bone research protocols

M E T H O D S I N M O L E C U L A R M E D I C I N E
TM
Bone
Research
Protocols
Edited by
Miep H. Helfrich, PhD
Stuart H. Ralston, MD
Bone
Research
Protocols
Edited by
Miep H. Helfrich, PhD
Stuart H. Ralston, MD
Human Osteoblast Culture 3
3
From:
Methods in Molecular Medicine, Vol. 80: Bone Research Protocols
Edited by: M. H. Helfrich and S. H. Ralston © Humana Press Inc., Totowa, NJ
1
Human Osteoblast Culture
James A. Gallagher
1. Introduction
Osteoblasts are the cells responsible for the formation of bone; they
synthesize almost all of the constituents of the bone matrix and direct its
subsequent mineralization. Once a phase of active bone formation is com-
pleted the osteoblasts do not become senescent but instead redifferentiate
into one of two other cell types: osteocytes and bone lining cells, both of
which play a major role in the regulation of calcium homeostasis and bone
remodeling.
Researchers have endeavored to culture osteoblasts from human bone for

several reasons:
1. To investigate the biochemistry and physiology of bone formation.
2. To investigate the molecular and cellular basis of human bone disease.
3. To investigate the role of cells of the osteoblastic lineage in regulating bone
resorption.
4. To screen for potential therapeutic agents.
5. To develop and test new biomaterials.
6. To use cell therapy in tissue engineering and bone transplantation.
The structure of bone tissue, the heterogeneity of cell types, the cross-
linked extracellular matrix, and the mineral phase combine to make bone
a difficult tissue from which to extract cells. Consequently, early attempts
to culture osteoblasts avoided human tissue and instead relied on enzy-
matic digestion of poorly mineralized fetal or neonatal tissue from
experimental animals. The first attempt to isolate cells from adult human
bone, using demineralization and collagenase digestion, was reported by
Bard and co-workers (1). The cultured cells were low in alkaline phos-
phatase and collagen synthesis, which were then regarded as the best
01/Gall/1-18/F1 2/26/03, 10:44 AM3
4 Gallagher
markers of the osteoblastic phenotype. Although the cells remained viable
for up to 2 wk they did not proliferate, and it was concluded that osteocytes
were the predominant cell type present. Mills et al. used the alternative
approach of explant culture and were successful in culturing cell populations
that included parathyroid hormone (PTH) responsive and alkaline phosphatase
positive cells (2).
The first successful attempts to isolate large numbers of cells that expressed
an osteoblastic phenotype from human bone were undertaken in Graham
Russell’s laboratory at the University of Sheffield in the early 1980s. The
defining characteristics of these studies were (1) the use of explant cultures,
which avoided the need for digestion of the tissue and (2) the availability of an

appropriate phenotypic marker. Successful culture of any cell type can be
achieved only if there is a specific marker of the phenotype that can be used to
confirm the identity of the cells in vitro. In this case, the marker was the then
recently discovered bone gla protein as measured by a radioimmunoassay
developed by Jim Poser (3,4). Nearly 20 yr later, bone gla protein, now known
as osteocalcin, undoubtedly remains the most specific marker of the osteoblas-
tic phenotype.
Although this culture system has been extensively modified by several
groups of researchers (see Note 1), the vast majority of published reports on
isolation of human osteoblasts still essentially use this simple but highly repro-
ducible explant technique. This technique and its modifications have been
described, compared, and reviewed elsewhere (5,6). The aim of this chapter is
to describe the basic methodology that is used in the author’s laboratory. This
is shown schematically in Fig 1. The nomenclature used by various research
groups to describe the isolated cells includes “human bone cells,” “human
osteoblasts in vitro,” “human osteoblastic cells,” and “HOBS.” We have pre-
ferred the conservative term “human bone derived cells” (HBDCs), and this is
used throughout this chapter.
HBDCs have been widely used to investigate the biology of the human
osteoblast, and their use has facilitated several major developments in our
understanding of the hormonal regulation of human bone remodeling. These
cells have also been used to investigate the cellular and molecular pathology of
bone disease. The major milestones in the culture of human osteoblasts are
summarized in Table 1. Figure 2 shows the increase in the application of
human osteoblast cultures since the initial reports in 1984.
Human bone cell culture is now becoming an important tool in tissue engi-
neering to test the biocompatibility and osteogenicity of novel biomaterials
and also for autologous transplantation of osteoblastic populations expanded
in vitro.
01/Gall/1-18/F1 2/26/03, 10:44 AM4

Human Osteoblast Culture 5
2. Materials
2.1. Tissue-Culture Media and Supplements
1. Phosphate-buffered saline (PBS) without calcium and magnesium, pH 7.4
(Invitrogen).
2. Dulbecco’s modification of minimum essential medium (DMEM) (Invitrogen)
supplemented to a final concentration of 10% with fetal calf serum (FCS), 2 mM
L-glutamine, 50 U/mL of penicillin, 50 µg/mL streptomycin. Freshly prepared 50
µg/mL of L-ascorbic acid should be added to cultures in which matrix synthesis
or mineralization is being investigated (see Note 2).
3. Serum-free DMEM (SFM).
4. FCS (see Note 3).
5. Tissue culture flasks (75 cm
2
) or Petri dishes (100-mm diameter) (see Note 4).
2.2. Preparation of Explants
1. Bone rongeurs from any surgical instrument supplier.
2. Solid stainless steel scalpels with integral handles (BDH Merck).
Fig. 1. Technique used to isolate cells expressing osteoblastic characteristics
(HBDCs) from explanted cancellous bone. E1, explant 1; E2, explant 2.
01/Gall/1-18/F1 2/26/03, 10:44 AM5
6 Gallagher
2.3. Passaging and Secondary Culture
1. Trypsin–EDTA solution: 0.05% Trypsin and 0.02% EDTA in Ca
2+
- and Mg
2+
-
free Hanks’ balanced salt solution, pH 7.4 (Invitrogen).
2. 0.4% Trypan blue in 0.85% NaC1 (Sigma Aldrich).

3. 70-µm “Cell Strainer” (Becton Dickinson).
4. Neubauer hemocytometer (BDH Merck).
5. Collagenase (Sigma type VII from Clostridium histolyticum).
6. DNase I (Sigma Aldrich).
Table 1
Phenotypic Milestones in the Culture and Characterization
of Osteoblastic Cells (HBCDs) from Human Bone
Isolation of viable cells from human bone Bard et al., 1972 (1)
Introduction of explant culture Mills et al., 1979 (2)
Production of osteocalcin Gallagher et al., 1984 (3)
Beresford et al., 1984a (4)
High alkaline phosphatase activity Gallagher et al., 1984 (3)
Beresford et al., 1984 (4)
Gehron-Robey and Termine 1985 (7)
Auf’molk et al., 1985 (8)
Responsiveness to PTH Beresford et al., 1984 (4)
MacDonald et al., 1984 (9)
Gehron-Robey and Termine 1985 (7)
Auf’mkolk et al., 1985 (8)
MacDonald et al., 1986 (10)
Synthesis of type I but not type III collagen Beresford et al., 1986 (11)
Synthesis of other bone matrix proteins Gehron-Robey and Termine 1985 (7)
Fedarko et al., 1992 (12)
Response to cytokines Beresford et al., 1984 (13)
Gowen et al., 1985 (14)
Response to oestrogen Vaishnav et al., 1984 (15)
Eriksen et al 1988 (16)
Expression of purinoceptors Schoefl et al., 1992 (17)
Bowler et al., 1995 (18)
Production of nitric oxide Ralston et al., 1994 (19)

