U.S. patent application number 12/995132 was filed with the patent office on 2011-07-07 for pufa covered implants.
Invention is credited to Jan Erik Ellingsen, Stale Petter Lyngstadaas, Marta Monjo, Christiane Petzold.
Application Number | 20110166670 12/995132 |
Document ID | / |
Family ID | 40874878 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110166670 |
Kind Code |
A1 |
Lyngstadaas; Stale Petter ;
et al. |
July 7, 2011 |
PUFA COVERED IMPLANTS
Abstract
A medical or dental implant which contains a metal material
selected from the group consisting of titanium or an alloy thereof,
wherein at least part of the surface of the metal material is
coated with a layer of a polyunsaturated fatty acids (PUFA). In a
preferred embodiment, the implant has been exposed to UV radiation
for at least 30 seconds before, simultaneously with and/or after
the coating with PUFA. Depending on the concentration of
polyunsaturated fatty acids on the surface, at least parts of the
implant exhibits improved effect on adhesion of mineralized and/or
hard tissue, such as on bone remodeling and/or improved
biocompatibility, or alternatively inhibits adhesion of mineralized
and/or hard tissue to the implant. The metal material is preferably
titanium, the polyunsaturated fatty acid is preferably EPA.
Inventors: |
Lyngstadaas; Stale Petter;
(Nessoddtanfen, NO) ; Monjo; Marta; (Palma de
mallorca, ES) ; Petzold; Christiane; (Osto, NO)
; Ellingsen; Jan Erik; (Bekkestau, NO) |
Family ID: |
40874878 |
Appl. No.: |
12/995132 |
Filed: |
May 29, 2009 |
PCT Filed: |
May 29, 2009 |
PCT NO: |
PCT/EP2009/056666 |
371 Date: |
February 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61056978 |
May 29, 2008 |
|
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|
Current U.S.
Class: |
623/23.53 ;
427/2.24 |
Current CPC
Class: |
A61P 19/08 20180101;
A61L 31/16 20130101; A61L 31/08 20130101; A61L 31/14 20130101; A61L
27/50 20130101; A61L 2430/02 20130101; A61K 31/593 20130101; A61L
2300/412 20130101; A61L 31/022 20130101; A61L 27/54 20130101; A61L
27/28 20130101; A61L 27/06 20130101; A61L 2300/428 20130101 |
Class at
Publication: |
623/23.53 ;
427/2.24 |
International
Class: |
A61F 2/28 20060101
A61F002/28; A61L 33/06 20060101 A61L033/06 |
Claims
1. A metal implant for controlled adhesion of mineralized and/or
hard tissue, wherein at least part of the surface of the implant is
coated with PUFA (polyunsaturated fatty acids) at a concentration
of up to 10 nanogram/mm.sup.2.
2. A metal implant according to claim 1, wherein the implant is
coated with PUFA at a concentration of up to 1
nanogram/mm.sup.2.
3. A metal implant for controlled adhesion of mineralized and/or
hard tissue, wherein at least part of the surface of the implant is
coated with PUFA (polyunsaturated fatty acids) at a concentration
of at least 1 .mu.g/mm.sup.2.
4. A metal implant according to claim 1, further characterized in
that at least the part of the implant coated with PUFA has been
exposed to UV radiation for at least 30 seconds.
5. A metal implant according to claim 4, wherein the implant has
been exposed to UV radiation for at least 10 minutes.
6. A metal implant according to claim 4, wherein the implant has
been exposed to UV radiation for at least 30 minutes.
7. A metal implant according to claim 4, wherein the implant has
been exposed to UV radiation before the implant is coated with
PUFA.
8. A metal implant according to claim 4, wherein the implant is
exposed to UV radiation after the implant is coated with PUFA.
9. A metal implant according to claim 4, wherein the implant is
exposed to UV radiation simultaneously to being coated with
PUFA.
10. A metal implant according to claim 1, wherein the implant
comprises titanium.
11. A metal implant according to claim 1, wherein the implant
comprises at least 90% by weight of titanium.
12. A metal implant according to claim 1, wherein the PUFA is
selected from the group consisting of n-3 and n-6 fatty acids.
13. A metal implant according to claim 1, wherein the PUFA
comprises EPA (eicosapentaenoic acid).
14. A metal implant according to claim 1, wherein the PUFA consists
of EPA (eicosapentaenoic acid).
15. A metal implant according to claim 1, wherein the implant
coating additionally comprises fat-soluble vitamins.
16. A metal implant according to claim 15, wherein the fat-soluble
vitamin is vitamin E.
17. A metal implant according to claim 1 wherein the implant
additionally comprises 7-dehydrocholesterol.
18. A metal implant produced by coating a metal implant with
7-dehydrocholesterol and subsequently irradiating said coated
implant with UV light whereby cholecalciferol (vitamin D.sup.3) is
formed.
19. (canceled)
20. A method of actively inhibiting adhesion of mineralized and/or
hard tissue to a metal implant comprising providing an implant
wherein at least part of the surface of the implant is coated with
PUFA (polyunsaturated fatty acids) at a concentration of up to 10
nanogram/mm.sup.2.
21. Method for manufacturing a metal implant comprising a) treating
the implant with a solution comprising PUFA; and b) irradiating at
least part of the surface of the implant with UV light for at least
30 seconds.
22. Method for manufacturing a metal implant comprising a)
irradiating at least part of the surface of the implant with UV
light for at least 30 seconds; and b) treating the implant with a
solution comprising PUFA.
23. Method for manufacturing a metal implant, comprising a) mirror
polishing and/or grit-blasting the implant, b) washing, c)
autoclaving, d) treating the implant with a solution comprising
PUFA; and e) irradiating at least part of the surface of the
implant with UV light for at least 30 seconds.
24. Method for manufacturing a metal implant, comprising a) mirror
polishing and/or grit-blasting the implant, b) washing, c)
autoclaving, d) irradiating at least part of the surface of the
implant with UV light for at least 30 seconds; and e) treating the
implant with a solution comprising PUFA.
25. Method for manufacturing a metal implant according to claim 21,
wherein the solution comprising PUFA comprises EPA.
26. Method for manufacturing a metal implant according to claim 21,
wherein the implant comprises titanium.
27. Method for manufacturing a metal implant according to claim 21,
wherein the surface is irradiated with UV light characterized by
Fluo.link, .lamda..=312 nm.
28. Method for manufacturing a metal implant according to claim 21,
wherein intensity of the UV light which the surface is irradiated
with is approximately 6 mW/cm.sup.2.
29. Method for manufacturing a metal implant according to claim 22,
wherein the solution comprising PUFA comprises EPA.
30. Method for manufacturing a metal implant according to claim 22,
wherein the implant comprises titanium.
31. Method for manufacturing a metal implant according to claim 22,
wherein the surface is irradiated with UV light characterized by
Fluo.link, .lamda..=312 nm.
32. Method for manufacturing a metal implant according to claim 22,
wherein intensity of the UV light which the surface is irradiated
with is approximately 6 mW/cm.sup.2.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a metal implant to be used
as medical and/or dental implant, which actively facilitates
controlled adhesion of hard and/or mineralized tissue to the
implant, e.g. which actively induces adhesion of hard and/or
mineralized tissue to the implant and/or exhibits improved effect
on bone remodeling and/or biocompatibility of the implant due to at
least part of its surface being coated with a low concentration
layer of polyunsaturated fatty acids (PUFA). The present invention
at the same time relates to a metal implant to be used as medical
and/or dental implant, which actively inhibits hard and/or
mineralized tissue adhesion to the implant, such as bone
attachment, due to at least part of its surface being coated with a
layer of polyunsaturated fatty acids (PUFA) in a high
concentration. The invention further relates to a method for
manufacturing said metal implant with either inducing or inhibiting
effect on hard and/or mineralized tissue adhesion and/or bone
remodeling, wherein the implant is coated with PUFA at a specific
concentration, or alternatively is coated with PUFA at a specific
concentration and irradiated with UV light.
BACKGROUND OF THE INVENTION
[0002] Medical implants, such as dental implants, orthopaedic
implants, prosthesis and vascular stents are commonly made of
titanium and/or a titanium alloy. Titanium is the material most
frequently used as implant in bone, as it has outstanding physical
and biological properties, such as low density, mechanical
strength, and chemical resistance against body fluids.
[0003] Dental implants are utilized in dental restoration
procedures in patients having lost one or more of their teeth. A
dental implant comprises a dental fixture, which is utilized as an
artificial tooth root replacement. Thus, the dental fixture serves
as a root for a new tooth. Typically, the dental fixture is a
titanium screw which has a roughened surface in order to expand the
area of tissue contact. The titanium screw is surgically implanted
into the jawbone, where after the bone tissue grows around the
screw. This process is called osseointegration, because osteoblasts
grow on and into the rough surface of the implanted screw. By means
of osseointegration, a rigid installation of the screw is
obtained.
[0004] Once the titanium screw is firmly anchored in the jawbone,
it may be prolonged by attachment of an abutment to the screw. The
abutment may, just as the screw, be made of titanium or a titanium
alloy. The shape and size of the utilized abutment are adjusted
such that it precisely reaches up through the gingiva after
attachment to the screw. A dental restoration such as a crown,
bridge or denture may then be attached to the abutment.
Alternatively, the titanium screw has such a shape and size that it
reaches up through the gingiva after implantation, whereby no
abutment is needed and a dental restoration such as a crown, bridge
or denture may be attached directly to the screw.
[0005] Orthopedic implants are utilized for the preservation and
restoration of the function in the musculoskeletal system,
particularly joints and bones, including alleviation of pain in
these structures. Vascular stents are tubular implants arranged for
insertion into blood vessels in order to prevent or counteract a
localized flow constriction, i.e. they counteract significant
decreases in blood vessel diameter.
[0006] As already mentioned above, titanium (Ti) is the implant
material of choice for use in dental and orthopaedic applications
and in vascular stents. The stable oxides that form readily on Ti
surfaces have been reported to attribute to its excellent
biocompatibility. However, it has also been reported that bone
response to implant surfaces was dependent on the chemical and
physical properties of Ti surfaces, thereby affecting implant
success. As such, attention has been focused on the surface
preparation of Ti implants.
[0007] The surface of Ti is only bioinert, thus current research on
modification of implant surfaces focuses on making virtual bioinert
materials become bioactive, or rather to influence the types of
proteins absorbed at the surface readily after implantation. The
assortment of surface modifications ranges from non-biological
coatings, such as carbide, fluorine, calcium, hydroxyl apatite or
calcium phosphate, to coatings that are to mimic the biological
surroundings using lipid mono- or bi-layers, growth factors,
proteins, and/or peptides.
[0008] E.g. several techniques, such as plasma spraying, laser
deposition, ion beam dynamic mixing, ion beam deposition, magnetic
sputtering, hot isostatic pressing, electrophoretic deposition,
sol-gel, ion implantation, NaOH treatment, and electrochemical
methods have been employed to deposit hydroxyapatite (HA) or
calcium phosphate coatings on Ti surfaces.
[0009] It has been reported that implants coated with
hydroxyapatite (HA) enhances osteoinduction. The superior
performance of these implants being attributed to more rapid
osseointegration and the development of increased interfacial
strength, which results from early skeletal attachment and
increased bone contact with the implant's surface.
[0010] Especially plasma spraying has been employed frequently,
however with numerous problems, including variation in bond
strength between the coating and the metallic substaret,
non-uniformity in the layer thickness, and poor adhesion between
the coating and the metal surface (Satsangi et al., 2004).
[0011] It has also been proposed to improve the biocompatibility of
prostheses or implants by binding or integrating various active
biomolecules to the surface of the prosthesis, e.g. on to the
metallic surface of a titanium prosthesis. It has been the aim with
implants prepared this way that they have improved fit; exhibit
increased tissue stickiness and increased tissue compatibility;
have a biologically active surface for increased cell growth,
differentiation and maturation; exhibit reduced immunoreactivity;
exhibit antimicrobial activity; exhibit increased biomineralisation
capabilities; result in improved wound and/or bone healing; lead to
improved bone density; have reduced "time to load" and cause less
inflammation. Such binding has often been proposed carried out
using for example chemical reactants having two reactive
functionalities such as formalin or glutaraldehyde, but the
reactive nature of these agents often leads to the biomolecules
becoming biologically inactive and/or with enhanced
immunoreactivity, which is of course undesirable.
[0012] An alternative surface modification is using phospholipids
coating which is reported to induce the deposition of calcium
phosphate. The role of phospholipids has also been suggested in the
initiation of calcium phosphate deposition in cartilage, bone,
healing fracture callus and calcifying bacteria. It has been
proposed that an implant surface coated with a
calcium-phospholipid-phosphate should be able to attract
hydroxyapaptite.
[0013] Surface coatings of lipid mono- or bilayers are presumed to
mimic cell surfaces and therewith to prevent foreign body
reactions. Lipid coatings have been shown to influence the
attachment of proteins to a surface that occurs immediately after
the implantation and were found to prevent cell adhesion and blood
clot formation (Kim et al., 2005). Certain phospholipids were
furthermore reported to decrease bacterial adhesion.
[0014] Lipid coatings are usually based on physical adhesion, where
ordered layers are obtained e.g. by using Langmuir-Blodgett
technique. Lipids of the cell membrane are not only forming passive
surroundings for the proteins incorporated into the membranes but
rather influence the cell metabolism actively. Chemical methods for
coating of metal substrates with a layer of biological molecules
usually involve a foregoing step for obtaining reactive groups on
surfaces (Khan W et al., 2007; Muller R et al., 2006) in order to
bind the biologically active molecules without altering their
structure and therewith possibly their function in the body.
[0015] Still, the coatings mentioned above all struggle from
several draw-backs, due to unresolved technical difficulties.
SUMMARY OF THE INVENTION
[0016] The present invention for the first time describes a metal
implant to be used as medical and/or dental implant, which actively
facilitates control of hard and/or mineralized tissue adhesion to
the implant, such as bone, cartilage or dentin addition to the
implant surface.
[0017] A typical implant of the present invention either actively
facilitates improved hard and/or mineralized tissue adhesion to the
implant, improved bone addition to the implant surface, improved
bone remodeling and/or biocompatibility of the implant, or it
actively inhibits and/or reduces hard and/or mineralized tissue
adhesion to the implant, such as inhibits and/or reduces bone
attachment to the implant. The effect of the implant on mineralized
and/or hard tissue adhesion being directly attributable to at least
part of its surface being coated with a low, or with a high
concentration layer of polyunsaturated fatty acids (PUFA).
[0018] The present invention at the same time relates to a metal
implant with improved biocompatibility which can facilitate a solid
incorporation into the bone, and to an implant which is easy to
remove again, wherein the high concentration of available double
bounds from the polyunsaturated fatty acids hinder tissue adherence
to a semi-permanent and/or temporary implant, a so called
"slippery" implant.
