U.S. patent application number 11/829829 was filed with the patent office on 2008-05-08 for method for the immobilization of mediator molecules on inorganic and metallic implant materials.
This patent application is currently assigned to Morphoplant GmbH. Invention is credited to Herbert Peter Jennissen.
Application Number | 20080107702 11/829829 |
Document ID | / |
Family ID | 7849669 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080107702 |
Kind Code |
A1 |
Jennissen; Herbert Peter |
May 8, 2008 |
Method for the Immobilization of Mediator Molecules on Inorganic
and Metallic Implant Materials
Abstract
A mediator molecule is immobilized on the surface of a metallic
or ceramic implant material. An anchor molecule (e.g., dialdehyde
or cyanogen bromide) having a functional group that covalently
binds the mediator molecule is covalently bound to the surface, and
the mediator molecule is coupled to the functional group of the
anchor molecule. The implant material may comprise titanium,
titanium alloy, aluminium or stainless steel or hydroxylapatite.
Oxide units on the implant material surface can be increased
preferably by treating with hot chromic-sulphuric acid for 0.5 to 3
hours at a temperature between 100 to 250.degree. C. prior to
binding the anchor molecule. Also, prior to binding the anchor
molecule, the surface of the implant material can be activated by
reacting with a silane derivative. Mediator molecules include BMP
protein, ubiquitin and antibiotics, and the implant material may be
an artificial joint or coronary vessel support such as a stent.
Inventors: |
Jennissen; Herbert Peter;
(Essen, DE) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
Morphoplant GmbH
|
Family ID: |
7849669 |
Appl. No.: |
11/829829 |
Filed: |
July 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10646913 |
Aug 21, 2003 |
7255872 |
|
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11829829 |
Jul 27, 2007 |
|
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09554972 |
May 23, 2000 |
6635269 |
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PCT/DE98/03463 |
Nov 24, 1998 |
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10646913 |
Aug 21, 2003 |
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Current U.S.
Class: |
424/422 ;
427/2.27 |
Current CPC
Class: |
Y10S 530/811 20130101;
A61L 31/10 20130101; A61L 31/16 20130101; A61L 27/30 20130101; A61L
31/10 20130101; A61L 27/50 20130101; A61L 27/54 20130101; A61L
31/082 20130101; A61L 27/34 20130101; A61L 2400/18 20130101; A61L
27/34 20130101; A61L 2300/414 20130101; A61L 27/28 20130101; A61L
27/12 20130101; A61L 31/08 20130101; A61L 27/06 20130101; A61L
27/28 20130101; A61L 31/08 20130101; A61L 31/047 20130101; A61L
27/227 20130101 |
Class at
Publication: |
424/422 ;
427/002.27 |
International
Class: |
A61F 2/30 20060101
A61F002/30; A61L 27/30 20060101 A61L027/30 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 1997 |
DE |
19752032.4 |
Claims
1. Method for the immobilization of mediator molecules on implant
materials, characterized in that in a first step anchor molecules
are covalently bound to the surface of the implant material,
wherein these anchor molecules have functional groups to which
further chemical compounds can be covalently bound, and in a second
step mediator molecules can be immobilized on the implant material
via these functional groups.
2. Method according to claim 1, characterized in that in an
intermediate step between the first and the second step spacer
molecules from the first step can be bound to the anchor molecules,
and the spacer molecules have further functional groups for the
covalent binding of further molecules, and in the second step the
mediator molecules are immobilized on the implant material via the
functional groups of the spacer molecules.
3. Method of claim 1 or claim 2, characterized in that at least a
part of the chemical bonds of the mediator molecules to the surface
of the implant material are modified such that the bonds can be
cleaved under physiological conditions.
4. Method according to claim 1, characterized in that the implant
material is composed of a material chosen from the group of metals,
metal alloys, ceramic materials or combinations thereof.
5. Method according to claim 1, characterized in that biologically
active substances such as bone growth factors from the class of the
BMP-proteins, antibiotics or mixtures thereof are used as mediator
molecules.
6. Method according to claim 5, characterized in that BMP-2 or
EMF-7 is used as the bone growth factor.
7. Method according to claim 1, characterized in that the surface
of the implant material is provided with an oxide layer prior to
the covalent binding of the anchor molecules.
8. Method according to claim 7, characterized in that the surface
of the implant material, chosen from titanium, titanium alloys or
stainless steel, is provided with an oxide layer by treatment with
chromic-sulfuric acid over a time span of 0.5 up 10 to 3 hours at
100 to 250.degree. C. prior to the covalent binding of the anchor
molecules.
9. Method for the application of an oxide layer to metallic
substrates, characterized in that the surface of the metallic 15
substrate is treated with chromic-sulfuric acid over a time span of
0.5 up to 3 hours at 100 to 250.degree. C.
10. Method according to claim 8 or 9, characterized in that the
chromic-sulfuric acid has a density of more than 1.40 g/cm3.
11. Method according to claim 2, characterized in that in a first
step anchor molecules are covalently bound to the implant surface,
in a second step spacer molecules are covalently bound to the
anchor molecules, wherein these spacer molecules reduce the
nonspecific absorption of the mediator molecules, and in a third
step the mediator molecules are covalently coupled to the spacer
molecules.
12. Method according to claim 11, characterized in that in a first
step aminoalkylsilane molecules are covalently bound to the implant
surface, in a second step agarose molecules are covalently bound to
the anchor molecules as spacer molecules, and in a third step BMP
or ubiquitin are covalently coupled to the agarose as mediator
molecules.
13. Implant, obtainable according to claims 1.
14. Implant according to claim 13, characterized in that the
implant material is composed of titanium, titanium alloys,
aluminium, stainless steel or hydroxylapatite.
15. Method for the immobilization of mediator molecules on implant
materials, characterized in that in a first step anchor molecules
are covalently bound to the chemically activated surface of the
implant material, wherein the anchor molecules have functional
groups to which further chemical compounds can be covalently bound
and in a second step mediator molecules are immobilized on the
implant material via these functional groups, wherein the implant
material is chosen from a material from the group of metals,
metallic alloys, ceramic materials or combinations thereof.
16. Method for the immobilization of mediator molecules on implant
materials, characterized in that in a first step anchor molecules
are covalently bound to the chemically activated surface of the
implant material, wherein these anchor molecules have functional
groups to which further chemical compounds can be covalently bound,
and in a second step mediator molecules are immobilized on the
implant material via these functional groups, wherein bone growth
factors from the class of the BMP proteins, ubiquitin, antibiotics
or mixtures thereof can be used as mediator molecules.
17. Method according, to claim 16, characterized in that the
implant material is chosen from a material from the group of
metals, metallic alloys, ceramic materials or combinations
thereof.
18. Method according to claim 15, characterized in that in an
intermediate step between the first and second step spacer
molecules are bound to the anchor molecules from the first step,
and these spacer molecules have further functional groups for the
covalent binding of further molecules, and in the second step the
mediator molecules are immobilized on the implant material via the
functional groups of the spacer molecules.
