Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Summary

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Abstract

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.

This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.

The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

Introduction

Bone is a dynamic connective tissue that has several functions, including supporting the muscles, protecting the internal organs and bone marrow, and storing and releasing calcium and growth factors^1,2. To maintain its integrity and proper function, bone tissue is continuously under the process of remodeling. In general terms, a cycle of bone remodeling can be divided into bone resorption and bone formation¹. An imbalance between these two phases of bone remodeling can lead to the development of bone pathologies. Also, diseases such as breast cancer often affect bone integrity; approxim....

Protocol

All mice used in these assays were obtained from BALB/c mice strains, using male and female mice indiscriminately. Previous culture experiments have also been performed using other strains, such as FVB, Swiss mice, CD-1, and CsA mice^11,12,14. All mice were housed according to National Institutes of Health (NIH) guidelines, Appendix Q. Procedures involving animal subjects have been approved by the Institutional Animals Care and U.......

Discussion

Here, we describe the protocol for a calvarial ex vivo model to evaluate bone formation or resorption and to study the interactions of cancer cells with calvarial mouse bone. The critical steps of this technique are the dissection, culture, embedding, and histomorphometrical analysis of the calvarias. During the dissection of the calvarias, it is crucial to cut the hemi-calvarias into a trapezoid, as it will strongly facilitate the orientation during the paraffin inclusion. When studying cancer cell interactions.......

Materials

Name	Company	Catalog Number	Comments
24 well cell culture	Corning	CLS3524
24 well non tissue culture	Falcon	15705-060
2 mL cryovial	SSI	2341-S0S
Antibiotics-Antimycotic	Corning	30-004-CI
BSA	Biowest	P6154-100GR
Centrifugue	Eppendorf	22628188	Centrifuge 5810R
Coverslips	Corning	2935-24X50
Cytoseal resin	Richard Allen	8310-10
DMSO		D2650-100ML
Dulbecco's Modification of Eagles Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate	Corning	10-017-CV
Dulbecco's PBS (10X)	Corning	20-031-CV
Ebedding Cassettes	Sigma	Z672122-500EA
EDTA	Golden	26400
Embedding Workstation	Thermo Scientific	A81000001
Eosin	Golden	60600
Ethanol absolute	JALMEK	E5325-17P
Fetal Bovine Serum	Biowest	BIO-S1650-500
Filters	Corning	CLS431229
Forceps and scissors	LANCETA HG	74165
Formalin buffered 10%	Sigma	HT501320
Glass slides 25 x 75 mm	Premiere	9105
Harris's Hematoxylin	Jalmek	SH025-13
High profile blades	Thermo Scientific	1001259
Histoquinet	Thermo Scientific	813150	STP 120
Insulin from bovine pancreas	Sigma	16634
Microscope	ZEISS	Axio Scope.A1
Microtome	Thermo Scientific	905200	MICROM HM 355S
Mouse food, 18% prot, 2018S	Harlan	T.2018S.15
Neubauer	VWR	631-0696
Orange G	Biobasic	OB0674-25G
Paraffin	Paraplast	39601006
Paraffin Section Flotation Bath	Electrothermal	MH8517X1
Petri dish	Corning	CLS430167
Phloxin B	Probiotek	166-02072
Trypan Blue	Sigma	T8154
Trypsin-EDTA	Corning	25-051-CI
Wax dispenser	Electrothermal	MH8523BX1
Xylene	Golden	534056-500ML