[0019] The invention further relates to a method for manufacturing
said metal implant with either inducing or inhibiting effect on
hard and/or mineralized tissue adhesion and/or bone remodeling,
wherein the implant is coated with PUFA at a specific
concentration, or alternatively is coated with PUFA at a specific
concentration and irradiated with UV light, either before,
simultaneously with, or after the coating step.
[0020] The invention consequently relates to a novel and simple
surface modification method to chemically bind PUFA molecules to a
surface comprising Ti or a titaniumoxide by utilizing UV
irradiation. A method is thus presented for manufacturing a metal
implant which facilitates improved hard and/or mineralized tissue
adhesion, improved bone addition, improved effect on bone
remodeling and/or biocompatibility, wherein the implant is coated
with PUFA at a specific concentration and irradiated with UV light,
before and/or after the coating.
[0021] Objects and features of the present invention will become
apparent from the claims and the following detailed description
considered in conjunction with the accompanying drawings. It is to
be understood, however, that the drawings are designed solely for
purposes of illustration and not as a definition of the limits of
the invention, for which reference should be made to the appended
claims.
FIGURE LEGENDS
[0022] FIG. 1: Modification of the 3 groups of coin surfaces. Each
group contains a control with no EPA on the surface, and surfaces
with 3.2 nmol EPA/mm.sup.2 or 16.3 nmol EPA/mm.sup.2 given to the
respective surfaces.
[0023] FIG. 2: Water contact angle against UV irradiation time
(average for 3 surfaces). After a slight increase for about 10 min
of irradiation the water CA decreased. The black point indicates
the contact angle after 30 min, where a decrease was found for all
the surfaces. After 60 min the decrease of the water CA
flattened.
[0024] FIG. 3: Contact angles measured on coins of the 3 different
groups with different concentrations of EPA solutions used for
surface modification. A--non-irradiated, unwashed surfaces with a
thick layer of physically bound EPA; B--non-irradiated, washed
surfaces with a thin layer of physically adsorbed EPA;
C--irradiated, washed surfaces with a thin layer of covalently
bound EPA.
[0025] FIG. 4: Profilometric observation of EPA drops on polished
Ti surfaces. 3.2 nmol EPA/mm.sup.2 was given to the surface and
pictures were taken a) before UV irradiation, and b) after 30 min
irradiation with UV light. The pictures were taken with 50.times.
magnification.
[0026] FIG. 5: Changes in the FTIR spectra of EPA and EPA after
irradiation for 30 min. The numbers from 1 to 8 highlight the main
changes in the structure as referred to in the text.
[0027] FIG. 6: Results for scintillation countings of .sup.14C-EPA
coated coins which were either non-irradiated or irradiated with UV
light for 30 min. The coins were unwashed, washed once, and washed
twice, respectively.
[0028] FIG. 7: Amount of cells on the surfaces after 24 h relative
to plastic (100%) as calculated from the amount of DNA on the
respective surfaces. A--non-irradiated, unwashed surfaces with a
thick layer of physically bound EPA; B--non-irradiated, washed
surfaces with a thin layer of physically adsorbed EPA;
C--irradiated, washed surfaces with a thin layer of covalently
bound EPA.
[0029] FIG. 8: Toxicity of surface modifications relative to
plastic (0% toxicity) and 0.1% Triton X-100 (100% toxicity) as
measured by LDH-level on the respective surfaces.
A--non-irradiated, unwashed surfaces with a thick layer of
physically bound EPA; B--non-irradiated, washed surfaces with a
thin layer of physically adsorbed EPA; C--irradiated, washed
surfaces with a thin layer of covalently bound EPA.
[0030] FIG. 9: Proposed mechanism for the photooxidation of EPA and
TiO.sub.2 which resulted in a thin layer of covalently bound EPA
and EPA photooxidation products. a) EPA is oxidized resulting in
generation of oxides and their radicals, peroxides and their
radicals, peroxy acids, and peresters within the EPA structure by
irradiation with UV light, and degradation of EPA molecules into
carboxylic and dicarboxylic acids; b) photooxidation of TiO.sub.2
surfaces led to hydroxidation as well as reduction of hydroxide
groups; c) bonding between reactive EPA-photooxidation products and
reactive groups on the irradiated TiO.sub.2 surfaces via formation
of ester, perester, ether, and peroxide groups. R1-4--remaining EPA
backbone of various length and with various numbers of double
bonds.
[0031] FIG. 10. Pull-out test measurements of tested surfaces after
10 weeks healing period. Boxplots represent the median value and
the distribution of the different measurements (n=6) of each
group.
[0032] FIG. 11. Changes in volumetric bone mineral density (vBMD)
of sub-implant cortical bone after 10 weeks of healing time using
micro-CT. Values represent means.+-.SEM (n=6).
[0033] FIG. 12. LDH activity measured in the wound fluid collected
from the implant site after 10 weeks of healing time.
[0034] FIG. 13. ALP activity measured in the wound fluid collected
from the implant site after 10 weeks of healing time.
[0035] FIG. 14. Total protein measured in the wound fluid collected
from the implant site after 10 weeks of healing time.
[0036] FIG. 15. Osteocalcin gene expression in the peri-implant
bone tissue attached to the modified titanium implants.
[0037] FIG. 16. IL-6 gene expression in the peri-implant bone
tissue attached to the modified titanium implants.
[0038] FIG. 17. TRAP gene expression in the peri-implant bone
tissue attached to the modified titanium implants.
[0039] FIG. 18. Changes of peak areas of different absorbances in
the FTIR spectrum caused by UV irradiation of PUFA.
[0040] FIG. 19. .alpha.-tocopherol and some of its possible
photooxidation products (after Yamauchi et al, 2002).
[0041] FIG. 20. Change of peak areas at typical absorbances for
.alpha.-tocopherol during UV irradiation.
[0042] FIG. 21. Changes of peak areas with UV irradiation time when
50 mol % of .alpha.-tocopherol was added to EPA or DHA illustrating
the antioxidative effect of .alpha.-tocopherol on PUFAs.
[0043] FIG. 22. Changes of peak areas with a maximum absorbance at
3300 cm.sup.-1 for PUFAs with different amounts of
.alpha.-tocopherol added.
[0044] FIG. 23. Scheme over the transformation from 7DHC to
1,25(OH).sub.2D.sub.3.
[0045] FIG. 24. FTIR spectra for 7-DHC that was irradiated with UV
light for different periods of time.
[0046] FIG. 25. FTIR spectra for cholecalciferol that was
irradiated with UV light for different periods of time.
[0047] FIG. 26. Changes of peak areas with UV irradiation time for
the absorbance spectra of 7-DHC and cholecalciferol.
[0048] FIG. 27. The spectra of the mixtures of 7-DHC+EPA on Ti
surfaces before and after UV irradiation for 15, 30, and 60
min.
[0049] FIG. 28. Changes in peak areas of the mixture of 7-DHC+EPA
on Ti surfaces during UV irradiation for up to 60 min compared to
the changes of peak areas of EPA alone and 7-DHC alone.
[0050] FIG. 29. Set-up for irradiating Ti samples with UV light
after the application of mixtures of PUFA and/or vitamins.
DETAILED DESCRIPTION
[0051] The present invention for the first time describes a novel
and simple surface modification method to chemically bind
polyunsaturated fatty acids (PUFA) molecules to a metal, such as to
a surface comprising Ti and/or a titaniumoxide by utilizing UV
irradiation.
[0052] In a particular embodiment, the PUFA used is
eicosapentaenoic acid 20:5 n-3 (EPA), which is a fatty acid with
positive effects on bone homeostasis in vivo, such as on bone
remodeling, bone formation and/or bone resorption. UV light is
herein employed to induce reactive binding sites on at least part
of one of the Ti surfaces of a medical implant (photocatalytic
effect) and/or within the EPA molecules, thus the UV radiation of
the implant is envisioned either to take place before and/or after
the coating of the Ti surface with PUFA.
[0053] As is documented in the experimental section (see e.g.
Example 2), the surface coating of an implant thus coated and
exposed to UV irradiation (chemically bound PUFA) has been
characterized by CA measurement, FTIR and scintillation counting.
Performance of the coated surfaces have further been tested in
vitro with cultivation of MC3T3-E1 cells for 24 h and been compared
to surfaces with physically adsorbed EPA (i.e. physically adsorbed
PUFA) or to non-modified Ti surfaces, as well as to Ti surfaces
that were exposed to UV irradiation without a pre-coating with
PUFA. Surprisingly, surfaces with chemically bound EPA were shown
to perform significantly better in terms of cell attachment
compared to non-modified or only UV irradiated Ti surfaces as well
as compared to surfaces with physically adsorbed EPA. What is more,
they even exhibited a comparably low toxicity.
[0054] In the present context, the phrase "implant" includes within
its scope any device intended to be implanted into the body of a
vertebrate animal, in particular a mammal such as a human.
Non-limiting examples of such devices are medical devices that
replace anatomy or restore a function of the body such as the
femoral hip joint; the femoral head; acetabuiar cup; vascular
stents, elbow including stems, wedges, articular inserts; knee,
including the femoral and tibial components, stem, wedges,
articular inserts or patellar components; shoulders including stem
and head; wrist; ankles; hand; fingers; toes; vertebrae; spinal
discs; artificial joints; dental implants; ossiculoplastic
implants; middle ear implants including incus, malleus, stages,
incus-stapes, malleus-incus, malleus-incus-stapes; cochlear
implants; orthopacdic fixation devices such as nails, screws,
staples and plates; heart valves; pacemakers; catheters; vessels;
space filling implants; implants for retention of hearing aids;
implants for external fixation; and also intrauterine devices
(IUDs); and bioelectronic devices such as intracochlear or
intracranial electronic devices. Medical implants may also be
denoted as medical prosthetic devices. Generally, a medical implant
is composed of one or several implant parts.
[0055] In the present context, the term "orthopedic implant"
includes within its scope any device intended to be implanted into
the body of a vertebrate animal, in particular a mammal such as a
human, for preservation and restoration of the function of the
musculoskeletal system, particularly joints and bones, including
the alleviation of pain in these structures.
[0056] In the present context, the term "dental implant" includes
within its scope any device intended to be implanted into the oral
cavity of a vertebrate animal, in particular a mammal such as a
human, in tooth restoration procedures. Dental implants may also be
denoted as dental prosthetic devices. Generally, a dental implant
is composed of one or several implant parts. For instance, a dental
implant usually comprises a dental fixture coupled to secondary
implant parts, such as an abutment and/or a dental restoration such
as a crown, bridge or denture. However, any device, such as a
dental fixture, intended for implantation may alone be referred to
as an implant even if other parts are to be connected thereto.
Dental implants are presently preferred embodiments.
[0057] The present invention relates to a metal implant to be used
as medical and/or dental implant, which actively facilitates
controlled adhesion of hard and/or mineralized tissue to the
implant, e.g. which actively induces adhesion of hard and/or
mineralized tissue to the implant and/or exhibits improved effect
on bone remodeling and/or biocompatibility of the implant due to at
least part of its surface being coated with a low concentration
layer of polyunsaturated fatty acids (PUFA). In a particular
embodiment, at least part of its surface is coated with a low
concentration layer of polyunsaturated fatty acids (PUFA) in
combination with a phospholipid, vitamin and/or antioxidant.
[0058] The present invention at the same time relates to a metal
implant to be used as medical and/or dental implant, which actively
inhibits hard and/or mineralized tissue adhesion to the implant,
such as bone attachment, due to at least part of its surface being
coated with a layer of polyunsaturated fatty acids (PUFA) in a high
concentration. It is furthermore understood that the later
embodiment of the present invention, i.e. the implant which
actively inhibits hard and/or mineralized tissue adhesion to the
implant, such as bone attachment, due to at least part of its
surface being coated with a layer of polyunsaturated fatty acids
(PUFA) in a high concentration, may still induce and/or actively
promote bone remodeling in tissue that is not in direct contact
with the implant. Thus, in such an embodiment, a slippery implant
is generated that is easily removable from the implantation site,
which still displays a beneficial effect on mineralized and/or hard
tissue growth in the general vicinity of the implantation site.
Without a wish to limit the scope of the present invention to a
particular theory, the above discussed beneficial effect of a high
concentration coating of PUFA of a slippery implant on close by
mineralized and/or hard tissue is most likely due to the natural
dilution of the PUFA once it is released into the surrounding
tissue of the implant, which will of course generate a sufficiently
lower concentration of PUFA at an easily calculatable distance from
the actual contact surface of the implant.
[0059] The invention further relates to a method for manufacturing
said metal implant with either inducing or inhibiting effect on
hard and/or mineralized tissue adhesion and/or bone remodeling,
wherein the implant is coated with PUFA at a specific
concentration, or alternatively is coated with PUFA at a specific
concentration and irradiated with UV light.
[0060] The device or implant according to the invention can be used
for a number of purposes. Examples of such purposes include use
for: inducing local hard and/or mineralized tissue (e.g. bone
tissue) formation at the implantation site; controlling microbial
growth and/or invasion at the implantation site or systemically;
reducing inflammation at the implantation site or systemically;
stimulating ligament repair, regeneration or formation; inducing
cartilage formation; nucleating, controlling and/or templating
biomineralization; improving attachment between implants and
tissues; improving osseointegration of implants; improving tissue
adherence to an implant; hindering tissue adherence to an (semi
permanent or temporary) implant; improving contact between tissues
or tissues and implants, improving tissue sealing of a (surgical)
wound; inducing apoptosis (cell death) in unwanted cells (e.g.
cancer cells); inducing specific cell differentiation and/or
maturation, increasing tissue tensile strength; improving wound
healing; speeding up wound healing; templating tissue formation;
guiding tissue formation; local gene therapy; stimulating nerve
growth; improving vascularisation in tissues adjacent to an
implant; stimulating local extracellular matrix synthesis;
inhibiting local extracellular matrix breakdown; inducing local
growth factor release; increasing local tissue metabolism;
improving function of a tissue or body-part; reducing local pain
and discomfort. The purpose will depend on the type of implant as
well as the nature and/or concentration of the PUFA.
[0061] Presently preferred embodiments may improve the
osseointegration of implants; i.e. they improve tissue adherence to
an implant, improve bone remodeling, hinder tissue adherence to a
(semi-permanent or temporary) implant, reduce bone remodeling,
and/or improve contact between tissues or tissues and implants. It
is presently envisioned that an implant does either display a
stimulating or a dampening effect on bone remodeling and/or
osseointegration, but it is of course also possible to produce an
implant that does display these contrary effects on different parts
of the implant.
[0062] The term "hard and/or mineralized tissue" is in the present
context employed to describe a variety of different naturally
occurring tissue types that have become mineralized, and/or tissue
having a firm intercellular substance. A hard and/or mineralized
tissue according to the present invention is preferably selected
from the group consisting of cartilage, bone, dental enamel,
dentine-like tissue, dental hard tissue, and cortical tissue.