Investigation of specific pathologies Marie et al., 1988 (20)
Walsh et al., 1995 (21)
Formation of mineralised nodules Beresford et al., 1993 (22)
Formation of bone in vitro and in vivo Gundle et al., 1995 (23)
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Human Osteoblast Culture 7
2.4. Phenotypic Characterization
1. 1,25-Dihydroxyvitamin D
3
[1,25-(OH)
2
D
3
] (Leo Pharmaceuticals or Sigma
Aldrich).
2. Menadione (vitamin K3) (Sigma Aldrich).
3. Alkaline phosphatase assay kit (Sigma Aldrich).
4. Staining Kit 86-R for alkaline phosphatase (Sigma Aldrich).
5. Osteocalcin radioimmunassay (IDS Ltd., Boldon, UK) (see Note 5).
6. Polymerase chain reaction (PCR) primers and reagents for a panel of osteoblastic
markers including osteocalcin (IDS Ltd., Boldon, UK).
2.5. In Vitro Mineralization
1. Dexamethasone (Sigma Aldrich).
2. Hematoxylin (BDH Merck).
3. L-Ascorbic acid (see Note 2).
4. Inorganic phosphate solution: Mix 500 mM solutions of Na
2
HPO
4
and NaH

2
PO
4
in a 4:1 (v/v) ratio. Sterile filter and store at 4°C prior to use.
2.6. Cryopreservation of Cells
1. Dimethyl sulfoxide (DMSO) (Sigma Aldrich).
2. Cryoampules.
3. Cell freezing container.
Fig. 2. Graph showing the increase in the application of human osteoblast cultures
since the initial reports in 1984.
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8 Gallagher
3. Methods
3.1. Establishing Primary Explant Cultures
A scheme outlining the culture technique is shown in Fig. 1.
1. Transfer tissue, removed at surgery or biopsy, into a sterile container with PBS or
serum-free medium (SFM) for transport to the laboratory with minimal delay,
preferably on the same day (see Note 6). An excellent source is the upper femur
of patients undergoing total hip replacement surgery for osteoarthritis. Cancel-
lous bone that would otherwise be discarded is removed from this site prior to the
insertion of the femoral prosthesis. The tissue obtained is remote from the hip
joint itself, and thus from the site of pathology, and is free of contaminating soft
tissue (see Note 7).
2. Remove soft connective tissue from the outer surfaces of the bone by scraping
with a sterile scalpel blade.
3 Rinse the tissue in sterile PBS and transfer to a sterile Petri dish containing a
small volume of PBS (5–20 mL, depending on the size of the specimen). If the
bone sample is a femoral head, remove cancellous bone directly from the open
end using sterile bone rongeurs or a solid stainless steel blade with integral
handle. Disposable scalpel blades may shatter during this process. With some

bone samples (e.g., rib), it may be necessary to gain access to the cancellous bone
by breaking through the cortex with the aid of the sterile surgical bone rongeurs.
4. Transfer the cancellous bone fragments to a clean Petri dish containing 2–3 mL
of PBS and dice into pieces 3–5 mm in diameter. This can be achieved in two
stages using a scalpel blade first, and then fine scissors.
5. Decant the PBS and transfer the bone chips to a sterile 30-mL “universal con-
tainer” with 15–20 mL of PBS.
6. Vortex-mix the tube vigorously three times for 10 sec and then leave to stand for
30 sec to allow the bone fragments to settle. Carefully decant off the supernatant
containing hematopoietic tissue and dislodged cells, add an additional 15–20 mL
of PBS, and vortex-mix the bone fragments as before. Repeat this process a mini-
mum of three times, or until no remaining hematopoietic marrow is visible and
the bone fragments have assumed a white, ivory-like appearance.
7. Culture the washed bone fragments as explants at a density of 0.2–0.6 g of tissue/
100-mm diameter Petri dish or 75-cm
2
flask (see Note 4) in 10 mL of medium at
37° in a humidified atmosphere of 95% air, 5% CO
2
.
8. Leave the cultures undisturbed for 7 d, after which time replace the medium with
an equal volume of fresh medium taking care not to dislodge the explants.
9. Check for outgrowth of cells at 7–10 d (see Note 8).
10. Replace the medium at 14 d and twice weekly thereafter until the desired cell
density has been attained.
3.2. Passaging Cells and Establishing Secondary Cultures
1. Remove and discard the spent medium.
2. Gently wash the cell layers three times with 10 mL of PBS without Ca
2+
and Mg

2+
.
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Human Osteoblast Culture 9
3. To each flask add 5 mL of freshly thawed trypsin–EDTA solution at room tem-
perature (20°C) and incubate for 5 min at room temperature with gentle rocking
every 30 sec to ensure that the entire surface area of the flask and explants is
exposed to the trypsin–EDTA solution.
4. Remove and discard all but 2 mL of the trypsin–EDTA solution, and then incu-
bate the cells for an additional 5 min at 37°C.
5. Remove the flasks from the incubator and examine under the microscope. Look
for the presence of rounded, highly refractile cell bodies floating in the trypsin–
EDTA solution. If none, or only a few, are visible tap the base of the flask sharply
on the bench top in an effort to dislodge the cells. If this is without effect, incu-
bate the cells for a further 5 min at 37°C.
6. When most of the cells have become detached from the culture substratum, trans-
fer to a “universal container” with 5 mL of DMEM with 10% FCS to inhibit
tryptic activity.
7. Wash the flask two to three times with 10 mL of SFM and pool the washings with
the original cell isolate.
8. Centrifuge at 250g for 5 min to pellet the cells.
9. Remove and discard the supernatant, invert the tube, and allow the medium to
drain briefly.
10. Resuspend the cell in 2 mL of SFM. If the cells are clumping see Note 9. If
required, the cell suspension can be filtered through a 70-µm “Cell Strainer”
(Becton Dickinson) to remove any bone spicules or remaining cell aggregates.
For convenience and ease of handling the filters have been designed to fit into the
neck of a 50-mL polypropylene tube. Wash the filter with 2–3 mL of SFM and
add the filtrate to the cells.
11. Take 20 µL of the mixed cell suspension and dilute to 80 µL with SFM. Add5 µL

of trypan blue solution, mix, and leave for 1 min before counting viable (round
and refractile) and nonviable (blue) cells in a Neubauer Hemocytometer. Using
this procedure, typically 1–1.5 × 10
6
cells are harvested per 75-cm
2
flask, of
which ≥75% are viable.
12. Plate the harvested cells at a cell density suitable for the intended analysis. We
routinely subculture at 5 × 10
3
–10
4
cells/cm
2
and achieve plating efficiencies
measured after 24 h of ≥70% (see Note 10).
3.3. Phenotypic Characterization
The phenotypic characterization of HBDCs is described in detail in ref. 5.
The simplest phenotypic marker to investigate is the enzyme alkaline phos-
phatase, a widely accepted marker of early osteogenic differentiation. Alkaline
phosphatase can be measured by simple enzyme assay or by histochemical
staining. Basal activity is initially low, but increases with increasing cell den-
sity. Treatment with 1,25-(OH)
2
D
3
increases alkaline phosphatase activity. The
most specific phenotypic marker is osteocalcin. This is a protein of Mr 5800
containing residues of the vitamin K-dependent amino acid γ-carboxyglutamic