19. Method according to claim 15, characterized in that at least a
part of the chemical bonds of the mediator molecules to the surface
of the implant material is modified such that the bonds can be
cleaved under physiological conditions.
20. Method according to claim 15, characterized in that BMP-2 or
BMP-7 is used as the bone growth factor.
21. Method according to claim 15, characterized in that the surface
of the implant material is provided with an oxide layer prior to
the covalent binding of the anchor molecules.
22. Method according to claim 21, characterized in that, prior to
the binding of the anchor molecules, the surface of the implant
material, chosen from titanium, titanium alloys, aluminum or
stainless steel, is provided with an oxide layer by treatment with
hot chromic-sulfuric acid over a time span of 0.5 up to 3 hours at
100 to 250.degree. C.
23. Method for the application of an oxide layer on metallic
substrates, characterized in that the surface of the metallic
substrate is treated with hot chromic-sulfuric acid over a time
span of 0.5 up to 3 hours at 100 to 250.degree. C.
24. Method according to claim 23, characterized in that the
chromic-sulfuric acid has a density of more than 1.40/cm.sup.3.
25. Method according to claim 24, characterized in that the
metallic substrate concerns an implant.
26. Method according to claim 15, characterized in that in a first
step anchor molecules are covalently bound to the implant surface,
in an intermediate step spacer molecules are covalently bound to
the anchor molecules, wherein the spacer molecules reduce the
nonspecific absorption of the mediator molecules, and in a second
step the mediator molecules are covalently coupled to the spacer
molecules.
27. Method according to claim 26, characterized in that in a first
step aminoalkylsilane molecules are covalently bound to the implant
surface, in a second step agarose molecules are covalently bound to
the anchor molecules as spacer molecules, and in a third step a
bone growth factor from the class of the BMP proteins or ubiquitin
is covalently coupled to the agarose as mediator molecules.
28. Implant, obtainable according to the process of claim 15.
29. Implant according to claim 28, characterized in that the
implant material is made of titanium, titanium alloys, aluminum,
stainless steel or hydroxylapatite.
30. Implant, obtainable according to the process of claim 16.
31. Implant according to claim 29, characterized in that the
implant material is made of titanium, titanium alloys, aluminum,
stainless steel or hydroxylapatite.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 10/646,913, which was filed on Aug. 21, 2003,
which is a divisional application of U.S. application Ser. No.
09/554,972, which was filed May 23, 2000, now U.S. Pat. No.
6,635,269, which is a national phase application of International
Application No. PCT/DE98/03463, filed Nov. 24, 1998.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a method for the
immobilization of mediator molecules on surfaces of metallic or
ceramic materials which are used for implants such as artificial
joints or also microimplants, for example so-called stents, as well
as implants produced according to the method.
[0003] The implantation of artificial joints or bones has gained
increasing importance in recent years, for example in the treatment
of joint dysplasias or joint dislocations or in sicknesses
resulting from joint attrition as a result of improper joint
positioning. 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 synthetic materials
such as teflon, have been continually improved, so that following a
successful healing process, implants exhibit lifetimes of 10 years
in 90-95% of all cases. Yet despite this progress and these
improved operational methods, an implantation still remains a
difficult and strenuous operation, particularly since it is
associated with a long process of healing-in of the implants, often
including month-long stays in clinics and health resorts, including
rehabilitation measures. In addition to the pain, the length of the
treatment period and the separation from familiar surroundings
represent heavy stresses for the affected patients. In addition,
the long healing process incurs high personal and treatment costs
due to the required intensive care.
[0004] The understanding of the molecular-lever processes required
for a successful growing-in of an implant has markedly increased in
recent years. Structural compatibility and surface compatibility
are crucial for the tissue tolerability of an implant.
Biocompatibility in a narrower sense depends only on the surface.
Proteins play a crucial role at all levels of integration. These
form an initially adsorbed protein layer as early as during the
implantation operation and thus, as explained below, since the
first cells will later colonize on this layer, determine the
further progression of the healing-in of the implant.
[0005] In the molecular interaction between implant, also referred
to as biomaterial, and tissue, a multitude of reactions take place
which seem to be strictly hierarchically ordered. The adsorption of
proteins on the surface of the biomaterial is the first biological
reaction which takes place. In the resulting protein layer, single
protein molecules are for example either transformed by
conformational changes to signal substances which are presented on
the surface, or protein fragments functioning as signal substances
are released by catalytic (proteolytic) reactions. Triggered by the
signal substances, cellular colonization takes place in the next
phase, and can include a multitude of cells such as leucocytes,
macrophages, immunocytes and finally also tissue cells
(fibroblasts, fibrocytes, osteoblasts, osteocytes). In this phase
other signal substances, so-called mediators such as for example
cytokines, chemokines, morphogens, tissue hormones and true
hormones play a decisive role. In the case of biocompatibility,
there is a final integration of the implant into the entire
organism, and one ideally obtains a permanent implant.
[0006] In light of work performed in recent years at the molecular
level of osteogenesis, chemical signal substances, the so-called
"bone morphogenic proteins" (BMP-1-BMP-13), which influence 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 the formation of new bones and bone healing by
effecting the proliferation and the differentiation of precursor
cells to osteoblasts. Furthermore they promote the formation of
hormone receptors, bone-specific substances such as collagen type
1, osteocalcin, osteopontin and finally mineralization. Here, the
BMP-molecules regulate the three key reactions chemotaxis, mitosis
and differentiation of the respective precursor cells. In addition,
the BMPs play an important role in embryogenesis; organogenesis of
bone and of other tissue, wherein osteoblasts, chondroblasts,
myoblasts and vascular smooth muscle cells (proliferation
inhibition by BMP-2) are known as target cells.
[0007] A particular aim in the immobilization method according to
the invention is a degree of stimulation (that is, surface
concentration of the immobilized protein) which allows a
multivalent interaction between surface and cell and enables the
effective control of bone and tissue formation.
[0008] To date, 13 BMPs including multiple isoforms are known. With
the exception of BMP-1, the BMPs belong to the "transforming growth
factor beta" (TGF-.beta.) superfamily, for which specific receptors
on the surface of the corresponding cells have been found. As the
successful use of recombinant human BMP-2 and/or BMP-7 in
experiments on defective healing processes in rats, dogs, rabbits
and monkeys has shown, no species-specificity seems to exist.
Previous attempts to exploit the bone formation-triggering
characteristics of the BMPs for implantation purposes, in which
BMP-2 and/or BMP-7 were noncovalently applied to metallic or
ceramic biomaterials, have however been largely unsuccessful.
SUMMARY OF THE INVENTION
[0009] The goal of the present invention is to produce improved
biomaterials for use as implants.
