Calcif Tissue Int (2010) 86:463469

DOI 10.1007/s00223-010-9362-3

The Role of Circulating Bone Cell Precursors in Fracture Healing

Patrizia DAmelio Maria Angela Cristofaro
Anastasia Grimaldi Marco Ravazzoli Fernanda Pluviano
Elena Grosso Gian Piero Pescarmona Giovanni Carlo Isaia

Received: 15 January 2010 / Accepted: 24 March 2010 / Published online: 14 April 2010
Springer Science+Business Media, LLC 2010

Abstract Fracture healing is a complex process that

involves several cell types; as a previous report suggested
an increase in osteoblast (OB) precursors in peripheral
blood during this process, this paper examines the role of
circulating bone cell precursors in this process in the light
of a prior suggestion that OB precursors are increased.
Nine healthy men less than 60 years old with traumatic
fractures were enrolled. The parameters circulating OB
precursors (osteocalcin?/alkaline phosphatase?/CD15cells) and osteoclast precursors (CD14?/CD11b?/vitronectin receptor ? cells) were measured by flow cytometry;
bone formation markers and TGFb1, by ELISA; and PTH,
by RIA in serum on arrival at the emergency department

The authors have stated that they have no conflict of interest.

P. DAmelio (&)
M. A. Cristofaro
A. Grimaldi

M. Ravazzoli
G. C. Isaia
Section of Gerontology, Department of Surgical and Medical
Disciplines, University of Torino, Corso Bramante 88/90,
10126 Torino, Italy
e-mail: [email protected]
F. Pluviano
Department of Imaging, Faculty of Medicine,
University of Torino, Torino, Italy
E. Grosso
Department of Orthopaedics, Traumatology,
CTO-CRF-ICORMA Hospitals, Torino, Italy
G. P. Pescarmona
Center for Experimental Research and Medical Studies
(CERMS), San Giovanni Battista Hospital, Torino, Italy
G. P. Pescarmona
Department of Genetics, Biology and Biochemistry,
University of Torino, Torino, Italy

(baseline) and 15 days after fracture. Bone cell precursors

behaved differently during healing. TGFb1 was inversely
correlated with OB number, but increased their degree of
maturation at baseline. Bone formation markers and
TGFb1 were increased after fracture, whereas PTH was
decreased. The TGFb1 increase was directly correlated
with age, whereas age was not correlated with the precursors. In conclusion, we confirm the role of TGFb1 in
fracture healing; and its possible role in the control of preOB homeostasis. There was no variation in circulating
precursor cells during healing, though the increase in
TGFb1 may suggest increased pre-OB maturation and
homing to the injured site.
Keywords Osteoblast precursors
Osteoclast precursors

Peripheral blood
TGFb
Fracture healing

Osteoclast lineage cells are present in the peripheral circulation and increased in bone lytic diseases (reviewed in
Ref. [1]). They are decreased by drugs active on bone
turnover such as bisphosphonates [2, 3]. Whether a comparable pool of circulating osteoblast (OB) lineage cells is
present and has a role in bone metabolism is uncertain [4].
It has been suggested that osteogenetic cells are present at
extremely low concentrations in the human peripheral
blood, express typical OB markers such as alkaline phosphatase (AP) and osteocalcin (OC), and form mineralized
nodules in vitro and bone tissue in vivo in nude mice [5,
6]. Their physiological role during skeletal growth and
fracture repair has also been proposed. Eghbali-Fatourechi
et al. [5] observed that cells positive for OC, but not for
AP, were over five times more numerous in the circulation
of adolescent boys compared to adult men, and that they
increased after fracture. A reduction of the circulating OB

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precursors pool has been described in postmenopausal

osteoporosis [7]; however, the physiological role of these
cells in bone metabolism remains to be clarified.
Fracture healing is still viewed as an unresolved cascade
of complex biological events involving intracellular and
extracellular molecular signaling for bone induction and
conduction [8]. Molecular mechanisms that regulate skeletal tissue formation in utero are repeated during this
process [9]. Many local and systemic regulatory factors,
including growth and differentiation factors, hormones,
cytokines, and extracellular matrix, interact with several
cell types, including bone- and cartilage-forming primary
cells recruited to the fracture site from the bone marrow
[10, 11] or the circulation [12]. One of the most studied
systemic factors is transforming growth factor b1 (TGFb1),
which is a potent chemotactic stimulator of mesenchymal
stem cells that enhances the proliferation of OB precursors
and chondrocytes, and may act in recruitment of bone cells
in the fractured bone [13]. It induces the production of
extracellular bone matrix proteins such as collagen, proteoglycans, osteopontin, osteonectin, and alkaline phosphatase (AP) [14] and regulates different cell types directly
involved in bone remodeling and fracture healing [1517].
A growing body of evidence indicates that intermittent
treatment with parathyroid hormone (PTH; 134) may
promote fracture healing [18] and suggests that endogenous
PTH may be equally involved.
This paper assesses the role of circulating bone precursor cells in fracture healing in relation to TGFb1 and PTH
in nine males.

