Figure 2.
Functional characterization of BMP-eBM. (A-E) HSC transplantation assay. (A) Schematic overview. BMP-eBM hematopoietic cells (1 × 107) were collected from CAG-EGFP mice and transplanted IV into lethally irradiated (12 Gy) C57BL6J mice. Control mouse represents no transplantation. (B) Survival curve of irradiated mice after 24 weeks (n = 4). (C) Frequency of donor contribution (GFP+) within CD45+ cells (n = 3). Recipient mice were euthanized 24 weeks after transplantation, and the peripheral blood, LB, Spleen, and thymus were harvested for FCM and histological analysis. (D) Frequency of GFP+ B cell (B220+), T cell (CD3+), and myeloid cells (CD11b+) in the peripheral blood, LB-BM, spleen and thymus after 24 weeks of transplantation (n = 3). (E) A frozen section of the femur showing engrafted GFP+ hematopoietic cells (green). The blue color represents DAPI staining. (F-H) HSC engraftment assay. (F) Schematic overview. BMP-2/β-TCP complexes were implanted subcutaneously into C57BL6J recipients. After 4 weeks of implantation, recipients were lethally irradiated (12 Gy) and transplanted with LB-BM cells (1 × 107) from C57BL6J-Ly5.1 mice. Eighteen weeks later, recipients were euthanized, and tissue samples were harvested for FCM analysis. (G) The number of CD45.1+ HSCs and Leptin receptor (LepR)+ stromal cells (CD45–Ter119–CD31–LepR+; n = 4). (H) Frequency of donor contribution (CD45.1+) in the HSC, B cell, T cell, and myeloid cell populations (n = 4). (I-J) Survival assay. (I) Schematic overview. BMP-eBM was collected from C57BL6J donor and transplanted subcutaneously into irradiated (LD60/30 dose, 7.5 Gy) C57BL6J recipient, with or without supplemental BMP-2 (2 μg). Control mouse represents BMP-2 (16 μg)/β-TCP (80 mg) transplantation. In this assay, BMP-eBM was produced from BMP-2 (16 μg)/β-TCP (80 mg). (J) Survival curve irradiated mice after 28 days (n = 10). (K-N) Retransplantation assay. BMP-eBM was produced from BMP-2 (8 μg)/β-TCP (40 mg) and immersed in 0.5 mM HCl (vehicle) or BMP-2 (1 μg) before retransplantation in other mice. (K) Photographs, (L) HE staining, and (M) immunostaining images of retransplanted BMP-eBMs 2 weeks after retransplantation (n = 3). In panel L, arrowheads indicate empty lacunae. For panel M: red, Emcn; blue, DAPI. (N) Quantification of Emcn+ area per total area of the photo shown in panel M (n = 3).

Functional characterization of BMP-eBM. (A-E) HSC transplantation assay. (A) Schematic overview. BMP-eBM hematopoietic cells (1 × 107) were collected from CAG-EGFP mice and transplanted IV into lethally irradiated (12 Gy) C57BL6J mice. Control mouse represents no transplantation. (B) Survival curve of irradiated mice after 24 weeks (n = 4). (C) Frequency of donor contribution (GFP+) within CD45+ cells (n = 3). Recipient mice were euthanized 24 weeks after transplantation, and the peripheral blood, LB, Spleen, and thymus were harvested for FCM and histological analysis. (D) Frequency of GFP+ B cell (B220+), T cell (CD3+), and myeloid cells (CD11b+) in the peripheral blood, LB-BM, spleen and thymus after 24 weeks of transplantation (n = 3). (E) A frozen section of the femur showing engrafted GFP+ hematopoietic cells (green). The blue color represents DAPI staining. (F-H) HSC engraftment assay. (F) Schematic overview. BMP-2/β-TCP complexes were implanted subcutaneously into C57BL6J recipients. After 4 weeks of implantation, recipients were lethally irradiated (12 Gy) and transplanted with LB-BM cells (1 × 107) from C57BL6J-Ly5.1 mice. Eighteen weeks later, recipients were euthanized, and tissue samples were harvested for FCM analysis. (G) The number of CD45.1+ HSCs and Leptin receptor (LepR)+ stromal cells (CD45–Ter119–CD31–LepR+; n = 4). (H) Frequency of donor contribution (CD45.1+) in the HSC, B cell, T cell, and myeloid cell populations (n = 4). (I-J) Survival assay. (I) Schematic overview. BMP-eBM was collected from C57BL6J donor and transplanted subcutaneously into irradiated (LD60/30 dose, 7.5 Gy) C57BL6J recipient, with or without supplemental BMP-2 (2 μg). Control mouse represents BMP-2 (16 μg)/β-TCP (80 mg) transplantation. In this assay, BMP-eBM was produced from BMP-2 (16 μg)/β-TCP (80 mg). (J) Survival curve irradiated mice after 28 days (n = 10). (K-N) Retransplantation assay. BMP-eBM was produced from BMP-2 (8 μg)/β-TCP (40 mg) and immersed in 0.5 mM HCl (vehicle) or BMP-2 (1 μg) before retransplantation in other mice. (K) Photographs, (L) HE staining, and (M) immunostaining images of retransplanted BMP-eBMs 2 weeks after retransplantation (n = 3). In panel L, arrowheads indicate empty lacunae. For panel M: red, Emcn; blue, DAPI. (N) Quantification of Emcn+ area per total area of the photo shown in panel M (n = 3).

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