Neurological heterotopic ossification following spinal cord injury is triggered by...

Journal of PathologyJ Pathol 2015; 236: 229–240Published online 26 March 2015 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.4519

ORIGINAL PAPER

Neurological heterotopic ossification following spinal cord injuryis triggered by macrophage-mediated inflammation in muscleFrançois Genêt,1,2,3† Irina Kulina,1,4† Cedryck Vaquette,5 Frédéric Torossian,6,7 Susan Millard,1 Allison R Pettit,1Natalie A Sims,8 Adrienne Anginot,6,7 Bernadette Guerton,6,7 Ingrid G Winkler,1 Valérie Barbier,1 Jean-JacquesLataillade,6,9 Marie-Caroline Le Bousse-Kerdilès,6,7 Dietmar W Hutmacher1 and Jean-Pierre Levesque1,4*

1 Blood and Bone Diseases Programme, Mater Research Institute, University of Queensland, Woolloongabba, Australia2 Department of Physical Medicine and Rehabilitation, Hôpital Raymond Poincaré, APHP, CIC-IT 1429, Garches, France3 Université Versailles Saint Quentin en Yvelines, END:ICAP U1179 INSERM, UFR des Sciences de la Santé–Simone Veil, Montigny leBretonneux, France4 School of Medicine, University of Queensland, Herston, Australia5 Institute of Health Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Australia6 Institut National de la Santé et de la Recherche Médicale, Unité 972, Villejuif, France7 Université Paris-Sud, Institut André Lwoff, Paris, France8 St Vincent’s Institute of Medical Research, Fitzroy, Australia9 Centre de Transfusion Sanguine des Armées, Clamart, France

*Correspondence to: JP Levesque, Blood and Bone Diseases Programme, Mater Research Institute, University of Queensland, Translational ResearchInstitute, 37 Kent Street, Woolloongabba, QLD 4102, Australia. E-mail: [email protected]

†These authors contributed equally to this study.

AbstractNeurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequenceof spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delaysrehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknownand consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models ofrare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently,we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formationof NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscularinflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both humanand mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblastdifferentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli.Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in theserum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reducedthe volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reducedthe size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocyticmacrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstratedthat a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: heterotopic ossification; spinal cord injury; macrophage; inflammation

Received 8 January 2015; Revised 17 February 2015; Accepted 19 February 2015

No conflicts of interest were declared.

Introduction

Heterotopic ossification (HO) is the abnormal forma-tion of bony material in soft tissues, a pathological con-dition first identified in spine-injured soldiers duringWorld War I [1]. Severe trauma is the main cause of HO,particularly in defence personnel, among whom> 50%of blast and gun-shot wound victims develop HO [2].HO is also frequent in civilians, affecting 20–29% ofpatients with spinal cord injuries (SCIs) and 5–20%

of those with traumatic brain injuries (TBIs) [3–6].Both these latter forms are termed ’neurological’ HO(NHO). NHOs are peri-articular, most frequently at thehip, elbow, knee and shoulder [7]. Because of theirlarge size (Figure 1A, B), NHOs are debilitating, caus-ing significant pain and gradual reduction in the rangeof motion of affected limbs, often progressing to com-plete ankylosis. This exacerbates functional disabilitiesby increasing difficulty in sitting, eating and dressing[8]. HO growth can also cause nerve and blood vessel

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 236: 229–240Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com

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compression, further increasing patient morbidity [9,10](Figure 1A). Treatment is currently limited to surgicalresection after NHOs have matured [7,8,11,12], a proce-dure that is challenging, particularly when ossificationsentrap large blood vessels and nerves (Figure 1A). Phar-macological interventions have, to date, shown limitedeffectiveness [4], reflecting the current limited knowl-edge of the aetiology and pathophysiology of NHO.

NHO is poorly understood for two reasons. First, stud-ies on humans are, by necessity, retrospective; conse-quently, no studies on the earliest stages of NHO haveoccurred to aid identification of the HO-initiating events.Second, there is no animal model of NHO and theonly existing animal models of non-neurological HOare limited to genetically modified mouse models of therare genetic disease fibrodysplasia ossificans progres-siva (FOP) [13–16]. Since pathological NHO occurswith relatively high incidence in SCI patients who aregenetically normal and had no sign of HO prior to theiraccidents, the relevance of genetic models of FOP totrauma-induced HO is questionable.

We now report the development of a physiologicallyrelevant animal model of NHO in genetically unmodi-fied mice. This model reveals fundamental mechanismslinking the original neurological lesion to the develop-ment of NHO and shows that, while substance P mayplay a role in mediating NHO growth, macrophages area critical contributor that may be targeted to prevent thegrowth of NHO.