Methods
Patients and Bone Turnover Markers
The study was approved by the Clinical Study Review
Committee of the San Giovanni Battista Hospital, Torino,
and all patients signed an informed consent statement prior
to recruitment. Nine healthy men aged 15 to 59 (mean, 33;
SD, 14) with a traumatic fracture (see Table 1) were
enrolled. None were on calcium and vitamin D, thyroid
hormones, corticosteroids, bisphosphonates, or PTH. The
presence of diseases that influence bone metabolism was
ruled out on the basis of the medical history and physical
examination. Forty milliliters of peripheral blood was
obtained upon arrival at the emergency department (baseline) and 15 days after the fracture.
Cell Isolation
Peripheral blood mononuclear cells (PBMCs) were
obtained with the FicolPaque method from 30 ml of blood

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P. DAmelio et al.: The Role of Circulating Bone Cell Precursors

Table 1 Types of fractures
Patient

Type of fracture

FG

Lumbar vertebra (L1) and ileopubic left branch

AC

Right femur diaphysis

LG

Tibial and peroneal diaphysis

FF

Left radius

AN

Left radius

GR

Tibial diaphysis and heel

PP

Tibial diaphysis

PP1

Ileopubic branch left and right

DS

Left femur diaphysis

in lithium heparin, as previously described [19]. The serum

was frozen at -80C until the measurements were done.
Flow Cytometry
To evaluate the role of circulating osteoclast and OB precursors in fracture healing, we measured them in the
PBMCs at baseline and 15 days after fracture. Briefly,
osteoclast precursors were evaluated by staining PBMCs
with fluorescein (FITC; supplied by B&D)-conjugated antiVNR, phycoerythrin (PE; supplied by B&D)-conjugated
anti-CD14, and allophycocyanin (APC; supplied by B&D)conjugated anti-CD11b mAb, or with the corresponding
isotype control, followed by incubation at 4C for 30 min
as previously described [2, 3, 20]. CD14?/CD11b?/
VNR- cells were regarded as early osteoclast precursors
[2, 3, 21, 22], and triple-positive (CD14?/CD11b?/
VNR?) cells as osteoclast precursors, according to the
literature [2, 3, 20, 2328].
OB precursors were evaluated by staining PBMCs with
FITC-conjugated anti-CD15 (to exclude granulocytes that
expressed AP; supplied by e-Bioscience), APC-conjugated
anti-AP (supplied by R&D Systems Inc.), PE-conjugated antiOC (supplied by R&D Systems Inc.), or the corresponding
isotype control, followed by incubation at 4C for 30 min as
previously described [5]. CD15/AP?/OC? cells were
regarded as OB precursors according to the literature [5].
Membrane antigen expression was analyzed with the
CellQuest software (Becton Dickinson & Co.) and displayed as bivariate dot plots or histograms. Each plot
depicts the results from 10,000 events representing viable
cells gated by cell size and granularity.
Cytokine Measurement
ELISA kits were used to measure bone AP (BAP; Instant
ELISA; Bender), OC (Instant ELISA; Bender) and TGF b1
(DuoSet; R&D Systems Inc.). PTH was measured with a
radioimmunologic assay (RIA-IRMA-Pantec).

P. DAmelio et al.: The Role of Circulating Bone Cell Precursors

465

Statistics
The normal distribution of each parameter was determined
with Kurtosis, since none were normally distributed; the
WilcoxonMannWhitney test was used to compare values
at baseline and 15 days after the fracture. Correlations
between parameters were performed by means of Spearmans coefficient or with partial correlation after correction
for age. The SPSS 17.0 software package was used to
process the data, with P \ 0.05 as the significance cutoff.

Results
Circulating OB and Osteoclast Precursors Do Not Vary
During Fracture Healing
Despite the presence of a clinically substantial callus, no
variation was observed in the percentage of circulating OB
and osteoclast precursors (Fig. 1a and b) or in their degree
of maturation as evaluated by the mean fluorescence
intensity (MIF) of the molecules studied (data not shown).
There was a high individual variability in the bone

precursor cells, especially in circulating pre-OB: OC?

