U.S. patent application number 13/177054 was filed with the patent office on 2011-11-24 for ceramic coating and method of preparation thereof.
Invention is credited to Haibo Qu, Mei Wei, Xiaohua Yu.
Application Number | 20110287167 13/177054 |
Document ID | / |
Family ID | 39563062 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110287167 |
Kind Code |
A1 |
Wei; Mei ; et al. |
November 24, 2011 |
CERAMIC COATING AND METHOD OF PREPARATION THEREOF
Abstract
A ceramic coating with gradient density/porosity and/or
incorporated biologically active agents can be fabricated on the
surface of substrates, including the surface of implantable medical
devices.
Inventors: |
Wei; Mei; (Conventry,
CT) ; Qu; Haibo; (Storrs, CT) ; Yu;
Xiaohua; (Willimantic, CT) |
Family ID: |
39563062 |
Appl. No.: |
13/177054 |
Filed: |
July 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11619659 |
Jan 4, 2007 |
8007854 |
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13177054 |
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60756039 |
Jan 4, 2006 |
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60828472 |
Oct 6, 2006 |
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60848045 |
Sep 27, 2006 |
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Current U.S.
Class: |
427/2.1 ;
118/600 |
Current CPC
Class: |
A61L 27/56 20130101;
C23C 18/1229 20130101; C23C 18/1208 20130101; C23C 18/1283
20130101; C23C 26/02 20130101; Y10T 428/26 20150115; C23C 18/127
20130101; A61L 27/306 20130101 |
Class at
Publication: |
427/2.1 ;
118/600 |
International
Class: |
B05D 1/00 20060101
B05D001/00; B05C 9/00 20060101 B05C009/00; B05C 11/10 20060101
B05C011/10 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The U.S. Government has certain rights in this invention
pursuant to Grant No. 0500269 awarded by the National Science
Foundation.
Claims
1. A method of coating a substrate with a gradient ceramic coating,
comprising: exposing a portion of a substrate to an aqueous system
at a temperature of about 20.degree. C. to about 100.degree. C. to
form a gradient ceramic coating on a surface of the substrate
having a density of about 75 to about 90% closest to the substrate
and a density of about 35 to about 60% at the gradient ceramic
coating surface as determined by scanning electron microscope;
wherein the aqueous system comprises water, Ca.sup.2+, Mg.sup.2+,
Na.sup.+, K.sup.+, Cl.sup.-, SO.sub.4.sup.2-, HPO.sub.4.sup.2-,
HCO.sub.3.sup.- and a buffer system; and wherein the aqueous system
has an initial pH of about 5.5 to about 7.5.
2. The method of claim 1, wherein the buffer system comprises
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid salts,
tris-hydroxymethylaminomethan, piperazine-1,4-bis(2-ethanesulfonic
acid), piperazine-1,4-bis(2-ethanesulfonic acid)salts, or
combinations thereof.
3. The method of claim 1, wherein the buffer system further
comprises an alkali metal carbonate.
4. The method of claim 1, wherein Ca.sup.2+ is present in an amount
of about 2.5 to about 15.0 mM; Mg.sup.2+ is present in an amount of
about 0.5 to about 5.0 mM; Na.sup.+ is present in an amount of
about 50.0 to about 300.0 mM; K.sup.+ is present in an amount of
about 2.0 to about 20.0 mM; Cl.sup.- is present in an amount of
about 50.0 to about 350.0 mM; SO.sub.4.sup.2- is present in an
amount of about 0 to about 2.0 mM; HPO.sub.4.sup.2- is present in
an amount of about 1.0 to about 10.0 mM; and HCO.sub.3.sup.- is
present in an amount of about 5.0 to about 100.0 mM.
5. The method of claim 1, wherein the aqueous system further
comprises silicate, strontium, zinc, silver, fluoride, or
combinations thereof.
6. The method of claim 1, wherein the gradient ceramic coating has
a total thickness of about 0.1 micrometers to about 70
micrometers.
7. The method of claim 1, wherein the exposing the substrate to the
aqueous system occurs for a time of about 10 hours to about 48
hours.
8. The method of claim 1, wherein the substrate comprises a metal,
a ceramic, a polymeric material, or silicon.
9. The method of claim 1, wherein the coating is performed in a
sealed container, wherein the sealed container comprises a pressure
valve.
10. The method of claim 9, wherein the sealed container has a
volume ratio of headspace to aqueous system of about 5 to about 15
at atmospheric pressure.
11. A method of incorporating a biologically active agent into a
ceramic coating on a substrate, comprising: exposing a portion of a
substrate to an aqueous system at a temperature of about 20.degree.
C. to about 100.degree. C. to form a ceramic coating on a surface
of the substrate; wherein the aqueous system comprises water,
Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, Cl.sup.-, SO.sub.4.sup.2-,
HPO.sub.4.sup.2-, HCO.sub.3.sup.-, a buffer system, and a
biologically active agent; wherein the aqueous system has an
initial pH of about 5.5 to about 7.5; wherein the ratio of aqueous
system volume to the substrate surface area is about 1 mm to about
50 mm; and wherein the biologically active agent concentration in
the aqueous system is less than about 1 mg/ml.
12. The method of claim 15, wherein the buffer system comprises
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid salts,
tris-hydroxymethyl aminomethan, piperazine-1,4-bis(2-ethanesulfonic
acid), piperazine-1,4-bis(2-ethanesulfonic acid)salts, or
combinations thereof.
13. The method of claim 11, wherein the buffer system further
comprises an alkali metal carbonate.
14. The method of claim 11, wherein the concentration of
biologically active agent in the aqueous system is less than about
0.5 mg/ml.
15. The method of claim 11, wherein Ca.sup.2+ is present in an
amount of about 2.5 to about 15.0 mM; Mg.sup.2+ is present in an
amount of about 0.5 to about 5.0 mM; Na.sup.+ is present in an
amount of about 50.0 to about 300.0 mM; K.sup.+ is present in an
amount of about 2.0 to about 20.0 mM; Cl.sup.- is present in an
amount of about 50.0 to about 350.0 mM; SO.sub.4.sup.2- is present
in an amount of about 0 to about 2.0 mM; HPO.sub.4.sup.2- is
present in an amount of about 1.0 to about 10.0 mM; and
HCO.sub.3.sup.- is present in an amount of about 5.0 to about 100.0
mM.
16. The method of claim 11, wherein the aqueous system further
comprises silicate, strontium, zinc, silver, fluoride, or
combinations thereof.
17. The method of claim 11, wherein the ceramic coating has a total
thickness of about 0.1 micrometers to about 70 micrometers.
18. The method of claim 11, wherein the ratio of the volume of the
aqueous system to the surface area of the substrate is about 10 mm
to about 40 mm.
19. The method of claim 11, wherein the biologically active agent
is a pharmaceutically active agent, an osteogenic factor, a
mitogen, a protein, or a gene.
20. A reactor for coating a substrate with a bioactive ceramic
coating, comprising: a liquid-holding container with a volume
sufficient to allow a ratio of the aqueous system volume to the
substrate surface area to be about 5 to about 50; and a gas valve
to control the rate of release of a gas from the container.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/619,659 filed Jan. 4, 2007, which claims the benefit of U.S.
Patent Application Ser. No. 60/756,039, filed Jan. 4, 2006; U.S.
Patent Application Ser. No. 60/828,472, filed Oct. 6, 2006; and
U.S. Patent Application Ser. No. 60/848,045, filed Sep. 27, 2006;
each of which is incorporated by reference herein in its
entirety.
BACKGROUND OF INVENTION
[0003] Implantable medical devices, such as orthopedic and dental
prostheses, can be made more permanent if the interface between the
existing bone and the device contains some natural bone growth to
knit the two components together. Such ingrowth has advantages over
the use of bone cement, both in terms of stability and
permanency.
[0004] "Bioactive" coatings on implantable medical devices allow
for the ingrowth of natural bone into and around the device,
forming chemical bonds between the device and natural bone.
Calcium-phosphate coatings have been prepared and have been shown
to promote direct bone apposition.
[0005] There are a variety of approaches to prepare a bioactive
ceramic coating on a substrate, for example electrophoresis, plasma
spray method, and the so-called biomimetic method. Several of these
approaches have their drawbacks, however. The electrophoresis
method, although a low-temperature coating technique, results in a
relatively low bond strength at the interface between the coating
and the substrate. Therefore, a post-sintering step is usually
necessary. The plasma spraying method does provide a relatively
strong bond, however due to the high temperatures involved in this
method, the hydroxyapatite coating decomposes during the coating
process. The biomimetic method results in carbonated
nano-crystalline apatite that is chemically bonded to a substrate
through the process of immersing the substrate in an aqueous
solution containing calcium, phosphate, and carbonate ions. Other
ions, such as sodium, potassium, magnesium, chloride, sulfate, and
silicate, may optionally be present in the solution.
[0006] The coatings achieved by previously disclosed methods,
however, do not have a gradient structure.
[0007] There have been attempts to incorporate different proteins
into the biomimetic apatite coatings by mixing the proteins with
the biomimetic coating solutions. However, only up to forty-five
percent of the protein in the solution could be incorporated into
the coating using the biomimetic method.
[0008] There remains a need in the art for improved bioactive
ceramic coatings in addition to processes to prepare such coatings.
There also remains a need in the art to improve the protein
incorporation efficiency into apatite coatings.
BRIEF DESCRIPTION OF THE INVENTION
[0009] Disclosed herein is a method of coating a substrate with a
gradient ceramic coating comprising exposing a portion of a
substrate to an aqueous system at a temperature of about 20.degree.
C. to about 100.degree. C. to form a gradient ceramic coating on a
surface of the substrate; wherein the aqueous system comprises
water, Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, Cl.sup.-,
SO.sub.4.sup.2-, HPO.sub.4.sup.2-, HCO.sub.3.sup.- and a buffer
system; and wherein the aqueous system has an initial pH of about
5.5 to about 7.5.
