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 contained in lacunae and canaliculi, osteocytes are also ultimately connected to the circulating system, therefore they can influence not only local but also systemic events, and vice versa their behavior can be regulated by both local and systemic changes².

The study of osteocytes has recently gained momentum, thanks to several technical advances, such as the generation of tissue- and cell-specific transgenic animals, the use of powerful microscopy techniques and of high-throughput molecular screening^3,4. However, knowledge of these cells is still incomplete, mainly due to the relative scarcity of adequate in vitro models. Actually, osteocytes have been always difficult to obtain and maintain in culture because of the deep location, and the low level of proliferation that characterizes such a differentiated cell type.

Along the years, a number of methods have been developed to isolate primary osteocytes^5-7, though they generally produce low cell yields and carry the risk that even a few contaminating fibroblasts will rapidly overgrow the osteocytes⁸. For this reason, most experimental in vitro work has been conducted so far on the well-established osteocyte cell line MLO-Y4 ⁹.

Additional in vitro methodologies could enhance the possibility of studying these cells and could improve the analysis of osteocyte biology and pathophysiology. To be largely adopted, such methods should be easy to reproduce, and would not require special instrumentation or very long times to reach cell maturation. Importantly, if applicable to primary cells, they would make it possible to take advantage of transgenic animals.  We recently described that treatment of the osteoblast cell line MC3T3-E1 and of primary osteoblasts with retinoic acid induces a rapid phenotype change leading to the development of a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes¹⁰.

All-trans retinoic acid (ATRA) is an active metabolic product of vitamin A which regulates gene transcription by binding the nuclear retinoic acid receptors (RARs). RARs bind to DNA as heterodimers with the retinoid X receptors (RXRs), ultimately leading to modulation of retinoic acid (RA)-responsive target genes¹¹. ATRA has been shown to modulate differentiation and maturation of several cell types, among them other ramified cells such as neuronal cells¹² and podocytes¹³.

The method here described is based on addition of ATRA to primary osteoblasts. To be effective, ATRA has to be added at a precise maturation stage on cells plated at a defined density; in our experience these conditions are critical to obtain the phenotype switch from osteoblasts to osteocytes.

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 red).
2. Use 3-4 day old mice (the procedure can be applied to as few as 1 mouse).
3. Sacrifice by decapitation. Spray the head with 70% ethanol and keep on ice. Remove skin and muscle layers by tweezers and dissecting/surgical scissors.
4. Gently remove the parietal bones and separate them by following the sagittal suture in two halves. Make sure bones are free of sutures and any adherent tissue and place individual bone pieces in well-plates containing HBSS medium until the procedure is completed.
5. Discard HBSS and add to each well the digesting medium for 15 min at 37 °C.
6. Discard the supernatant.
7. Add again the digesting medium for 15 min at 37 °C.
8. This time save the supernatant and place it in alpha-MEM (Minimum Essential Medium) supplemented with 10% FBS (Fetal Bovine Serum) and 1% streptomycin/penicillin.
9. Repeat steps 1.7 and 1.8 2x.
10. Pool the three collected fractions, spin cells by centrifuging at 425 g for 5 min, discard the supernatant, add 3 ml of supplemented alpha-MEM to resuspend the cell pellet, and transfer to a 25 cm² flask.
11. Change the medium after 24 hr to remove debris and dead cells.
12. Add 50 µg/ml ascorbic acid (AA) and 3 mM glycerol 2-phosphate disodium salt hydrate (GP) to the alpha-MEM medium.
13. Thereafter, change the medium (alpha-MEM plus AA/GP) every other day.

2. ATRA Protocol

1. Conduct all experiments with ATRA (All-Trans Retinoic Acid) in a darkened room to prevent inactivation.
2. Prepare a 1X trypsin solution in HBSS medium and keep at 37 °C until use.
3. Remove the medium from subconfluent cells and carefully wash them with HBSS.
4. Add 1 ml trypsin, gently rotate the flask to allow all cells to be covered by trypsin, and incubate for 3 min at 37 °C.
5. Check for the actual detachment of cells under an inverted microscope.
6. Add 3 ml of alpha-MEM medium to inactivate trypsin.
7. Recover the cells and centrifuge for 15 min at 425 g.
8. Resuspend the cell pellet in fresh medium.
9. Place a drop in a hemocytometer counting chamber, allow to settle for a few min and count the cell number.
10. Place cells in 25 cm² flasks at a density of 10,000 cells/cm² with alpha-MEM plus AA/GP. Be aware that cell density is relevant to the optimal development of cell processes and intercellular contacts. A starting density higher than 10,000 cells/cm² impairs proper development of cell processes, but lower cell numbers result in premature cell death, due to the absence of intercellular contacts.
11. Add 5 µM ATRA to the medium every 24 hr.
12. Monitor the cells daily to assess morphology: morphological changes are generally observed after 48 hr. If, after the first 24-48 hr, cells are still proliferating, replate at the above density. A homogeneous population of mature osteocytes is present after 4-5 days. Depending on the needs, use cells at any time point, up to 12-15 days.
13. When cells are plated on other supports (such as coverslips or well-plates), be sure that the above density is maintained and continue to add ATRA every 24 hr until use. Leave cells to adhere to the new support for at least 24 hr before the experiment.

3. Immunostaining

1. For immunostaining, plate the cells on coverslips and follow established methodologies (see for instance Burry¹⁵).
2. For example, for indirect immunofluorescence, fix in 4% paraformaldehyde or cold acetone, incubate sequentially with the primary antibody at RT for 1 hr in a humidified chamber, then with the secondary fluorescent-labeled antibody in the dark, at RT for 30 min, in a humidified chamber.
3. Use DAPI or analogues for nuclear counterstaining, and mount in anti-fading medium.

4. Alizarin Red Staining and Extraction

1. Plate the cells on coverslips.
2. Fix the cells by immersing the coverslip in 70% ethanol for 10 min.
3. Cover the cells with some drops of alizarin red staining solution (1 mg/ml, pH 5.5) for 30 min.
4. Wash the coverslips with tap water. Mount with a drop of glycerol on glass slides and take images using an upright microscope at 10X and 20X magnification.
5. After the above procedure, recover the coverslips by immersing the slides in tap water.
6. Put each coverslip in a well plate.
7. Add 60% perchloric acid (the quantity has to be enough to completely cover the cells) and leave O/N at 4 °C with gentle agitation.
8. Read the optical density of the supernatant by spectrophotometry at 450 nm wavelength.
9. To normalize values for cell number, add Janus Green (0.2% solution in PBS) to cover the cell preparation for 10 min.
10. Wash carefully with ultrapure water.
11. Add 0.5 N HCl (the quantity has to be enough to cover all cells) and shake gently by hand for a few sec.
12. Read the absorbance at 615 nm by spectrophotometry.

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