Materials and methods

Human studiesThis study was approved by the institutional reviewboard of the Comité de Protection des Personnes XI,Ile de France. It was a non-interventional study withthe usual standard of care, without additional proce-dures. In France, patient consent is not needed for anony-mous surgical waste collection (only information andnon-opposition notes); the need for consent for thisstudy was waived (No. 10 005; 2010).

Mouse treatmentsAll mouse procedures were approved by the AnimalExperimentation Ethics Committee of the Universityof Queensland. All experiments were performed on5–6 week-old female C57BL/6 mice, obtained from theAnimal Resource Centre (Perth, Australia). Mice wereanaesthetized by intraperitoneal injection of 75 mg/kgketamine, 10 mg/kg xylazine in sterile saline. A laminec-tomy was performed on the dorsal spine and the spinalcord transected with a scalpel blade between T7 andT8. The muscles and skin were sutured. Control animalswere anaesthetized and sham-operated with incision ofthe dorsal skin.

Immediately after surgery, while still anaesthetized,inflammation in the right hamstring muscles was

induced by intramuscular injection of 12.5 μg car-diotoxin (CDTX) from Naja mossambica, diluted in75 μl phosphate-buffered saline (PBS). As a contralat-eral control, the left hamstring muscles were injectedwith 75 μl PBS without CDTX. At the end of surgery,the mice received a subcutaneous injection of 20 mg/kgciprofloxacin; they were left to recover on a heat pad at37 ∘C until awake and returned to their cages.

As SCI causes paraplegia, the mouse bladders wereemptied manually by gentle massage of the bladdertwice daily throughout the experiments. As prophylaxisfor bladder infections, the mice were given 125 mg/lciprofloxacin in drinking water following spinal cordtransections. The mice were euthanized by CO2 asphyx-iation between day 3 and week 3 after surgery, tomeasure heterotopic bone volumes by micro-computedtomography (μCT), immunohistology and additionalanalyses.

Clodronate (dichloromethylene bisphosphonate), agift from Roche Diagnostics (Mannheim, Germany),was packaged into liposomes, as previously described[17]. Empty liposomes were prepared under the sameconditions in PBS without clodronate. Phagocyticmacrophages were depleted in vivo by retro-orbitallyinjecting 100 μl/20 g body weight clodronate-loadedliposome suspension immediately after surgery andinjection of CDTX, and then every second day for 2weeks. Control mice were injected with an equivalentvolume of saline or PBS-loaded liposomes preparedunder the same conditions but without clodronate.

Zoledronate (Zometa, Novartis Pharmaceuticals) wasinjected intraperitoneally at 50 μg/kg daily for the first5 days after surgery, and then at 100 μg/kg every thirdday from day 6 to day 12. The mice were euthanized foranalysis 14 days after surgery.

RP67580 (Tocris), a selective antagonist of the sub-stance P receptor NK1R [18], was injected intraperi-toneally twice daily at a dose of 2 mg/kg in 2% ethanolin saline (vehicle). Control mice were injected in parallelwith an equivalent volume of vehicle.

Mouse tissue samplingAt specified time points after surgery and CDTX injec-tion, mice were euthanized by CO2 asphyxiation. Theskin was removed and the whole mouse body wasput into a 50 ml tube filled with freshly made 4%paraformaldehyde in PBS. After 24 h fixation on a rota-tor in the cold room at 4 ∘C, the tubes were emptied, themouse carcasses rinsed once in PBS and preserved in atube full of PBS containing 0.1% NaN3 until processingfor μCT or immunohistology.

Blood was harvested in heparinized tubes by cardiacpuncture following anaesthesia by isofluorane inhala-tion. The mice were then immediately euthanized byCO2 asphyxiation and further processed, as describedabove. Blood samples were centrifuged twice at 800× gat 4 ∘C for 20 min to harvest blood plasma. Blood plasmasamples were stored at −80 ∘C.