01/Gall/1-18/F1 2/26/03, 10:44 AM9
10 Gallagher
acid. In humans its synthesis is restricted to mature cells of the osteoblast lin-
eage. It is an excellent late stage markers for cells of this series despite the fact
that its precise function in bone has yet to be established. Osteocalcin can be
measured by one of the many commercially available kits. 1,25-(OH)
2
D
3
increases the production of osteocalcin in cultures of HBDCs, but not fibro-
blasts obtained from the same donors. More recently, researchers have adopted
the use of reverse transcription (RT)-PCR to look at the expression of osteo-
blastic markers in HBDCs. PCR primers for a panel of osteoblastic markers
including osteocalcin are shown in Table 2.
3.4. Phenotypic Stability in Culture
As a matter of routine we perform all of our studies on cells at first passage.
Other investigators have studied the effects of repeated subculture on the phe-
notypic stability of HBDCs and found that they lose their osteoblast-like char-
acteristics. In practical terms this presents real difficulties, as it is often
desirable to obtain large numbers of HBDCs from a single donor. As an alter-
native to repeated subculture, trabecular explants can be replated at the end of
primary culture into a new flask (see Fig. 1). Using this technique, it is possible
to obtain additional cell populations that continue to express osteoblast-like
characteristics, including the ability to mineralize their extracellular matrix,
and maintain their cytokine expression profile (6). Presumably, these cultures
are seeded by cells that are situated close to the bone surfaces, and that retain
Table 2
PCR Primers for a Panel of Osteoblastic Markers
Osteoblastic
phenotype

marker Primer pairs T
m
(°C) Product size (bp)
Osteocalcin 5'-ccc tca cac tcc tcg ccc tat-3'
5'-tca gcc aac tcg tca cag tcc -3' 65 246
PTH receptor 5'-agg aac aga tct tcc tgc tgc a-3'
5'-tgc atg tgg atg tag ttg cgc gt-3' 55 571
Alkaline 5'-aag agc ttc aaa ccg aga tac aag-3'
phosphatase 5'-ccg agg ttg gcc ccg at-3' 68 715
CBFA1 5'-ccc cac gac aac cgc acc-3'
5'-cac tcc ggc cca caa atc tc-3' 60 388
Osteoprotegerin 5'-ggg cgc tac ctt gag ata gag tt-3'
5'-gag tga cag ttt tgg gaa agt gg-3' 60 760
RANKL 5'-act att aat gcc acc gac atc-3'
5'-aaa aac tgg ggc tca atc ta-3' 54 462
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Human Osteoblast Culture 11
the capacity for extensive proliferation and differentiation. The continued sur-
vival of these cells may be related to the gradual release over time in culture of
the cytokines and growth factors that are known to be present in the extracellu-
lar bone matrix, many of which are known to be produced by mature cells of
the osteoblast lineage. The addition of 25 µM
L-ascorbic acid (50 µg/mL) (see
Note 2) to HBDCs in secondary culture (E1P1) produces a sustained increase
in the deposition of matrix due to an increase in the synthesis of collagen and
noncollagenous protein and bone sialoprotein and osteocalcin.
3.5. Passaging Cells Cultured in the Continuous Presence
of Ascorbate
Because of their synthesis and secretion of an extensive collagen-rich extra-
cellular matrix, HBDCs cultured in the continuous presence of ascorbate can-

not be subcultured using trypsin–EDTA alone. They can, however, be
subcultured if first treated with purified collagenase. The basic procedure is as
follows:
1. Rinse the cell layers twice with SFM (10 mL/75-cm
2
flask).
2. Incubate the cells for 2 h at 37°C in 10 mL of SFM containing 25 U/mL of puri-
fied collagenase (Sigma type VII) and 2 mM additional calcium (1:500 dilution
of a filter-sterilized stock solution of 1 M CaCl
2
).
3. Gently agitate the flask for 10–15 sec every 30 min.
4. Terminate the collagenase digestion by discarding the medium (check that there
is no evidence of cell detachment at this stage).
5. Gently rinse the cell layer twice with 10 mL of Ca
2+
- and Mg
2+
-free PBS. To
each flask add 5 mL of freshly thawed trypsin–EDTA solution, pH 7.4, at room
temperature (20°C).
6. Typically this procedure yields ~3.5–4 × 10
6
cells/75-cm
2
flask after 28 d in pri-
mary culture. Cell viability is generally ≥90%.
3.6. Setting Up Mineralizing HBDC Cultures
The function of the mature osteoblast is to form bone. Despite the over-
whelming evidence that cultures of HBDCs contain cells of the osteoblast lin-

eage, initial attempts to demonstrate the presence of osteogenic (i.e., bone
forming) cells proved unsuccessful. Subsequently, several authors reported that
culture of HBDCs in the presence of ascorbate and millimolar concentrations
of the organic phosphate ester β-glycerol phosphate (β-GP) led to the forma-
tion of mineralized structures resembling the nodules that form in cultures of
fetal or embryonic animal bone derived cells (reviewed in ref. 22). These have
been extensively characterized and shown by a variety of morphological, bio-
chemical, and immunochemical criteria to resemble embryonic/woven bone
formed in vivo. An alternative to the use of β-GP is to provide levels of inor-
01/Gall/1-18/F1 2/26/03, 10:44 AM11
12 Gallagher
ganic phosphate sufficient to support the process of cell-mediated mineraliza-
tion in vitro, and the preferred method when studying HBDCs, is supplementa-
tion of the culture medium with inorganic phosphate (Pi; see Note 11). The
protocol for inducing matrix mineralization in cultures of HBDCs is as follows:
1. Prepare fragments of human trabecular bone as described in Subheading 3.1.,
steps 1–6 (see Note 12).
2. Culture the washed bone fragments in medium supplemented with 100 µM
L-ascorbic acid 2-phosphate and either 200 nM hydrocortisone or 10 nM dexam-
ethasone.
3. Culture for 4–5 wk until the cells have attained confluence with medium changes
twice weekly.
4. When the cells have synthesized a dense extracellular matrix, subculture using
the sequential collagenase/trypsin–EDTA protocol and plate the cells in 25-cm
2
flasks at a density of 10
4
viable cells/cm
2
.

