Supplementry Informtion PDGF-BB secreted y preosteoclsts induces ngiogenesis during coupling with osteogenesis Hui Xie, Zhung Cui, Long Wng, Zhuying Xi, Yin Hu, Lingling Xin, Chngjun Li, Ling Xie, Jnet Crne, Mei Wn, Gehu Zhen, Qin Bin, Bin Yu, Weizhong Chng, To Qiu, Mureen Pickrski, Le Thi Duong, Jolene J. Windle, Xinghng Luo, Eryun Lio & Xu Co Nture Medicine: doi:.8/nm.8
TRAP + cell Mo/mc PDGF-BB (pg ml ) c TRAP PDGF-BB 8 Durtion of tretment (dys) RANKL (ng ml ) 7 7 Durtion of 8 tretment (dys) RANKL (ng ml ) d TRAP DAPI PDGF-BB DAPI TRAP PDGF-BB DAPI BM TB Supplementry Figure. Secretion of PDGF-BB decreses grdully during the fusion process of TRAP + cells. Imges of TRAP stining of cells treted with ng ml of M-CSF nd or ng ml of RANKL for different dys for the development of different stges of TRAP + cells. Scle r, μm. () ELISA nlysis of PDGF-BB levels in the conditioned medium from monocytes nd mcrophges (cells t dy) nd different stges of TRAP + cells. Dt shown s men s.d. n = ; P <. (ANOVA). (c) In vitro cultured monocytes nd mcrophges (Mo/mc) nd TRAP + cells were stined for PDGF-BB (rown) nd further sujected to TRAP stining (red). Blck rrows indicted cells tht re stined positively for oth TRAP nd PDGF-BB. Scle r, μm. (d) Immunostining imges of TRAP (red) nd PDGF-BB (green). DAPI stins nuclei (lue). Dshed lines outline one surfce. TB: treculr one; BM: one mrrow. Scle r, μm. Nture Medicine: doi:.8/nm.8
TRAP F/8 DAPI F/8 PDGF-BB DAPI P P CB CB BM CB BM CB Supplementry Figure. F/8 + OsteoMcs re not the sme cells s preosteoclsts. Immunostining imges of TRAP (red) nd F/8 (green) (), s well s F/8 (Red) nd PDGF-BB (green) () in femorl endosteum (ottom) nd diphysel periosteum (top). DAPI stins nuclei (lue). Dshed lines outline one surfce. P: periosteum; CB: corticl one; BM: one mrrow. Scle rs, μm. Nture Medicine: doi:.8/nm.8
T. BV/TV (%) Ct. Th (μm) Ps. Pm (mm) WT TRAP-Cre Pdgf flox/flox TRAP-Cre;Pdgf flox/flox M M M M M M Supplementry Figure. Neither TRAP-Cre trnsgene nor Pdgf flox llele ffect corticl nd treculr one mss. Quntittive μct nlysis of the treculr one volume frction (T. BV/TV), corticl thickness (Ct. Th) nd periostel perimeter (Ps. Pm). For -month-old ( M) mice, n = 8 (Pdgf flox/flox ), n = 7 (other groups). For -month-old ( M) mice, n = 8 (Pdgf flox/flox nd TRAP-Cre;Pdgf flox/flox ), n = 7 (TRAP-Cre), n = (wild-type; WT). Dt shown s men s.d. P <. versus WT, TRAP-Cre, or Pdgf flox/flox mice (ANOVA). Nture Medicine: doi:.8/nm.8
BM PDGF-BB reltive percentges (%) PDGF-BB (pg) BM PDGF-BB reltive percentges (%) BM PDGF-BB reltive percentges (%) Pdgf flox/flox BMCs c Preosteoclsts Pdgf / ECs from BMCs BM ECs Other BM cells 8 8 8 8 7 Supplementry Figure. The reltive contriution of preosteoclsts nd endothelil cells to PDFG-BB production in one mrrow. () The percentge of one mrrow (BM) PDGF-BB concentrtions in -month-old mle Pdgf / versus Pdgf flox/flox mice. n = 7 (Pdgf / ), n = 8 (Pdgf flox/flox ). () Totl PDGF-BB mount (left) nd the percentge of PDGF-BB levels (right) in sorted endothelil cells (ECs) versus one mrrow cells (BMCs) from -month-old mle Pdgf / mice. n =. (c) Estimted reltive contriution of preosteoclsts, endothelil cells nd other cells to the one mrrow PDGF-BB production sed on () nd (). Nture Medicine: doi:.8/nm.8
