Note: Descriptions are shown in the official language in which they were submitted.
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WO 98/36782 - 1 - PCT/EP98/00742
Bone replacement material with a surface coating of
peptides with the RGD amino acid sequence
The invention relates to bone replacement material
based on a porous polymer material which has a surface
coating of peptides with the RGD amino acid sequence.
Bone replacement materials mean materials used as
implants for replacing or reconstituting bony
structures on account of defects after surgical
operations occasioned by disease or accident. Examples
which may be mentioned are shaped implants such as bone
prostheses of a wide variety of types, bone-connecting
elements, for example in the form of medullary nails,
bone screws and osteosynthesis plates, implant
materials for filling in spongy bone defects or dental
extraction cavities, and for plastic surgical treatment
of contour defects in the maxillofacial region.
Implant materials which are regarded as particularly
favourable for the incorporation process are those
having a high bioactivity, namely such that they are
accepted by the body and integrated into it. In the
case of bone replacement material, this means firm and
permanent adhesion to endogenous tissue, in particular
to bone, should take place soon.
It is known that hitherto the most favourable
incorporation results are in practice achieved only
with endogenous materials, that is to say with bone
transplants. The availability of bone transplants is
limited by their nature. Autologous transplants, that
is to say transplants from the same individual, can, if
in fact available in a suitable shape and quantity, be
removed only by at least one additional surgical
operation, in turn occasioning an additional healing
process at the site of removal. The same also applies
in principle to homologous transplants, that is to say
transplants from donor individuals of the same species.
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With the latter there are also problems of
compatibility and moreover the risk of infection with
viruses such as, in particular, hepatitis and HIV
viruses, which still cannot be completely precluded.
Furthermore, storage of donated material in bone banks
is costly and, in the final analysis, only limited in
time.
Implant materials for bone replacement made of
synthetic materials not related to the body or of
materials related to the body may show a behaviour
ranging from bioinert to bioactive, depending on their
nature and composition. The results of incorporation of
endogenous bone transplants have not, however, to date
been achievable by any synthetic implant material.
Recent findings show that integration of the implant
material into bone must be preceded by cellular
colonization of the surface. This is followed by
deposition of extracellular matrix and formation of new
bone tissue. The complete process is multifactorial and
is considerably influenced by the properties of the
bone replacement material, the vitality of the
substrate bone and the biomechanical circumstances.
It is known that good or very good osteoconductive
properties are possessed by calcium phosphate ceramics,
hydroxyapatite-containing bone cement and specific
polymers which are distinguished, in particular, by a
hydrophilic surface. However, the good
osteoconductivity of these materials often cannot be
combined with optimized biomechanical properties, so
that the ceramics in particular are brittle and have
low adaptability to the elastic requirements in the
bone.
One possibility of stimulating cellular adhesion to
surfaces was found with the discovery of integrins
(proteins in the cell membrane). Integrins recognize
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amino acid sequences, for example the RGD sequence, on
structural proteins and bind thereto. This controls the
adhesion of cells in the body.
The furnishing of implant surfaces with synthetically
available peptides with RGD sequences with the aim of
speeding up the incorporation of implants is known.
Implants disclosed to date are mostly metallic
prostheses, in particular made of titanium or titanium
alloys. However, no convincing implants of this type
with results which might approach the results of
incorporation of endogenous bone transplants are yet
available.
The invention was therefore based on the problem of
providing a bone replacement material which is able not
only to bring about cellular adhesion but also to be
integrated into bone more quickly, and thus has a
biological activity which comes as close as possible to
that of endogenous bone transplants.
It has now been found that this is achieved by a bone
replacement material which is essentially composed of a
porous polymer material with a surface coating of
peptides with the RGD amino acid sequence.
The furnishing of implant surfaces with synthetically
available .peptides with RGD sequences is known. The
implants disclosed and tested to date are mostly
metallic prostheses, in particular made of titanium or
titanium alloys. It is furthermore known (for example
from WO 91-05036) to treat surfaces, such as those of
polymers, metals or else ceramic materials, with
peptides which may, inter alia, also have RGD
sequences. In this case, however, these peptides are
specifically covalently bonded. The said surfaces are
appropriately activated with reactive groups and
reacted with the peptides using a coupling reagent,
whereupon the peptides are covalently bonded. However,
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it contains no hints which would lead to the porous
polymeric bone replacement materials according to the
invention, which are loaded (that is to say no specific
formation of covalent bonds) with peptides which
stimulate cell adhesion on the surface of the implants.
