Harris, Gerald

Jameson, John

Molthen, Robert

Slavens, Brooke

Smith, Peter

English

epublications@Marquette

Marquette Universitye-Publications@MarquetteBiomedical Engineering Faculty Research andPublications Biomedical Engineering, Department of8-18-2010Validation of a Micro-CT Approach forCharacterization of Murine and Human Bone inOsteogenesis ImperfectaJohn JamesonMarquette University, john.jameson@marquette.eduBrooke SlavensMarquette University, brooke.slavens@marquette.eduRobert MolthenMarquette University, robert.molthen@marquette.eduPeter SmithOthopaedic and Rehabilitation Engineering CenterGerald HarrisMarquette University, gerald.harris@marquette.eduPublished version. 34th Annual Meeting of the American Society of Biomechanics, Providence, RI, August18-21. 2010, (August 2010). Publisher Link. © 2010 American Society of Biomechanics. Used withpermission.VALIDATION OF A MICRO-CT APPROACH FOR CHARACTERIZATION OF MURINE AND HUMAN BONE IN OSTEOGENESIS IMPERFECTA  1,2John Jameson, 1-3Brooke Slavens, 1,2,4Robert Molthen, 2,3Peter Smith, and 1-3Gerald Harris  1Department of Biomedical Engineering, Marquette University, Milwaukee, WI, USA 2Orthopaedic and Rehabilitation Engineering Center (OREC), Milwaukee, WI, USA 3Shriners Hospitals for Children, Chicago, IL, USA 4Zablocki VA Medical Center, Milwaukee, WI, USA email: john.jameson@marquette.edu, web: http://www.orec.org  INTRODUCTION  Osteogenesis imperfecta (OI) is a rare genetic, musculoskeletal disease characterized by mutations in type I collagen, ultimately leading to abnormal collagen synthesis and assembly [1]. Although it is highly heterogeneous, common symptoms include frequent bone fractures, decreased stature, and progressive spinal and limb deformities. Currently patients are classified into eight clinical groups, where Type III is the most severe form compatible with life. To date, there is little biomechanical data of OI bone. Because of the limited availability and small size of human OI specimens, most research has focused on animal models. One commonly studied murine model of Type III OI is the homozygous oim B6C3Fe a/a-Col1a2oim/J strain (oim/oim). The goal of this study was to validate a system for the evaluation of oim/oim and human OI bone samples using micro-computed tomography (μCT).  METHODS  Animals. All animals were obtained from Jackson Laboratory (Bar Harbor, ME) and studies were performed under approval of an Institutional Animal Care and Use Committee (IACUC) protocol. Five oim/oim and five wild type (+/+) femora were harvested from male mice and fresh frozen at -70°C until μCT evaluation. Average ages for the oim/oim and +/+ strains at tissue dissection were 9 and 10 weeks, respectively.  Imaging. All scans were performed using a μCT system developed at the Zablocki VA Medical Center (Milwaukee, WI) [2]. The system is composed of a 100.50 X-ray source (3-mm focal spot; Comet North America), an AI-5830-HP image intensifier (North American Imaging) coupled to a Silicon Mountain Design SMD1M-15 CCD camera (DALSA), and a specimen micromanipulator stage, all mounted on a precision rail. Femora were thawed, placed in 1.5-mL Eppendorf tubes filled with saline, mounted on the specimen stage, and scanned in continuous mode (33 kVp, 242 μA, 7-frame average) at two magnification levels. Reconstructions were performed using a standard Feldkamp algorithm, with resulting voxel sizes of 17 and 34 μm, corresponding to high and low magnification, respectively.  Geometrical analysis. Cortical and trabecular regions of interest (ROIs) were isolated and analyzed for several structural morphometric parameters using ImageJ (v1.42; NIH) and MicroView (v2.1.2; GE Healthcare). Femoral length was measured using MicroView’s line tool. Longitudinal cortical regions were selected from the low magnification scans, where each ROI extended distally 2.5 mm from the femoral midpoint. These regions were analyzed for cross-sectional area (CSA) and cortical thickness (Ct.Th). Cylindrical trabecular regions were located in the high magnification scans just proximal to the distal femoral growth plate, extending proximally 1.5 mm with a diameter of 1 mm. After applying a local threshold (determined using MicroView’s auto-threshold option), trabecular ROIs were evaluated for bone volume fraction (BV/TV) and trabecular number (Tb.N), thickness (Tb.Th), and spacing (Tb.Sp).  