U.S. patent application number 12/298889 was filed with the patent office on 2011-06-23 for bone tissue implant comprising lithium ions.
This patent application is currently assigned to Astra Tech AB. Invention is credited to Stig Hansson, Ingela Petersson.
Application Number | 20110151026 12/298889 |
Document ID | / |
Family ID | 38736015 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110151026 |
Kind Code |
A1 |
Hansson; Stig ; et
al. |
June 23, 2011 |
BONE TISSUE IMPLANT COMPRISING LITHIUM IONS
Abstract
The present invention is based on that local administration of
lithium ions in bone tissue has been found to improve the bone
formation and bone mass upon implantation of a bone tissue implant
in said bone tissue. In particular, the invention relates to a bone
tissue implant having an implant surface covered by an oxide layer
comprising lithium ions and a method for the manufacture thereof. A
blasting powder comprising lithium ions, a method for locally
increasing bone formation, and the use of lithium ions or a salt
thereof for manufacturing a pharmaceutical composition for locally
increasing bone formation are also provided by the present
invention.
Inventors: |
Hansson; Stig; (Askim,
SE) ; Petersson; Ingela; (Goteborg, SE) |
Assignee: |
Astra Tech AB
Molndal
SE
|
Family ID: |
38736015 |
Appl. No.: |
12/298889 |
Filed: |
July 8, 2008 |
PCT Filed: |
July 8, 2008 |
PCT NO: |
PCT/EP2008/058858 |
371 Date: |
January 21, 2009 |
Current U.S.
Class: |
424/722 ;
106/286.8; 205/333; 623/23.53 |
Current CPC
Class: |
A61L 27/306 20130101;
A61F 2/30767 20130101; A61F 2310/00598 20130101; A61F 2/3094
20130101; A61F 2002/30929 20130101; A61F 2310/00023 20130101; A61P
19/08 20180101; A61C 8/0012 20130101; A61P 19/00 20180101; A61L
2430/02 20130101 |
Class at
Publication: |
424/722 ;
623/23.53; 205/333; 106/286.8 |
International
Class: |
A61F 2/28 20060101
A61F002/28; C25D 9/06 20060101 C25D009/06; C09D 1/00 20060101
C09D001/00; A61K 33/00 20060101 A61K033/00; A61P 19/00 20060101
A61P019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 2007 |
EP |
07112079.4 |
Claims
1. An implant for implantation into bone tissue having a surface;
said surface being covered by an oxide layer characterized in that
said oxide layer comprises lithium ions.
2. An implant according to claim 1, wherein said implant is a
metallic implant.
3. An implant according to claim 2, wherein said metallic implant
comprises titanium or an alloy of titanium.
4. An implant according to claim 1, wherein said implant is a
non-metallic implant; said surface being an applied metallic
implant layer.
5. An implant according to claim 4, wherein said metallic implant
layer comprises titanium or an alloy of titanium.
6. An implant according to claim 1, wherein said oxide layer has a
thickness within the range of from 2 to 100 nm.
7. An implant according to claim 6, wherein said oxide layer has a
thickness within the range of from 2 to 18 nm.
8. An implant according to claim 6, wherein said oxide layer has a
thickness within the range of from 2 to 6 nm.
9. An implant according to claim 1, wherein said oxide layer is a
metal oxide layer formed from said surface of said implant.
10. An implant according to claim 1 further comprising a deposit on
top of said oxide layer, wherein said deposit comprises a bone
stimulating agent.
11. An implant according to claim 10, wherein said bone stimulating
agent is selected from the group consisting of lithium, strontium,
magnesium and calcium or a combination thereof.
12. An implant according to claim 10, wherein said deposit is a
salt precipitation comprising any one or a combination of ions
selected from lithium, strontium, magnesium and calcium.
13. An implant according to claim 12, wherein said salt
precipitation is a lithium salt precipitation.
14. An implant according to claim 1, wherein said implant lacks a
coating comprising a calcium phosphate compound.
15. An implant according to claim 1, wherein said surface of said
implant comprises a micro roughness.
16. An implant according to claim 1, wherein said surface of said
implant comprises fluorine.
17. A method for manufacturing a bone tissue implant having an
implant surface covered by an oxide layer comprising lithium ions,
said method comprising the steps of: a) providing an implant having
an implant surface; b) forming an oxide layer covering said implant
surface; c) forming negatively charged ions on said oxide layer;
and d) bringing said oxide layer into contact with lithium
ions.
18. A method according to claim 17, wherein said oxide layer is
formed spontaneously.
19. A method according to claim 17, wherein said negatively charged
ions on said oxide layer are formed by subjecting said implant
surface to an alkaline environment.
20. A method according to claim 19, wherein said alkaline
environment is formed by subjecting said implant surface to an
alkaline solution.
21. A method according to claim 17, wherein said negatively charged
ions on said oxide layer are formed by applying a potential within
the range of -0.5 V to -3.5 V.
22. A method according to claim 17, wherein said step c and said
step d are performed simultaneously.
23. A method according to claim 17, wherein said oxide layer is
brought into contact with lithium ions by subjecting said oxide
layer to a solution comprising lithium ions.
24. A method according to claim 23, wherein said solution comprises
lithium hydroxide.
25. A method according to claim 24, wherein said solution comprises
lithium hydroxide in a concentration of 5.3 M or less.
26. A method according to claim 25, wherein said solution comprises
lithium hydroxide in a concentration in the range of from 0.05 to 2
M.
27. A method according to claim 17, further comprising the step of
forming a deposit comprising a bone stimulating agent on top of
said oxide layer.
28. A method according to claim 27, wherein said bone stimulating
agent is selected from the group consisting of lithium, strontium,
calcium and magnesium or a combination thereof.
