we had shown that the proinflammatory environ-

ment had a direct negative effect on cell division (Fig. 4B) andinduced senescence (Fig. 4 C and E–G). If salicylate treatmentreduces this chronic inflammatory milieu, theoretically, cell se-nescence should decrease as a result. We performed an SA-β-galassay with cells from young, middle-aged, and middle-agedsalicylate-treated mice. Similar to our previous results (Fig. 4G),with aging, the senescent cell fraction increased (Fig. 6E);however, after salicylate treatment, the percentage of SA-β-gal–positive cells significantly decreased (Fig. 6E). This was con-firmed in the heterochronic serum assay. Young SSPCs treatedwith serum from middle-aged antiinflammatory-treated mice exhibi-ted a senescence phenotype similar to the young homochronic group(SI Appendix, Fig. S6). If senescence is reduced in response to salic-ylate treatment, does this lead to an increase in the SSPC frequencywithin the bone marrow? By using FACS for LepR, we showed thataging resulted in a significant decrease in SSPC number and thatsalicylate treatment partially recovered this loss of SSPC number (Fig.

Fig. 5. Young Nfkb1−/− mice mimic aging phenotype of SSPCs. (A) qRT-PCR of bone tissue from 30-wk-old Nfkb1−/− and age-matched WT mice revealingproinflammatory SASP-like phenotype in Nfkb1−/− mice (n = 3, P < 0.05). (B) Nfkb1−/− mice show characteristic decline of SSPC number at younger age (n = 4,P < 0.05). (C) Proliferation assay of WT and Nfkb1−/− SSPCs showing linear increase in cell number for WT cells and a steady state for Nfkb1−/− cells (n = 4). (D)Alizarin red staining of mineralization assay for WT and Nfkb1−/− cells in osteogenic media (OM) and GM. (E) Quantification of Alizarin red and alkalinephosphatase staining showing reduced osteogenic differentiation of Nfkb1−/− cells (n = 4, ***P < 0.001). (F) Expression analysis for osteogenic genes of WTand Nfkb1−/− cells treated with osteogenic differentiation media (n = 4, **P < 0.01 and ***P < 0.001).

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6F). Cfu-forming assay analysis confirmed the reversal of decreasedcfu formation after salicylate treatment (Fig. 6G). These analysesfurther confirmed that it is the inflammatory component ofinflamm-aging and not the chronological aging component thatleads to a decrease in SSPC number.

RNA Sequencing Analysis Reveals Rejuvenation of the SSPC Pool AfterAntiinflammatory Treatment. Number and function of SSPCs arecritical for successful bone regeneration. Having established thatSSPC number declines with age and that this decrease can be haltedby modulating the age-associated proinflammatory environment, wenext sought to understand the effect of changes in the immuneenvironment on the transcriptome of SSPCs. We first used an un-biased sequencing approach to compare the transcriptome of youngand middle-aged SSPCs. Hierarchical cluster analysis revealed a

stark separation between young and middle-aged SSPCs (Fig. 7A).In line with our FACS analysis showing a rescue of SSPC frequency,we observed a shift of the transcriptional profile of middle-agedSSPCs to that of the young SSPCs after antiinflammatory treat-ment. We then used gene-set enrichment analysis (GSEA) to fur-ther understand this potential rejuvenation of the SSPC pool (Fig.7B). SSPCs from young animals enriched for genes associated withstemness, as did SSPCs from middle-aged antiinflammatory-treatedmice, again supporting a reversal of the aging phenotype. Similarly,genes associated with osteogenesis and decreased adipogenesiswere enriched in the young SSPCs, and again immunomodulationwith an antiinflammatory agent led to an increased enrichment forthese gene sets in middle-aged SSPCs (Fig. 7B). From these data,we conclude that, on a transcriptional level, modulation of the