[0010] According to the invention this goal is achieved by the
provision of a method for the immobilization of mediator molecules
on metallic and ceramic materials. In the method according to the
invention, in a first step a chemical compound is covalently bound
to the surface of the implant material as an anchor molecule,
wherein this chemical compound has a functional group which can
either be bound itself as a spacer molecule or to another compound
serving as a spacer molecule. In a second step a mediator molecule
such as a bone growth factor can be immobilized on the implant
material via functional groups, for example free amino groups or
carboxylate groups by means of a covalent bond. In this way it is
possible to form a chemotactic and/or biologically active implant
surface (a so-called juxtacrine surface), which leads to the
colonization, proliferation and differentiation of bone cells.
[0011] The method according to the invention for the immobilization
of the mediator molecules is distinguished by the fact that the
implant material used is composed of metallic materials such as
pure titanium or metallic titanium alloys such as
chrome/nickel/aluminium/vanadium/cobalt-alloys (for example TiAlV4,
TiAlFe2.5), stainless steels (for example V2A, V4A, chrome-nickel
316 L) or ceramic materials such as hydroxylapatite, aluminium
oxide or of a combination, in which for example metallic material
is coated with ceramic material. Synthetic polymer materials are
also suited for use as the implant material.
[0012] Further subject matter of the invention is the therapeutic
prevention or alleviation of the late complication restenosis
elicited by a proliferation of smooth vessel muscle cells by
coating a coronary vessel support (so-called coronary stent, length
approximately 10 mm) with the help of a biomolecule or a mediator,
for example BMP-2, in order to promote healing-in and
tolerability.
[0013] According to the invention the mediator molecules can be
biomolecules which are advantageous for the biocompatibility of the
implant in that they hinder a possible rejection of the implant
and/or promote growing-in of the implant.
[0014] Preferred mediator molecules which can be used in the
present method are bone growth-promoting proteins from the class of
bone growth factors "bone morphogenic proteins" or also ubiquitin.
It can be advantageous for the immobilization to use one protein of
this class alone, in combination with other members of this class
or also together with biomolecules such as proteins of other
classes or low molecular weight hormones or also antibiotics to
improve immunoresistance. Here, these molecules can also be
immobilized on the surface via bonds which are cleavable in the
biological environment.
[0015] According to the invention the surface of implant material
is chemically activated, wherein the activation takes place via a
silane derivative such as for example
.gamma.-aminopropyltriethoxysilane or a trimethylmethoxy- or
trimethylchlorosilane derivative or
3-glycidoxypropyltrimethoxysilane and the reaction is performed not
only in an aqueous but also in an organic solvent. In a second step
a spacer molecule serving as a spacer can be covalently coupled to
the surface activated in this way. A dialdehyde such as glutaric
dialdehyde, an isothiocyanate derivative or a triazine derivative
can for example serve as the spacer. A dicarboxylic acid or a
corresponding derivative such as succinic acid can be used as the
spacer molecule. Following possible activation of the coupling
group present in the spacer molecule, for example a carbonyl
functionality, by way of a common method for this purpose, the bone
growth-promoting protein is bound to the implant material via amino
groups accessible on its surface.
[0016] According to the invention it is also possible to use an
aryl amine as a spacer molecule. This can for example be obtained
by reaction of the implant material activated by a silane compound
with a benzoic acid chloride substituted with nitro groups such as
for example p-nitrobenzoylchloride followed by reduction of the
nitro group. In this case the covalent linking of the mediator
protein takes place via three carboxyl groups which can be
activated according to standard procedures for this purpose.
[0017] The present method further includes coupling of the mediator
molecule via anchor molecules only, without prior activation of the
implant surface by silane as described above by way of example,
wherein cyanogen bromide can for example be used for this purpose.
In this case the covalent immobilization of the mediator molecule
can take place via three amino groups of the protein.
[0018] The method according to the invention includes the coupling
of a bone growth factor to the surface of the implant via spacer
molecules, the covalent bonds of which are not cleaved under
physiological conditions. As an advantageous development, a bone
growth factor is coupled to the surface of the implant via spacer
molecules, the covalent bonds of which are cleavable under
physiological conditions for a limited release of the mediator
protein. Alternatively it is also possible to couple the bone
growth factors without the help of the spacer molecule, for example
by way of the carbodiimide method, to the activated surface of the
implant.
[0019] According to another further development of the method, two
or more spacer molecules are used for the immobilization of at
least one bone growth factor.
[0020] The loading density of the mediator protein immobilized on
the implant material according to the method of the invention is
generally 0.03 to 2.6 .mu.g/cm2 (for example 1-100 pmol/cm2 BMP-2).
In this loading range, a multivalent interaction between a cell
(for example 10 .mu.m diameter) and the BMP-molecules on a
biologicalized surface can be achieved, since approximately 106-108
immobilized protein molecules are located in the adhesion site.
[0021] The inventors have performed extensive experiments to
elucidate the mechanism of the binding of the protein molecules to
the surface. In the course of this, they found that with metallic
surfaces such as for example with titanium the binding takes place
via covalent bonds via the titanium dioxide molecules formed on the
metal surface, which are preferably transformed into hydroxyl
groups by treatment with dilute nitric acid.
[0022] In contrast to the methods known in the prior art, in which
biomolecules are for example deposited onto polymer surfaces or
inorganic bone materials and remain on the surface of the substrate
only via affinity interactions with the polymer molecules, the
inventors have been successful here in covalently anchoring the
biomolecules to the surface and, in this way, providing them for a
longer time on the surface of the implant.
[0023] Further investigations by the inventor have shown that the
anchoring of the mediator molecules on the surface can be
qualitatively and quantitatively improved by increasing the number
of the accessible metallic oxide units on the surface. It was found
by the inventors that the number of oxide groups can surprisingly
be increased by treating the surface of the metal with hot,
preferably sediment-free chromic-sulfuric acid. In contrast to the
expectation that the metal dissolves under these conditions, a
relatively uniform oxide layer is generated on the surface of the
metal by the use of this acid. The method is so mild that even
coronary vessel supports, so-called stents (which can for example
be fashioned from stainless steel or titanium) can be coated
without destroying the thin sensitive meshing (50-150 .mu.m
diameter). In this way the oxide layer can reach a thickness of 10
.mu.m up to 100 .mu.m and can be relatively "smoothly" constructed
without pits or holes. Pure titanium or titanium alloys (for
example TiAlV4, TiAlFe2.5), aluminium or stainless steel (for
example V2A, V4A, chrome nickel 316 L) can be used as the metal for
the implant. A common commercial chromic-sulfuric acid of 92% by
weight H2SO4, 1.3% by weight CrO3 and with a density of 1.8 g/cm3
as for example available from the company Merck is preferably used
to achieve a thin smooth layer of metal oxide. In order to achieve
this, the metal substrate is placed in the chromic-sulfuric acid
and is treated over a time span of 1 up to 3 hours at 100 to
250.degree. C., preferably 30 min at 240.degree. C., is
subsequently carefully rinsed with water, is boiled in water or in
a solution of 1-4 mM EDTA (ethylenediaminetetraacetate), preferably
4 mM EDTA for 30 min, in order to remove the chrome ions remaining
on the surface, and is then dried.