cells increased in five patients, and OC?/AP? cells in
three (Fig. 2).
There were no evident clinical differences between these
patients and those who did not display such increases. By
contrast, bone formation markers increased in all nine
patients during fracture healing (Fig. 3a and b). There were
no correlations between these markers and OB precursors.
TGFb1 Increases During Fracture Healing
Since TGFb1 plays an important role in the local recruitment of osteogenic cells during fracture healing [13], we
measured it in serum and found that it increases after
fracture (Fig. 3c). As age could influence TGFb levels [29,
30], we correlated this parameter with OB precursors after
correction for age; TGFb was inversely correlated with OB
precursors (R = -0.77, P = 0.025) and directly correlated
with the OC MIF (R = 0.86, P = 0.027): these correlations disappeared during healing. These data suggest that
systemic TGFb1 may influence the degree of maturation of
OB precursors under physiological conditions, while the
increase in TGFb1 after fracture may influence the

A-OSTEOBLAST PRECURSORS
BASAL

AFTER FRACTURE
20

10

10

103

103

102
101

% of positive cells

OC-PE

OC-PE

102
101

0
1010
0

101

103
102
AP-APC

100100

104

101

102
AP-APC

103

10

104

BASAL

AFTER FRACTURE

10
VNR-PE

VNR-PE

10

30

% of positive cells

104

102
101
100

102
101
100

100

101

103
102
CD11b-APC

104

AP+/OC+

Basal
After fracture

B-OSTEOCLAST PRECURSORS
104

OC+

100

101

Fig. 1 Bone cell precursors during fracture healing: FACS analysis

of circulating osteoblast and osteoclast precursors from PBMCs of
patients at baseline and 15 days after fracture. a Dot plots represent
OC?/AP?/CD15- cells gated on monocytes (OB precursors). The
graph represents the percentage of OC? cells and AP?/OC? cells at
baseline and 15 days after fracture. Bars show the mean and SE for all
patients, the differences are not significant according to the

103
102
CD11b-APC

104

20

10

Early precursors
(CD14+/CD11b+)

Osteoclast precursors
(CD14+/CD11b+/VNR+)

WilcoxonMannWhitneys test. b Dot plots represent VNR? and

CD11b? cells gated on CD14? cells (osteoclast precursors). The
graph represents the percentage of CD14?/CD11b?/VNR- cells
(early osteoclast precursors) and the percentage of CD14?/CD11b?/
VNR? cells (osteoclast precursors) at baseline and 15 days after
fracture. Bars show the mean and SE for all patients; differences are
not significant according to the WilcoxonMannWhitney test

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466
40

AP+/OC+ cells (%)

7.5

OC+ cells (%)

Fig. 2 OB precursors at
baseline and after fracture.
Graphs show the percentage of
OC? cells (left) and of AP?/
OC? cells (right) for each
patient at baseline and 15 days
after fracture. Patients initials
are indicated

P. DAmelio et al.: The Role of Circulating Bone Cell Precursors

30

20

10

Basal

To assess the role of endogenous PTH in fracture healing,

we measured its level at baseline and after fracture: PTH
values were always within the physiological range (10
65 pg/ml) and decreased, although not significantly, during
healing (Fig. 3d). PTH is inversely correlated with the
degree of maturation of OB precursors at baseline, whereas
this correlation disappears during healing (Fig. 4).
None of the measured parameters correlates with
osteoclast precursors (data not shown).

Basal

After fracture

Discussion
Fracture healing is a complex process that involves a
variety of cells, such as hematopoietic cells, platelets,
immune system cells, fibroblasts, and bone cells. On the
strength of a recent suggestion [5] that circulating OB
precursors are involved in fracture healing, we evaluated
both OB and osteoclast precursors in the peripheral blood
with respect to bone formation markers, TGFb1, and PTH
at baseline and 15 days after a traumatic fracture. The
period of 15 days was chosen because of its clinical

Age Affects TGFb1 and BAP During Fracture Healing

Since fracture healing slows with age and gene expression
in callus varies accordingly [29], we evaluated the

B 15

15

p=0.007

BAP (IU/L)

Osteocalcin (ng/mL)

2.5

relationship among age, bone cell precursors, and variation

in bone formation markers and TGFb1 and PTH levels.
Age was not significantly correlated with bone cell precursors (data not shown). Figure 5 shows that age is
directly correlated with the increase in BAP and TGFb1
after fracture but not with the decrease in PTH.

PTH Decreases During Fracture Healing

5.0

After fracture

recruitment of osteogenic cells from the circulation into the

fracture site by enhancing their homing ability.

