U.S. patent application number 10/343520 was filed with the patent office on 2004-06-10 for process for the preparation of bioactive implant surfaces.
Invention is credited to Chatzinikolaidou, Maria, Jennissen, Herbert P., Rumpf, Heike.
Application Number | 20040109937 10/343520 |
Document ID | / |
Family ID | 7651198 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040109937 |
Kind Code |
A1 |
Jennissen, Herbert P. ; et
al. |
June 10, 2004 |
PROCESS FOR THE PREPARATION OF BIOACTIVE IMPLANT SURFACES
Abstract
The invention relates to a method for producing bioactive
implant surfaces consisting of metallic or ceramic materials, to be
used for implants such as artificial joints or very small implants
such as so-called stents. The invention also relates to implants
produced according to this method.
Inventors: |
Jennissen, Herbert P.; (Alte
Klosterstr 19, DE) ; Chatzinikolaidou, Maria; (Essen,
DE) ; Rumpf, Heike; (Marl, DE) |
Correspondence
Address: |
Harris F Brotman
Procopio Cory Hargreases & Savitch
530 B Street
Suite 2100
San Diego
CA
92101
US
|
Family ID: |
7651198 |
Appl. No.: |
10/343520 |
Filed: |
August 15, 2003 |
PCT Filed: |
August 1, 2001 |
PCT NO: |
PCT/DE01/02893 |
Current U.S.
Class: |
427/2.26 ;
623/23.57; 623/23.59 |
Current CPC
Class: |
A61L 2300/414 20130101;
A61L 31/08 20130101; A61L 2300/406 20130101; A61L 31/10 20130101;
A61L 27/50 20130101; A61L 27/227 20130101; A61L 2300/802 20130101;
A61P 19/00 20180101; A61L 27/28 20130101; A61L 27/54 20130101 |
Class at
Publication: |
427/002.26 ;
623/023.57; 623/023.59 |
International
Class: |
B05D 003/10 |
Claims
1. A process for the production of bioactive implant surfaces of
metallic or ceramic materials in which, in a first step, anchor
molecules having hydrophobic radicals are covalently bonded to the
surface of the implant material and, in a second step, mediator
molecules which, as a result of non-covalent interactions between
the mediator molecules and the hydrophobic radicals of the anchor
molecules, are immobilized, are added to the implant material
treated in this way, where in the first step the loading density of
the anchor molecules on the implant surface is chosen, depending on
the chain length of the hydrophobic radical of the anchor molecule,
such that the anchor molecules do not interact with one another
themselves and, depending on the covered surface on the implant
material, which is covered by an individual mediator molecule
absorbed in the second step, at least 10, preferably 15, contact
sites are formed between the hydrophobic radicals of the anchor
molecules for hydrophobic interaction with the individual mediator
molecule.
2. The process as claimed in claim 1, in which the hydrophobic
radicals of the anchor molecules are radicals having 1 to 30,
preferably 3 to 20, particularly preferably 3 to 8, carbon atoms,
which can also be replaced by silicon or heteroatoms such as N, O
or S in the chain, where the hydrophobic radicals can also
optionally be substituted by one or more substituents from halogen,
alkoxy, hydroxyl, thiol, amino, alkyl-amino, dialkylamino or
trialkylamino groups, where the alkyl groups of the substituent
preferably have 1-6 carbon atoms and can be straight-chain or
branched.
3. The process as claimed in claim 2, in which the hydrophobic
radicals are branched carbon chains optionally substituted as above
and having 1 to 30, preferably 3 to 20, particularly preferably 3
to 8 carbon atoms.
4. The process as claimed in any of the preceding claims, in which
the implant material consists of a material selected from the group
consisting of the metals, the metallic alloys, the ceramic
materials or combinations thereof.
5. The process as claimed in any of the preceding claims, in which
the mediator molecules used are biologically active substances such
as bone growth factors from the class consisting of the BMP
proteins, antibiotics or mixtures thereof.
6. The process as claimed in claim 5, in which the bone growth
factor used is BMP-2 or BMP-7.
7. The process as claimed in any of the preceding claims, in which
the surface of the implant material is provided with a hydrophilic
coating before the covalent bonding of the anchor molecules.
8. The process as claimed in claim 7, in which the hydrophilic
coating is a hydrophilic oxide layer.
9. The process as claimed in claim 7 or 8, in which the surface of
the implant material, selected from titanium, titanium alloys,
aluminum or stainless steel, is provided with an oxide layer before
the covalent bonding of the anchor molecules by treatment with
chromosulfuric acid for a period of 0.5 up to 3 hours at 100 to
250.degree. C.
10. The process as claimed in claim 9, in which the chromosulfuric
acid has a density of more 1.40 g/cm.sup.3.
11. The process as claimed in any of the preceding claims, in
which, in the first step, as anchor molecules, hydrocarbon
radicals, which can be straight-chain or branched, having 1 to 30,
preferably 1 to 20 carbon atoms, particularly preferably 1 to 15
carbon atoms, which can also optionally be substituted by one or
more substituents from halogen, alkoxy, hydroxyl, thiol, amino,
alkyl or dialkylamino groups, are covalently bonded to the surface
of the implant material and, in the second step, bone growth
factors which, as a result of noncovalent interactions between the
bone growth factors and the hydrophobic radicals of the anchor
molecules, are immobilized, are added to the implant material
treated in this way, where, in the first step, the loading density
of the anchor molecules on the implant surface is chosen, depending
on the chain length of the hydrophobic radical of the anchor
molecule, such that the anchor molecules do not interact with one
another themselves and, depending on the covered surface on the
implant material, which is covered by an individual bone growth
factor molecule absorbed in the second step, at least 10,
preferably 15 contact sites are formed between the hydrophobic
radicals of the anchor molecules for hydrophobic interaction with
the individual bone growth factor molecule.