[0024] If a thicker metal oxide layer and/or an oxide layer with
small micro- and nanopores is to be provided on the metal surface,
the chromic-sulfuric acid described above is diluted with water to
a density of 1.5 to 1.6 g/cm3. In a subsequent treatment of the
surface of the metal implant as described above with the acid
diluted in this way, a "rough" surface layer with pits and pores is
formed, so that the surface available for loading with mediator
molecules is increased. It is therefore possible to apply a
multitude of different oxide layers with different characteristics
to metal surfaces with high adhesion by tuning to various densities
of chromic-sulfuric acid. The invention is therefore also directed
to such a method for forming a thermodynamically unified metal
oxide layer (no contact angle hysteresis) on the implant material
by means of hot chromic-sulfuric acid.
[0025] The metal oxide layer on the implant material made of the
materials cited above can then be activated via treatment with
dilute nitric acid (approximately 5% by weight) and subsequent
coupling of a silane derivative, optionally additionally of a
spacer molecule, as described above. The mediator molecules can
then be anchored via the molecules of the silane derivative or of
the spacer via coupling methods such as for example by way of
carbonyldiimidazole on the implant surface.
[0026] In order to exclude the nonspecific adsorption of the
mediator molecules, which can be up to 30% of the adsorbed mediator
molecules on the metal surface, it is further preferred in the
scope of the present invention to first couple an
adsorption-preventing layer of spacer molecules such as for example
agarose to the surface of the implant on which the metal oxide
layer is provided, to which adsorption-preventing layer the
mediator molecules can then be coupled. A prevention of nonspecific
adsorption can make sense in order to for example preclude a
blocking of BMP-receptors as a result of conformational changes of
the BMP-proteins following nonspecific adsorption to the surface.
The invention is therefore also directed to such a method for the
formation of a nonspecific binding-preventing coating on the metal
oxide layer and subsequent coupling of the mediator molecules. The
use of a coating of agarose for this purpose is preferred.
[0027] A ceramic material such as for example hydroxylapatite can
be used as the implant material. Here, the hydroxylapatite should
first be activated by treatment with aminoalkylsilane and then
reacted with a coupling agent such as carbodiimidazole. In the next
step a coupling of the mediator molecules such as BMP or ubiquitin
to the surface can take place. When using hydroxylapatite, the use
of spacer molecules is not necessarily required.
[0028] In the case that the mediator molecules used are not easily
soluble under the coupling conditions, the solubility can be
increased by addition of surfactants/detergents and the reaction
can be performed. In this way, difficultly soluble bone growth
factors and other mediators can be kept in solution at
pH-values>6 without losing biological activity by ionic and
nonionic detergents in the concentration range of 0.05-10%,
preferably 1-5% by weight, in particular 0.066% SDS at
pH-values>6, in particular pH 8-10 for the covalent coupling
method at alkaline pH.
[0029] The influence of materials modified by the method of the
invention on bone cells or on osteoblast cell lines (MC3T3-E1) were
studied in cell culture systems, wherein the modified materials
were presented in flake form for this purpose. It was observed
that, following application of the cells, confluent cell lawns
formed and functional changes by BMP-2 (for example synthesis of
alkaline phosphatase) on the materials took place.
BRIEF DESCRIPTION OF THE FIGURES
[0030] This patent or application contains a color image. Copies of
this patent or patent application publication with the color image
will be provided by the Office upon request and payment of
necessary fee.
[0031] FIG. 1 is a photograph showing various substrates with
oxidized TiO.sub.2 flakes.
[0032] FIG. 2 is a graph showing hysteresis measurements of various
surfaces.
[0033] FIG. 3 is a graph showing the change in contact angle and
hysteresis with non-oxidized titanium flakes following
APS-modification and protein coupling.
[0034] FIG. 4 is a graph showing the change in contact angle and
hysteresis with oxidized titanium flakes following APS-modification
and protein coupling.
[0035] FIG. 5 is a graph showing the reduction of non-specific
adsorption of fibrinogen by agarose coating of quartz glass
plates.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention will now be further explained with the
help of the following examples. The experiments were performed with
highly pure human BMP2 as well as ubiquitin produced in house by
genetic engineering or commercially obtained (company: Biochrom
KG/Seromed, Berlin).
EXAMPLE 1
Immobilization of BMP on Powdered Titanium with Spacer
[0037] a) Production of a Implant Surface Capable of Reaction
[0038] 0.5 g titanium powder (particle diameter 50-100 .mu.m) are
added to 9 ml distilled water and, depending on the degree of
substitution, 0.2-2 ml 10% (v/v) .gamma.
aminopropyltriethylethoxysilane are added and the pH of this
reaction batch is adjusted to a value between 3 and 4 by addition
of 6 N HCl while stirring. After regulation of pH, the reaction
solution is incubated in a water bath for 2 h at 75.degree. C.
Subsequently the activated metal is separated by vacuum filtration,
is washed with approximately 10 ml distilled water and is dried in
a drying cabinet at 115.degree. C.
[0039] b) Activation of the Implant Surface and Insertion of a
Spacer Molecule
[0040] 0.5 g of the metal powder derivatized with the
aminoalkylsilane is added to 12.5 ml 2.5% glutaraldehyde in 50 mM
NaH2PO4, pH 7.0. The reaction is carried out to conversion or until
a change of color is observed. The reaction product is subsequently
separated over a filter and is washed with copious amounts of
distilled water.
[0041] c) Immobilization of Protein
[0042] To the washed reaction product with glutaraldehyde is added
BMP in an amount of 0.1-3.0 mg/g titanium powder, 0.066% sodium
dodecyl sulfate (SDS) at neutral pH followed by reaction overnight
at 4.degree. C.
EXAMPLE 2
Immobilization of BMP on Powdered Titanium without a Spacer
[0043] a) Production of an Implant Surface Capable of Reaction
[0044] The production of an implant surface capable of reaction
took place in the same way as in Example 1.
[0045] b) Activation of the Surface of the Implant
[0046] 1.0 g of the metal derivatized with the aminoalkylsilane
derivative is added to 50 ml 0.03 M H3PO4 with a pH adjusted to
4.0. To this were added 100-200 mg of a water soluble carbodiimide,
for example 1-cyclohexyl-3-(2-morpholinoethyl)
carbodiimide-methoxy-p-toluene sulfonate.
[0047] c) Immobilization of the Protein
[0048] BMP is added directly to the activated titanium powder
mentioned above in an amount of 0.1-3.0 mg/g titanium powder and is
incubated overnight at 4.degree. C.