Macrophages drive heterotopic ossification after spinal cord injury 231

Figure 1. Heterotopic ossification requires both SCI and local inflammation in a mouse model of NHO. (A) Example of NHO in a patientwith SCI. (B) Fragment of mature NHO resected from a SCI patient. (C) μCT of a representative mouse with SCI and CDTX in the right leghamstring and saline in the left hamstring. (D) Quantification of HO volumes by 3D μCT reconstitution at indicated time points followingSCI and intramuscular CDTX injection; each symbol represents a separate mouse (in the SCI-alone group, three mice at day 8 and three atday 15); bars and errors bars represent mean± SD for each experimental group; SCI and CDTX injection had significantly higher HO volumesthan mice with SCI alone (p= 0.002 for each time point). (E) Illustrative μCT of right legs at indicated time points following SCI and CDTXinjection. (F) X-ray radiographs of a SCI patient at different stages of NHO proximal to the hip at indicated times post-injury

Microcomputed tomography (μCT)After removal of the bowels and viscera, whole mousebodies were analysed by μCT. The scans were per-formed in a μCT scanner (μCT40, SCANCO MedicalAG, Brüttisellen, Switzerland) at a resolution of 30 μmand three-dimensional (3D) images of the lower partsof the mouse bodies were reconstructed from the scansusing the μCT system software package. Quantitativeassessments of bone volumes in the muscular mass bya subtraction technique of orthotopic mouse skeleton(hip, femur, tibia, fibula) were measured to detect andquantify HO.

Histology of mouse and human tissuesDetailed methods for the histology of fixed mouse mus-cle tissues and human HO biopsies are provided in Sup-plementary materials and methods (see supplementarymaterial).

Mouse muscle cell isolation, culture and osteogenicdifferentiationIsolation and sorting of progenitor cells for mouse mus-cle and bone marrow, and their subsequent culture underosteogenic conditions with or without mouse plasma, are


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described in Supplementary materials and methods (seesupplementary material).

Human muscle cell isolation, cultureand osteogenic differentiationMuscles attached to HO biopsies were cut out fromthe bone mass, minced into small pieces and cul-tured in α-minimal essential medium (α-MEM) with10% fetal calf serum (FCS) at 37 ∘C. When outgrow-ing fibroblast-like cells reached subconfluence, the cellswere trypsinized and seeded at 4000 cells/cm2 for cul-ture. An aliquot was analysed by flow cytometry, usinghuman CD45-FITC, human CD90-PE (BD Pharmin-gen), human CD73-PE (BD Pharmingen) or humanCD105-PE (R&D Systems) for 30 min, and analyseswere performed on a BD Fortessa flow cytometer.

For osteoblastic differentiation, cells were seeded at3000 cells/cm2 in α-MEM supplemented with 10% FCSat 37 ∘C. After cell adhesion, the medium was removedand cells were cultured in α-MEM supplemented with10% FCS and with 0.1 μM dexamethasone, 44 μM ascor-bic acid and 10 mM β-glycerophosphate for 3 weeks.

After fixation in 4% paraformaldehyde for 10 min,cells were stained with alizarin red, as described above.

qRT–PCRMouse muscle and bone marrow progenitors, sortedas described above, were directly collected into tubescontaining 2 ml Trizol. RNA was isolated, reverse-transcribed and qPCR performed, as previouslydescribed [19], using either TaqMan® UniversalPCR Master Mix (Applied Biosystems, Life Technolo-gies) for Pax7, Prrx1, Pthr1 and Sp7, or SYBR GreenPCR Master Mix for mouse β-actin. TaqMan GeneExpression Assay primer and probe sets for mousePax7 (Mm01354484_m1), Prrx1 (Mm00440932_m1),Pthr1 (Mm00441046_m1) and Sp7 (Mm04209856_m1)were purchased from Applied Biosystems. Sequencesfor β-actin were: forward 5′-AGCACTGTGTTGGCATAGAGGTC-3′; reverse 5′-CTTCTTGGGTATGGAATCCTGTG-3′. Results were expressed relative to β-actin mRNA concentration.

Quantification of substance P and phosphatein plasmaBlood samples from healthy donors or from patientswith HO were collected in tubes containing citratesolution and centrifuged at 2000 rpm for 10 min at4 ∘C. Plasma fractions were stored at −80 ∘C. Sub-stance P concentrations were measured using SubstanceP Parameter Assay Kit (R&D Systems), according to themanufacturer’s instructions.

At specified time points, mice were anaesthetized withisofluorane and blood was collected by cardiac puncturein tubes containing 30 U sodium heparin. After two cen-trifugations at 2000× g, plasma samples were stored at−80 ∘C. Phosphate concentrations were measured usinga colorimetric assay from Sigma Chemicals (cat. no.

MAK 030), following the manufacturer’s instructions,after 200-fold dilution of mouse plasma samples inMilli-Q water in order to keep phosphate concentrationswithin the 0–30 μM linear detection range.

Statistical analysesTwo-sided non-parametric Mann–Whitney test wasused to compare medians of HO bone volumes, bloodphosphate or substance P concentrations betweendifferent groups. The significant association betweenco-occurrence of SCI and muscular inflammation inmice was calculated using Fisher’s exact test. Allcalculations were performed using GraphPad Prism 5software.

