Research ArticleBONE HEALING

A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix

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Science Translational Medicine  24 Apr 2019:
Vol. 11, Issue 489, eaar4953
DOI: 10.1126/scitranslmed.aar4953
  • Fig. 1 Amino acid sequence alignment of BMP-2, BMP-6, activin A, and chimera monomers and their corresponding ribbon structures.

    (A) Amino acid (AA) sequence alignment for BMP-2 (blue), BMP-6 (magenta), activin A (yellow), BV-260, BV-261, BV-262, and BV-265 monomers. Amino acid sequences substituted into the chimeras are represented by the corresponding colors of the parental molecules. Positions of β-strands 1 to 9, PHL, and the α-helix 3 (α3) in BMP-2 are located above the amino acid sequence of BMP-2. Ribbon diagrams of glycosylated (B) BMP-6 (pink/magenta), (C) BMP-2 (cyan/blue), (D) activin A (yellow/orange), (E) BV-260, (F) BV-261, (G) BV-262, and (H) BV-265 in the “butterfly” orthogonal view. Sections of the chimera ribbon diagrams representing amino acid sequences originating from the BMP-2, BMP-6, and activin A are depicted in the color of the corresponding parental molecule. Labels indicating the location of the type I and II BMP receptor binding domains for each monomer, the relative positions of β-strands 1 to 9 (β1 to β9), the PHL, and the α-helix 3 (α3) are located next to or superimposed on the BMP-2 ribbon diagram (monomer 1, dark blue; monomer 2, blue). Ribbon diagrams of BMP-2, BMP-6, activin A, and BV-261 were generated from the published crystal structure (RCSB Protein Data Bank codes: 6OMN, 6OMO, 2ARV, and 6OML, respectively). The BMP-2 ribbon diagram was used as a surrogate for BV-260. The BV-261 ribbon diagram was used as a surrogate for BV-262 and BV-265. Only one BV-261 monomer displayed a resolved glycan in the crystal structure. The glycan of the second monomer was disordered because of crystal packing artifact. Detailed annotation of the amino acids involved in type I and II receptor binding and glycan tethering can be found in fig. S1A.

  • Fig. 2 Structural and cell-based support for increased BV-261/AKL2 binding due to glycan tethering and the effect of noggin on ACTRIIB/BMP/chimera binding and ALP activity.

    (A) Magnified ribbon diagram of BMP-2 and (B) BV-261 demonstrating glycan tethering by R16 and E109 (hydrogen bonds represented by green and orange dotted lines, respectively). (C) Ribbon diagram of a BV-261 monomer composed of amino acid sequences from the PHL and α3 helix regions of BMP-6 (red) substituted into BMP-2 (blue; Fig. 1, A and F) superimposed on BMP-2 (cyan) and (D) BMP-6 (pink). The position of the PHL of BV-261 compared to BMP-2 or BMP-6 is indicated by the blue and black arrows, respectively. The position of the N terminus is designated by N. (E) Alkaline phosphatase (ALP) activity in C2C12 cells as a function of BMP-2, endoglycosidase H (Endo Hf)–treated BMP-2, BV-261, and Endo Hf–treated BV-261 concentration (n = 3 and 2 repetitions per plate). (F) Sensorgrams comparing BV-262 (25 nM) and (G) BV-265 (25 nM) binding to ACTRIIB immobilized on the biosensor (nanometer shift) as a function of noggin concentration (0.125 nM). (H) ALP activity in C2C12 cells after treatment with BMP-2, BMP-6, BMP-2/6, BV-262, or BV-265 in the presence of increasing noggin concentration (n = 3 and 2 repetitions per plate). Data are means ± SD (data file S1).

  • Fig. 3 Comparison of BMPs and chimeras in cell-based and rodent bone induction assays.