[0063] In general, mineralized tissue is vital to many
characteristic adaptive phenotypes in vertebrates. Three primary
tissues, enamel (enameloid), dentin, and bone, are found in the
body armor of ancient agnathans and mammalian teeth, suggesting
that these two organs are homologous. Mammalian enamel forms on
enamel-specific proteins such as amelogenin, whereas dentin and
bone form on collagen and many acidic proteins, such as SPP1,
coordinately regulate their mineralization.
[0064] In a presently preferred embodiment, the implant comprises
at least 90% of weight of a metal material.
[0065] It is presently preferred that the metal material is
titanium or an alloy thereof, e.g. an alloy with zirconium,
tantalum, hafnium, niobium, aluminum, vanadium, chrome, cobalt,
magnesium, iron, gold, silver, copper, mercury, tin or zinc. In a
particularly preferred embodiment, the metal material is
titanium.
[0066] Also, preferably the metal material is zirconium, hafnium,
tantalum, niobium or mixtures of two or more of these. The metal
material preferably also is a metal hydride, such as TiH, metal
hydroxide, such as TiOH, a hydride of an alloy, or a hydroxide of
an alloy. Alternatively the material may be an oxide of a metal.
Also, the implant material may be aluminium, gold or surgical steel
nickel.
[0067] The term "cp" is well known to the person skilled in the art
and stands for "commercially pure" and relates to the level of
pureness of the employed metal, such as Ti.
[0068] When the metal material is an alloy of titanium, zirconium,
tantalum, hafnium or niobium, it may be an alloy between one or
more of these metal elements; or it may be an alloy containing one
or more other metals such as aluminium, vanadium, chrome, cobalt,
magnesium, iron, gold, silver, copper, mercury, tin or zinc; or
both.
[0069] In a presently preferred embodiment, the implant comprises
at least 90% of weight of titanium and/or an alloy of titanium.
[0070] In one embodiment of the invention, the Ti comprising
implant is exposed to UV radiation prior, simultaneously and/or
after coating with a PUFA. TiO.sub.2 is a well known photocatalyst
(Nakamura et al., 2002). If the surface is irradiated with UV
light, electron-hole pairs are generated, reactive oxygen compounds
are released, and water molecules dissociate and adsorb at the
surface. Those hydroxide groups cause an increased hydrophilicity
of the surface. The efficacy of UV irradiation has been shown
previously by contact angle (CA) measurements and, to a certain
extent, by FTIR measurements (Nakamura et al., 2002; Miyauchi et
al., 2002).
[0071] In one embodiment, the present invention relates to a method
to coat a cp Ti medical implant with a thin layer of PUFA, e.g. as
demonstrated in Examples 1 and 2 with eicosapentaenoic acid (EPA),
alternatively the implant is additionally exposed to UV irradiation
and washing (chemically bound PUFA coating) or only washed
(physically adsorbed PUFA coating). In the presently presented
examples, the changed surface characteristics of the cp Ti implant
are then detected and characterize with physical and chemical
methods.
[0072] In the present context PUFA stands for polyunsaturated fatty
acids, a term well known to the person skilled in the art to
include a well defined group of fatty acids.
TABLE-US-00001 TABLE 1 Common Fatty Acids Chemical Names and
Descriptions of some Common Fatty Acids Carbon Double Common Name
Atoms Bonds Scientific Name Sources Butyric acid 4 0 butanoic acid
butterfat Caproic Acid 6 0 hexanoic acid butterfat Caprylic Acid 8
0 octanoic acid coconut oil Capric Acid 10 0 decanoic acid coconut
oil Lauric Acid 12 0 dodecanoic acid coconut oil Myristic Acid 14 0
tetradecanoic acid palm kernel oil Palmitic Acid 16 0 hexadecanoic
acid palm oil Palmitoleic Acid 16 1 9-hexadecenoic acid animal fats
Stearic Acid 18 0 octadecanoic acid animal fats Oleic Acid 18 1
9-octadecenoic acid olive oil Ricinoleic acid 18 1
12-hydroxy-9-octadecenoic acid castor oil Vaccenic Acid 18 1
11-octadecenoic acid butterfat Linoleic Acid 18 2
9,12-octadecadienoic acid (n-6) grape seed oil Alpha-Linolenic Acid
18 3 9,12,15-octadecatrienoic acid (n-3) flaxseed (ALA) (linseed)
oil Gamma-Linolenic 18 3 6,9,12-octadecatrienoic acid (n-6) borage
oil Acid (GLA) Arachidic Acid 20 0 eicosanoic acid peanut oil, fish
oil Gadoleic Acid 20 1 9-eicosenoic acid fish oil Arachidonic Acid
20 4 5,8,11,14-eicosatetraenoic acid liver fats (AA) (n-6) EPA 20 5
5,8,11,14,17-eicosapentaenoic fish oil acid (n-3) Behenic acid 22 0
docosanoic acid rapeseed oil Erucic acid 22 1 13-docosenoic acid
rapeseed oil DHA 22 6 4,7,10,13,16,19-docosahexaenoic fish oil acid
(n-3) Lignoceric acid 24 0 tetracosanoic acid small amounts in most
fats
[0073] As is well known in the art, a typical PUFA may be described
by the following formulae:
H.sub.3C-n.sub.x-((n.sub.x(.dbd.)n.sub.x).sub.y-n.sub.x-((n.sub.x(.dbd.)-
n.sub.x).sub.y-n.sub.x-COOH
wherein: n is any natural and/or artificially modified C and/or any
other natural and/or artificial element that can form a double
binding, and (.dbd.) represents a double binding, and x is a number
between 0 and x, and y is a number between 1 and x, and wherein the
position of the double binding is variable along the chain of
n.
[0074] The double bounds can be either all in cis or all in trans,
or in mixed configurations.
[0075] Polyunsaturated fatty acids have effects on diverse
physiological processes impacting normal health and chronic
diseases. The predominant sources of PUFA are vegetable oils and
fish. Chemically, PUFA belong to the class of simple lipids, as are
fatty acids with two or more double bonds in cis position. The
location of the first double bond, counted from the methyl end of
the fatty acid, is designated by the omega- or n-number.
[0076] PUFA occur throughout animal, plant, algae, fungi and
bacteria and is found widely in many lipid compounds such as
membranes, storage oils, glycolipids, phospholipids, sphingolipids
and lipoproteins. PUFA is produced commercially from selected seed
plants, and some marine sources.
[0077] There are two main families of PUFA, n-3 and n-6 (see Table
1).
[0078] Typical PUFA are:
3-Series PUFA, n-3: Alpha-linolenic acid (ALA), eicosapentaenoic
acid (EPA), docosahexaenoic acid (DHA) 6-Series PUFA, n-6: linoleic
acid, gamma-linolenic acid (GLA), arachidonic acid (AA)
[0079] Preferably, the PUFA is selected from the group consisting
of n-3 and n-6 fatty acids.
[0080] Dietary PUFA have long been recognized as being able to
exert unique influences on metabolic pathways and cellular growth.
For example, they can act as hormones and control the activity of
transcription factors. What is more, PUFA were found to elicit
changes in gene expression that precede changes in membrane
composition by directly governing the activity of nuclear
transcription factors. The effects exerted by PUFA are most likely
mediated via changes in membrane composition, altered hormone
release or signalling, and/or ligand/receptor interaction.
[0081] In general, fatty acids (FA), the basic modules of lipids
have a great influence on properties of the cell membrane (e.g.
fluidity) and on cell metabolism. Dietary FA have prior been shown
to influence bone modelling and remodelling. Especially the n-6/n-3
ratio of polyunsaturated fatty acids (PUFA) is of implicit
importance. EPA (eicosapentaenoic acid 20:5 n-3) is a precursor in
the production of eicosanoids in the body, a group of signalling
molecules that can as well be synthesized from other n-3 and n-6
PUFA, such as AA (arachidonic acid 20:4 n-6) and DHA
(docosahexaenoic acid 22:6 n-3). Those PUFA have opposing effects
on, amongst others, bone metabolism and inflammation.
[0082] Preferably, the PUFA used in the present invention comprises
or consists of EPA.
[0083] Although in one presently preferred embodiment, EPA was
chosen as coating substance for a metal surface, such as a Ti
surface of an implant, it is well documented in the field that DHA
acts in a similar way with regards to bone tissue stimulation. Thus
in another embodiment, DHA or mixtures between EPA and DHA are
envisioned as surface coating as well.
[0084] Bone is a multifunctional organ that consists of a
structural framework of mineralized matrix and contains
heterogeneous populations of chondrocytes, osteoblasts, osteocytes,
osteoclasts, endothelial cells, monocytes, macrophages, lymphocytes
and hemopoietic cells. Bone growth is regulated by complex
interactions between different intercellular and extracellular
players. Without the intention to be bound by a specific scientific
hypothesis, it is envisioned that PUFA in a low concentration have
a beneficial effect on bone growth, formation, resorption and/or
adhesion, whereas a high concentration of PUFA is repellent to and
actively inhibits bone growth, formation and/or adhesion.
[0085] Thus, the present invention relates to a metal implant to be
used as medical and/or dental implant, which actively facilitates
controlled adhesion of bone to the implant, e.g. which actively
induces adhesion of bone to the implant and/or exhibits improved
effect on bone remodelling and/or biocompatibility of the implant
due to at least part of its surface being coated with a low
concentration layer of polyunsaturated fatty acids (PUFA). The
present invention at the same time relates to a metal implant to be
used as medical and/or dental implant, which actively inhibits bone
adhesion to the implant, such as bone attachment, due to at least
part of its surface being coated with a layer of polyunsaturated
fatty acids (PUFA) in a high concentration. The invention further
relates to a method for manufacturing said metal implant with
either inducing or inhibiting effect on bone adhesion and/or
remodelling, wherein the implant is coated with PUFA at a specific
concentration, or alternatively is coated with PUFA at a specific
concentration and irradiated with UV light.
[0086] The invention also relates to a method for manufacturing a
metal implant with an improved effect on adhesion of mineralized
and/or hard tissue to the implant, comprising
a) treating the implant with a solution comprising PUFA; and b)
irradiating at least part of the surface of the implant with UV
light for at least 30 seconds.
[0087] Also, the invention is related to a method for manufacturing
a metal implant with an improved effect on adhesion of mineralized
and/or hard tissue to the implant, comprising
a) irradiating at least part of the surface of the implant with UV
light for at least 30 seconds; and b) treating the implant with a
solution comprising PUFA.
[0088] The invention is also related to a method for manufacturing
a metal implant with an improved effect on adhesion of mineralized
and/or hard tissue to the implant, comprising
a) mirror polishing and/or grit-blasting the implant, b) washing,
c) autoclaving, d) treating the implant with a solution comprising
PUFA; and e) irradiating at least part of the surface of the
implant with UV light for at least 30 seconds.
[0089] Further, the invention is related to a method for
manufacturing a metal implant with an improved effect on adhesion
of mineralized and/or hard tissue to the implant, comprising [0090]
a) mirror polishing and/or grit-blasting the implant, [0091] b)
washing, [0092] c) autoclaving, [0093] d) irradiating at least part
of the surface of the implant with UV light for at least 30
seconds; and [0094] e) treating the implant with a solution
comprising PUFA.
[0095] In the methods for manufacture of an implant of the
invention, only part of the implant may be treated with a solution
comprising PUFA.
[0096] Preferably the at least part of the surface of the implant
irradiated with UV light is the part treated with a solution
comprising PUFA.
[0097] In the methods of the invention for manufacturing a metal
implant with an improved effect on adhesion of mineralized and/or
hard tissue to the implant the solution comprising PUFA preferably
comprises EPA.
[0098] Preferably, in the methods for manufacturing a metal implant
with an improved effect on adhesion of mineralized and/or hard
tissue to the implant, the implant preferably comprises
titanium.
[0099] In a presently preferred embodiment, the invention thus
relates to a method for manufacturing a metal implant with an
improved effect on adhesion of mineralized and/or hard tissue to
the implant, wherein the surface is irradiated with UV light
characterized by Fluo.link, .lamda.=312 nm.
[0100] In an equally preferred embodiment, the invention further
relates to a method for manufacturing a metal implant with an
improved effect on adhesion of mineralized and/or hard tissue to
the implant, wherein intensity of the UV light which the surface is
irradiated with is approximately 6 mW/cm.sup.2.
[0101] In vitro experiments documented in the experimental section
(see Example 1) were carried out to test cytotoxicity and cell
attachment of MC3T3-E1 cells to EPA-modified titanium implants. The
surface characterization of the implants described herein show that
the surface structure of titanium was not changed during the
procedure, while chemical properties of the titanium surfaces and
of FA were changed in order to form chemical bindings. Significant
amounts of .sup.14C-EPA were found to be left on surfaces of
UV-irradiated implants compared to non-irradiated implants after
thorough washing with methanol. In vitro results on MC3T3-E1 cells
showed that a high amount of EPA was toxic to the cells. However,
surfaces with lower amounts of EPA bound to the surface with UV
light showed lower toxicity and higher cell attachment compared to
untreated implants and uncoated implants.
[0102] In conclusion, the present results demonstrate that UV light
is a suitable method to bind PUFA, e.g. EPA, and to create a thin
layer of PUFA on the surface of e.g. titanium implants, which has
shown to improve the cell viability and attachment of osteoblastic
cells.
[0103] UV light in the range of UV C (100 nm-280 nm) and/or UV B
light (280 nm-315 nm) is preferred for the present invention. For
example, 254 nm and 302 nm have been proven to work well for the
present invention. The activity of titanium is reduced from about
350 nm to 400 nm. Consequently UV B is a safe area for TiO.sub.2
activation (S.-M. Oh et al. 2003).
[0104] EPA is susceptible to light-induced oxidation due to
unsaturations in the structure. Especially the highly energetic UV
light leads to oxidation and generation of reactive oxide groups.
As is shown in Example 2, analysis of FTIR spectra showed that
changes occurred in the EPA structure. Measurements of the amount
of .sup.14C-labelled EPA on surfaces showed that a measurable
amount of .sup.14C-EPA was left on the non-irradiated and
irradiated surfaces. However, the significant difference in
measured .sup.14C signals indicated that the irradiated surface
coating was stable against thorough washing and therewith
chemically bound. The fatty acid film on irradiated surfaces may
consist of a combination of chemically and physically bound EPA.
Surface roughness of the polished cp Ti samples precluded detailed
analysis of the coating structure with for example grazing
incidence x-ray reflectometry, where a S.sub.a not higher than 1 nm
is required; such a low surface roughness could e.g. be obtained by
sputtering a thin layer of Ti onto smooth Si surfaces.
