Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Summary

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models. 

Abstract

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.

The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.

After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.

Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models.

The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Introduction

Osteocytes, the most abundant bone cell type, are terminally differentiated, highly ramified cells deeply located within the skeleton. The cell body of mature osteocytes are contained in bone lacunae and have diverse shapes; osteocytes with elongated cell bodies are found in the cortical bone, whereas rounded osteocytes are more frequent in the trabecular bone¹. Branched dendrites extend from the cell body and reside in tiny channels called canaliculi, forming an intricate web that makes multiple contacts not only with other osteocytes, but also with other bone cell types, bone marrow, blood vessels and associated pericytes. Through the interstitial fluid c....

Protocol

All animal experiments were performed according to the National and European current regulations regarding the protection of animals used for scientific purposes and were reviewed and approved by the ethical committee of Milan University.

1. Isolation of Primary Osteoblasts

The method, with minor modification, follows the procedure described by Dodig et al¹⁴.

1. Prepare digesting medium, by adding 0.1% Collagenase P and 0.05% Trypsin (starting from 2.5% Trypsin 10X, no phenol red) to HBSS medium (Hanks Balanced Salt Solution, no calcium, magnesium, or phenol....

Results

Results were obtained from 5 to 10 independent experiments.

Cell Morphology

AA/GP treated primary cells have mostly cobblestone-like features, characteristic of mature osteoblasts. Interspersed ramified cells (as indicated by red arrows in Figure 1A) can be found, which likely represent some osteocytes.

With ATRA treatment, cells rapidly start displaying ramifications, which are generally observed after 2 days. As shown in

Discussion

In the last years the osteocyte has emerged as the most central cell in the bone. Research advances are progressively revealing a number of previously unsuspected or unproven osteocyte properties, which are of enormous value for designing novel and better treatment for a variety of bone diseases. However, investigation of osteocyte biology has been suffering from the limited availability of in vitro models to such an extent that osteoblasts have been used as surrogate cells over a long period before the osteocyt.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase P	Roche Applied Science	11213857001
Trypsin	Gibco, Life Technologies	15400054
HBSS	Gibco, Life Technologies	14175129	Pre-warm at 37 °C before use
Alpha-MEM	Invitrogen, Life Technologies	22571038	Pre-warm at 37 °C before use
FBS	Sigma-Aldrich	F4135
Streptomycin/Penicillin	Sigma-Aldrich	P4333
Ascorbic acid	Sigma-Aldrich	A4403
Glycerol 2-phosphate disodium salt hydrate	Sigma-Aldrich	G9422
ATRA	Sigma-Aldrich	R2625	Protect ATRA from light
Paraformaldehyde	Sigma-Aldrich	P6148	Dissolve in PBS and filter before use. Always work under a chemical hood.
DAPI	Sigma-Aldrich	32670	Can be added to the secondary antibody
Alizarin red	Sigma-Aldrich	A5533
Janus Green	Sigma-Aldrich	201677
Perchloric acid	Sigma-Aldrich	176745	Use with caution (skin and eye protection are recommended)
HCl	Sigma-Aldrich	320331	Use with caution (skin and eye protection are recommended)
Glycerol	Sigma-Aldrich	G5516
Fluorsave	Calbiochem - Merck	345789
Economy Tweezers #7, 0.40 x 0.5 mm tips	World Precision Instruments	501981
Economy Tweezers #4, 0.40 x 0.45 mm tips	World Precision Instruments	501978
Dissecting Scissors, straight, 10 cm curved	World Precision Instruments	14394
Surgical Scissors, 14 cm, straight, S/S	World Precision Instruments	501218
Culture flasks	Corning	430168
Well-plates	Corning	3335
Thermanox coverslips	Thermo Scientific Nunc	12-565-27
Microscope	Zeiss Apotome
Spectrometer	Safas Xenius