[0010] In another embodiment, a coated substrate comprises a
gradient ceramic coating, wherein the gradient ceramic coating is
prepared by exposing a portion of a substrate to an aqueous system;
wherein the aqueous system comprises water, Ca.sup.2+, Mg.sup.2+,
Na.sup.2+, K.sup.+, Cl.sup.-, SO.sub.4.sup.2-, HPO.sub.4.sup.2-,
HCO.sub.3.sup.- and a buffer system.
[0011] In another embodiment, a method of incorporating a
biologically active agent into a ceramic coating on a substrate
comprises exposing a portion of a substrate to an aqueous system at
a temperature of about 20.degree. C. to about 100.degree. C. to
form ceramic coating on a surface of the substrate; wherein the
aqueous system comprises water, Ca.sup.2+, Mg.sup.2+, Na.sup.+,
K.sup.+, Cl.sup.-, SO.sub.4.sup.2-, HPO.sub.4.sup.2-,
HCO.sub.3.sup.-, a buffer system, and a biologically active agent;
wherein the aqueous system has an initial pH of about 5.5 to about
7.5; wherein the ratio of aqueous system volume to the substrate
surface area is about 5 to 50; and wherein the biologically active
agent concentration in the aqueous system is less than about 1
mg/ml.
[0012] In yet another embodiment, a coated substrate comprises a
ceramic coating comprising a biologically active agent, wherein the
ceramic coating is prepared by exposing a portion of a substrate to
an aqueous system, wherein the exposing is performed at a
temperature of about 20.degree. C. to about 100.degree. C.; wherein
the aqueous system comprises water, Ca.sup.2+, Mg.sup.2+, Na.sup.+,
K.sup.+, Cl.sup.-, SO.sub.4.sup.2-, HPO.sub.4.sup.2-,
HCO.sub.3.sup.-, a buffer system and a biologically active agent;
and wherein the aqueous system has an initial pH of about 5.5 to
about 7.5.
[0013] In still another embodiment, a reactor for coating a
substrate with a bioactive ceramic coating comprises a
liquid-holding container with a volume sufficient to allow a ratio
of the aqueous system volume to the substrate surface area to be
about 5 to about 50; and a gas valve to control the rate of release
of a gas from the container.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIG. 1 illustrates a schematic of a gradient coating (20) on
a substrate (10).
[0015] FIG. 2 illustrates an ESEM image of a coating surface
closest to the substrate surface having a dense morphology.
[0016] FIG. 3 illustrates an ESEM image of a coating surface
furthest from the substrate having a less dense and more porous
morphology.
[0017] FIG. 4 illustrates pH versus time of immersion of the
aqueous systems having an initial pH of a) 6.56, b) 6.45 and c)
6.40.
[0018] FIG. 5a illustrates pH versus immersion time of three
volumes of aqueous system.
[0019] FIG. 5b is a graphic illustration of the total inorganic
carbon (TIC) content in the three aqueous systems of varying
volumes before and after soaking the substrates.
[0020] FIG. 6 is a schematic illustration of a general reactor used
to produce coatings on irregular shaped substrates.
[0021] FIG. 7 is a schematic illustration of a reactor to produce a
coating on the surface of a hip acetabular cap.
[0022] FIG. 8 biologically active agent release behavior from
apatite coating.
DETAILED DESCRIPTION
[0023] Disclosed herein are methods of forming gradient ceramic
coatings and/or ceramic coatings containing biologically active
agents; coatings prepared therefrom; and articles prepared
therefrom.
[0024] The methods described herein allow for a mild and convenient
approach to form a gradient ceramic coating or apatite coating on
the surface of a variety of substrates. The methods involve
immersing a substrate or portion of a substrate into an aqueous
system under controlled conditions of temperature, pH, ion
concentration, and buffer to result in the formation of a gradient
ceramic coating or a bone-like apatite layer on the substrate
surface. The gradient morphology improves the bioactivity of the
ceramic coating, as the portion of the coating in direct contact
with the substrate is dense, allowing a strong bond to be formed
between the coating and substrate. The surface portion of the
ceramic coating is less dense/more porous. When used in implantable
medical device applications, the porous surface allows for bone
ingrowth as bone cells can penetrate the porous coating surface to
form a strong bond between the substrate and existing bone.
[0025] Also disclosed herein is a method of coating a substrate
with a ceramic coating comprising a biologically active agent. The
advantage of the present method is that biologically active agent
can be co-precipitated with apatite crystals onto a substrate
without losing its biological activity. In addition, the
biodegradation of these biomimetic coatings in vivo can lead to
gradual release of the incorporated biologically active agents. As
a result, these coatings have great potential as drug-carriers in
orthopedic and dental applications.
[0026] Furthermore, the methods described herein allow for a mild
and convenient approach to incorporate biologically active
molecules into a ceramic coating or apatite coating on the surface
of a variety of substrates. A significant amount (about 50 to 100%)
of the biologically active agent is incorporated into the ceramic
coating. The methods involve immersing a substrate or a portion of
a substrate into an aqueous system containing biologically active
agent under controlled conditions of temperature, pH, ion
concentration, and buffer to result in the formation of a ceramic
coating or a bone-like apatite layer on the substrate surface
containing significant amounts of the biologically active agent.
When used in implantable medical device applications, the ceramic
coating allows for strong bone fixation quickly. Furthermore, the
biologically active agents can aid the regeneration and healing of
bone tissue.
[0027] The method of incorporating biologically active agents into
a ceramic coating on a substrate with high yield and high
efficiency comprises exposing a portion of a substrate to an
aqueous system to form a ceramic coating on a surface of the
substrate; wherein the aqueous system comprises water, Ca.sup.2+,
Mg.sup.2+, Na.sup.+, K.sup.+, Cl.sup.-, SO.sub.4.sup.2-,
HPO.sub.4.sup.2-, HCO.sub.3.sup.-, a biologically active agent, and
a buffer system. In general, the ratio of the aqueous system volume
to the substrate surface area is about 5 to about 50 in order to
achieve about 50 to about 100% incorporation efficiency of the
biologically active agent in the coating. The concentration of the
biologically active agent in the aqueous system is less than about
1.0 mg/ml, specifically less than about 0.75 mg/ml, and more
specifically less than about 0.5 mg/ml. The initial pH and the
concentration of buffer in the aqueous system are selected and
controlled in order to produce the desired bioactive ceramic
coating.
[0028] As used herein "exposing a portion of a substrate" means any
portion or all of the substrate is exposed to the aqueous
system.
[0029] As used herein "biologically active agent" means an active
pharmaceutical ingredient (e.g. an antibiotic) or other
biologically active molecule such as a protein (e.g. a growth
factor, osteoclacin, etc.), a gene, an osteogenic factor, a
mitogen, and the like.
[0030] As used herein "ratio of the aqueous system volume to the
substrate surface area" means the volume of the aqueous system in x
unit of length cubed divided by the substrate surface area in x
unit of length squared. For example for a 5 millimeter.times.5
millimeter square substrate exposed to a 5 milliliter (5
milliliters=5000 cubic millimeters) volume of aqueous system would
have a ratio of the aqueous system volume to the substrate surface
area of 200 (dropping the remaining unit of length in
millimeters).
[0031] In another embodiment, a ceramic coating contains a
biologically active agent, wherein the efficiency of the
incorporation of the biologically active agent from the aqueous
system into the ceramic coating is greater than 50%.
[0032] The coating methods are performed at low temperatures
suitable for temperature sensitive substrates such as polymeric
materials and hydrogels, or temperature sensitive biologically
active agents. The coating process can be performed at a relatively
short amount of time. Furthermore, the methods can be used to coat
porous substrates and substrates having complex geometries.
Additional embodiments are directed to the ceramic coatings
themselves as well as articles prepared from substrates comprising
the ceramic coatings. In general, the ceramic coating can be
prepared by exposing a portion of a substrate to an aqueous system
comprising inorganic ions. The substrate is exposed for a period of
time and at a temperature to allow for the formation of the ceramic
coating on the exposed surface of the substrate. Exposing can
include immersion of the substrate or portion of the substrate to
the aqueous system. The resulting ceramic coating is generally a
bone-like apatite.
[0033] The aqueous system generally comprises the following
inorganic ions Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, Cl.sup.-,
SO.sub.4.sup.2-, HPO.sub.4.sup.2- and HCO.sub.3.sup.-. The aqueous
system can be prepared by dissolving in an aqueous solvent salts
that when disassociated will result in the particular ions
Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, Cl.sup.-, SO.sub.4.sup.2-,
HPO.sub.4.sup.2- and HCO.sub.3.sup.-. The aqueous solvent can be
deionized and purified water. Exemplary salts include those that
result in an aqueous solution of the desired ions, for example,
alkali metal halides, alkaline earth metal halides, alkali metal
hydrogen carbonates, alkali metal phosphates, and alkali metal
sulfates. Specific salts include, NaCl, KCl, K.sub.2HPO.sub.4,
MgCl.sub.2, Na.sub.2SO.sub.4, CaCl.sub.2 and NaHCO.sub.3.
[0034] The particular concentrations of each of the above-described
ions initially present in the aqueous system can be as follows:
[0035] Ca.sup.2+ at about 2.5 to about 15.0 mM, specifically about
4.0 to about 12.0, and more specifically about 8.0 to about 10.0
mM;
[0036] Mg.sup.2+ at about 0.5 to about 5.0 mM, specifically about
1.0 to about 4.5 mM, and more specifically about 1.5 to about 3.0
mM;
[0037] Na.sup.+ at about 50.0 to about 300.0 mM, specifically about
80.0 to about 200.0 mM, and more specifically about 100.0 to about
150.0 mM;
[0038] K.sup.+ at about 2.0 to about 20.0 mM, specifically about
5.0 to about 15.0 mM, and more specifically about 7.0 to about 10.0
mM;
[0039] Cl.sup.- at about 50.0 to about 350.0 mM, specifically about
100.0 to about 200.0 mM, and more specifically about 120.0 to about
150.0 mM;
[0040] SO.sub.4.sup.2- at about 0 to about 2.0 mM, specifically
about 0.1 to about 1.0 mM, and more specifically about 0.2 to about
0.5 mM;
[0041] HPO.sub.4.sup.2- at about 1.0 to about 10.0 mM, specifically
about 3.0 to about 8.0 mM, and more specifically about 5.0 to about
7.5 mM; and
[0042] HCO.sub.3.sup.- at about 5.0 to about 100.0 mM, specifically
about 10.0 to about 50.0 mM, and more specifically about 20.0 to
about 40.0 mM.