12. The process as claimed in claim 11, in which the carbon
radicals are immobilized in the first step, depending on the degree
of branching of the radical used, in a number of at least 3,
preferably at least 5 and particularly preferably at least 10
radicals to 10 nm.sup.2 of the implant surface.
13. The process as claimed in claim 11 or 12, in which the carbon
radicals are immobilized in the first step, depending on the degree
of branching of the radical used, in a number of at most 100,
preferably at most 60 radicals per 10 nm.sup.2 of the implant
surface.
14. An implant, obtainable by the process as claimed in any of
claims 1-13.
15. The implant as claimed in claim 14, in which the implant
material consists of titanium, titanium alloys, aluminum, stainless
steel, steel alloys or hydroxyapatite.
16. The implant as claimed in claim 14 or 15, in which the implant
is a joint or bone prosthesis, a stent or a dental implant.
Description
[0001] The present invention relates to a process for the
production of bioactive implant surfaces of metallic or ceramic
materials which are used for implants such as artificial joints,
dental implants or alternatively very small implants, e.g.
"stents", and to implants produced by the process, which, as an
"active device", permit a controlled release of the bioactive
molecules from the implant materials.
[0002] The implantation of artificial joints or bones has gained
increasing importance in recent years, e.g. in the treatment of
arthrodysplasia or joint luxation or in diseases which can develop
on the wear of joints as a result of malarticulation. The function
of the implants and the materials used for their production, which
in addition to metals such as titanium or metal alloys can also
include ceramics or plastic materials such as Teflon, have
continually been improved such that implants can have service lives
of 10 years in 90-95% of cases after a successful course of
healing. Regardless of these advances and improved operative
procedures, an implantation still remains a difficult and irksome
intervention, in particular since it is associated with a prolonged
healing process of the implant, which often comprises stays for
months in clinics and health resorts including rehabilitation
measures. In addition to the pain, the length of the treatment
period and separation from the familiar environment are great
burdens here for the patients affected. Furthermore, the prolonged
healing process causes high personnel and care costs due to the
intensive care being necessary.
[0003] The understanding of the processes at the molecular level
which are necessary for successful ingrowth of an implant has
expanded significantly in recent years. Structural compatibility
and surface compatibility are crucial for the tissue compatibility
of an implant. Biocompatibility in the narrower sense is limited
solely by the surface. At all levels of integration, proteins play
a decisive role. As explained below, they decide about the further
course of the implant healing even during the implantation
operation due to the formation of an initial adsorbed protein
layer, since the first cells later settle on this layer.
[0004] In the molecular interaction between implant, which is also
designated as a biomaterial, and tissue, a large number of
reactions take place which appear to be strictly hierarchically
ordered. As the first biological reaction, the adsorption of
proteins on the surface of the biomaterial takes place. In the
protein layer resulting thereby, subsequently individual protein
molecules are converted, for example either by means of
conformational changes to give signal substances which are
presented on the surface, or protein fragments acting as signal
substances are released by means of catalytic (proteolytic)
reactions. Triggered by the signal substances, in the next phase
cell population takes place, which can include a multiplicity of
cells such as leucocytes, macrophages, immunocytes, and finally
also tissue cells (fibroblasts, fibrocysts, osteoblasts,
osteocytes). In this phase, other signal substances, "mediators",
such as, for example, cytokines, chemokines, morphogens, tissue
hormones and true hormones play a crucial role. In the case of
biocompatibility, integration of the implant into the entire
organism takes place, and ideally a permanent implant is
obtained.
[0005] In the light of studies which have been carried out in
recent years at the molecular level of osteogenesis, chemical
signal substances, the "bone morphogenic proteins" (BMP-1-BMP-14),
which have an influence on bone growth, have gained increasing
importance. BMPs (in particular BMP-2 and BMP-4, BMP-5, BMP-6,
BMP-7) are osteoinductive proteins, which stimulate bone
regeneration and healing of the bone by bringing about the
proliferation and differentiation of precursor cells to
osteoblasts. Moreover, they promote the formation of alkaline
phosphatase, hormone receptors, bone-specific substances such as
collagen type 1, osteocalcin, osteopontin and finally
mineralization. The BMP molecules in this case regulate the three
key reactions chemotaxis, mitosis and differentiation of the
respective precursor cell. Moreover, BMPs play an important role in
embryogenesis, organogenesis of the bone and other tissue, known
target cells being osteoblasts, chondroblasts, myoblasts and
vascular smooth muscle cells (inhibition of proliferation by
BMP-2).
[0006] 14 BMPs including multiple isoforms are now known. Except
for BMP-1, the BMPs belong to the "transforming growth factor beta"
(TGF-) superfamily, for which specific receptors have been detected
on the surfaces of the corresponding cells. As the successful
employment of recombinant human BMP-2 and/or BMP-7 in experiments
relative to defect healing processes in rats, dogs, rabbits and
monkeys has shown, no species specificity appears to be present.