29. A method according to claim 27, wherein said deposit is formed
by precipitating a salt comprising any one or a combination of the
ions selected from lithium, strontium, calcium and magnesium on
said oxide layer.
30. A method according to claim 29, wherein said salt is lithium
hydroxide.
31. A method according to claim 29, wherein said deposit is formed
by applying a potential which is more negative than -3.5 V.
32. A method according to claim 29, wherein said deposit is formed
by subjecting said implant surface to an alkaline solution
comprising lithium hydroxide in a concentration of more than 5.3
M.
33. A method according to claim 17, further comprising the step of
creating a micro roughness on said surface of said implant after
step a).
34. A method according to claim 17, further comprising the step of
applying fluorine to said surface of said implant.
35. A blasting powder comprising a metal oxide, wherein said metal
oxide comprises lithium ions.
36. A blasting powder according to claim 35, wherein said metal
oxide is titanium dioxide.
37. A blasting powder according to claim 35, wherein said metal
oxide is lithium oxide.
38. A method for locally increasing bone formation by administering
a composition comprising lithium ions or a salt thereof and a
pharmaceutically acceptable carrier to a person in need
thereof.
39. A method according to claim 38, wherein said composition is
administered at an implantation site upon implantation of an
implant into bone tissue at said implantation site before,
simultaneously with and/or after said implant is placed in a cavity
in the bone tissue at said site.
40. (canceled)
41. (canceled)
42. A kit for implantation of an implant into bone tissue
comprising an implant characterized in that said kit further
comprises a composition comprising lithium ions or a salt thereof
and a pharmaceutically acceptable carrier.
Description
TECHNICAL FIELD
[0001] The present invention relates to an implant for implantation
into bone tissue and a method for manufacturing thereof.
[0002] The invention also relates to a blasting powder and a method
for locally increasing bone formation.
TECHNICAL BACKGROUND
[0003] A one-stage procedure is nowadays often used for implanting
orthopaedic or dental implants, generally metallic implants, into
bone tissue.
[0004] In the one-stage procedure, a first implant part, such as a
dental fixture, is surgically placed into the bone tissue, and a
healing cap or a secondary implant part, such as an abutment, is
then attached to the first implant part directly after the surgical
operation. The soft tissue is thereafter allowed to heal around the
healing cap or the secondary implant part. When a healing cap is
used, the cap is removed after a few weeks or months without any
surgical procedure, and secondary implant parts, such as an
abutment and a provisional crown, are attached to the first implant
part. The one-stage procedure is for instance described in L Cooper
et al: "A multicenter 12-month evaluation of single-tooth implants
restored 3 weeks after 1-stage surgery", The International Journal
of Oral & Maxillofacial Implants, Vol 16, No 2 (2001).
[0005] The two-stage procedure, which is another known implantation
procedure, involves in a first stage surgically placing a first
implant part, such as a dental fixture, into the bone tissue, where
it is then allowed to rest unloaded and immobile for a healing
period of three months or more in order to allow the bone tissue to
grow onto the implant surface to permit the implant to be well
attached to the bone tissue, the cut in the soft tissue covering
the implant site being allowed to heal over the implant, and in a
second stage opening the soft tissue covering the implant and
attaching secondary implant parts, such as a dental abutment and/or
a restoration tooth, to the first implant part, such as said
fixture, forming the final implant structure. This procedure is for
instance described by Branemark et al: "Osseointegrated Implants in
the Treatment of the Edentulous Jaw, Experience from a 10-year
period", Almquist & Wiksell International, Stockholm,
Sweden.
[0006] However, the fact that the implant not should be loaded
during the healing period means that the secondary implant parts
may not be attached to the first implant part and/or used during
the healing period of three months or more. In view of the
discomfort associated with this, it is desirable to minimize the
time period necessary for the above-mentioned first stage or even
perform the entire implantation procedure in a single operation,
i.e. to use the one-stage procedure.
[0007] For some patients, it might be considered better to wait at
least three months before functionally loading the implant, both
for one- and two-stage procedures. However, an alternative using
the one-stage procedure is to put the implant in function directly
after implantation (immediate loading) or a few weeks after
implantation (early loading). These procedures are, for instance,
described by D M Esposito, pp 836-837, in Titanium in Medicine,
Material Science, Surface Science, Engineering, Biological
Responses and Medical Application, Springer-Verlag (2001).
[0008] It is essential that the implant establishes a sufficient
stability and bond between implant and bone tissue to enable the
above disclosed immediate or early loading of the implant. It shall
also be noted that an immediate or early loading of the implant may
be beneficial to bone formation.
[0009] Some of the metals or alloys, such as titanium, zirconium,
hafnium, tantalum, niobium, or alloys thereof, that are used for
bone implants are capable of forming a relatively strong bond with
the bone tissue, a bond which may be as strong as the bone tissue
per se, sometimes even stronger. The most notable example of this
kind of metallic implant material is titanium and alloys of
titanium whose properties in this respect have been known since
about 1950. This bond between the metal and the bone tissue has
been termed "osseointegration" (Albrektsson T, Branemark P I,
Hansson H A, Lindstrom J, "Osseointegrated titanium implants.
Requirements for ensuring a long-lasting, direct bone anchorage in
man", Acta Orthop Scand, 52:155-170 (1981)).
[0010] It may be noted that in contact with oxygen, titanium,
zirconium, hafnium, tantalum, niobium and their alloys are
instantaneously covered with a thin oxide layer. This native oxide
layer on titanium implants mainly consists of titanium(IV) dioxide
(TiO.sub.2) with minor amounts of Ti.sub.2O.sub.3, TiO and
Ti.sub.3O.sub.4.
[0011] Although the bond between the (oxidised) metal, e.g.
titanium, and the bone tissue may be comparatively strong, it is
desirable to enhance this bond.
