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Effects of platelet factors on biodegradation and osteogenesis inmetaphyseal defects filled with nanoparticular hydroxyapatite—anexperimental study in minipigs
OLAF KILIAN1, SABINE WENISCH1, VOLKER ALT1, MARKUS LAUER1,
ROSEMARIE FUHRMANN2, ELVIRA DINGELDEIN3, TARJA JONULEIT4,
REINHARD SCHNETTLER1, & RALF-PETER FRANKE2
1Department of Trauma Surgery, University of Giessen, Germany, 2Department for Biomaterials, University of Ulm, Germany,3Osartis GmbH, Obernburg, Germany, and 4CellMed AG, Alzenau, Germany
(Received 26 July 2007; revised 18 September 2007; accepted 18 September 2007)
AbstractThere are no studies on the cellular activity in the early phase of biodegradation and bone healing of bone substitutes loadedwith platelet factors (PLF). The purpose of this study was to evaluate the cellular effects of PLF in combination withnanoparticulate hydroxyapatite (HA) on the biodegradation and bone formation after 20 days. Autogenous PLFs wereobtained by centrifugation of miniature pig blood samples and subsequent degranulation of platelets by calcium andthrombin. A cylindrical bone defect with a diameter of 8.9 mm was created in the distal femoral condyle of 20 miniature pigs.Four of the defects were left empty, 8 were filled with HA with loading and 8 with HA loaded with PLF. The distal femur washarvested after 20 days and TRAP-staining, cathepsin-K and CD44 staining and scanning electron microscopy wereperformed for cellular assessment of biodegradation was done. Histomorphometry of new bone formation and ofbiodegradation of HA material was performed. PLF loading of HA led to statistically significant more TRAP-positive cellswith enhanced biodegradation of the nanoparticulate HA but no statistically enhanced new bone formation compared tounloaded HA. Furthermore, there was a higher number of CD44 and cathepsin-K positive cells by PLF-loading. In summary,PLF led to stimulation of the cellular process of the biodegradation of HA.
Keywords: Platelet factors, hydroxyapatite, animal model, cell activation, biodegradation
Introduction
When bone regeneration is impaired in congenital or
acquired bone defects, implantation of autogenous
spongiosa or bone replacement materials may become
mandatory. Increasingly, proteins such as growth
factors or constituents of the extracellular matrix are
added to bone replacement materials in an attempt to
improve the biological properties.
Based on research findings on calcium- and
thrombin-dependent platelet aggregation (aggluti-
nation), degranulation, and release of growth factors,
e.g. platelet-derived growth factor (PDGF) and
transforming growth factor (TGF), platelet rich
plasma is topically applied to improve healing of
bone defects. In the literature, the effects of platelet
factors (PLF) on de novo bone formation remain
controversial (Marx et al. 1998; Kassolis et al. 2000;
Shanaman et al. 2001; Fennis 2002; Furst et al. 2003;
Kovacs et al. 2003; Zhang et al. 2003; Roldan et al.
2004; Wiltfang 2004; Yazawa et al. 2004).
Studies on the cellular mechanisms of bone defect
healing and on the relationship between signal and
regulator proteins and cell-to-matrix and cell-to-cell
interactions are fundamental for understanding
osteointegration of biomaterials.
The initial phases of fracture healing are thought
to parallel those of wound healing and are characterized
ISSN 0897-7194 print/ISSN 1029-2292 online q 2007 Informa UK Ltd.
DOI: 10.1080/08977190701687585
Correspondence: O. Kilian, Department of Trauma Surgery, University of Giessen, Rudolf Buchheim Street 7, 35392 Giessen, Germany.Tel: 49 641 9944601. Fax: 49 641 9944609. E-mail: [email protected]
Growth Factors, June 2007; 25(3): 191–201
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by inflammation, matrix protein expression, resorp-
tion, and angiogenesis. Reparative and regenerative
processes occurring during the subsequent phases are
tied to specific cells, e.g. osteoblasts and osteoclasts.
