Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Summary

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Abstract

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

Introduction

Osteosarcoma (OS) usually occurs in actively growing areas, the metaphysis of long bones, during adolescence. More than 80% of the OS-affected sites have preference for the metaphysis of proximal tibia and proximal humerus as well as both distal and proximal femur, corresponding to the location of the growth plate¹. OS comprises multiple cell subtypes with mesenchymal properties and considerable diversity in histologic features and grade. Evidences support mesenchymal stem cells (MSCs), osteoblasts committed precursors and pericytes as the cells of origin^2,3,

Protocol

Animal care and use are conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication NO.80-23, revised in 1996) after approval from the Animal Ethics Committee of Sun Yat-sen University.

1. Bone preparation

1. Obtain 4 to 6-week-old BALB/c mice (without sex-specific requirement). Euthanize a mouse aseptically by cervical dislocation and cut off fresh fibula, tibia and femur from a hindlimb with sterile surgical scissors. Pe.......

Discussion

Generally, OS can be classified as osteoblastic, chondroblastic, and fibroblastic subtypes depending on its dominant histologic component. Its prognosis is dependent not only on histologic parameters but also on its anatomic site. It may occur inside the bones (in the intramedullary or intracortical compartment), on the surfaces of bones, and in extraosseous sites¹⁹. The emergence and heterogeneity of OS can be elucidated as a conjugation of oncogenic events and an adequate microenvironmenta.......

Materials

Name	Company	Catalog Number	Comments
15 mL centrifuge tube	Greiner	188271
50 mL centrifuge tube	Greiner	227270
6 cm cell culture dish	Greiner	628160
6-well plate	Greiner	657160
Ampicillin	Sigma-Aldrich	A9393
C57-BL/6J mouse	Sun Yat-sen University Laboratory Animal Center
CO2 incubator	SHEL LAB	SCO5A
Dibasic sodium phosphate	Guangzhou Chemical Reagent Factory	BE14-GR-500G
DMEM/F12	Sigma-Aldrich	D0547
Fetal bovine serum	Hyclone	SH30084.03
Hemocytometer	BLAU	717805
Kanamycin	Sigma-Aldrich	PHR1487
MG-63	Chinese Academy of Science, Shanghai Cell Bank		Human osteosarcoma cell line
MNNG/HOS	Chinese Academy of Science, Shanghai Cell Bank		Human osteosarcoma cell line
Phenol red	Sigma-Aldrich	P4633	A solution of phenol red is used as a pH indicator: its color exhibits a gradual transition from yellow to red over the pH range 6.6 to 8.0.
Potassium chloride	Sangon Biotech	A100395
Potassium Phosphate Monobasic	Sangon Biotech	A501211
Sodium chloride	Sangon Biotech	A501218