    (A) ALP activity in C2C12 cells as a function of BMP and chimera concentration. Data are means ± SD (n = 3 repetitions of duplicate determinations). Mean values for EC50: BMP-2 = 4 ± 1 ng/mlD, BV-262 = 5 ± 3 ng/mlB,D, BV-261 = 7 ± 1 ng/mlB,C,D, BV-265 = 8 ± 3 ng/mlB,C,D, BV-260 = 61 ± 20 ng/mlA,C, BMP-6 = 36 ± 68 ng/mlA,B, and BMP-2 = 257 ± 99 ng/mlA. Mean values for EC50 not sharing the same letter are significantly different [Kruskal-Wallis rank sum test group effect, P > 0.0001; Dunn post hoc test, P < 0.05 (group comparison P values in data file S1)]. (B) Mineralization of C3H10T1/2 cells as a function of BMP-2, BV-262, and BV-265 concentration after 10 days (n = 3). (C) mRNA expression of ALP, SOX9, OSX, and RUNX2 normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hPDCs induced by growth media (GM), BMP-2, BMP-2/6, BV-262, and BV-265 (100 ng/ml) after 14 days determined by reverse transcription polymerase chain reaction (RT-PCR). (D) ALP activity in hPDCs after 14 days of stimulation by growth media, BMP-2, BMP-2/6, BV-262, and BV-265 (100 ng/ml; top, fast blue stained wells; bottom, relative staining intensity; n = 4). Mean values not sharing the same letter are significantly different [analysis of variance (ANOVA) group effect, P < 0.0002 and 0.0001, respectively; Tukey honestly significant difference (HSD) post hoc test, P < 0.05 (group comparison P values in data file S1)]. (E) Mineralization of hPDCs 23 days after stimulation with BMP-2, BV-262, or BV-265 (100 mg/ml) (top, Alizarin red–stained wells; bottom, OsteoImage-stained wells; n = 4). (F) Radiographs and μCT 3D and slice images of bone induction 16 days after injection of either BMP-2/buffer or BV-265/buffer (0.22 mg each) into muscle adjacent to the femur in rats (n = 4). (G) Histological appearance of a BMP-2/buffer–treated and (H) a BV-265/buffer–treated intramuscular explant demonstrating a bone nodule within the surrounding muscle (M) consisting of trabecular bone (TB) within highly vascularized connective tissue 16 days after injection (Safranin-O/Fast Green stain). Higher-magnification images in figs. S17 to S19. Individual data can be found in data file S1.

  • Fig. 4 Evaluation of BMP-2, BMP-2/6, BV-260, BV-261, and BV-262/ACS in macaque fibula osteotomy models.

    (A, C, E, and G) Radiographic time series and (B, D, F, and H) corresponding ex vivo μCT images of representative macaque bilateral fibula 0.5-cm wedge osteotomies treated with BMP-2/ACS (0.5 mg/cm3) on one side and BMP-2/6/ACS (0.5 mg/cm3) on the contralateral side (10 weeks; n = 6), bilateral 1-mm fibula transverse osteotomies treated with BMP-2/ACS (0.5 mg/cm3) on one side and BV-260/ACS (0.5 mg/cm3) on the contralateral side (8 weeks; n = 6), bilateral 1-mm fibula osteotomies treated with BMP-2/ACS (0.5 mg/cm3) on one side and BV-261/ACS (0.5 mg/cm3) on the contralateral side (8 weeks; n = 7), and bilateral fibula 0.5-cm wedge osteotomies treated with BMP-2/ACS (0.5 mg/cm3) on one side and BV-262/ACS (0.5 mg/cm3) on the contralateral side (10 weeks; n = 6). Black arrows locate the position of the osteotomy immediately after surgery (Post Op).

  • Fig. 5 Evaluation of BMP-2, BV-262, and BV-265/CPM in macaque fibula osteotomy models.

    (A and D) Radiographic time series, (B and E) corresponding ex vivo μCT images, and (C and F) histology (Goldner’s trichrome stain) of representative macaque bilateral fibula 0.5-cm wedge osteotomies treated with BMP-2/CPM (1.5 mg/cm3) on one side and BV-262/CPM (0.5 mg/cm3) on the contralateral side (10 weeks; n = 6) and bilateral fibula 1-mm oblique osteotomies treated with BV-262/CPM (0.5 mg/cm3) on one side and BV-265/CPM (mg/cm3) on the contralateral side (8 weeks; n = 8). Radiodense CPM is visible immediately after surgery (Post Op, white arrows). Black arrows locate the position of the osteotomy.

  • Fig. 6 CM components, cytokine retention profiles, and rat intramuscular explants.