[0043] In one embodiment, the particular concentrations of ions
initially present in the aqueous system can be as follows:
Ca.sup.2+ is present in an amount of about 4.0 to about 12.0 mM;
Mg.sup.2+ is present in an amount of about 1.0 to about 4.5 mM;
Na.sup.+ is present in an amount of about 80.0 to about 200.0 mM;
K.sup.+ is present in an amount of about 5.0 to about 15.0 mM;
Cl.sup.- is present in an amount of about 100.0 to about 200.0 mM;
SO.sub.4.sup.2 is present in an amount of about 0.1 to about 1.0
mM; HPO.sub.4.sup.2 is present in an amount of about 3.0 to about
8.0 mM; and HCO.sub.3.sup.- is present in an amount of about 10.0
to about 50.0 mM.
[0044] In another embodiment, the particular concentrations of ions
initially present in the aqueous system can be as follows:
Ca.sup.2+ is present in an amount of about 8.0 to about 10.0 mM;
Mg.sup.2 is present in an amount of about 1.5 to about 3.0 mM;
Na.sup.+ is present in an amount of about 100.0 to about 150.0 mM;
K.sup.+ is present in an amount of about 7.0 to about 10.0 mM;
Cl.sup.- is present in an amount of about 120.0 to about 150.0 mM;
SO.sub.4.sup.2- is present in an amount of about 0.2 to about 0.5
mM; HPO.sub.4.sup.2+ is present in an amount of about 5.0 to about
7.5 mM; and HCO.sub.3.sup.- is present in an amount of about 20.0
to about 40.0 mM.
[0045] An additional component present in the aqueous system is a
buffer system. The buffer system can contain HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or
N'-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Molecular
formula: C.sub.8H.sub.17N.sub.2SO.sub.3; CAS No: 7365-45-9) and an
alkali metal hydrogen carbonate (e.g. NaHCO.sub.3, KHCO3, etc.)
which are added to the aqueous system in amounts to substantially
stabilize the aqueous system. The concentration of HEPES present in
the aqueous system can be at about 5.0 grams per liter (g/L) to
about 80.0 g/L, specifically about 10.0 g/L to about 60.0 g/L, and
more specifically about 12.0 g/L to about 48.0 g/L.
[0046] Additional buffer systems are also suitable and can be
tailored to provide a desired property of the coating, which in
some cases is a gradient morphology. The additional buffer system
may include tris-hydroxymethyl aminomethan (TRIS), HEPES salts,
piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), PIPES salts,
combinations of the foregoing with an alkali metal carbonate, and
combinations thereof.
[0047] In one embodiment, the buffer system is not a
carbonate-bicarbonate buffer system prepared by bubbling carbon
dioxide into the aqueous system.
[0048] In another embodiment, the aqueous system is stable and no
visual precipitation occurs throughout the coating formation.
[0049] The aqueous system may optionally contain additional ionic
components such as silicate, strontium, zinc, silver, fluoride,
combinations thereof, and the like.
[0050] FIG. 1 illustrates a schematic of a gradient ceramic coating
(20) on a substrate (10). The coating nearest the substrate surface
has a greater density and lower porosity as compared to the
density/porosity of the surface of the ceramic coating. As
previously mentioned, the dense coating at the interface with the
substrate provides a strong bond between the coating and the
substrate. The more porous surface of the ceramic coating, when
present on implantable medical devices for example, will induce
bone ingrowth and thereby will integrate with the natural bone when
implanted in a patient. As such, the ceramic coatings having a
gradient density/porosity can be considered bioactive.
[0051] As used herein, "gradient ceramic coating" means a
progressively increasing or decreasing difference in the density or
porosity of the ceramic coating over the distance of the thickness
of the coating.
[0052] As used herein, the term "functionally gradient bioactive
ceramic coating" means the dense coating at the interface will
provide a strong bonding strength with the substrate, while the
porous portion at the surface will induce bone ingrowth and thereby
well integrate with the natural bone.
[0053] As used herein "bioactive" means the ceramic coating can
induce bone ingrowth resulting in the formation of a strong bond
across the interface between the coating and the natural bone.
[0054] The density/porosity of the ceramic coating can be adjusted
by several parameters including amount of NaHCO.sub.3, initial pH
of the aqueous system, amount of buffer, temperature of the coating
process, calcium concentration, and phosphate concentration.
[0055] The density/porosity of the ceramic coating can be adjusted
by carefully choosing the initial pH of the aqueous system. Over
time, the pH of the aqueous system increases due to the bicarbonate
ions in the solution naturally decomposing into hydroxyl groups and
carbon dioxide. The initial formation of the gradient coating is
formed when the aqueous system has an initial pH of about 5.5 to
about 7.5. The initial stage of the coating process is slower as
HCO.sub.3.sup.- inhibits the crystal growth of the coating.
Therefore, the coating will grow slower and denser at the initial
stages of the coating process as the concentration HCO.sub.3.sup.-
is initially high. As the HCO.sub.3.sup.- ions decompose, the rate
of coating formation increases and the inhibitory effect of the
bicarbonate ions is less pronounced. The increased rate of coating
formation results in the gradient morphology.
[0056] The amount of buffer in the aqueous system will also alter
the pH change profile during the coating process. When there is
less buffer in the aqueous system, more HCO.sub.3.sup.- will be
present in the system when the pH range for apatite formation is
achieved. In the absence of buffer in the aqueous system, the
coating that forms exhibits minimal gradient morphology, as the
density/porosity of the resulting coating is substantially the same
throughout the entire thickness of the coating.
[0057] The calcium and phosphate concentrations can also be chosen
to obtain the optimal pH range for apatite formation.
[0058] If needed, the initial pH of the aqueous system can be
adjusted by the addition of an inorganic acid or inorganic base. An
exemplary inorganic acid includes halo acids (e.g. hydrochloric
acid). Exemplary inorganic bases include alkali metal hydroxides
(e.g. NaOH, KOH, etc.). The initial pH of the aqueous system can be
about 5.5 to about 7.5, specifically about 6.0 to about 6.60, more
specifically about 6.10 to about 6.45, and yet more specifically
about 6.20 to about 6.38. As used herein, "initial pH" means the pH
of the aqueous system prior to contact with the substrate to be
coated.
[0059] The initial pH of the aqueous system and the type and amount
of buffer system can be selected to generate a desired gradient
ceramic coating. After the desired aqueous system is prepared, the
substrate is exposed to the aqueous system at a particular
temperature to allow for the formation of the gradient coating. The
substrate can be exposed to the aqueous system for a time
sufficient for the formation of a gradient coating of sufficient
thickness. Coatings having sufficient thickness can be formed in
less than about 3 days. Specifically, the substrate can be exposed
in the aqueous system for about 4 to about 48 hours, specifically
about 10 to about 40 hours, more specifically about 12 to about 35
hours, and yet more specifically about 20 to about 30 hours until
the desired thickness of coating is formed.
[0060] To prepare a ceramic coating comprising a biologically
active agent the substrate can be exposed to the aqueous system for
a time sufficient for the formation of a coating of sufficient
thickness. Coatings having sufficient thickness can be formed in
less than about 3 days. Specifically, the substrate can be exposed
in the aqueous system for about 4 to about 48 hours, specifically
about 10 to about 40 hours, more specifically about 12 to about 35
hours, and yet more specifically about 20 to about 30 hours until
the desired thickness of coating is formed.
[0061] The temperature of the aqueous system during the coating
process can be about 20 to about 100.degree. C., more specifically
about 25 to about 60.degree. C., yet more specifically about 35 to
about 45.degree. C., and still yet more specifically about 38 to
about 42.degree. C. In one embodiment, the temperature of the
aqueous system can be varied during the coating process to create a
gradient coating. At different temperatures, the optimal pH range
for apatite formation will also be different as the rate of
HCO.sub.3.sup.- decomposition is affected by temperature. By
increasing the temperature, the greater the rate of HCO.sub.3.sup.-
decomposition as compared to lower temperatures for same time
period.
[0062] In one embodiment, a gradient coating can be formed from an
aqueous system containing about 18 mM NaHCO.sub.3, 12.5 mM
Ca.sup.+, 5 mM HPO.sub.4.sup.2-, and 44 g/L HEPES. The initial pH
of the aqueous system is about 6.02 and the coating process
performed at a temperature of about 42.degree. C.
[0063] In another embodiment, the temperature of the aqueous system
during the coating process can be about 20 to about 100.degree. C.,
the initial pH of about 5.5 to about 7.5, the HCO.sub.3.sup.- at
about 10 to about 150 mM, HPO.sub.4.sup.2- at about 1 to about 10
mM, Ca.sup.2+ at about 2.5 to about 15 mM, and HEPES at about 5 g/L
to about 80 g/L.
[0064] In yet another embodiment, the temperature of the aqueous
system during the coating process can be about 25 to about
60.degree. C., the initial pH is about 5.5 to about 7.5,
HCO.sub.3.sup.- at about 20 to about 100 mM, HPO.sub.4.sup.2- at
about 3 to about 8 mM, Ca.sup.2+ at about 4 to about 13 mM, and
HEPES at about 10 g/L to about 50 g/L.
[0065] In yet another embodiment, the temperature of the aqueous
system during the coating process can be about 35 to about
45.degree. C., the initial pH is about 6.38 to about 6.45,
HCO.sub.3.sup.- at about 30 to about 40 mM, HPO.sub.4.sup.2- at
about 2.5 to about 3.5 mM, Ca.sup.2 at about 7 to about 9 mM, and
HEPES at about 10 g/L to about 14 g/L.
[0066] In still yet another embodiment, the temperature of the
aqueous system during the coating process can be about 35 to about
45.degree. C., the initial pH is about 6.00 to about 6.10,
HCO.sub.3.sup.- at about 60 to about 80 mM, HPO.sub.4.sup.2- at
about 4.5 to about 5.5 mM, Ca.sup.2 at about 11 to about 13 mM, and
HEPES at about 42 g/L to about 45 g/L.