5. After a further 14 d, supplement the medium with 0.01% phosphate solution (see
Subheading 2.5.4.).
6. After 48–72 h, wash the cell layers two to three times with 10 mL of SFM.
7. Fix with 95% ethanol at 4
°
C (see Note 13).
3.7. Measuring Alkaline Phosphatase Activity
1. Add 2.5 mL of staining solution from the Sigma 86-R Staining Kit to each flask
of cells (or enough to coat the surface).
2. Place specimens in a humidified chamber and incubate for 1 h at 20°C in the dark.
3. Wash under running tap water and counterstain the nuclei for 15 sec with hema-
toxylin.
4. Mineral deposits can be stained using a modification of von Kossa’s technique.
5. Prior to examination, mount sections in DPX and cell layers in flasks covered
with glycerol.
3.8. Cryopreservation of HBDC
If required, HBDCs can be stored frozen for extended periods in liquid
nitrogen or in ultralow temperature (–135°) cell freezer banks. We use the fol-
lowing protocol:
1. Passage the cells using trypsin–ETDA as described in Subheading 3.2.,
steps 1–5.
2. Pellet the cells by centrifugation at 250g for 5 min and pour off the supernatant.
3. Resuspend the cell pellet in FCS, adjust to a density of 1–2 × 10
6
cells/mL in a
volume of 900 µL and transfer to a cryoampule.
4. Swirl the ampule in an ice water bath.
5. Add 100 µL of DMSO gradually while holding the ampule in the iced water.
6. Close ampules tightly and freeze at 5°C/min to 4°C, followed by 1°C/min
to –80°C in a cell freezing container.

7. Transfer the cells to liquid nitrogen for long-term storage.
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Human Osteoblast Culture 13
3.9. Thawing Cells
1. Retrieve the cells from liquid nitrogen and place in a water bath set at 37°C.
2. Transfer the cells to a universal container to which at least 20 volumes of pre-
heated culture medium has been added.
3. Centrifuge at 250g for 2 min to pellet the cells and pour off the supernatant.
4. Resuspend the cells in approx 10 mL of the medium and place into culture for 24 h.
5. Replace the medium after 24 h and culture for 2–3 wk.
4. Notes
1. Although most investigators have used the original explant method with only
minor modifications, others have developed alternative techniques for the isola-
tion and culture of HBDCs. Gehron-Robey and Termine used prior digestion of
minced bone with clostridial collagenase and subsequent culture of explants in
medium with reduced calcium concentrations (7). In contrast, Wergedal and
Baylink have used collagenase digestion to liberate cells directly (24). Marie and
co-workers have used a method in which explants are first cultured on a nylon
mesh (20). These alternative methods are described in greater detail in ref. 5.
2. Beresford and co-workers introduced the more stable analogue L-ascorbic acid 2-
phsophate (Wako Pure Chemical Industries Ltd.) which does not have to be added
daily (see ref. 6 for details).
3. Batches of serum vary in their ability to support the growth of HBDCs. It is
advisable to screen batches and reserve a large quantity of serum once a suitable
batch has been identified. HBDCs will grow in autologous and heterologous
human serum, but as yet no comprehensive studies have been performed to iden-
tify the effects on growth and differentiation.
4. The authors have obtained consistent results with plasticware from Sarstedt and Becton
Dickinson. Smaller flasks or dishes can be used if the amount of bone available is <0.2 g.
5. Several other assays for osteocalcin are commercially available.

6. Bone can be stored for periods of up to 24 h at 4°C in PBS or SFM prior to culture
without any deleterious effect on the ability of the tissue to give rise to popula-
tions of osteoblastic cells.
7. Bone cells have also been cultured successfully from many other anatomical sites
including tibia, femur, rib, vertebra, patella, and digits.
8. With the exception of small numbers of isolated cells, which probably become
detached from the bone surface during the dissection, the first evidence of cellu-
lar proliferation is observed on the surface of the explants, and this normally
occurs within 5–7 d of plating. After 7–10 d, cells can be observed migrating
from the explants onto the surface of the culture dish (see Fig. 3). If care is taken
not to dislodge the explants when feeding, and they are left undisturbed between
media changes, they rapidly become anchored to the substratum by the cellular
outgrowths. The typical morphology of the cells is shown in Fig. 4, but cell shape
varies between donors, from fibroblastic to cobblestone-like. Cultures generally
attain confluence 4–6 wk post plating, and typically achieve a saturation density
of 29,000 ± 9000 cells/cm
2
(mean + SD, N = 11 donors).
01/Gall/1-18/F1 2/26/03, 10:44 AM13
14 Gallagher
9. If the cells are clumping, resuspend in 2 mL of SFM containing 1 µg/mL of DNase I
for each dish or flask treated with trypsin-EDTA, and using a narrow-bore
2-mL pipet, repeatedly aspirate and expel the medium to generate a cell suspen-
sion.
10. In our experience the minimum plating density for successful subculture is
3500 cells/cm
2
. Below this the cells exhibit extended doubling times and often
fail to grow to confluence. Note that cells can be passaged successfully onto a
Fig. 3. Migration of cells expressing osteoblastic characteristics (HBDCs) from

explanted cancellous bone.
Fig. 4. Typical morphology of cells expressing osteoblastic characteristics
(HBDCs) from explanted cancellous bone (see Note 14).
01/Gall/1-18/F1 2/26/03, 10:44 AM14
Human Osteoblast Culture 15
range of substrates. Recently HBDC culture has been used to investigate
biocompatibility and osteogenicity of novel biomaterials. Figure 5 shows HBDCs
passaged onto nanotopography textured titanium coated silicon and subsequently
stained with antibodies specific for β-tubulin.
11. Mineralizing cultures: HBDCs cultured in the continuous presence of glucocorti-
coids and the long-acting ascorbate analogue produce a dense extracellular matrix
that mineralizes extensively following the addition of Pi. This is the case for the
original cell population (E1P1) and that obtained following replating of the tra-
becular explants (E2P1), which further attests to the phenotypic stability of the
cultured cells. Cells cultured in the continuous presence of ascorbate and treated
with glucocorticoids at first passage show only a localized and patchy pattern of
mineralization, despite possessing similar amounts of extracellular matrix and
alkaline phosphatase activity. Cells cultured without ascorbate, irrespective of
the presence or absence of glucocorticoids, secrete little extracellular matrix, and
do not mineralize. The ability of the cells to mineralize their extracellular matrix
is dependent on ascorbate being present continuously in primary culture. The
addition of ascorbate in secondary culture, even for extended periods, cannot
compensate for its omission in primary culture. This finding provides further
Fig. 5. Human primary derived osteoblasts cultured (7 d) on nanotopography tex-
tured titanium-coated silicon immunostained for β-tubulin to visualize the microtubles
for specific orientations correlating to the stimulus of nanotopography. Image taken
using a CLSM310 Zeiss confocal microscope. (J. M. Rice, J. A. Hunt and J. A.
Gallagher, unpublished.)
01/Gall/1-18/F1 2/26/03, 10:44 AM15
16 Gallagher

