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Housing in the Animal Enclosure Module Spaceflight Hardware Increases Trabecular Bone Mass in Ground-Control Mice

Shane A. Lloyd
Shane A. Lloyd
, 
Virginia S. Ferguson
Virginia S. Ferguson
, 
Steven J. Simske
Steven J. Simske
, 
Alexander W. Dunlap
Alexander W. Dunlap
, 
Eric W. Livingston
Eric W. Livingston
 y 
Ted A. Bateman
Ted A. Bateman
   | 01 jul 2013
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Article Category: Research Article
Publicado en línea: 01 jul 2013
Páginas: 2 - 19
DOI: https://doi.org/10.2478/gsr-2013-0001
© 2013 Shane A. Lloyd et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

Photograph of the Animal Enclosure Module (AEM) hardware. The AEM has water and food delivery systems, a 12-hour light/dark cycle, wire mesh walls to allow for animal gripping during spaceflight, and a constant airflow mechanism to remove floating waste. The AEM can support up to 10 adult mice. Image used with permission of the National Aeronautics and Space Administration Ames Research Center.
Photograph of the Animal Enclosure Module (AEM) hardware. The AEM has water and food delivery systems, a 12-hour light/dark cycle, wire mesh walls to allow for animal gripping during spaceflight, and a constant airflow mechanism to remove floating waste. The AEM can support up to 10 adult mice. Image used with permission of the National Aeronautics and Space Administration Ames Research Center.

Figure 2.

Cortical quantitative histomorphometric parameters. Femur mid-diaphysis cross-sections were collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Vivarium and AEM mice received a single injection of the bone label Calcein (20 mg/kg) approximately 22 hours prior to the start of the study. Measured parameters included mineralizing surface normalized to bone surface (MS/BS) at both the (A) periosteal surface (Ps.MS/BS) and (B) endocortical surface (Ec.MS/BS). MS/BS was used as an index of bone formation. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05).
Cortical quantitative histomorphometric parameters. Femur mid-diaphysis cross-sections were collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Vivarium and AEM mice received a single injection of the bone label Calcein (20 mg/kg) approximately 22 hours prior to the start of the study. Measured parameters included mineralizing surface normalized to bone surface (MS/BS) at both the (A) periosteal surface (Ps.MS/BS) and (B) endocortical surface (Ec.MS/BS). MS/BS was used as an index of bone formation. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05).

Figure 3.

Representative images of trabecular microstructure at the proximal tibia. These images illustrate the greater amount of trabecular bone in mice housed in the Animal Enclosure Module (AEM) versus standard Vivarium housing. Images were selected based on group mean for trabecular bone volume fraction (BV/TV). Images were acquired using a μCT20 (Scanco Medical AG; Brüttisellen, Switzerland) with an isotropic voxel size of 9 m and represent a three dimensional reconstruction of 100 slices (0.9 mm total) of the tibia, immediately distal to the epiphyseal plate.
Representative images of trabecular microstructure at the proximal tibia. These images illustrate the greater amount of trabecular bone in mice housed in the Animal Enclosure Module (AEM) versus standard Vivarium housing. Images were selected based on group mean for trabecular bone volume fraction (BV/TV). Images were acquired using a μCT20 (Scanco Medical AG; Brüttisellen, Switzerland) with an isotropic voxel size of 9 m and represent a three dimensional reconstruction of 100 slices (0.9 mm total) of the tibia, immediately distal to the epiphyseal plate.

Figure 4.

Trabecular microstructural and histomorphometric parameters. Tibias were collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Microstructural parameters assessed by microCT included (A) trabecular bone volume fraction (BV/TV) and (B) trabecular connectivity density (Conn.D). Vivarium and AEM mice received a single injection of the bone label Calcein (20 mg/kg) approximately 22 hours prior to the start of the study. Measured trabecular histomorphometric parameters included (C) trabecular mineralizing surface normalized to bone surface (MS/BS). MS/BS was used as an index of bone formation. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).
Trabecular microstructural and histomorphometric parameters. Tibias were collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Microstructural parameters assessed by microCT included (A) trabecular bone volume fraction (BV/TV) and (B) trabecular connectivity density (Conn.D). Vivarium and AEM mice received a single injection of the bone label Calcein (20 mg/kg) approximately 22 hours prior to the start of the study. Measured trabecular histomorphometric parameters included (C) trabecular mineralizing surface normalized to bone surface (MS/BS). MS/BS was used as an index of bone formation. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Figure 5.

Trabecular osteoblast and osteoclast surface parameters. Histological sections were prepared from the trabecular bone of the tibia and L5 lumbar vertebrae collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. We assessed (A) tibia trabecular osteoblast surface normalized to bone surface (Ob.S/BS) and (B) tibia trabecular osteoclast surface normalized to bone surface (Oc.S/BS). These measurements were also made at the L5 vertebrae (C,D). Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).
Trabecular osteoblast and osteoclast surface parameters. Histological sections were prepared from the trabecular bone of the tibia and L5 lumbar vertebrae collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. We assessed (A) tibia trabecular osteoblast surface normalized to bone surface (Ob.S/BS) and (B) tibia trabecular osteoclast surface normalized to bone surface (Oc.S/BS). These measurements were also made at the L5 vertebrae (C,D). Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Figure 6.