The invention therefore relates to a bone replacement
material based on a porous polymer material which has a
surface coating of peptides with the RGD amino acid
sequence.
The invention relates in particular to a bone
replacement material of this type which is in the form
of a shaped implant.
The invention also relates to an implantation kit
consisting of two or more separate components, one
component of which comprises a bone replacement
material according to the invention and another
component of which comprises a liquid preparation of a
peptide with the RGD amino acid sequence.
The invention furthermore relates to the use of
peptides with the RGD amino acid sequence for loading
the surface of a porous and/or surface-textured polymer
material for bone replacement, whereby biological
activation takes place through stimulation of cell
adhesion to this surface.
The invention further relates to a method for the
biological activation of bone replacement materials
based on a porous polymer material by stimulating cell
adhesion to the surface thereof, which is characterized
in that the surface thereof is coated with a liquid
preparation of a peptide with the RGD amino acid
sequence.
Polymer materials represent a material which is
otherwise of low biocompatibility and, although their
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mechanical properties can be adapted to that of bone,
they have not to date been employed as bone replacement
because they do not unite with the bone.
The relevance of the present invention is now that this
polymer material of low biocompatibility, which would
be very desirable as bone replacement for mechanical
reasons, is optimized by the loading with RGD peptides
and biocompatibility is achieved.
Porous polymeric materials preferred in this connection
are essentially polyacrylates and/or polymethacrylates,
polymethylmethacrylates (PMMA), polyethylenes (PE),
polypropylenes (PP) and/or polytetrafluoroethylenes
(PTFE). It is, of course, also possible to employ
copolymers of the said polymers with one another, and
copolymers of these polymers with other polymers. The
production of polymer materials of these types is
generally known to the skilled person and need not be
explained in detail here.
In a preferred embodiment, the porous polymer material
itself is in the form of a shaped implant in the bone
replacement material according to the invention or, in
another preferred embodiment, it forms the surface or a
surface coating of a shaped implant.
Shaped articles according to the invention which are
particularly preferred are those having a partly or
completely interconnected pore system. Polymers with
such pore systems can be produced, for example, in
analogy to the procedure described in the patent
application EP 0 705 609. However, the skilled person
is furthermore well aware of the general process for
producing porous polymer materials, and there is
therefore no need to pursue this any further here. In
addition, materials of this type are also commercially
available. The skilled person is familiar with their
composition and the way of processing them.
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Also preferred in this connection are polymers or
composites of polymers and mineral or metallic
additives, in particular in particulate or fibrous
form.
If the polymer material itself is in the form of a
porous implant, it can be produced, for example, by the
process described in the abovementioned EP 0 705 609 by
spot fusion of polymethylmethacrylate (PMMA) particles.
This process is essentially carried out by mixing three
different components together. The first component
thereof is a solid component consisting of a fine-
particle polymer of acrylic and/or methacrylic esters
(these polymers are commercially available) and, where
appropriate, other additives such as polymerization
catalysts, X-ray contrast media, fillers and dyes. The
second component is a liquid component consisting of
acrylic and/or methacrylic ester monomers, where
appropriate with additives such as polymerization
accelerators and stabilizers. The third component
consists of coarse-particle granules, of a
biocompatible material with a maximum particle diameter
of from 0.5 to 10 mm. Preferred materials are based on
polyacrylates and/or polymethacrylates, polyolefins,
copolymers of acrylates with styrene and/or butadiene,
and epoxy resins. The three main components are
combined and mixed together. After the components have
been intimately mixed, the polymerization starts owing
to the catalyst which is present; the composition
remains liquid or capable of plastic deformation for a
period of some minutes, after which the cured final
product is present. It is thus possible in this way to
produce porous implants from bone cement particles
which preferably have an interconnecting porosity.
These materials are then loaded according to the
invention with RGD-containing peptides. This porous
bone replacement material can be used in a conventional
way during the liquid or plastic stage as bone cement
for implanting bone prostheses. The surgeon is also
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able to convert the composition into shaped articles of
any shape and size and, after curing, implant them for
reconstruction of bone defects or as local active
substance depots into the body regions to be treated.
In a preferred embodiment, the porous polymer materials
have an average pore width of from 0.05 mm to 2.50 mm,
particularly preferably 0.10 mm to 1.25 mm.
It is thus necessary according to the invention for the
surface of the shaped implant to have a porous form,
which can be achieved, for example, by providing a
composite material or a bone cement with a porous
surface coating or a corresponding roughened surface.
