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Osteocalcin enhances bone remodeling around
hydroxyapatite/collagen composites
Stefan Rammelt,1 Mirjam Neumann,1 Uwe Hanisch,2 Antje Reinstorf,3 Wolfgang Pompe,3 Hans Zwipp,1
Achim Biewener1
1Department of Trauma and Reconstructive Surgery, University Hospital “Carl Gustav Carus,” Dresden, Germany
2Institute of Legal Medicine, University Hospital “Carl Gustav Carus,” Dresden, Germany
3Institute of Materials Science, University of Technology, Dresden, Germany
Received 4 September 2004; accepted 27 October 2004
Published online 30 March 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30263
Abstract: The effect of osteocalcin (OC), an extracellular
bone matrix protein, on bone healing around hydroxyapa-
tite/collagen composites was investigated. Cylindrical
nanocrystalline hydroxyapatite implants of 2.5-mm diame-
ter containing 2.5% biomimetically mineralized collagen
type I were inserted press-fit into the tibial head of adult
Wistar rats. To one implant group, 10 �g/g OC was added.
Six specimens per group were analyzed at 2, 7, 14, 28, and 56
days. After 14 days, newly formed woven bone had reached
the implant surface of the OC implants whereas a broad
fibrous interface could still be observed around controls.
Woven bone was formed directly around both implant
groups after 28 days and had been replaced partially by
lamellar bone around the OC implants only. No significant
differences in total bone contact were seen between both
groups after 56 days. The higher number of phagocytosing
cells and osteoclasts characterized immunohistochemically
with ED1, cathepsin D, and tartate-resistant alkaline phos-
phatase around the OC implants at the early stages of bone
healing suggests an earlier onset of bone remodeling. The
earlier and increased expression of bone-specific matrix pro-
teins and multifunctional adhesion proteins (osteopontin,
bone sialoprotein, CD44) at the interface around the OC
implants indicates that OC may accelerate bone formation
and regeneration. This study supports the observations from
in vitro studies that OC activates both osteoclasts and osteo-
blasts during early bone formation. © 2005 Wiley Periodi-
cals, Inc. J Biomed Mater Res 73A: 284–294, 2005
Key words: hydroxyapatite; collagen; osteocalcin; implants;
bone substitutes
INTRODUCTION
Self-setting calcium phosphate bone cements are
considered useful in the filling of large osseous defects
after trauma, infection, or tumor resection.1 The addi-
tion of type I collagen (Coll) increases the fracture
toughness, improves the pasty behavior and the in
vivo properties of such materials.2,3 The osteoconduc-
tive properties of both hydroxyapatite (HA) and Coll
have been investigated extensively and HA/Coll com-
posites have been proposed to achieve a biological
bone substitute.2,4,5 Osteocalcin (OC) is an extracellu-
lar matrix protein produced by osteoblasts which con-
stitutes 2% of the total protein content in bone. It is
distributed in cement lines of both cortical and trabec-
ular bone.6 OC is thought to have a role in the early
stages of bone healing.7 It seems to be crucial in reg-
ulating osteoblast activity and binding of HA.8,9 In
previous experiments, OC has been shown to enhance
adhesion of osteoblast-like cells (SAOS-2) on the sur-
face of HA/Coll composite materials in vitro.3 Because
many of the proposed properties of extracellular ma-
trix proteins have so far remained speculative,6,8 our
goal was to investigate the effect of OC on bone–
implant interaction and bone remodeling in vivo.
Few investigators have added components of the
organic bone matrix other than Coll to potential bone
substitutes. Johnson et al.10 used a composite alloim-
plant of human bone morphogenetic protein and au-
tolysed allogeneic bone containing a mixture of extra-
cellular matrix proteins including OC. They achieved
bony union in 24 of 25 cases of resistant nonunions in
a clinical trial. However, the addition of such potent
osteoinductive factors such as bone morphogenetic
Correspondence to: S. Rammelt, Klinik und Poliklinik fu¨r
Unfall- und Wiederherstellungschirurgie, Universita¨tsklini-
kum “Carl Gustav Carus” der TU Dresden, Fetscherstrasse
74, 01307 Dresden, Germany; e-mail: strammelt@
hotmail.com
Contract grant sponsor: Deutsche Forschungsgemein-
schaft (German Research Foundation, DFG); contract grant
number: FOR 308/2-1
© 2005 Wiley Periodicals, Inc.