Signal proteins such as cytokines and growth factors
control and regulate cell function and cellular
metabolism through the phases of wound and fracture
healing (Einhorn et al. 1995; Olmedo et al. 1999).
Inflammation and vascular injury lead to extravasa-
tion of blood components and platelets adhere to
injured vessel walls. Upon clotting, platelet alpha-
granules were shown to release proteins including
growth factors such as PDGF and TGF-b. These
proteins can initiate intracellular signal cascades
(Bolander 1992).
a-Granules also store cytokine IL-1 b, IL-6, and IL-
11, as well as other inflammatory mediators. These are
released with platelet degranulation and have an
impact on the severity of inflammation.
The fibrin plug formed during the initial phases of the
coagulation cascade is rich in released signal proteins
and represents a transitional extracellular matrix for
cellular chemotaxis and for the migrationof cells into the
wound or fracture zone. Cells invading the fibrin mesh
are acted upon by these growth factors and cytokines.
In addition to hematopoetic cells, the initial
inflammatory phase targets, e.g. monocytes/macro-
phages, fibroblasts and myofibroblasts. Cell migration
into the fibrin mesh was shown to be induced by
chemotactic stimuli due to platelet growth factors, to
fragments of the extracellular matrix, and by specific
proteins such as the monocyte chemoattractant
protein-1 (MCP-1) (Carnevale and Cathcart 2003).
The cells of the phagocytic system were shown to
play a fundamental role in the inflammatory phase.
Activation of mononuclear monocytes/macrophages
leads to resorption of necrotic tissue and foreign
particles.
Mononuclear macrophages can fuse and turn into
multinucleated macrophages. These cells can attach
to foreign particles and to necrotizing tissue and
phagocytize these substances. Monocyte/macrophage
activation is associated with expression of, e.g. colony-
stimulating factor 1 (CSF-1), tumor necrosis factor a
(TNF-a), and PDGF. In turn, these factors can
activate additional monocytes/macrophages (Singer
and Clark 1999).
The inflammatory cascade can also initiate prolifer-
ation and differentiation of endothelial cells. This was
shown to be mediated by monocytes/macrophages,
platelets, mast cells and leukocytes when angiogenetic
factors such as vascular endothelial growth factor
(VEGF), angiopoetin-1, basic fibroblast growth factor
(bFGF), TGF-b1, PDGF, TNF-a, and insulin-like
growth factor-1 (IGF-1) were excreted (Carmeliet
and Jain 2000).
Growth factors can regulate cellular activities by
binding to and interacting with specific transmembrane
receptors known as tyrosine kinases, which initiate
various intracellular signal cascades.
Implantation of a platelet-enriched biomaterial can
be associated with platelet growth factors acting upon
the inflammatory phase. It is hypothesized that
stimulation of intracellular cascades and synthesis of
pro-inflammatory factors is continued into the
granulation phase, which is important for defect
healing. During the granulation phase, early minerali-
zation can be expected to start on approximately the
20th day post biomaterial implantation.
The purpose of the current study was to analyze the
effect of PLF on: (1) biodegradation of a nanoparti-
cular hydroxyapatite (HA) paste implanted into a
critical-size defect of the distal femur in minipigs, and
(2) on the osteogenic processes 20 days after
implantation.
Material and methods
Study design
The study protocol was approved by the Ethics
committee of the University of Giessen. Twenty nine-
months old, male Lewe miniature pigs (SBMF
Laboratories, Dresden, Germany) with a body weight
between 46 and 56 kg were used for the current
randomized study in which a critical size defect was
created in the femur condyle. The miniature pigs were
divided into three groups. The defects in pigs of
group 1 (n ¼ 8) were filled with pure hydroxyapatite
Ostimw 25% (HA/PLF(0)). The animals of group 2
(n ¼ 8) received a composite of hydroxyapatite,
enriched with PLF (HA/PLF(þ)) in a 10:1 ratio of
HA to PLF (1.8 ml Ostimw 33% þ 0.3 ml platelet
factor). The defects in pigs of group 3 (n ¼ 4) were not
filled. Bone biopsy of the respective contralateral
subchondral region of the femur condyle was used as
negative control for immunohistochemical staining.