Fig. 6. Antiinflammatory treatment reverts inflamm-aging phenotype and increases SSPC pool. (A) Serum cytokine levels of IFN-γ (P < 0.05), TNF-α (P < 0.01),and IL-6 (P < 0.01) in young and middle-agedWT mice (n = 10). (B) Antiinflammatory drug treatment with salicylate reverses inflamm-aging phenotype (n = 6, P <0.05). (C) Immunofluorescence for NF-κBp65 reveals nuclear localization of NF-κBp65 after treatment with middle-aged serum and a decrease in nuclear local-ization of NF-κBp65 in cells subjected to serum from middle-aged NSAID-treated mice. Quantification confirms increase in NF-κB activation with aging anddecrease to juvenile levels after NSAID treatment (n = 4, P < 0.01). (D) Western blot for p-p65 confirming the decreased NF-κB activation in response to NSAIDtreatment (n = 3, *P < 0.05 and **P < 0.01). (E) SA-β-gal staining of young, middle-aged, and salicylate-treated middle-aged SSPCs show reversal of senescencephenotype in aging animals (n = 3, P < 0.05). (F) Graph showing SSPC frequency in young, middle-aged, and salicylate-treated middle-aged mice (n = 11, **P <0.01 and ***P < 0.001). (G) Cfu-forming assay confirms increase of SSPC frequency after salicylate treatment (n = 3, P < 0.001). tx, treated; wo, weeks old.

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proinflammatory environment in the aging animal leads to a re-versal of the aging phenotype.

Antiinflammatory Drug Treatment Abrogates the Aging Phenotype ofSSPCs and Restores Regenerative Potential. The previous set of ex-periments using unbiased sequencing strongly suggest that SSPCaging can be reversed. Next, we set out to interrogate the cellular

function of the rejuvenated SSPC in an in vitro and in vivo envi-ronment. First, we evaluated the bone marrow compartment as awhole. Gene expression levels of Osx and Osteocalcin (Oc) sig-nificantly decreased in response to aging (Fig. 8A). We next ana-lyzed bone marrow cells from middle-aged mice treated withsalicylate, and here Osx, Oc, and Alp significantly increased com-pared with middle-aged untreated animals, and even reachedlevels equal to or higher than cells from young animals (Fig. 8A).To test the functional differentiation capacity of SSPCs, we ex-posed SSPCs to osteogenic differentiation media in vitro and thenquantified mineralization as a readout for osteogenic differentia-tion. Young SSPCs demonstrated robust mineralization, whereasmiddle-aged SSPCs showed only some isolated foci of minerali-zation (Fig. 8B). SSPCs harvested from salicylate-treated middle-aged mice recovered their osteogenic function, which resulted inmineralization comparable to that of young mice (Fig. 8B). Aginghas been associated with fatty degeneration of the bone marrowcompartment (27). To directly test whether inflammation associ-ated with aging can be attributed to this fatty degeneration, wesubjected young and middle-aged SSPCs to adipogenic differen-tiation media. As expected, middle-aged cells were more prone todifferentiate into adipocytes, as shown by increased Pparg andFabp4 expression, and this was reversed in cells from NSAID-treated mice (Fig. 8C). We confirmed this expression pattern byusing a functional adipogenic differentiation assay and againdemonstrated an increase in adipogenesis with aging (Fig. 8D).However, SSPCs frommiddle-aged salicylate-treated mice exhibitedless adipogenic differentiation (Fig. 8D), suggesting that inflamm-aging plays an active role in fatty degeneration of the bone marrow.We then evaluated tissue sections of young, middle-aged, andmiddle-aged NSAID-treated mice and observed a significant in-crease in adipocyte number with aging, which was reversed in micetreated with sodium salicylate (Fig. 8E).Next, we investigated whether the suppression of low-grade

inflammation had a direct effect on homeostasis of the skeletonand bone regeneration in vivo. First, we analyzed bone homeo-stasis and performed μCT analysis of lumbar spine segments andhumeri of young, middle-aged, and middle-aged salicylate-treated mice. BV/TV, Tb.N, and Tb.Th were significantly re-duced in aged animals compared with young animals, whereasTb.Sp was increased, confirming an age-appropriate bone loss(SI Appendix, Fig. S7). Middle-aged animals treated with salic-ylate showed a skeletal phenotype comparable to the untreatedmiddle-aged control animals, indicating that salicylate treatmentover a 12-wk time course in this age group did not affect bonehomeostasis.Finally, we set out to examine whether the decrease in cell se-

nescence, increase in SSPC number, and shift of the osteogenic/adipogenic balance toward osteogenesis in response to antiin-flammatory treatment resulted in a measurable proregenerativeeffect in vivo. To avoid masking effects of an endogenous healingresponse in a skeletal injury, we elected to use an ectopic trans-plantation model, as this represents the most stringent readout of invivo bone formation of SSPCs (34). Here, we used a subrenalcapsule transplantation assay to test whether suppression of chronicinflammation in the middle-aged animal successfully restores oste-ogenic capacity of the SSPCs in an in vivo setting. The subrenalcapsule assay represents an ideal functional assay because cellstransplanted between the renal capsule and the parenchyma receivesufficient blood supply and nutrients while being devoid of proos-teogenic or prochondrogenic stimuli that would confound thereadout (35). SSPCs from young, middle-aged, and middle-agedsalicylate-treated mice were transplanted under the renal capsule.After 3 wk, histological staining and histomorphometry revealed adecrease in bone formation in the group containing middle-agedSSPCs compared with young SSPCs (Fig. 8 F and H). In starkcontrast, cells transplanted from salicylate-treated middle-aged miceexhibited a robust osteogenic response, similar to that observed with