Results

Establishment of a mouse model of NHO followingSCIWe performed spinal cord transections between T7 andT8 (SCI) in 102 anaesthetized wild-type C57BL/6 miceand followed the mice for up to 4 weeks for HO. Noneof these mice developed HO, despite paraplegia (seesupplementary material, Table S1). We then reasonedthat additional factors must contribute to NHO and,based on the increased NHO incidence with degreeof trauma sustained at time of injury and associationwith peri-articular inflammation [7,20,21], hypothesizedthat muscular or peri-articular inflammation may triggerNHO following SCI.

Cohorts of mice underwent SCI alone, or SCI com-bined with muscular inflammation by intramuscularinjection of CDTX in the right hamstring muscles.Control mice underwent sham operation alone orsham operation with CDTX intramuscular injection.No mice with SCI alone or CDTX alone developedNHO, as assessed by μCT (see supplementary material,Table S1). In contrast, 112 of 113 mice with SCI andCDTX injection developed NHO (Figure 1C; see alsosupplementary material, Table S1). Remarkably, withinthis experimental group, NHO always developed in thelimb injected with CDTX, and never in the PBS-injectedcontralateral limb. NHO occurrence was therefore sig-nificantly associated with the combination of both SCIand CDTX-induced inflammation (p< 10−4).

In longitudinal studies of the model, discrete min-eralized foci were detected disseminated through thehamstring muscle as early as 3 days after SCI andCDTX injection. These nodules expanded and fusedthroughout most of the hamstring muscles by 2 weeks(Figure 1D, E). Control mice that underwent SCIwithout CDTX injection did not develop NHO duringthis time period. The progressive pattern of miner-alization in the muscle, including fusion into largermineralized masses, was similar to growing HO inSCI patients (Figure 1F). Of note, HO was accompa-nied by the formation of resorption pits on skeletal



bones, particularly affecting pelvic bones and vertebrae(Figure 1F). Osteopenia/osteoporosis in skeletal bonesis also observed in NHO patients [22–24].

To test whether HO required the CNS lesion asso-ciated with the SCI, or only hind limb immobilizationcaused by the paraplegia, cohorts of mice underwentbilateral sciatic nerve section with intramuscular injec-tion of CDTX in the right hind leg and PBS in the controlleft leg. Although paraplegic, none of these mice devel-oped NHO detectable by μCT (results not shown), con-firming that hind limb immobilization is not sufficient tosupport NHO.

Histology of undecalcified muscles 3 weeks afterSCI and CDTX injection demonstrated that the nod-ules detected by μCT were bona fide mineralizedbone nodules (black-stained extracellular matrix usingvon Kossa staining) within the muscle mass, contain-ing osteocytes within lacunae in the bone matrix aswell as bone marrow, osteoblasts and osteoclasts inpseudo-medullary cavities created within the largerbone nodules (Figure 2A). Haematoxylin and eosin(H&E) staining of sections collected 3 weeks aftereither CDTX injection alone (Figure 2B, top left) orSCI and CDTX injection (Figure 2B, top middle)demonstrate striking differences between these groups.In CDTX injection alone, only minor inflammatoryinfiltration persisted within the muscle and regenerationof muscle was apparent, including the presence of newmultinucleated myofibres (Figure 2B, top left, arrows).Immunohistochemistry demonstrated that F4/80+

macrophages were only rarely detected within CDTXinjection-alone samples (unpublished data). In contrast,woven bone-like structures were common within mus-cle from SCI and CDTX-injected mice, including somecontaining a marrow rich in diverse leukocytes, sug-gesting haematopoietic activity (Figure 2B, top middle),as observed in human NHO. Immunohistochemistryin a serial section series (Figure 2B) clearly showedformation of osteocalcin+ woven heterotopic bone thathad numerous associated osterix/Sp7+ osteoblast-likecells, some of which also expressed osteocalcin. Theaffected muscle regions were infiltrated with a largenumber of inflammatory F4/80+Mac-2+ macrophages,and some F4/80+Mac-2–/dim osteomacs were observed,forming a canopy over osteoblast-like cells within theneo-formed heterotopic bone. Contralateral musclesinjected with PBS were negative for F4/80, Mac-2,osterix/Sp7 and osteocalcin (not shown).

The features of NHO in our mouse model weresimilar to mature NHO surgically resected from patients(Figure 2C), where mineralized heterotopic bone residesin direct contact with adjacent muscle fibres. The het-erotopic bones contained mature haematopoietic tissuesimilar to bone marrow, with adipocytes, leukocytes,polyploid megakaryocytes (Figure 2C) and cuboidalosteoblasts lining mineralized heterotopic bone surfaces(Figure 2C). Analogous to our mouse model, biopsies ofresected mature human NHO displayed accumulationof CD68+ macrophages in the muscle adjacent to theheterotopic bone (cf Figure 2B, C).