[0067] In one embodiment, the gradient coating has a density of
about 75 to about 90% closest to the substrate while the surface of
the coating away from the substrate has a density of about 35 or
about 60%, which is demonstrated by scanning electron microscope
(SEM) observations.
[0068] In another embodiment, the coating has a density of about 50
to about 90%, specifically about 65 to about 85%, and more
specifically about 70 to about 80%.
[0069] Generally, the longer the substrate is exposed to the
aqueous system, the thicker the resulting ceramic coating will be.
Coatings having a total thickness of about 0.1 to about 70
micrometers can be formed, specifically about 1 to about 50
micrometers, yet more specifically about 5 to about 40 micrometers,
and still yet more specifically about 10 to about 25 micrometers.
The crystal size of the resulting coating is less than about 1
micrometer.
[0070] In one embodiment, the coating has a bonding strength
between the coating and the substrate of about 5 to about 25 MPa as
determined using a modified ASTM C-633 method as provided in Kim
H-M, Miyaji F, Kokubo T, Nakamura T. "Bonding strength of bonelike
apatite layer to Ti metal substrate." Journal of Biomedical
Materials Research 1997; 38(2):121-127, which is incorporated
herein in its entirety. More specifically, the bonding strength
between the coating and the substrate is about 8 to about 20 MPa,
and more specifically about 10 to about 19 MPa. In a further
embodiment, the bonding strength between the coating and the
substrate is equal to or greater than the cohesive strength within
the coating.
[0071] In another embodiment, a method of coating a substrate with
a gradient ceramic comprises exposing a portion of a substrate to
an aqueous system in a closed system, e.g., a sealed container, at
a temperature of about 20.degree. C. to about 100.degree. C. to
form a gradient ceramic coating on a surface of the substrate,
wherein the aqueous system comprises water, Ca.sup.2+, Mg.sup.2+,
Na.sup.+, K.sup.+, Cl.sup.-, HPO.sub.4.sup.2-, HCO.sub.3.sup.- and
a buffer system, wherein the aqueous system has an initial pH of
about 5.5 to about 7.5, and wherein the closed system comprises a
volume ratio of headspace to aqueous system of about 5 to about 15
at atmospheric pressure.
[0072] In yet another embodiment, a method of coating a substrate
with a gradient ceramic comprises exposing a portion of a substrate
to an aqueous system in a closed system, e.g., a sealed container,
at a temperature of about 30.degree. C. to about 60.degree. C.,
specifically about 35.degree. C. to about 45.degree. C., to form a
gradient ceramic coating on a surface of the substrate, wherein the
aqueous system comprises water, 7.5 millimolar (mM) Ca.sup.2+, 3 mM
HPO.sub.4.sup.2-, 142.0 mM Na.sup.+, 5.0 mM K.sup.+, 1.5 Mg.sup.+,
103.0 mM Cl.sup.-, 27.0 mM HCO.sub.3.sup.-, and 0.5 mM
SO.sub.4.sup.2-, HEPES (11.928 g per 1000 ml water), and 1M HCl
(6.5 ml per 1000 ml water), and wherein the closed system comprises
a volume ratio of headspace to aqueous system of about 5 to about
15 at atmospheric pressure.
[0073] When preparing a ceramic coating containing a biologically
active agent, the incorporation efficiency of the biologically
active agent into the ceramic coating can be adjusted by carefully
choosing the ratio of aqueous system volume to substrate surface
area for the coating process. When substrates are soaked in the
aqueous system, both Ca.sup.2+ and HPO.sub.4.sup.2 ions are
adsorbed onto the surface of the substrate to form apatite
coatings. The bicarbonate (HCO.sub.3.sup.-) ion in the solution has
a two-fold function. These ions act as an inhibitor to slow down
the apatite formation while at the same time, they decompose into
CO.sub.2 and OH.sup.- during the coating process, as shown in
equation (1).
HCO.sub.3.sup.-.fwdarw.CO.sub.2+OH.sup.- (1)
[0074] By carefully controlling both the ratio of aqueous system
volume to substrate surface area and the HCO.sub.3.sup.-
decomposition rate (the CO.sub.2 release rate), the amount of
calcium and phosphate ions depositing on the surface of the
substrate can be maximized while minimizing the amount of
HCO.sub.3.sup.- remaining in the aqueous system. The coating
formation process can be expedited by the depletion of
HCO.sub.3.sup.-. As the apatite has a strong affinity for the
biologically active agent, the more apatite is formed, the greater
the amount of biologically active agent that can be incorporated
into the ceramic coating.
[0075] To achieve a high efficiency of the incorporation of the
biologically active agent in the ceramic coating, the ratio of
aqueous system volume to substrate surface area for the coating
process can be about 1 to about 50, specifically about 4 to about
40, more specifically about 5 to about 30, and yet more
specifically about 10 to about 20.
[0076] The efficiency of the incorporation of the biologically
active agent from the aqueous system into the ceramic coating can
be about 50% or greater, specifically about 60% or greater, more
specifically about 70% or greater, and yet more specifically about
75% or greater.
[0077] The temperature of the aqueous system during the coating
process to form the ceramic coating containing a biologically
active agent can be about 20 to about 100.degree. C., more
specifically about 25 to about 60.degree. C., yet more specifically
about 35 to about 45.degree. C., and still yet more specifically
about 38 to about 42.degree. C. At different temperatures, the
optimal pH range and aqueous system volume for apatite formation
will also be different as the rate of HCO.sub.3.sup.- decomposition
is affected by temperature and aqueous system volume. By increasing
the temperature, the greater the rate of HCO.sub.3.sup.-
decomposition will be increased as compared to lower temperatures
for same time period.
[0078] In another embodiment, the temperature of the aqueous system
during the coating process to form a ceramic coating containing a
biologically active agent can be about 20 to about 100.degree. C.,
the initial pH can be about 5.5 to about 7.5, the HCO.sub.3.sup.-
at about 10 to about 150 mM, HPO.sub.4.sup.2- at about 1 to about
10 mM, Ca.sup.2 at about 2.5 to about 15 mM, and HEPES at about 5
g/L to about 80 g/L.
[0079] In yet another embodiment, the temperature of the aqueous
system during the coating process to form a ceramic coating
containing a biologically active agent can be about 25 to about
60.degree. C., the initial pH can be about 5.5 to about 7.5,
HCO.sub.3.sup.- at about 20 to about 100 mM, HPO.sub.4.sup.2- at
about 3 to about 8 mM, Ca.sup.2 at about 4 to about 13 mM, and
HEPES at about 10 g/L to about 50 g/L.
[0080] In yet another embodiment, the temperature of the aqueous
system during the coating process to form a ceramic coating
containing a biologically active agent can be about 35 to about
45.degree. C., the initial pH can be about 6.38 to about 6.45,
HCO.sub.3.sup.- at about 30 to about 40 mM, HPO.sub.4.sup.2- at
about 2.5 to about 3.5 mM, Ca.sup.2 at about 7 to about 9 mM, and
HEPES at about 10 g/L to about 14 g/L.
[0081] In yet another embodiment, the biologically active agent
incorporation efficiency is above 80% when the biologically active
agent concentration in the aqueous system is less than 0.1
mg/ml.
[0082] In still yet another embodiment, the temperature of the
aqueous system during the coating process to form a ceramic coating
containing a biologically active agent can be about 35 to about
45.degree. C., the initial pH is about 6.00 to about 6.10,
HCO.sub.3.sup.- at about 60 to about 80 mM, HPO.sub.4.sup.2- at
about 4.5 to about 5.5 mM, Ca.sup.2 at about 11 to about 13 mM, and
HEPES at about 42 g/L to about 45 g/L.
[0083] Exemplary substrates that can be coated with the described
ceramic coating include implantable medical devices useful in
biomedical applications, including orthopedic applications (e.g.,
joint prostheses) and devices and appliances for orthodontic
applications and dental implants. The aqueous system lends itself
to the uniform application of a ceramic coating even to substrates
having surfaces of complex geometries. Additional applications in
the biomedical field include drug/protein delivery devices. In
addition, this coating system can also be used to load living
cells.
[0084] The coatings can be used to prepare medical, surgical,
reconstructive, orthopedic, orthodontic, prosthodontic, endodontic
or dental devices, implants, appliances, or a component thereof
(e.g., a screw or other attaching connector, etc.).
[0085] The substrates can be made from a wide variety of material
types, including metal, ceramic, polymeric materials, silicon,
glass, and the like. When used in biomedical applications, the
material should be biocompatible. As used herein, "biocompatible"
means being biologically compatible in that a toxic, injurious, or
immunological response is not produced in living tissue. Suitable
material for the substrate includes, for example, titanium,
stainless steel, nickel, cobalt, niobium, molybdenum, zirconium,
tantalum, chromium, alloys thereof and combinations thereof.
Exemplary polymeric material include polylactide (PLA),
poly(glycolic acid) (PGA), poly(methyl methacrylate) (PMMA), other
biocompatible polymeric material, and the like. Exemplary ceramic
materials include alumina, titania, and zirconia, glasses, and
calcium phosphates, such as hydroxyapatite and tricalcium
phosphate.
[0086] Prior to the coating step, the surface of the substrate can
be prepared to improve the adhesion of the coating. The substrate
can be cleaned or treated to remove any surface contaminants. The
metal substrates can be surface treated by sand-blasting, scoring,
polishing, and grinding to increase the surface roughness.
Alternatively, the metal substrate can undergo chemical surface
treatments prior to coating to provide a level of surface
roughness. Exemplary chemical treatments for metal substrates
include, acid etchings with strong mineral acids, such as
hydrofluoric, hydrochloric, sulfuric, nitric and perchloric acids;
treatment with strong alkalis, such as sodium hydroxide, potassium
hydroxide; treatment with oxidizing agents such as peroxyhalogen
acids, hydroxyperoxides, or hydrogen peroxide to form a metal oxide
layer. Washing with deionized or purified water can effect removal
of surface contaminants due to the surface treatment.
[0087] In one embodiment, the coating methods described herein do
not involve bubbling carbon dioxide or a gaseous weak acid into the
aqueous system to control the pH of the aqueous system.
