Full Text

Assesment of the Biological Activity of Chemically
Immobilized rhBMP-2 on Titanium surfaces in vivo
AbschaÈtzung der biologischen AktivitaÈt von chemisch immobilisiertem rhBMP-2
auf TitanoberflaÈchen in vivo
G. Voggenreiter, K. Hartl, S. Assenmacher1,
M. Chatzinikolaidou2, H. M. Rumpf2
and H. P. Jennissen2
Previously it has been shown that recombinant human bone morphogenetic protein (rhBMP-2) can be chemically immobilized by
ªanchor moleculesº on titanium surfaces for serving as a drug delivery device. This opened the question of whether the insoluble
immobilized rhBMP-2 retained its activity in comparison to the
same amount of soluble rhBMP-2 included with the implant samples. Electropolished titanium miniplates (10 6 0.8 mm) were
ªsurface-enhancedº by a novel treatment with chromosulfuric acid
and then coated with a total amount of 150 ± 200 ng rhBMP-2 prepared by recombinant technology. Periosteal flaps (7 20 mm)
were detached and isolated from the anterior surface of the tibiae
of adult rabbits and wrapped around the titanium sample plates
which were then implanted in the M. gastrocnemius. In the first
experimental group various controls without rhBMP-2 were combined (n ˆ 12). In the second experimental group implants with
chemically immobilized rhBMP-2 (n ˆ 8) were compared with implants to which non-immobilized soluble rhBMP-2 was added
(n ˆ 8). Animals were sacrificed after 28 days and a quantitative
evaluation was carried out by means of serial sections. Untreated
control plates showed bone formation in 2/12 implants, rhBMP-2
coated implants in 6/8 and implants with free rhBMP-2 administered subperiostally in 8/8 cases. In the case of rhBMP-2 coated
implants the induced bone had direct contact to the implant in
all cases while in the group with free administered rhBMP-2 the
bone had no contact to the implant in two cases, but was separated
by a fibrous capsule. Bone volume, bone surface area, and trabecular number displayed no difference between the two rhBMP-2groups. However, in the biocoated group a tendency to an increase
in the bone-implant contact area was evident. No differences in osteoid area, osteoid perimeter and eroded perimeter were detected.
We conclude that in the case of non-immobilized rhBMP-2 there is
the danger for formation of fibrous tissue between the implant and
the newly formed bone and in addition the generation of ectopic
bone at inappropriate places. In contrast chemically immobilized
rhBMP-2 does not have these drawbacks and at the same time displays a biological activity on surfaces similar to that of soluble
rhBMP-2 demonstrating that biomaterial surfaces can be tailored
for a selective and specific interaction with the target tissue.
In bisherigen Arbeiten konnte gezeigt werden, dass rekombinantes humanes Bone Morphogenetic Protein (rhBMP-2), welches in
vitro aktiv ist, kovalent auf TitanoberflaÈchen immobilisiert werden
kann. Es erhebt sich die Frage, ob das in dieser Form immobilisierte
rhBMP-2 seine biologische AktivitaÈt in vivo behaÈlt. Die OberflaÈche
von elektropolierten TitanplaÈttchen (10 6 0.8 mm) wurde
durch Behandlung mit ChromschwefelsaÈure veredelt und die PlaÈttchen wurden anschlieûend mit einer Gesamtmenge von 150 ±
200 ng rhBMP-2 beschichtet, das mittels rekombinanter DNATechnologie in E. coli gewonnen wurde. Von der Vorderkante
der Tibia erwachsener Kaninchen wurde ein 7 20 mm groûer Perioststreifen entnommen, die TitanplaÈttchen damit umwickelt und
das Komposit dann in den M. gastrocnemius implantiert. Die Experimente wurden in zwei Hauptgruppen aufgeteilt. In der ersten
Versuchsgruppe wurden verschiedene Kontrollen in Abwesenheit
von rhBMP-2 zusammengefasst (n ˆ 12). In der zweiten Versuchsgruppe wurde die biologische Reaktion auf chemisch immobilisiertes rhBMP-2 (n ˆ 8) mit nicht-immobilisiertem loÈslichem rhBMP2 in der Periosttasche (n ˆ 8) verglichen. Die Versuchsdauer betrug
28 Tage. Die quantitative Analyse der Knochenneubildung wurde
an Serienschnitten durchgefuÈhrt. WaÈhrend es in der ersten Versuchsgruppe bei Implantaten ohne rhBMP-2 wie zu erwarten nur
in 2/12 FaÈllen zu einer ganz geringen Knochenneubildung kam,