Hydroxyapatite matrix
The HA used in the present study is a fully synthetic
injectable nanocrystalline paste Ostimw (Coripharm,
Obernburg, Germany) and consists of a suspension of
pure hydroxyapatite in water prepared by a wet
chemical reaction. The needle shaped HA crystals
with a size of 21 nm in a-direction and of 36 nm in
c-direction form agglomerates (Tadic and Epple
2004). Phase purity of the HA was determined by
X-ray diffraction which showed conformity with pure
HA and an average crystallite size of 18 nm. The
atomic ratio of calcium:phosphorus is 1.67. Ostimw
paste does not harden after application into the bone
and is free of endothermal heating in contrast to
calcium phosphate bone cements (Tadic et al. 2002).
O. Kilian et al.192
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Isolation of platelet factors
About 150 ml of peripheral blood of the external vena
jugularis was centrifuged at 100g for 10 min at room
temperature. Platelet rich plasma was separated and
again centrifuged at 300g for 10 min, followed by
withdrawal of the plasma and another centrifugation
step at 1000g for 30 min. The platelet-rich plasma
supernatant was carefully transferred into a pre-
connected bag for further preparation of the growth
factors. About 1000 IE thrombin (Gentrac Inc.,
Middleton, USA) and 10 ml of 8.4% calcium-
gluconate (Braun, Melsungen, Germany) were
added for aggregation and degranulation of the
platelets. After degranulation, the liquid supernatant
was filled into tubes and stored at 2208C. Plasma was
heat-inactivated at 658C for 30 min and again
centrifuged at 1000g for 30 min. The liquid super-
natant was then filled into tubes and stored at 2208C.
Surgical procedure
A cylindrical defect with a diameter of 8.9 mm and a
depth of 10 mm was created in the subchondral region
of the right femur condyle in 20 miniature pigs using a
saline cooled diamond bone-cutting system (Merck,
Darmstadt, Germany). Filling of the defects was done
according to the study design. Post-operative weight-
bearing was not limited. After 20 days the animals
were sacrificed and the distal femurs were harvested.
Immunohistochemical staining
Demineralized samples were paraffin-embedded and
5mm slides were performed with a rotator microtome
(Leica, Bensheim, Germany). The samples were
incubated overnight with primary goat-anti-Kathepsin
K antibody (Dako, Glostrup, Denmark, dilution:
1:50) and rat-anti-CD44 antibody (Serotec, Oxford,
UK; dilution: 1:50) at 48C. The secondary biotiny-
lated anti- goat and anti- rat antibodies (Dako,
Glostrup, Denmark, dilution 1:50) in 3% FCS and
12% pig serum were applied for 30 min at room
temperature. Rinsing with PBS was followed by
marking with the ABC complex/HRP (Dako,
Glostrup, Denmark) for another 30 min. The chro-
mogen Nova Red (Vector Laboratories, Burlingame,
USA) was used for visualization. Counterstaining was
done by hematoxylin (Shandon Scientific Ltd,
Cheshire, UK).
Transmission electron microscopy
Bone samples of the implants were removed and fixed
by immersion in Yellow-Fix (4% paraformaldehyde,
2% glutaraldehyde, 0.04% picric acid). After rinsing
(3 £ 5 min) in 0.1 M phosphate buffer (pH 7.2)
the specimens were postfixed for 2 h in 1% osmium
tetroxide (OsO4), washed carefully and repeatedly in
0.1 M phosphate buffer (pH 7.2), and dehydrated in
series of graded ethanol. Subsequently, the samples
were embedded in Epon (Serva, Heidelberg,
Germany). Polymerization was performed at 608C
for 20 h. Thin sections were cut with a diamond knife
(458, Diatome, Switzerland) on an Ultracut (Reichert-
Jung, Germany). Semithin sections (1mm) were
stained with Richardson (1% methylene blue, 1%
borax, 1% azure II). Ultrathin sections (80 nm) were
counterstained with uranyl acetate and lead citrate
(Reichert Ultrostainer, Leica, Germany) and exam-
ined in a Zeiss EM 109 transmission electron
microscope.
