Rat benzo[a]pyrene-induced femoral intramedullary osteosarcoma generated here is fit for translational research.

Abstract

INTRODUCTION. For translational research, animal models of osteosarcoma (OS) must share with humans similar histological and ultrastructural alterations to enable useful comparisons when developing new treatment strategies. We developed a rat model of femoral OS (F-OS) with genotoxic benzo[a]pyrene (BZ[a]P) in Sprague Dawley (SD) rats to simulate human OS of long rapidly growing bones in children and adolescents. Here we describe our histological and ultrastructural findings, and suggest their possible functional implications. 

OBJECTIVE. To induce F-OS in rats by applying BZ[a]P perifemorally daily for 4 wks, to analyze the morphology of the lesioned tissues at the 6^th wk post-induction, and to identify any related functional implications   

METHODOLOGY. This project (95/17) was approved by our institutional research committee; animals were handled according to Mexican law (NOM 062-ZOO-1999).  F-OS was induced in the right limb of young male SD rats (200 g, bw) with 25mg/kg of BZ[a]P dissolved in dimethyl sulfoxide (approx. 300 microliters) in a fume hood. The toxic solution was applied perifemorally every 24h/4 wks under isoflurane anesthesia to ensure researchers’ safety and accuracy during drug application. Rats’ femur response to BZ[a]P was recorded by radiography 3 times in 6 wks, under im ketamine/xylazine (25 mg/kg – 5 mg/kg, respectively) anesthesia, at which time they were then euthanized. Dissected femurs were fixed in 10% formaldehyde in Sorensen´s buffer (pH 7.2), decalcified in 10% HCl, and prepared for histology: three-micron-thick sagittal sections were obtained with a RM2245 Leica microtome, and stained with H&E and Masson´s stain. Soft intramedullary specimens from these same femurs, were re-fixed in 2.5% glutaraldehyde and 1% osmium tetroxideh in Sorensen´s buffer; propylene-oxide-treated specimens were included in epoxy resin for transmission electron microscopy (TEM): eighty-nm-thick sections were obtained with a Leica/Reichert Ultracut S ultramicrotome and contrasted with uranyl acetate and lead citrate.

RESULTS. BZ[a]P induced an aggressive invasive (possibly metastatic) femoral mesenchymal intramedullary neoplasm producing osteoid. Highly pleomorphic malignant OBS (mOBS) were seen showing atypical nuclei (some large, even geometric structures), lipid droplets, and atypical mitoses: spindle-shaped, ss-mOBS, small epithelioid e-mOBS, with poorly defined membranes, and plasmacytoid, p-mOBS cells were both osteoblastic and osteolytic: eosinophilic osteoblastic pOBSs were found embedded alongside malignant osteoid; ss-mOBS appeared invading non-neoplastic medullary tissue, and e-mOBS broke through cortical bone (CB) epithelia. With TEM, the clear (unstained) perinuclear areas of large mOBS seen by histology, showed dilated, hyperplastic/stressed endoplasmic reticula (h/|sER) associated with severely swollen mitochondria (rounded and elongated), lacking their unique inner membrane folds (cristae).

CONCLUSION. The histological and ultrastructural description of the chemically induced intramedullary F-OS rat model we generated here, fits many of the key morphological features that have been described for conventional intramedullary human OS (80% of OS diagnoses), and with it we expect to validate new pharmaceuticals for the treatment of OS.