zeigte sich eine deutliche Knochenneubildung bei 6/8 Implantaten
mit chemisch immobilisiertem rhBMP-2 und bei 8/8 Implantaten
mit freiem rhBMP-2. Bei immobilisiertem rhBMP-2 hatte der neugebildete Knochen in allen FaÈllen einen unmittelbaren Kontakt zur
ImplantatoberflaÈche, waÈhrend bei freiem rhBMP-2 der Knochen in
2 FaÈllen keinen Kontakt zum Implantat aufwies, sondern von einer
Bindegewebsschicht getrennt war. Hinsichtlich Knochenvolumen,
KnochenoberflaÈche und Trabekelzahl ergab sich kein Unterschied
zwischen immobilisiertem und freiem rhBMP-2. Die Parameter
OsteoidflaÈche, Osteoidumfang und erodierter Umfang zeigten
ebenfalls keine Gruppenunterschiede. Wir schlieûen, dass im Falle
des nicht-immobilisierten rhBMP-2 die Gefahr eines fibroÈsen Interfaces zwischen Implantat und neugebildetem Knochen und der Induktion von ektopischem Knochen an ungewollter Stelle besteht.
Im Gegensatz dazu zeigt kovalent immobilisiertes rhBMP-2 auf TitanoberflaÈchen eine aÈhnlich hohe biologische AktivitaÈt wie loÈsliches rhBMP-2 ohne die obengenannten Gefahren. OberflaÈchen
von Biomaterialien koÈnnen somit durch rhBMP-2 Beschichtungen
so veraÈndert werden, dass eine spezifische Interaktion mit dem
Zielgewebe induziert wird.
1 Introduction
1
2
Department of Trauma Surgery, University Hospital Mannheim
Department of Trauma Surgery, Bethesda Hospital Duisburg
Institute of Physiological Chemistry, University Hospital Essen
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0933-5137/01/1212-0942$17.50 ‡ .50/0
A variety of osteoinductive biomaterials has been developed for reconstruction of skeletal defects in the past years
and potentially they can play a major role in the future [1].
These investigations focussed on growth factors and cytokines
Mat.-wiss. u. Werkstofftech. 32, 942±948 (2001)
Ó WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
as well as bone morpogenetic protein-2 (rhBMP-2), which
were generally applied in soluble form together with a delivery system or with a carrier such as demineralized bone matrix, collagen or polyglycol/ and polylactide acide matrices
[2 ± 5]. In several investigations it was demonstrated, that
rhBMP-2 retains its biological activity in this form of application resulting in bone formation [3].
The surface properties of biomaterials are of great importance in regard to biocompatibility and to the biological response of the organism to an implant. Therefore besides
the well known effect of accelerating healing of bone defects,
rhBMP-2 may improve the osseointegration of alloarthroplasties. This has not been a matter of investigation so far. To make
the implantation of a biocoated prostheses clinically practicable, it is desirable to achieve a stable immobilization of significant amounts of proteins such as rhBMP-2 on the implant
surface.
Recently a concept for biocoating implants with biomolecules on the basis of chemotactic-juxtacrine surfaces was developed [6, 7]. Titanium plates were first surface enhanced by
treatment with chromosulfuric acid (CSA) at 200 ± 250 8C,
leading to an increase in the thickness and hydrophilicity
of the metal oxide layer and an enhancement of the binding
capacity for chemical modification reactions [6]. Then
rhBMP-2 was immobilized in amounts of ca. 70 ± 380 ng/
cm2 on the metal surface [6].
The rhBMP-2 prepared in our laboratory [6] has been
shown to possess a high biological activity in vitro [8]. Since
the treatment of metals with chromosulfuric acid leads to ultra-hydrophilic micro- and nanostructured metallic surfaces of
a darker color [9] it was not possible to test the biological activity of immobilized rhBMP-2 by classical colorimetric cellular in vitro assays. In the present report the osteoinductive
properties of rhBMP-2 biocoated titanium miniplates were
therefore investigated in an animal model. Special emphasis
was given to the question of whether biocoating results in a
biocompatible implant and whether rhBMP-2 retains its biological activity. It is demonstrated here in an in vivo model that
rhBMP-2 chemically immobilized on CSA-enhanced titanium
surfaces shows a biological activity comparable to that of a
similar amount of soluble rhBMP-2.