[0088] Although the coatings have been discussed in terms of its
application for implantable medical devices, the coatings can be
used for a wide variety of uses, such as a hydroxyapatite
chromatography sorbent useful for the separation of biomolecules,
for example.
[0089] Also disclosed herein is a reactor for coating a substrate
with a bioactive ceramic coating comprising a liquid-holding
container and a gas valve ("pressure valve") to control the rate of
release of a gas from the container. The liquid-holding container
can be prepared from a non-reactive or inert material such as glass
or Teflon.TM..
[0090] The shape of the interior of the liquid-holding container
can be a regular shape such as a cube, a cone, a sphere, a
cylinder, or the like. Optionally, the shape of the interior of the
liquid-holding container can be similar to the shape of the
substrate to be coated. In order to have the container with a shape
similar to the substrate, the container can be molded to follow the
shape of the substrate to be coated.
[0091] In one embodiment, the shape of the interior of the
liquid-holding container is hemispheric and the substrate is a hip
acetabular cap.
[0092] The liquid-holding container can optionally have a volume
sufficient to allow a ratio of the aqueous system volume to the
substrate surface area to be about 5 to about 50.
[0093] The gas valve is a gas-releasing valve used to control the
rate of release of a gas, such as carbon dioxide, from the
liquid-holding container. The gas valve can be a manual gas valve,
a pressure-responsive gas valve, or an automated gas valve.
[0094] FIG. 6 is a schematic illustration of a general reactor used
to produce coatings on irregular shaped substrates. In one
embodiment, the reactor (100) for coating a substrate (140) with a
ceramic coating containing a biologically active agent is a
container with an aqueous system volume to substrate surface area
ratio of 5 mm to 50 mm such that the coating achieves about 50% to
about 100% incorporation efficiency for biologically active agent.
The reactor (100) of FIG. 6 includes a gas valve (120) to control
the CO.sub.2 release rate from the aqueous system (130). The
reactor container can be a double-jacketed container or it can be
placed in an incubator to maintain a constant temperature in the
container. With a suitable aqueous system volume to substrate
surface area ratio and an appropriate CO.sub.3.sup.- decomposition
rate (or CO.sub.2 release rate), which can be controlled by the gas
valve, the decomposition of HCO.sub.3.sup.- can be controlled to a
point that only a trace amount of HCO.sub.3.sup.- remains in the
aqueous system after the apatite nucleation on the substrate
surface. The coating formation process can be expedited due the
depletion of the coating formation inhibitor (HCO.sub.3.sup.-).
[0095] FIG. 7 is a schematic illustration of a reactor (150) to
produce a coating (160) on the surface of a hip acetabular cap
(140). The reactor (150) comprises of a reactor container (110) to
house the aqueous system (130). Depending on the size of hip
acetabular cap (140), the radius of the container internal chamber
is about 5 to about 50 mm. The reactor also comprises a gas valve
(120) to control the CO.sub.2 release rate. The container can be
either a cubic or hemisphere (as shown FIG. 7) which is similar to
the shape of the hip acetabular cap such that the ratio of the
aqueous system volume to the substrate surface area is 5 to 50.
EXAMPLES
Example 1
Effect of the Temperature on the Calcium-Phosphate Coating
Formation Process
[0096] A simulated body fluid (SBF) solution was prepared
containing 7.5 millimolar (mM) Ca.sup.2+, 3 mM HPO.sub.4.sup.2-,
142.0 mM Na.sup.+, 5.0 mM K.sup.+, 1.5 Mg.sup.2+, 103.0 mM
Cl.sup.-, 27.0 mM HCO.sub.3.sup.-, and 0.5 mM SO.sub.4.sup.2-;
prepared from NaCl, NaHCO.sub.3, Na.sub.2CO.sub.3, KCl,
K.sub.2HPO.sub.4.3.sup.H.sub.2O, MgCl.sub.2H.sub.2O, HEPES (11.928
g per 1000 mL water), CaCl.sub.2, Na.sub.2SO.sub.4, and 1M HCl (6.5
mL per 1000 mL water). SLA.RTM. titanium discs provided by
Straumann were used as the substrates in this study. The discs were
sandblasted, gritted, and acid etched. The titanium discs were
thoroughly washed with de-ionized water before immersion into the
SBF solution. The formation of the coating was carried out at three
different temperatures: 20.degree. C., 40.degree. C. and 60.degree.
C. After soaking the discs at each temperature for 24 hours (h),
the discs were removed from the solution, gently washed using
de-ionized water and dried at 60.degree. C. in an oven overnight.
The coatings were characterized using X-ray diffraction (XRD) and
Fourier transform infra-red (FTIR) to determine the composition of
the coating. Environmental electron scanning microscope (ESEM) was
also used to examine the surface morphology of the coatings.
[0097] It was found that a calcium-phosphate coating having a
reasonable thickness (about 10 to about 40 micrometers) was formed
on the surfaces of the SLA discs under all three operating
temperatures after only soaking in the SBF for 24 hours. XRD
patterns of the calcium-phosphate coatings formed at different
temperatures showed peaks between about 35 and 41 degree (O) that
are attributed to the substrate. At 20.degree. C., except for the
sharp peaks attributed to the titanium substrate, a "glass bulge"
is present with no sharp peaks discernable, suggesting that the
calcium-phosphate coating formed was an amorphous material. As the
temperature increased to 40.degree. C., a slight bulge, in
combination with some peaks, was observed indicating a poorly
crystallized calcium-phosphate coating was formed. The
crystallinity of the coating improved with the increase of the
temperature to 60.degree. C., as a relatively crystallized
calcium-phosphate coating was formed as evidenced by the XRD
pattern.
[0098] It was found that the surface morphology of the coating also
varied with the temperature of the coating step. ESEM images of the
SLA titanium disc before and after coating at 20.degree. C.,
40.degree. C., and 60.degree. C. were obtained. It has been
observed that the calcium-phosphate coatings were uniformly
deposited on the surface of SLA discs. At a relatively low
temperature, such as 20.degree. C., the coating has a dense
feature. When the temperature of the coating step was increased,
the coating became increasingly porous.
[0099] As indicated by the results, bioactive ceramic coatings
prepared from calcium-phosphate can be made on titanium substrates.
The ceramic coating density/morphology and crystallinity can be
tailored by the temperature of the coating step. Furthermore, a
short deposition time was required to achieve a homogenous coating
with a reasonable thickness.
Example 2
Preparation of a Bioactive Ceramic Coating Using an Aqueous System
Stabilized with HEPES
[0100] SLA.RTM. titanium discs available from Straumann were used
as the substrates in this example. The discs were cleaned with
deionized (DI) water prior to the coating process. An aqueous
system used as the coat forming solution was prepared by dissolving
2.701 grams (g) of NaCl, 0.736 g of NaHCO.sub.3, 0.112 g of KCl,
0.595 g of K.sub.2HPO.sub.4.3H.sub.2O, 0.155 g of
MgCl.sub.2H.sub.2O, 24 g of HEPES, 0.733 g of CaCl.sub.2 and 0.036
g of Na.sub.2SO.sub.4 in 500 milliliters (ml) of DI water. The
initial pH of the aqueous system was adjusted to 6.40 by adding 0.5
ml of 1 molar (M) NaOH at room temperature (21.degree. C.). The
resulting aqueous system was clean and free of any visible
precipitation.
[0101] A 100 ml aliquot of the aqueous system was transferred into
a 200 ml Pyrex.RTM. glass bottle. Two SLA.RTM. titanium discs were
added to two bottles. The bottles were then closed, capped, and
placed into a temperature controlled water bath set at 40.degree.
C. The discs were soaked in the aqueous system for about 24 hours
to effect coating formation. The coated SLA discs were then removed
from the aqueous system, rinsed with DI water, and dried at room
temperature.
[0102] The resulting coated discs were then characterized with XRD,
FTIR, and ESEM. The analyses revealed a dense morphology to the
coating nearest to the substrate surface (FIG. 2). The coating
surface furthest from the substrate surface, however, was less
dense and more porous (FIG. 3).
Example 3
Exploration of NaHCO.sub.3 Concentration on the Morphology of the
Resulting Ceramic Coating
[0103] Aqueous systems having different NaHCO.sub.3 concentration
were tested to study the effect of the HCO.sub.3.sup.- on the
ceramic coating morphology. The amount of NaHCO.sub.3 was varied as
follows: 0.736 g of NaHCO.sub.3 in 500 ml DI water and 1.472 g of
NaHCO.sub.3 in 500 ml DI water. The amounts of the remaining
components were the same as described in Example 2 above, and the
coating process was performed as described above for Example 2. It
was determined that the solution containing 0.736 g of NaHCO.sub.3
in 500 ml DI water was capable of forming a coating having a
gradient morphology.
Example 4
Exploration of the Initial pH of the Aqueous System on the
Morphology of the Resulting Ceramic Coating
[0104] Aqueous systems having different initial pH were tested to
study the effect on the ceramic coating morphology. The initial pH
was varied as follows: 6.40, 6.46, and 6.52, each at room
temperature (42.degree. C.). The coating procedure and aqueous
system were the same as described in Example 2 except that the
initial pH of the system was adjusted. ESEM images were obtained
showing that only the aqueous system having an initial pH at 6.40
formed a porous gradient coating.
[0105] A second experiment was performed exploring the initial pH
of the aqueous system on the morphology of the resulting ceramic
coating. The aqueous system according to Example 2 was used having
a Ca.sup.2+ ion concentration of 7.5 mM and an HPO.sub.4.sup.2- ion
concentration of 3.0 mM. Analytical grade reagents NaCl,
NaHCO.sub.3, MgCl.sub.2, K.sub.2HPO.sub.4 and CaCl.sub.2 were
dissolved into de-ionized water with desired amounts. Hepes
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was chosen to
buffer the solution. The concentrations of the remaining ions are
provided in Table 1 below.
TABLE-US-00001 TABLE 1 Aqueous system Ion concentration Ion (mM)
Na.sup.+ 109.5 K.sup.+ 6.0 Mg.sup.2+ 1.5 Ca.sup.2+ 7.5 Cl.sup.-
110.0 HCO.sub.3.sup.- 17.5 HPO.sub.4.sup.2- 3.0 SO.sub.4.sup.2-
0
[0106] The aqueous systems were prepared having an initial pH of
6.56, 6.45 and 6.40, respectively, using addition of hydrochloric
acid.