[0012] There are to date several methods for treating metallic
implants in order to obtain a better attachment of the implant, and
thus improved osseointegration. Some of these involve altering the
morphology of the implant, for example by creating irregularities
on the implant surface in order to increase the surface roughness
in comparison to an untreated surface. It is believed that an
increased surface roughness, which gives a larger contact and
attachment area between the implant and the bone tissue, provides a
better mechanical retention and strength between implant and bone.
It is well-known within the art that a surface roughness can be
provided by, for example, plasma spraying, blasting or acid
etching.
[0013] Other methods for obtaining a better attachment of the
implant to the bone tissue involve alteration of the chemical
properties of the implant surface.
[0014] Several methods involve the application of a layer of
ceramic material, such as hydroxyapatite, to the implant surface,
inter alia in order to improve the bonding of the implant to bone
since hydroxyapatite is chemically related to bone. A disadvantage
with coatings comprising hydroxyapatite is, however, that they may
be brittle and may flake or break off from the implant surface,
which may in turn lead to an ultimate failure of the implant.
[0015] Other methods for altering the chemical properties of the
implant involve application of fluorine and/or fluoride on the
implant surface (WO 94/13334, WO 95/17217, WO 04/008983, and WO
04/008984).
[0016] WO 2006/004297 discloses an osseoinductive metal implant,
such as titanium or an alloy thereof, comprising a layer of metal
oxide and a layer of a bio-active material composed of any one or
more of Li, Na, K, Rb, Cs, Fr, Mg, Ca, Sr, Ba, Ra, Sc, Y, Lu, Ti,
Zr, Hf, Nb, Ta, Cr, Mo, W, Mn, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd,
Pt, Cu, Ag, Au, Zn, Ga, In, Ti, Sn and Bi formed thereon. A working
example is, however, only described for a titanium implant
comprising calcium as the bioactive layer.
[0017] Mention can also be made of WO 2002/096475 referring to a
titanium implant comprising calcium, phosphor or sulphur in the
titanium oxide layer, and WO 2005/084577 referring to a titanium
implant comprising magnesium in the titanium oxide layer.
[0018] Although implants which provide a comparatively strong bond
between the implant surface and the bone exist, there is a need in
the art to enhance this bond, i.e. to improve the
"osseointegration" process of an implant in bone tissue.
[0019] Thus, there is a need in the art to provide an implant
having a desired rate of attachment and which has the ability to
form a mechanically strong bond between the bone and the implant
upon implantation thereof in bone tissue.
SUMMARY OF THE INVENTION
[0020] It is an object of the invention to meet the above mentioned
needs.
[0021] Thus, a biocompatible implant intended for implantation into
bone tissue is to be provided.
[0022] The inventors have found that lithium ions locally
administered in bone tissue has a local effect on the bone
formation and bone mass in the bone tissue.
[0023] It has further been found that an implant comprising a
surface oxide layer comprising and/or releasing lithium ions
induces an increased production of alkaline phosphatase in
osteoblasts, which is crucial for further differentiation and
mineralisation. Hence, an improved rate of bone formation and an
improved rate of attachment between bone tissue and the implant may
be achieved, further improving the possibility of immediate or
early loading of the implant.
[0024] Furthermore, it has been found that an implant comprising a
surface oxide layer comprising and/or releasing lithium ions
provides an increased proliferation of osteoblasts and an increased
production of osteoprotegerin, in comparison to a metallic implant
comprising a surface oxide layer containing, for instance, calcium
or magnesium ions. An improved bone mass is thereby provided which
implies a mechanically stronger bond between the implant and the
bone tissue.
[0025] Accordingly, it has been found that locally administered
lithium ions improve the osseointegration process of an implant in
bone tissue.
[0026] According to a first aspect of the invention, the above
objects are achieved with an implant for implantation into bone
tissue which has a surface covered by an oxide layer, wherein said
oxide layer comprises lithium ions.
[0027] According to a second aspect of the invention, a method for
manufacturing a bone tissue implant having the above mentioned
characteristics is provided. The method comprises the steps of:
[0028] a) providing an implant having an implant surface;
[0029] b) forming an oxide layer covering said implant surface;
[0030] c) forming negatively charged ions on said oxide layer;
and
[0031] d) bringing said oxide layer into contact with lithium
ions.
[0032] The method of the invention is inexpensive and easy to carry
out, thereby enabling mass production. Furthermore, it is easy to
sterilize and to store.
[0033] According to a third aspect of the present invention, a
blasting powder comprising a metal oxide comprising lithium ions is
provided.
[0034] According to a fourth aspect of the invention, a method for
locally increasing bone formation is provided. Said method
comprises administering a composition comprising lithium ions or a
salt thereof and a pharmaceutically acceptable carrier to a person
in need thereof.
[0035] A fifth aspect of the invention relates to the use of
lithium ions or a salt thereof for manufacturing a pharmaceutical
composition for locally increasing bone formation.
[0036] A sixth aspect of the invention relates to a kit for
implantation of an implant into bone tissue comprising an implant
and a composition comprising lithium ions or a salt thereof and a
pharmaceutically acceptable carrier.
[0037] Other features and advantages of the present invention will
become apparent from the following description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a TOF-SIMS image illustrating the presence and the
distribution of lithium for a sterilized titanium sample after
reduction in LiNO.sub.3. (Lithium is Shown as White Dots.)
[0039] FIG. 2 illustrates the presence of lithium, (peak no 7) on a
sterilized titanium sample (FIG. 2a) compared to the reference
sample of an oxalic acid treated sterilized titanium sample (FIG.
2b) (from TOF-SIMS measurements).
[0040] FIG. 3 is a TOF-SIMS image illustrating the presence and the
distribution of lithium for a sterilized titanium sample after
anodization in LiOH.