2 Materials and Methods
2.1 Implant Biocoating
Biologically active recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared as previously described [6]. Rectangular, zero-hole, electropolished titanium
miniplates (cp titanium, grade 2; GB Implantattechnologie,
Essen) 10 6 0.8 mm in size corresponding to a total
area of ca. 1.2 cm2 [6, 7] were employed throughout. Unmodified pure titanium miniplates were cleaned and treated with
5% HNO3 as described earlier [6]. For biocoating with
rhBMP-2 the miniplates were first surface-enhanced by a novel procedure with chromosulfuric acid (CSA) [6, 7]. Characteristically the treatment of titanium with chromosulfuric acid
(Ti-CSA) leads to ultra-hydrophilic (contact angles 0 ± 108, no
hysteresis) [9], bioadhesive surfaces. rhBMP-2 was immobilized by chemical substitution of the metal surface with anchor-molecules (ANC) such as 3-aminopropylthriethoxysilane and carbonyldiimidazole (Ti-CSA-ANC) [6]. After silanization of the titanium surface with 3-aminopropylthrieMat.-wiss. u. Werkstofftech. 32, 942±948 (2001)
thoxysilane the terminal amino group (Ti-APS, Ti-CSAAPS) was activated by carbonyldiimidazole which on incubation with 0.3 mg/ml rhBMP-2 forms covalent bonds to the eamino group of lysine residues in BMP-2 (Ti-CSA-ANCBMP-2). The amount of rhBMP-2 immobilized, which was
determined with 125I-rhBMP-2 [6] in separate experiments,
ranged from 150 ± 200 ng/cm2 [7]. Although the rhBMP-2 immobilized in this manner cannot be washed off by 1% SDS
buffer solutions (pH 7.0 ± 7.4) it cannot be excluded that
non-covalently adsorbed rhBMP-2 coexists with covalently
bound molecules on the chemically modified surface. As
we have shown, proteins immobilized in this manner can
be removed by washing in hot (60 8C) strongly alkaline
(0.1 M NaOH) 1% SDS solutions [6]. Employing this drastic
procedure it cannot, however, be excluded that a hydrolysis of
the carbamate linkage between the propylamine silane residue
and the protein occurs. Therefore at the moment it is not possible to quantitatively discriminate between the covalently
and non-covalently bound species of rhBMP-2. In consideration of these problems we therefore speak of a chemical immobilization of rhBMP-2 by anchor molecules which denotes
a primary covalent linkage to rhBMP-2 with simultaneously
non-covalently bound rhBMP-2 on the surface. Thus the noncovalently bound rhBMP-2 would display chemotactic activity and the covalently bound species juxtacrine activity in
agreement with our chemotactic-juxtacrine surface model
[6]. The miniplate specimens prepared as described above
and employed in the animal experiments are shown in Table 1.
The biological activity of soluble rhBMP-2 was tested with
MC3T3-E1 cells by the activation of the de novo synthesis
of alkaline phosphatase [8].
2.2 Operative Procedure
Appropriate consideration was given to the guidelines for
care and use of laboratory animals of the government of
Nordrhein-Westfalia, Germany. Under general anesthesia (ketamine/xylazine) the skin at the anterior edge of the tibia was
dissected longitudinally in adult rabbits (CHbb:CH, Fa. Thomae, Biberach/Riû, Germany; body weight 3 ± 3.5 kg) and a
periosteal strip of the dimension 7 20 mm was harvested.