[0107] A commercially available titanium plate (McMaster-Carr) was
cut into small plates with a size of 15.times.15.times.1 mm These
small plates were polished using a series of silicon carbide papers
(grade 600-1200), and then rinsed with de-ionized water in an
ultrasonic bath. The metal plates were dried at room temperature
overnight. The clean titanium alloy plates were then soaked in 5 M
NaOH solution at 60.degree. C. for 3 days. After alkaline
treatment, the titanium plates were gently cleaned with de-ionized
water and immersed in 100 ml of each of the aqueous systems. The
formation of the coating was carried out at a temperature of
40.degree. C. After soaking for 24 hours, the plates were removed
from each solution, gently washed and air-dried overnight. The pH
value change in each aqueous system with the time was recorded
using a pH meter (Accumet Excel XL15). The composition of the
ceramic coating was evaluated using X-ray diffraction analysis
(XRD) (BRUKER AXS D5005), and the surface morphology of the coating
was observed using an environmental scanning electron microscopy
(ESEM) (Philips ESEM 2020).
[0108] The aqueous system with the highest initial pH value, 6.56,
started to form colloidal precipitates after 4 hours of soaking.
The pH-time profile of this solution revealed that the pH value
reached the peak (6.69) after 4 hours of immersing the specimen
(FIG. 4). In contrast, the aqueous systems having an initial pH of
6.45 and 6.40 remained stable and clear throughout the experiment.
The highest pH values of these two pH-time profiles were 6.64 and
6.55, respectively (FIG. 4). These results suggest that there is a
pH range, above which the colloidal precipitation of apatite was
yielded in the solution. The aqueous systems remained relative
stable below this pH range.
[0109] ESEM images obtained showed that the ceramic coating was
uniformly deposited on the surface of the titanium plates. The
coating formed in the aqueous system having an initial pH of 6.45
was denser but with cracks, while the coating formed in the aqueous
systems with initial pH values of 6.56 and 6.40 were rougher and
more porous.
[0110] XRD results showed that pure apatite was formed at all three
initial pH conditions. A broad peak around 31.degree.-33.degree.
suggests that poorly crystallined apatite was formed for all three
aqueous systems. The coating formed in the aqueous system with an
initial pH of 6.56 had the lowest relative intensity
(25.degree.-27.degree., 31.degree.-33.degree.) to the substrate,
while the coatings formed in aqueous systems with initial pH values
of 6.45 and 6.40 had higher relative intensities. These results
indicated that a relatively denser coating was formed for the
aqueous system with a lower initial pH. Two chemical reactions
occurred during the apatite formation in the aqueous system. First,
both Ca.sup.2+ and HPO.sub.4.sup.2 ions reacted to form into
apatite coatings at an appropriated pH range. Meanwhile, the
bicarbonate ions (HCO.sub.3) in the aqueous system decomposed into
CO.sub.2 and OH.sup.- and thereby increased the pH of the solution
(HCO.sub.3.sup.-.fwdarw.CO.sub.2+OH.sup.-). The apatite coating
forms when the aqueous system is within an appropriate pH range.
When the pH of the solution is above the pH range (e.g., initial pH
of 6.56), the apatite nucleates on the surface of the titanium
substrates as well as in the aqueous system solution. As a result,
less coating was generated on the surface of the substrate due to
the competition between the two processes, as evidenced by the XRD
results.
[0111] Due to the difference in initial pH values, the
decomposition rate of the bicarbonate in the aqueous systems was
different; therefore the remaining bicarbonate ion concentration in
the aqueous systems was different. The higher the bicarbonate ion
concentration in the aqueous system, the denser the coating formed.
ESEM images revealed that the ceramic coating formed in an aqueous
system with a lower initial pH (6.40) is more porous than the
coating formed in an aqueous system with a higher initial pH
(6.45). Without wishing to be bound by theory, these results are
possibly due to the different bicarbonate ion concentrations in the
solutions.
Example 5
Exploration of the Effect of Soaking Temperature on the Morphology
of Ceramic Coating
[0112] Aqueous systems having different soaking temperatures were
tested to study the effect of temperature on the ceramic coating
morphology. The coating procedure was the same as described in
Example 2, but with varied soaking temperatures. The soaking
temperatures explored were 20.degree. C., 30.degree. C., and
40.degree. C. ESEM images showed that a gradient coating was formed
at 40.degree. C., while dense coatings were formed at lower
temperatures.
Example 6
Exploration of the Effect of Aqueous System Volume on the Ceramic
Coating Quality
[0113] Aqueous systems having different volumes, (50, 100, and 200
ml), were employed to study the ceramic coating quality of the
resulting ceramic coatings, particularly bonding strength between
the coating and substrate. Commercially available titanium discs,
15 mm in diameter and 2 mm in thickness, were used as the
substrates in this study. These discs were sandblasted, gritted and
acid etched. They were thoroughly washed with de-ionized water
before immersion in the aqueous systems. The aqueous system was
prepared based on the procedure described in Example 2, where the
Ca.sup.2+ and HPO.sub.4.sup.2- concentrations were adjusted to 12.5
mM and 5 mM, respectively. The aqueous system was buffered using
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and the
initial pH of the solution was adjusted using hydrochloric acid.
The ion concentration of the resulting aqueous systems is provided
in Table 2 below.
TABLE-US-00002 TABLE 2 Ion Aqueous system Na.sup.+ 127.0 K.sup.+
10.0 Mg.sup.2+ 3.0 Ca.sup.2+ 12.5 Cl.sup.- 123.0 HCO.sub.3.sup.-
35.0 HPO.sub.4.sup.2- 5.0 SO.sub.4.sup.2- 0
[0114] Three different volumes of the aqueous system, 50 ml, 100 ml
or 200 ml, were placed in a 200 ml bottle to prepare type I, II and
III apatite coatings, respectively. Substrate discs were added to
each bottle and the bottles were sealed. The formation of the
coating was carried out at a temperature of 40.degree. C. After
soaking in the aqueous system for 24 hours, the discs were removed
from each solution, gently washed and air-dried for overnight.
[0115] The change of pH with time in the three aqueous systems was
measured using a pH meter (accumet Excel XL15). The pH of the
solution was measured every 15 min, and a pH profile against time
was plotted for the three solutions. The results are provided in
FIG. 5a.
[0116] A two-stage pH profile was observed for the 50 ml aqueous
system. At stage one, the pH of the 50 ml aqueous system increased
at an initial rate of 0.075 pH unit/hour, and peaked at 4.5 hours.
At stage two, the pH of the solution started to drop at 8 hours.
Similarly, a two-stage pH profile was also observed for the 100 ml
aqueous system. The pH of the solution increased at an initial rate
of 0.040 pH unit/hour, peaked at 6-10 hours, and dropped
afterwards. In contrast, a single stage pH profile was observed for
the 200 ml aqueous system, where the pH of the solution dropped at
an extremely low rate, 0.003 pH unit/hour, throughout the entire
experiment.
[0117] The total inorganic carbon (TIC) content in the three
aqueous systems before and after soaking the specimens was assessed
using a total organic/inorganic carbon (TOC/TIC) analyzer (OI
Analytical 700 TOC analyzer). Two soaking time points were studied,
12 and 24 hours, and each point was repeated 3 times. The results
are provided in FIG. 5b.
[0118] The carbonate/bicarbonate ion content in all three aqueous
systems at different time points was measured using a total
inorganic carbon analysis. The TIC measurement revealed that the
carbonate/bicarbonate ions decreased with the increase of the
soaking time for both the 50 ml and the 100 ml aqueous systems
during the coating formation (FIG. 5b). In the first 12 hours,
approximately 70% and 50% of carbonate/bicarbonate ions were
released from the 50 ml and the 100 ml aqueous systems,
respectively. After 24 hours of reaction, about 80% and 70% of the
carbonate/bicarbonate ions were released from the 50 ml and the 100
ml aqueous systems, respectively. In contrast, the TIC content
varied little during the entire coating process for the 200 ml
aqueous system.
[0119] The amount of coating was determined using the following
procedure. Coating was formed on both sides of titanium substrates.
The bottom side of the apatite coating was gently removed using
hydrochloric acid. After being dipped into 1M HCl, a cotton Q-tip
was used to gently rub across the coating to dissolve the apatite
coating. The substrates were then cleaned with DI water and dried
at room temperature. After the bottom side of the coating was
removed, each of the top coating was also removed by dissolving in
10 ml 1M HCl solution for 10 min. The weight of the top coating was
calculated as the difference between the weight of the substrates
before and after removing the coating.
[0120] The coatings were examined using X-ray diffractometer
(BRUKER AXS D5005) with a copper target. The voltage and current
setup were 40 kV and 40 mA, respectively. A step size of
0.02.degree. and a scan speed of 0.5.degree./min were used. The XRD
pattern of the coatings prepared from the 50 ml, 100 ml, and 200 ml
aqueous systems suggest that pure apatite coatings were obtained
for all systems. The bulge at around 31.degree.-33.degree. (an
overlap of 3 major peaks ((211), (112) and (300) of hydroxyapatite)
suggests that poorly crystallined apatite was formed for all three
coating systems. The relative intensity of this
bulge)(31.degree.-33.degree. increased as the aqueous system volume
increased, and the highest density was observed for the 200 ml
aqueous system.
[0121] The coating thickness and surface morphology of the apatite
coatings was evaluated using an environmental scanning electron
microscope (ESEM) (ESEM 2020 Philips). The densities of different
types of coatings were also evaluated using weight and thickness of
the top coatings. ESEM images indicate that uniform ceramic
coatings were formed on titanium discs for all three aqueous
systems, yet the coating morphologies were different. The coating
obtained from the 50 ml aqueous system (type I) was the least
dense, crack-free, and uniformly composed of numerous apatite
globules of 30-50 .mu.m in diameter. The coating obtained from the
100 ml aqueous system (type II) was denser than type I coating, and
its apatite globules had a much smaller size, 20-30 .mu.m in
diameter. The coating obtained from the 200 ml aqueous system (type
III) was the densest among the three coatings. The size of apatite
globules on type III apatite coating was the smallest, 10-20 .mu.m
in diameter. The properties of weight, thickness, and average
density of the three types of coatings are provided in Table 3. As
illustrated, by increasing the volume of the aqueous system, the
three parameters of weight, thickness, and density are all
increased.