[0041] FIG. 4 illustrates the degree of proliferation of MG-63
cells after 3, 7 and 14 days culture on cell treated polystyrene
with and without lithium in different concentrations.
[0042] FIG. 5 illustrates the production of alkaline phosphatase in
the cell culture medium after 3, 7 and 14 days culture on cell
treated polystyrene with and without lithium in different
concentrations.
[0043] FIG. 6 shows the degree of proliferation of MG-63 cells on
reference surfaces 1 and 2 after 7 days of culture with and without
lithium in different concentrations.
[0044] FIG. 7 shows the production of alkaline phosphatase on
reference surface 1 comprising lithium at a concentration of 1 mM
compared to the unstimulated surface comprising no lithium.
[0045] FIG. 8 is a scanning electron microscopy image illustrating
the morphology of MG-63 cells cultured on reference surface 1 after
36 h.
[0046] FIG. 9 is a scanning electron microscopy image illustrating
the morphology of MG-63 cells cultured on reference surface 2 after
36 h.
[0047] FIG. 10 is a scanning electron microscopy image illustrating
the morphology of MG-63 cells cultured on reference surface 1
comprising lithium after 36 h.
[0048] FIG. 11 illustrates the differences in MG-63 cell
proliferation between reference surface 1, reference surface 2, and
reference surface 1 comprising lithium, calcium and magnesium,
respectively.
[0049] FIG. 12 shows the amount of osteoprotegerin measured after 7
and 14 days of cell culture on reference surface 1 comprising
lithium, calcium, and magnesium, respectively.
[0050] FIG. 13 illustrates the removal torque test (RTQ) values
after 6 weeks of implantation in rabbit tibia of an implant
comprising lithium according to the invention compared to an
implant with a commercially available Osseospeed.TM. surface.
DETAILED DESCRIPTION OF THE INVENTION
[0051] As used herein the term "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. Implants may be
used to replace anatomy and/or restore any function of the
body.
[0052] Generally, an 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 restoration tooth. 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.
[0053] As used herein the term "implant (intended) for implantation
into bone tissue" refers to implants intended for at least partial
implantation into bone tissue, such as dental implants, e.g
one-piece implants, orthopaedic implants, and the like. An implant
for implantation into bone tissue may also be referred to as a bone
tissue implant.
[0054] As used herein the term "implant surface" refers to at least
one defined surface region of an implant. Thus, the defined surface
region may include the entire surface area of the implant or
portions thereof.
[0055] An example of an implant surface intended for implantation
into bone tissue is the surface of a dental fixture that is
intended for implantation into the jawbone of a patient and to be
in contact with bone tissue.
[0056] Another example of an implant surface intended for
implantation into bone tissue is the surface of a hip joint implant
that is intended for implantation into the femur of a patient.
[0057] The present invention relates to an implant for implantation
into bone tissue having a surface; said surface being covered by an
oxide layer, wherein said oxide layer comprises lithium ions.
[0058] An implant according to the invention is biocompatible and
has a local effect on the bone formation and bone mass in the bone
tissue. Furthermore, the inventive implant causes an increased
proliferation of osteoblasts, and an increased production of
alkaline phosphatase and osteoprotegerin in bone tissue. Alkaline
phosphatase is an enzyme produced by osteoblasts which plays a
major role in the mineralization of bone, and osteoprotegerin is a
cytokine known to increase the bone mineral density and bone volume
in the bone tissue. The production of alkaline phosphatase and
osteoprotegerin clearly indicates that the implant according to the
invention has a positive effect on bone remodelling.
[0059] An implant according to the invention provides an improved
implant stability and bone tissue response as measured by removal
torque (RTQ) tests (FIG. 13).
[0060] The oxide layer covering the implant surface comprises
lithium ions which are dispersed in at least part of the oxide
layer.
[0061] Lithium is a small, positively charged, non-toxic, light
weight ion which has been found to be easily dispersed in the oxide
layer covering the surface of the implant.
[0062] Although a therapeutic effect of lithium salts is known for
manic-depressive disorders, studies have shown that lithium therapy
also affects the bone mineral metabolism (for instance, Baran et
al, "Lithium Inhibition of Bone Mineralization and Osteoid
Formation", J Clin Invest, pp 1691-1696 (1978); Nordenstrom et al,
"Biochemical Hyperparathyroidism and Bone Mineral Status in
Patients Treated Long-Term with Lithium", Metabolism, vol 43, No
12, pp 1563-1567 (1994); and Mak et al, "Effects of Lithium Therapy
on Bone Mineral Metabolism: A Two-Year Prospective Longitudinal
Study", J Clin Endocrin and Metabol, vol 83, No 11, pp 3857-3859
(1998)).
[0063] Recently, oral administration of lithium chloride has been
suggested for treatment of disorders of low bone mass, such as
osteoporosis, since it has been concluded that lithium enhances
bone formation and improves bone mass via activation of the
canonical Wnt pathway resulting in, for instance, stimulation of
pre-osteoblast replication, induction of osteoblastogenesis and
inhibition of osteoblast and osteocyte apoptosis (Clement-Lacroix
et al, "Lrp5-independent Activation of Wnt Signalling by Lithium
Chloride Increases Bone Formation and Bone Mass in Mice", PNAS, vol
102, No 48, pp 17406-17411 (2005) and Krishnan et al, "Regulation
of Bone Mass by Wnt Signaling", J Clin Invest, vol 116, No 5, pp
1202-1209 (2006).
[0064] The Wnt signalling pathways is an attractive target for the
treatment of osteoporosis, and in general for bone anabolic drug
discovery (Rawadi G et al, "Wnt signalling pathway: a new target
for the treatment of osteoporosis", Expert. Opin. Ther. Targets,
Vol 9, pp 1063-1077 (2005))). The fact that lithium activates the
Wnt pathway makes the present invention highly suitable for
implantation into bone tissue.