The titanium miniplates were mounted on a special fixation
device and wrapped with the periosteal strip (Fig. 1). The strip
was secured with two 5 ± 0 Polydigoxanone sutures (Fa. Ethikon, Norderstedt, Germany). Then a 7 mm wide and 10 mm
deep pocket was made in the muscle belly of the M. gastrognemius and the periost-implant composit was inserted. The
specimens were implanted in the following six groups (see
Table 1): (i) unmodified titanium miniplates (n ˆ 2); (ii)
CSA-treated miniplates (Ti-CSA, n ˆ 2), (iii) silanized
non-CSA-treated miniplates (Ti-APS, n ˆ 4); (iv) silanized
CSA-treated miniplates (Ti-CSA-APS, n ˆ 4); (v) soluble
rhBMP-solution instilled with unmodified CSA-miniplates
(n ˆ 8) and (v) chemically immobilized rhBMP-2 on CSAtreated miniplates (Ti-CSA-ANC-BMP-2, n ˆ 8). For application of free soluble rhBMP-2 (group v) 20 ll of a
rhBMP-2 solution (50 lg/ml rhBMP-2 in 50 mM sodium bglycerophosphate, 0.066% SDS, pH 7.0) was injected between periosteum and implant (Ti-APS-CDI). To avoid loss
of BMP-2 solution, the injection between periost and miniplate was performed after insertion of the composite into
the muscle pouch. Animals were sacrificed after 28 days
by an overdose of T61 (Fa. HoÈchst, Frankfurt, Germany).
Titanium
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Table 1. Experimental groups and surface treatment
Experimental Group
n
Comments
(i) unmodified Ti miniplates
2
1 specimen with low-degree bone formation
(ii) Ti-CSA miniplates
2
±
(iii) Ti-APS miniplates
4
1 specimen with low-degree cartilage formation
(iv) Ti-CSA-APS miniplates
4
1 specimen with low-degree bone formation
(v) unmodified Ti CSA-miniplates ‡ free rhBMP-2
8
bone induction 8/8, 2 cases of fibrous capsule on
miniplate
(vi) rhBMP-2 biocoated miniplates (Ti-CSA-ANC-BMP-2)
8
bone induction 6/8, bone-metal contact 6/6
Experimental Group 1 (controls)
Experimental Group 2 (rhBMP-2)
Ti: titanium
Ti-CSA: chromosulfuric acid treated titanium
APS: 3-aminopropyltriethoxysilane
ANC: anchor molecules
For further details see the text
#1000 ± 4000, Struers, Kopenhagen, Denmark) to a thickness
of 60 lm (Knuth-Rotor-3, Struers, Kopenhagen, Denmark).
Then the specimens were stained with basic fuchsine.
2.4 Quantitative analysis
Fig. 1. Wrapping of a titanium plate with a periosteal strip forming
an implant composite
Abb. 1. Umwickelung eines TitanplaÈttchens mit einem Perioststreifen zu einem Implantat-Komposit
A strip of periosteum (7 mm 20 mm) was isolated from the front
of the tibia and wrapped around the titanium miniplate. The periostminiplate composite was then implanted into the gastrocnemius
muscle of the rabbit.
2.3 Radiological and histological technique
The specimens were harvested with a surrounding layer of
muscle, fixed in 40% ethanol, run through the alcohol series
and embedded in methylmetacrylate. Serial sections of
150 lm thickness were made using a rotating saw (InnenlochsaÈge 1600, Leica Instruments GmbH, Nusslach, Germany).
The serial sections were placed on high resolution plates (INTAS( High Resolution Plates 2 2 0.65 inch, Intas, GoÈttingen, Germany) and microradiographs were obtained using a
table radiographic system (FaxitronÒ Radiographic Inspection
System Model 43805, Hewlett Packard, Inc., Mc Minnville,
USA). The sections were mounted with a cyanoacrylate based
glue (RotiÒcoll 1 Cyanacrylate glue, Carl Roth GmbH, Karlsruhe, Germany) on glass slides and grounded (Sic paper
944
G. Voggenreiter et al.
The morphometric analysis for estimation of the total
amount of newly formed bone was performed by means of
the microradiographs of all sections of every specimen. For
evaluation, images were obtained with an analogue camera
(XC-77CE CCD Video Camera Module, Sony, Inc., Japan)
at a 7-fold magnification (Leica Wild M420, Leica AG, Heerbrugg, Switzerland) and were analysed digitally by means of
an image analysing system (KS 400 Vs.2, Fa. Kontron, Eching, Germany). The following parameters were evaluated:
Bone volume (mm3), Bone area (mm2), Trabecular number
and Implant-bone contact area (mm2).