If the porous polymer material forms the surface or the
surface coating of a shaped implant, they can consist
of all known and conventional implant materials as long
as they can be coated with a layer of porous polymer.
Implant materials can be divided into the classes of
mineral, in particular ceramic, materials,
physiologically acceptable metallic materials,
physiologically acceptable polymer materials and
composite materials of two or more materials of the
type mentioned.
Examples of suitable mineral materials are materials
based on calcium-containing materials such as, in
particular, calcium carbonate, calcium phosphates and
systems derived from these compounds. Ones to be
mentioned as preferred from the group of calcium
phosphates are hydroxyapatite, tricalcium phosphate and
tetracalcium phosphate.
However, mineral-based implant materials usually ensure
high mechanical stability only if they are employed as
ceramics, that is to say in the form of materials or
workpieces which have been sintered at sufficiently
high temperatures.
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Details on bone ceramics and particularly favourable
processes for producing them can be found, for example,
in the patent documents DE 37 27 606, DE 39 03 695, DE
41 00 897 and DE 40 28 683.
The metallic material employed is mainly titanium or a
titanium alloy. Also of particular interest are
combined materials whose mechanical properties cover a
considerably wider range than do the pure polymers.
Thus, besides the use of polymers coated with RGD
peptides as bone replacement, combination of materials
of this type with other implant compone [sic] is also
very important.
An example which may be mentioned of such a combination
is combining a metal prosthesis (for example titanium)
with a porous polymer treated according to the
invention. For this purpose, for example, the titanium
implant is treated in a manner known per se for
combining with the polymer. This can take place, for
example, by the Kevloc process or the Sulicoater
process (described in DE 42 25 106). A layer of porous
polymer is then applied to the pretreated titanium
surface, for example in a manner analogous to that
described in EP 0 705 609. The polymer-coated part of
the prosthesis is then subsequently coated with the RGD
peptide.
Another preferred embodiment of this invention is
represented by the following implant material. In place
of the titanium implant, a corresponding implant made
of a fibre composite material (carbon fibre and epoxy
resin) is coated with a porous layer of, for example,
PMMA, after which the peptide coating takes place. An
implant of this type has the advantage of elasticity
adapted to bone, creating a bone-implant interface
without boundary layer, and achieving optimal force
transmission from the implant into the bone.
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Clinically, this avoids bone resorption through stress
shielding, and the prosthesis is held for longer.
The porous polymer layer applied to an appropriate
implant material preferably has a layer thickness of
from 0.2 mm to 25 mm, particularly preferably from
2.0 mm to 20 mm.
The average pore width is preferably in the range from
0.05 mm to 2.50 mm and the range is particularly
preferably from 0.10 mm to 1.25 mm.
Suitable peptides with the RGD sequence which can be
employed according to the invention are all peptides
and their compounds with non-peptide substituents which
contain the amino acid sequence arginine-glycine-
aspartic acid (RGD) and which are able to adhere via
their peptide and non-peptide substituents to the
polymer surfaces.
The following lists of preferred peptides and peptide
compounds are intended to have merely an exemplary and
by no means limiting character, with the following
abbreviations being used:
Asp = Aspartic acid
Gly = Glycine
Arg = Arginine
Tyr = Tyrosine
Ser Serine
=
Phe = Phenylalanine
RGD (Arg-Gly-Asp), GRGD (Gly-Arg-Gly-Asp), GRGDY (Gly-
Arg-Gly-Asp-Tyr), RGDS (Arg-Gly-Asp-Ser), GRGDS (Gly-
Arg-Gly-Asp-Ser), RGDF (Arg-Gly-Asp-Phe), GRGDF (Gly-
Arg-Gly-Asp-Phe), compounds of peptides with fatty
acids or else acrylate-substituted RGD peptides. The
peptides can be either linear or cyclic.
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The coating of the bone replacement material according
to the invention with a peptide compound or a peptide
with the RGD sequence is not difficult per se. It
preferably starts from a suitable liquid solution of
the appropriate peptide, into which the material to be
loaded is immersed. It has been possible to show in
this connection that the eventual coating of the
implant surface is relatively independent of the
concentration of the solution over a wide range. It is
possible with very low concentrations to achieve just
as complete loading of the surface by appropriate
prolongation of the exposure time.
The preferred concentration range for the peptide
solution can be stated to be 10 ng - 100 ~g/ml. The
exposure time is preferably from 10 minutes to
24 hours.
