Bone loss and structural changes in a sickle cell disease mouse … · 2017-01-30 · Title:...
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Bone loss and structural changes in a sickle cell disease mouse model + 1 Smith, RA; 2 Shackelford, H; 3 Pestina, T; 3 Persons, DA; 2 Gartner, TK; 1 University of Tennessee; Department of Orthopaedic Surgery-Campbell Clinic, 2 University of Memphis, Department of Biological Sciences, 3 St. Jude Children’s Research hospital, Department of Hematology Email: [email protected] Introduction A variety of sickle cell disease (SCD) mouse models have been developed that allow detailed studies of the disease in a controlled manner. Unlike the other transgenic SCD mouse models, the Berkeley model sustains knockouts for mouse alpha and beta hemoglobins and has been extensively characterized. A complete comparison of the model relative to SCD patients is required to know which features of SCD can be studied in the mouse model. Bone abnormalities for the Berkeley SCD mice have not been documented although they are a common effect of SCD. We present here a preliminary study analyzing the bone abnormalities found in the Berkeley mouse model. We provide quantitative characterizations of bone abnormalities in SCD mice, thereby providing new insights into SCD and demonstrating that these mice are ideal subjects for the study of SCD-elicited bone abnormalities. Materials and Methods Five homozygous (+/+), C57BL/6 SCD 6 month old male mice, and 5 age-matched heterozygous (+/-) congenic female control mice were used for this study. A GE Lunar PIXImus2 peripheral dual-energy x-ray absorptiometer (DEXA, Lunar Corp., Madison, WI) unit was used to measure the bone mineral density (BMD, mg/cm 2 ) and content (BMC, mg) of the SCD and control skeletons. High resolution micro-computed tomography (Micro-CT, µCT 40 Scanco Medical, Basserdorf, Switzerland) was used to determine three-dimensional bony architecture. Mechanical testing was done using an Instron Universal Test System (Instron Corp., Norwood MA, model 5567) to evaluate the strength of the bones. Results A B Bone Mineral Density WB C L F/T 0.00 0.01 0.02 0.03 0.04 0.05 0.06 grams/cm2 C57Bl/6 M C57Bl/6 F SCD +/- SCD +/+ Bone Mineral Content WB C L F/T 0.00 0.02 0.04 0.06 0.08 0.10 0.18 0.24 grams SCD +/- SCD +/+ Figure 1. SCD decreases BMC more than BMD of the vertebrae relative to the long bones. (A) DEXA BMD data for the whole body (WB), cervical (C) and lumber vertebrae (L), and the femur and tibia complexes (F+T). (B) DEXA BMC data (note that the ordinate is discontinuous) for the WB, C, L, and F+T. DEXA screening demonstrated that bone mineral density (BMD) and bone mineral content (BMC) deficiencies occurred throughout the skeletons (WB, excluding the skulls) of the SCD mice, including the cervical (C) and lumbar vertebrae (L) and the femur and tibia (F+T) complexes (Fig. 1). The BMD values in Fig. 1A understate the SCD related differences because the values are not corrected for the male biased BMD gender difference of C57BL/6 mice (Fig. 1A). Since there were significant differences in DEXA values between the groups, micro-CT was used to assess the bone geometry and obtain volumetric BMD on the body of the lumbar vertebrae and the femurs. Reconstructed images and their supporting data from micro-CT scans of L revealed that the SCD vertebrae have 19% fewer trabeculae, 46% less trabecular connectivity, 38% greater separation between the trabeculae, 50% less apparent BMD and 45% less bone volume/total volume than the control vertebrae (Fig. 2A and B). The cortical bone of the SCD femur is 25% thinner, has 6% less apparent BMD, 12% less cortical volume, and 36% greater marrow volume than the bones from SCD+/- mice (Fig. 2C). C SCD+/- SCD+/+ D SCD+/- SCD+/+ A SCD+/- SCD+/+ B SCD+/- SCD+/+ Figure 2. SCD causes deleterious bone abnormalities in cortical and trabecular bone of the lumbar vertebrae and femurs. (A) Reconstructed cross-sectional images from micro-CT scans of whole lumbar vertebrae. (B) Reconstructed micro-CT images of lumbar vertebrae trabecular bone (100 slices taken from the anterior end of the vertebral body). (C) Reconstructed cross-sectional, mid-diaphyseal region images