Fig. 7. RNA-seq analysis reveals a shift toward increased stemness and osteo-genesis and decreased adipogenesis in middle-aged antiinflammatory-treatedmice. (A) Heat map representing gene-expression values of the top 133 geneswith a false discovery rate-adjusted P value less than 0.01 (q > 0.01) across allsamples. Hierarchical clustering of these genes reveals that LepR+ SSPCs isolatedfrom 52-wk-old antiinflammatory-treated mice cluster with 12-wk-old LepR+

SSPCs. Columns indicate single samples, and rows indicate genes. (B) GSEA plotsdemonstrate that young SSPCs and middle-aged treated SSPCs positively cor-relate with gene sets for stemness (BOQUEST STEM CELL UP), osteogenesis(SKELETAL DEVELOPMENT), and decreased adipogenic potential (TSENG ADI-POGENIC POTENTIAL DN).

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juvenile SSPCs (Fig. 8 G and H), indicating that suppression ofchronic inflammation in the middle-aged animal can restore theregenerative capacity of SSPCs.

DiscussionChronic inflammation in elderly subjects has been linked to avariety of diseases (20). Here, we provide evidence that SSPCsrespond to elevated levels of proinflammatory cytokines withincreased senescence, decreased stem/progenitor cell number,and decreased functionality. In a pharmacological rescue experi-ment, we show that reduction of age-related chronic inflammationleads to a functional restoration of bone regeneration through adecrease in stem/progenitor cell senescence, increase in stem/pro-genitor cell number, and osteogenic gene expression (Fig. 9).Chronic inflammation in elderly subjects has been attributed

to a constant decay of extracellular macromolecules and in-tracellular organelles, resulting in an initiation and maintenanceof an immune response (29). Degeneration of aged cells leads tosecretion of reactive molecules, proinflammatory enzymes, andmediators that in turn will lead to further deterioration of thetissue, starting a vicious cycle that is characteristic of this chroniccondition. Although all cells are affected by this proinflammatorymilieu, the impact on the resident stem/progenitor cell, responsiblefor regeneration in response to injury, is likely the most detrimental,as it jeopardizes maintenance of tissue integrity. The toxic media-tors within the tissue environment result in DNA damage, proteindegradation, and organelle injury of the stem/progenitor cell,causing cellular senescence, which in turn results in further stimu-lation of the chronic inflammatory status through the cell’s secretoryphenotype known as the SASP (29, 36). Here, we aimed at dis-rupting this vicious cycle by modulating the proinflammatory envi-ronment by using a mild antiinflammatory drug. As shown by serumcytokine analysis and gene-expression analysis of bone marrow cells,

the described approach resulted in an inflammatory milieu com-parable to that of a young animal, thus allowing us to study whetherthe detrimental effects of inflamm-aging on SSPCs can be reversed.The effect of inflamm-aging on SSPCs can be broken up into

cell-intrinsic and cell-extrinsic changes. Whereas the proinflammatoryenvironment of the aged skeleton exerts its negative effect on thestem/progenitor cell through cell-extrinsic mechanisms such asmodulation of signaling pathways [Wnt, Notch, BMPs (37)], cell-intrinsic mechanisms include self-renewal defects and induction ofstress-induced pathways that lead to cellular senescence (13).Whereas cell-intrinsic defects such as senescence and self-renewaldefect are considered irreversible, extrinsically induced defects maybe reparable. As shown here, modulation of the inflammatory milieuby using a pharmacological compound resulted in a restoration ofregenerative capacity. Interestingly, we observed that a short expo-sure of middle-aged SSPCs to a young systemic environment, as seenin the renal capsule transplantation assay, did not lead to a functionalrestoration. This suggests that the brief exposure to the young envi-ronment alone is not sufficient to functionally rejuvenate SSPCs orthat the molecular microenvironment repressed the properties of theresident stem/progenitor cells, and, when modulated with an antiin-flammatory drug, this repression was reversed, resulting in return ofregenerative potential. This finding is supported by heterochronictransplantation assays and parabiosis experiments, which showed thatthe aging of a variety of stem cells was largely driven by cell-extrinsicmechanisms of the surrounding environment (38). In these assays,introduction of circulation from young animals restored the re-generative potential of the aged skeleton (39), indicating that thedetrimental effects of aging on the progenitor pool may be reversible(40). A prime target for the mechanism of action observed in theparabiosis model is the inflammatory cytokine environment. Here, weprovide strong evidence that inflammation, not chronological aging,is the main driver of SSPC dysfunction, and we demonstrate that