Enzymehistochemical detection of tartrate-resistant acidic
phosphatase
Activity of tartrate-resistant acid phosphatase
(TRAP) was investigated by incubation of the slices
in a solution of naphtol AS-BI phosphate (Sigma
Chemical, Germany) and fast red violet LB salt
(Sigma Chemical, Germany) in 0.2 M acetate buffer
(pH 5.0) containing 50 mM (þ) tartaric acid for
20 min at 378C. Then the slices were counterstained
with hematoxylin. Detection of alkaline phospatase
was performed by means of a “ready for use
substrate” (KPL 2 Cessna Count, Gaitherburg,
Maryland; USA) composed of NBT (nitroblue-
tetrazolium) and BCIP (5-bromine-4-chlorine-3
indolyle-phosphate). The slices were incubated in
this solution for 2 h at 378C.
Computer assisted quantification of TRAP positive regions
In TRAP stained paraffin sections, mono and multi-
nucleated macrophages were visualized with a Stemi
SV 11 (Carl Zeiss, Jena) stereo microscope. Images of
diagnostically relevant regions of each specimen were
acquired with a Kappa DX 30 (Kappa, Gleichen,
Germany) camera and segmentally analyzed. For
quantitation, the image processing software Image Pro
Plus version 4.5 (Medica Cybernetics Inc., Silver
Spring, USA) was utilized.
In granulation tissue, there was an annular
arrangement of TRAP positive cells around the bone
defects. By using the quantification software, an area
of interest (AOI) encompassing the complete defect
circumference was defined for each slide (12 AOI per
section). Pixels were automatically quantitated. The
ratio of the “positive” region to the total AOI was
expressed as a percentage.
For the statistical analysis of the ratios of TRAP
positive areas in the 12 AOI within each experimental
group (n ¼ 8), the Wilcoxon test was used.
Effects of platelet factors 193
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Quantitating de novo woven bone formation
The ratio of toluidine positive pixels was assessed in
nine thin ground sections per animal within each
experimental group (n ¼ 8) using the Optima Version
6.5 (MediaCybernetics) image processing software.
Automatic area segmentation was initiated with a
preset threshold. Pixel counts for each resultant area
were exported to Microsoft Exel. In a subsequent step,
areas containing HA particles were marked by
deep blue staining, pixel counts calculated and the
data exported to Excel.
For analysis, the difference between “total area pixel
count” and “total pixel count in the marked areas” was
calculated with an Excel macro. The subsequently
calculated ratio of this difference and the total number
of pixels of the CCD camera chip represents the
fraction of “bone formed de novo” within the total
area. The two-tailed t-test was used for statistical
analysis.
Results
Semithin section
Nanoparticular hydroxyapatite bulk is degraded by
mononuclear macrophages and multinucleated giant
cells at 20 days after implantation. After phagocytosis,
these cells separate from the surface of the HA
agglomerates and migrate through the stroma
(Figure 1).
Tartrate resistant acid phosphatase (TRAP)
In both, HA/PLF(0) group and HA/PLF(þ) group,
TRAP positive multinucleated cells could be observed
along the surfaces of the HA particles and within the
granulation tissue localized between HA particles
(Figure 2B,C). The multinucleated giant cells which
attached to HA frequently contained large numbers of
intracytoplasmic vacuoles corresponding in size of the
cell nuclei. Sometimes, the numerous small vacuoles
caused a foamy appearance of the cytoplasma of the
multinucleated cells (Figure 2D). Multinucleated cells
localized within the granulation tissue between HA
particles were more dense in appearance and were
filled with a smaller number of vacuoles.
Compared to the HA/PLF(0) group (median:
3.903), a significant larger number ( p , 0.01) of
multinucleated giant cells were TRAP positive in the
HA/PLF(þ) group (median: 8.349) (Figure 2E).