Previous experiments to utilize the bone formation-inducing
properties of the BMPs specifically for implantation purposes by
applying BMP-2 and/or BMP-7 noncovalently to metallic or ceramic
biomaterials have, however, very largely proceeded
unsuccessfully.
[0007] The object of the present invention consists in making
available improved biomaterials for use as implants, which are
distinguished by an increased loading density with mediator
molecules, in particular BMPS, and a prolonged long-term release
into the tissue surrounding the implants.
[0008] According to the invention, this object is achieved by a
process for the production of bioactive implant surfaces of
metallic or ceramic materials being made available in which in a
first step anchor molecules having hydrophobic radicals are
covalently bonded to the surface of the implant material and in a
second step mediator molecules which, as a result of noncovalent
interactions between the mediator molecules and the hydrophobic
radicals of the anchor molecules, are immobilized, are added to the
implant material treated in this way, where in the first step the
loading density of the anchor molecules on the implant surface is
chosen, depending on the chain length of the hydrophobic radical of
the anchor molecule, such that the anchor molecules do not interact
with one another themselves and, depending on the covered surface
on the implant material, which is covered by an individual mediator
molecule absorbed in the second step, at least 10, preferably 15,
contact sites are formed between the hydrophobic radicals of the
anchor molecules for hydrophobic interaction with the mediator
molecule.
[0009] The undesired interaction between the anchor molecules is
primarily to be understood as meaning a steric interaction, which
is not desired here, in order that the anchor molecules can
interact with the mediator molecules in a manner which is
sterically unhindered by one another.
[0010] Contact site is to be understood according to the invention
as meaning the site of the greatest hydrophobic interaction between
a radical of the anchor molecules and the mediator molecule. In
this case, a number of contact sites can be present on one radical
due to branching of the radical. Thus, a carbon chain terminally
substituted by a methyl group can have at least two contact sites.
The inventors have recognized that the immobilization of the
mediator molecules by hydrophobic interaction depends crucially on
the number of the contact sites for the hydrophobic interaction
between the radicals and the mediator molecule. In this case,
adjacent contact sites which are as close as possible are
advantageous, so that more strongly branched radicals are
preferred, since a number of adjacent contact sites are available
here. For example, a terminal trimethyl group on a hydrophobic
radical is preferred compared with a straight-chain unbranched
chain having the same total number of carbon atoms.
[0011] In the immobilization process according to the invention, a
degree of substitution of the anchor molecule is in particular
achieved, therewith indirectly (i.e. surface concentration of the
immobilized protein), which permits a multivalent interaction
between surface and cell and makes it possible effectively to
control bone or tissue formation.
[0012] In the process according to the invention, in a first step
alkyl, alkenyl or alkynyl or aryl radicals having 1 to 30,
preferably 3 to 20, particularly preferably 3 to 8, carbon atoms,
which can also be replaced by silicon in the alkyl chain and/or
heteroatoms such as N, O or S in the alkyl chain and/or in the aryl
ring, preferably in a branched chain, which can also optionally be
substituted by one or more substituents from halogen, alkoxy,
hydroxyl, thiol, amino, alkyl- or dialkylamino groups, where the
alkyl groups of the substituent preferably have 1-6 carbon atoms
and can be straight-chain or branched, but are preferably
unsubstituted and particularly preferably branched, are preferably
covalently bonded to the surface of the implant material. This
bonding of the radicals can in each case take place by means of a
coupling via a silyl group, a bromocyano group or an amino group,
for example of an aminoalkane.
[0013] In a second step, mediator molecules such as bone growth
factors can be immobilized on the implant material by means of
noncovalent bonding, presumably on account of hydrophobic
interactions on the implant material. It is thereby made possible
to form a chemotactically acting and/or biologically active,
"juxtacrine", implant surface, which leads to the colonization,
proliferation and differentiation of bone cells. Thus, "active
implants" can be made available which in the case of molecules
released from the surface show a chemotactic action on cells, in
the case of BMPs on osteoblasts, at a distance of 500 to 1000
.mu.m.
[0014] The determination of the loading density of the implant
surface with anchor molecules, which as a rule only have one
hydrophobic radical with, depending on the degree of branching, at
least one contact site, is as a rule carried out starting from the
size estimation of the mediator molecule, which is usually present
as an ellipsoid. Subsequently, after perusal of the surface area of
the mediator molecule projected onto the implant surface, the
number of necessary contact sites is determined as at least 10 and,
as a function thereof, the chain length and the degree of branching
of the anchor molecules is established. The loading density is then
calculated from this.
[0015] Initial investigations of the inventors showed that after
modification of titanium surfaces with amino-propylsilane (APS),
the number of immobilized amino groups determined using the
Bolton-Hunter reagent showed values in the range from 1.0-2.5
nmol/cm.sup.2. Taking into consideration Loschmidt's number, about
60 molecules/10 nm.sup.2 result at 1 nmol/cm.sup.2. From this
value, a mean distance of the APS molecules from one another of
about 0.4-0.5 nm can be calculated, which appears to be a
reasonable value.