TABLE-US-00003 TABLE 3 Coating Property Type I Type II Type III
Weight (mg) 4.5 8.5 11.5 Thickness (.mu.m) 12 .+-. 2 19 .+-. 1 24
.+-. 3 Density (% of Theoretical 67% 80% 85% density) (d =
weigh/(thickness*surface area).sub.--
[0122] The bonding strength of the three types of apatite coatings
to the substrates was evaluated using a modified ASTM C-633 method
as provided in Kim H-M, Miyaji F, Kokubo T, Nakamura T. "Bonding
strength of bonelike apatite layer to Ti metal substrate." Journal
of Biomedical Materials Research 1997; 38(2):121-127. Both sides of
the substrates (with apatite coating on one side) were bonded to a
cylindrical stainless steel fixture (15 mm in diameter and 15 mm in
length) using a super-glue (Henkel, Loctite Superglue, USA). The
tensile load was applied normal to the substrates using an Instron
testing machine (Instron 5869) at a crosshead speed of 1 mm/min
until fracture occurred. For each type of coating, five specimens
were tested. The fracture surface of the specimens was examined
using an environmental scanning electron microscope (ESEM) (Philips
ESEM 2020).
[0123] The average bonding strength for the three aqueous systems
using the tensile strength test were 8.52.+-.2.41, 10.36.+-.2.78
and 17.23.+-.2.55 MPa for types I, II, and III apatite coatings,
respectively. The average bonding strength of type I coating was
slightly lower than that of type II coating, although the
difference was not significant. In contrast, the bonding strength
of type III coating was significantly higher than those of both
types I and II coatings (p<0.01).
[0124] ESEM images showed the fracture surface of substrate and the
attached fixture for all apatite coatings. No glue penetration was
observed for all coating systems. Apatite was observed on the
surfaces of both the substrate and the fixture for all three types
of apatite coatings. However, the amount of apatite observed on the
substrate decreased in the following order: type I>type
II>type III. This suggested that the bonding strength within the
apatite coating became stronger with the increase of the aqueous
system volume, and the coating became less and less likely to fail
cohesively within the apatite coating. For type I and II coatings,
the ESEM images exhibited that most of the fractures occurred
within the apatite coating. While for type III coating, most of the
fractures occurred at the interface between the coating and
substrate. When titanium substrates were soaked in the aqueous
system, two chemical reactions occurred during the apatite coating
formation process. First, both Ca.sup.2+ and HPO.sub.4.sup.2- ions
were adsorbed onto the surface of the substrate to form into
apatite coatings. During the further growth of the apatite coating,
the pH of the solution decreased due to H.sup.+ release or OH.sup.-
consumption according to the general reaction
5Ca.sup.2++3HPO.sub.4.sup.2-+4OH.sup.-.fwdarw.Ca.sub.5(PO.sub.4).sub.3OH+-
H.sub.2O (equation (1)). In addition, the bicarbonate ions
(HCO.sub.3) in the solution decomposed into CO.sub.2 and OH.sup.-,
as shown in equation HCO.sub.3.sup.-.fwdarw.CO.sub.2+OH.sup.-
(equation (2)), increasing the pH of the aqueous system.
[0125] In general, the apatite coating forms when the aqueous
system is within an appropriate pH range. When apatite is formed,
the pH of the aqueous system decreases, as shown in equation (1).
To have continuous apatite formation, the pH of the aqueous system
can be increased by the decomposition of HCO.sub.3.sup.-, as shown
in equation (2). Not wishing to be bound by theory, but the coating
formation process is controlled by the decomposition of HCO.sub.3
which decomposes at different rates depending upon the headspace
volume in the closed system. Both types I and II aqueous systems
demonstrated a two-stage pH profile. At the first stage, the pH of
the solution was increased to the pH range for apatite formation by
the decomposition of HCO.sub.3. According to Henry's Law the amount
of CO.sub.2 in the aqueous system is in direct proportion to the
partial pressure of the CO.sub.2 above the aqueous system in the
sealed container. In this study, three different volumes of aqueous
system were used with the following order:
Vt.sub.ypeI<V.sub.typeII<V.sub.typeIII. The lower the volume
of the aqueous system, the higher the volume of the air in the
headspace of the sealed container, and the more CO.sub.2 is needed
to build up a high CO.sub.2 partial pressure in the sealed
container. In this study, the type I system (50 ml aqueous system)
had the largest space above aqueous system among the three systems.
As a result, more CO.sub.2 was expected to release from the type I
solution than type II solutions before the CO.sub.2 partial
pressure in the space above the aqueous system reached equilibrium.
The TIC results support the above assumptions. It was found that
after 12 hours, only about 30% and 50% carbonate/bicarbonate ions
remained in the types I and II aqueous systems, respectively.
Further, based on Henry's Law and equation (2), a more rapid pH
increase at stage I was shown for type I solution than that of type
II as more CO.sub.2 released from the type I solution. The pH
profile of these two solutions showed that it took less time (4-5
hours) for type I solution to reach the peak pH value than type II
solution (6 hours). In addition, the total pH increase (0.19 unit)
at the first stage for type I solution was higher than that for
type II solution (0.13 unit). Due to faster pH increase at the
first stage of type I solution, a relatively lower initial pH was
introduced for this system to avoid the pH overshot at the peak
range. At the second stage, the pH of both types I and II solutions
decreased. A pH decrease was also observed for type III solution
from the beginning to the end. The pH decrease suggests that more
OH.sup.- ions were consumed to form apatite than those decomposed
by HCO.sub.3.sup.-. Unlike the sharp pH drop in both types I and II
solutions after reaching the peak points, the pH of type III
solution decreased very slowly throughout the whole experiment. The
TIC result indicated that there was more than 95%
carbonate/bicarbonate ions remained in the type III solution after
24 hours, while only about 20% and 30% carbonate/bicarbonate ions
remained in type I and type II solutions, respectively. The
combination of the TIC results and pH profiles suggest that the pH
change in type III solution is attributed to the apatite
formation.
[0126] Besides HCO.sub.3.sup.- decomposition, HCO.sub.3.sup.-
itself can also affect apatite formation. The carbonate in the
aqueous system can contribute to the formation of carbonated
apatite, and the high HCO.sub.3.sup.- content could render a dense
apatite coating. Not wishing to be bound by theory, the combination
of the above two factors suggest that the apatite coating becomes
more porous with the decrease of the HCO.sub.3.sup.- content in the
aqueous system. The HCO.sub.3.sup.- content in the three aqueous
systems increased in the order of type I<type II<type III,
and the density of apatite coating formed on the titanium
substrates increased in the order of aqueous system volume: type
III (85%)>type II (80%)>type I (67%) (Table 3). Also the
surface morphology of apatite coatings revealed that the coatings
were getting denser as aqueous system volume increased.
Accordingly, it can be concluded from the results of density and
morphology of the apatite coatings that the apatite coating grew
denser as the aqueous system volume increased.
[0127] The volume of aqueous system also affects the bonding
strength of the apatite coating. Most of the type I and II coatings
failed within the apatite coating, suggesting the cohesive strength
within the coating (8-10 MPa) was lower than the bonding strength
at the interface between the coating and the substrate. In
contrast, most of the type III coating failed at the interface
between the coating and the substrate, indicating that the cohesive
strength within the coating was stronger than the interfacial
bonding strength, about 17 MPa. Based on the examination of the
failure sites and the bonding strength of different types of
apatite coatings, the bonding strength of the coating could be
significantly improved by reducing the rate of coating formation to
form a dense coating.
Example 7
Exploration of the Effect of the Ratio of Solution Volume to the
Substrate Surface Area on the Coating Formation Process
[0128] A simulated body fluid solution was prepared containing 7.5
millimolar (mM) Ca.sup.2+, 3 mM HPO.sub.4.sup.2-, 142.0 mM
Na.sup.+, 5.0 mM K.sup.+, 1.5 Mg.sup.2+, 103.0 mM Cl.sup.-, 27.0 mM
HCO.sub.3.sup.-, and 0.5 mM SO.sub.4.sup.2-; prepared from NaCl,
NaHCO.sub.3, Na.sub.2CO.sub.3, KCl, K.sub.2HPO.sub.4.3H.sub.2O,
MgCl.sub.2H.sub.2O, HEPES (11.928 g per 1000 mL water), CaCl.sub.2,
Na.sub.2SO.sub.4, and 1M HCl (6-10 mL per 1000 mL water).
High-grade titanium plates (7 mm.times.7 mm) were cleaned with
ethanol and de-ionized water. The titanium plates were then soaked
in 5M NaOH solution at 60.degree. C. for 3 days. The titanium
plates were thoroughly washed with de-ionized water before
immersion into the aqueous system. The formation of the coating was
carried out at three different volumes: 0.5, 1.5 or 2.5 ml in a 7.5
ml vial (the ratio of aqueous system volume to the plate surface
area is 10 mm, 20 mm and 30 mm, respectively). After soaking in the
aqueous system for 24 hours at 40.degree. C., the plates were
removed from the aqueous system, gently washed (rinsed) for about
two minutes at room temperature using about 500 ml of de-ionized
water and dried at room temperature for overnight. Both of the
amount of calcium left in the aqueous system and the amount of
calcium that incorporated into the coating were measured using
atomic absorption spectrophotometry (AAS). Environmental electron
scanning microscope (ESEM) was used to examine the surface
morphology of the coatings.
[0129] It was found that the surface morphology of the coating, and
the calcium and phosphate incorporation rate varied with the volume
of the aqueous system. ESEM images showed that at a low volume
solution, such as 0.5 ml, the coating had a porous feature. When
the volume of the solution increased, the coating became
increasingly dense. With larger aqueous system volume, it took a
longer time for the HCO.sub.3.sup.- to completely decompose.