[0105] In Example 2, the 2 reference groups with physically
adsorbed EPA on their surfaces showed that large quantities of EPA
(group A) had a toxic effect on the cells, and small quantities of
EPA (group B) had no significant effect on cell attachment. For
group C it was found that UV-irradiation of TiO.sub.2 alone
(control in group C) had a slightly negative effect on cell
attachment while toxicity was not increased.
[0106] Chemically bound EPA in group C had a positive effect on
cell attachment and was shown to be nontoxic. Therefore it can be
suggested that EPA is effective even though it is chemically bound
to the surfaces and therefore not directly available for the cells.
A surface coating with EPA may change the characteristics of the
protein layer readily adsorbed onto the surfaces of the samples
when in contact with protein-containing media. A possible
explanation for this phenomenon is that EPA is prone to
autoxidation on air due to its unsaturations; thus the results of
the in vitro tests, which showed a positive effect of the coating
by irradiation.
[0107] Furthermore, studies are included which investigate Ti
surfaces, which alone are activated with UV-irradiation and such
surfaces to which the EPA is given to the surfaces subsequently. By
this specific measure, the alteration of the fatty acid molecules
may be reduced.
[0108] Implant surfaces are exposed to high friction forces during
implantation; thus a surface coating should be thin and firm to
stand high abrasion forces. Thus the present invention in a
presently preferred embodiment relates to implants with a chemical
surface coating of PUFA.
[0109] As was shown in CA measurement in the experimental section,
surface roughness parameters did not change due to surface
modification procedure, indicating that all changes of surface
contact angles discussed were most likely caused by changes in
surface chemistry.
[0110] A preferred cleaning solvent, such as methanol, leaves a
thin layer of carboneous residues on surfaces. Higher carbon
content on metal surfaces has prior been reported to increase
hydrophobicity of those surfaces. This explains the increase in CA
for the positive control in group B. The comparably lower CA of the
surface coated coins in group B indicated that most probably, and
in accordance with the amount of .sup.14C-EPA measured on those
surfaces, a thin layer of physically adsorbed EPA was left after
washing with methanol. For chemical coating (group C), reactive
sites had to be generated both, on the Ti surface and within EPA
molecules to create a thin and stable surface layer. The
photocatalytic effect of UV light on TiO.sub.2 was utilized for
generation of reactive hydroxide groups on the surfaces of the Ti
coins. As is well known in the art, TiO.sub.2 becomes amphiphilic
due to UV-irradiation, with a structure containing of coexisting
hydrophilic and lipophilic phases. In the presently presented
experiments, CA measurements with water showed that irradiated
TiO.sub.2 surfaces became more hydrophilic.
[0111] Analysis with the Profilometer showed that EPA was unevenly
distributed on non-irradiated surfaces (group A); drop-like
structures, or rather regions with more or less thick EPA layer
were observed (data not shown). However, the fatty acids were
evenly spread on the surfaces after irradiation of the samples,
which was most probably due to lipophilic properties of the
surfaces.
[0112] Consequently, the present invention in one aspect relates to
a medical implant, which is at least in part coated with an evenly
spread thin layer of PUFA. Such an implant is in a presently
preferred embodiment coated with chemically bound PUFA, such as
with EPA, AA and/or DHA. An implant corresponding to said
particular embodiment is further characterized by exhibiting
improved effect on hard and/or mineralized tissue adhesion and/or
bone formation, remodeling, addition and/or biocompatibility.
[0113] In the present context, a thin layer of PUFA represents a
monolayer of a PUFA, e.g. EPA, representing about
1-5.times.10.sup.15 bound unsaturated bindings per mm.sup.2
titanium surface, such as approximately 3.times.10.sup.15 bound
unsaturated bindings per mm.sup.2 titanium surface.
[0114] Typically, a high concentration of PUFA per mm.sup.2 metal,
e.g. titanium surface represents about 1-5 nanomol PUFA/mm.sup.2
titanium surface, such as 3.2 nanomol PUFA/mm.sup.2 titanium
surface. A high concentration coating thus comprises about 0.1-5
.mu.g PUFA/mm.sup.2 titanium surface, such as at least 1 .mu.g
PUFA/mm.sup.2 titanium surface. Typically said PUFA is EPA.
[0115] In turn, a low concentration of PUFA per mm.sup.2 titanium
surface represents less than 0.5 nanomol PUFA/mm.sup.2 titanium
surface. A low concentration coating thus comprises about 1-100
nanogram PUFA/mm.sup.2 titanium surface, such as at the most 10
nanogram PUFA/mm.sup.2 titanium surface or at the most 1
nanogram/mm.sup.2 titanium surface. Typically said PUFA is EPA.
[0116] A presently preferred embodiment is thus a metal implant for
controlled mineralized tissue adhesion, wherein at least part of
the surface of the implant is coated with PUFA (polyunsaturated
fatty acids) at a concentration of less than 0.01 .mu.mol, such as
less than 0.001 .mu.mol.
[0117] In the experiment data, it is in Example 1 shown a better
attachment and cell differentiation in vitro.
[0118] In contrast, it is shown in vivo in Example 3, a decreased
bone attachment strength with the implants that were UV irradiated
(with and without PUFA). The results indicates that surface
treatment with UV light, in presence or absence of EPA, reduces
bone attachment and volumetric bone mineral density in the
peri-implant bone tissue. None of the presented surface treatments
showed increased LDH activity levels as a result of tissue necrosis
on the adjacent bone. In addition, gene expression of bone
formation markers show higher mRNA levels in UV irradiated (with
and without PUFA). It can be concluded from this study that surface
functionalisation of Ti implants with UV light and EPA is a
biocompatible coating to reduce bone bonding ability of Ti and
maintain new bone formation.
[0119] The goal of Example 3 was to produce Ti surfaces with a
layer of chemically bound EPA using UV irradiation that maintain
the healing process of cortical bone while having a low attachment,
to be applicable as bone fracture plates. These applications
require the development of surfaces that prevent soft tissue
attachment and irritation, allow tissue gliding, but maintain their
biocompatible properties. Pure titanium metal is one of the most
widely chosen materials for bone plates, because of its excellent
biocompatibility and corrosion resistance. The only available
strategy today to minimise bony integration is using surfaces with
minimal roughness by polishing titanium fixation plates. Although
surface roughness has been considered the major determinant in
osseointegration, surface chemistry plays an important role as
well. Coating Ti with a uniform layer of EPA using UV irradiation
provides an example to investigate how surface chemistry
modification interact in their effect on cells and influence
osseointegration.
[0120] Furthermore, another preferred embodiment is a metal implant
coated with PUFA and further characterized in that at least the
part of the implant coated with PUFA has been exposed to UV
radiation for at least 30 seconds, such as for at least 10 minutes,
or for at least 30 minutes. The UV treatment may take place before,
after or simultaneously the coating of the implant with PUFA.
[0121] In one embodiment, the implant's functionality is further
improved by binding and/or integrating one or more various active
elements and/or substances to the surface of the implant, either
before, simultaneously, and/or after the PUFA coating and
alternative UV treatment of the implant. Such bindings are
preferably carried out using for example chemical reactants having
two reactive functionalities such as formalin or glutaraldehyde.
Typically, an element or substance to be bound to the surface of
the implant is selected from the group consisting of vitamins (such
as vitamin E, C, D, A), in particular fat-soluble vitamins,
antioxidants, ions (such as fluoride, calcium, phosphates,
carbonates), and antibiotics (such as fat soluble antibiotics,
microclines, tetracycline).
[0122] Vitamin E is preferably present on the implant or device
during UV irradiation as it prevents oxidation of PUFA (as
demonstrated in Example 4).
[0123] 7-Dehydrocholesterol (7-DHC, provitamin D.sub.3) is
converted to cholecalciferol (vitamin D3) via previtamin D.sub.3 in
the human skin. Further conversion to 25(OH)D.sub.3 is mainly
reported to happen in the liver. However, since the hydroxylase
enzyme CYP27A1 is ubiquitously expressed in other tissues, the
system for local production may be also available in bone (Aiba I
et al., 2006). Besides the kidneys, 1.alpha.,25(OH).sub.2D3 has as
well been shown to be synthesised locally in bone from
25(OH)D.sub.3 (Ichikawa et al., 1995; Atkins et al., 2007). A
schematic overview of the conversion can be found as FIG. 23. 1
.alpha.,25(OH).sub.2D.sub.3 is the biological active form of
vitamin D3 and acts as a hormone to regulate serum calcium and
phosphate levels and is an important factor during bone growth and
mineralisation (St-Arnaud R, 2008).
[0124] Vitamin-D deficiency has been considered as systemic risk
factor for reduced bone mineral density, osteoporosis, and impaired
osseointegration. Ageing results in a number of changes in bone and
calcium metabolism which can potentially affect osseointegration.
The secretion of parathyroid hormone increases with age.
Conversely, there is a decrease in calcitonin and Vitamin D
absorption with age. The main problem with vitamin D deficiency is
that is very difficult to calculate from dietary intake and
sun-exposure what the blood level will be.
[0125] Therefore, coating 7-DHC to titanium implants and activating
the conversion from 7-DHC to cholecalciferol with UV light, with or
without PUFA, may affect bone healing positively and improve
peri-implant bone healing in normal and osteoporotic patients.
Cholecalciferol will be then converted to calcidiol and calcitriol
in the bone with their own hydroxylases when needed. 7-DHC has the
advantage of having a much lower toxicity compared to
cholecalciferol and hydroxilated forms of vitamin D. Besides,
irradiation of 7-DHC gives the right end product that can be
modified further in the body, while calcitriol might oxidize to
other compounds on the titanium surfaces.
[0126] Consequently, the present invention also relates to a metal
implant wherein the implant additionally comprises
7-dehydrocholesterol. The invention also relates to an implant
coated with 7-dehydrocholesterol and subsequently irradiated with
UV light to form cholecalciferol (vitamin D.sub.3). Such an implant
may or may not also have PUFA on its surface. Titanium (Ti)
surfaces were coated with either 7-DHC (with or without PUFA in the
form of EPA) or cholecalciferol to analyse their transition
initiated by UV light in Example 5.
[0127] For 7-DHC conversion, the most effective irradiation has
wavelengths from 270 to 300 nm although the wave length range of UV
C (100 nm-280 nm) and UV B (280 nm-315 nm) also are preferred.
[0128] Typically, the device or implant specimen is produced
aseptically and/or sterile, allowed to air-dry and is then packaged
in a sterile, airtight plastic bag in which it is stored until use
for implantation. However, sometimes a wet storage system might be
desired, e.g. like canning or storage in a fluid like saline or
simply an electrolyte from the manufacturing process. Although the
coating can be run under aseptic or even sterile conditions, the
need for doing this may be avoided by including a sterilization
step prior to use, using conventional methods such as ionizing
radiation, heating, autoclaving, or ethylene oxide gas etc. The
choice of method will depend on the specific characteristics and
properties of the implant.
[0129] Prior to the coating treatment, the implant should be
thoroughly cleaned. This may typically consist of the implant being
mechanically pre-treated by electropolishing or sandblasting to
modify surface structure if desired, and subsequently thoroughly
cleaned using hot caustic soda followed by a de-greasing step, e.g.
in concentrated tri-chloro-ethylene, ethanol or methanol, before
being treated in a pickling solution, e.g. hydrofluoric acid, to
remove undesired oxides and impurities on the surface. After
pickling, the implant specimen is washed thoroughly in hot, double
distilled ion-exchanged water.
[0130] Although the present invention is mainly concerned with the
coating of metal implants with PUFA at specific high or low
concentrations, it is equally envisioned that for example a variety
of naturally occurring lipids characterized by comprising multiple
double bindings can be used in a similar way. In this context, a
thin layer of naturally occurring lipids would be considered to
represent a monolayer of a naturally occurring lipid, representing
about 1-5.times.10.sup.15 bound unsaturated bindings per mm.sup.2
titanium surface, such as 3.times.10.sup.15 bound unsaturated
bindings per mm.sup.2 titanium surface.
[0131] The invention is further illustrated by the following,
non-limiting examples.
Experimental Section
Example 1
Rough Surface Implant
Matrix:
TABLE-US-00002 [0132] Type Concentration of coin of EPA Treatment
N.sup.o of coins Polished 0 mM irradiated with UV light in vitro 6
5 = 30 10 mM no irradiation char. 6 3 = 18 50 mM Grit- 0 mM
irradiated with UV light in vitro 6 5 = 30 blasted 10 mM no
irradiation char. 6 3 = 18 50 mM Total number of coins 96
Cleaning
[0133] 1. Coins rinsed with pure water 2. Coins washed with ethanol
3. Ultrasonication of coins in water for 5 min 4. Again coins
rinsed with pure water 5. Coins allowed to dry on sterile bench 6.
Autoclaving of coins
Surface Modification
[0134] 1. Work in sterile bench 2. EPA solution is filtered (0.2
.mu.m pore size) to sterilise it 3. 10 .mu.l of respective solution
of EPA in methanol is given on the surface of the coins 4. Coin is
allowed to dry on sterile bench 5. Part of the coins are irradiated
with UV light after the solvent evaporated .fwdarw.Fluo.link,
.lamda.=312 nm .fwdarw.Time of irradiation: 30 min (first test
showed that the contact angle is increasing up to 10 min
irradiation and decreases subsequently) .fwdarw.Intensity of
irradiation ca 6 mW/cm.sup.2 6. Wash UV irradiated coins with
methanol to remove unbound fatty acids
Characterization
[0135] 1. Profilometer: 150.times., 3 coins, 3 places on each coin,
measurement area of 200.times.160 .mu.m.sup.2 (i.e. square of
3.times.3), important are the picture from pl.mu.and the surface
roughness parameter calculated with SensoMap 2. Contact angle: Drop
of 3 .mu.l on coin, CA is measured, the drop is enlarged 2.times.
with 1 .mu.l of pure water, after each enlargement the surface CA
is measured, the CA is not measured after drying the coin as this
could change the structure of the fatty acids on the surface of the
coin and therewith the CA 3. FT IR (DRIFT) to characterize the
fatty acids on the surface, difference before and after UV
irradiation.
Example 2
Smooth Surface Implant
[0136] The aim of this study was to investigate the effect of EPA
coating on Ti surface on osteoblast cell response in vitro. Methods
to either physically adsorb or chemically bind the layer with EPA
were compared, the latter one being produced by UV irradiation of
TiO.sub.2 surfaces and EPA. Those surface coatings were detected
and characterized by physical and chemical analytical methods. In
vitro experiments were carried out to test cytotoxicity and ability
of EPA-modified Ti surfaces to promote cell attachment and
osteogenic differentiation of MC3T3-E1 cells.
Materials and Methods
Chemicals
[0137] EPA was purchased from Sigma (St. Louis, Mo., USA),
radioactive labelled [.sup.14C]-EPA was obtained from American
Radiolabeled Chemicals, Inc. (St. Louis, Mo., USA).