[0016] In the case of the coupling of the protein ubiquitin (m=8.5
kDa), the inventors obtained maximum values of 1-2 g/cm.sup.2. On
calculation using 1 g/cm.sup.2, 3.85.times.10-11 mol/cm.sup.2 are
obtained. The conversion to molecules then gives 2.3 molecules of
ubiquitin per 10 nm.sup.2, thus a mean distance of the molecules
with the assumption of a point size of 6.7 nm, which in the case of
an estimated actual size of 3-4 nm diameter for the ubiquitin
molecule means a quite high packing density in the form presumably
of a monolayer on the surface. Since in the case of the adsorption
of ubiquitin similarly high values (as in the case of the coupling
of BMP-2) in the range from 1-3 g/cm.sup.2 (=2-6 mol of
ubiquitin/10 nm.sup.2) are obtained, the inventors were able to
calculate that on average to one molecule of ubiquitin 10-30
molecules APS (60/6 and 60/2) are available for an interaction
reaction. The inventors were thus able to estimate that one
molecule of ubiquitin covers an area ("footprint") which contains
approximately this number of APS molecules, i.e. at most 10-30 APS
molecules can theoretically react with one molecule of ubiquitin,
where a random reaction is to be assumed.
[0017] With the assumption of a hydrophobic adsorption, according
to determination of the inventors not all (i.e. 30) propyl radicals
can react with the ubiquitir, since it does not have so many
"hydrophobic patches" for a geometrically defined bond on one side
of the molecule. According to estimation of the inventors, at most
4-10 alkyl radicals on the ubiquitin are therefore able to find a
specific binding site and actually lead to the adsorption of
ubiquitin.
[0018] If the degree of substitution is now reduced, i.e. the
number of alkyl radicals/10 nm.sup.2, the distance becomes so great
that sufficient radicals can no longer react with the ubiquitin,
and adsorption no longer takes place. On the other hand, if the
alkyl chain length is increased, the binding energy of an alkyl
chain with the protein is increased, and only a few alkyl radicals
are needed in order to bind a molecule of ubiquitin.
[0019] When using BMP-2 (m=26 kDa) having a size of about
4.times.4.times.8 nm (bonding to longitudinal side), the inventors
initially assumed a maximum occupation of approximately 0.5-1
molecule per 10 nm.sup.2. This means that for BMP-2 on the basis of
an approximately twice as large "footprint" only approximately half
the number of the molecules are absorbed, BMP-2 instead also can
cover approximately twice as many immobilized alkyl radicals
(20-50), of which also, in turn, according to calculation of the
inventors, presumably only at most 8-20 are available sterically
for interactions with the BMP-2.
[0020] From experiments of the inventors with hexylagaroses which
have only approximately 7-8 alkyl radicals/10 nm.sup.2, it is known
to them that an adsorption of BMP-2 is not possible with this low
number of interaction partners. Experiments of the inventors have
therefore shown that only in a higher range from about 10-60 alkyl
radicals/10 nm.sup.2 at a calculated distance of the radicals of
0.5-3 nm is a satisfactory adsorption of BMP-2 possible. An
adsorption with a half-life of release of 90-100 days is, according
to the knowledge of the inventors, only possible if a number of at
least 8-15 alkyl radicals per BMP-2 molecule can be available for
the reaction at specific sites. This interaction will, however,
probably only be poor statistically according to calculation of the
inventors at a degree of substitution of below 10 alkyl radicals/10
nm.sup.2, such that higher degrees of substitution are more
promising.
[0021] On the part of the inventors, it was found that a dependence
of the chain length of the alkyl radicals employed and of the
distance of the alkyl radicals from one another for the
best-possible adsorption of mediator molecules exists. On the one
hand, the length of the chains must not be so large that the
radicals are tangled together on the implant surface, on the other
hand the distance of the radicals to one another must be so great
that these do not interact with one another. Depending on the size
of the absorbed mediator molecule, best-possible values for the
occupation of the surface of the implant with respect to the chain
length, the degree of branching of the chain and the distance of
the radicals thus result for the individual case. For the
adsorption of the BMP-2, the inventors have determined an
occupation of 10 to 60 radicals per 10 nm.sup.2, preferably 10 to
30 radicals per 10 nm.sup.2, at a chain length of between 1 to 30,
preferably 1 to 20, particularly preferably 1 to 8, carbon atoms,
preferably in a chain which can also optionally be substituted by
one or more substituents from halogen, alkoxy, hydroxyl, thiol,
amino, alkyl or dialkylamino groups.
[0022] In a preferred variant, to increase the interaction between
the radicals on the surface of the implant and the mediator
molecules, the surface of the implant is first hydrophilized by
applying a hydrophilic coating, in a further step the hydrophobic
radicals on the surface modified in this way are then immobilized
and then the mediator molecules are added to the surface for the
noncovalent hydrophobic interaction with the radicals, the ratios
of chain length and degree of occupation indicated above preferably
being used for BMP-2.
[0023] The process according to the invention for the
immobilization of the mediator molecules is distinguished in that
the implant material employed consists of metallic materials such
as pure titanium or metallic titanium alloys such as
chromium/nickel/aluminum/vanadium/cobalt alloys (e.g. TiAlV4,
TiAlFe2.5), stainless steels (e.g. V2A, V4A, chrome nickel 316L) or
ceramic materials such as hydroxyapatite, alumina or of a
combination, in which, for example, metallic material is coated
with ceramic material. Synthetic polymer materials are also
suitable for use as implant material.
[0024] The invention also relates to therapeutically preventing or
alleviating, by coating a coronary stent (length about 10 mm) with
the aid of a biomolecule or of a mediator, e.g. BMP-2, the late
complication restenosis, which is caused by a proliferation of
vascular smooth muscle cells, in order thus to promote healing and
compatibility.