Therefore more HCO.sub.3.sup.- remained in the solution and, as a
result, slowed down the apatite formation. Calcium and phosphate
measurements revealed that increasingly more calcium and phosphate
had contributed to the formation of apatite coating as the aqueous
system volume decreased. Table 4 provides the aqueous system volume
effect on calcium and phosphate incorporation efficiency.
TABLE-US-00004 TABLE 4 Coating Solution 0.5 ml 1.5 ml 2.5 ml Ratio
of solution volume to the 10 mm 30 mm 50 mm plate surface area %
Phosphate incorporated about 100 84.5 63.2 % Calcium incorporated *
about 67 60.1 48.9 * Note: The stoichiometric Ca:P ratio of
hydroxyapatite is 1.67 while the Ca:P ratio of the aqueous system
is 2.5. Therefore the maximum calcium incorporation efficiency of
the aqueous system is around 67%.
Example 8
Exploration of the Initial pH of the Aqueous System on the
Morphology of the Resulting Ceramic Coating
[0130] Aqueous systems having different initial pH were tested to
study the effect of pH on the ceramic coating morphology. The
initial pH was varied as follows: 6.20, 6.31, 6.40, 6.52, each at
room temperature (23.degree. C.). The ratio of solution volume to
the plate surface area was 10 mm. The coating procedure and aqueous
system were the same as described in Example 7 except that the
initial pH of the system was varied. The aqueous system having
initial pHs at 6.20 and 6.31 formed coating without precipitation.
Such precipitations are the result of nucleation of hydroxyapatite
in solution rather than on the substrate surface. The precipitation
in the solution is undesirable as a large amount of the
biologically active agent can remain in the solution by absorbing
to the precipitations in the solution instead of incorporating into
the coating on the substrate. As a result, the incorporation
efficiency of the biologically active agents in such systems is
low.
Example 9
Exploration of the Effect of Biologically Active Agent
Concentration on Biologically Active Agent Incorporation
Efficiency
[0131] Aqueous systems with different concentrations of bovine
serum albumin (BSA) were used to study the biologically active
agent incorporation efficiency. The BSA concentration was varied as
follows: 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml and 0.0001 mg/ml. The
formation of the coating was carried out by adding 0.5 ml of the
aqueous system in a 7.5 ml vial with an initial pH of 6.20 at
40.degree. C. for 24 hours. The aqueous system and coating
procedures were the same as described in Example 8. Coatings were
formed for all systems. Table 5 illustrates the incorporation
efficiency of BSA with different initial BSA concentrations. The
results show that almost all BSA (>85%) was incorporated into
the ceramic coating. The BSA concentration that can be achieved is
about 40 .mu.g/cm.sup.2. It has been reported that bone
morphogenetic protein (BMP) concentration over 250 .mu.g/implant
can significantly improve the bone growth of primate (baboon and
monkey). Therefore, in order to achieve a medically useful level of
BMP concentration, only small amounts (about 6-7 ug/cm.sup.2
coating) would be needed.
TABLE-US-00005 TABLE 5 Coating 0.1 0.01 0.001 0.0001 Solution mg/ml
mg/ml mg/ml mg/ml Initial BSA 40 5 0.5 0.05 concentration
(.mu.g/cm.sup.2) % BSA incorporated 85 about 100 about 100 about
100
Example 10
Exploration of the Effect of Aqueous System Volume on Biologically
Active Agent Incorporation Efficiency
[0132] Commercially available titanium plates (20 mm.times.20
mm.times.1 mm) were used as the substrates. The plates were
sandblasted with 800# sand paper, gritted and then treated with 5M
NaOH at 60.degree. C. for 24 h. The plates were thoroughly washed
with de-ionized water before immersion in the aqueous system.
[0133] The aqueous system was prepared according to Example 7 above
wherein the Ca.sup.2+ and HPO.sub.4.sup.2- concentrations of the
solution were adjusted to 7.5 mM and 3.0 mM, respectively. Aliquots
of the aqueous system were placed in sealed 40 ml bottles to
prepare solutions containing BSA. The pretreated titanium plates
were horizontally placed within individual beakers containing 3 mL
of the aqueous system and Fluorescein-isothiocyanate (FITC)-labeled
BSA (FITC-BSA)(Fraction V, >98%, Sigma, USA) at 100 .mu.g/ml
(n=4). Samples were incubated in a water bath, maintained at
42.degree. C. for 24 hours. The coated plates were then rinsed with
de-ionized water and air dried at ambient temperature.
[0134] FESEM micrographs of the coated plates containing the
FITC-BSA were compared to a coated plate free of FITC-BSA
(control). Both the coatings with and without BSA were composed of
crystals of about 0.3 .mu.m thick and 2 .mu.m across, but the
shapes of the crystal plates of the two kinds of coatings were
quite different. The observed differences between the two kinds of
coatings indicate that not only is BSA absorbed to the surface of
the coating, but also affects the lattice structure and orientation
of crystals of the coating.
[0135] To visualize the spatial distribution of FITC-BSA in the
coating, FITC-BSA incorporated coatings were compared with a
non-protein coating (negative control) using confocal microscopy
(Leica SP2 Spectral Confocal Microscope) at an excitation
wavelength of 488 nm using a 40.times. magnification oil immersion.
A side depth profile through the thickness of the mineral layer on
each of the coatings was obtained by stacking the series of images.
Results indicate that the BSA incorporated into the coating
distributed homogeneously through the whole thickness of the
coating.
[0136] The amount of FITC-BSA in the coating was determined using
an indirect method: the aqueous systems remaining after the coating
processes were collected and the concentrations of FITC-BSA were
measured by a microplate reader (Molecular Devices M2 plate reader)
with a fluorescence absorbance mode: Ex: 490 nm; Em: 530. The
concentrations of FITC-BSA in the aqueous systems before the
coating process were also measured. A calculation was then
performed to compute the incorporation rate of BSA into the
coatings:
R.sub.incorporation(%)=(C.sub.total-C.sub.remaining)/C.sub.total.times.1-
00% [0137] R.sub.incorporation: FITC-BSA incorporation rate into
coating [0138] C.sub.total: FITC-BSA concentration in the aqueous
system before coating [0139] C.sub.remaining: FITC-BSA
concentration remaining in the aqueous system after coating
[0140] For the aqueous system with a BSA concentration of 100
.mu.g/ml, about 76% BSA incorporated into the coating. This result
suggests that a high incorporation rate can be achieved using
biomimetic co-precipitation. The high incorporation rate of BSA
into the coating was due to the smaller volume of aqueous system
used in this study.
[0141] Calcium and phosphate remaining in the aqueous system after
the coating process were quantified to calculate the incorporation
rate of Calcium and Phosphate, respectively. Calcium concentrations
were measured by atomic absorbance spectromatography (AAS);
phosphate concentrations were obtained by molybdenum blue chemistry
method: a reagent of pure water, 2.5% ammonium molybdate reagent,
10% wt ascorbic acid (v:v:v=5:1:1) was prepared by orderly adding
in all the components; the samples were mixed with the reagent by
1:4 (v/v) then incubated in 60.degree. C. water bath for 15 min.;
all the samples were measured under a microplate reader (Biotek,
MQX200) under a wavelength of 830 nm. A simple calculation was
performed to compute the incorporation rate of calcium and
phosphate into the coatings:
R.sub.incorporation(%)=(C.sub.total-C.sub.remaining)/C.sub.total.times.1-
00% [0142] R.sub.incorporation: calcium or phosphate incorporation
rate into coating [0143] C.sub.total: calcium or phosphate
concentration in the aqueous system before coating [0144]
C.sub.remaining: calcium or phosphate concentration remaining in
the aqueous system after coating
[0145] Calcium and phosphate incorporation rate of the examined
aqueous system were relatively high. The phosphate incorporation
rate was around 90% which meant that most of phosphate in the
aqueous system had been consumed for the formation of the coating.
Compared to phosphate, the incorporation rate of calcium was about
60%. The lower rate is believed to be due to the high initial
calcium ion concentrations in the aqueous system. The remaining
calcium in the aqueous system would not decrease after the
phosphate was almost used up to form the coating.
[0146] To characterize the protein release kinetics, the coated
titanium plates were soaked in 3 ml of phosphate buffered saline
(PBS) at pH=7.4 in sealed beakers in a 37.degree. C. incubator. At
certain time intervals, 3 ml of the immersion solution was taken
out for FITC-BSA concentration measurement. The same volume of
fresh PBS was refilled into the beaker at each time point. The
FITC-BSA concentration was measured as previously described.
[0147] The results of the release analysis reveals that the
incorporated BSA was gradually released as a function of soaking
time. Two release stages were observed, as shown in FIG. 8. An
initial burst release of BSA was observed during the first 48
hours, whereas a sustained release was demonstrated for the
following 48 h. After 96 hours of release study, 55% of the BSA had
released from the coating for the system with an initial dose of
109.5 .mu.g BSA, while 45% BSA was released for the system with an
initial dose of 77.4 .mu.g. These results show a high BSA release
rate compared to the results reported by other researchers.
[0148] The terms "a" and "an" herein do not denote a limitation of
quantity, but rather denote the presence of at least one of the
referenced item. The suffix "(s)" as used herein is intended to
include both the singular and the plural of the term that it
modifies, thereby including one or more of that term (e.g., the
metal(s) includes one or more metals). Ranges disclosed herein are
inclusive and independently combinable (e.g., ranges of "up to
about 25 wt %, or, more specifically, about 5 wt % to about 20 wt
%", is inclusive of the endpoints and all intermediate values of
the ranges of "about 5 wt % to about 25 wt %," etc).
[0149] While the invention has been described with reference to a
preferred embodiment, it will be understood by those skilled in the
art that various changes may be made and equivalents may be
substituted for elements thereof without departing from the scope
of the invention. In addition, many modifications may be made to
adapt a particular situation or material to the teachings of the
invention without departing from essential scope thereof.
Therefore, it is intended that the invention not be limited to the
particular embodiment disclosed as the best mode contemplated for
carrying out this invention, but that the invention will include
all embodiments falling within the scope of the appended
claims.
* * * * *