No TRAP positive cells could be seen within the
granulation tissue of the defects which were not filled
with HA, whereas few TRAP positive multinucleated
cells were localized at the surfaces of the host bone
(Figure 2A).
Figure 1. (A) Degradation of HA by multinucleated giant cells (black arrows, scale bar ¼ 50mm), and (B) migration of giant cells filled with
hydoxyapatite through the stroma (scale bar ¼ 50mm). TEM of (C) mononuclear macrophage (scale bar ¼ 5mm), and (D) multinucleated
giant cell (scale bar ¼ 5mm), GC ¼ giant cell, HA ¼ hydroxyapatite, N ¼ nucleus, G ¼ golgi apparatus.
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Cathepsin-K immunohistochemistry
In the HA/PLF(0) group, numerous multinucleated
giant cells revealing cathepsin-K immunoreactivity
were found along the surface of the host bone. Some of
these cells were flat and elongated in shape, while
others, especially those located within resorption
lacunae of the host bone, were more spherical in
shape. Additionally, numerous cathepsin-K positive
multinucleated cells adhered at the surfaces of the HA
particles, and frequently these cells could be found
along the entire circumference of the implanted
particle. The cytoplasm of multinucleated cells
contained many vacuoles.
Regarding HA particles which were partly sur-
rounded by newly formed bone, the occurrence of
multinucleated giant cells were restricted to the free
surfaces. Positively stained giant cells could also be
demonstrated in granulation tissue localized between
HA particles. The majority of the cells revealed
compact spherical shape and contained numerous
cytoplasmatic vacuoles. In addition, cathepsin-K
positive mononuclear cells of the granulation tissue
could be identified in close vicinity to the implanted
HA. In comparison, cathepsin-K positive multi-
nucleated giant cells and mononuclear cells were
more frequent in the HA/PLF(þ) implant group than
in the HA/PLF(0) group (Figure 3B,C).
Within the granulation tissue of the empty defects
only mononuclear cells of the macrophage lineage
revealed cathepsin-K immunoreactivity (Figure 3A).
CD44 immunoreactivity
CD44 positive mononuclear cells were abundant in
the granulation tissue of the HA/PLF(0) group. A
large number of these cells were localized in close
Figure 2. Enzymehistochemical detection of TRAP in multinucleated giant cells (A) on the host bone surface in unfilled defect, (B) on the
HA/PLF(0) and (C) HA/PLF(þ) implant surfaces (black arrows, scale bar ¼ 100mm). (D) TRAP-marked multinucleated giant cells (scale
bar ¼ 10mm). (E) Box-and-whisker plot of tartrate-resistant acidic phosphatase positive areas in HA/PLF(0) (median: 3.903), and
HA/PLF(þ) (median: 8.349) implants (quartiles, median, lower and upper quartile). * p , 0.01. HB ¼ host bone, HA ¼ hydroxyapatite,
GC ¼ giant cell.
Effects of platelet factors 195
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proximity to the small capillaries. Regarding the
cellular staining pattern of the mononucleated cells,
immunreactivity became visble along the entire
circumference of the plasma membrane, whereas the
cytoplasm remained unstained.
The basal aspects of the cell membranes of
multinucleated giant cells which adhered at the
surfaces of the implant particles were also strongly
CD44 positive, while the apical aspects of the
membranes revealed no signs of immunoreactivity. In
contrast to this staining pattern, the cell membranes
of multinucleated giant cells located within granulation
tissue showed a strong CD44 signal along
their entire circumference (Figure 4E). The number
of CD44 positive multinucleated giant cells
was increased in the HA/PLF(þ) implant group
(Figure 4B,C).
Within the granulation tissue of the empty defects,
only mononuclear cells revealed positive membrane-
associated CD44 staining. Frequently, these cells
could be identified in close vicinity to capillaries. No
CD44 positive multinucleated giant cells could be
observed in the granulation tissue of the empty defects
(Figure 4A).