evidence to support the hypothesis that maintenance of adequate levels of ascor-
bate during the early stages of explant culture is of critical importance for the sur-
vival of cells that retain the ability to proliferate extensively and give rise to
precursors capable of undergoing osteogenic differentiation. HBDCs cultured con-
tinuously in the presence of ascorbate and glucocorticoids retain the ability to form
bone when implanted in vivo within diffusion chambers in athymic mice (23).
12. For studies of in vitro mineralization, it is preferable to obtain trabecular bone
from sites containing hematopoietic marrow such as the upper femur or iliac crest.
13. Fixation of mineralized cultures: This can be done in situ, for viewing en face, or
if sections are to be cut following detachment of the cell layer from the surface of
the flask using a cell scraper. Great care is needed if the cell layer is to be har-
vested intact, particularly when mineralized.
14. The available evidence indicates that cultures of HBDCs contain cells of the
osteogenic lineage at all stages of differentiation and maturation. This conclu-
sion is consistent with the expression of both early (alkaline phosphatase) and
late (osteocalcin, bone sialoprotein) stage markers of osteoblast differentiation.
In addition, in ascorbate-treated cultures there is a small subpopulation (≤5%) of
cells that express the epitope recognized by the monoclonal antibody (MAb)
STRO-1 (25), which is a cell-surface marker for clonogenic, multipotential mar-
row stromal precursors capable of giving rise to cells of the osteogenic lineage in
vitro. The presence of other cell types, including endothelial cells and those
derived from the hematopoietic stem cell, has been investigated using a large
panel of MAbs and flow cytometry and/or immunocytochemistry. The results of
these studies reveal that at first passage there are no detectable endothelial, lym-
phoid, or erythroid cells present. A consistent finding, however, is the presence
of small numbers of cells (≤5%) expressing antigens present on cells of the mono-
cyte/macrophage series. In the absence of added calcitriol, particularly in SFM or
FCS that has been depleted of endogenous calcitriol by charcoal treatment, the
amount of ostecalcin produced by HBDCs is below the limits of detection in
most assays (3,4). The same applies to the detection of steady-state levels of

osteocalcin mRNA. An exception to this general rule is when HBDCs are cul-
tured for extended periods in the presence of L-ascorbate or its stable analog, L-
ascorbate-2-phosphate.
Acknowledgments
I am grateful to Jane Dillon and Paula Finnigan for their assistance in pre-
paring this chapter and to Dr. John Hunt and Dr. Judith Rice, UKCTE, Clinical
Engineering, University of Liverpool for Fig. 3.
References
1. Bard, D. R., Dickens, M. J., Smith, A. U., and Zarek, J. M. (1972) Isolation of
living cells from mature mammalian bone. Nature 236, 314–315.
2. Mills, B. G., Singer, F. R., Weiner, L. P., and Hoist, P. A. (1979) Long term culture
of cells from bone affected with Paget’s disease. Calcif. Tissue Int. 29, 79–87.
01/Gall/1-18/F1 2/26/03, 10:44 AM16
Human Osteoblast Culture 17
3. Gallagher, J. A., Beresford, J. N., McGuire, M. K. B., et al. (1984) Effects of
glucocorticoids and anabolic steroids on cells derived from human skeletal and
articular tissues in vitro. Adv. Exp. Med. Biol. 171, 279–292.
4. Beresford, J. N., Gallagher, J. A., Poser, J. W., and Russell, R. G. G. (1984) Pro-
duction of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3,
parathyroid hormone and glucocorticoids. Metab. Bone Dis. Rel. Res. 5, 229–234.
5. Gallagher, J. A., Gundle, R., and Beresford, J. N. (1996) Isolation and culture of
bone forming cells (osteoblasts) from human bone, in Human Cell Culture Proto-
cols (Jones, G. E., ed.), Humana Press Totowa, NJ.
6. Gundle, R., Stewart, K., Screen, J., and Beresford, J. N. (1998) Isolation and cul-
ture of human bone derived cells, in Marrow Stromal Cell Culture (Beresford, J.
and Owen, M., eds.), Cambridge University Press, Cambridge, UK.
7. Gehron Robey, P. and Termine, J. D. (1985) Human bone cells in vitro. Calcif.
Tissue Int. 37, 453–460.
8. Auf’mkolk, B., Hauschka, P. V., and Schwartz, R. (1985) Characterisation of
human bone cells in culture. Calcif. Tissue Int. 37, 228–235.

9. MacDonald, B. R., Gallagher, J. A., Ahnfelt-Ronne, I., Beresford, J. N., Gowen,
M., and Russell, R. G. G. (1984) Effects of bovine parathyroid hormone and
1,25(OH)2D3 on the production of prostaglandins by cells derived from human
bone. FEBS Lett. 169, 49–52.
10. MacDonald, B. R., Gallagher, J. A., and Russell, R. G. G. (1986) Parathyroid
hormone stimulates the proliferation of cells derived from human bone. Endocri-
nology 118, 2245–2449.
11. Beresford, J. N., Gallagher, J. A., and Russell, R. G. G. (1986) 1,25-Dihydroxy-
vitamin D
3
and human bone derived cells in vitro: effects on alkaline phosphatase,
type I collagen and proliferation. Endocrinology 119, 1776–1785.
12. Fedarko, N. S., Vetter, U., Weinstein, S., and Robey, P. G. (1992) Age-related
changes in hyaluronan, proteoglycan, collagen and osteonectin synthesis by
human bone cells. J. Cell Physiol. 151, 215–227.
13. Beresford, J. N., Gallagher, J.A., Gowen, M., et al. (1984) The effects of mono-
cyte-conditioned medium and interleukin 1 on the synthesis of collagenous and
non-collagenous proteins by mouse bone and human bone cells in vitro. Biochim.
Biophysica. Acta Gen. Subj. 801, 58–65.
14. Gowen, M., Wood, D. D., and Russell, R. G. (1985) Stimulation of the prolifera-
tion of human bone cells in vitro by human monocyte products with interleukin-1
activity in J. Clin. Invest. 4, 1223–1229.
15. Vaishnav, R., Gallagher, J. A., Beresford, J. N., Poser, J. W., and Russell, R. G.
G. (1984) Direct effects of stanozolol and oestrogen on human bone cells in cul-
ture, in Osteoporosis. Proceedings of Copenhagen International Symposium, pp.
485–488.
16. Eriksen, E. F., Colvard, D. S., Berg, N. J., et al. (1988) Evidence of estrogen
receptors in normal human osteoblast-like cells. Science 241, 84–86.
17. Schoefl, C., Cuthbertson, K. S. R., Walsh, C. A., et al. (1992) Evidence for P2-
purinoceptors on osteoblast-like cells. J. Bone Min. Res. 7, 485–591.