Muscle progenitors isolated from naïve musclesdisplay osteogenic potentialTo determine whether muscle-derived mesenchy-mal cells contribute to NHO, we sorted musclesatellite cells from hamstring muscles dissectedfrom naïve C57BL/6 mice as CD45−Ter119−CD31−

Sca1−CD34+ cells, muscle interstitial cells as CD45−

Ter119−CD31−Sca1+CD34+ cells, as previouslyreported [25,26], and CD45−Ter119−CD31−Sca1−CD34− muscle progenitor cells. We also sorted CD45−

Ter119−CD31−Sca1+ and CD45−Ter119−CD31−Sca1−

CD34− mesenchymal progenitor cells (MPCs) frompassage 2 cultured adherent bone marrow stromal cells(see supplementary material, Figure S1). We confirmedthe identity of these sorted populations by qRT–PCR,using primers specific for Pax7 (satellite cells), Prrx1(MPCs), Pthr1 and Sp7 (osteoblasts and their progeni-tors) mRNA (see supplementary material, Figure S2).

When cultured in osteogenic conditions, satellite,interstitial and muscle progenitor cells isolated from thenaïve hamstring muscle were all capable of depositingmineral after 2–3 weeks of culture, as detected by vonKossa and alizarin red staining, similar to MPCs sortedfrom the bone marrow (Figure 3A, B); hence, residentprogenitors located within the muscle have osteogenicpotential. Likewise, we isolated plastic-adherent MPCsfrom the muscle immediately adjacent to NHO resectedfrom patients. After culture, these muscle cells dis-played phenotypic characteristics of human MPCs,lacking expression of the pan-leukocyte marker CD45and expressing CD90, CD73 and CD105 antigens[27] (Figure 3C). Under osteogenic culture conditions,these muscle-derived human MPCs also deposited min-eral, as detected by alizarin red staining (Figure 3D).Therefore both mouse and human muscles harbourprogenitors with osteogenic potential, suggesting thatthe osteoblasts responsible for NHO in muscles couldbe derived from local mesenchymal progenitors.

Systemic factors promoting ossification arereleased upon SCITo test whether SCI could cause systemic release offactors promoting osteogenic differentiation in inflamedmuscles, we injected CDTX in the non-paralysed frontlimb above the SCI. NHO formed in the CDTX-injectedfront limb (Figure 4B), supporting the hypothesis thatsystemic signals prime NHO development independentof paralysis. Muscle interstitial cells were also sortedfrom the hamstring muscles of naïve mice, expanded inculture until confluent and then cultured without addi-tion of osteogenic factors, but in the presence of 10%blood plasma from mice that had undergone either shamoperation, SCI or a combination of SCI plus intramus-cular CDTX. Alizarin red staining showed that plasmafrom mice that had undergone SCI together with intra-muscular injection of CDTX enhanced mineralization ofmuscle interstitial cells in culture (Figure 4B). Together,these findings suggest that systemic factors released into


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Figure 2. Histology of NHO in mice and humans. (A) von Kossa staining of mouse right hind limb section following SCI and injection of CDTX:legs were sampled 3 weeks post-surgery and CDTX injection; mineralized hydroxyapatite is stained black; (middle panel) magnification ofthe yellow rectangle in the left panel and shows an example of HO containing marrow-like tissue; (right panel) HO with distinct osteocytes(yellow arrows) entrapped within lacunae of the mineralized matrix. (B) H&E staining or immunohistochemistry on serial sections of a mousehind limb with antibodies specific for F4/80, Mac-2, osteocalcin and osterix/Sp7: legs were sampled 3 weeks post-surgery and CDTX injection;arrows in top left image show multinucleated regenerating myofibres; in two bottom left sections (F4/80 and Mac-2), arrowheads showF4/80+Mac-2− osteomacs; arrows show F4/80+Mac-2+ inflammatory macrophages. (C) H&E staining and CD68 immunohistochemistryof human NHO biopsies: micrographs show junction between muscle (Mu) and heterotopic bone (top left panel); cuboidal osteoblasts(OB) covering heterotopic bone surfaces (top right panel); haematopoietic cells with adipocytes (AD) (bottom left panel) are found in theheterotopic bone marrow; in immunohistochemistry (bottom right panel), CD68+ macrophages are stained brown

the circulation following the combination of SCI withintramuscular inflammation promote the mineralizationof muscle-derived progenitor cells.