De novo woven bone formation
In both, the HA/PLF(0) and the HA/PLF(þ )
group subsets of HA particles were partly or even
completely surrounded by newly formed woven bone,
and sometimes small bridges of woven bone could be
detected between neighbouring HA particles. In
general, the osteoid-synthesizing osteoblasts were
closely associated with the surfaces of the implant
whose particles were scattered throughout the whole
defect area. Frequently, small capillaries were seen in
close proximity to the newly formed osteoid
(Figure 5A–C).
Within the empty defects, woven bone formation
was restricted to the surfaces of the host bone. No
signs of bone formation could be observed within
the granulation tissue which was well vascularized
and contained large numbers of collagen
syntheszing fibroblasts (Figure 6A). Different from
the HA/PLF(0) group, the HA particles in the
HA/PLF(þ) group appeared to be in a more soluble
phase, as the HA surfaces were no longer circum-
script and were merging with the woven bone.
Numerous vessels were detectable in the granulation
tissue between HA and newly formed bone. After
staining, the interface between HA and woven
bone of the HA/PLF(0) implants appeared signifi-
cantly different compared to that of HA/PLF(þ)
implants.
Osseous healing of the defect originated from the
host bone and extended toward the center of the
defect, but at 20 days after implantation bone
formation only localized near the interface between
host bone and biomaterial remained incomplete in
both HA groups (Figure 6B–D).
Figure 3. In unfilled defect (A) cathepsin-K (black arrows) synthesized by multinucleated cells was seen only along the host bone, abundant
numbers of cathepsin-K positive giant cells appeared in the HA/PLF(0) (B) and HA/PLF(þ) (C) implants, (D) negative control (scale
bar ¼ 100mm), HA ¼ hydroxyapatite, HB ¼ host bone, CT ¼ connective tissue.
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Discussion
The numbers of multinucleated giant cells located
both in proximity to HA particles and in the
granulation tissue as well as the numbers of
mononuclear macrophages differed between both
experimental groups. Increased numbers of these
cells were observed near HA/PLF(þ) implants.
Growth factors such as PDGF and TGF-b released
from activated platelets have been reported to activate
maturation and chemotaxis (Sanchez et al. 2003).
The increased quantity of macrophages in the
granulation tissue around HA/PLF(þ ) implant
particles can be explained by the addition of platelet
lysate in this group, because this was the methodical
difference between both experimental groups.
The relatively large spatial distances between
individual implant particles in the fibrovascular tissue
filling the defect probably were caused by the material
being degraded by physicochemical processes (halis-
teresis). After the initial break down, nanoparticulate
HA disintegrated into small clumps. This process
assumingly was enhanced by the lowered pH resulting
from a poorly vascularized environment.
A positive effect of this degradation is the space
gained for cells to migrate into and for the formation
of granulation tissue (Shi et al. 2002). The autors
demonstrate the osteoconductive properties of the
implant, despite the wide spacing of the individual HA
clumps. Apposition of implant and newly formed
bone can illustrate this.
Figure 4. In unfilled defect (A) CD44 staining (black arrows) was only weakly. Large numbers of CD44 positive stained giant cells appeared
in the HA/PLF(þ) (C) implants compared to the HA/PLF(0) implants (B), (D) negative control (scale bar ¼ 100mm). (E) The level of
CD44 antigen stain was high in giant cell membranes in proximity to implants (black arrows), however, only weak staining (white arrows) was
found on the cell membrane opposed to the hydroxyapatite surface (scale bar ¼ 10mm). (F) Fusion of mononuclear macrophages was
indicated by close plasmalemmal contact of neighbouring cells (thin arrows), and partly intact cell membranes (black arrow), TEM (scale
bar ¼ 1mm), HA ¼ hydroxyapatite, HB ¼ host bone, CT ¼ connective tissue, GC ¼ giant cell, N ¼ nucleus, M ¼ mitochondria.