The surface coating with peptides preferably amounts to
50~ to 100 of the free surface.
The peptide substance moreover adheres firmly to the
polymer surface without further treatment. The implants
are sterilized in a customary way, for example by
'y irradiation, heat or ethylene oxide, and are then
ready for implantation.
In a preferred embodiment, the bone replacement
material according to the invention is in the form of
an implantation kit which is ready for use and consists
of two or more separate components, in which one
component comprises a porous polymer material,
preferably as shaped implant, and another component
comprises a liquid preparation of a peptide with the
RGD sequence. An embodiment of this type is
particularly advantageous for effectively countering
possible stability problems which might occur on long-
term storage of finished bone replacement materials
according to the invention. The bone replacement
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materials according to the invention in the form of an
implantation kit of this type are used by loading the
porous polymeric implantation material with the RGD
peptide-containing solution in the prescribed manner
shortly before or during the surgical operation.
Thus, depending on the embodiment, the bone replacement
material according to the invention represents an at
least equivalent substitute for homologous and
autologous bone transplants, or is a considerable
improvement in respect of the incorporation behaviour
for other types of bone replacement.
Not only do the bone replacement materials according to
the invention bring about cellular adhesion, owing to
the immobilization of peptides with the RGD sequence on
a porous polymer implant, but it has also been possible
to demonstrate in experiments that integration of these
implant materials in bone takes place significantly
faster than with untreated implants.
The beneficial effect of the RGD coating on the
incorporation behaviour of implants for bone
replacement is, as already mentioned, applicable to
virtually all types of bone replacement materials and
implant materials as long as they are of such a nature
or design either that they consist wholly or partly of
porous polymer material, or that the implants are
coated with such a porous polymer layer. This
requirement is also met by, for example, implants whose
surface has a porous structure or is at least
roughened.
It is possible in principle for the bone replacement
materials according to the invention to be in the form
not only of shaped implants but also of powders,
granules, particles or fibres, depending on the
requirements of the site of insertion and the purpose
of use .
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It is assumed even without further explanation that a
skilled person is able to utilize the above description
in the widest sense. The preferred embodiments are
therefore to be interpreted merely as a descriptive and
by no means as in any way a limiting disclosure.
The complete disclosure of all the applications,
patents and publications mentioned hereinbefore and
hereinafter is incorporated into this application by
reference.
The following examples are representative of the
present invention.
Example 1
a) Production of a porous polymer shaped article
A low-viscosity bone cement (composition: 31 g of
polymethylmethacrylate/polymethylacrylt [sic]
(94/6) copolymer, 6 g of hydroxyapatite powder,
3 g of zirconium dioxide) is stirred with 30 ml of
methylmethacrylate monomer in a conventional way.
The components comprise the dibenzoyl
peroxide/dimethyl-p-toluidine starter system.
100 g of pure, cylindrical polymethylmethacrylate
granules (diameter 2 mm, length 3 mm) are added to
this ,paste and thoroughly mixed with the bone
cement paste. The mixed composition is put in
polypropylene moulds and left to cure for about
15 min. The result is an article with
inteconnecting [sic] pores and a porosity of 20~.
b) Loading of the polymeric shaped article with RGD
peptide
The shaped article obtained as in a) is immersed
in a solution of the tetrapeptide GRGD-
concentration 100 ~.g/ml, exposure time about 60
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minutes, for loading with this RGD peptide, and is
finally dried. The success of the coating was then
measured in a cell adhesion test. The results show
that the unloaded cylinders are virtually not
colonized by cells, whereas the materials
according to the invention show a dense cell lawn
extending deep into the pores.
Example 2
Experimental investigations
Species: Rabbit
Implants: a) porous PN~IA shaped article
b) PMMA shaped article according to
the invention coated with GRGD
Both implants were sterilized by y
irradiation and implanted into rabbit
f emora .
Implantation site: Into the patellar sliding bearing
of the left and right femora.
After 2 weeks, the new bone formation and the
mineralization are assessed by histological
examination.
Result:
a) PMMA
The implant bed shows only a thin circular ring of
newly formed trabecular bone permeated with
connective tissue. There is no evident direct
superimposition of the trabecular bone on the
cement beads.
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b) PMMA + GRGD
Extensive new trabecular bone formation can be
found in this case and envelops three quarters of
the entire implant; the trabecular bone is
directly superimposed on the cement beads.