[0065] Furthermore, the fact that lithium previously has been used
for the treatment of manic depressive disorders implies that the
toxicological picture and the side effects upon systemic
administration are well known.
[0066] In addition, lithium has a relatively simple chemistry and
is generally indestructible and unaffected by e.g.
sterilization.
[0067] The incorporation of lithium ions into the oxide layer may
disrupt the oxide structure, thereby making the oxide more
reactive. When the oxide layer is incorporated with positively
charged lithium ions, an increased positive surface charge density
is provided on the oxide surface (implant surface). Hence, electron
rich proteins in the bone tissue may be electrically attracted to
the surface. Incorporated ions can also affect the conductivity of
the oxide which may have a positive effect on osseointegration and
hemocompatibility.
[0068] At least part of the oxide layer should comprise lithium
ions, and the desired osseoinductive effect of lithium in an
implant of the invention may be achieved by the presence of the
ions in the oxide layer. However, it may also be achieved by the
release of lithium ions from the oxide layer into the physiological
fluid surrounding the implant.
[0069] Preferably, lithium ions are homogenously distributed in the
oxide layer. Such homogenous distribution is illustrated in FIG.
1.
[0070] The implant according to the present invention is suitably a
metallic implant, such as an implant made of titanium or an alloy
of titanium.
[0071] In embodiments however, the implant can be a non-metallic
implant comprising e.g. a ceramic, a plastic or a composite
material. In such embodiments, the implant surface is a metallic
implant layer applied on the non-metallic implant, whereby a partly
metallic implant surface is provided. The metallic implant layer
preferably comprises titanium or an alloy of titanium.
[0072] However, the metallic implant, and the metallic implant
layer are not limited to a specific metal, but may be made of any
biocompatible metallic material, such as zirconium or an alloy
thereof, hafnium or an alloy thereof, niobium or an alloy thereof,
tantalum or an alloy thereof, a chromium-vanadium alloy, or any
combination of these materials.
[0073] The oxide layer covering the surface of the implant has a
thickness within the range of from 2 to 100 nm.
[0074] In contact with oxygen, titanium, zirconium, hafnium,
tantalum, niobium and their alloys are instantaneously covered with
a thin oxide layer. This native oxide layer on titanium implants
mainly consist of titanium(IV) dioxide (TiO.sub.2) with minor
amounts of Ti.sub.2O.sub.3, TiO and Ti.sub.3O.sub.4.
[0075] In preferred embodiments, the oxide layer is an oxide layer
which is formed spontaneously, e.g. in contact with air. The
thickness of such a spontaneously formed oxide layer is within the
range of from 2 to 18 nm, for example within the range of from 2 to
6 nm.
[0076] The oxide layer according to the invention does not grow
substantially thicker over time and protects the underlying
metallic surface from reacting with any surrounding agent.
[0077] Metal implants surfaces covered by oxide layers are known in
the art. However, several prior art documents stress the importance
of providing a thick oxide layer, preferably above 600 nm onto the
implant surface (Sul et al, "Resonance frequency and removal torque
analysis of implants with turned and anodized surface oxides",
Clin. Oral. Impl. Res., Vol 13, pp 252-259 (2002); Sul et al,
"Qualitative and quantitative observations of bone tissue reactions
to anodised implants", Biomaterials, Vol 23, No 8, pp 1809-1817
(2002)). Such implants require an additional step of oxidation
since oxide layers of the above mentioned thickness are not
obtainable spontaneously.
[0078] The present inventors have found that an oxide layer having
a thickness of less than 100 nm, preferably an oxide layer having
the thickness of a native oxide layer, i.e. a spontaneously formed
oxide layer, of less than 18 nm is more suitable for implantation
into bone tissue since thick oxide layers may be very brittle.
Furthermore, thick oxide layers may lead to cracking and peeling
during longer periods of implantation of an implant in bone
tissue.
[0079] This finding is in contrast to Xiropaidis et al who states
that titanium implants with native oxide layers are considered less
osteoconductive. (Xiropaidis et al, "Bone-implant contact at
calcium phosphate-coated and porous titanium oxide
(TiUnite.TM.)-modified oral implants", Clin. Oral. Impl. Res, No
16, pp 532-539 (2005)).
[0080] An oxide layer according to the invention does not interfere
with, or modify the topography of the implant surface. Furthermore,
an implant comprising an oxide layer having a thickness of less
than 100 nm, e.g. less than 18 nm, e.g. between 2 and 6 nm is
biocompatible making it suitable for incorporation into the human
body.
[0081] Hence, an oxide layer comprising lithium ions according to
the invention is suitable for any geometry or any substrate.
[0082] The implant surface of the implant according to the
invention is preferably a metallic implant surface comprising a
metal oxide layer on its surface.
[0083] In particular, the implant, in particular the surface of the
implant according to the invention comprises titanium or an alloy
thereof. Such an implant surface is thus covered by a titanium
oxide layer.
[0084] Accordingly, in an implant of the present invention, the
oxide layer covering the surface of the implant is a metal oxide
layer formed on the metallic surface of the implant.
[0085] In embodiments, the implant according to the invention may
further be provided with a deposit on top of the oxide layer. Such
a deposit may comprise a bone stimulating agent, such as lithium,
strontium, calcium, magnesium or any other ion having a bone
stimulating effect. Typically, the deposit comprises lithium
ions.
[0086] As used herein the term "deposit" relates to a continuous or
discontinuous film provided on top of the oxide layer. Such a
deposit may have any thickness and is not incorporated into the
oxide layer, but is provided thereon.
[0087] Typically, the deposit is a salt precipitation comprising
any one or a combination of ions selected from lithium, strontium,
magnesium and calcium.
