Supplementary Materials

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).