Serum markers of bone turnover. Levels of the serum bone formation marker (A) osteocalcin and (B) alkaline phosphatase were assessed. In addition, we measured the serum bone resorption marker (C) tartrate-resistant acid phosphatase 5b (TRAP5b) as well as serum levels of (D) calcium and (E) phosphorus. Blood was collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).
Serum markers of bone turnover. Levels of the serum bone formation marker (A) osteocalcin and (B) alkaline phosphatase were assessed. In addition, we measured the serum bone resorption marker (C) tartrate-resistant acid phosphatase 5b (TRAP5b) as well as serum levels of (D) calcium and (E) phosphorus. Blood was collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Mineral composition analysis of the femur. Tissue was collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Baseline Vivarium AEM
Total Dry Mass (mg) 28.1 ± 0.5A,V 31.1 ± 0.3 31.3 ± 0.3
Total Organic Mass (mg) 10.6 ± 0.2A,V 11.3 ± 0.1 11.6 ± 0.1
Total Mineral Mass (mg) 17.5 ± 0.3A,V 19.8 ± 0.2 19.7 ± 0.2
Total Mineral Content (%) 62.3 ± 0.3V 63.7 ± 0.2 63.0 ± 0.3
Diaphysis Mineral Content (%) 64.0 ± 0.3V 65.5 ± 0.4# 64.4 ± 0.2#
Metaphysis Mineral Content (%) 60.1 ± 0.4 61.0 ± 0.5 61.3 ± 0.5

Trabecular microarchitectural parameters of the tibia and humerus. Tissue was collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. SMI=structure modeling index, Tb.N=trabecular number, Tb.Th=trabecular thickness, Tb.Sp=trabecular separation, BV/TV=trabecular bone volume fraction, Conn.D=trabecular connectivity density. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Baseline Vivarium AEM
Proximal Tibia
SMI (no units) 3.08 ± 0.06 2.98 ± 0.06# 2.63 ± 0.07#
Tb.N (1/mm) 4.07 ± 0.07 4.01 ± 0.09# 4.44 ± 0.1#
Tb.Th (mm) 0.036 ± 0.007 0.039 ± 0.006 0.040 ± 0.004
Tb.Sp (mm) 0.247 ± 0.005 0.252 ± 0.006# 0.226 ± 0.006#
Proximal Humerus
BV/TV (%) 10.4 ± 0.5V 8.5 ± 0.4# 10.0 ± 0.6#
Conn.D (1/mm3) 80 ± 7V 53 ± 8 73 ± 9

Femur cortical histomorphometric parameters. Data was obtained from femur mid-diaphysis cross-sections collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Vivarium and AEM mice received an injection of the bone label Calcein (20 mg/kg) approximately 22 hours prior to the start of the study. BV=bone volume, TV=tissue volume, Ma.V=medullary volume, Av.Ct.Th=average cortical thickness, Ec=endocortical, Ec.ES=endocortical eroded surface, BS=bone surface. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Baseline Vivarium AEM
BV (mm3) 0.65 ± 0.01A,V 0.68 ± 0.01 0.70 ± 0.01
TV (mm3) 1.54 ± 0.02A,V 1.60 ± 0.02 1.59 ± 0.01
Ma.V (mm3) 0.90 ± 0.01 0.91 ± 0.02 0.89 ± 0.01
Av.Ct.Th (µm) 170 ± 6A 176 ± 5 194 ± 5
Ec.ES/BS (%) - 19.9 ± 2.9 24.5 ± 2.3

Tibia trabecular histomorphometric parameters. Data was obtained from tibia metaphysis cross-sections collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Vivarium and AEM mice received an injection of the bone label Calcein (20 mg/kg) approximately 22 hours prior to the start of the study. OV/BV = osteoid volume normalized to bone volume, OS/BS = osteoid surface normalized to bone surface, O.Th = osteoid thickness, ES/BS = eroded surface. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Baseline Vivarium AEM
OV/BV (%) 4.6 ± 0.4A 4.1 ± 0.3 1.2 ± 0.2#
OS/BS (%) 19 ± 1A 18 ± 1 8 ± 1#
O.Th (µm) 3.1 ± 0.2A 3.0 ± 0.1 2.5 ± 0.1#
ES/BS (%) - 4.5 ± 0.6 1.4 ± 0.3#

Mechanical, material, and structural properties of the femur. Tissue was collected from female C57BL/6J mice. Mice were housed for 13 days in standard vivarium cages or flight hardware (Animal Enclosure Module; AEM), with n=12 per group. Baseline control animals (n=12) were sacrificed on the first day of the study. Data are presented as mean ± SEM. Statistical significance between vivarium and AEM is indicated with a hash mark (#; p<0.05); differences between vivarium or AEM and baseline are indicated with V or A, respectively (p<0.05).

Baseline Vivarium AEM
Stiffness (N/mm) 42.3 ± 2.5V,A 59.1 ± 2.7# 50.3 ± 2.1#
Elastic Force (N) 8.6 ± 0.4V,A 11.2 ± 0.4 11.4 ± 0.3
Maximal Force (N) 10.3 ± 0.3V,A 12.6 ± 0.3 12.5 ± 0.3
Failure Force (N) 8.6 ± 0.4 7.9 ± 0.6 9.0 ± 0.7
Imax (mm4) 184 ± 9A 213 ± 8 238 ± 6
Imin (mm4) 97 ± 4 106 ± 4 113 ± 3
Micro-Hardness (kgf/mm²) 67.6 ± 1.7 71.5 ± 1.3 72.9 ± 1.3
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