[0025] According to the invention, the mediator molecules can be
biomolecules which are advantageous for the biocompatibility of the
implant, in that they counteract a possible rejection of the
implant and/or promote the ingrowth of the implant.
[0026] Mediator molecules which can be used in the present process
are preferably proteins promoting bone growth from the class
consisting of the bone growth factors `bone morphogenic proteins`
or alternatively ubiquitin. Advantageously, for immobilization a
protein of this class on its own, in combination with further
members of this class or alternatively together with biomolecules
such as proteins of other classes or low molecular weight hormones
or alternatively antibiotics, can be employed for the improvement
of immune defense. In this case, these molecules can also be
immobilized on the surface by means of bonds which can be cleaved
in the biological medium.
[0027] According to the invention, the surface of the implant
material is preferably chemically activated, the activation taking
place by means of a silane derivative such as, for example,
-aminopropyltriethoxysilane or a trimethylmethoxy- or
trimethylchlorosilane derivative or
3-glycidoxypropyltrimethoxysilane and the reaction being carried
out both in an aqueous solvent and in an organic solvent. In a
second step, mediator molecules can be immobilized on the implant
material by means of noncovalent bonding to the surface activated
in this way.
[0028] The process is characterized in that for the hydrophobic
interaction the stationary insoluble phase used is a carrier on
which a monomolecular, entropically ordered water structure is
formed on apolar groups arranged gridlike situated thereon. A
similar ordered monomolecular water layer is present on the
hydrophobic areas of the protein (BMP). If the two molecules (e.g.
alkyl radicals and BMP-2) come into contact with one another with
their monomolecular water layers, the water layers are destroyed by
a more unordered system of individual water molecules becoming of
the ordered water structure. The free energy of the interaction
thus results due to an increase in the entropy of the water
molecules. At relatively high temperature, these hydrophobic
interactions become stronger (greater free energy).
[0029] The BMP must be brought to hydrophobic interaction with a
suitable hydrophobic carrier. Such a carrier consists, for example,
of an insoluble phase and hydrophilic and hydrophobic chemical
structures situated thereon. In particular, suitable carriers are
all solid phases having hydrophilic surfaces which carry additional
hydrophobic/apolar groups.
[0030] Specific examples for such carriers of organic and inorganic
type are celluloses, agaroses or appropriate polymer particles
coated with carbohydrates or polyhydroxycarbon chains, i.e.
hydrophilically, and silica, zeolite or aluminum hydroxide
particles.
[0031] Novel carriers are hydrophilic metal surfaces which are
occupied/substituted gridwise appropriately with alkyl or aryl
groups, for example, in a later process. On such solid phases,
suitable degrees of substitution with hydrophobic groups lie in a
range from 0.01 to 3.0 nmol/cm.sup.2, preferably in a range from
0.01 to 2.0 nmol/cm.sup.2, where the ratios indicated above should
be kept to, in particular when using BMP-2.
[0032] A hydrophilic solid phase suitable for substitution with
hydrophobic groups is preferably metal surfaces cleaned in dilute
acid or metal surfaces enhanced using chromosulfuric acid having
contact angles of between 0-90.degree., preferably 0-20.degree.. In
particular, suitable hydrophobically interacting metal surfaces
cleaned with dilute acid are those such as titanium, steel, steel
alloys such as Cr/Mo steel or steel or titanium surfaces enhanced
using chromosulfuric acid, which has been substituted with methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, dodecyl or
hexadecyl groups (chain length 1-30, preferably 1-20, particularly
preferably 1 to 8 carbon atoms, preferably in a chain, which can
also be substituted by one or more substituents such as methyl,
ethyl, methoxy or ethoxy groups or halogen atoms such as chlorine,
fluorine). The hydrophobic alkyl interaction can be strengthened by
combination with a sulfur atom, for example in the form of a
thioether bond or as a thiol such as (mercaptopropyl radicals).
[0033] Particular forms of hydrophobic interaction can also be
achieved using immobilized aromatic radicals (phenyl or tolyl
radicals, 6-7 C atoms), in particular in combination with sulfur
atoms (phenylthiosilane, or thienyl radicals, 4-6 C atoms).
[0034] The hydrophobic interaction at the contact sites takes place
at temperatures from 0.degree.-100.degree. C., preferably at
5-50.degree. C. at a pH of 3.0-11.0, preferably at pH 6-10.
Preferably suitable degrees of substitution with the radicals are
0.1-2.5 nmol/cm.sup.2, which corresponds to a lattice distance of
the alkyl or aryl groups covalently coupled on the surface of 0.2-5
nm, preferably 0.3-1 nm.
[0035] With a molecule of 5-6 nm diameter, for example of BMP-2, at
high degrees of substitution and thus small lattice distances
(0.2-1 nm), a number of such radicals could react with the molecule
and bond it firmly. The bonding strength (affinity) of the surface
is thus proportional to the chain length of the alkyl radical and
the degree of substitution and increases greatly with these
parameters. A preferable bonding of BMP-2 takes place from a chain
length of C-1, preferably C-3 (propyl) with a degree of
substitution of 0.01-2.5 nmol/cm.sup.2, preferably of 0.2-1
nmol/cm.sup.2. With shorter alkyl chains, higher degrees of
substitution, and with longer chains, lower degrees of substitution
are preferred as minimum sizes.