EXAMPLE 3
Immobilization of BMP on Flake Shapes Titanium with Spacer
[0049] a) Production of an Implant Surface Capable of Reaction
[0050] The activation of the implant surface took place in the same
way as in Example 1. Instead of titanium powder the same amount of
titanium flakes was simply used.
[0051] b) Activation of the Implant Surface and Insertion of a
Spacer Molecule
[0052] The metal flake activated with the aminoalkylsilane
derivative is added to 12.5 ml 2.5% glutaraldehyde in 50 mM
NaH2PO4, pH 7.0. Reaction is carried out until a change of color is
observed. Subsequently the reaction product is separated over
filter and is washed with copious amounts of distilled water.
[0053] c) Immobilization of the Protein
[0054] BMP in an amount of 0.1-3.0 mg/g titanium flakes is added to
the washed reaction product at neutral pH and is incubated
overnight at 4.degree. C.
EXAMPLE 4
Immobilization of BMP on Flaked Titanium without Spacer
[0055] a) Production of an Implant Surface Capable of Reaction
[0056] The activation of the implant surface took place in the same
way as in Example 1. Instead of titanium powder the same amount of
titanium flakes was simply used.
[0057] b) Activation of the Implant Surface
[0058] The metal flakes derivatized with the aminoalkylsilane are
added to 50 ml 0.03 M H3PO4 with a pH adjusted to 4.0. To this were
added 100-200 mg of a water soluble carbodiimide for example
1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-methoxy-p-toluene
sulfonate).
[0059] c) Immobilization of the Protein
[0060] BMP was added directly to the coupling batch mentioned above
in an amount of 0.3-3.0 mg/g titanium flakes and is incubated
overnight at 4.degree. C.
EXAMPLE 5
Slow-Release-Immobilization of BMP on Flaked Titanium without
Spacer
[0061] a) Activation of the Implant Surface
[0062] 1 titanium flake (0.5.times.1.0 cm) with a thickness of 0.1
to 0.5 mm is added to 25 ml distilled water. The pH is adjusted to
10-11 and 1 g CNBr is added while maintaining the pH at 10-11 and
while maintaining the temperature at 15-20.degree. C. When the
pH-value no longer changes, the reaction is completed and the metal
flake is washed with 100 ml H2O.
[0063] b) Immobilization of the Protein
[0064] BMP is added to the metal plate activated with CNBr in an
amount of 0.1-3.0 mg/g flake in 0.066% SDS and is incubated
overnight at pH 9.0 and 4.degree. C. The coupling reaction can also
be carried out at pH 7.0. The flake is thoroughly washed after the
coupling. The covalent bond between the metal flake and BMP
hydrolyzes with a half-life of about 1-4 weeks so that soluble BMP
is released.
EXAMPLE 6
Immobilization of BMP on Flaked Hydroxylapatite without Spacer
[0065] a) Production of an Implant Surface Capable of Reaction
[0066] Hydroxylapatite is reacted overnight in 10% solution of
aminopropyltriethylethoxysilane in toluene under reflux conditions.
After this the hydroxylapatite is washed with toluene and is
dried.
[0067] b) Activation of the Implant Surface
[0068] 1.0 g of the apatite made capable of reaction with the
aminoalkylsilane derivative is added to 50 ml 0.03 M H3PO4 with a
pH adjusted to 4.0. To this are added 100-200 mg of a water soluble
carbodiimide, for example
1-cyclohexyl-3-(2-morpholinoethlyl)-carbodiimide-methoxy-p-toluene
sulfonate.
[0069] c) Immobilization of the Protein
[0070] BMP is added directly to the coupling batch mentioned above
in an amount of 1-10 mg/g of hydroxylapatite and is incubated
overnight at 4.degree. C.
EXAMPLE 7
Immobilization of BMP on Flaked Hydroxylapatite with Spacer
[0071] a) Production of an Implant Surface Capable of Reaction
[0072] Hydroxylapatite is reacted overnight in a 10% solution of
>aminopropyltriethylethoxysilane in dry toluene under reflux
conditions. After this hydroxylapatite is washed with toluene and
is dried.
[0073] b) Activation of the Implant Surface and Insertion of a
Spacer Molecule
[0074] 0.5 g of the apatite made capable of reaction with the
aminoalkylsilane derivative is added to 12.5 ml 2.5% glutaraldehyde
in 50 mM NaH2PO4, pH 7.0. The reaction is carried out to conversion
or until a change in color is observed. Subsequently the reaction
product is separated over a filter is washed with copious amounts
of distilled water.
[0075] c) Immobilization of the Protein
[0076] BMP is added directly to the coupling batch mentioned above
in an amount of 1-10 mg/g hydroxylapatite and is incubated
overnight at pH 7.0 at 4.degree. C.
[0077] In place of the methods given in the production examples
under 2a, 4a and 6a, an implant surface capable of reaction can
also be provided in the following way. For this, 0.5 g of metal
powder, 1 metal flake or 1 g apatite is allowed to react overnight
in a 2% solution of 3-glycidoxypropyltrimethoxysilane (GPS) in dry
toluene under reflux conditions. After this the respective sample
material is washed with toluene and is dried under vacuum. 15 ml
the acetic acid/H.sub.2O (90:10) containing 0.83 g sodium periodate
were added to the above amounts of GPS to form a primary hydroxy
derivative capable of reaction from the epoxy derivative. The batch
is mixed for 2 h at room temperature and incubated. The liquid
phase is then removed and is washed with water, acetone and
diethylether (20 ml, respectively). It can then be incorporated
into one of the above mentioned activation reactions.
[0078] Instead of the methods given in the production examples
under 2b, 4b and 6b, the activation of the implant surface can also
take place in the following way. For this 0.5 g of the metal powder
(2a) derivatized with the aminoalkylsilane or a metal flake
derivatized with aminoalkylsilane (4a) or 1.0 g of the apatite (6a)
made capable of reaction with the aminoalkylsilane derivative are
washed with 50 ml water-free acetone (<0.3%). Then, 10 ml of a
solution of 3% carbonyldiimidazole/acetone are added to the
silane-derivatized material and are incubated 30 min at room
temperature. Washing with 20 ml acetone follows, and then the
coupling with the protein BMP can take place.
EXAMPLE 8
Checking of the Biological Activity of Immobilized BMP in Cell
Culture According to Bingmann
[0079] In this test the biological efficacy of BMP in vitro on
primary cultures of bone explants (guinea-pig calvaria cells) is
investigated: adhesion number, growth, proliferation, functional
changes in the hormone stimulability and in the spreading of
reinduced ionic signals (for example calcium ions and H+-ions). The
metal samples (flakes) are coated with BMP in such a way that one
half of the flake is biologicalized and the other half serves as a
control. Initial results prove that the flakes coated with BMP
effect a marked functional change of the bone cells.