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proteins did not allow any conclusions on the perfor-
mance of OC alone. The goal of the present study was
to investigate whether the addition of OC enhances
bone healing around HA/Coll composites in a rat
tibia model. To our knowledge, this is the first study to
investigate the effect of OC on bone healing around
HA/Coll implants in an animal model.
MATERIALS AND METHODS
Preparation of the implants
A commercially available calcium phosphate cement (Cal-
cibon�, BIOMET Merck Biomaterials, Darmstadt, Germany)
containing 58 wt % Ca3(PO4)2, 24 wt % CaHPO4, 8.5 wt %
CaCO3, and 8.5 wt % precipitated HA, which sets under
formation of nanocrystalline HA11 was used as starting ma-
terial. It was mixed with 2.5 wt % biomimetically mineral-
ized freeze-dried bovine type I Coll12 to obtain a fiber-
reinforced material. Ten micrograms of human recombinant
OC was added to 1 g of the HA/Coll composite3 in one
implant group (OC) whereas the group without OC served
as control (CO). Cylinders with a 2.5-mm diameter and
5-mm length were prepared for implantation and sterilized
by �-radiation with 32.7 Gy.
Animal experiments
Permission for conducting the experiments was obtained
by the local animal ethics committee. The guidelines of the
Federal State of Saxony for the care and use of laboratory
animals have been observed. Sixty male adult Wistar rats
were operated under injectional anesthesia with a combina-
tion of 0.7 mL/kg ketamine and 0.4 mL/kg medetomidine.
Before injection, a general O2/CO2 anesthesia was applied.
The animals were placed in a supine position, the hindlimbs
shaved, disinfected, and draped free. A longitudinal stab
incision was made over the anterior aspect of the tibial head.
A circular defect was made with a 2.5-mm drill (Synthes,
Bochum, Germany) centered over the tibial head 2 mm
below the knee joint. The cylindrical cement implants of
2.5-mm diameter were introduced press-fit into the defects
in the tibial head (Fig. 1). The incision was closed with a
single suture. The animals were allowed to walk free imme-
diately. For postoperative analgesia, tramadone was added
to the drinking water over the first 24 h after surgery. Six
animals of each group were killed at 2, 7, 14, 28, and 56 days
after general O2/CO2 anesthesia. The tibiae were then ex-
cised and stripped from all adherent soft tissue.
Histological workup
Specimens were fixed with 4% buffered formaldehyde for
8 h at room temperature, washed, decalcified for 4 weeks in
ethylenediaminetetraacetic acid at pH 7.4–7.6, dehydrated
overnight in a Shandon Hyper Center tissue processor
(Rankin Biomedical Corp., Clarkston, MI), embedded in
paraplast, and dried overnight. Sections of 2 �m were cut
and mounted on silane-coated slides for conventional stain-
ing techniques (hematoxylin and eosin, Goldner-Masson
trichrome) and enzyme-histochemistry [tartate-resistant
acid phosphatase (TRAP) and naphthyl acetate choline es-
terase].
After washing in phosphate-buffered saline solution (pH
7.4), the sections were dewaxed and treated with 0.3% hy-
drogen peroxide for 10 min. For immunohistochemical char-
acterization, the sections were treated with blocking agent
and incubated for 1 h at 37°C with primary antibodies. The
antibodies were detected with biotinylated secondary anti-
bodies followed by a streptavidin/biotin-peroxidase com-
plex (Dako, Glostrup, Denmark). Peroxidase activity was
visualized with 3�-3�-diaminobenzidine. For characteriza-
tion of the interface region, we used primary antibodies
against the prevalent intermediate filaments of mesenchy-
mally derived cells (vimentin), inflammatory cell receptors
(CD3), multifunctional adhesion proteins (CD44), bone-spe-
cific extracellular matrix products (osteopontin, bone sialo-
protein), macrophages (ED1, cathepsin D), osteoclasts
(TRAP), and von Willebrand factor as a marker for angio-
neogenesis. Nuclei were stained with hematoxylin. Finally,
sections were dehydrated and covered. Control procedures
without adding primary antibodies were performed.