[0088] Usually, the deposit is a lithium salt precipitation, i.e. a
lithium salt which is precipitated on top of the oxide layer of the
implant surface.
[0089] Examples of suitable lithium salts are lithium hydroxide,
lithium fluoride, lithium chloride, lithium sulphate, lithium
nitrate, lithium carbonate. However, any lithium salt capable of
being at least partly dissolved in the physiological fluid
surrounding the implant may be used. Such salts are known to a
person skilled in the art.
[0090] Upon implantation, a deposit comprising a salt precipitation
dissolves easily and rapidly in the surrounding fluid such that the
bone stimulating ions are released from the implant. An implant
provided with such a deposit on its surface may be particularly
beneficial in situations where an implant needs to integrate more
rapidly, e.g. in bone of poor quality and quantity.
[0091] An advantage associated with an implant comprising a deposit
of the above mentioned kind on its surface is that bone stimulating
ions, e.g. lithium ions are easily and efficiently released into
the physiological fluid surrounding the implant. Hence, a higher
dose of bone stimulating ions, e.g. lithium ions may be released
into the surrounding fluid.
[0092] Accordingly, the desired effect of lithium may be obtained
both from ions present in the oxide in the oxide layer on the
implant surface and ions released therefrom.
[0093] An implant according to the present invention suitably lacks
a coating comprising a calcium phosphate compound. As outlined in
the introduction, such implants are more prone to flake or break
off from the implant surface, which may lead to an ultimate failure
of the implant.
[0094] In embodiments of the invention, the implant surface may
further comprise a micro-roughness having a root-mean-square
roughness (R.sub.q and/or S.sub.q) of .ltoreq.250 nm (i.e. a
micro-roughness comprising pores having a pore diameter of
.ltoreq.1 .mu.m and a pore depth of .ltoreq.500 nm) on at least a
part of the implant surface. As used herein the term "nano- or
micro-roughness" refers to a surface roughness comprising surface
irregularities having dimensions smaller than 1 .mu.m
[0095] Such surface roughness is likely to give a larger contact
and attachment area between the implant and the bone tissue, and
provide a better mechanical retention and strength between implant
and bone. In alternative embodiments, the implant surface comprises
fluorine and/or fluoride, such as 0.2-20 at %, and optionally also
a micro-roughness having a root-mean-square roughness (R.sub.q
and/or S.sub.q) of 250 nm, on at least a part of the implant
surface.
[0096] Optionally, the surface of the implant according to the
invention may comprise a macro-roughness. As used herein the term
"macro-roughness" refers to a surface roughness comprising surface
irregularities having dimensions greater than 1 .mu.m.
[0097] It shall also be noted that the implant surface may be
either threaded or unthreaded or it may be given other use
dependent topographical features.
[0098] Furthermore, the present invention relates to a method for
manufacturing a bone tissue implant having the characteristics
outlined above, comprising the steps of:
[0099] a) providing an implant having an implant surface;
[0100] b) forming an oxide layer covering said implant surface;
[0101] c) forming negatively charged ions on said oxide layer;
and
[0102] d) bringing said oxide layer into contact with lithium
ions.
[0103] As previously mentioned, the implant may be a metallic
implant, or it may be a non-metallic implant provided with a
metallic surface. When non-metallic implants are used in the
present invention, a metallic implant surface may be provided by
any suitable technique known to those skilled in the art. For
example, any suitable electrochemical treatment can be used.
[0104] An oxide layer covering the surface of the implant is
preferably formed spontaneously, e.g. in contact with air. Such a
layer is passive and inert, i.e. it is stable and prevents the
underlying metallic surface from further reaction.
[0105] It is however possible to use any conventional oxidization
techniques in the method above. Hence, the method is not limited to
the spontaneous formation of an oxide layer. For instance, an oxide
layer can be formed on a metallic implant surface by anodic
oxidation of the implant in an electrolyte, such as an aqueous
solution of an organic acid. An oxide layer can also be formed on a
metallic implant surface by heating in air at, for instance,
150-1300.degree. C. Moreover, an oxide layer can be formed on a
metallic implant surface by precipitating the oxide on the implant
surface from a suitable solution.
[0106] As already mentioned, an oxide layer covering said implant
surface is preferably formed spontaneously, which is advantageous
as no additional step of oxidation is actually required.
[0107] Referring to step c) in the method outlined above,
negatively charged ions may be formed on said oxide layer by
subjecting the implant surface to an alkaline environment.
[0108] In contact with an aqueous solution the metal oxide, e.g.
titanium oxide surface will disrupt the water molecule structure in
its close vicinity, and, depending on the pH, become either
positively or negatively charged. When the surface is uncharged and
no ions are adsorbed on the surface, the pH is called the point of
zero charge pH.sub.PZC. The pH.sub.pzc for titanium oxide is
between 5-7.
[0109] Hence, when the titanium oxide surface is surrounded by an
aqueous, alkaline environment, e.g. an alkaline solution having a
pH over 7, the surface becomes slightly negatively charged due to
the formation of surface bound, negatively charged hydroxide
groups. Positively charged lithium ions, which may be present in a
surrounding solution can thus be electrically attracted to the
oxide surface (implant surface) and thereby become incorporated
into at least part of the oxide layer, typically in the upper part
of the oxide layer. Preferably, the lithium ions are homogenously
distributed in the oxide layer.
[0110] An alkaline environment may be achieved locally on the
surface of the oxide; i.e. negatively charged ions may be formed on
the oxide layer by applying a potential which is more negative than
-0.5 V; typically in the range of from -0.5 to -3.5 V. The
application of such a potential will increase the disruption of
water molecules, generating the formation of hydrogen gas, and
surface bound, negatively charged hydroxide groups on the implant
surface.