Statistical analysis. Data from each genotype was pooled and compared for statistical significance using an unpaired student’s t-test. RESULTS AND DISCUSSION  Structural data. Femoral length was not significantly different between the two groups. However, oim/oim cortical and trabecular indices were generally inferior to controls (Table 1) and agreed well with OI mouse literature [3,4]. Oim/oim cortical geometry was more flattened and ellipsoidal in appearance (Figure 1B). Mid-shaft oim/oim cortices showed a 16% reduction in CSA, suggesting decreased resistance to bending loads, as found in a prior study [3]. Oim/oim trabecular regions appeared more open in structure (Figure 1B). This was confirmed by our findings of significantly reduced BV/TV and Tb.N, with a corresponding increase in Tb.Sp (Table 1). These results agree with prior findings that OI mice have decreased trabecular bony tissue that is also of reduced mechanical viability [3,4].  Extension to human OI bone. After establishing agreement with murine literature, we applied our methods to a human OI bone specimen. Pediatric OI patients suffer frequent fractures that require routine surgical intervention. Small bone fragments are often removed during these procedures. One such specimen was collected (under written consent and IRB approval) and fresh frozen until μCT imaging was conducted. The bone was scanned at 24-µm voxel resolution and analyzed using similar methods to the animal study. The wedge-shaped sample was composed almost exclusively of trabecular bone (Figure 2); thus, only applicable parameters were calculated. Surprisingly, the specimen showed increased BV/TV and Tb.N, but decreased Tb.Sp (Table 1) compared to healthy femoral head tissue [5]. One possible explanation is that this patient was treated with bisphosphonates, which have been shown to have similar effects in murine studies [3,4]. To our knowledge, this is the first application of μCT to determine morphometric parameters in human OI bone.   Figure 1: Representative surface-shaded renderings of mouse femora from μCT data. (A) Whole femur. (B) Cortical and trabecular ROIs.   Figure 2: Human OI bone sample. (A) Wedge-shaped specimen with dimensions 10.5 x 6.25 x 5.56 mm. (B) Cylindrical trabecular ROI with 3-mm diameter and 2.5-mm height.  REFERENCES  1.Chiasson RM, et al. Interdisciplinary treatment approach for children with osteogenesis imperfecta, Shriners Press, 2004. 2.Karau KL, et al. Am J Physiol 281, H1447-H1457, 2001. 3.Misof BM, et al. Bone 36, 150-158, 2005. 4.Uveges TE, et al. JBMR 24, 849-859, 2009. 5.Hildebrand T, et al. JBMR 14, 1167-1174, 1999.  ACKNOWLEDGEMENTS  This work was supported by OREC, the Dr. Ralph and Marian Falk Medical Research Trust, and NIDRR Grant H133G050201. Table 1: Summary of calculated bone parameters. * p < 0.06 between +/+ and oim/oim. † p < 0.05 between +/+ and oim/oim. Genotype  Morphometric Parameters  CSA (mm2) Ct.Th (mm) BV/TV Tb.N (mm-1) Tb.Th (mm) Tb.Sp (mm) Control (+/+) 0.87 ± 0.15 0.20 ± 0.03 0.25 ± 0.05 6.89 ± 1.71 0.036 ± 0.006 0.12 ± 0.04 Oim/oim 0.73 ± 0.10* 0.19 ± 0.01 0.14 ± 0.03† 3.67 ± 1.03† 0.038 ± 0.005 0.28 ± 0.09† Healthy Human [5] - - 0.26 ± 0.08 1.60 ± 0.29 0.194 ± 0.033 0.64 ± 0.11 Human OI - - 0.45 2.90 0.154 0.19 +/+ OIM Cortical Trabecular A B A B 

Validation of a Micro-CT Approach for Characterization of Murine and Human Bone in Osteogenesis Imperfecta

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Validation of a Micro-CT Approach for Characterization of Murine and Human Bone in Osteogenesis Imperfecta

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