Figure 1. Schematic drawing of the position of the cylin-
drical implants (2.5-mm diameter, 5-mm length) in the me-
taphyseal portion of the rat tibia.
OSTEOCALCIN ENHANCES BONE REMODELING 285
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Figure 2. Scanning electron microscopic images of the change in conformation of the HA/Coll cement (a) from a plate-like
crystalline structure (particle size 200–500 nm) to a finer microstructure with a finer particle population of less than 200 nm
in diameter (b). Original magnification �10,000.
Figure 3. Morphological changes in the bone/implant interface over the entire time course (hematoxylin and eosin stain)
around controls (left) and OC implants (right). Original magnification �200 (a, b), �100 (c–h), and �25 (i, j). See Results
section for details. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
286 RAMMELT ET AL.
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Statistical analysis
Cells stained with selected intracellular markers of bone
resorption and bone formation (ED1, cathepsin D, TRAP,
osteopontin) were evaluated with respect to total cell count
per low-power field (25 mm2) with a standard 10 � 10 grid
at an original magnification of �100 chosen randomly in
three subsequent histological slices per animal. The speci-
mens were blinded for the cell counts. Taken together, three
low-power fields accounted for approximately two-thirds of
the whole bone/implant interface. For evaluation of the
bone/implant contact at the final stages of the experiment,
histomorphometrical analysis was performed with Quan-
timed software (Leica, Bensheim, Germany). The whole
bone/implant interface was evaluated for direct bone con-
tact with the implant surface at an original magnification of
�25. Data were analyzed with SPSS for Windows (Chicago,
IL) software using Student t test. Normality for the data was
evaluated with the �2 test. Statistical significance of differ-
ences between the two implant groups was assumed at p �
0.05.
RESULTS
Material properties after addition of OC
The HA supplemented with 2.5 wt % type I Coll
shows a mixture of plate-like and needle-like crystal-
line microstructure with a particle size ranging from
200 to 500 nm [Fig. 2(a)]. Addition of OC after the
setting reaction resulted in a significantly finer micro-
structure with particles of less than 200 nm in size [Fig.
2(b)]. The compressive strength of the obtained mate-
rial averaged 25 MPa as assessed with an Instron
material testing device. The properties of the HA/Coll
composite with different amounts of added OC have
been described in detail previously.3
Morphological findings
On the second postoperative day, both implants
were surrounded by a broad fibrous interface with
abundant erythrocytes, neutrophils, lymphocytes, and
early granulation tissue [Fig. 3(a,b)]. Around the OC
group, a beginning fibrous organization of the inter-
face from the local lamellar bone, rich in fibroblasts
and fibroblast-like cells, could be observed [Fig. 3(b),
arrow]. Neither osteoclasts nor multinucleated giant
cells were found at the interface of either implant.
Slightly more monocytes and mononuclear macro-
phages were seen around the OC implants than
around controls.
On the seventh postoperative day, the interface
around the OC implants was rich in mononuclear cells
Figure 3. (Continued from the previous page)
OSTEOCALCIN ENHANCES BONE REMODELING 287
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[Fig. 3(d)], whereas around the controls the interface
was more fibrous with predominately fibroblasts and
few neutrophils [Fig. 3(c)]. Small, newly formed os-
teoid seams were observed around both implants [Fig.
3(c,d), arrows]. More osteoclasts and osteoblasts were
seen directly at the interface of OC implants. Small
capillaries were seen in the interface around both im-
plants.