Fig. 8. SSPC function is restored in aging animals after antiinflammatory treatment. (A) qRT-PCR of SSPCs shows increased osteogenic gene expression afterrepression of inflamm-aging (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001). (B) Representative images and quantification of osteogenic differentiationanalyzed by Alizarin red staining (n = 3, P < 0.001). (C) Adipogenic differentiation analyzed by qRT-PCR for adipogenic markers (n = 3, P < 0.05) and (D) OilRed O staining (representative images and quantification; n = 3, **P < 0.01 and ***P < 0.001). (E) Histomorphometry for adipocyte number in the tibial bonemarrow of young, middle-aged, and middle-aged NSAID-treated mice (n = 5, *P < 0.05 and ***P < 0.001). (F and G) Pentachrome histology of renal capsule(rc) transplants of 52-wk-old (wo) untreated and salicylate-treated SSPCs into young WT host mice. (F) Histomorphometry shows a decrease in regenerate sizein middle-aged mice and restoration of regenerative function after salicylate treatment (n = 4, P < 0.05). bg, bone graft; tx, treated.

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NF-κB activation in young animals can mimic the aging phenotype inrespect to SSPC number and function. This is further supported bythe expression of the senescence-associated genes Cdkn1a (p21) andCdkn2a (p16). Recent data suggest distinct roles for Cdkn1a andCdkn2a. Kim et al. (41) and Baker et al. (42) suggest that expressionof Cdkn1a is associated with senescence induced by chronologicalaging, whereas Cdkn2a expression is associated with stress-inducedsenescence (43). In our experiments, dysfunctional SSPCs frommiddle-aged mice exhibit heightened Cdkn2a expression (Figs. 4Hand 5A), possibly indicating a stress response to the increased in-flammatory environment rather than an age effect.Our findings provide strong support for a central role of NF-κB

as a mediator of inflamm-aging. Chen et al. (44) previouslydemonstrated a link between aging and NF-κB activation in aprogeroid mouse model during bone homeostasis, supporting ourfindings during bone regeneration. During homeostasis, NF-κB–mediated inflammation promotes osteoclastogenesis (44) in theaging animal, which may contribute to the osteoporotic phenotypecharacteristic of aging animals. Although osteoclasts play an im-portant role during regeneration, their main function is centeredaround the remodeling phase of repair, which occurs at laterstages when bone matrix deposition has been completed. Becauseour focus aimed at the early stages of regeneration rather thanremodeling and homeostasis, we did not further examine theconnection between NF-κB and osteoclastogenesis.There are caveats to this work. Usually, 52-wk-old mice are

not considered aged yet; however, as demonstrated here, theyclearly exhibit an increased proinflammatory cytokine profileconsistent with the process of aging. We selected this age grouprather than studying older animals for two reasons. First, SSPCnumber is already significantly decreased at 1 y of age, renderingtranscriptional analyses of this cell population difficult. At 2 y,when mice are considered aged, SSPC number is very low,making it impossible to study the effects of inflamm-aging on thiscell population. Second, we chose this middle-aged group tobetter represent the human age cohort most often debilitated by

orthopedic conditions, the baby-boomer generation (45). It is thispopulation that would most benefit from a translational approachthat improves bone regeneration. Septa- and octogenarians, on thecontrary, often undergo replacement procedures (i.e., hip hemi-arthroplasty, shoulder arthroplasty) rather than being subjected toprocedures that rely on functional bone regeneration. Thus, ourstudy was executed by using 52-wk-old mice in an attempt tosimulate the physiology of middle-aged orthopedic patients.Sodium salicylate, an antiinflammatory drug with similar antiin-

flammatory potency as aspirin, exerts its effect through indirectinhibition of prostaglandin biosynthesis (reviewed in ref. 46).Although this nonspecific inhibitory effect undoubtedly reversed theinflamm-aging phenotype of the SSPCs in these murine models,future work will focus on a more targeted suppression of inflamm-aging using specific Cox2 inhibitors, small-molecule antagonistsagainst epigenetic regulators of the innate immune system, andmodulators of NF-κB activity.In summary, we present compelling data that age-associated

inflammation, regulated by NF-κB and triggered by increasedcell senescence, leads to a functional decline of SSPCs, which canbe overcome and reversed by suppression of inflammation byusing a low-grade antiinflammatory drug.