Titanium Implants and Treatments
[0138] Cp Ti implants with a diameter of 6.25 mm and a height of 2
mm were machined from cp Ti rods and subsequently grinded and
polished (Phoenix 4000, Buehler GmbH, Dusseldorf, Germany) in seven
sequences. A special holder was used to polish 96 implants at the
time to avoid batch's differences in future surface
characterisation. The silicon carbide papers, the porous neoprene
for final polishing and the abrasive colloidal silica suspension
(OP--S) were supplied by Struers GmbH (Willich, Germany). The first
step consisted in grinding all the implants with P500 in water
until they were levelled, with 65 N of pressure and a
contra-rotation at 250 rpm. The grinding time was then set down to
10 min and the grain size decreased with papers: P800; P1200 and
P2500. For the P4000 polishing paper, the OP--S polishing
suspension was used in addition. For the last step, a special
porous neoprene (MD-Chem) was used at 200 rpm and 50 N of pressure
for 9 min in co-rotation with OP--S suspension, and 1 min in
co-rotation with water.
[0139] After polishing, titanium coins were cleaned before
sterilization. They were washed together in a glass beaker with
deionised water for 30 s, with 70% ethanol for 30 s, and in
ultrasonic bath at 40.degree. C. for 5 min in deionised water. The
implants were subsequently placed in 40% NaOH solution in a water
bath of 40.degree. C. for 10 min, sonicated in deionised water for
5 min, and then washed with deionised water until the pH reached 6.
Afterwards the implants were sonicated in deionised water at
50.degree. C. for 5 min, placed in 50% HNO.sub.3 solution at
50.degree. C. for 10 min, and sonicated in deionised water for
another 5 min. The implants were washed with deionised water until
pH reached 6 and were stored in 70% ethanol until use. Before use,
the coins were rinsed with water, rinsed with ethanol, sonicated
for 5 min at room temperature and rinsed with deionised water. The
titanium coins were then sterilised by autoclaving at 121.degree.
C. for 15 min.
[0140] For the surface modification of titanium implants, EPA was
dissolved in methanol to 10 mM and 50 mM solution. The EPA
solutions were filtered with a 0.22 .mu.m pore size filter before
use. Three groups of coins were prepared; non-irradiated and
unwashed coins (group A), non-irradiated and washed coins (group
B), and UV-irradiated and washed coins (group C). An overview for
the 3 different groups is given in Table 2 and FIG. 1.
TABLE-US-00003 TABLE 2 Modification of the different groups of
coins as used for in vitro experiments. Group Modification Washing
Amount of EPA A non-irradiated -- 0 .mu.mol unwashed 0.1 .mu.mol
0.5 .mu.mol B non-irradiated 2 .times. 1 ml methanol 0 .mu.mol
washed 0.1 .mu.mol 0.5 .mu.mol C irradiated 2 .times. 1 ml methanol
0 .mu.mol washed 0.1 .mu.mol 0.5 .mu.mol
[0141] Those 3 groups were chosen to show the effects of a thick
layer of non-irradiated and therewith physically adsorbed EPA (A),
to show the effect of a thin layer of non-irradiated and therewith
physically bond EPA (B), and the effect of a thin layer of
irradiated and therewith chemically bond EPA (C). For each group a
cp Ti coin without any EPA surface coating served as a reference.
If EPA was given to the surface, 10 .mu.l of the respective EPA
solution was used, which equals an amount of 3.2 nmol EPA/mm.sup.2
or 0.99 .mu.g EPA/mm.sup.2 for the 10 mM solution and 16.3 nmol
EPA/mm.sup.2 or 4.93 .mu.g EPA/mm.sup.2 for the 50 mM solution. The
coins were allowed to dry on air for 15 min in a sterile flow
bench. Coins of group C were irradiated with UV light for 30 min
(Fluo.link, .lamda.=312 nm, I=6 mW/cm.sup.2). Coins of group B and
C were washed with methanol to remove unbound EPA. Group B was
washed after drying and group C after irradiation for 30 min. For
washing, 1 ml of methanol was given into a microcentrifuge tube,
the coin was added and vortexed for 10 s. This was repeated once
with fresh methanol. The coins were allowed to air-dry for 15 min
in the sterile flow bench and were used immediately for further
analysis or cell cultivation.
Profilometry
[0142] The surfaces of the coins were visualized using the blue
light profilometer PL.mu. 2300 (Sensofar, Terrassa, Spain).
Pictures were taken at 50.times. magnification and the final
picture consists of 3.times.3 assembled pictures (663.times.497
.mu.m.sup.2). Three non-overlapping pictures were taken on each
coin at random positions.
Contact Angle Measurements
[0143] The contact angles (CA) of the surfaces of the different
titanium implants were analyzed using a video-based contact angle
system (OCA 20, DataPhysics Instruments GmbH, Filderstadt,
Germany). The fluid used was deionised water. A drop of 6 .mu.l was
given to the surface with a velocity of 0.6 .mu.l s.sup.-1. The
drop shape was recorded with a camera (4.17 pictures s.sup.-1) and
contact angles were measured afterwards from the recorded pictures.
The measurements took place at 22.degree. C. Three samples were
analysed for each modification group. One drop was placed on each
sample.
Diffuse reflectance Infrared Fourier Transform (DRIFT)
spectroscopy
[0144] Thick films of lipids and the changes caused by UV
irradiation were analysed with FTIR Spectrum 100, Perkin Elmer
(USA) The diffuse reflectance unit (DRIFT) was used to collect the
spectra. A cleaned, sterilised, but unmodified polished Ti coin
served as reference. 10 .mu.l of 50 mM EPA solution was given to
the surface of a polished Ti coin. The FTIR spectra were collected
before and after irradiation with UV light at different time points
of irradiation. The spectra were collected from 4000 cm.sup.-1 to
450 cm.sup.-1 with a resolution of 2 cm.sup.-1. Each spectrum was
the result of 8 single spectra. The spectra were baseline corrected
with the program Spectrum (Version 6.1.0) from PerkinElmer.
Amount of EPA on Implant Surfaces
[0145] In order to determine the amount of .sup.14C labelled EPA on
the surfaces of polished coins in the different groups, their
surfaces were modified with 10 .mu.l of 10 mM EPA solution which
contained 1% [.sup.14C]-EPA. The coins were either non-irradiated
or irradiated for 30 min with UV light before washing. Three coins
were produced for each group. Each coin was transferred to 5 ml of
Insta-gel II Plus liquid scintillation fluid (Packard, Groningen,
Netherlands), incubated over night and then measured for 3 min in a
liquid scintillation counter (1500 Tri-Carb Liquid scintillation
Analyzer, Packard Instrument Co., USA).
Cell Cultures
[0146] The murine osteoblast cell line MC3T3-E1 (DSMZ,
Braunschweig, Germany) was used as in vitro model. Cells were
routinely cultured at 37.degree. C. in a humidified atmosphere of
5% CO.sub.2 and maintained in alpha-MEM (PAA Laboratories GmbH,
Austria) supplemented with 10% fetal calf serum (PAA Laboratories
GmbH, Austria) and 50 IU penicillin/ml and 50 .mu.g streptomycin/ml
(Sigma, St. Louis, Mo., USA). Cells were subcultured 1:4 before
reaching confluence using PBS (PAA Laboratories GmbH, Austria) and
trypsin/EDTA (Sigma, St. Louis, Mo., USA). To test the different
surface modification of titanium implants with EPA, coins were
placed in a 96-well plate and 10.sup.4 cells were seeded on each
coin. The same number of cells was cultured in parallel in plastic
in all the experiments.
Cell Viability Study
[0147] Lactate dehydrogenase (LDH) activity was used as an index of
cytotoxicity in the culture media. After 24 h, the culture media
was collected, centrifuged at 500.times.g for 5 min at 4.degree. C.
The supernatant was stored at 4.degree. C. LDH activity was
determined spectrophotometrically according to the manufacturer's
kit instructions (Cytotoxicity Detection kit, Roche Diagnostics,
Mannheim, Germany), and presented relative to the LDH activity in
the medium of cells seeded on plastic (low control, 0% of cell
death) and on plastic adding 1% Triton X-100 (high control, 100%
cell death), after subtracting the absorbance value obtained in the
culture medium alone without cells (background control), using the
equation:
Cytotoxicity (%)=(exp. value-low control)/(high control-low
control).times.100
Cell Attachment Study
[0148] The number of cells attached to each coin was measured using
DNeasy Tissue Kit (Qiagen Inc, Valencia, Calif., USA), which uses
silica-gel--membrane technology for rapid and efficient
purification of total cellular DNA without organic extraction or
ethanol precipitation. The buffer system is optimized to allow
direct cell lysis followed by selective binding of DNA to the
DNeasy membrane. The number of cells attached to the implants was
calculated after measuring DNA concentration at 260 nm using a
Nanodrop spectrophotometer (Nanoprop Technologies, Wilmington,
Del., USA) and taking into account that the DNA content in 10.sup.6
mouse cells is about 5.8 .mu.g.
RNA Isolation and Real-Time RT-PCR
[0149] Total RNA was isolated using a monophasic solution of phenol
and guanidine isothiocyanate (Trizol, Invitrogen Life Technologies,
Carlsbad, Calif., USA), following the instructions of the
manufacturer. RNA was quantified at 260 nm using a Nanodrop
spectrophotometer (Nanoprop Technologies, Wilmington, Del.,
USA).
[0150] The same amount of total RNA (0.5 .mu.g) from each sample
was reverse transcribed to cDNA at 42.degree. C. for 60 min in a
final volume of 40 using iScript cDNA Synthesis kit (BioRad
Laboratories, Hercules, Calif., USA) that contains both oligo(dT)
and random hexamers. Each cDNA was diluted 1/5 and aliquots were
frozen (-20.degree. C.) until the PCR reactions were carried out.
Real-time PCR was performed for two housekeeping genes: 18S
ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), and five target genes: osterix (Osx), alkaline phosphatase
(ALP), collagen type I (coll-I), osteocalcin (OC), and bone
sialoprotein (BSP). Sequences of sense and antisense primers were
as follows: 5'-GTAACCCGTTGAACCCCATT-3' (SEQ ID NO:1) and
5'-CCATCCAATCGGTAGTAGCG-3' (SEQ ID NO:2) for 18S rRNA;
5'-ACCCAGAAGACTGTGGATGG-3' (SEQ ID NO:3) and
5'-CACATTGGGGGTAGGAACAC-3' (SEQ ID NO:4) for GAPDH;
5'-AGAGCATGACCGATGGATTC-3' (SEQ ID NO:5) and
5'-CCTTCTTGAGGTTGCCAGTC-3' (SEQ ID NO:6) for coll-I;
5'-ACTGGCTAGGTGGTGGTCAG-3' (SEQ ID NO:7) and
5'-GGTAGGGAGCTGGGTTAAGG-3' (SEQ ID NO:8) for Osx;
5'-AACCCAGACACAAGCATTCC-3' (SEQ ID NO:9) and
5'-GAGAGCGAAGGGTCAGTCAG-3' (SEQ ID NO:10) for ALP;
5'-CCGGGAGCAGTGTGAGC-TTA-3' (SEQ ID NO:11) and
5'-TAGATGCGTTTGTAGGCGGTC-3' (SEQ ID NO:12) for OC;
5'-GAAAATGGAGACGGCGATAG-3' (SEQ ID NO:13) and
5'-ACCCGAGAGTGTGGAAAGTG-3' (SEQ ID NO:14) for BSP.
[0151] Real-time PCR was performed in the iCycler (BioRad
Laboratories, Hercules, Calif., USA) using SYBR green detection.
Each reaction contained 5 .mu.l of cDNA, 500 nM of the sense and
antisense specific primers (for all, except for collagen-I which
was 300 nM), 12.5 .mu.l of 2.times.1Q SYBR Green Supermix in a
final volume of 25 .mu.l. The amplification program consisted of a
preincubation step for denaturation of the template cDNA (3 min
95.degree. C.), followed by 40 cycles consisting of a denaturation
step (15 s 95.degree. C.), an annealing step (15 s 60.degree. C.;
for all, except for ALP which was 65.degree. C.) and an extension
step (30 s 72.degree. C.). After each cycle, fluorescence was
measured at 72.degree. C. A negative control without cDNA template
was run in each assay. Samples were run in duplicate.
[0152] Real-time efficiencies were calculated from the given slopes
in the iCycler software using serial dilutions, showing all the
investigated transcripts high real-time PCR efficiency rates, and
high linearity (r>0.99) when different concentrations were used.
PCR products were subjected to a melting curve analysis on the
iCycler and subsequently 2% agarose/TAE gel electrophoresis to
confirm amplification specificity, T.sub.m and amplicon size,
respectively.
Statistics
[0153] All data are presented as mean values.+-.SEM. Differences
between groups were assessed by Students t-test using the program
SPSS.RTM. for Windows, version 14.0. Results were considered
statistically significant at p<0.05 (indicated as *), and
p<0.01 (indicated as **).
Results
Effect of UV Irradiation on Surface Hydrophobicity
[0154] Effect of UV irradiation on CA of TiO.sub.2 surfaces was
examined to gain information about the irradiation time that should
be applied in later experiments. Water CA was measured before
irradiation and after an irradiation time of 1, 5, 10, 30, 60, 120,
180, and 240 min. The average CA for the 3 coins at each time point
is given in FIG. 2. The CA was increased during early irradiation
and decreasing subsequently. After 30 min of irradiation the CA was
lower than the initial CA for all the coins measured. Therefore, an
irradiation time of 30 min was used in the subsequent
experiments.
Effect of EPA Surface Modification on Roughness and
Hydrophobicity
[0155] Changes in surface hydrophobicity caused by surface
modification were detected by CA measurements. CA values for the 3
groups are given in FIG. 3.
[0156] The water CA of cleaned, autoclaved Ti surfaces (control A)
was 75.5.degree..+-.7.0.degree.. If 3.2 nmol EPA/mm.sup.2 was added
to those surfaces no significant changes in CA were found for
non-irradiated surfaces (73.8.degree..+-.0.3.degree.); however, a
significantly higher CA of 100.5.degree..+-.5.5.degree. was
measured for surfaces with 16.3 nmol EPA/mm.sup.2 added.
[0157] The surfaces without EPA in group B (control B) had a CA of
88.9.degree..+-.7.5.degree., which was higher compared to control
A. Ti coins that were coated with EPA and washed with methanol
subsequently had a CA of 68.6.degree..+-.9.3.degree. and
68.4.degree..+-.8.2.degree. for 3.2 nmol EPA/mm.sup.2 and 16.3 nmol
EPA/mm.sup.2, respectively; a CA that was lower compared to the
surface of control B.
[0158] As expected from previous experiments, irradiation with UV
light for 30 min followed by washing with methanol led to a slight
decrease in the surface contact angle to
54.5.degree..+-.2.4.degree. (control C) compared to control A.