01/Gall/1-18/F1 2/26/03, 10:44 AM17
18 Gallagher
18. Bowler, W. B., Gallagher, J. A., and Bilbe, G. (1995) Identification and cloning
of human P2U purinceptor present in osteoclastoma, bone and osteoblasts. J. Bone
Miner. Res. 10, 1137–1145.
19. Ralston, S. H., Todd, D., Helfrich, M., Benjamin, N., and Grabowski, P.S. (1994)
Human osteoblast-like cells produce nitric oxide and express inducible nitric oxide
synthase. Endocrinology 135, 330–336.
20. Marie, P. J., Sabbagh, A., De Vernejoul, M. C., and Lomri, A. (1988) Osteocalcin
and deoxyribonucleic acid synthesis in vitro and histomorphometric indices of
bone formation in postmenopausal osteoporosis. J. Clin. Invest. 69, 272–279.
21. Walsh, C. A., Birch, M. A., Fraser, W. D., et al. (1995) Expression and secretion
of parathyroid hormone-related protein by human osteblasts in vitro: effects of
glucocorticoids. J. Bone Miner. Res. 10, 17–25.
22. Beresford, J. N., Graves, S. E., and Smoothy, C. A. (1993) Formation of
mineralised nodules by bone derived cells in vitro: a model of bone formation?
Am. J. Med. Genet. 45, 163–178.
23. Gundle, R. G., Joyner, C. J., and Triffitt, J. T. (1995) Human bone tissue forma-
tion in diffusion chamber culture in vivo by bone derived cells and marrow stro-
mal cells. Bone 16, 597.
24. Wergedal, J. E. and Baylink, D. J. (1984) Characterisation of cells isolated and
cultured from human trabecular bone. Proc. Soc. Exp. Biol. Med. 176, 60–69.
25. Walsh, S., Jefferiss, C., Stewart, K., Jordan, G. R., Screen, J., and Beresford, J. N.
(2000) Expression of the developmental markers STRO-1 and alkaline phos-
phatase in cultures of human marrow stromal cells: regulation by fibroblast growth
factor (FGF)-2 and relationship to the expression of FGF receptors 1-4. Bone 27,
185–195.
01/Gall/1-18/F1 2/26/03, 10:44 AM18
Bone Cells from Calvariae and Long Bones 19
19

From:
Methods in Molecular Medicine, Vol. 80: Bone Research Protocols
Edited by: M. H. Helfrich and S. H. Ralston © Humana Press Inc., Totowa, NJ
2
Osteoblast Isolation from Murine Calvariae
and Long Bones
Astrid Bakker and Jenneke Klein-Nulend
1. Introduction
When conducting in vitro research on bone, a choice has to be made between
using bone organ or bone cell cultures. When one decides to use the latter, the
question is whether to use primary cells or cell lines. The advantage of using
cell lines over freshly isolated cells lies in the ready availability of large num-
bers of cells, the homogeneity of the cell cultures, and the expected invariabil-
ity of the phenotype. In the long run, however, cell lines appear unstable to
some extent. In addition, their clonal selection has favored rapidly growing
cells, but has not necessarily selected for the whole range of bone-specific gene
expression characteristic of primary bone cells. This means that in certain
experiments, the use of primary bone cells is preferred to the use of cell lines.
Peck and co-workers initiated the use of primary bone cell cultures in 1964
(1). They isolated cells from frontal and parietal bones of fetal and neonatal rat
calvariae by collagenase digestion of the uncalcified bone matrix. The isolated
cells were viable, proliferated during culture, and exhibited high activity of the
osteoblast marker alkaline phosphatase (ALP). The real nature of the cells,
however, especially the amount of contamination with connective tissue fibro-
blasts, could not be defined unambiguously (1). Wong and Cohn (1974) tried
to isolate a better defined and more homogeneous cell population by removing
the outer layers of the periosteum with successive collagenase treatments (2).
Although this method led to cell cultures that were more osteoblastic in nature,
these were not free from other cell types, such as osteoclast precursors, either
(3). Other investigators have tried to improve the osteoblastic character of the

isolated bone cell populations by removing the fibroblastic outer periosteum
before using enzymatic digestion to isolate the cells from the calvarium (4,5).
02/Nulend/19-28/F1 2/26/03, 10:44 AM19
20 Bakker and Klein-Nulend
This method resulted in two cell populations, one of which was still osteogenic
after prolonged culture time (osteoblastic cells), and another one that was not
(periosteal fibroblasts) (6). These studies led to a broad range of methods for
obtaining well-defined osteoblast-like cells in vitro, which are at present widely
used as a tool to improve our knowledge of bone biology (7–9).
This chapter describes the isolation of primary mouse bone cells from adult
mouse calvariae and long bones, as well as the process of isolation of bone
cells from neonatal mouse calvariae. Owing to their difference in origin and
method of isolation, it is to be expected that each of the primary bone cell
cultures described will have its own characteristics. For example, it has been
shown that neonatal cells show a higher basal release of nitric oxide and a
higher response to 1,25-dihydrogxyvitamin [D
3
1,25-(OH)
2
D
3
] treatment than
bone cells obtained from adult bone (10). Because vitamin D3 stimulates
immature bone cell differentiation, this supports the notion that neonatal cell
cultures contain more immature, rapidly growing cells than cultures from adult
bone. Thus, for in vitro studies investigating the cellular behavior of adult bone,
it seems advisable to use cells from adult bone fragments to reproduce best the
inherent cellular properties of the adult tissue.
Another example relates to mechanosensitivity of bone cells. Because the
prevailing mechanical strains in the skull are much lower than those in the

axial and appendicular skeleton, the question has arisen whether cells from
calvariae or long bones should be used in such studies. We addressed this
issue by studying the nitric oxide production of the bone cells in response to
mechanical stimulation in the form of fluid flow. We found no difference in the
responsiveness of osteoblasts from adult mouse calvariae or adult mouse long
bones (12). These results suggest that the cellular mechanosensitivity of
calvariae and long bone cells is not intrinsically different and that either cell
culture can be used for these sorts of experiments.
2. Materials
2.1. Tissues
1. Cells are obtained from the long bones and the calvariae of adult (age 9 wk or
older) mice, or the calvariae from neonatal mice pups (age 3–4 d).
2.2. Instruments
All of the following materials have to be sterile.
1. Polystyrene plate and needles for fixing the mice.
2. Scalpels (no. 10 and 11), scissors, tweezers, and curved forceps.
3. 5-mL and 10-mL syringes, 27G1/2 needles, disposable cell scrapers, and 0.2-µm
disposable filter units.
02/Nulend/19-28/F1 2/26/03, 10:44 AM20
Bone Cells from Calvariae and Long Bones 21
4. 25-cm
2
tissue culture flasks (Nunc), six-well tissue plates (Costar), 94/16-mm
cellstar Petri dishes (Greiner), and 145/20-mm cellstar (large) Petri dishes
(Greiner).
5. 100 × 16 mm (10 mL) conical base test tubes with screw cap (Bibby Sterilin Ltd.,
Staffordshire, UK).
2.3. Media and Solutions
1. Phosphate-buffered saline (PBS): 137 mM NaCl, 1.5 mM KH
2