from micro-CT scans of femurs. (D) Reconstructed trabecular bone images from micro-CT scans from the heads of femurs The cortical bone was thinner with less BMD in the SCD femurs compared with control and were much weaker than the controls as it took significantly less (1/3) force to break the SCD femurs in the mid- diaphyseal region (table 1A). The heads of the SCD femurs have significantly less bone volume, fewer (16%) trabeculae, (19%) greater trabecular separation and less connectivity than the control femurs (Fig. 1B). Table 1A: Cortical Bone (Femur) Thickness (µm) BMD (mg/cm 3 ) Flexure load (N) SCD +/- 253 ± 6.15 1284 ± 4.16 29.3 ± 3.3 SCD +/+ 189 ± 3.78 1223 ± 8.92 9.0 ± 1.1 P values 0.001 0.003 0.002 Table 1B: Trabecular Bone (Femur) BV/ TV (%) Tb.N (1/mm) Conn-Dens (mm 3 ) SCD +/- 7.64 ± 0.81 3.55 ± 0.17 95.5 ± 16.0 SCD +/+ 5.29 ± 0.58 3.01 ± 0.13 26.1 ± 6.9 P values 0.01 0.04 0.01 Discussion The data presented here demonstrate the extensive deleterious effects of SCD on bone structure, and lead to two interesting conclusions that provide new insight into the effects of SCD on bone structure and metabolism. First, SCD has a greater effect on vertebral bodies, than on the femurs. Second, SCD diminishes net bone formation (BMC) of the vertebrae substantially more (50%) than it decreases BMD of the vertebrae (16%). Although bone and bone marrow infarction, the related avascular necrosis, and osteomyelitis are documented bases of SCD related bone abnormalities, SCD is a systemic inflammatory disorder. Specifically, hemolysis of red blood cells releases hemoglobin that indirectly scavenges nitric oxide, which along with repeated episodes of vascular occlusion and reperfusion facilitates inflammation, and inflammation contributes to bone damage. Thus, much of the bone abnormality documented here may be the result of enhanced circulating levels of inflammatory cytokines resulting from inflammation in SCD patients. Therefore, the differential effects of SCD on bone metabolism discussed above may be, at least in-part, the result of inflammation. Comparable human SCD bone data are not available at this time. Poster No. 0552 • ORS 2012 Annual Meeting
Transcript of Bone loss and structural changes in a sickle cell disease mouse … · 2017-01-30 · Title:...
Bone loss and structural changes in a sickle cell disease mouse model +1Smith, RA; 2Shackelford, H; 3Pestina, T; 3Persons, DA; 2Gartner, TK;
1University of Tennessee; Department of Orthopaedic Surgery-Campbell Clinic, 2University of Memphis, Department of Biological Sciences, 3St. Jude Children’s Research hospital, Department of Hematology
Email: [email protected]
Introduction A variety of sickle cell disease (SCD) mouse models have been developed that allow detailed studies of the disease in a controlled manner. Unlike the other transgenic SCD mouse models, the Berkeley model sustains knockouts for mouse alpha and beta hemoglobins and has been extensively characterized. A complete comparison of the model relative to SCD patients is required to know which features of SCD can be studied in the mouse model. Bone abnormalities for the Berkeley SCD mice have not been documented although they are a common effect of SCD. We present here a preliminary study analyzing the bone abnormalities found in the Berkeley mouse model. We provide quantitative characterizations of bone abnormalities in SCD mice, thereby providing new insights into SCD and demonstrating that these mice are ideal subjects for the study of SCD-elicited bone abnormalities. Materials and Methods Five homozygous (+/+), C57BL/6 SCD 6 month old male mice, and 5 age-matched heterozygous (+/-) congenic female control mice were used for this study. A GE Lunar PIXImus2 peripheral dual-energy x-ray absorptiometer (DEXA, Lunar Corp., Madison, WI) unit was used to measure the bone mineral density (BMD, mg/cm2) and content (BMC, mg) of the SCD and control skeletons. High resolution micro-computed tomography (Micro-CT, µCT 40 Scanco Medical, Basserdorf, Switzerland) was used to determine three-dimensional bony architecture. Mechanical testing was done using an Instron Universal Test System (Instron Corp., Norwood MA, model 5567) to evaluate the strength of the bones. Results