Furtheron the statical histomorphometric parameters were
evaluated from the stained sections. Images were obtained
using a digital camera (Leica DC 200, Leica Microscopy Systems AG, Heerbrugg, Switzerland) and a light microscope
(Leica DM LB, Leica Microsystems GmbH, Wetzlar, Germany) at a magnification of 100. Parameters were determined
according to the suggestions of the histomorphometry-committee of the American Society of Bone and Mineral Research
(8): Osteoid area (O.Ar), Osteoid perimeter (O.Pm), Eroded
perimeter (E.Pm).
2.5 Statistics
Statistical analysis including mean values (MV) and standard deviation (SD), was performed using SPSS 7.5 (SPSS,
Inc., Chicago, Il). Comparisons between groups were done
using unifactorial analysis of variance. When the analysis
of variance detected significant differences, groups were compared with the ScheffeÁ test.
Mat.-wiss. u. Werkstofftech. 32, 942±948 (2001)
3 Results
3.1 Histology
No intra- or postoperative complications were observed. In
the conventional light microscopy there was no histological
evidence of round cell infiltrations or giant-cell reactions at
any site in the implantation series after four weeks. In a first
experimental group (controls, see Table 1) unmodified, CSAtreated, silanized non-CSA treated, and silanized CSA-treated
titanium miniplates were implanted. The implants were surrounded by the implanted periosteum and a thin capsule of
collagenous fibrous tissue containing spindle shaped cells
with few blood vessels (Fig. 2). We detected no difference regarding the tissue reaction between these various controls (see
Table 1). No local or generalized (fever, shock) immuno-inflammatory reactions were observed, however no immunohistochemistry has been performed. Additionally the application
of soluble as well as chemically immobilized BMP exhibited
excellent biocompatibility.
In the experimental group 2 (with rhBMP-2, see Table 1)
also no local or generalized (fever, shock) immuno-inflammatory reactions were observed. While implants without rhBMP2 exhibited bone formation in only 2/12 cases, bone formation
was observed in 6/8 implants with chemically immobilized
rhBMP-2 and in 8/8 implants with free soluble rhBMP-2.
In implants with immobilized rhBMP-2 the newly formed
bone had direct contact to the implant surface in all (6/6)
cases, while in implants with rhBMP-2 applied freely the
bone had no contact to the implant in two (2/8) cases. These
Fig. 2. Silanized CSA-treated implant at 4 weeks
Abb. 2. Silanisiertes CSA-behandeltes Implantat nach 4 Wochen
The silanized CSA-treated miniplate was surrounded by the implanted periosteum (not shown) and covered a thin layer of collagenous fibrous tissue. No evidence of inflammatory reactions could
be detected (fuchsine stain, magnification 100).
differences are shown in microradiographies in Fig. 3. In
Fig. 3A (free soluble rhBMP-2) the induced bone formed inappropriately, separated from the miniplate by a fibrous capsule. In Fig. 3B (immobilized rhBMP-2) the newly formed
bone is in direct contact with the titanium miniplate. This difference between the two types of application of rhBMP-2 is
Fig. 3. Microradiographical comparison of implants with soluble and chemically immobilized rhBMP-2
Abb. 3. Mikroradiographischer Vergleich von Implantaten mit freiem
und chemisch immobilisiertem und
rhBMP
A direct bone contact was missing in two (2/8) of the implants (Fig. 3A) with soluble rhBMP-2 applied in free form. The newly formed
bone is separated from the titanium implant by fibrous tissue forming a capsule around the miniplate. In implants with chemically immobilized rhBMP-2 a direct contact of the bone was evident in all animals with bone formation (6/8) after 4 weeks (Fig. 3B), ( 7 magnification). For further details see Fig. 4.
Table 2. Histomorphometric parameters
Group
3
Bone volume (mm )
Bone surface (mm2)
Trabecular number
Implant-bone area (mm2)
Osteoid area (mm2)
Osteoid perimeter (mm)
Eroded perimeter (mm)
Controls
Biocoated BMP
Free BMP
0.3
8
5
0
0.8
0.09
0.003
2.1
54
61
6.3
12.2
3.6
0.25
1.9
62
72
4.0
8.7
2.7
0.2
0.2
7
6
0.6
0.07
0.004
2.1*
62*
72*
6.0 *
11.6*
3.2*
0.35*
1.8*
78*
114*
4.7*
8.2*
2.2*
0.33*
Given are mean values and standard deviation
* P < 0.05 compared to control
Mat.-wiss. u. Werkstofftech. 32, 942±948 (2001)
Titanium
945
BMP-groups. In these cases the outer surface of the induced
bone was covered by the inner periosteal layer. This indicates
that the periosteum is the source of osteoprogenitor cells,
being the target cells for the applied BMP.