PO
4
, 2.7 mM KCl,
and 8.1 mM Na
2
HPO
4
. Adjust the pH to 7.4.
2. Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Paisley, UK): Add 2.2 g
NaHCO
3
/L; adjust the pH to 7.4.
3. Complete culture medium (cCM): DMEM, supplemented with 100 U/mL of peni-
cillin (Sigma), 50 µg/mL of streptomycin sulfate (Gibco), 50 µg/mL of
gentamycin (Gibco), 1.25 µg/mL of fungizone (Gibco), 100 µg/mL of ascorbate,
and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA; see Note 1).
Make fresh and filter sterilize.
4. Collagenase solution: 2 mg of collagenase II (Sigma) per milliliter of DMEM.
Make fresh and filter sterilize.
5. Trypsin solution: 0.25% trypsin 1:250 (Difco, Detroit, MI, USA) and 0.10%
EDTA in PBS; filter sterilize.
6. Digestion solution: Add 1 mL of trypsin solution and 3.2 mg of collagenase II to
4 mL of PBS. Make fresh.
7. Vitamin D medium (VDM): DMEM, supplemented with 100 U/mL of penicillin,
50 µg/mL of streptomycin sulfate, 50 µg/mL of gentamicin, 1.25 µg/mL of
fungizone, 100 µg/mL of ascorbate, 0.2% bovine serum albumin (Sigma) and 10
–8
M
1,25-(OH)
2

D
3
. Make fresh and shield away from direct light.
8. Vitamin D control medium (VDCM): Composition is the same as vitamin D
medium, except that the 1,25-(OH)
2
D
3
is replaced by an equal amount of vehicle.
9. BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA).
3. Methods
Normal techniques for working under sterile conditions (use of sterile media
and instruments and working in a flow cabinet) should be used to keep the cell
cultures sterile.
3.1. Isolation and Culture of Primary Bone Cells from Adult
Mouse Long Bones
1. Euthanize one or two adult mice by means of cervical dislocation.
2. Fix the mouse in a supine position on a polystyrene plate or in a large Petri dish,
and clean the abdomen and extremities using 70% ethanol.
3. Make a single incision through the skin, starting at the top of the sternum and
ending a few millimeters above the genitals, using a no. 10 scalpel. Make a sec-
ond incision starting from the top of the first incision and ending at the wrist of
02/Nulend/19-28/F1 2/26/03, 10:44 AM21
22 Bakker and Klein-Nulend
the upper left extremity. Repeat this procedure with the other paws. Carefully
remove the skin from the abdomen with a blade.
4. Change your blade for a sterile no. 11 scalpel. Remove the muscles from the long
bones in the limb (femur; tibia and fibula; or humerus, radius, and ulna), and
scrape the bone with a scalpel until it is clean (see Note 2). Excise the long bone
and place it in a Petri dish with PBS.

5. When all the long bones have been removed, cut off the epiphyses.
6. Flush out the bone marrow with PBS, using a 5-mL syringe and a 27-gauge needle.
7. Cut the clean diaphyses into small pieces of approx 1–2 mm
2
using scissors.
8. Wash the bone pieces with PBS, and incubate in 4 mL of collagenase solution at
37
°
C in a shaking water bath to remove all remaining soft tissue and adhering
cells.
9. After approx 1 h, vigorously shake the solution by hand.
10. After 2 h, add 4 mL of cCM containing 10% FBS to inhibit further collagenase
activity, and rinse the bone pieces three times with cCM.
11. Transfer the bone pieces to 25-cm
2
flasks, containing 5 mL of cCM, at a density of
about 20–30 fragments per flask. Replace culture medium three times per week.
12. Adult mouse bone cells will start to migrate from the bone chips after 3–5 d. On
average the cell monolayer growing from the bone fragments will reach
confluency after 11–15 d.
13. To obtain more cells, trypsinize the monolayer by incubating the cells with 1 mL of
trypsin solution at 37°C for 10 min.
14. Plate the cells at 25 × 10
3
cells per well in six-well culture dishes containing
3 mL of cCM per well.
15. Change medium three times per week, and after approx 7–10 d cells will reach
subconfluency, upon which they can be used for experiments (see Note 3 and
Subheading 3.4.). The average number of cells thus obtained lies between
4 x 10

6
and 6 × 10
6
cells.
3.2. Isolation and Culture of Primary Bone Cells from Adult Mouse
Calvariae
1. Euthanize two adult mice and fix them on a polystyrene plate, or in a large Petri dish.
2. Clean the head using 70% ethanol, and make a cut through the skin at the base of
the skull, using scissors.
3. Make an incision starting at the nose bridge, and ending at the base of the skull.
Remove the skin from the top of the head (see Fig. 1).
4. Use scissors to cut through the bone at the base of the neck. Cut the calvariae
loose, while holding the head with curved forceps placed in the orbita.
5. Transfer the calvariae to a Petri dish with PBS and remove the soft tissues using
tweezers or by scraping with a knife (see Note 2).
6. Remove the sutures, using scissors, and chop the remaining bone into small frag-
ments of approx 1–2 mm
2
.
7. Incubate the fragments for 30 min in 4 mL of collagenase solution at 37° in a
shaking water bath.
02/Nulend/19-28/F1 2/26/03, 10:44 AM22
Bone Cells from Calvariae and Long Bones 23
8. Remove the collagenase solution and replace with fresh collagenase solution. Incu-
bate another 30 min, then replace the collagenase solution for trypsin solution.
9. Incubate in trypsin for 30 min. Replace by 4 mL of collagenase solution for the
fourth and final incubation step of 30 min.
10. Add 4 mL of cCM to the collagenase to inhibit collagenase activity. Rinse the
bone pieces three times with cCM.
11. Transfer the bone pieces to 25 cm

2
flasks, containing 5 mL of cCM, at a density
of approx 20–30 fragments per flask.
12. Change the medium three times per week. Adult mouse bone cells will start to
migrate from the bone chips after 3–5 d. On average the cell monolayer growing
from the bone fragments will reach confluency after 11–15 d, upon which the
monolayer is trypsinized by incubating the cells with 1 mL of trypsin solution at
37°C for 10 min.
13. Plate the cells at 25 × 10
3
cells per well in six-well culture dishes containing
3 mL of cCM per well.
14. After approx 7–10 d cells will reach subconfluency, upon which they can be used
for experiments (see Note 3 and Subheading 3.4.). The average number of cells
thus obtained lies between 4 × 10
6
and 6 × 10
6
cells.
3.3. Isolation and Culture of Bone Cells from Neonatal Mouse
Calvariae
1. Euthanize 20–30 neonatal mice pups (2–3 liters) by decapitation or halothane
inhalation, and place the heads in a Petri dish with PBS (see Note 4).
2. Grasp the head by the nape of the neck, and cut the skin away using scissors.
Fig. 1. Schematic presentation of mouse calvariae. (A) Make a cut through the skin
at the base of the skull, using scissors (short dashed line). For adult tissue, make an
incision starting at the nose bridge, ending at the base of the skull (long dashed line),
and remove the skin from the top of the head (arrows). Use scissors to cut away the
skin from the top of the neonatal mouse heads. (B) Dissect calvariae as indicated by
area C and remove as much soft tissue as possible. Do not include the shaded area near

