Title

Development and characterization of a murine spine loading model

Date of Completion

January 2006

Keywords

Biology, Genetics|Biology, Cell|Biology, Animal Physiology

Degree

Ph.D.

Abstract

It is generally accepted that bone initially responds to mechanical loading by activating resident mature osteoblasts and that prolonged loading results in the generation of new osteoblasts from a progenitor population. This model predicts a heterogeneous mixture of cells that are acting in a concerted fashion, but cannot be easily appreciated with current methods of identification. We have been building promoter-GFP reporter constructs designed to reflect different levels of osteoblast differentiation. For this study we crossed pOBCol3.6GFPcyan (3.6cyn) transgenic mice, whose reporter is active in preosteoblasts and osteoblasts, with pOBCol2.3GFPemerald (2.3emd) transgenic mice, whose reporter is active in osteoblasts and osteocytes. 5-6 month old "2-color" mice were then subjected to an orbital loading protocol that produces an isometric load on the skeleton as the animals resist the motion of the cage. The animals were injected with xylenol orange (XO) two days prior to sacrifice after 7, 21 or 35 days of loading. Quantitation of fluorescent signals utilized histology generated via Cryojane™ frozen sections taken from non-demineralized bone. Images were generated on a Zeiss-Improvision workstation using a series of dichromic filters that allowed for the isolation of multiple of fluorescent signals from the same sample. Fluorescent ELF-97 stain under two different conditions was employed to visualize alkaline phosphatase (AP) and tartate resistant acid phosphatase (TRAP) activity. All results were compared to unexercised littermates. After only 7 days of the protocol there was a 30-fold increase in female XO labeling that continued to be high throughout the exercise protocol. Five different cell populations on the bone surface were identified and quantitated: an immature osteoblast population (3.6cyn +, 2.3emd -, AP+, XO-), a dual osteoblast population (3.6cyn ++, 2.3emd ++, AP ++, XO +), a mature osteoblast population (3.6cyn -, 2.3emd +, AP +, XO+), a non-GFP cell population (3.6cyn -, 2.3emd -, AP +, XO+) and a preosteoclast population (3.6cyn +, 2.3emd -, TRAP+). The size of each cell population changed with continued orbital loading. With further refinements to this histological approach, it may be possible to fully appreciate the different bone lining populations and their response to perturbations affecting bone mass. ^