3.2 Histomorphometry
Compared to the control group without rhBMP-2
chemically
immobilized
rhBMP-2
(0.3 0.2 mm3),
(2.1 2.1 mm3) as well as free rhBMP-2 (1.9 1.8 mm3)
lead to a significant increase in induced bone volume (Table 2). Corresponding results have been found for trabecular
number and bone surface. While the bone had no contact to the
implant in the control group, a tendency towards an increased
bone-implant contact area in immobilized rhBMP-2 compared to free rhBMP-2 was detected (6.3 6.0 vs.
4.0 4.7 mm2). The statistical histomorphometric parameters osteoid area, osteoid perimeter and eroded perimeter
displayed no significant difference between immobilized
and free rhBMP-2 (Fig. 5). There was no difference in the
quality of the induced bone between the two forms of application.
4 Discussion
Fig. 4. Microphotographical comparison of stained implant preparations implanted with soluble free (A) and chemically immobilized (B) rhBMP-2
Abb. 4. Mikrophotographischer Vergleich von gefaÈrbten ImplantatpraÈparationen, die mit loÈslichem freiem (A) oder chemisch immobilisiertem (B) rhBMP-2 implantiert wurden
A. Capsule formation between bone and implant after employment
of free soluble rhBMP-2 in the composite.
B. Direct bone-implant contact with chemically immobilized
rhBMP-2 in the composite.
For further details se Fig. 3 (fuchsine stain, magnification 20).
demonstrated in stained micrographs shown in Fig. 4. In
Fig. 4A the dense fibrous capsule separating the bone from
the miniplate is easily observed. In contrast in Fig. 4B the induced bone has come into direct contact with the miniplate
surface. In all cases of bone formation the newly formed
bone was woven bone (Fig. 4 & 5) and only occasionally remnants of cartilage were observed indicating enchondral ossification.
The newly induced bone was located between the implanted periosteum and the surface of the implant in both
946
G. Voggenreiter et al.
In the present investigation we were able to demonstrate in
vivo, that rhBMP-2 immobilized to an implant surface by
chemical means retains its biological activity. In an animal
model of ectopic new bone formation no difference regarding
quantity and quality of the induced bone compared to an
equivalent amount of free applied rhBMP-2 were evident.
However although not being statistically significant, immobilization of rhBMP-2 compared to free rhBMP-2 there is a tendency towards an increase in the bone-implant contact area.
We used the model of ectopic bone formation by means of
periosteal flap transplantation to estimate the net amount of
newly formed bone independently from the capacity of regeneration of the surrounding bone. Implantation of specimens
into muscles, enables us to test the sole osteoinductive capacity of a bone substitute or implant [10]. We have demonstrated, that implantation of surface enhanced plates (TiCSA) wrapped with periosteum exhibit no increased induction
of bone and showes incapsulation by formation of dense fibrous tissue containing spindle shaped cells. Surface enhancement by chromosulfuric acid treatment yields a thickened
oxide layer and a very hydrophilic surface [6, 9]. In combination with rhBMP-2 (Ti-CSA-ANC-BMP-2) a proliferation
and differentiation of mesenchymal progenitor cells of the
cambium layer of the periosteum and a differentiation of cells
of the osteoblast lineage is initiated [11]. This results in
enchondral bone formation.
Another important result was that in 25% of the cases where
soluble rhBMP-2 was instilled with the miniplates a formation
of fibrous tissue between the specimen and the induced bone
was observed. This strongly indicates that without immobilization of the rhBMP-2 on the implant surface there is the danger of incapsulation by formation of a fibrous interface and
ectopic bone induction at a distance of the implant even if
only the small quantity of 1 lg of rhBMP-2 is applied. In contrast immobilized rh-BMP seems to minimize the risk of incapsulation by the tendency of increasing the bone implant
interface.