[0111] Alternatively, an alkaline environment is achieved by
subjecting the implant surface to an alkaline solution, e.g. by
soaking the implant surface in an alkaline solution. Such an
alkaline solution should have a pH higher than 7, e.g. higher than
10; and typically higher than 11. The soaking time may be less than
30 minutes, e.g. less than 20 minutes, typically between 10 and 15
minutes.
[0112] The implant surface is then brought into contact with
positively charged lithium ions; e.g. by subjecting the implant
surface to a solution comprising lithium ions. The step of bringing
said oxide layer into contact with lithium ions may be performed
simultaneously with or after the step of forming negatively charged
ions on said oxide layer. Preferably the steps c) and d) of the
method according to the invention are performed simultaneously.
[0113] For example, by applying a potential within the range of
from -0.5 V to -3.5 V in a solution comprising lithium, negatively
charged hydroxide groups will be formed, leading to an
electrostatic interaction between surface bound hydroxide groups
and lithium ions present in the solution. This electrostatic
interaction results in that lithium ions are incorporated into the
oxide layer. This is further described in Example 1.
[0114] The solution comprising lithium ions may be a solution
comprising lithium hydroxide in a concentration of 5.3 M or less.
The concentration of lithium hydroxide should not exceed 5.3 M,
which represents the solubility product of lithium hydroxide. When
the concentration of LiOH exceeds 5.3 M, salt crystals will be
formed and precipitate on the surface of the oxide. In step c) of
the method according to the invention, it is desired that the ions
become incorporated into the oxide, and hence the concentration of
LiOH should not exceed the solubility product.
[0115] A preferred concentration of lithium hydroxide is within the
range of from 0.05 to 2 M.
[0116] The steps of forming negatively charged ions on the oxide
surface and bringing said oxide layer into contact with lithium
ions, thereby incorporating lithium ions into the oxide layer
covering the surface of the implant is not limited to a specific
method but may be achieved by any suitable method, or any
combination of methods. For example, the implant surface may be
anodized in an alkaline solution comprising lithium ions. Example 1
illustrates the incorporation of lithium ions by anodizing in
lithium hydroxide.
[0117] By subjecting the implant surface to an anodization step,
the thickness of the oxide layer will be affected. However, as the
anodizing is preferably performed with a relatively low scan rate,
e.g. below 6 mV/s until reaching 8V, the thickness of the oxide
will not grow thicker than 100 nm.
[0118] Hence, lithium ions are incorporated into the oxide layer by
means of the electrostatic interaction between negatively charged
hydroxide groups formed on the implant surface and positively
charged lithium ions present in a surrounding solution.
[0119] Optionally, the method according to the invention may
comprise the step of rinsing and/or cleaning said implant surface
after step d). Furthermore, the implant surface may be dried and
sterilized after said rinsing step.
[0120] In embodiments, the method according to the invention
further comprises the step of forming a deposit comprising a bone
stimulating agent such as lithium, strontium, calcium, magnesium on
top of said oxide layer, e.g. by precipitating a salt comprising
the above mentioned ions on the surface of the implant; i.e. on the
oxide layer covering said surface.
[0121] The salt may be any suitable salt of the ions above which is
at least partly soluble in the physiological fluid surrounding the
implant. The precipitation of a salt on the implant surface will
form a continuous or a non-continuous film on the surface. The
thickness of the deposit will depend on the amount of salt
precipitated.
[0122] Such a salt deposit dissolves easily and rapidly in contact
with the physiological fluid surrounding the implant such that the
desired bone stimulating effect is achieved by the release of bone
stimulating ions from the implant surface.
[0123] When the deposit is a lithium salt precipitation, the step
of forming such a deposit may be achieved by modifying the above
described methods for forming negatively charged ions on the
surface of the oxide layer. For example, a potential more negative
than -3.5 V can be applied. Such a negative potential gives rise to
a significantly enhanced hydrogen gas development and an increased
disruption of water molecules. Hence, an excess of negatively
charged, surface bound hydroxide groups are formed at the oxide
surface resulting in a deposit, i.e. a precipitate of lithium
hydroxide on top of the oxide layer. See example 2 for further
description.
[0124] Furthermore, by subjecting the implant surface to a solution
comprising lithium hydroxide at a concentration above the
solubility product of 5.3 M, a lithium salt deposit will be formed
on the oxide surface (implant surface). This is also due to the
excess of hydroxide groups in the surrounding.
[0125] However, the step of forming a deposit of e.g. a lithium
salt is not limited to any specific method, but any method may be
used. Neither is it limited to a specific lithium salt, but any
salt which is at least partly soluble in the physiological fluid
surrounding an implant may be used.
[0126] Furthermore, any method for forming a salt precipitation
comprising any or a combination of the ions selected from lithium,
strontium, magnesium and calcium can be used, e.g. the solution
which comprises lithium may also comprise any or a combination of
the above mentioned ions. In such cases, a small amount of these
ions may also become incorporated into the oxide layer.
[0127] The step of forming a deposit may also be performed by a
combination of the above mentioned methods.
[0128] It should however be noted that known methods for ion
incorporation and deposit formation on an implant surface may also
be used in the present invention. Such methods include e.g.: [0129]
plasma deposition, for instance using plasma source ion
implantation or metal plasma immersion ion implantation, [0130] any
electrochemical treatment, for instance voltametry in an
electrolyte comprising lithium ions, [0131] treatment of the
implant with an aqueous and/or non-aqueous solution comprising
lithium ions, for instance by dipping said implant in said
solution, [0132] treatment of the implant with a sol-gel technique,
[0133] beam ion implantation, [0134] vacuum arc, [0135] filtered
vacuum arc, [0136] metal vapour vacuum arc, [0137] ion plating,
[0138] chemical vapour deposition, [0139] plasma assisted chemical
vapour deposition, [0140] sputtering, [0141] laser ablation, [0142]
providing a coating, such as a calcium phosphate-containing coating
or a silane coating, on the implant surface, in or to which lithium
ions can be incorporated or attached,
[0143] any combination of these methods or the like. The method
according to the invention may further comprise the step of
creating a micro roughness on the implant surface.