Fig. 3 Effects of fracture

healing on bone formation
markers, TGFb1 and PTH. a
Serum OC, b serum BAP, c
serum TGFb1, and d serum
PTH at baseline and 15 days
after fracture. Bars show the
mean and SE for all patients.
Significance p-values calculated
with the WilcoxonMann
Whitney test are indicated

DS
PP1
LG
GF
AN
PP
FF
GR
AC

10

Baseline

10

After fracture

Baseline

After fracture

Baseline

After fracture

p=0.021

75

1000

123

D
PTH (pg/mL)

TGF 1 (pg/mL)

C 2000

Baseline

After fracture

50
25
0

P. DAmelio et al.: The Role of Circulating Bone Cell Precursors

467

Basal

After fracture

R =-0.50, p=0.023

500

150

MIF OC

MIF OC

250
0
25

50

75

100

100

50

PTH

-250

-500

10

20

30

40

50

60

70

80

PTH
Fig. 4 Correlations between PTH and degree of OB precursor
maturation at baseline and after fracture. Linear correlation between
PTH and mean fluorescence intensity (MIF) of OC at baseline (left)
and 15 days after fracture (right). Dotted lines represent the

500

R=0.73, p=0.026

BAP fold change to

baseline(%)

TGF 1 fold change to

baseline(%)

250

-250
10

20

30

40

50

60

70

Age (years)

500

R=0.79, p=0.006
250

-250
0

PTH fold change to

baseline(%)

Fig. 5 Correlations between

the percentage-fold changes in
TGFb1, BAP, and PTH between
baseline and 15 days after
fracture and age. Linear
correlation between the
percentage-fold change in a
TGFb1, b BAP, and c PTH
between baseline and 15 days
after fracture. Dotted lines
represent the confidence
intervals. Significant R- and pvalues calculated by
Spearmans test are indicated

confidence intervals. R- and p-values calculated by Spearmans test

are indicated. The MIF indicates the degree of maturation toward
osteoblasts of OB precursor cells

10

20

30

40

50

60

70

Age (years)

500
400
300
200
100
0
-100
0

10

20

30

40

50

60

70

Age (years)

significance in the evaluation of callus formation and, also,

because of the previous observation of an increase in OB
precursors 2 weeks after fracture [5].
As expected, there was an increase in bone formation
markers. There was no significant variation in the number
of circulating bone cell precursors, nor was there any
correlation between the bone formation markers and OB
precursors. Eghbali-Fatourechi et al. [5], however,
observed a striking increase in the percentage of OB precursors 2 weeks after fracture in three healthy men but,
these cells may either decrease or increase after fracture.
We conclude that there are evidently significant betweensubject differences in the extent to which the number of OB
precursors is influenced by fracture.
The reduction in PTH level, although not significant,
and its correlation with the degree of OB precursor

maturation suggest that endogenous PTH is involved in the

physiological control of fracture healing and in OB precursor maturation.
TFGb1 levels increased after fracture. Platelets have been
shown to release this factor during the initial inflammatory
phase of bone healing [31, 32], and it may thus be involved in
initiating callus formation. Recent studies have suggested
that it may act with IGF-1 released by bone implants in
stimulating callus formation [33]. Here we demonstrate an
increase in TGFb1 15 days after fracture, corroborating its
role in human fracture healing. We also found a strong
inverse correlation between TGFb1 and OB precursors at
baseline, whereas TGFb1 is positively correlated with the
degree of maturation of OB precursors (OC MIF). This
positive correlation may account for the increased homing
ability of circulating precursors [7]. The homing ability of

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circulating osteogenic cells during fracture healing was

elegantly demonstrated by Kumagai et al. in a parabiotic
mice model [12]. They showed that these cells are physiologically mobilized to contribute to callus formation and
fracture repair. Enahanced homing ability may account for
the absence of a universal increase in our patients.
It has been shown (mainly in animal models) that the time
needed for bone formation to bridge a fracture gap increases
with age [34]. The reason for this slowing of fracture healing
is not fully understood. Up-regulation of genes related to
fracture healing has recently been shown in the callus of
older animals. Meyer et al. [29, 30] reported up-regulation of
TGFb in the callus of older rats. This can be seen as an
attempt by the tissue to accelerate the healing process. Our
data confirm these findings, as in older patients we observe a
greater increase in TGFb1 and BAP after fracture, whereas
age is not correlated with bone cell precursors. TGFb1 is
directly correlated with the degree of OB precursor maturation even after correction for age. Its enhancement of OB
precursors homing ability is presumably not age dependent.
In conclusion, although the cohort observed was small,
we suggest PTH to be a possible player in the fracture
healing process and confirm the role of TGFb1 in the
recruitment of osteogenic cells to the fracture site and its
increase as a function of age. On the contrary, we found no
significant variation in the number of circulating bone cell
precursors after fracture.
Further studies are needed to clarify the complex process of fracture healing and the role of osteogenic cell
recruitment from the circulation.
Acknowledgments This work was supported by a grant from the
Fondazione Internazionale Ricerche Medicina Sperimentale (FIRMS)
Compagnia San Paolo. P. DAmelio was supported by a fellowship
from the Regione Piemonte.

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