A! B!Bone Mineral Density
WB C L F/T
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SCD +/-SCD +/+
Figure 1. SCD decreases BMC more than BMD of the vertebrae relative to the long bones. (A) DEXA BMD data for the whole body (WB), cervical (C) and lumber vertebrae (L), and the femur and tibia complexes (F+T). (B) DEXA BMC data (note that the ordinate is discontinuous) for the WB, C, L, and F+T. DEXA screening demonstrated that bone mineral density (BMD) and bone mineral content (BMC) deficiencies occurred throughout the skeletons (WB, excluding the skulls) of the SCD mice, including the cervical (C) and lumbar vertebrae (L) and the femur and tibia (F+T) complexes (Fig. 1). The BMD values in Fig. 1A understate the SCD related differences because the values are not corrected for the male biased BMD gender difference of C57BL/6 mice (Fig. 1A). Since there were significant differences in DEXA values between the groups, micro-CT was used to assess the bone geometry and obtain volumetric BMD on the body of the lumbar vertebrae and the femurs. Reconstructed images and their supporting data from micro-CT scans of L revealed that the SCD vertebrae have 19% fewer trabeculae, 46% less trabecular connectivity, 38% greater separation between the trabeculae, 50% less apparent BMD and 45% less bone volume/total volume than the control vertebrae (Fig. 2A and B). The cortical bone of the SCD femur is 25% thinner, has 6% less apparent BMD, 12% less cortical volume, and 36% greater marrow volume than the bones from SCD+/- mice (Fig. 2C).
C SCD+/- ! SCD+/+! D SCD+/- ! ! SCD+/+!
A SCD+/- ! SCD+/+! B SCD+/- ! ! SCD+/+!
Figure 2. SCD causes deleterious bone abnormalities in cortical and trabecular bone of the lumbar vertebrae and femurs. (A) Reconstructed cross-sectional images from micro-CT scans of whole lumbar vertebrae. (B) Reconstructed micro-CT images of lumbar vertebrae trabecular bone (100 slices taken from the anterior end of the vertebral body). (C) Reconstructed cross-sectional, mid-diaphyseal region images from micro-CT scans of femurs. (D) Reconstructed trabecular bone images from micro-CT scans from the heads of femurs The cortical bone was thinner with less BMD in the SCD femurs compared with control and were much weaker than the controls as it took significantly less (1/3) force to break the SCD femurs in the mid-diaphyseal region (table 1A). The heads of the SCD femurs have significantly less bone volume, fewer (16%) trabeculae, (19%) greater trabecular separation and less connectivity than the control femurs (Fig. 1B). Table 1A: Cortical Bone (Femur)
Thickness (µm) BMD (mg/cm3) Flexure load (N) SCD +/- 253 ± 6.15 1284 ± 4.16 29.3 ± 3.3 SCD +/+ 189 ± 3.78 1223 ± 8.92 9.0 ± 1.1 P values 0.001 0.003 0.002 Table 1B: Trabecular Bone (Femur)
BV/ TV (%) Tb.N (1/mm) Conn-Dens (mm3) SCD +/- 7.64 ± 0.81 3.55 ± 0.17 95.5 ± 16.0 SCD +/+ 5.29 ± 0.58 3.01 ± 0.13 26.1 ± 6.9 P values 0.01 0.04 0.01 Discussion The data presented here demonstrate the extensive deleterious effects of SCD on bone structure, and lead to two interesting conclusions that provide new insight into the effects of SCD on bone structure and metabolism. First, SCD has a greater effect on vertebral bodies, than on the femurs. Second, SCD diminishes net bone formation (BMC) of the vertebrae substantially more (50%) than it decreases BMD of the vertebrae (16%). Although bone and bone marrow infarction, the related avascular necrosis, and osteomyelitis are documented bases of SCD related bone abnormalities, SCD is a systemic inflammatory disorder. Specifically, hemolysis of red blood cells releases hemoglobin that indirectly scavenges nitric oxide, which along with repeated episodes of vascular occlusion and reperfusion facilitates inflammation, and inflammation contributes to bone damage. Thus, much of the bone abnormality documented here may be the result of enhanced circulating levels of inflammatory cytokines resulting from inflammation in SCD patients. Therefore, the differential effects of SCD on bone metabolism discussed above may be, at least in-part, the result of inflammation. Comparable human SCD bone data are not available at this time.
Poster No. 0552 • ORS 2012 Annual Meeting