[0036] Suitable substances for the synthesis of alkyl- or
aryl-loaded metal surfaces are alkyltrichlorosilanes
(methyltrichlorosilane, ethyltrichlorosilane,
propyl-trichlorosilane, etc.), dialkyldichlorosilanes
(di-methyldichlorosilane, diethyldichlorosilane,
dipropyl-dichlorosilane etc.), trialkylchlorosilanes
(trimethyl-chlorosilane, ethyldimethylchlorosilane, propyl- etc.),
alkyltrimethoxysilanes, methyltrimethoxysilane, ethyl-, propyl-
etc.), alkyltriethoxysilanes (methyltriethoxy-silane, ethyl-,
propyl- etc.), phenyltrichlorosilane, phenyldimethylchlorosilane,
phenylthiotrimethylsilane, p-tolyltrichlorosilane.
[0037] Continuing investigations of the inventors have shown that
the anchoring of the alkyl or aryl radicals to the surface of the
implant material can be improved qualitatively and quantitatively
by providing on the implant surface a hydrophilic coating, for
example agarose, polyacrylate, or preferably by increasing the
number of metal oxide units available on the surface.
[0038] On the part of the inventors, it has been found that the
number of oxide groups can surprisingly be increased by treating
the surface of the metal with hot, preferably sediment-free,
chromosulfuric acid. In contrast to the expectation that the metal
dissolves under these conditions, when using this acid a novel
essentially uniform hydrophilic oxide layer 5-50 nm thick is
produced on the surface of the metal. The process is so gentle that
even coronary stents (which can be manufactured, for example, from
stainless steel or titanium) can be coated without destruction of
the thin sensitive lattice work (50-150 m diameter). In the case of
large implants, the hydrophilic oxide layer can achieve a thickness
of 10 m up to 100 m and can be built up relatively "smoothly"
without hollows or holes. The metal employed for the implant can in
this case be pure titanium or titanium alloys (e.g. TiAlV4,
TiAlFe2.5), aluminum or stainless steel (e.g. V2A, V4A, chrome
nickel 316L, Cr/Mo steel). A commercially available chromosulfuric
acid containing 92% by weight of H.sub.2SO.sub.4, 1.3% by weight of
CrO.sub.3 and having a density of 1.8 g/cm.sup.3, obtainable, for
example, from Merck, is preferably used to achieve a thin smooth
layer of metal oxide. For this, the metal substrate is inserted
into the chromosulfuric acid and treated for a period of 1 up to 3
hours at 100 to 250.degree. C., preferably for 30 to 60 minutes at
240.degree. C., then carefully rinsed with water, then boiled for
30 min in water or a solution of 1-4% EDTA (ethylenediamine
tetraacetate) pH 7.0, preferably 2% EDTA pH 7.0, in order to remove
heavy metal ions, e.g. chromium ions, remaining on the surface and
then dried.
[0039] If a thicker metal oxide layer is to be provided on the
metal surface and/or preferably an oxide layer having small micro-
and nanopores, the chromosulfuric acid described above is diluted
with water to a density of 1.5 to 1.6 g/cm.sup.3. In a subsequently
following treatment of the metal implant surface as described above
with the acid diluted in this way, a "rough" surface layer having
hollows and pores is formed, such that the surface available for
loading with mediator molecules is enlarged. By adjustment of
different densities of chromosulfuric acid and different treatment
times and temperatures, it is therefore possible to apply a
multiplicity of various oxide layers of different properties to
metal surfaces with high adhesiveness. The invention is therefore
also directed at such a process for the formation of a
thermodynamically uniform metal oxide layer (no contact angle
hysteresis) on the implant material by means of hot chromosulfuric
acid.
[0040] The metal oxide layer on the implant material of the
abovementioned materials can then be activated by means of
treatment with dilute nitric acid (about 5% by weight) and
subsequent coupling of a silane derivative.
[0041] The mediator molecules can then be anchored noncovalently to
the implant surface via the molecules of the silane derivative.
[0042] The implant material used can also be a ceramic material
such as, for example, hydroxyapatite. The hydroxyapatite should in
this case first be activated by treatment with aminoalkylsilane and
the anchor molecules should then be anchored. According to the
invention, anchor molecules are to be understood as meaning those
molecules which are anchored to the surface of the implant and show
noncovalent interactions with the mediator molecules if in the next
step a noncovalent bonding of the mediator molecules, such as BMP,
to the surface takes place.
[0043] If, under the coupling conditions, the mediators employed
are poorly soluble in the medium, the solubility can be increased
by addition of surfactants/detergents and the reaction can be
carried out. Thus, at pHs of >6, poorly soluble bone growth
factors and other mediators can be kept in solution by ionic or
nonionic detergents in the concentration range 0.05-10%, preferably
1-5%, by weight, in particular in 0.066% SDS and pHs of >6, in
particular at pH 8-10 for noncovalent bonding processes in the
alkaline pH range without loss of the biological activity.
[0044] The influence of the materials modified by the process
according to the invention on bone cells was investigated in animal
experiments, the modified materials for this purpose having been
prepared in platelet or dumbbell form. It was observed here that 4
weeks after the incorporation into the animals accelerated bone
formation with contact to the implant surface by BMP-2 occurred on
the materials.
[0045] The present invention is illustrated further with the aid of
the following examples.