Coating with EPA, irradiation for 30 min and washing with methanol
led to an increased CA of 65.8.degree..+-.5.2.degree. for coating
with 3.2 nmol EPA/mm.sup.2, and 66.8.degree..+-.1.5.degree. for
coating with 16.3 nmol EPA/mm.sup.2, compared to control C.
[0159] Analysis of the surfaces with Laser Profilometry showed that
the surface roughness parameters did neither change by irradiation
nor by coating of the surfaces with a thin layer of EPA (i.e. all
surfaces of group B and group C, and Control A). Average roughness
S.sub.a for those surfaces was 19 nm.+-.2 nm. However, coating with
a thick layer of EPA increased S.sub.a to 54.2 nm.+-.13 nm when 3.2
nmol EPA/mm.sup.2 was applied to the surface. Profilometric
observations of EPA drops show that EPA was unevenly distributed on
non-irradiated surfaces (group A) having differently sized
drop-like structures (FIG. 4a). However, EPA was evenly spread over
the whole surface after irradiation with UV light for 30 min (FIG.
4b).
Effect of UV Irradiation on the IR-Spectrum of EPA
[0160] Changes in IR spectra were used to obtain information about
how the chemical structure of EPA was changed due to UV
irradiation. The absorbance spectra for non-irradiated EPA and EPA
which was irradiated with UV light for 30 min are given in FIG. 5.
Of special interest are the changes in the region .about.3680-2900
cm.sup.-1 (1 in FIG. 5), the changes of the peaks 1850-1550
cm.sup.-1 (2-4 in FIG. 5) and changes of some absorbances in the
fingerprint region (5-8 in FIG. 5).
[0161] The peak 1 at 3680-2900 cm.sup.-1 was not found in the
spectrum of non-irradiated EPA but appears in the spectrum of the
EPA that was irradiated for 30 min. The peak in the region
1850-1550 cm.sup.-1 appears to be the result of three absorbances.
Two of those three absorbances shifted their position or changed
their intensity slightly due to the irradiation. Peak 2 with a
maximum at .about.1730 cm.sup.-1 was only a shoulder of medium
intensity in the non-irradiated sample. However its intensity was
increased after irradiation for 30 min with UV light leading to
broadening of the apparent peak. Peak 3, with a maximum at
.about.1713 cm.sup.-1 was very strong both before and after
irradiation with UV light. Neither intensity nor position of this
absorption changed. Thirdly, a peak of weak to medium intensity
with a maximum at .about.1650 cm.sup.-1 was found in absorbance
spectrum of non-irradiated EPA, in FIG. 5 referred to as peak 4.
This peak was not visible as a defined peak after irradiation but
contributed to a broadening appearance of peak 3.
[0162] Many differences in the absorbance spectra of non-irradiated
and irradiated EPA were detected in the fingerprint region of the
spectra. Most prominent were the increased intensities at 1170
cm.sup.-1 (peak 5) and 1070 cm.sup.-1 (peak 6) after UV
irradiation, the increased absorbance at -970 cm.sup.-1 (peak 7),
and the decreased absorbance at 710 cm.sup.-1 (peak 8).
Amount of EPA on Implant Surfaces
[0163] Scintillation counting of .sup.14C-labelled EPA was used to
quantify the amount of EPA on the Ti surfaces and to investigate
the efficacy of UV light to chemically bind EPA (FIG. 6). The
results show that after the first washing in methanol the amount of
EPA decreased to the same level for both non-irradiated and
irradiated coins. After the second washing in methanol, EPA was
still present on non-irradiated and irradiated coins, but there was
significantly more EPA on the irradiated surfaces compared to the
non-irradiated surfaces.
Cell Attachment to Implant Surfaces
[0164] The number of MC3T3-E1 cells attached to the implants was
quantified by measuring DNA content (FIG. 7). No significant
differences in the amount of DNA were detected between the 3
modifications of group A and group B. The relative amount of cells
present on surfaces of group C that did not contain EPA was
slightly lower compared to pure Ti surfaces of group A. However,
the relative amount of cells on the surfaces of group C coated with
3.2 nmol EPA/mm.sup.2 and with 16.3 nmol EPA/mm.sup.2 was
significantly higher compared to the control of group C (p=0.002
and p=0.003, respectively) and also significantly higher when
compared to control in group A (p=0.004 and p=0.009 for surfaces
coated with 3.2 nmol EPA/mm.sup.2 or 16.3 nmol EPA/mm.sup.2,
respectively). The cell amount was highest on surfaces coated with
3.2 nmol EPA/mm.sup.2.
Effect of EPA Surface Modification on Cell Viability
[0165] To determine whether different surface modifications with
EPA could affect osteoblast cell survival, the LDH activity was
measured after 24 h of cultivation (FIG. 8). The results showed a
significant increase in LDH activity only in group A with implants
having a thick layer of EPA on their surfaces, while thin layers of
either physically or chemically bound EPA were nontoxic to the
cells.
Discussion
[0166] In this study we compared different methods to coat Ti
implant surfaces with EPA by physical adsorption and covalent
binding using UV light. We demonstrated that UV light is a suitable
method to generate reactive binding sites on TiO.sub.2 and within
the structure of the EPA molecules. Moreover, in vitro testing
showed that implants with UV-bound EPA were able to enhance cell
attachment of MC3T3-E1 cells.
[0167] Implant surfaces are exposed to high friction forces during
implantation; thus to be clinically significant a surface coating
should be thin and firm to withstand high abrasion forces. A
covalent surface coating could therefore be an interesting option.
Such a thin layer of covalently attached molecules may change the
surface properties of the material, such as hydrophobicity, pH
close to the surface, and structure of the adjacent water layer.
Those properties strongly influence the interaction between
biomolecules and the surfaces shortly after implantation and may
thus facilitate osteoblast attachment and differentiation, which
would improve the performance of implants.
[0168] TiO.sub.2 surfaces became amphiphilic by irradiation with UV
light for 30 min, which agreed with previous findings 29. CA
measurements with water showed an increased hydrophilicity after an
initial decrease. The decrease in hydrophilicity to after 15 min of
irradiation with UV light has not yet been described in literature
and could be explained by contamination of the surfaces with
carbon, an increase in the thickness of the oxygen layer, or slight
changes in surface roughness untraceable with profilometry.
Profilometric pictures showed that drops of EPA spread on the
TiO.sub.2 surfaces after UV irradiation for 30 min indicating
increased lipophilicity of TiO.sub.2 surfaces. Those results showed
that 30 min was an appropriate time for surface irradiation with UV
light as the EPA, which was unevenly distributed on the TiO.sub.2
surfaces before irradiation, was covering the whole sample surface
after 30 min of UV-irradiation.
[0169] Surface CA is dependent on chemistry and roughness of
surfaces. As average surface roughness S.sub.a did not change due
to UV irradiation, coating of the surfaces with EPA and subsequent
washing with methanol (such as in group B and C), all changes of
surface contact angles measured for those groups were most probably
caused by changes in surface chemistry. EPA molecules are
hydrophobic and thin layers of EPA were thus increasing the water
CA of surfaces. Thick layers of EPA (3.2 nmol EPA/mm.sup.2 and 16.3
nmol EPA/mm.sup.2 in group A) resulted in soft but rougher surfaces
with EPA being unevenly distributed. EPA molecules dissolved in
water in small quantities, which decreased the surface tension of
water used for the measurements and decreased therewith the
resulting CA. Cleaning solvents such as methanol leave a thin layer
of carboneous residues on surfaces (Brunette et al., 2001). Higher
carbon content on metal surfaces was reported to increase
hydrophobicity of surfaces (Hirani et al., 2007). This explains the
increased CA for the control in group B that was washed with
methanol.
[0170] The comparably higher hydrophilicity of the surface coated
coins in group B in accordance with the amount of .sup.14C-EPA
measured on those surfaces indicated that a thin layer of
physically adsorbed EPA was left after washing with methanol. The
decrease in hydrophilicity for the EPA-coated surfaces compared to
their control and together with the scintillation counting of
.sup.14C-EPA coated surfaces indicated that a thin layer of
covalently bound EPA was left on the surfaces after UV
irradiation.
[0171] Analysis of FTIR spectra showed the changes that occurred in
the EPA structure by irradiation with UV light. Absorbance in the
3680-2900 cm.sup.-1 region was strongly related to the appearance
of hydroxide groups (--OH) as well as to the generation of peroxide
(--OO--) and peroxy acid groups (--CO--OOH) (Socrates et al., 2001)
caused by UV irradiation. Absorbance at .about.1730 cm.sup.-1 was
caused by C.dbd.O stretching vibration of esters (Socrates et al.,
2001). The increase of the ester peak indicated formation of ester
groups but as well the appearance of perester groups (CO--O--O--)
after irradiation with UV light (Socrates et al., 2001). Absorbance
at 1713 cm.sup.-1 was caused by C.dbd.O stretching vibrations of
carboxylic acid groups and did not change in intensity or position
(Guillen et al., 1997).
[0172] Absorbance with an intensity maximum at .about.1650
cm.sup.-1 was associated with stretching vibrations of C.dbd.C
bonds and its changes in the spectra indicated the appearance of
trans double bonds caused by UV-irradiation (Socrates et al.,
2001). Increased absorbance at 1170 cm.sup.-1 and 1070 cm.sup.-1
again showed the generation of ester groups (CO--O--C asymmetric
and symmetric stretch, respectively) by UV irradiation (Socrates et
al., 2001). Increased absorbance at 970 cm.sup.-1 together with a
decreased absorbance at 710 cm.sup.-1 indicated the transformation
of cis C.dbd.C double bonds to trans C.dbd.C double bonds caused by
UV irradiation (Socrates et al., 2001). All together, reactive
groups, i.e. oxides and their radicals, peroxides and their
radicals, peroxy acids, and peresters were generated within the EPA
structure by irradiation with UV light. EPA is susceptible to auto-
and photooxidation due to unsaturations in the structure.
Especially the highly energetic UV light leads to oxidation and
generation of reactive oxide groups (Guillen et al., 1997, Cheng et
al., 2003, Melo et al., 1988). Photooxidation of EPA may change the
chemical structure of the molecules in several ways. One
possibility is the formation of oxide groups via peroxide radicals
and peroxides in position 5, 6, 8, 9, 11, 12, 14, 15, 17, or 18,
together with formation of trans double bonds in the respective
positions (Chacon et al., 2000). Another way is degradation of EPA
via oxide radicals that are formed in the same positions as for the
first possibility, just followed by fragmentation into aldehydes,
and the subsequent formation of carboxylic acids and dicarboxylic
acids (Rontani et al., 1998) or aldehydes such as malondialdehyde,
the volatile end product of fatty acid oxidation that was reported
to have negative effects on cell metabolism e.g. by reaction
proteins in vivo (Refsgaard et al., 2000). EPA was reported to
protect the skin of mice in vivo against UV irradiation, and
thereby to protect against photocarcinogenesis (Moison et al.,
2001). Those reactive groups may have caused reduction of hydroxide
groups and generation of radical oxide groups on the TiO.sub.2
surfaces. We hypothesise, that part of the EPA peroxides or their
decomposition products that were generated by UV irradiation bound
covalently to the TiO.sub.2 surfaces via ester, perester, peroxide,
and ether bonds (FIG. 9).
[0173] Measurements of the amount of .sup.14C-labelled EPA on
surfaces showed that a measurable amount of .sup.14C-EPA was left
on both, non-irradiated and irradiated surfaces. However, the
significant difference in measured .sup.14C signals indicated that
the irradiated surface coating was more stable against thorough
washing than the coating on non-irradiated surfaces. The fatty acid
film on irradiated surfaces may consist of a combination of
chemically and physically bound EPA and EPA photooxidation
products. The data did not support any speculation about how the
EPA layer is structured or about how exactly the UV-irradiated EPA
is attached to the surface. Surface roughness of the polished cp Ti
samples precluded detailed analysis of the coating structure with
for example grazing incidence x-ray reflectometry, where a S.sub.a
of about 1 nm is required; such low surface roughness could be
obtained by sputtering a thin layer of Ti onto smooth Si surfaces
(Kim et al., 2004).
[0174] Measurements of LDH values and amount of DNA on the surfaces
showed that large quantities of EPA (3.2 nmol EPA/mm.sup.2 and 16.3
nmol EPA/mm.sup.2 in group A) had a toxic effect on the cells,
while small quantities of physically bound EPA (group B) had no
toxic effect but also significant effect on cell attachment. The
toxic effect of high doses of EPA on cell viability has been
reported in other cell lines and agrees with the results of the
present study.
[0175] Chemically bound EPA in group C had a positive effect on
cell attachment and was shown to be nontoxic. It can therefore be
suggested that EPA could have affected cell attachment, even though
it was chemically bound to the surfaces and therefore not directly
available for the cells. Surfaces coated with EPA may have changed
the characteristics of the protein layer readily adsorbed onto the
surfaces of the samples when in contact with protein-containing
media. Dependence of adsorption of proteins and cells on surface
modifications has been examined by other groups as well (Kim et
al., 2005, Cooper et al., 2001, Svedhem et al., 2003, Iwasaki et
al., 2003, Willumeit et al., 2007). Auto- and photooxidized lipids
were reported to have negative effects on cells if ingested, as
peroxides and photooxidation end products (such as malondialdehyde,
4-hydroxyalkenals) cause oxide radicals which in turn cause damage
to DNA and proteins (Refsgaard et al., 2000, Cheung et al., 2007).
Epoxides of AA on the other hand, another eicosanoid, have been
shown to have important biological functions (Capdevila et al.,
2000). EPA, just as AA, is prone to auto- and photooxidation on air
due to its unsaturations in the carbon chain; thus the results of
the in vitro tests, which showed a positive effect of the coating
by irradiation, are of interest and will be further investigated in
long term in vitro tests and in vivo studies. In later studies a
coating method could be tested where the Ti surfaces alone are
activated with UV irradiation and the EPA is given to the surfaces
subsequently. By this the alteration of the fatty acid molecules
may be reduced, and results compared with the present
investigation.
Example 3
[0176] Experiments performed in vivo with titanium implants
containing a high dosage of physical adsorbed EPA and a low dosage
of chemically-bound EPA.
Materials and Methods
Titanium Coins
[0177] Commercially pure (cp) machined titanium implants with a
diameter of 6.25 mm and a height of 1.95 mm were cleaned and
sterilized before use. Briefly, implants were washed together in a
glass beaker with deionised water for 30 s, then with 70% ethanol
for 30 s, and then with ultrasonic bath at 40.degree. C. for 5 min
in deionised water. The implants were subsequently placed in 40%
NaOH solution in a water bath of 40.degree. C. for 10 min,
sonicated in deionised water for 5 min, and then washed with
deionised water until the pH reached 6. Afterwards the implants
were sonicated in deionised water at 50.degree. C. for 5 min,
placed in 50% HNO.sub.3 solution at 50.degree. C. for 10 min, and
sonicated in deionised water for another 5 min. The implants were
washed with deionised water until reached pH=6 and were stored in
70% ethanol. Before use, the coins were rinsed with water, rinsed
with ethanol, sonicated for 5 min at room temperature and rinsed
with deionised water. The titanium coins were then sterilised by
autoclaving at 121.degree. C. for 15 min.