EXAMPLE 9
Coding of Titanium Powder with Protein
[0080] a) Hydroxylation with Nitric Acid
[0081] 2 g titanium powder (atomized<60 .mu.m) is stirred for 2
h at 80.degree. C. in 5% HNO3 under reflux. Afterwards the powder
is separated over a frit and is washed with 500 ml water (pH=6-7).
The powder is further washed with 30 ml dry ethanol.
[0082] b) Silanization with 3-aminopropyltriethoxysilane (APS)
[0083] 1 g hydroxylated titanium powder is suspended in 45 ml dry
toluene and is treated with 5 ml APS under nitrogen as a protective
gas (working in an atmosbag). The suspension is boiled for 4 h
under reflux. Separation over a frit and washing with 200 ml
toluene and 100 ml ethanol follows. The substance is dried with
acetone.
[0084] c) Activation of the Silane Powder with Carbonyldiimidazole
(CDI)
[0085] 750 mg of CDI are dissolved in 15 ml of dry acetone and are
treated with 300 mg of the product of 2). The mixture is stirred at
room temperature for 3 h and then separated over a frit. Further
washing with 50 ml acetone and 50 ml water follows.
[0086] d2) Coupling with 125I-Ubiquitin
[0087] Ubiquitin is 125-iodinated with the help of Chloramine T
according to a known method. 100 mg of the silane powder of 3) are
suspended in 1 ml of a buffer solution of 50 mM Na-phosphate
buffer, pH 10.0, in which 1 mg/ml 125I-ubiquitin of a specific
radioactivity of 5000-20000 cpm/.mu.g is dissolved. The ubiquitin
concentration can be between 0.01 and 1.0 mg/ml. The mixture is
rotation-stirred (German: am Rad geruhrt) 2 h at room temperature
and is then stirred overnight. The supernatant is pipetted off.
Washing three times with 1 ml buffer follows. Washing four times
with a solution of 0.1 M NaOH, 1% sodium dodecyl sulfate (SDS) and
two more times with buffer and two times with water follows. The
titanium powder coated with 125I-ubiquitin is mixed in a small
Eppendorf tube with 1 ml acetone. The supernatant is pipetted off
and the powder is dried overnight under oil-pump vacuum.
[0088] Controls are carried out with the activated and/or
nonactivated product of 2) (see Table 1).
[0089] d2) Coupling with 125I-BMP-2
[0090] The coupling of BMP-2 to ubiquitin takes place analogously,
with the difference that 50 mM Na-borate, 0.066% SDS at pH 10 is
used as the buffer. The concentration of BMP-2 was between 0.01-1
mg/ml. TABLE-US-00001 TABLE 1 Protein coupling to titanium powder
by way of example of the protein 125I-ubiquitin Immobilized
125I-ubiquitin on titanium powder (2400 cm2/g) .mu.g/cm2 A.
Adsorption: Ubiquitin on pure titanium powder 0.638 Ubiquitin on
APS-modified titanium powder 0.417 after NaOH/SDS treatment 0.107
B. Covalent coupling of ubiquitin* Experiment method 1: Control
0.107 covalent coupling 0.122 Experiment method 2: control 0.035
covalent coupling 0.094 *Definition of the covalently bound
protein: the amount of protein which is measured following washing
with 0.1 M NaOH/1% SDS (see method).
EXAMPLE 10
Coating of Titanium Flakes with an Oxide Layer to Increase the
Protein Binding Capacity
[0091] The oxidation of the titanium flakes (each about 0.5.times.1
cm) is carried out in boiling chromic-sulfuric acid at a
temperature of 190-200.degree. C. for 1.5 h. The flakes, having
become grey by the oxidation, are thoroughly rinsed with water.
After this the flakes are boiled in water for 30 min. The flakes
are dried at room temperature (RT) in the air (see FIGS. 1 and
2--the flakes 1 and 2 shown in FIG. 1 are untreated, the flakes 3
and 4 are treated with chromic-sulfuric acid of density 1.8 g/cm3,
the flakes 5 and 6 are treated with chromic-sulfuric acid of
density 1.6 g/cm3). An EDX-analysis (Energy Dispersive Analysis of
X-rays) under scanning electron microscope control of the new layer
yielded up to 90% TiO2.
[0092] As shown in FIG. 1, the oxidized TiO2-flakes are clearly
more darkly colored and have completely lost their
metallic-shine.
[0093] The hysteresis-diagrams shown in FIG. 2 provide proof of the
successful oxidation treatment. The test of the different surfaces
of the titanium flakes took place here by way of the Wilhelmy Plate
Method. The values for the single plates A, B and C are as
follows:
A. Not Cleaned: .theta.Vor=76.2.degree., .theta.Ruck=18.2.degree.,
hysteresis: large
B. cleaned: .theta.Vor=36.5.degree., .theta.Ruck=21.1.degree.,
hysteresis: small
C. Oxidized: .theta.Vor=20.0.degree., .theta.Ruck=15.0.degree.,
hysteresis: none
(Translator Note: The German subscripts "Vor" and "Ruck" indicate
forward and backward directions, respectively)
[0094] The advance angle (.theta.Vor) and the hysteresis are
crucial. One can see that the flake (A) which was not cleaned, with
an advance angle (.theta.Vor) of 76.degree., is very hydrophobic.
The large hysteresis surface is an indication of impurities. The
cleaned polished flakes (B) show improved characteristics with a
significantly smaller contact angle of 36.5.degree. and a marked
decrease in the hysteresis. The best results were however achieved
with the oxidized flakes (C), which have a contact angle of only
20.degree. without visible hysteresis, in other words a
thermodynamically unified surface.
EXAMPLE 11
Covalent Protein Coating of Titanium Flakes
[0095] a) Hydroxylation with Nitric Acid
[0096] For the purposes of comparison, oxidized and nonoxidized
titanium flakes were heated under reflux for 2 h at 80.degree. C.
in 5% HNO3. Afterwards the flakes were washed with 500 ml water
(pH=6-7). The flakes were further washed with 30 ml dry
ethanol.
[0097] b) Silanization
[0098] The nonoxidized or (as described above) oxidized titanium
flakes were placed in heated containers for the silanization
reaction. The containers should cool down in advance in a dry
environment, preferably under nitrogen in a dessicator. 50 ml dry
toluene and 2.5 ml APS are mixed under inert gas in an atmosbag
(nitrogen). The container is loaded with the flakes as quickly as
possible in air and is placed under inert gas in the round bottom
flask with the APS/toluene mixture. It is closed and heated for 3 h
under reflex. (Contact thermometer 140.degree. C.) The flakes are
rinsed three times with 10 ml trichloromethane, acetone and
methanol. The flakes are dried in air.
[0099] c) Activation with Carbonyldiimidazole
[0100] After this the flakes in the container are placed in a
solution of acetone (dried) and carbonyldiimidazole. The solution
contains 50 ml acetone and 2.5 g CDI. The round bottom flask is
closed under inert gas and is stirred for 4 h at room temperature.