    (A) CM composed of CDHA granules embedded in a fenestrated, polymer mesh–reinforced, macroporous rhCollagen matrix. (B) Top/bottom rhCollagen coating. (C) Scanning electron microscopy (SEM) image of CDHA granules within the macroporous rhCollagen matrix. (D) SEM image of a CDHA granule demonstrating high surface roughness. (E) SEM of a CDHA granule demonstrating internal interconnected porosity. (F) Fluorescence image of a sectioned CDHA granule demonstrating penetration of Alexa Fluor 488 fluorescent–conjugated BV-265 (cyan pseudo-color) into the interior of the granule. (G) In vivo retention of 125I-labeled BV-265 delivered in CDHA granules and in the CM compared to 125I-labeled BMP-2 implanted in CPM and ACS or injected in buffer into a rodent muscle pouch determined as a function of time (days). Data are means ± SD (n = 4 to 6 animals per group). (H) Histological evaluation of CM alone and (I) 0.45 μg, (J) 2.26 μg, and (K) 11.3 μg BV-265/CM–treated rat intramuscular implants at 14 days (Goldner’s trichrome stain). Trabecular bone is visible in the 2.26 and 11.3 μg BV-265/CM–treated explants. Higher-magnification images of 11.3 μg BV-265/CM–treated explants at (L) 3 days, (M) 5 days, (N) 9 days, and (O) 14 days. M, muscle; CG, CDHA granules; CF, collagen fibers; RBC, red blood cells; SC, stromal cells; BV, blood vessels; MOC, multinucleated osteoclasts.

  • Fig. 7 Evaluation of BV-265/CM in a macaque fibula 2-cm critical-sized ostectomy.

    (A) Radiographic image of a representative untreated macaque 2-cm ostectomy demonstrating failure to bridge at 12 weeks (n = 3). (B to D) Radiographic time series, (E to G) μCT 3D reconstructions/slice images, and (H to J) histology (Goldner’s trichrome stain) of a representative macaque 2-cm ostectomy treated with CM alone (n = 3), BV-265/CM (0.05 mg/cm3), and BV-265/CM (0.15 mg/cm3) (n = 6).

  • Fig. 8 Evaluation of BV-265/CM in baboon fibula 2.5-cm critical-sized ostectomy and fibula wedge osteotomy models.

    (A and D) Radiographic time series, (B and E) μCT 3D reconstructions/slice images, and (C and F) histology (Goldner’s trichrome stain) of a representative baboon fibula 2.5-cm ostectomy treated with BV-265/CM (0.15 mg/cm3, 26 weeks; n = 6) and a representative baboon fibula 1.0-cm wedge osteotomy treated with BV-265/CM (0.15 mg/cm3, 12 weeks; n = 6). OB, osteoblasts; TO, trabecular osteoid; GO, granule osteoid.

  • Table 1 BMP receptor binding affinity.

    ReceptorReceptor binding affinity KD (nM)
    BMP-2BMP-6BMP-2/6Activin ABV-260BV-261BV-262BV-265TGF-β1
    Type 1ALK1*NBNB
    ALK2NB700255NBNB2.22.00.9
    ALK31.111.01.7NB1.03.32.01.7
    ALK4NBNB
    ALK5NBNBNB
    ALK61.120.00.5NB0.80.81.00.4
    Type IIACTRIIA52.73.22.50.82.140.02.0<0.1
    ACTRIIB8.00.71.1<0.10.55.00.5<0.1
    BMPRII26.73.98.01.33.682.03.5<0.1
    TΒRIINBNB<0.1

    *BMP-9–positive control KD for ALK1 = 0.5 nM.

    †Not performed.

    ‡NB = no specific binding (>200 nM).

    • Table 2 Torsional biomechanical measurements of the cynomolgus macaque bilateral fibula osteotomy models.

      Torsional biomechanical measurements of the cynomolgus macaque bilateral fibula osteotomy models.. Values presented as means ± SD. Mean values not sharing the same letter are significantly different (P < 0.05). Torsional mechanical properties data and equal variance paired two-tailed t test P values in data file S1. Torsional mechanical properties data and ANOVA group effect P values for all comparisons in data file S1. Tukey HSD post hoc test individual comparisons P values in data file S1.