Mat.-wiss. u. Werkstofftech. 32, 942±948 (2001)
Fig. 5. Histological comparison of the
effect of soluble and chemically immobilized rhBMP-2 on bone formation
Abb. 5. Histologischer Vergleich des
Effektes von loÈslichem und chemisch
immobilisiertem rhBMP-2 auf die
Knochenbildung
rhBMP-2 applied in free form (A) as well as chemically immobilized rhBMP-2 (B) induced the formation of woven bone. The statistical
histomorphometric parameters, osteoid perimeter, osteoid area and eroded perimeter displayed no differences between the two groups
(fuchsine stain, 100 magnification). For further details see Table 2.
A limitation of the present study is that we have chosen only
one period of implantation (28 days) and only one dose range
of rhBMP-2 (150 ± 200 ng immobilized; 1 lg soluble rhBMP2). It was the aim of the present study to investigate, whether
rhBMP-2 retains its biological activity after chemical immobilization. In this respect we can conclude that the experiments were very successful. This of course allows no conclusions on the possible improvement of osseointegration by such
biocoated implants. Thus further work is necessary before a
general clinical application.
BMP improves the primary stability of implants in maxillofacial surgery. At three weeks after implantation the reverse
torque strength of titanium screws has been increased from
32 Ncm to 74 Ncm by implantation of a composite of collagen
type I carrier with bovine BMP. In addition the bone contact
was increased from 17% to 82%. The differences between the
groups, however, were much smaller after 12 weeks [12]. Lind
et al [13] tested the integration of specimens in the femoral
condyle of canines. In a push out test osteogenic protein 1
(OP-1, BMP-7) in combination with a collagen matrix was
superior to the matrix without OP-1 after 6 weeks. Compared
to the untreated control collagen as well as the collagen/OP-1
composite demonstrated a threefold increase of bone formation.
On metal surfaces rhBMP-2 can covalently [6] and noncovalently bound [7, 14] . It has been possible to show in
an in vivo gap-healing model that non-covalently immobilized
rhBMP-2 on titanium implants leads to an excellent circumferential osseointegration of the implant within 4 weeks [7].
Adsorbed rhBMP-2 can be slowly released from the surface
making the latter chemotactically active for attracting osteoprogenitor cells for bone induction. Here we show (Figs. 3, 4)
that a surface with simultaneously covalently and non-covalently immobilized rhBMP-2 (chemotactic-juxtacrine surface
[6]) is also very biocompatible and capable of enhanced bone
induction in an ectopic composite periostal flap healing model. Osteoinduction by release of BMP-2 to target cells from
retained protein at the site of application has been described
in a different model by Uludag et al. [15, 16]. Since the biocoated implants employed in this paper introduced a smaller
amount of rhBMP-2 in immobilized form to the periost flap
pocket in comparison to the amount of rhBMP-2 applied in
soluble form it appears surprising that the biological effects
are similar. This discrepancy can probably be explained by
the fact that soluble rhBMP-2 may be lost in the periost pocket
Mat.-wiss. u. Werkstofftech. 32, 942±948 (2001)
by diffusion, degradation and binding to other structures and
proteins. Thus the biological activity is probably reduced by a
factor of 2 ± 5 due to these processes leading to a biologically
effective amount of free rhBMP-2 of the same magnitude as
immobilized on the implant. This would also be in agreement
with the fact that the induced bone was nearly identical in
quantity and quality in both cases. These questions should
be further clarified in future studies.
In conclusion, we were able to demonstrate, that rhBMP-2
retains its biological activity after immobilization on implant
surfaces. The amount of the induced bone was identical in the
groups with rhBMP-2 applied freely and rhBMP-2 coupled
chemically to the implant surface. Therefore surfaces of biomaterials can be modeled by BMP-2-biocoating and a specific
interaction with the target tissue is induced which may be of
great value in future clinical implant applications.
References
1. Barnes, G. L., Kostenuik, P. J., Gerstenfeld, L. C., Einhorn, T.
A., Growth factor regulation of fracture repair. J. Bone Miner.
Res. 14 (1999) 1805 ± 1815.
2. Nimni, M. E., Polypeptide growth factors: targeted delivery
systems. Biomaterials 18 (1997) 1201 ± 1225.
3. Riley, E. H., Lane, J. M., Urist, M. R., Lyons, K. M., Lieberman,
J. R., Bone morphogenetic protein-2: biology and applications.
Clin. Orthop. 324 (1996) 39 ± 46.