As excessive calcification, particularly of arteries, hasbeen linked to hyperphosphataemia in chronic kidneydisease patients [28], we measured plasma phosphateconcentration in a cohort of mice that underwent SCIand CDTX intramuscular injection, and a cohort of con-trol mice with sham surgery. Plasma phosphate lev-els remained in the normal range in both experimentalgroups (Figure 4C).

Substance P, a neuropeptide mediating physiologicalresponse to pain and produced by neurons, osteoblastsand macrophages, has been reported to promote HOin genetically altered mouse models of FOP [29].Therefore, we measured substance P concentration inthe plasma of NHO patients and healthy volunteers.Substance P concentration was significantly higherin plasma from NHO patients compared to healthyvolunteers (Figure 4D). To test the role of substanceP in NHO, mice that had undergone SCI and intramus-cular injection of CDTX were injected twice daily with



Figure 3. Muscle satellite cells and interstitial cells can mineralize matrix in vitro. (A) von Kossa staining of muscle interstitial cells (1),muscle satellite cells (2), Sca1−CD34− muscle progenitor cells (3), sorted bone marrow Sca1+ MPCs (4), sorted bone marrow Sca1−CD34−

MPCs (5) and mouse osteoblastic cell line MC3T3 (6) cultured for 3 weeks in osteogenic medium or without osteogenic factor; blackstaining shows mineralized phosphate deposited at the bottom of the wells. (B) Quantification of the alizarin red staining absorbance ofcells cultured under osteogenic conditions (grey bars) or in non-osteogenic expansion medium (empty bars); data are mean± SD of fourwells of a typical experiment out of five independent sorts and experiments. (C) Flow cytometry expression profile of human muscle cellsisolated from HO: cultured human HO muscle cells are negative for the pan-leukocyte marker CD45 and positive for MPC markers CD90,CD73 and CD105. (D) Alizarin red staining after 3 weeks of culture of human muscle cells isolated from HO; cells were cultured in osteogenicdifferentiation (OD) medium or non-osteogenic control culture (CRTL) medium

the selective substance P receptor NK1R antagonistRP67580 or vehicle alone. RP67580 administrationsignificantly reduced the volume of NHO by 30%(Figure 4E), indicating that substance P may contributeto the development and severity of NHO following SCI.

Phagocytic macrophages are key drivers of NHOAs muscular inflammation is necessary for NHO for-mation following SCI in our mouse model, and F4/80+

macrophages were a prominent component of themuscle inflammatory infiltrate, we further investigatedthe importance of macrophages to NHO pathogenesis.Mice with SCI and intramuscular CDTX injectionwere injected intravenously with clodronate-loadedliposomes to deplete phagocytic macrophages [30–32].Control mice underwent SCI and intramuscular injec-tion of CDTX but received PBS-loaded liposomes.

Clodronate-loaded liposomes significantly reducedthe volume of NHOs by 11-fold compared to PBS–liposome controls (Figure 5A, B), and in some miceprevented NHO development.

Since clodronate-loaded liposomes can also depleteosteoclasts [31] and osteoclasts can promote osteoblastformation and activity via coupling mechanisms [33],we tested the effect of osteoclast depletion alone byinjecting zoledronate in combination with SCI andCDTX. Efficacy of this zoledronate regimen was con-firmed by the absence of bone resorption pits on the iliaccrest and vertebrae of mice with SCI (Figure 5A). Inter-estingly at day 14, in direct opposition to the effect ofclodronate-loaded liposomes, zoledronate increased theaverage bone volume of NHO three-fold compared toSCI plus CDTX mice injected with saline (Figure 5A,C), possibly due to the absence of osteoclasts to resorb


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Figure 4. NHO requires systemic factors released upon SCI. (A) Quantification of HO volumes after 3D μCT reconstitution in non-paralysedright front limb 8 days after SCI and injection of CDTX in the right front limb. (B) Alizarin red staining of muscle interstitial cells sortedfrom naïve mice after 2 weeks of culture in the absence of mouse plasma or in the presence of 10% blood plasma from mice 4 days aftersham operation alone, SCI alone, or SCI together with CDTX intramuscular injection; data are mean± SD or values obtained from fourseparate plasma samples from four separate mice in each experimental group. (C) Phosphataemia in mice that underwent SCI with CDTXintramuscular injection (developing HO) or sham operation (no HO); data are mean± SD of four mice/experimental group and for each timepoint. (D) Substance P concentration in blood plasmas from healthy donors or patients with NHO. (E) Effect of the NK1 receptor antagonistRP67580 on HO volumes after SCI and CDTX intramuscular injection; HO volumes were measured by 3D μCT reconstitution in right hindlimb 14 days after SCI and injection of CDTX in the right hind limb. (A, D, E) Each dot represents a separate individual; bars and error barsrepresent mean± SD; p values were calculated using the Mann–Whitney test

developing NHO. Collectively, this suggests that phago-cytic macrophages, not osteoclasts, recruited in theinflamed muscle, trigger the development of NHO sub-sequent to SCI.