      ComparisonModel
      (no. of animals)
      Duration
      (weeks)
      NonunionCallus
      volume
      (mm3)
      Torsional stiffness
      (Nm/degree)
      (% intact fibulae)
      Maximum
      torque
      (Nm)
      (% intact fibulae)
      BMP-2/ACS
      (0.5 mg/cm3)
      Wedge
      (6)
      103/6869.1 ± 342.9A0.043 ± 0.032A,*
      68.5%
      0.92 ± 0.36A,*
      44.2%
      BMP-2/6/ACS
      (0.5 mg/cm3)
      3/6847.9 ± 213.1A0.031 ± 0.017A,*
      64.9%
      0.87 ± 0.23A,*
      31.9%
      BMP-2/ACS
      (0.5 mg/cm3)
      Transverse
      (6)
      84/6438.9 ± 265.4A0.041 ± 0.024A,*
      56.1%
      0.75 ± 0.29A,*
      44.1%
      BV-260/ACS
      (0.5 mg/cm3)
      4/6454.9 ± 300.3A0.032 ± 0.013A,*
      51.7%
      0.69 ± 0.24A,*
      32.5%
      BMP-2/ACS
      (0.5 mg/cm3)
      Transverse
      (7)
      84/7378.4 ± 110.6A0.029 ± 0.012A,*
      30.4%
      0.65 ± 0.15A,*
      48.8%
      BV-261/ACS
      (0.5 mg/cm3)
      6/7572.8 ± 178.2B0.044 ± 0.018A,*
      45.8%
      0.82 ± 0.29A,*
      60.9%
      BMP-2/ACS
      (0.5 mg/cm3)
      Wedge
      (6)
      106/6876.8 ± 182.5A0.048 ± 0.019A,*
      50.2%
      0.92 ± 0.20A,*
      68.4%
      BV-262/ACS
      (0.5 mg/cm3)
      6/61,368.4 ± 266.9B0.063 ± 0.019B
      65.2%
      1.17 ± 0.27B
      87.6%
      BMP-2/CPM
      (1.5 mg/cm3)
      Wedge
      (6)
      106/61,227.2 ± 280.3A0.053 ± 0.021A,*
      55.1%
      0.89 ± 0.20A,*
      66.3%
      BV-262/CPM
      (0.5 mg/cm3)
      6/61,219.9 ± 174.0A0.055 ± 0.019A,*
      57.3%
      0.89 ± 0.37A,*
      66.7%
      BV-262/CPM
      (0.5 mg/cm3)
      Oblique
      (8)
      87/81,244.2 ± 280.3A0.049 ± 0.025A,*
      50.4%
      0.71 ± 0.33A,*
      52.9%
      BV-265/CPM
      (0.5 mg/cm3)
      8/81,329.7 ± 240.1A0.067 ± 0.025B
      69.2%
      1.10 ± 0.22B
      81.9%
      Intact fibulae(24)0.096 ± 0.0271.34 ± 0.27

      *Torsional mechanical properties significantly different from intact fibulae (P < 0.05).

      • Table 3 Comparison of BMP-2 and BV-265 retention in multiple carriers.

        AUC (fraction*days) = area under the % retention versus time curve for 0 to 7 days, 0 to 14 days, and 0 to 21 days. Individual data located in data file S1.

        Treatmentt1/2
        (days)
        AUC0–7 days
        (fraction*days)
        AUC0–14 days
        (fraction*days)
        AUC0–21 days
        (fraction*days)
        AUC0–28 days
        (fraction*days)
        BMP-2/buffer (n = 4)0.711.40
        BMP-2/ACS (n = 4)2.693.033.29
        BMP-2/CPM (n = 6)8.125.128.6610.6411.95
        BV-265/CDHA (n = 3)5.145.188.2210.46
        BV-265/CM (n = 4)6.034.897.659.3910.67
      • Table 4 Mechanical properties of the CM and ACS.

        Values presented as means ± SD. Mean values not sharing the same letter are significantly different (P < 0.05). Compressive mechanical properties equal variance two-tailed t test (P < 0.00001). Tensile mechanical properties ANOVA group effect for all comparisons (P < 0.00001). Tukey HSD post hoc test CM containing mesh versus CM without mesh and ACS (P < 0.0001). CM without mesh and ACS (P > 0.95). Data and individual P values located in data file S1.

        CarrierNo. per groupStrength*
        (kPa)
        Modulus
        (kPa)
        Compressive mechanical properties
          ACS60.8 ± 0.2A3.6 ± 0.5 A
          CM982.7 ± 15.2B1,402.4 ± 195.0B
        Tensile mechanical properties
          ACS636.5 ± 5.0A103.9 ± 6.0A
          CM without a
        mesh
        618.3 ± 2.7A94.8 ± 6.1A
          CM containing a
        mesh
        6262.5 ± 65.0B708.4 ± 98.1B

        *Compressive strength determined at 50% strain.