After this the flakes are rinsed three times with 10 ml acetone and
water. The flakes are dried in air.
[0101] d1) Coating of Protein with 125I-Ubiquitin
[0102] After this the flakes are added individually to a buffer
solution of 50 mM Na-phosphate buffer pH 10 containing a
concentration of 1 mg/ml 125I-ubiquitin of a specific radioactivity
of 5000-20000 cpm/.mu.g. (The ubiquitin concentration can be
between 0.01-1.0 mg/ml with or without 0.066% SDS.) The flakes are
shaken for 12-14 h at room temperature. After this the flakes are
washed four times each in phosphate buffer, a solution of 1.0 M
NaOH, 1% sodium dodecyl sulfate (SDS) at room temperature and are
then incubated 15 min at 60.degree. in a solution of 0.1 M NaOH, 1%
sodium dodecyl sulfate. Thorough washing with water follows (see
Table 2 and FIG. 3-4).
[0103] d2) Protein Coating with 125I-BMP-2
[0104] BMP-2 is radioactively labeled (specific radioactivity
5000-20000 cpm/.mu.g) using the known Bolton-Hunter Method in a
buffer of 125 mM sodium borate, 0.066% SDS, pH 8.4. The coupling of
125I-BMP-2 takes place in a buffer with 50 mM sodium borate, 0.066%
SDS at pH 10. The concentration of 125I-BMP-2 can be between
0.01-1.0 mg/ml. The flakes are shaken for 12-14 h at room
temperature. After this the flakes are washed four times each in
phosphate buffer, a solution of 0.1 M NaOH, 1% sodium dodecyl
sulfate (SDS) at room temperature and are then incubated 15 min at
60.degree. in a solution of 0.1 M NaOH, 1% sodium dodecyl sulfate.
Thorough washing with water follows. TABLE-US-00002 TABLE 2 Protein
coupling to titanium flakes by way of example of the protein
125I-ubiquitin Immobilization of 125I-ubiquitin polished with
titanium flakes titanium coated with oxide flakes .mu.g/cm2
.mu.g/cm2 A B Adsorption experiment APS-flakes 0.800 1.06 0.914
Covalent coupling experiment "irreversible" nonspecific Adsorption
(control) 0.040 0.177 0.114 App. covalent 0.114 0.500 0.604
Coupling 0.106 0.446 0.589
[0105] TABLE-US-00003 TABLE 3 Comparative coupling of the proteins
125I-ubiquitin and 125I-BMP-2 to oxidized titanium flakes (treated
with chromic-sulfuric acid, density 1.84) in the presence of 0.066%
SDS .mu.g/cm2 Immobilization of 125I-ubiquitin Covalent coupling
experiment Oxide-coated titanium flakes A. "Irreversable"
nonspecific Adsorption (control) 125I-ubiquitin (0.01 mg/ml) 0.003
125I-BMP-2 (0.01 mg/ml) 0.005 125I-ubiquitin (1.0 mg/ml) 0.172
125I-BMP-2 (1.0 mg/ml) 0.1-0.2* B. Covalent 125I-ubiquitin (0.01
mg/ml) 0.009 125I-BMP-2 (0.01 mg/ml) 0.010 125I-ubiquitin (1.0
mg/ml) 0.570 125I-BMP-2 (1.0 mg/ml) 0.4-0.6* *approximated
[0106] All derivatives of titanium flakes depicted in Table 2 have
been tested in cell culture with osteoblasts descendants (MC3T3).
Confluent cell lawns stimulable by BMP-2 formed on all flakes. The
oxidized flakes yielded approximately twice as high stimulation
rates. The results allow the conclusion that the flakes do not
exhibit any toxicity, whereby the oxidized flakes were clearly
better then the nonoxidized flakes.
[0107] FIG. 3 shows the change in contact angle and in hysteresis
with nonoxidized (polished) titanium flakes following
APS-modification and protein coupling. One can qualitatively
monitor the coating, however no quantitative conclusions can be
drawn. The values for the individual flakes A, B and C are as
follows: TABLE-US-00004 A. Cleaned .theta.Vor = 36.5.degree.,
.theta.Ruck = 21.1.degree., hysteresis: small B. APS-modified
.theta.Vor = 68.6.degree., .theta.Ruck = 22.6.degree., hysteresis:
large C. 125I-ubiquitin .theta.Vor = 46.1.degree., .theta.Ruck =
17.4.degree., hysteresis: none
(Translator Note: The German subscripts "Vor" and "Ruck" indicate
forward and backward directions, respectively)
[0108] FIG. 4 shows changes in contact angle and hysteresis with
oxidized titanium flakes following APS-modification and protein
coupling. One can similarly monitor the coating qualitatively here,
however, no quantitative conclusions can be drawn. The values for
the individual flakes A, B and C are as follows: TABLE-US-00005 A.
Cleaned .theta.Vor = 36.5.degree., .theta.Ruck = 21.1.degree.,
hysteresis: small B. APS-modified .theta.Vor = 76.7.degree.,
.theta.Ruck = 15.9.degree., hysteresis: large C. 125I-ubiquitin
.theta.Vor = 76.9.degree., .theta.Ruck = 48.2.degree., hysteresis:
large
(Translator Note: The German subscripts "Vor" and "Ruck" indicate
forward and backward directions, respectively)
EXAMPLE 12
Coating of Titanium Flakes with Agarose to Reduce Nonspecific
Protein Adsorption (=Protein-Repellent Layer)
[0109] a) Oxidation of the Agorose with Sodium Periodate to
Dialdehyde-Agorose
[0110] The reduction batch of 19 g 4% agarose-gel spheres
(diameter: 40-190 .mu.m) for example sepharose 4B, Pharmacia, 100
ml distilled water, 2.5 ml 0.4 M sodium periodate solution was
treated as follows:
[0111] The agarose-gel spheres are first washed in a Buchner funnel
with distilled water and are then shortly sucked dry by vacuum
filtration. The moist gel cake is then taken up in 100 ml water.
After addition of 2.5 ml 0.4 M sodium periodate, the agorose-gel
suspension is stirred for 4 h in the dark in an ice bath and then
overnight at room temperature. After this the product is washed
with distilled water, 3% sodium thiosulfate solution and again with
distilled water, and water is finally removed with acetone. The
finished agarose is subsequently dried under oil-pump vacuum at
30.degree. C. Like the native agarose, the dialdehyde-agarose still
has the ability to gel. Under these conditions, 1% of all
agarobiose units are oxidized.
[0112] b) Coupling of Dialdehyde-Agarose to Aminopropylsilyl
Titanium Flakes
[0113] Reaction batch per flake:
4 ml of a solution of dry dialdehyde-agarose in potassium-phosphate
buffer (0.1 M; pH=7.0) at 80.degree. C.
Reaction batch a): 0.7% solution.