the neck, as this will result in heavy fibroblast contamination of the cultures. Sf, Skin
flap; C, Calvaria.
02/Nulend/19-28/F1 2/26/03, 10:44 AM23
24 Bakker and Klein-Nulend
3. Hold the head with curved forceps placed through the orbita, cut the calvariae
loose along the edge, and place it in a Petri dish with PBS (see Fig. 1 for a dia-
gram illustrating how to dissect the calvariae).
4. Pin the calvariae down with tweezers and cut away the edges and sutures with a
small scalpel. Transfer the calvariae halves to a 25-mL tube with PBS and wash
twice with PBS.
5. Incubate the calvariae in 4 mL of digestion solution at 37°C in a shaking water
bath. After 10 min shake the calvariae by hand for a few seconds.
6. Incubate for a total of 20 min, and then transfer the supernatant, containing cells,
to a 10-mL tube. Add 700 µL of fetal calf serum (FCS) to the cell suspension to
inhibit collagenase and trypsin activity.
7. Wash the calvariae with 3 mL of DMEM (without FBS!), shake well, and add the
supernatant to the tube containing the cell suspension. This is population no. 1.
8. Add new digestion solution to the calvariae, and repeat the previous three steps
to obtain population no. 2. During the 20 min that the calvariae have to incubate
in the water bath, centrifuge cell population no. 1 for 5 min at 300g Discard the
supernatant, resuspend the cell pellet in 1 mL of cCM, and add to 17 mL of cCM.
Pipet in a six-well plate at 3 mL of cell suspension per well.
9. Repeat the entire procedure for a total of four times, to obtain population
nos. 1–4.
10. The culture medium is changed 1 d after isolation of the bone cells.
11. Within approx 5 d cells will reach subconfluency, upon which they are trypsinized
by incubation with 200 µL of trypsin solution per well, at 37°C for 10 min.
12. Population nos. 1 and 2, resembling osteoblast progenitor cells, are pooled, as
well as population nos. 3 and 4. This latter pooled cell population is enriched
with cells exhibiting biochemical characteristics associated with differentiated

osteoblasts, such as high ALP activity and osteopontin expression. Both pooled
populations can be used directly for experiments (see Note 5).
13. The number of cells obtained using this method varies between 6 × 10
6
and
10 × 10
6
cells
.
see

Note 5

for an alternative method for osteoblast isolation from
neonatal calvariae.
3.4. Characterization of the Osteoblast Phenotype
by Determination of ALP Activity
Primary bone cell cultures are not 100% pure and may contain some fibro-
blasts and other nonosteoblastic cell types. It is therefore advisable to check
routinely the osteoblastic phenotype of the cultures. Because vitamin D
3
stimu-
lates the differentiation of immature bone cells, leading to enhanced ALP
activity (11), the osteoblastic phenotype of the primary mouse bone cell cul-
tures can be determined as follows:
1. Take a subconfluent cell culture in a six-well plate, wash the cells once with
PBS, and replace the medium by 3 mL of either VDM or VDCM per well. After
3 d of incubation, remove the medium and wash the cell layer with PBS.
02/Nulend/19-28/F1 2/26/03, 10:44 AM24
Bone Cells from Calvariae and Long Bones 25

2. Put the cells on ice and add 1 mL of cold milliQ water to the cells. Harvest the
cells with a cell scraper, and transfer the cell suspension to a 10-mL tube. Soni-
cate on ice for 10 min, and then centrifuge for 10 min at 500 g. Transfer the
supernatants for determination of ALP activity and total protein content.
3. Determine ALP activity by using p-nitrophenyl phosphate as a substrate at pH 10.3,
according to the method described by Lowry (12). Read the absorbance at 405 nm.
4. Measure the protein content of the homogenate using a BCA Protein Assay
Reagent Kit according to the manufacturer’s protocol. Read the absorbance at
570 nm. On average the incubation of adult mouse bone cell cultures with 1,25-
(OH)
2
D
3
will result in a twofold induction of ALP production. In neonatal mouse
calvarial cultures the induction of ALP production by 1,25-(OH)
2
D
3
is on aver-
age sixfold.
4. Notes
1. Addition of serum to the medium is necessary for the survival and stimulation of
proliferation of the primary mouse bone cells. “Serum” is not a constant and
homogeneous product, however, and the growth rate of primary bone cells can
vary considerably between several batches of serum. It is therefore recommended
to test several batches of serum on their cell proliferative ability and continue to
use the one that produces the best results.
2. Sometimes the primary bone cultures can contain fibroblasts, which grow faster
than the bone cells and can quickly overgrow the primary bone cell cultures. If
this problem occurs care should be taken to remove all soft tissues better by scrap-

ing the bones with a knife before starting the collagenase treatment. Also make
sure the collagenase is not expired, and that the collagenase solution is made
fresh every time.
3. The exact nature of the bone cells that are isolated from adult long bones and
adult calvariae has not yet been determined. Because the cell isolation protocols
involve removing the soft tissues and all adhering cells by means of incubation
with collagenase, the cells that are isolated from the bones might represent osteo-
cytes that reverted to proliferation after several days of exposure to fetal calf
serum. The appearance of the isolated bone cells, however, is mostly osteoblastic
(Fig. 2), and several osteoblast specific markers are expressed by these cells.
Absence of staining for von Willebrand factor (factor VIII) shows that the bone
cell cultures do not contain endothelial cells.
4. Smaller numbers of calvariae can be used successfully, leading to a proportion-
ately lower yield of osteoblasts.
5. We prefer the use of collagenase type II; however, other groups have
reported use of crude collagenase type IA (Sigma C9891), which is cheaper
and intrinsically contains trypsin as a contaminant. An alternative protocol
for isolation of osteoblasts from neonatal mouse calvariae, which uses
alternate collagenase and EDTA incubations to remove as much mineral-
ized matrix as possible and increase cellular yield, is given below in the
following subheadings.
02/Nulend/19-28/F1 2/26/03, 10:44 AM25
26 Bakker and Klein-Nulend
Materials
1. Stock solution of collagenase type I at 10 mg/mL in Hanks’ balanced salt solu-
tion (HBSS, Gibco). Filter sterilize and freeze aliquots for single use at –20°C.
Dilute in HBSS to 1 mg/mL just before use. HBSS contains calcium, which
increases the activity of the collagenase.
2. 4 mM EDTA in PBS without calcium and magnesium. Filter sterilize and store at 4°C.
3. Conical 15-mL centrifuge tubes (polypropylene, to reduce cell loss by minimiz-

ing adhesion to tube). Conical 25-mL centrifuge tubes for incubations. Sterile
Petri dishes for dissection (ideally glass).
4. Sterile small curved forceps and spring bow scissors.
Method
1. Dissect calvariae as described in Subheading 3.3., steps 1–3 and collect them in HBSS.
2. All incubations are carried out in 3 mL of solution (making sure calvaria are
completely covered) in a 25-mL centrifuge tube in a shaking water bath at 37°C.
Fig. 2. Phase contrast microscopy of primary mouse bone cell cultures. (A) Adult
mouse bone cells growing out of the bone chips, d 6 of culture. (B) Subconfluent layer
of adult mouse bone cells, first passage. (C) Neonatal mouse calvarial cells, popula-
tion nos. 1 and 2, d 3 of culture. (D) Neonatal mouse calvarial cells, population nos. 3
and 4, d 3 of culture. Note the cuboidal morphology of the osteoblasts.
02/Nulend/19-28/F1 2/26/03, 10:44 AM26