Materials and MethodsPatients and Specimens. All experiments involving human subjects were ap-proved by the New York University (NYU) School of Medicine InstitutionalReview Board. After informed consent was obtained, specimens wereobtained during routine ICBG harvest. One cubic centimeter of ICBG wasimmediately transferred into a microcentrifuge tube and placed on ice.Samples were dissociated and stained with antibodies against CD45 andCD271. FACS analysis was performed by using a BD LSRII cell analyzer withhigh-throughput sampler. CD45-negative and CD271-positive SSPCs weredisplayed as percentages of total single cell number (8–11). Patient data,including radiographic and clinical time to union, were extracted from aprospective patient database at the NYU Langone Orthopedic Hospital.

Isolation of SSPCs. Tibiae and femurs were harvested as previously described(47). Dissociated cell samples were stained with antibodies against CD31,CD45, Ter-119, and LepR for purification by flow cytometry (Moflo XDP;Beckman-Coulter). CD31−CD45−Ter-119−LepR+ cells were identified as SSPCs(12, 34).

Isolation and Culture of SSPCs. For the in vitro experiments, tibial and femoralSSPCs were isolated by centrifugation (48). SSPCs were resuspended in GM(DMEM containing 10% FBS and 1% penicillin/streptomycin; Thermo FisherScientific) and then plated in 75-mL tissue culture flasks. Media was changedevery 2 d. All cellular assays described were performed with SSPCs at passage1 from at least three different mice in three technical replicates.

Statistical Analysis. A priori power analysis to obtain statistical significance(P = 0.05, power 80%) resulted in n = 4 for each group after body-size adjust-ment, expecting a 25% difference between the groups. Prism 7 (GraphPadSoftware) was used for statistical computations. A Student’s t test was used forall comparisons in which there were two groups; ANOVA followed by Holm–�Sidák correction for post hoc testing was applied for analyses in which therewere two or more comparisons being made. Error bars in the figures representSEMs. An asterisk denotes a P value <0.05 unless denoted otherwise in thefigure legend.

Further standard materials and methods including animals,antiinflammatory treatment, monocortical defects, renal capsule transplants, his-tology and histomorphometry, μCT analyses, multiplex ELISA, SDS/PAGE andWestern blots, cfu assay, proliferation assay, osteogenic and adipogenicdifferentiation, RNA isolation and quantitative real-time PCR, in vitrohomochronic and heterochronic serum treatment, identification of senes-cent cells, cell-cyle analysis, and RNA sequencing (RNA-seq) analysis aredescribed in the SI Appendix.

ACKNOWLEDGMENTS. We thank Ripa Chowdhury (NYU College of Den-tistry) for assistance with the μCT imaging, funded through NIH Grant S10OD010751. Cell sorting/flow cytometry technologies were provided by NYULangone’s Cytometry and Cell Sorting Laboratory, and RNA-seq and analysiswas performed in the Genome Technology Center, both of which are sup-ported by NIH/National Cancer Institute Grant P30CA016087. This work was

Fig. 9. Schematic illustration of the cellular mechanism leading to impairedbone regeneration in elderly subjects. During aging, senescent SSPCs secreteSASP factors, which result in activation of NF-κB in adjacent SSPCs, inducingthem to undergo senescence. This accumulation of senescent SSPCs leads todecreased SSPC self-renewal and proliferation, resulting in a decrease inoverall number. Together with a decline in osteogenic differentiation, thisleads to impaired bone regeneration in elderly subjects. NSAID treatmentinhibits NF-κB activation, thereby blocking the effect of the SASP factors onsurrounding SSPCs, which leads to a decrease in SSPC senescence, increase inSSPC number, improved osteogenic differentiation, and, finally, enhancedbone regeneration.

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supported by NIH/National Institute on Aging Grant 1R01AG056169; NIH/National Institute of Arthritis and Musculoskeletal and Skin GrantK08AR069099 (to P.L.); and a grant from the Orthopaedic Research and

Education Foundation and the Orthopaedic Trauma Association, fundedin part by Zimmer Biomet, Depuy Synthes, and the Society of MilitaryOrthopaedic Surgeons.

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