        • Table 5 Characterization of CM, BV-265/CM, and BMP-2/CM explants.

          Characterization of CM, BV-265/CM, and BMP-2/CM explants.. Values presented as means ± SD. Mean values for the dose-ranging study and mean values for the paired BMP-2 versus BV-265/CM comparison not sharing the same letter are significantly different (P < 0.05) (n = 4 per group). Explant nodule area for the 0.0 μg group was the result of residual CDHA granules with no bone identified. ANOVA group effect for total explant nodule area and bone area/total explant nodule area (P < 0.000003 and P < 0.00056, respectively). Tukey HSD hoc test explant nodule area (P < 0.05) (group comparison P values in data file S1). Equal variance two-tailed paired t test explant 11.3 μg BMP-2 versus BV-265/CM area and bone area/explant area (P < 0.045 and P > 0.006). Individual data located in data file S1.

          Treatment:
          Concentration
          (μg, mg/cm3)
          Explant nodule area
          (mean ± SD)
          Bone area/explant
          nodule area
          (mean ± SD)
          BV-265/CM: 0.0 μg,
          0.0 mg/cm3
          6.9 ± 1.8 mm2, A,B0.0 ± 0.0%A
          BV-265/CM: 0.45 μg,
          0.004 mg/cm3
          10.0 ± 2.7 mm2, B7.4 ± 6.3%A
          BV-265/CM: 2.26 μg,
          0.02 mg/cm3
          10.4 ± 2.9 mm2, B25.6 ± 13.3%B
          BV-265/CM: 11.3 μg,
          0.1 mg/cm3
          23.9 ± 2.6 mm2, C26.7 ± 3.1%B
          BMP-2/CM: 11.3 μg,
          0.1 mg/cm3
          12.5 ± 4.3 mm2, A35.8 ± 0.1%A
          BV-265/CM: 11.3 μg,
          0.1 mg/cm3
          20.7 ± 4.3 mm2, B31.8 ± 0.1%B
        • Table 6 Torsional biomechanical measurements of the nonhuman primate bilateral fibula ostectomy and osteotomy models.

          Values presented as means ± SD. Mean values not sharing the same letter are significantly different (P < 0.05). ANOVA group effect for macaque fibula ostectomy callus area (P < 0.002). Tukey HSD post hoc test (P < 0.05) (group comparison P values in data file S1). Equal variance paired two-tailed t test comparison of 0.05 versus 0.15 mg/cm3 BV-265/CM torsional stiffness and maximum torque (P > 0.10 and P < 0.016, respectively). ANOVA group effect for macaque fibula ostectomy torsional stiffness and maximum torque versus intact fibulae (P < 0.00002). Tukey HSD post hoc test (P < 0.05) (group comparison P values in data file S1). Equal variance unpaired two-tailed t test comparison of baboon 2.5-cm ostectomy and baboon 1.0-cm wedge osteotomy BV-265/CM (0.15 mg/cm3) versus intact fibulae torsional stiffness and maximum torque (P > 0.65 and P < 0.001, respectively, and P > 0.0007 and P < 0.0009, respectively). Individual data located in data file S1.

          Treatment
          (mg/cm3)
          No. of animalsDuration
          (weeks)
          Callus
          volume
          (mm3)
          Torsional stiffness
          (Nm/degree)
          Maximum torque
          (Nm)
          Macaque BV-265/CM 2.0-cm fibula ostectomy model
          Untreated defect312No bone presentInsufficient stiffness or strength to allow
          mechanical testing
          CM alone3133.7 ± 28.3A
          BV-265/CM
          (0.05 mg/cm3)
          6 (bilateral)12422.8 ± 211.1B0.033 ± 0.019A,*0.70 ± 0.12A,*
          BV-265/CM
          (0.15 mg/cm3)
          573.8 ± 43.2B0.063 ± 0.024A,*1.04 ± 0.24B
          Intact macaque fibulae0.096 ± 0.0271.34 ± 0.27
          Baboon BV-265/CM 2.5-cm fibula ostectomy model
          BV-265/CM
          (0.15 mg/cm3)
          6260.188 ± 0.1084.13 ± 0.56*
          Baboon BV-265/CM bilateral 1.0-cm fibula wedge osteotomy model
          BV-265/CM
          (0.15 mg/cm3)
          3 (bilateral)120.316 ± 0.046*5.62 ± 1.27*
          Intact baboon fibulae50.163 ± 0.0562.69 ± 0.47

          *Significantly different from intact fibulae.