4. Zegzula, H. D., Buck, D. C., Brekke, J., Wozney, J. M., Hollinger, J. O., Bone formation with use of rhBMP-2 (recombinant
human bone morphogenetic protein-2). J. Bone Joint Surg. Am.
79 (1997) 1778 ± 1790.
5. Heckman, J. D., Ehler, W., Brooks, B. P., Aufdemorte, T. B.,
Lohmann, C. H., Morgan, T., Boyan, B. D., Bone morphogenetic protein but not transforming growth factor-beta enhances
bone formation in canine diaphyseal nonunions implanted with
a biodegradable composite polymer. J. Bone Joint Surg. Am. 81
(1999) 1717 ± 1729.
6. Jennissen, H. P., Zumbrink, T., Chatzinikolaidou, M., Steppuhn,
J., Biocoating of Implants with Mediator Molecules: Surface
Enhancment of Metals by Treatment with Chromosulfuric
Acid. Materialwiss. Werkstofftech. 30 (1999) 838 ± 845.
7. Jennissen, H. P., Chatzinikolaidou, M., Rumpf, H. M., Lichtinger, T., MuÈller, R., Modification of Metal Surfaces and Biocoating of Implants with Bone-Morphogenetic Protein 2 (BMP-2).
DVM Bericht 313 (2000) 127 ± 140.
Titanium
947
8. Wiemann, M., Rumpf, H. M., Bingmann, D., Jennissen, H. P.,
The Binding of rhBMP-2 to the Receptors of viable MC3T3
Cells and the Question of Cooperativity. Materialwiss. Werkstofftech. 32 (2001) 931 ± 936.
9. Jennissen, H. P., Ultra-Hydrophile metallische Biomaterialien.
Biomaterialien 2 (2001) 45 ± 53.
10. Assenmacher, S., Voggenreiter, G., Fischbacher, M., Nast-Kolb,
D., Knochenbildung durch freie Periostlappentransplantation
im Knochenersatzstoff aus poroÈsem Gips. Chirurgisches Forum 29 (2000) 433 ± 437; Springer, Berlin, Heidelberg, New
York.
11. Lian, J. B., Stein, G. S., Canalis, E., Robey, P.-G., Boskey, A. L.
(1999) Bone Formation: osteoblast lineage cells, growth factors, matrix proteins, and the mineralization process. In Primer
on the metabolic bone diseasees and disorders of mineral metabolism. (Favus, M. J., ed), pp. 14 ± 29.
12. Bessho, K., Carnes, D. L., Cavin, R., Chen, H. Y., Ong, J. L.,
BMP stimulation of bone response adjacent to titanium implants in vivo. Clin. Oral Implants. Res. 10 (1999) 212 ± 218.
13. Lind, M., Overgaard, S., Song, Y., Goodman, S. B., Bunger, C.,
Soballe, K., Osteogenic protein 1 device stimulates bone healing to hydroxyapaptite- coated and titanium implants. J. Arthroplasty 15 (2000) 339 ± 346.
948
G. Voggenreiter et al.
14. Jennissen, H. P., Verfahren zur Herstellung bioaktiver ImplantatoberflaÈchen. Patent Application DE 100 37 850.1 ± 45, pp.
1 ± 16, German Patent Office Munich , 2000.
15. Uludag, H., Gao, T., Porter, T. J., Friess, W., Wozney, J. M.,
Delivery systems for BMPs: factors contributing to protein retention at an application site. J. Bone Joint Surg. Am. 83-A
Suppl 1 (2001) S128 ± S135.
16. Uludag, H., D'Augusta, D., Golden, J., Li, J., Timony, G., Riedel, R., Wozney, J. M., Implantation of recombinant human
bone morphogenetic proteins with biomaterial carriers: A correlation between protein pharmacokinetics and osteoinduction
in the rat ectopic model. J. Biomed. Mater. Res. 50 (2000)
227 ± 238.
Anschrift: Priv.-Doz. Dr. med. Gregor Voggenreiter, Department of
Trauma Surgery, University Hospital Mannheim, University of
Heidelberg, 68165 Mannheim, Germany, Tel.: ‡49-6 21-3 8323 35, Fax: ‡49-6 21-3 83-20 09, e-mail: gregor.voggenreiter
@uch.ma.uni-heidelberg.de
Received: 9/20/01
[T 455]
Mat.-wiss. u. Werkstofftech. 32, 942±948 (2001)