Fig 1.
(A) Image of the RH used in the ground control and spaceflight conditions (credit Dominic Hart, NASA).
(B) Each experimental condition of the NASA RR-1 study showing the biologically relevant independent variables examined. Adapted from Choi, et al. [11]. (C) Representative studied sites of μCT analysis with type of bone and the thickness of the selected VOI.
Fig 2.
Bone loss in the mouse femoral head is associated with weightlessness in microgravity.
Characterization metrics of cancellous bone in the femoral head show that (A) BV/TV is decreased in FL. Bones from FL mice have significantly lower (B) Tb.N and of those trabeculae, spaceflight reduces (C) Tb.Th. Both age and spaceflight increase (D) Tb.Sp, whereas only FL has an increase in (E) Tb.Pf. (F) Conn.D is not significantly affected; however, VIV and FL have similarly reduced Conn.D. VIV bones experience another similar increase in magnitude of Tb.Pf, which was not statistically significant. Data shown are the mean ± standard deviation with a scatter plot (ns: non-significant, * : p < 0.033, **: p < 0.002, ***: p < 0.0002, ****: p < 0.0001). (G) μCT volumetric reconstructions of a representative sample from each group show evident decreased cancellous bone in FL.
Fig 3.
The loss of bone in the femoral distal epiphysis is affected by housing type and weightlessness conditions in microgravity.
Cancellous bone measurements in the distal epiphysis exhibit a housing-related dependency on (A) BV/TV as well as an effect from spaceflight. (B) Tb.N is decreased in FL following the same patterns as BV/TV. Interestingly, (C) Tb.Th. is not affected by age, cage type, or spaceflight, however age and spaceflight significantly increase (D) Tb.Sp (E) Tb.Pf is only effected in FL. (F) Conn.D is decreased in FL. Data shown are the mean ± standard deviation with a scatter plot (ns: non-significant, * : p < 0.033, **: p < 0.002, ***: p < 0.0002). (G) μCT volumetric reconstructions of a representative sample from each group show visibly decreased presence of bone in VIV and FL.
Fig 4.
Femoral neck marrow cavity is enlarged in spaceflight but not significantly changed with age and habitat conditions.
Cortical bone characterization metrics in the femoral neck indicate FL has less (A) BA/TA than VIV. Nonsignificant trends in the data show an increase in (B) Ct.Th in VIV that is opposite in FL. (C) E.Pm is significantly increased in VIV, GC, and FL from BL, though (D) P.Pm experiences no changes. Data shown are the mean ± standard deviation with a scatter plot (ns: non-significant, * : p < 0.033, **: p < 0.002, ***: p < 0.0002). (E) μCT volumetric reconstructions of a representative sample from each group show similar presence of bone in BL and GC, while VIV and FL demonstrate erosion in the endosteal surface.
Fig 5.
In the femoral midshaft, spaceflight abrogates increases in bone parameters BA/TA and Ct.Th in controls.
Cortical bone characterization metrics in the femoral midshaft indicate the effect of age increasing (A) BA/TA and (B) Ct.Th, which is undone in FL. (C) E.Pm and (D) P.Pm are unchanged between groups. Data shown are the mean ± standard deviation with a scatter plot (ns: non-significant, **: p < 0.002, ***: p < 0.0002). (E) μCT volumetric reconstructions of a representative sample from each group show similar presence of bone in BL and GC, while FL demonstrates erosion in the endosteal surface.
Fig 6.
Vertebral cancellous tissueμCT parameters are not significantly affected by aging from 16 to 21 weeks, housing type, or microgravity.
Cancellous bone characterization metrics in the L2 vertebrae demonstrate no differences between groups in (A) BV/TV, (B) Tb.N, (C) Tb.Th, (D) Tb.Sp, (E) Tb.Pf, or (F) Conn.D. Nonsignificant trends implicate an effect of spaceflight reducing BV/TV, increasing DA, and reducing Tb.N. Data shown are the mean ± standard deviation with a scatter plot (ns: non-significant). (G) μCT volumetric reconstructions of a representative sample from each group show a slight decrease in bone parameters from FL mice, which are not deemed significant by statistical testing.
Fig 7.
Bone loss in microgravity versus 1g controls in the RH is specific to weight-bearing skeletal sites.
Using one-sample and paired t-tests, findings revealed (A) vertebrae showed no significant change, while the (B) femoral head and (C) distal epiphysis experienced significant bone loss in spaceflight, suggesting that microgravity unloading, but not other systemic factors, caused bone loss in LEO. (D-F) The vivarium cage housing, known to provide reduced topological enrichment, also elicited bone losses of smaller magnitude than spaceflight, but only in weight-bearing bone sites.
Fig 8.
Spaceflight in microgravity accelerates secondary endochondral ossification of the proximal epiphysis in the femur.
FL bones had 50% of their cohort ossified in the (A) femoral head, over twice as many as in GC cohort. Differences in ossification at BL (16 weeks) and the other groups (21 weeks) in the (B) distal epiphysis appear independent of aging. (C) μCT volumetric reconstructions of the femoral head from a representative sample from each group show increased cavitation in the mineralized cartilage of the VIV and FL groups.
Fig 9.
Spaceflight in microgravity reduces sGAG content in the femoral head and distal epiphysis.
Representative histology of Safranin-O staining on femoral heads, with the (A) BL as the control, show a decrease of proteoglycan content in (B) VIV, a loss of the distinct tidemark in (C) GC, and complete deficit in an ossified sample from (D) FL. The distal epiphyses show ossification-dependent changes to the growth plate. During pre-ossification at 21 weeks (F, VIV), proteoglycan content decreases and is upregulated during ossification (GC, FL) to finalize remodeling into the dual-walled gap characteristic of distal epiphysis secondary ossification.