          Supplementary Materials

          • stm.sciencemag.org/cgi/content/full/11/489/eaar4953/DC1

            Materials and Methods

            Fig. S1. Amino acid sequence alignment of BMP-2, BMP-6, activin A, and chimera monomers and their corresponding ribbon structures.

            Fig. S2. BMPs and chimera amino acid sequence and corresponding C2C12 ALP activity EC50.

            Fig. S3. Expression of BMP type I and II receptors in human, nonhuman primate, canine, rat, and mouse bone and muscle.

            Fig. S4. BMP-2 and BV-265 heparin binding.

            Fig. S5. Ribbon diagram of glycosylated BMP-2 and BMP-6.

            Fig. S6. SDS-PAGE analysis of BMP-2 and BV-261 and the role of glycosylation in increased BV-261/ALK2 binding.

            Fig. S7. BMP and BV-261 monomer surface charge diagrams and their corresponding glycans superimposed on ALK3 ribbon diagrams.

            Fig. S8. Chimera/noggin binding and the effect of gremlin on BMP/chimera-induced ALP activity.

            Fig. S9. BMP versus activin A/TGF-β signaling in C2C12 cells.

            Fig. S10. BMP versus activin A/TGF-β signaling in hMSCs.

            Fig. S11. BMP receptors in hPDCs and BMP versus activin A/TGF-β signaling in hPDCs and hMSCs.

            Fig. S12. Cytokine-induced C2C12 CAGA-luciferase and HEK-Blue TGF-β SEAP activity.

            Fig. S13. Smad 1/5/8 immunohistological staining of BMP-2– and BV-262/ACS–treated rat intramuscular explants.

            Fig. S14. BMP-2, BMP-6, and BMP-2/6 activity in C3H10T1/2 cells and in a rat intramuscular explant.

            Fig. S15. Angiogenic gene expression and secreted proteins in BMP and chimera-treated hMVECs and hPDCs.

            Fig. S16. Receptor and osteogenic gene expression in BMP and chimera-treated hUVECs and hMVECs.

            Fig. S17. BV-265/buffer–treated rat intramuscular explant.

            Fig. S18. BMP-2/buffer–treated rat intramuscular explant.

            Fig. S19. Endochondral and direct bone formation induced by BV-265/buffer in a rat intramuscular explant.

            Fig. S20. Bone nodule vascular support in a BV-265/buffer–treated rat intramuscular explant.

            Fig. S21. Vascular support in a BMP-2/buffer–treated rat intramuscular explant.

            Fig. S22. Histological appearance of BMP-2/ACS–treated versus BMP-2/6/ACS–treated and BMP-2/ACS–treated versus BV-262/ACS–treated nonhuman primate fibula osteotomies.

            Fig. S23. Granule shedding in an uncoated and a rhCollagen-coated CM.

            Fig. S24. Histological appearance of CM alone and BV-265/CM–treated rat intramuscular implants.

            Fig. S25. Time course histological appearance of 11.3 μg BV-265/CM and CM–treated rat intramuscular implants.

            Fig. S26. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 1 day after treatment.

            Fig. S27. Histological evaluation of a BV-265-2/CM–treated rat intramuscular implant 1 day after treatment.

            Fig. S28. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 3 days after treatment.

            Fig. S29. Histological evaluation of a BV-265/CM–treated rat intramuscular implant 3 days after treatment.

            Fig. S30. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 5 days after treatment.

            Fig. S31. Histological evaluation of a BV-265/CM–treated rat intramuscular implant 5 days after treatment.

            Fig. S32. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 9 days after treatment.

            Fig. S33. Histological evaluation of a BV-265/CM-treated rat intramuscular implant 9 days after treatment.

            Fig. S34. Histological evaluation of a BMP-2/CM-treated rat intramuscular implant 14 days after treatment.

            Fig. S35. Histological evaluation of a BV-265/CM-treated rat intramuscular implant 14 days after treatment.

            Fig. S36. Analysis of the BMP-2 sequence using iTope.

            Fig. S37. Analysis of the BV-262 sequence using iTope.