Preparation of Implants
[0178] For the surface modification of titanium implants, EPA was
dissolved in methanol to 10 mM solution. The EPA solution was
filtered with a 0.22 .mu.m pore size filter to sterilise it before
use. Four groups of coins were prepared; non-irradiated and
unwashed coins (Control), high dosage of EPA (EPA), UV-irradiated
coins (UV) and low dosage of EPA after UV irradiation and washing
(UV+EPA). When EPA was given to the surface, 10 .mu.l of the
respective 10 mM EPA solution was used, which equals total 3.2 nmol
EPA/mm.sup.2. The coins were allowed to dry on air for 15 min in a
sterile flow bench (all four groups of coins). Coins of UV and
UV-EPA groups were then irradiated with UV light for 30 min
(Fluo.link, .lamda.=312 nm, I=6 mW/cm.sup.2). These last groups of
coins (UV and UV-EPA groups) were washed afterwards with methanol
to remove unbound EPA, and to follow the same procedure for the
control coins irradiated with UV and without EPA. The coins were
allowed to air-dry for 15 min in the sterile flow bench and packed
in sterile micro-centrifuge tubes.
Materials and Methods for XPS Analysis
[0179] Kratos Axis Ultra.sup.DLD XPS instrument (Kratos,
Manchester, UK) was used to perform surface analysis of the
modified implants' surfaces. Monochromatic Al K.alpha. x-rays were
used with a current of 10 mA and a voltage of 15 kV. An area of
300.times.700 .mu.m.sup.2 was analysed on one sample per each
group, with a pass energy of 80.0 eV for survey scans and 20.0 eV
for high energy resolution elemental scans of Ti 2p, C 1s, O 1s,
and N 1s. The peak areas were calculated with the program CasaXPS
3.2.12. The binding energy scale was calibrated by assigning the
hydrocarbon peak to a binding energy of 284.8 eV. Shirley
background was used to quantify the survey spectra, and mixed
Shirley and linear backgrounds were used for the quantification of
the detailed elemental scans.
Animal Study and Pull-Out Analysis
[0180] Six New Zealand White female rabbits, 6 months old and a
weight of 3.0-3.5 kg, were used in this study (ESF ProdukterEstuna
A B, Norrtalje, Sweden).
[0181] The implants with high dosage of EPA (n=6) and their
controls (n=6), and implants with a low dosage of EPA chemically
bound after UV light irradiation (n=6) and their UV-irradiated
controls (n=6) were placed in calibrated cortical bone defects in
the tibia of rabbits (New Zealand White). The methods used were all
according to a standardized and validated model established for the
study of bone attachment to titanium implant surfaces (Ronold and
Ellingsen, 2002). Each rabbit received four implants, two in each
tibia bone. Location of test and control implants was randomized
and the operator was blinded. At 10 weeks after implantation the
rabbits were sacrificed and the tibia bones with the implants
attached were excised. Directly after excising the tibia bone was
fixed in a specially designed jig, and the implants were detached
using a calibrated pull-out procedure. The load was applied until
loosening of the implant and recorded on a load versus time plot.
The remaining tibial bone was used for studying volumetric bone
mineral density (vBMD) using micro-computed tomography (micro-CT).
Wound fluid was collected from the implant site after removal of
implants for LDH, ALP activity and total protein. The implants were
directly transferred to a sterile tube and processed for RNA
extraction, for gene expression analysis of inflammatory and bone
markers.
Micro-Tomography Analysis
[0182] The tibia was fixated in 4% neutral buffered formaldehyde
and then kept in 70% ethanol. Bone samples were scanned with a
commercially available desktop micro-CT scanner. Bone mineral
density was studied in the sub-implant cortical bone after implant
removal, to study changes in mineralization in the peri-implant
bone, from new bone (lower in vBMD) to more mature bone (higher in
vBMD).
Wound Fluid Analyses: LDH Activity, Alp Activity and Total
Protein
[0183] LDH activity and total protein was analysed in the wound
fluid collected from the implant site following a 10 week healing
period. The release of LDH is a sensitive marker for tissue
necrosis (Williams et al., 1983) and thus the biocompatibility of
the implants. ALP activity has been proposed to play an important
role in the initiation of mineralization process around titanium
implants. The amount of total protein was used to indicate the
amount of wound fluid present at the interface with the implants,
and to correlate with the bone-to-implant attachment strength.
In Vivo Gene Expression of Inflammatory and Bone Markers: IL-6,
Osteocalcin and TRAP
[0184] Gene expression of IL-6, osteocalcin and TRAP was studied
using real-time RT-PCR as an indication of inflammation (IL-6) bone
formation (osteocalcin) or bone resorption (TRAP) in the
peri-implant bone tissue attached to the surface of the different
groups of implants.
Conclusion
[0185] The elemental compositions of the surfaces were calculated
from the survey spectra of the respective sample and are given in
Table 3. The C content increased with addition of EPA to the
surfaces, while the Ti signals decreased for those samples,
indicating that a thin layer of EPA was left on the surfaces after
washing. The survey showed as well a clear reduction of N signal
after UV irradiation. N and various contaminants such as Na, Zn,
Mg, and Si were detected in traces, probably due to surface
contaminations from air. The detail spectra revealed further
details about the binding states of elements (for C1s see Table 4
and for O1s see Table 5). Up to 4 peaks (3 for Control, UV, and
EPA) were fitted into the detail spectra of C is. C--C, and C--H
signals were detected at a BE (binding energy) of 284.8 eV (Han et
al., 2008; Viornery et al., 2002). The intensity of this signal
decreased slightly for the EPA surface, and more clearly for the
surface UV+EPA. The intensity of the C-0 signal at BE=285.8 eV (Han
et al., 2008; Viornery et al., 2002) remained approximately similar
for all analysed surfaces. A signal for C.dbd.O binding states at
287.9 eV [22] was only detected for the surfaces of the group
UV+EPA. The signal assigned to --O--C.dbd.O at 288.8 eV (Han et
al., 2008; Viornery et al., 2002) was low on control surfaces,
increased for all the other surfaces, and highest for UV+EPA
surfaces. Up to 5 peaks (4 peaks for Control and UV) were fitted in
the detail spectra of 01s. The TiO.sub.2 signal was assigned to
BE=529.9 eV (Viornery et al., 2002; Lu et al., 2000). It was
decreased for the EPA surface and even lower for the UV+EPA
surface. TiO and TiOH gave a signal at the same BE; 531.2 eV
(Viornery et al., 2002; Lu et al., 2000). It was slightly increased
for the surface of the UV group and about 2-fold increased for the
surface of the UV+EPA group. The percentage of the peak for C--O,
C.dbd.O, and C--OH at 532.4 eV (Viornery et al., 2002) was slightly
decreased on UV surfaces compared to Control, increased on EPA
surfaces, and decreased on UV+EPA surfaces compared to Control. O
that was bound in combinations containing --O--C.dbd.O was fitted
at 533.7 (Beamson et al., 1992). This variation was present only in
surfaces of EPA and UV+EPA.
TABLE-US-00004 TABLE 3 Results of elemental analysis from XPS
survey spectra on the differently modified implant surfaces.
Element composition [at. %] Sample C O Ti N Contaminants Control
52.2 31.8 12.6 1.4 2.1 EPA 60.4 27.4 7.2 1 3.8 UV 46.5 35.2 13.5 ND
4.8 UV + EPA 62.6 28.3 6.3 ND 2.8 ND--Not detected
TABLE-US-00005 TABLE 4 Percentage of area for the peaks fitted into
the detail spectra of the C1s peak. Percentage areas of the
deconvoluted peaks Binding Energy [eV] 284.8 eV 285.8 eV 287.9 eV
288.8 eV Possible bonding states Sample C--C/C--H C--O C.dbd.O
--O--C.dbd.O Control 71.3 26.5 ND 2.2 EPA 69.2 25.8 ND 5.0 UV 71.4
24.3 ND 4.3 UV + EPA 66.8 25.0 2.9 5.4
TABLE-US-00006 TABLE 5 Percentage of area for the peaks fitted into
the detail spectra of the O1s peak. Percentage areas of the
deconvoluted peaks Binding Energy [eV] 529.8 eV 531.2 eV 532.4 eV
533.8 eV Possible bonding states Sample TiO2 TiO/TiOH
C--O/C.dbd.O/C--OH --O--C.dbd.O Control 61.7 23.1 15.2 ND EPA 47.4
19.5 26.0 7.1 UV 59.0 28.1 12.9 ND UV + 36.8 47.4 7.9 7.8 EPA
[0186] The implants coated with a low dosage of EPA chemically
bound after UV light irradiation and their UV-irradiated controls
showed a lower attachment to the cortical bone. A lower vBMD of the
cortical bone in both type of implants was observed, which could be
related with the differences in the biomechanical properties of
peri-implant cortical bone found with the pull-out values. The
amount of total protein in the wound fluid, higher in the implants
coated with a low dosage of EPA chemically bound after UV light
irradiation and their UV-irradiated controls, could also be related
with their lower pull-out values. No differences in LDH activity
(biocompatibility) were observed with the four different groups of
implants, but an increase in the gene expression of the
inflammatory marker IL-6 was observed in the group of implants with
high dosages of EPA. No differences were found in the gene
expression of TRAP, suggesting the bone resorption was not affected
differently with the implants tested. The implants coated with a
low dosage of EPA chemically bound after UV light irradiation and
their UV-irradiated controls showed a higher expression of the
pro-osteogenic marker osteocalcin and a higher ALP activity,
indicating that osteoblastic cell activity and on-going bone
formation was improved in these samples. The results of this study
are shown in FIGS. 10-17.
Example 4
[0187] Titanium (Ti) surfaces were coated with two different n-3
polyunsaturated fatty acids (PUFAs), without and in combination
with .alpha.-tocopherol (vitamin E) to analyse the antioxidative
properties of .alpha.-tocopherol on PUFAs during UV irradiation.
.alpha.-tocopherol is a substance that is considered safe by the
authorities as a food additive. .alpha.-tocopherol has been shown
to have anti-inflammatory effects in vitro by inhibiting IL-1.beta.
release from monocytes. At the same time, a decrease in cell
adhesion was observed (Reno et al, 2005). We hypothesized that
coating of Ti surfaces with PUFAs in combination with
.alpha.-tocopherol could prevent oxidation of PUFA and result in a
Ti surface with improved properties.
Materials and Methods
[0188] The coating substances were purchased from Sigma-Aldrich
with the highest grade of purity available. Surfaces of Ti (c.p.
grade IV) disks, 6.25 mm in diameter, were coated with different
substances and mixtures thereof. The surfaces were dried on air and
subsequently irradiated with UV light (.lamda.=302 nm, P=6 W,
distance to surfaces 43 mm, lamp purchased from VWR, Oslo, Norway).
The samples were analysed with FTIR spectroscopy (DRIFT) after 0
min, 15 min, 30 min, and 60 min of irradiation. An equally
irradiated, uncoated Ti disk was used as a background for the FTIR
measurements. Mixtures were applied to the implants and irradiated
for 0 min, 15 min, 30 min, and 60 min. The spectra obtained by FTIR
spectroscopy were analysed for typical absorbances connected with
photooxidation of the surface coatings. Typical peak areas were
quantified and will be compared and discussed in this document.
[0189] The substances used as coating were:
EPA (eicosapentaenoic acid; 20:5, n-3); 10 .mu.l of 50 mM solution
in ethanol DHA (docosahexaenoic acid; 22:6, n-3); 10 .mu.l of 50 mM
solution in ethanol EPA+DHA; 1:1-10 .mu.l in total
.alpha.-tocopherol; 10 .mu.l of 50 mM solution in ethanol
EPA+.alpha.-tocopherol; 1:1-10 .mu.l in total
DHA+.alpha.-tocopherol; 1:1-10 .mu.l in total
EPA+DHA+.alpha.-tocopherol; 1:1:1-10 .mu.l in total
[0190] The presentation of the results of the analysis is organised
as follows:
1. UV irradiation of the n-3 PUFAs EPA, DHA, and EPA+DHA 2. UV
irradiation of .alpha.-tocopherol 3. Combination of
.alpha.-tocopherol and the n-3 PUFAs
1. UV Irradiation of the N-3 PUFAs EPA, DHA, and EPA+DHA
[0191] The most important absorbances that showed changes in the
n-3 PUFA chemical structure due to UV irradiation are given in the
following table (Table 6):
TABLE-US-00007 TABLE 6 Important changes of absorbances in FTIR
spectroscopy for PUFAs FIG. 18 shows how the mentioned absorbencies
are changing. The values shown are calculated from the absorbance
peak areas and are given as % compared to non-irradiated sample.
Very interesting are the opposing trends of the absorbances of
C.dbd.C cis and C.dbd.C trans, which indicate the formation of
trans fatty acids from cis fatty acids. Oxidation caused by UV
irradiation was observed. Wavenumber Changes caused by (max
absorbance) Group UV irradiation 3300 cm-1 --OH increase 1710 cm-1
C.dbd.O increase 1065 cm-1 CO--O--C change 974 cm-1 C.dbd.C trans
appearance and increase 710 cm-1 C.dbd.C cis decrease
2. UV Irradiation of .alpha.-Tocopherol
[0192] The Chemistry of the oxidation process of .alpha.-tocopherol
was described by Wang X, 1999: TOH.fwdarw.TO
(.alpha.-tocopherol.fwdarw..alpha.-tocopheroxyloxyl) and Yamauchi
R, 2002 (see FIG. 19): According to those sources, the following
absorbances are of interest as they might be changing during UV
irradiation (Table 7):
TABLE-US-00008 TABLE 7 Important changes of absorbances in FTIR
spectroscopy for .alpha.-tocopherol Wavenumber Changes caused (max
absorbance) Group by UV irradiation 1676 cm-1, 1646 cm-1 Quinones
appearance/increase 1620 cm-1 C.dbd.C aromatic increase 1250 cm-1
C--O of epoxides appearance/increase
[0193] As can be seen from FIG. 20, the absorbances of quinones
were increased during UV irradiation while the absorbance areas for
epoxides did not change, indicating that no epoxides were formed
from .alpha.-tocopherol during UV irradiation, but quinones, such
as .alpha.-tocopherylquinone. The absorbance of aromatic C.dbd.C
groups decreased during UV irradiation, indicating that those
bindings were reduced by photooxidation.