PDGF-BB (pg ml ) Migrted MSC numers/fv Migrted EPC numers/fv L- Ctsk / 8 + + L- Mo/mc POC CM CM Ctsk / NS + + + + POC CM POC CM +PDGF- BB A Mo/mc CM POC CM +VPC c 8 Ctsk / NS L- + + + + Mo/mc POC POC POC CM CM CM CM +PDGF- +VPC BB A Supplementry Figure. Inhiition of CTSK increses secretion of PDGF-BB from preosteoclsts to stimulte migrtion of MSCs nd EPCs. () PDGF-BB concentrtions y ELISA in conditioned medium (CM) of vehicle- or cthepsin K inhiitor (L-)-treted monocytes nd mcrophges (Mo/mc) or preosteoclsts (POC) from or Ctsk / mice. (,c) Trnswell ssys for migrtion of vehicle- or SP, ntgonist (VPC)-treted MSCs () or EPCs (c) induced y Mo/mc or POC CM with or without ddition of PDGF-BB neutrlizing ntiody. Bone slices were included in ll the culture systems for preprtion of CMs. Dt shown s men s.d. n = ; P <., P <. (ANOVA). Nture Medicine: doi:.8/nm.8
MAR (μm dy ) BFR/BS (μm μm yer ) N. osteoclcin + /BS (N mm ) Serum osteoclcin (ng ml ) T. BV/TV (%) T. N (N mm ) Ct. Th (μm) Ps. Pm (mm) + L Ctsk / L- + L- + Ctsk / 8 8 7 9 L- + L- + c 7 8 8 L- + + L- + + L- + + L- + TB PB TB PB Supplementry Figure. Inhiition of CTSK increses osteolst one formtion. (,) μct imges of femor from mle wild-type mice dministered with vehicle ( ) or L- ( + L) or n ge-mtched mle Ctsk knockout (Ctsk / ) mouse (), nd quntittive nlysis of treculr one frction (T. BV/TV), treculr numer (T. Th), corticl thickness (Ct. Th), nd periostel perimeter (Ps. Pm). (). Scle r, mm. n = 8. (c) Quntittive nlysis of minerl pposition rte (MAR) nd one formtion rte (BFR) t treculr one (TB) nd periostel one (PB). n =. (d) Quntittive nlysis of the numer of immunostined osteoclcin + cells on TB nd PB surfce. n = 8. (e) ELISA of serum osteoclcin. n = 8. Dt shown s men s.d. P <., P <. (ANOVA). Nture Medicine: doi:.8/nm.8 d Ctsk / Ctsk / Ctsk / TB PB e
Ct. Th (μm) Ps. Pm (mm) Es. Pm (mm) T. BV/TV (%) T. Th (μm) T. N (N mm ) Emcn Ki7 DAPI Uterus weight (mg) SHAM OVX SHAM 8 OVX c SHAM OVX d 8 7 8 9 SHAM 9 8 7 OVX.8... e SHAM OVX Supplementry Figure 7. OVX mice exhiit one loss nd decresed endothelil cell prolifertion. () Imges of uterus from shm-operted (SHAM) or ovriectomized (OVX) mice. Scle r, cm. () Quntittive nlysis of uterus weight. (c) μct imges of femor from mice. Scle rs, mm. (d) Quntittive μct nlysis of treculr one frction (T. BV/TV), treculr thickness (T. Th), treculr numer (T. N), corticl thickness (Ct. Th), periostel perimeter (Ps. Pm) nd endostel perimeter (Es. Pm). (e) Immunostining of Endomucin (red) nd Ki7 (green) showing proliferting endothelil cells in SHAM nd OVX mice. Dt shown s men s.d. n = ; P <., P <. versus SHAM (t test). Nture Medicine: doi:.8/nm.8
Ct. Th (μm) Ps. Pm (mm) Emcn Ki7 DAPI T. BV/TV (%) T. Th (μm) Vehicle PDGF-BB L- 8. 8 Es. Pm (mm) T. N (N mm )... Supplementry Figure 8. Incresing PDGF-BB stimultes endothelil cell prolifertion nd preserves one mss in OVX osteoporotic mice. () Immunostining of Endomucin (red) nd Ki7 (green) showing proliferting endothelil cells in OVX mice treted with either vehicle, PDGF-BB or L-. DAPI stins (lue) nuclei. () μct quntifiction of treculr one frction (T. BV/TV), treculr thickness (T. Th), treculr numer (T. N), corticl thickness (Ct. Th), periostel perimeter (Ps. Pm) nd endostel perimeter (Es. Pm). Dt shown s men s.d. n =. P <., P <. (ANOVA). Nture Medicine: doi:.8/nm.8