[0046] Modification of Metals (Titanium, 316 L Stainless
Steel):
[0047] Either mechanically polished/electropolished, anodic-ally
oxidized small titanium plates or small titanium alloy plates
plasma-sprayed with porous titanium alloy with or without
chromosulfuric acid enhancement are employed. To the same extent,
stainless mechanically polished/electropolished steels with or
without chromosulfuric acid enhancement can be employed.
[0048] Cleaning Processes
[0049] Before each use, the metals are cleaned by heating to
80.degree. C. in 5% HNO.sub.3 for 2 hours. After washing again in
water, the small plates were dried by washing in 30 ml of dry
methanol. Afterwards, they were either used directly or enhanced
with chromosulfuric acid.
[0050] Chromosulfuric Acid Enhancement
[0051] In the chromosulfuric acid enhancement, the small titanium
plates were incubated in chromosulfuric acid (92% H.sub.2SO.sub.4,
1.35 CrO.sub.3) at 190-240.degree. C. for 30-90 min and the small
steel plates at 190-230.degree. C. for 30-90 min. Afterwards, the
metal samples were washed copiously with water and then boiled in
2% EDTA pH 7.0 and subsequently in water for 30 min in each case.
After washing again in water, the small plates were dried by
washing in 30 ml of dry methanol.
[0052] Loading of Surfaces with Aminopropyltriethoxysilane:
[0053] The cleaned carriers (5-10 small titanium plates) were
treated under inert gas with or without chromosulfuric acid
enhancement with 47.5 ml of toluene and 2.5 ml of
aminopropyltriethoxysilane in a Teflon holder and sealed. The batch
was then boiled under reflux and with slow stirring for 3-3.5
hours. The small plates were then washed 3 times with 10 ml of
trichloromethane, acetone and methanol and then air-dried. At the
aminopropyltriethoxysilane concentration indicated, it was possible
with the aid of the Bolton-Hunter method to determine a surface
concentration of amino groups of 1.5 to 2.5 nmol/cm.sup.2.
[0054] Loading of Surfaces with Trialkylmonochlorosilanes:
[0055] The cleaned carriers (small metal plates) were treated with
or without chromosulfuric acid enhancement with a 5% strength
trialkylsilane solution (v/V) in dry toluene which additionally
contains 5% of pyridine (v/v) with or without chromosulfuric acid
enhancement. After a reaction time of 1-3 h, they are washed with
ethanol, 0.01 M hydrochloric acid and dist. water. If required, the
carriers can be dried in vacuo at 60-110.degree. C.
[0056] Loading of Surfaces with Alkyltrimethoxysilanes:
[0057] The cleaned carriers (small metal plates) were treated with
or without chromosulfuric acid enhancement with a 5% strength
solution of alkyltrimethoxysilane solution (v/V) in dry
trichloroethylene. After a reaction time of 12 h at room
temperature, they are washed with trichloroethylene, acetone and
ethanol. In the case of mercaptopropyltrimethoxysilane, UV light
must be excluded. If required, the carriers (without SH groups) can
be dried in vacuo at 100-110.degree..
[0058] Loading of Surfaces with Dichlorodialkyl- and
Trichloroalkylsilanes:
[0059] The cleaned carriers (small metal plates) were treated in
dry toluene with or without chromosulfuric acid enhancement with a
5-10% strength dichlorodialkyl- or trichloroalkylsilane solution
(v/V). After a reaction time of 1-3 h, they are washed with ethanol
and dist. water. If required, the carriers can be dried in vacuo at
60-110.degree. C.
[0060] Binding of rhBMP-2 to a Propylamine-Titanium Binding
Lattice:
[0061] The propylamine-coated small titanium plates were washed
with 125 mM Na borate buffer, 0.066% sodium dodecyl sulfate, pH
10.0 and equilibrated. They were then treated with an rhBMP-2
solution (recombinant human BMP-2) (0.2-0.3 mg/ml in 125 mM Na
borate buffer, 0.066% sodium dodecyl sulfate, pH 10.0) and
incubated with shaking at room temperature for 12-14 hours. They
were then washed 4.times. with borate buffer and subsequently with
water.
[0062] Binding of rhBMP-2 to electropolished titanium: 10-30
ng/cm.sup.2
[0063] Binding of rhBMP-2 to chromosulfuric acid-enhanced titanium:
2-10 ng/cm.sup.2
[0064] Binding of rhBMP-2 to chromosulfuric acid-enhanced
propylamine-titanium binding lattice: 100-270 ng/cm.sup.2
[0065] Similarly high values can also be obtained for
chromosulfuric acid-enhanced titanium with a clean propyltitanium
binding lattice. It is to be observed here that the hydrophobically
adsorbed BMP-2, however, cannot be washed off by extensive washing
with buffer solutions or water.
[0066] As indicated above, surprisingly the noncovalently bonded
loading with BMP-2 was also not able to be removed by use of a
surfactant such as with a 1% SDS solution, which allows it to be
concluded that there are extremely strong adsorption forces. These
hydrophobic interactions can be strengthened by charge transfer
complexes, H bond formation and charge weakening, while
substitution of the chain with hydroxyl or thiol groups and charge
strengthening by, for example, ammonium radicals leads to the
weakening of the hydrophobic interactions.