Two weeks after implantation, the interface around
both implant groups was smaller and abundant in
newly formed Coll fibers [Fig. 3(e,f)]. Newly formed
osteoid was seen directly at the implant surface in
both groups, the contact area being greater in the OC
group. Around the control implants, numerous mac-
rophages were detected within the fibrous interface
[Fig. 3(e)]. Around the OC implants, osteoid reached
the surface on several spots [Fig. 3(f)]. Macrophages
and osteoclasts were seen on the opposite side of the
osteoid seams but not in the interface, indicating a
remodeling of the newly formed bone. Osteoblasts
were observed directly at the surface of the OC im-
plants only, whereas around the control implants a
greater number of osteoclasts were seen [see Statistical
Analysis (Cell Counts) for details].
At four weeks, bone healing displayed different
patterns around the two implants. Around the control
implants, newly formed woven bone emanated from
the cancellous bone of the metaphysis toward the
implantation site [Fig. 3(g)]. The remaining interface
was rich in fibrocytes and mononuclear cells. In con-
trast, around OC implants, woven bone was already
seen directly at the implant surface and transformed
partly into lamellar bone. Whereas some osteoclasts
were still present at the interface of the control im-
plants, almost no osteoclasts were observed around
OC implants. At this stage, more osteoblasts were
detected around the control implants, than around OC
implants. Inflammatory cells such as granulocytes or
lymphocytes were not observed around either im-
plant.
At the end point of the experiment, 56 days after
implantation, the woven bone had been replaced by
lamellar bone around both implants, resembling sec-
ondary callus formation [Fig. 3(i,j)]. The remaining
interface was wider and richer in fibroblasts around
the control implants [Fig. 3(i)]. Few osteoblasts and
almost no osteoclasts were seen around both types of
implants.
Histomorphometrical findings
Fifty-six days after implantation, a considerable
portion of the implant surface was in direct contact
with the newly formed bone whereas the remaining
implant surface was covered by a fibrous interface.
The extent of newly formed bone varied considerably
within each tested group. On histomorphometrical
analysis, the direct bone/implant contact averaged
40.2% (range 21–56%, SD 16.6) around the OC im-
plants and 49.2% (range 28–67%, SD 16.8) around the
control implants. With the numbers available, these
differences were not statistically significant (p �
0.475).
Histochemical and immunohistochemical findings
Inflammatory cells, predominately neutrophils
staining positive for naphthyl acetate choline esterase,
were observed abundantly around both implant types
at day 2. These cells decreased more rapidly around
the OC implants than in the controls, where they
could be observed until day 14 after implantation.
For all immunohistochemical staining procedures,
negative controls without primary antibodies dis-
played no reaction. Fibroblast-like cells and mesen-
chymal stromal cells, staining positive for vimentin,
were seen abundantly around both implants from day
2 to 14 and then decreased gradually with a more
fibrous organization of the remaining interface. The
latter was smaller around the OC implants. At day 56
after implantation, no mesenchymal cells were seen
around both implants. Immunohistochemical staining
for CD44, indicating interaction of fibroblasts and os-
teoblasts, was observed as a small seam around the
implants at day 2 and increased until day 14, more
markedly around the OC implants [Fig. 4(a,b)]. Reac-
tions against CD44 decreased gradually after 2 weeks
and were negative at the interface of both implants at
8 weeks.
Mononuclear cells of the monocyte-macrophage lin-
eage as specified with cathepsin D and ED1 displayed
a fairly different pattern in the two study groups [Fig.
4(c,d)]. Around the OC implants, a considerable num-
ber of ED1- and cathepsin D-positive cells appeared
directly at the implant surface at day 2. Both cathepsin
D- and ED1-positive cells were abundant at day 7 and
decreased thereafter until both reactions were barely
discernible at day 56 after surgery. In contrast, few
cells stained for cathepsin D and no ED1-positive cells
were seen in the interface around the control implants
at day 2. Immunohistochemical reaction against ED1
and cathepsin D was increased on days 7 and 14 and
decreased thereafter.
Osteoclasts, as characterized morphologically and
with enzymatic staining against TRAP, were detected
at the interface of both implants from the seventh
postoperative day onward. The osteoclasts were
higher in number and closer to the implant in the OC
group at that time [Fig. 5(a,b)]. In this group, the
reactivity against TRAP decreased markedly thereaf-
288 RAMMELT ET AL.
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