Discussion

We have developed a clinically relevant mouse modelof NHO following SCI that does not depend ongenetic manipulation and displays the pathophysiol-ogy observed in patients with NHO (Table 1). Unlikegenetically altered or bone morphogenetic protein(BMP)-implant models of FOP [13–16], this new modelprovides a powerful tool for determining the aetiologyof NHO and, in particular, assessment of preventativetherapies. In this model, while prophylactic inhibitionof substance P provides partial benefit (30%), prophy-lactic depletion of macrophages reduced NHO size by90%, with complete prevention in some animals. This

supports the hypothesis that local macrophage-driveninflammation is a dominant trigger in developmentof NHO, and that targeting inflammatory pathwaysassociated with phagocytic macrophage recruitmentand/or function in the inflamed muscle is a promisingtherapeutic approach for preventing NHO development.

The involvement of local inflammation andmacrophages is common to our model and NHOin patients. The only three single-nucleotide polymor-phisms (SNPs) known to be associated with increased ordecreased HO following trauma are in genes regulatinginflammation (TLR4 and CFH) or neurotransmission(ADRB2) [34]. While these SNP studies are correlative,they provide circumstantial evidence that inflamma-tion/innate immunity and the nervous system areimportant for NHO following SCI. Our SCI–NHOmodel demonstrates for the first time that it is thecombination of SCI and local muscle inflammation thatis the underlying mechanism in NHO development,with either insult alone being insufficient. Therefore,



Figure 5. NHO needs macrophages in the inflamed muscle upon SCI. (A) μCT of right hind limbs after SCI and injection of CDTX; the micewere additionally injected with either PBS or clodronate-loaded liposomes to deplete macrophages, or zoledronic acid to deplete osteoclasts.(B, C) HO volumes after 3D μCT reconstitution; each symbol represents a separate mouse; bars and errors bars represent mean± SD foreach experimental group; p values were calculated using the Mann–Whitney test

SCI could prime bone marrow or muscle mesenchymalprogenitor cells towards osteogenic differentiationby releasing factors that include, but are not lim-ited to, substance P, whilst local macrophage-dependentinflammation triggers and drives site-specific osteogenicdifferentiation, leading to heterotopic ossification. OurSCI–NHO model is highly consistent with clinicalobservations, as SCI and TBI patients who developNHO are those who were victims of the most violentimpacts with co-trauma or infections [7], including amuch higher incidence in war casualties, who are morelikely to have these co-morbidities than civilians [2].

The effects of macrophage inhibition in theSCI–NHO model is consistent with our previous obser-vations that specific subsets of resident macrophagesare critical to the maturation of osteoblasts and boneformation in skeletal bones [31,35]. In long bones,macrophage depletion induces a rapid loss of matureosteoblasts on bone surfaces, arrests bone formationand mineralization in adult mice [31,35] and impairsbone repair following fracture [32,36]. Inflammatorymacrophages can promote osteoblast and bone for-mation [37]. Macrophages can express a large arrayof molecules that can have pro-anabolic actions [33],including BMP-2 [38,39] and oncostatin M [33,37], andmay be involved in the pathogenesis of NHO. Whethermacrophages directly or indirectly support ossificationin NHO, and the identity of the macrophage-derived

molecular mediators, are the subjects of ongoingresearch.

The finding that muscular inflammation in the frontlimb, above the SCI, also promoted NHO suggeststhat factors priming resident muscle progenitor cellsto osteogenic fate could be released in the circulationor induced locally by a feedback mechanism from theinjured CNS. Consistent with this, plasma from TBI andSCI patients has been reported to induce expression ofosterix by primary skeletal muscle cells [40], and toenhance osterix, Runx2 and alkaline phosphatase mRNAlevels in the human osteoblastic cell line FOB1.19 [41].

From a therapy viewpoint, depletion of macrophageswas effective at reducing or, in some cases, completelypreventing NHO development in mice. This is con-sistent with the reported role of osteal macrophagesin supporting osteoblast differentiation and function[31,32,35,36]. Further identification of the mechanismsby which muscle-infiltrating macrophages support NHOdevelopment after SCI is warranted in order to developtherapies that target developing HO without affectingimmunity or the normal repair of the muscle tissue.