Effects of platelet factors 197
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Figure 5. (A) In addition to HA degradation processes by multinucleated giant cells, osteoid rims with adherent osteoblasts were detected
(white arrows) at 20 days after implantation, semithin section (scale bar ¼ 50mm). Cluster of osteoblasts (white arrows) on the
hydroxyapatite surface showed osteoid synthesis and immediate mineralisation (black arrows) at 20 days after implantation, (B) semithin
section (scale bar ¼ 50mm), and (C) TEM (scale bar ¼ 10mm), HA ¼ hydroxyapatite, VE ¼ vessel, GC ¼ giant cell, CT ¼ connective
tissue, MB ¼ mineralized bone.
Figure 6. (A) Newly formed woven bone (black arrows) was seen (A) only on host bone surfaces in defects not filled with biomaterial, (B) in
the hydoxyapatite filled defects de novo bone formation (black arrows) was aligned between the HA particles; some HA particles were
completely surrounded by woven bone, (C) Quantitation of de novo woven bone formation by assessment of the strong toluidine staining of
HA, semithin section (scale bar ¼ 100mm). (D) Relative amounts of de novo bone formation near the interface in the implant groups at
20 days postoperatively, p , 0.05.
O. Kilian et al.198
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A break down of implant material into smaller
fragments was shown to activate macrophages and
multinucleated foreign body giant cells followed by an
inflammatory response (Dupraz et al. 1998; Gunther
et al. 1998).
Anderson et al. (2001) showed in vitro that
hydroxyapatite particles less than 10mm in size
stimulated macrophage activity. Sabokar et al.
(2003) reported that macrophages and multinu-
cleated giant cells phagocytized small particles (up to
1mm in size) while resorptive cells became attached to
larger particles. In vitro studies revealed that particu-
late HA debris increased the synthesis of proinflam-
matory cytokines (interleukin 1 and 6, tumor necrosis
factor a) in human fibroblasts (Ninomiya et al. 2001),
thereby showing a direct link between hydroxyapatite
and the induction of an inflammatory response in the
surrounding connective tissue.
In granulation tissue, all multinucleated giant cells
contained the proteinase cathepsin-K, which is
expressed by osteoclasts as well as by multinucleated
giant cells. It is of interest that the antigen markers of
giant cells located in granulation tissue were fre-
quently decreased. Buhling et al. (2001) reported that
cathepsin-K is also a marker of macrophage activation
and differentiation.
In analogy to the observation made by Bonnema
et al. (2003), we could demonstrate that in the
vicinity of biomaterial implants, macrophages and
multinucleated giant cells often respond strongly to
anti-CD44 antibody. Especially the free walls of
multinucleated giant cells attached to the implant
vividly interacted with the tested antibody, whereas
the cell walls facing the implant did not react. Sterling
et al. (1998) explained this phenomenon with
alignment of the CD44 membrane proteins towards
neighboring macrophages available for fusion with
the giant cell. The membrane region attaching the
giant cell to hydroxyapatite remains CD44 negative,
while the free basolateral sections of the cell do show
a CD44 immune response. It is conceivable that the
membrane-bound receptor CD44 hampers attach-
ment of the cell to HA, so that during attachment,
the receptor needs to be redistributed to the
basolateral cell wall. Adhesion proteins such as
integrines can be anchored to the apical wall which
remains free of CD44. This is where giant cells attach
to HA.
Macrophages located in connective tissue displayed
a positive CD44 signal on their entire cell membrane.
According to Vignery’s (2000) definition, these cells
must reach a “critical density” in order to fuse.
Macrophages in close proximity to one another
exhibited a decreased or even no immunoreactivity
at all at those parts of the cell membrane facing
neighboring macrophages. The more or less imminent
cell fusion could explain this phenomenon, as it
involves a partial reorganisation of the cell walls.
Osteointegration of the biomaterial and stimulation
of bone consolidation for defect-healing are the actual
goals of using bone replacement materials. A faster
osteointegration of implants and their long-term
function due to enrichment with platelet growth
factors would be desirable. While evaluating defect-
healing by microradiography after implantation of a
bovine collagen matrix in landrace pigs, Schlegel et al.
(2004) were unable to demonstrate that enrichment
with platelet growth factors (PLF) induced an
increase of mineralization within 2 weeks. After 4
and 12 weeks, the mineralization was slightly higher in
the PLF than in the collagen groups. Furst et al.