Reaction batch b): 1.4% solution
Reaction batch c): 2.1% solution
Reaction batch d): 4.0% solution
[0114] The dry dialdehyde-agarose is first dissolved in the buffer
in the desired concentration (0.7-4%) at 80.degree. C. The
aminopropylsilyl titanium flakes (for production see above) are
then placed in the solution in a holder, and stirring for 2 h at
80.degree. C. follows. After 20 minutes 400 mg of sodium
cyanoborohydride are added to reduce the Schiff bases formed. The
product is finally washed with 15 ml each of 4M sodium chloride
solution and water at 80.degree. C. and finally with water at room
temperature to remove excess agarose. Water is removed from the
flakes with acetone, and these are then dried overnight at
30.degree. C. under vacuum. The agarose layer on the titanium
flakes can finally be activated as described with
carbonyldiimidazole to couple primary amines (for example to
aminoacids or proteins).
[0115] c) Activation of the Agarose Layer with
Carbonyldiimidazole
[0116] 150 mg carbonyldiimidazole are dissolved in 3 ml acetone and
are then added to the agarose-coated titanium flake. The flake is
incubated for 2 h at room temperature and is then thoroughly rinsed
with acetone and distilled water.
[0117] d1) Protein Coating with 125I-Ubiquitin
[0118] After this the agarose flakes are added individually to a
buffer solution of 50 mM sodium phosphate buffer pH 10 containing a
concentration of 1 mg/ml 125I-ubiquitin with the specific
radioactivity of 5000-20000 cpm/.mu.g. (The ubiquitin concentration
can be between 0.01 and 1.0 mg/ml.) The flakes are shaken for 12-14
h at room temperature. The reaction of the flakes by incubation
with 40 mg/ml glycin in 50 mM sodium phosphate buffer pH 10 at room
temperature is then timed for 4 h. Washing with 15 ml each of
water, 1 M sodium chloride and water follows. Washing with 1% SDS
at room temperature is also possible if required.
[0119] d2) Protein Coating with 125I-BMP-2
[0120] BMP-2 is radioactively labeled (specific radioactivity of
5000-20000 cpm/.mu.g) using the known Bolton-Hunter Method in a 125
mM sodium borate buffer. The coupling of 1251-BMP-2 takes place in
a buffer with 50 mM sodium borate, 0.066% SDS at pH 10. The
concentration of 125I-BMP-2 can be between 0.01-1.0 mg/ml. The
flakes are shaken 12-14 h at room temperature. The reaction of the
flakes by incubation with 40 mg/ml glycin in 50 mM sodium phosphate
buffer pH 10 at room temperature is then timed for 4 h. Washing
follows with 15 ml each of water, 1 M sodium chloride and water.
Washing with 1% SDS at room temperature is also possible if
required.
[0121] d3) Derivatization of Quartz Glass Plates:
[0122] In an analogous method quartz glass plates can also be
coated with agarose. The protein repellent effect
(fibrinogen-adsorption, TIRF--(Total Inner Reflection Spectroscopy)
method) can be especially well visualized on these flakes. FIG. 5
shows the reduction of the nonspecific adsorption of fibrinogen by
agarose coating of quartz glass plates measured independent of time
in the TIRF-Online-Method. The adsorption of fibrinogen
(concentration 0.01 mg/ml) was carried out in 50 mM tris/HCl, 150
mM NaCl, 0.1 mM EDTA, pH 7.4. The fluorescence of tryptophan was
excited at 290 nm and the emission was measured at 350 nm with a
fluorescence spectrophotometer (Spex Fluorolog 112XI) under
TIRF-conditions. The agarose was covalently bound in monomeric form
to the amino function of the aminopropylsilyl moiety. cps: counted
photons per second. The curves here have the following
meanings:
curve 1: aminopropylsilyl-modified quartz glass plate
curve 2: unmodified quartz glass plate (control)
curve 3: quartz glass plate covalently coated with 0.7% agarose
curve 4: quartz glass plate covalently coated with 4% agarose
EXAMPLE 13
Protein Coating of Porous Hydroxylapatite
A Material for Replace the Bone
[0123] a) Preparation of the Hydroxylapatite
[0124] The following materials were used:
[0125] a. Porous hydroxylapatite (isolated from bovine bone) for
example endobon, Merck, density: 1.289 g/cm3
[0126] b. 125I-Ubiquitin or 125I-BMP-2
[0127] 3 ml dry toluene were mixed under nitrogen with 0.15 ml
aminopropyl silane (APS). The porous hydroxylapatite (150 mg) is
added and is boiled for 5 h under reflux. After this the
hydroxylapatite is rinsed three times with acetone, three times
with chloroform and three times with methanol. The porous
hydroxylapatite is then poured into a solution of dry acetone (3
ml) and 150 mg of carbonyldiimidazole under nitrogen and is stirred
for 3 h at room temperature. Rinsing three times with 10 ml acetone
follows.
[0128] b1) Coupling of Protein with 125I-Ubiquitin
[0129] The hydroxylapatite from a) is transferred in 1 ml phosphate
buffer (50 mM) pH 10. 20 .mu.l of ubiquitin (approximately: 50
mg/ml) and 10 .mu.l radioactive ubiquitin (specific radioactivity
of the final solution: 32600 cps/.mu.g) are added to the phosphate
buffer. The solution is mixed and is first rotation-stirred
(German: am Rad geruhrt) for 2 h at room temperature. Further
stirring for 24 h at 4.degree. C. follows. After this the modified
hydroxylapatite is rinsed three times with water, and then four
times with a solution of 0.1 M NaOH, 1% sodium dodecyl sulfate
(SDS) and then three times with water. The radioactivity is
measured in a gamma counter and the degree of substitution is
determined. Controls with washed hydroxylapatite and/or with
hydroxylapatite coated with APS are carried out (see Table 3).
[0130] b2) Coupling of Proteins with 125I-BMP-2
[0131] The hydroxylapatite from a) is transferred in 1 ml 50 mM
Na-borate buffer, 0.066% SDS, pH 10. The coupling of 125I-BMP-2
(specific radioactivity see above) takes place in the same buffer
(50 mM sodium borate, 0.066% SDS at pH 10) with incubation for 2 h
at room temperature. Further stirring for 24 h at 4.degree. C.
follows. After this the modified hydroxylapatite is rinsed three
times with water, then four times with a solution of 0.1 M NaOH, 1%
sodium dodecyl sulfate (SDS) and then three times with water. The
radioactivity is measured in a gamma counter and the degree of
substitution is determined. Controls are carried out with washed
hydroxylapatite and/or hydroxylapatite coated with APS. The
concentration of 125I-BMP-2 in the coupling can be between 0.01-1.0
mg/ml. TABLE-US-00006 TABLE 4 Coupling of protein to porous
hydroxylapatite by way of example of the protein 125I-ubiquitin
Porous hydroxylapatite coupled ubiquitin .mu.g/g Control 6
125I-ubiquitin 24 30
* * * * *