[0067] In this case, the inventors found in their experiments that
a controlled release of BMP-2 can be decisively influenced by a
positive charge present on the alkyl radical. In this case, the pK
of the alkaline group --NH.sub.2 plays an important role, which can
lie at pK 8-12 and can be strongly influenced by substitution of
the nitrogen, for example to give the quaternary ammonium ion, such
that a charge-influenced adsorption dependent on the pH and later
release of the BMP-2 on the surface take place.
[0068] Even at a pH of 7.0, in the physiological range, the
noncovalent bond between the hydrophobic ligands immobilized on the
metal and the BMP-2 is extraordinarily stable, such that at most
0.1-1% of the adsorbed BMP is released per day. Since in the case
of groups substituted with amino groups on the implant surface both
the amino groups and the BMP are positively charged at a pH of 7.0,
an electrostatic adsorption is virtually excluded in this case.
[0069] The experiments described above were carried out under
appropriately adjusted conditions using the other compounds
included in the table. These are mean values of in each case 4
experiments with standard deviation. The small plates
(5.times.10.times.1 mm;=1 cm.sup.2), after pre-cleaning with
HNO.sub.3 or after pretreatment with chromosulfuric acid, were
individually washed 4.times.in 125 mM borate, 0.066% SDS, pH 10,
for 15 min. The adsorption conditions were as follows:
.sup.125I-BMP-2 solution: C.sub.bmp=0.1 mg/ml in 125 mM borate,
0.066% SDS, pH 10; 12-14 h at 5.degree. C.
[0070] The abbreviations used in the table have the following
meaning:
1TABLE Noncovalent immobilization of rhBMP-2 on alkyl-,
fluoroalkyl-, phenyl- and fluorophenyl- modified titanium surfaces
Titanium Titanium chromosulfuric Ti-EP (electropolished)
acid-treated Ti-EP Ti-CSB T.sub.1/2 Silanizing agent ng/cm.sup.2
ng/cm.sup.2 V, .degree. R, .degree. V, .degree. R, .degree. (days)
1 Ti control 1 29 .+-. 4 2 .+-. 2 40 17 0 0 (nonspecific
adsorption) 34 12 3 4 33 16 0 3 48 8 0 0 40 12 2
1C.sub.9H.sub.23NO.sub.3Si aminopropyltriethoxysilane (APS) 21 .+-.
2 105 .+-. 14 87 86 19 18 87 87 29 31 67 3
2C.sub.2H.sub.6C.sub.12Si dimethyldichlorosilane (DDS) 69 .+-. 29
228 .+-. 16 77 87 58 52 89 87 47 48 100 4 3C.sub.3H.sub.7C.sub.13Si
n-propyltrichlorosilane (PTC) 71 .+-. 5 121 .+-. 25 87 87 53 56 86
87 60 61 5 4C.sub.6H.sub.16O.sub.3- Si propyltrimethoxysilane (PTM)
68 .+-. 10 121 .+-. 27 81 85 22 18 88 86 23 25 6
5C.sub.5H.sub.13ClSi propyldimethylchlorosilane (PDMC) 43 .+-. 2 68
.+-. 10 87 84 33 7 39 38 1 2 7 6C.sub.6H.sub.15ClSi
n-butyldimethylchloro- silane (BDMC) 31 .+-. 2 64 .+-. 3 72 70 12 4
74 75 4 5 8 7C.sub.6H.sub.13Cl.sub.3- Si hexyltrichlorosilane (HTC)
81 .+-. 8 218 .+-. 16 87 87 7 13 63 54 6 0 96 K Ti control 2 15
.+-. 3 5 .+-. 1 (nonspecific adsorption) 9
8C.sub.14H.sub.32O.sub.3Si n-octyltriethoxysilane (C8) 59 .+-. 2
119 .+-. 31 85 87 38 37 57 52 1 6 10 9C.sub.18H.sub.40O.sub.3Si
n-dodecyltriethoxysilane (C12) 25 .+-. 1 76 .+-. 7 86 87 61 60 72
73 8 9 11 10C.sub.24H.sub.52O.sub.3Si n-octadecyltriethoxysilane
(C18) 14 .+-. 3 51 .+-. 11 87 87 60 60 87 86 57 32 12
11C.sub.14H.sub.19F.sub.13O.sub.3Si (Tridecafluoro-1,1,2,2-
tetrahydrooctyl)triethoxy- silane (F13) 24 .+-. 4 62 .+-. 11 83 86
61 60 86 86 21 21 13 12C.sub.16H.sub.19F.sub.17O.sub.3Si
(heptadecafluoro-1,1,2,2- tetrahydrodecyl)triethoxy- silane (F17)
24 .+-. 7 57 .+-. 16 90 90 17 67 87 86 60 59 14
13C.sub.12H.sub.20O.sub.3Si phenyltriethoxysilane (Phe) 44 .+-. 12
67 .+-. 20 54 52 15 12 43 35 12 7 15 14C.sub.12H.sub.15F.sub.5Si
Pentafluorophenylpropyl- trimethoxysilane (5FPP) 50 .+-. 7 105 .+-.
13 77 80 17 18 57 45 6 5 Ti-EP: electropolished metal Ti-CSB: metal
treated with chromosulfuric acid v: advance angle (peripheral angle
measurement according to Wilhelmy R: withdrawal angle (peripheral
angle measurement according to Wilhelmy t.sub.1/2: half-life of the
release of .sup.125I-rhBMP-2
* * * * *