3. Combination of .alpha.-Tocopherol and the N-3 PUFAs
[0194] Addition of .alpha.-tocopherol to EPA, DHA, and EPA+DHA was
tested. FIG. 21 shows the changes of peak areas of the
characteristic absorbances for the averages of peak areas of EPA
and DHA with .alpha.-tocopherol added. The changes of peak areas
with UV irradiation time were very slight compared with changes of
peak areas for the PUFAs alone, indicating a protective effect of
.alpha.-tocopherol.
[0195] The antioxidative effect of .alpha.-tocopherol was dependent
on the amount of .alpha.-tocopherol added, as could be shown for
instance on the example of OH absorbances at 3300 cm.sup.-1 (FIG.
22). The increase of peak area was much more pronounced for the
PUFAs alone compared to 1:1 mixtures with 50% .alpha.-tocopherol
(EPA+.alpha.-tocopherol, DHA+.alpha.-tocopherol) or 1:1:1 mixtures
(EPA+DHA+.alpha.-tocopherol, 33 mol % .alpha.-tocopherol).
Conclusion
[0196] UV irradiation was shown to change the molecular structure
of photosensitive PUFAs such as EPA and DHA in a similar manner. As
for a typical photooxidation process, the absorbance intensities
increased for OH, CO--O--C, C.dbd.O, and C.dbd.C trans groups,
while the absorbance intensity decreased for the C.dbd.C cis group.
.alpha.-tocopherol seemed to be less affected by UV irradiation up
to 1 h, only the appearance of a weak quinone absorbance was
observed. This is in accordance to one oxidation pathway proposed
by Yamauchi et al. Addition of .alpha.-tocopherol to the PUFAs in 2
different concentrations indicated a concentration dependent
protective effect of the .alpha.-tocopherol.
Example 5
[0197] 7-Dehydrocholesterol (7-DHC, provitamin D.sub.3) is
converted to cholecalciferol (vitamin D.sub.3) via previtamin
D.sub.3 in the human skin. Further conversion to 25(OH)D.sub.3 is
mainly reported to happen in the liver, but since the hydroxylase
enzyme CYP27A1 is ubiquitously expressed in other tissues, the
system for local productions is also available in bone (Aiba I et
al., 2006). 1.alpha.,25(OH).sub.2D.sub.3 has as well been shown to
be synthesised locally in bone from 25(OH)D.sub.3 (Ichikawa et al.,
1995; Atkins et al., 2007). A schematic overview of the conversion
can be found as FIG. 23. 1.alpha.,25(OH).sub.2D.sub.3 is the
biological active form of vitamin D.sub.3 and acts as a hormone to
regulate serum calcium and phosphate levels and is an important
factor during bone growth and mineralisation (St-Arnaud R, 2008).
Thus, adding 7-DHC as a surface coating to titanium implants
irradiated with UV light, with or without PUFA, might affect bone
healing positively.
[0198] Titanium (Ti) surfaces were coated with either 7-DHC or
cholecalciferol to analyse their transition initiated by UV light.
We aimed to specify how specific the UV initiated conversion from
7-DHC to cholecalciferol was and which irradiation time would be
the most appropriate. Furthermore, the surfaces were coated with a
mixture of EPA (representing an n-3 PUFA) and 7-DHC to analyse
possible interactions between the 2 substances during UV
irradiation.
Materials and Methods:
[0199] EPA, 7-DHC and cholecalciferol were purchased from
Sigma-Aldrich with the highest grade of purity available. Surfaces
of Ti (c.p. grade IV) disks, 6.25 mm in diameter, were coated with
either 7-DHC or cholecalciferol, or a mixture of 7-DHC+EPA
(1:1).The surfaces were dried on air and subsequently irradiated
with UV light (.lamda.=302 nm, P=6 W, distance to surfaces 43 mm,
lamp purchased from VWR, Oslo, Norway). The samples were analysed
with FTIR spectroscopy (DRIFT) after 0 min, 15 min, 30 min, and 60
min of irradiation. An equally irradiated, uncoated Ti disk was
used as a background for the FTIR measurements. The spectra
obtained by FTIR spectroscopy were analysed for typical absorbances
connected with photooxidation of the surface coatings. Typical peak
areas were quantified if possible and will be compared and
discussed in this document.
Results
[0200] The most important absorbances that showed changes in the
chemical structure of 7-DHC and cholecalciferol due to UV
irradiation are given in the following table (Table 8):
TABLE-US-00009 TABLE 8 Important changes of absorbances in FTIR
spectroscopy for 7-DHC and cholecalciferol Changes caused by UV
irradiation of Wavenumber 7-DHC and (max absorbance) Group
cholecalciferol 7-DHC + EPA 3300 cm-1 --OH no changes increase 1730
cm-1 C.dbd.O ester groups increase 1710 cm-1 C.dbd.O carboxylic
increase acids 1680 cm-1 C.dbd.C trans appearance and increase 1650
cm-1 C.dbd.C cis increase 1625 cm-1 C.dbd.C aromatic increase
a) UV Irradiation of 7-DHC and Cholecalciferol
[0201] The following FIGS. 24 and 25 show how the absorbance
spectra of 7-DHC and cholecalciferol were changing with UV
irradiation time. As the chemical structures of the 2 substances
are very alike, the absorbance spectra appeared to be quite
similar. Also their behaviour with UV irradiation time appeared to
be comparable (Table 8).
[0202] From the absorbance spectra and the changes of peak areas
measured (FIG. 26), we can assume that no --OH groups were
generated due to the irradiation. The peak area at 3300 cm.sup.-1
was stable with UV irradiation time. The peak area at about 1700
cm.sup.-1 (including the area from 1850 cm.sup.-1 to 1550
cm.sup.-1) was increasing clearly for both substances, indicating
the formation of 0=0 double bonds (aromatic, cis- and trans-), as
well as the formation of C.dbd.O ester and carboxylic groups.
b) UV Irradiation of 7-DHC+EPA
[0203] The spectra of the mixture of 7-DHC and EPA seemed to
represent both substances in their typical absorbances (FIG. 27).
The changes of the peak areas (FIG. 28) confirm this observation.
At 3300 cm.sup.-1 we found an increase in peak area that lies in
between the values for 7-DHC (no changes) and the values for EPA
(clear increase). At 1700 cm.sup.-1 we found the same result, which
only becomes clear in the absolute values which again are in
between the values measured for 7-DHC (slow increase from small
peak area) and EPA (steep increase from large peak area).
Conclusions
[0204] None of the absorbance spectra of 7-DHC after UV irradiation
was completely similar to the absorbance spectrum of non-irradiated
cholecalciferol. A likely explanation for this is that a mixture of
several photooxidation products is formed due to UV irradiation as
described in the literature (see FIG. 23). The conversion from
7-DHC to cholecalciferol is about 0.8% only (Olds et al., 2008) and
thus may not be detectable with FTIR. This is not a problem, since
all the photooxidation products produced by UV irradiation also
evolve in the human body and are biological inactive, thus
preventing hypervitaminosis of vitamin D.sub.3.
[0205] Combining equal amounts of 7-DHC and EPA on Ti surfaces did
not result in any chemical interactions between the 2 substances
that could be measured with FTIR spectroscopy. The irradiation of
the mixture resulted in spectra that had the appearance and
absorbance peak areas that were a combination from the spectra and
peak areas of 7-DHC and EPA. Using other PUFAs than EPA is not
expected to result in findings different from this.
Example 6
[0206] This example demonstrates how smooth and rough implants with
PUFA and/or vitamins may be manufactured.
A.1. Smooth Implants:
[0207] Commercially pure (cp) titanium disks with a diameter of
6.25 mm and a height of 2 mm were grinded and polished (Phoenix
4000, Buehler GmbH, Duesseldorf, Germany) in seven sequences. The
silicon carbide papers, the porous neoprene for final polishing and
the abrasive colloidal silica suspension (OP--S) were supplied by
Struers GmbH (Willich, Germany). The first step consisted in
grinding all the implants with P500 in water until they were
levelled, with 65 N of pressure and a contra-rotation at 250 rpm.
Then, the grinding time was set down to 20 min and the grain size
decreased with papers: P800; P1200 and P2500. For the P4000
polishing paper, the OP--S polishing suspension was used. For the
last two steps, a special porous neoprene (MD-Chem) was used at 200
rpm and 50 N of pressure, 14 min in co-rotation with OP--S
suspension, and then 1 min in co-rotation with water. The
grinding/polishing were necessary to create uniform plane and clean
surfaces prior to modification. After polishing, all the disks were
washed alternately with NaOH at 40 vol. % and HNO.sub.3 at 50 vol.
% in ultrasonic bath. Then washed with deionised water to reach a
neutral pH, and stored at room temperature in 70 vol. %
ethanol.
A. 2. Rough Implants:
[0208] Commercially pure (cp) titanium disks with a diameter of
6.25 mm and a height of 2 mm were blasted with titanium dioxide
(TiO.sub.2) particles with a particle size of 180-220 .mu.m
(Blasmaster.TM. oxide powder, F. J. Brodmann&CO, L.L.C., Los
Angeles, USA) after polishing. The distance from the implants to
the jets was approximately 20 mm during blasting procedure and the
TiO2 particle stream hit the surface with an angle of 90.degree..
The air pressure used for blasting was set to 0.5 Mpa. After
polishing and blasting, all the disks were washed alternately with
NaOH at 40 vol. % and HNO.sub.3 at 50 vol. % in ultrasonic bath.
Then washed with deionised water to reach a neutral pH, and stored
at room temperature in 70 vol. % ethanol.
B. Surface Modification of Smooth and Rough Implants with Pufa
and/or Vitamins
[0209] For the surface modification of smooth and rough implants
with PUFA and/or vitamins:
1. Work in sterile bench. 2. Solutions of the respective substances
are prepared with ethanol. 3. Working solutions are filtered (0.2
.mu.m pore size) to sterilise them. 4. 10 .mu.l of respective
solution is given to the surface of the coins (with the selected
concentration and combination of PUFA/vitamins). 5. Coins are
allowed to dry on sterile bench for 15 min. 6. The samples are
irradiated with UV light (UV C 100 nm-280 nm, and UV B light 280
nm-315 nm). The set-up is given in FIG. 29. One problem is to
irradiate the samples from all sides. A reflecting Al foil can be
bended and used to access the samples from down and the sides if
the samples are placed on a UV transparent surface (e.g. quartz).
FIG. 29 shows an examplary set-up for irradiating Ti samples with
UV light. D. Characterization of Smooth and Rough Implants Modified
with Pufa and/or Vitamins: [0210] 1. Profilometer: a blue light
laser profilometer (Sensofar Pl.mu.2300, Terrassa, Spain) is
utilised to scan 200.times.160 .mu.m2 areas with a
150.times.(150.times./0.95 Lu Plan Apo, EPI) Nikon (Nikon, Tokyo,
Japan) objective. The roughness and waviness parameters from each
coin were then calculated with advanced software (SensoMap Plus
4.1, Sensofar, Terrassa, Spain), and representative pictures of the
different surfaces are chosen. [0211] 2. Contact angle: measured at
least 24 h after end of UV irradiation. The contact angles (CA) of
the surfaces of the different titanium implants are analyzed using
a video-based contact angle system (OCA 20, DataPhysics Instruments
GmbH, Filderstadt, Germany). The fluid used was deionised water. A
drop of 3 .mu.l was given to the surface with a velocity of 0.6
.mu.l s-1. The drop shape is recorded with a camera (4.17 pictures
s-1) and contact angles were measured afterwards from the recorded
pictures. A drop of 3 .mu.l on coin, CA is measured, the drop is
enlarged 2.times. with 1 .mu.l of pure water, after each
enlargement the surface CA is measured, the CA is not measured
after drying the coin as this could change the structure of the
PUFA on the surface of the coin and therewith the CA. [0212] 3. FT
IR (diffuse reflectance) to characterise the difference before and
after UV irradiation of PUFA and vitamins. Films of PUFA and/or and
the changes caused by UV irradiation were analysed with FTIR
Spectrum 100, Perkin Elmer (USA) The diffuse reflectance unit
(DRIFT) is used to collect the spectra. A cleaned, sterilised, but
unmodified polished Ti coin served as reference. The FTIR spectra
is collected before and after irradiation with UV light at
different time points of irradiation. The spectra were collected
from 4000 cm.sup.-1 to 450 cm.sup.-1 with a resolution of 2
cm.sup.-1. Each spectrum was the result of 8 single spectra. The
spectra were baseline corrected with the program Spectrum (Version
6.1.0) from PerkinElmer. [0213] 4. FTIR-microscopy is performed to
answer the following questions: [0214] How are the components
distributed before and after UV irradiation? [0215] Is there a
difference if the components are alone on the surface or in
different combinations? [0216] How is the crystalline structure of
the Ti coins? [0217] Is there any correlation between the
distribution of components and the crystals? [0218] 5. XPS is
performed to get information about the atomic composition of the
surfaces. Kratos Axis UltraDLD XPS instrument (Kratos, Manchester,
UK) is used to perform surface analysis of the modified implants'
surfaces. Monochromatic Al K.alpha. x-rays were used with a current
of 10 mA and a voltage of 15 kV. An area of 300.times.700 .mu.m2
was analysed on one sample per each group, with a pass energy of
80.0 eV for survey scans and 20.0 eV for high energy resolution
elemental scans of Ti 2p, C.sub.1s, O 1s, and N 1s. The peak areas
were calculated with the program CasaXPS 3.2.12. The binding energy
scale was calibrated by assigning the hydrocarbon peak to a binding
energy of 284.8 eV. Shirley background was used to quantify the
survey spectra, and mixed Shirley and linear backgrounds were used
for the quantification of the detailed elemental scans.
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Sequence CWU 1
1
14120DNAArtificialPrimer 1gtaacccgtt gaaccccatt
20220DNAArtificialPrimer 2ccatccaatc ggtagtagcg
20320DNAArtificialPrimer 3acccagaaga ctgtggatgg
20420DNAArtificialPrimer 4cacattgggg gtaggaacac
20520DNAArtificialPrimer 5agagcatgac cgatggattc
20620DNAArtificialPrimer 6ccttcttgag gttgccagtc
20720DNAArtificialPrimer 7actggctagg tggtggtcag
20820DNAArtificialPrimer 8ggtagggagc tgggttaagg
20920DNAArtificialPrimer 9aacccagaca caagcattcc
201020DNAArtificialPrimer 10gagagcgaag ggtcagtcag
201117DNAArtificialPrimer 11ccgggagcag tgtgagc
171221DNAArtificialPrimer 12tagatgcgtt tgtaggcggt c
211320DNAArtificialPrimer 13gaaaatggag acggcgatag
201420DNAArtificialPrimer 14acccgagagt gtggaaagtg 20
* * * * *