The analysis of cells isolated from the SCI–NHOmodel suggests that the osteoblasts responsible for NHOin muscles may not necessarily be derived from MPCsrecruited from the bone marrow. Although this remainsto be demonstrated genetically with lineage-trackingexperiments, muscle satellite cells, interstitial cells or


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Table 1. Comparison between the pathophysiological features of human NHO and mouse NHO modelMouse model Human NHO

Conditions to develop NHO Local Inflammation alone No NoSCI alone No Not applicableSCI+ one or more essential risk

factors (RFs; inflammation,bacteriuria, infections,spasticity, autonomichyper-reflexia, completelesions, etc.)

Yes Yes (but specific RFs areundetermined)

SCI+ local muscle inflammationbelow the lesion

Yes with cardiotoxin More frequent with concomitantlocal inflammation

SCI+ local muscle Inflammationabove the lesion

Yes with cardiotoxin Yes if associated local injuryfracture, muscle or joint lesion

Peripheral lesion (bilateral sciaticnerve section) alone

No Not described

Peripheral lesion (bilateral sciaticnerve section)+ local muscle;inflammation

No Not described

Initial location of the NHO In the inflamed muscle In muscles around the joint (hip,elbow, knee and shoulder)

Structural and functional features Presence of bone marrow Yes YesHaematopoiesis activity Morphological appearance but not

functionally demonstratedYes, functionally demonstrated

(unpublished data)Presence of osteocytes Yes YesPresence of macrophages in

adjacent muscleYes Yes

Skeletal bones Presence of osteoporosis Yes Yes

muscle progenitor cells sorted from the muscle of naïvemice, which all have the potential to regenerate myofi-bres in vivo following muscle injury [25,26], were allcapable of mineralizing a matrix in vitro, as previouslyreported in mouse [42] and human [43]. Therefore, thisopens the possibility that inappropriate signalling fromthe CNS and innate immune system can reprogrammemuscle progenitor cells to osteogenesis instead of mus-cle repair.

In conclusion, a significant advantage of theSCI–NHO model over existing HO models [13,15,16]is that it can be induced in genetically unmodified micewithout the introduction of exogenous BMP (in theform of a transgene or BMP-overexpressing cells), acti-vating mutations of BMP receptors or pro-osteogenicengineered biomaterials (such as demineralized bonematrix). This is the first preclinical model of NHO thatreliably reproduces disease pathophysiology without thenecessity of creating genetic susceptibilities that maynot be clinically relevant to a large proportion of patientssuffering from injury-associated NHO. The SCI–NHOmodel will enable the use of powerful genetic tools tofurther elucidate the mechanisms that lead to NHO.Importantly, it provides a clinically relevant animalmodel to test therapeutics for their ability to reduce orprevent NHO.

Acknowledgements

IGW and JPL are supported by a Career Develop-ment Fellowship (No. 1033736) and a Research Fel-lowship (No. 1044091), respectively, from the National

Health and Medical Research Council (NHMRC) ofAustralia. This study was supported in part by theMater Foundation (to JPL), a NHMRC Project Grant(to NAS), Fondation BNP–Paribas (to FG), AssistancePublique – Hôpitaux de Paris (to FG), Université deVersailles Saint Quentin (to FG), Institut National de laSanté et de la Recherche Médicale (to MCLBK), Servicede Santé des Armées (to JJL) and travel fellowships fromthe Forum for European–Australian Science and Tech-nology Cooperation, IPSEN, MERZ and ALLERGAN(to FG).

Author contributions

FG, IGW and JPL conceived the study; FG, JJL, IGW,MCLBK, DWH and JPL designed the study; FG, IKand VB designed and performed experiments on miceand analysed the results; CV performed μCT and 3Dreconstruction; FG, IK and CV analysed the results; SM,ARP and NAS performed immunohistology on mousetissues and analysed results; FG provided clinical dataand samples; FT, AA, BG, JJL and MCLBK performedanalyses on human samples and analysed results; andFG, ARP, NAS, IGW, JJL, MCLBK, DWH and JPLwrote and edited the manuscript.

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SUPPLEMENTARY MATERIAL ON THE INTERNETThe following supplementary material may be found in the online version of this article:

Supplementary materials and methods

Figure S1. Strategy to sort satellite cells and interstitial cells from mouse skeletal muscles and MPCs from mouse bone marrow stromal cells

Figure S2. Validation by qRT–PCR of progenitor cells sorted from muscle and bone marrow

Table S1. Development of NHO in the mouse requires both SCI and muscular inflammation


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