(2004) examined bone consolidation in mandibular
osseous defects in 8 minipigs which had been
implanted collagen I plus PLF or just collagen I
alone. Histomorphometrically, the authors showed
that after four and eight weeks in the collagen I group,
bone consolidation was significantly ( p , 0.001)
higher than in the group where collagen I plus PLF
had been used. Using a minipig model and micro-
radiography, Wiltfang et al. (2004) found that defect-
healing after 14 days was significantly better in the
group where PLF had been used in combination with
spongiosa compared to the control group. Combining
PLF with b-tricalcium-phosphate ceramics did not
stimulate osteogenesis. After 4 weeks, no differences
were detectable between the spongiosa þ PLF
group and the group where only PLF had been used.
There were also no differences between osteointegra-
tion of PLF-enriched biomaterials and biomaterials
without such enrichment.
Fennis et al. (2002), however, describe that PLF
stimulated healing of corticospongious grafts in
mandibular defects in a goat model.
A comparison of literature reports is often
hampered by the various graft materials used and the
variability in the preparation of PLF and platelet
growth factors, respectively. The amount of platelet
growth factors released from alpha granules depends
on the platelet concentration which significantly varies
from one study to the other and especially on the
platelet activation induced by the platelet processing.
A comparison of various studies reveals that the
methods to separate platelets and to activate and
degrade them differ tremendously.
Osteoblasts which were found in large quantities in
all groups in tissues close to the defect borders and
which formed finger-like patterns pointing towards
the defect center are thought to be an indication of
osteoneogenesis. This phenomenon was most pro-
nounced in the HA/PLF(þ) implant group. In
addition, also the cross-linking between implant
particles and osteoid was the strongest in the
HA/PLF(þ) group. This could be explained by the
effect of platelet growth factors. The osteoblasts that
migrated from the host bone into the defect where
they surrounded implant particles and formed osteoid
Effects of platelet factors 199
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are assumed to be an indicator of osteoconductive
effects of the nanoparticulate hydroxyapatite. When
the specimens where taken, the implant particles
surrounded by osteoid appeared to be completely
linked with the newly formed bone and thereby
protected from additional degradation.
Numerous HA particles surrounded by osteoblasts
and osteoid were found in granulation tissue. The fact
that osteoid embedded HA particles were not
contiguous with host bone osteoid illustrates the HA
particles’ osteoconductive effect. No substantial
osteoinductive effect of platelet growth factors was
evident, a fact most probably caused: (1) by the
limited biological activity of exogenous platelet growth
factors, which also have a short half-life, and (2) by a
paucity of target cells as precursors in ectopic sites
during the early inflammatory phase.
Increased ossification in proximity to host bone
seen with HA/PLF(þ) implants is probably caused
by increased fibrin cross-linking due to the effect
of the topically applied platelet lysate on the
coagulation cascade. This can enhance the attach-
ment of hydroxyapatite crystals to the surface of
periimplant bone.
Increased fibrin binding and cross-linking at the
surface is thought to translate into improved proper-
ties of the extracellular matrix and into accelerated
migration of inflammatory and mesenchymal pre-
cursor cells. This results in accelerated degradation of
the HA particles during the earliest phase of bone
defect healing.
In conclusion, composite material consisting of a
biodegradable, nanoparticular hydroxyapapite
enriched with autologous PLF has shown its positive
effect regarding biodegradation and initial osteogen-
esis in early stage of bone healing which was
comparable to the results for pure hydroxyapatite in
current study. The local application of the growth
factors induces an increasing resorptive cell activation
in the reparative phase of bone defect healing. Major
number of osteoblast in the platelet factor enriched
implant was seen in the inferface space between host
bone and biomaterial, but no substantial osteoinduc-
tive effect of platelet growth factors was evident.
Acknowledgements
This work was supported by Federal Ministry for
Education and Research (BMBF; Tissue engineering,
No: PTJ-BIO/0312741), Bonn, Germany.
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