Figure 1.
Schematic illustration of the experimental design.
A) Illustration of the irradiation and transplantation setup. B) Overview of the timeline and sample collection times from the 12 week and 28 week experiments.
Figure 2.
Engraftment analysis and in vitro bone resorption.
A) Flow cytometry analysis of peripheral blood samples stained with an antibody against CD45.2 to quantify the level of engraftment. Flow cytometry was conducted in samples from all mice (see Methods section) and at the time points indicated. B-D) Splenocytes were isolated and cultured on bovine cortical bone in the presence of RANKL and M-CSF. At day 10 bone resorption was measured by CTX-I (B) and calcium release (C) release and osteoclast numbers measured by TRACP activity in the supernatants (D). Osteoclast cultures are representative of two individual experiments with 6 replicates of each condition.
Figure 3.
A) µCT analysis of the vertebrae from both the 12 and the 28-week experiment. For comparison the control group was normalized to 100%. 1) Bone volume/Total Volume (BV/TV) in % of control, 2) Trabecular Thickness (Tb.Th.) in % of control, and 3) Degree of Mineralization of the Bone (DMB) in % of control. B) Bone histomorphometry on vertebrae from the 12-week experiment. 1) Bone volume/Total Volume (BV/TV), 2) Trabecular Thickness (Tb.Th.), 3) Trabecular Number (Tb.N.), and 4) Trabecular Spacing (Tb.Sp.) C) µCT analysis of the femur diaphysis from both the 12 and the 28-week experiment. For comparison the control group was normalized to 100%. 1) Cortical Bone Volume (Ct.BV) in % of control, 2) Cortical Thickness (Ct.Th.) in % of control, 3) Cortical Degree of Mineralization of Bone (DMB) in % of control, 4) Endocortical Diameter (Ec.Dm.) in % of control, 5) Endocortical Marrow Volume (Ec.M.V.) in % of control, 6) Periosteal Diameter (P.Dm.) in % of control. µCT was conducted on all bones from mice having completed the study (see methods section).
Figure 4.
Biochemical markers of bone turnover.
Serum samples were collected in both experiments and CTX-I (A), TRACP 5b (B), CTX/TRACP 5b (C), ALP (D), P1NP (E) were measured at baseline and at week 6, 10, 18, 22 and 28, post transplantation. The oc/oc data (gray squares) are plotted as percent of control (black circles) at all time points, and when samples from both experiments were present they were pooled after normalization. The biomarker measurements were conducted in samples from all mice, and for the samples collected during the first 12 weeks on pooled data from both experiments as described in the Methods section.
Figure 5.
At termination of the 12-week experiment vertebrae were collected for bone histomorphometry. No significant differences were observed in osteoclast surface per unit bone surface (Oc.S/BS), number of osteoclasts per unit bone perimeter (N.Oc.Pm), osteoblast surface per unit bone surface (Ob.S/BS), number of osteoblasts per unit bone perimeter (N.Ob.Pm), bone formation rate (BFR/BV), mineral appositional rate (MAR), mineralizing surface (MS/BS) or osteoid volume (OV/BV). Bone histomorphometry was conducted on all specimens from the 12-week experiment (see Methods section).
Figure 6.
Maximal force achieved at failure (Fmax) as determined by 3-point bending test of the femoral cortex (A) or femoral neck (B). In C Fmax was determined by vertebral compression. Bone strength testing was conducted on all bone specimens collected as described in the methods section.
Figure 7.
Bone structure was assessed using polarized light microscopy as described in the methods section.