EFFECT OF VITAMIN D3 METABOLITES ON BONE INTEGRITY

Participants: E.A. Smith, E.P. Frankenburg, S.K. Volkman, A. Norman, S.A. Goldstein

Keywords: bone mechanics, Vitamin D3, drug treatment

Introduction

Vitamin D3 is a steroid hormone that is of paramount importance in bone development and bone mass regulation. Vitamin D3 must be converted into active metabolites, 1a,25(OH)2D3 and 24,25(OH)2D3, which are instrumental in regulating calcium homeostasis; both its absorption in the small intestine and resorption of bone. It is apparent that misexpression of Vitamin D3, its metabolites, or their receptors can have profound effects on the metabolism and properties of bone. While the effects of 1,25(OH)2D3 on bone have been widely studies, the effects of 24,25(OH)2D3 on bone properties are not as well understood. The goal of this study is to examine the effects of an additional Vitamin D3 metabolite, 1a,24,25(OH)2D3 on the mechanical and geometric characteristics of bone, and compare these to the effects of 1a,25(OH)2D3 and 24,25(OH)2D3.

Materials and Methods

Newly hatched male White Leghorn chicks were either fed a Vitamin D3-deficient diet, or given one of three doses of a Vitamin D3 metabolite. The chicks were divided into the following experimental groups based on treatment: Group A: Vitamin-D deficient, B: Vitamin D3 (150 nmole/kg diet); C: 1a,24,25(OH)2D3 (31.5 nmole/kg diet); D: 1a,24,25(OH)2D3 (94.5 nmole/kg diet); E: 1a,24,25(OH)2D3 (285 nmole/kg diet); F: 24,25(OH)2D3 (31.5 nmole/kg diet); G: 24,25(OH)2D3 (94.5 nmole/kg diet); H: 1a,25(OH)2D3 (3.5 nmole/kg diet); I: 1a,25(OH)2D3 (10.5 nmole/kg diet); J: 1a,25(OH)2D3 (31.5 nmole/kg diet). Chicks were sacrificed after 4 weeks of treatment, and both legs were scanned on the µCT system. Three-dimensional images were reconstructed at a resolution of 40µm, and cross-sectional area (CSA), cortical thickness (CT), and moments of inertia (Ixx, Iyy, and J) were measured. Specimens will then be mechanically tested in four-point bending on a servohydraulic MTS machine, and load-displacement data will be used to determine stiffness and load at yield and failure.

Progress

A subset of 118 specimens have been µCT scanned and the images reconstructed. A custom thresholding algorithm was developed to threshold each image slice individually. Approximately 25 specimens remain to be µCT scanned before mechanical testing.

Preliminary Results

These results show a profound decrease in geometric properties due to Vitamin D3 deficiency when compared to all treated groups (see Table 1). Treatment with the lowest dose of 1a,24,25(OH)2D3 (Group C) resulted in a geometry that was not significantly different from Vitamin D3-treated controls (Group B), while treatment with the highest dose (Group E) yielded a significantly greater cortical thickness and a significantly lower polar moment of inertia. Treatment with the lowest dose of 24,25(OH)2D3 produced a significantly smaller geometry, while increasing the dose of 24,25(OH)2D3 returned the geometry to control levels. In general, treatment with low levels of 1a,25(OH)2D3 (Groups H and I) did not significantly change the geometry of the bones, while increasing the dose of 1a,25(OH)2D3 (Group J) resulted in an increase in CT and decrease in J. These results suggest an interesting role of Vitamin D3 metabolites in regulation and distribution of bone mass, but further geometric analyses including all groups, and characterization of the mechanical integrity of these bone specimens will provide further insight into their effects on bone.

Table 1: Geometrical Properties

1 p<0.05 vs. B, C, E, F, G, H, I

2 p<0.05 vs. A, B, C, G, H, I, J

3 p<0.05 vs. A, B, C, F, G, I

4 p<0.05 vs. A, B, C, F, G

5 p<0.05 vs. A, B, C, G, H, I

6 p<0.05 vs. A, B, G, H, I