MECHANICAL AND GEOMETRIC CHARACTERISTICS OF BONE IN BIGLYCAN DEFICIENT MICE

Participants: T. Adachi, E.A. Smith, K. Kozloff, M. Young, S.A. Goldstein

Keywords: bone mechanics, extracellular matrix, transgenic animal

Introduction

Biglycan (BGN) and decorin are he most predominant proteoglycans in the extracellular matrix of bone. BGN is most abundant in developing bone, and is usually localized to pericellular locales. Because of their ability to bind and modulate the activity of TGF-b, both decorin and biglycan have been hypothesized to influence cell proliferation and differentiation. The precise role of either proteoglycan is, however, not well understood. Transgenic animals are widely used to elucidate the roles of these bone matrix proteins and their effects on mechanical properties. The goal of this study was, therefore, to characterize the mechanical and geometrical properties of bone from a BGN deficient animal.

Materials and Methods

The right femur from a total of 40 BGN -/0 (KO) or BGN +/0 (WT) wild-type mice at 3 months (KO: n=5, WT: n=5), 6 months (KO: n=9, WT: n=8) and 9 months (KO: n=8, WT: n=5) of age were acquired from collaborators at the Bone Branch of the NIDR, NIH. The femora of 30 mice were scanned on a µCT system, and 3-dimensional images were reconstructed at a resolution of 20 µm. Cross-sectional area (CSA), cortical thickness (CT), and moments of inertia (Ixx, Iyy, and J), as well as inner and outer fiber lengths in the AP and ML directions of were determined for the mid-diaphyseal region. All femora were then tested to failure in four-point bending on an MTS servohydraulic testing machine at a constant displacement rate of 1.0 mm/sec. Load-displacement data was acquired, and used to determine stiffness, and loads and displacements to both yield and failure. A two-way Analysis of Variance (ANOVA) was used to compare differences between experimental group and age. Tukey's post-hoc test was used for comparisons between groups with p<0.05 considered statistically significant. In a parallel study, the 4^th caudal vertebrae are being evaluated in a similar way. Briefly, the specimens are dissected free, scanned on the µCT system and then subjected to mechanical testing. Uniaxial compression to failure is implemented using a custom servo-controlled system. Load to yield and failure, stiffness and energy measures are documented.

Progress

All mechanical testing and µCT analyses for the femurs have been completed. A manuscript entitled "Targeted Disruption of the Biglycan Gene Leads to an Osteoporosis-like Phenotype in Mice" was published in Nature Genetics 20: 78-82, Sept. 1998. The vertebral studies are continuing and final data analysis is underway.

Results

Results showed a trend toward both decreased mechanical and geometrical properties in the BGN -/0 groups when compared to the wild-type controls (Tables 1 and 2). BGN deficiency, however, only produced statistically significant alterations in yield load and cortical thickness that change with age. The yield load of the 3 month specimens were significantly greater than that 6 month and 9 month specimens in each treatment group. The CT of specimens in the 3 month KO group are only significantly different than that of the 9 month KO group. These results were accompanied by a general decrease in bone growth rate and bone mass in the mutant animals. This indicates a potentially important, yet still unclear, role of BGN in bone development and bone mass regulation. The vertebral data appears to be following a similar trend, except that differences were found at all times. The complete analysis will be ready in the near future.

Table 1: Mechanical Properties

1p<0.05 vs. KO, 6mo; KO, 9mo.

2p<0.05 vs. WT, 6mo.; WT, 9mo.

Table 2: Geometrical Properties

ap<0.05 vs. 9 mo. KO