            Fig. S38. Analysis of the BV-265 sequence using iTope.

            Fig. S39. CM implanted in a cynomolgus macaque fibula 2-cm ostectomy.

            Fig. S40. Histological appearance of a CM-treated macaque fibula 2-cm ostectomy 12 weeks after treatment.

            Fig. S41. Histological appearance of a BV-265/CM (0.05 mg/cm3)–treated macaque fibula 2-cm ostectomy neocortex 12 weeks after treatment.

            Fig. S42. Histological appearance of the medullary region of a BV-265/CM (0.05 mg/cm3)–treated macaque fibula 2-cm ostectomy 12 weeks after treatment.

            Fig. S43. Histological appearance of a BV-265/CM (0.15 mg/cm3)–treated macaque fibula 2-cm ostectomy neocortex 12 weeks after treatment.

            Fig. S44. Histological appearance of the medullary region of a BV-265/CM (0.15 mg/cm3)–treated macaque fibula 2-cm ostectomy 12 weeks after treatment.

            Fig. S45. Positive TRAP-stained osteoclasts in macaque fibula ostectomies.

            Fig. S46. Radiographic time series of a baboon fibula 2.5-cm ostectomy treated with BV-265/CM (0.15 mg/cm3).

            Fig. S47. Histological appearance of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy neocortex 26 weeks after treatment.

            Fig. S48. Histological appearance of the medullary region of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy 26 weeks after treatment.

            Fig. S49. Positive TRAP-stained osteoclasts in baboon fibula ostectomies.

            Fig. S50. Histological appearance of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy neocortex 12 weeks after treatment.

            Fig. S51. Histological appearance of the medullary region of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy 12 weeks after treatment.

            Fig. S52. Histological appearance of the wedge region of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy 12 weeks after treatment.

            Table S1. Data processing, refinement statistics, and Procheck analysis for glycosylated BMP-2, BMP-6, and BV-261.

            Table S2. Summary of nonhuman primate BMP/chimera comparisons.

            Table S3. Cytokine CD4+ T cell assays (50 patient donors).

            Table S4. Collagen CD4+ T cell assays (50 patient donors).

            Data file S1. Individual datasets for included study data (Excel file).

          • The PDF file includes:

            • Materials and Methods
            • Fig. S1. Amino acid sequence alignment of BMP-2, BMP-6, activin A, and chimera monomers and their corresponding ribbon structures.
            • Fig. S2. BMPs and chimera amino acid sequence and corresponding C2C12 ALP activity EC50.
            • Fig. S3. Expression of BMP type I and II receptors in human, nonhuman primate, canine, rat, and mouse bone and muscle.
            • Fig. S4. BMP-2 and BV-265 heparin binding.
            • Fig. S5. Ribbon diagram of glycosylated BMP-2 and BMP-6.
            • Fig. S6. SDS-PAGE analysis of BMP-2 and BV-261 and the role of glycosylation in increased BV-261/ALK2 binding.
            • Fig. S7. BMP and BV-261 monomer surface charge diagrams and their corresponding glycans superimposed on ALK3 ribbon diagrams.
            • Fig. S8. Chimera/noggin binding and the effect of gremlin on BMP/chimera-induced ALP activity.
            • Fig. S9. BMP versus activin A/TGF-β signaling in C2C12 cells.
            • Fig. S10. BMP versus activin A/TGF-β signaling in hMSCs.
            • Fig. S11. BMP receptors in hPDCs and BMP versus activin A/TGF-β signaling in hPDCs and hMSCs.
            • Fig. S12. Cytokine-induced C2C12 CAGA-luciferase and HEK-Blue TGF-β SEAP activity.
            • Fig. S13. Smad 1/5/8 immunohistological staining of BMP-2– and BV-262/ACS–treated rat intramuscular explants.
            • Fig. S14. BMP-2, BMP-6, and BMP-2/6 activity in C3H10T1/2 cells and in a rat intramuscular explant.
            • Fig. S15. Angiogenic gene expression and secreted proteins in BMP and chimera-treated hMVECs and hPDCs.
            • Fig. S16. Receptor and osteogenic gene expression in BMP and chimera-treated hUVECs and hMVECs.
            • Fig. S17. BV-265/buffer–treated rat intramuscular explant.
            • Fig. S18. BMP-2/buffer–treated rat intramuscular explant.
            • Fig. S19. Endochondral and direct bone formation induced by BV-265/buffer in a rat intramuscular explant.
            • Fig. S20. Bone nodule vascular support in a BV-265/buffer–treated rat intramuscular explant.
            • Fig. S21. Vascular support in a BMP-2/buffer–treated rat intramuscular explant.
            • Fig. S22. Histological appearance of BMP-2/ACS–treated versus BMP-2/6/ACS–treated and BMP-2/ACS–treated versus BV-262/ACS–treated nonhuman primate fibula osteotomies.
            • Fig. S23. Granule shedding in an uncoated and a rhCollagen-coated CM.
            • Fig. S24. Histological appearance of CM alone and BV-265/CM–treated rat intramuscular implants.
            • Fig. S25. Time course histological appearance of 11.3 μg BV-265/CM and CM–treated rat intramuscular implants.
            • Fig. S26. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 1 day after treatment.
            • Fig. S27. Histological evaluation of a BV-265-2/CM–treated rat intramuscular implant 1 day after treatment.
            • Fig. S28. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 3 days after treatment.
            • Fig. S29. Histological evaluation of a BV-265/CM–treated rat intramuscular implant 3 days after treatment.
            • Fig. S30. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 5 days after treatment.
            • Fig. S31. Histological evaluation of a BV-265/CM–treated rat intramuscular implant 5 days after treatment.
            • Fig. S32. Histological evaluation of a BMP-2/CM–treated rat intramuscular implant 9 days after treatment.
            • Fig. S33. Histological evaluation of a BV-265/CM-treated rat intramuscular implant 9 days after treatment.
            • Fig. S34. Histological evaluation of a BMP-2/CM-treated rat intramuscular implant 14 days after treatment.
            • Fig. S35. Histological evaluation of a BV-265/CM-treated rat intramuscular implant 14 days after treatment.
            • Fig. S36. Analysis of the BMP-2 sequence using iTope.
            • Fig. S37. Analysis of the BV-262 sequence using iTope.
            • Fig. S38. Analysis of the BV-265 sequence using iTope.
            • Fig. S39. CM implanted in a cynomolgus macaque fibula 2-cm ostectomy.
            • Fig. S40. Histological appearance of a CM-treated macaque fibula 2-cm ostectomy 12 weeks after treatment.
            • Fig. S41. Histological appearance of a BV-265/CM (0.05 mg/cm3)–treated macaque fibula 2-cm ostectomy neocortex 12 weeks after treatment.
            • Fig. S42. Histological appearance of the medullary region of a BV-265/CM (0.05 mg/cm3)–treated macaque fibula 2-cm ostectomy 12 weeks after treatment.
            • Fig. S43. Histological appearance of a BV-265/CM (0.15 mg/cm3)–treated macaque fibula 2-cm ostectomy neocortex 12 weeks after treatment.
            • Fig. S44. Histological appearance of the medullary region of a BV-265/CM (0.15 mg/cm3)–treated macaque fibula 2-cm ostectomy 12 weeks after treatment.
            • Fig. S45. Positive TRAP-stained osteoclasts in macaque fibula ostectomies.
            • Fig. S46. Radiographic time series of a baboon fibula 2.5-cm ostectomy treated with BV-265/CM (0.15 mg/cm3).
            • Fig. S47. Histological appearance of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy neocortex 26 weeks after treatment.
            • Fig. S48. Histological appearance of the medullary region of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy 26 weeks after treatment.
            • Fig. S49. Positive TRAP-stained osteoclasts in baboon fibula ostectomies.
            • Fig. S50. Histological appearance of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy neocortex 12 weeks after treatment.
            • Fig. S51. Histological appearance of the medullary region of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy 12 weeks after treatment.
            • Fig. S52. Histological appearance of the wedge region of a BV-265/CM (0.15 mg/cm3)–treated baboon fibula 2.5-cm ostectomy 12 weeks after treatment.
            • Table S1. Data processing, refinement statistics, and Procheck analysis for glycosylated BMP-2, BMP-6, and BV-261.
            • Table S2. Summary of nonhuman primate BMP/chimera comparisons.
            • Table S3. Cytokine CD4+ T cell assays (50 patient donors).
            • Table S4. Collagen CD4+ T cell assays (50 patient donors).
            • Legend for data file S1

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            Other Supplementary Material for this manuscript includes the following:

            • Data file S1. Individual datasets for included study data (Excel file).

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