[0144] Before, simultaneously with and/or after the provision of
lithium ions or a salt thereof on the implant surface, a nano-
and/or micro-roughness can be optionally provided on the implant
surface using, for instance, mild etching, micro-fabrication,
anodization, flame spraying, electrochemical treatment, laser,
spark erosion, or any other suitable method of surface
modification. Reference can be made to WO 04/008983 and WO
04/008984, wherein suitable methods for obtaining such an implant
surface are disclosed. It is however preferred that the nano-
and/or micro-roughness is provided after step a) in the method
according to the invention.
[0145] The method of the invention may also comprise the step of
applying fluorine to the implant surface. Reference can be made to
WO 04/008983, wherein suitable methods for obtaining such an
implant surface are disclosed.
[0146] A suitable method, according to WO 04/008983, for providing
fluorine and/or fluoride and a micro-roughness having a
root-mean-square roughness (R.sub.q and/or S.sub.q) of .ltoreq.250
nm on at least a part of an implant surface is by treatment of the
implant with an aqueous solution of hydrofluoric acid having a
concentration of less than 0.5 M, such as 0.1 M, for an etching
period of up to 180 sec, such as 60 s, at room temperature (see WO
04/008983 for more information).
[0147] In addition, a macro-roughness can be optionally provided on
the implant surface prior to providing lithium ions or a salt
thereof, and prior to optionally providing the micro-roughness,
thereon. A macro-roughness can, for instance, be provided by
blasting, e.g. with titanium dioxide particles, etching,
micro-fabrication, anodization, flame spraying, any electrochemical
treatment, laser, spark erosion, machining, knurling, or any other
suitable method of surface modification.
[0148] It shall also be noted that the implant surface may be
either threaded or unthreaded or be given other use dependent
topographical features.
[0149] The method for manufacturing a bone tissue implant according
to the invention is not limited to the incorporation of lithium
ions, but may be applied to incorporate positively charged ions
into the implant surface in general. Hence, such a method involves
the steps of:
[0150] a) providing an implant having an implant surface;
[0151] b) forming an oxide layer covering said implant surface;
[0152] c) forming negatively charged ions on said oxide layer;
and
[0153] d) bringing said oxide layer into contact with positively
charged ions.
[0154] The present invention further relates to a blasting powder,
which comprises a metal oxide, wherein the metal oxide comprises
lithium ions. The metal oxide may be a metal oxide selected from
the group consisting of titanium oxide, zirconium oxide, hafnium
oxide, tantalum oxide, and niobium oxide. Preferably the blasting
powder comprises titanium oxide into which lithium ions are
incorporated.
[0155] It is also possible to simply use lithium oxide as the
blasting powder of the present invention.
[0156] It may be possible to use said blasting powder in the method
according to the invention to further enhance the incorporation of
lithium ions into the oxide layer covering the implant surface.
However, a blasting powder may be used by itself to incorporate
lithium ions into any oxide layer provided on any implant
surface.
[0157] The present invention also relates to a method for locally
increasing bone formation. Such a method comprises administering a
composition comprising lithium ions or a salt thereof and a
pharmaceutically acceptable carrier to a person in need thereof.
Preferably, the composition comprising lithium ions or a salt
thereof and a pharmaceutically acceptable carrier is administered
at an implantation site upon implantation of an implant into bone
tissue at said implantation site before, simultaneously with and/or
after said implant is placed in a cavity in the bone tissue at said
site.
[0158] The composition comprising lithium ions, or a salt thereof,
can be administered in and/or nearby said cavity in the bone
tissue.
[0159] Examples of suitable pharmaceutically acceptable carriers
for use in said composition are a physiological saline solution;
disintegrated autologous bone; a demineralised bone matrix;
liposomes; nano- or microparticles of biodegradable polymer(s),
such as polylactic acid (PLA) and polyglycolic acid (PGA);
hyaluronic acid; collagen; chondroitin sulfate; a hydrogel, such as
alginate; chitosan; a scaffold of polyester and tricalcium
phosphate; and the like.
[0160] A specific example of a suitable carrier is PepGen P-15
PUTTY.TM., which are particles of hydroxyapatite enhanced with
P-15, a synthetic peptide that mimics the cell-binding region of
Type-I collagen, suspended in sodium hyaluronate.
[0161] The composition according to the invention can either be an
immediate release, a delayed release or a controlled release
formulation.
[0162] The invention also relates to the use of lithium ions, or a
salt thereof, for manufacturing a pharmaceutical composition (as
disclosed above) for locally increasing bone formation.
[0163] The composition may be locally administered at an
implantation site upon implantation of an implant into bone tissue
at said site.
[0164] According to the present inventors, local administration of
lithium ions or a salt thereof directly into the bone tissue is
preferred over systemic administration. Foreign agents often have a
variety of effects on the human body, which are both known and
unknown. Local administration of lithium or a salt thereof in bone
tissue is beneficial as the bone stimulating effect will be
achieved, while side effects are avoided.
[0165] In addition, the invention relates to a kit for implantation
of an implant into bone tissue comprising an implant and a
composition (as disclosed above) comprising lithium ions, or a salt
thereof, and a pharmaceutically acceptable carrier.
[0166] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent for
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope
thereof.
[0167] The invention will now be illustrated by means of the
following non-limiting examples.
EXAMPLES
Example 1
Incorporation of Lithium Ions into a Titanium Oxide Layer
Reduction in LiNO.sub.3
[0168] Titanium samples were reduced by potential step technique in
0.1M LiNO.sub.3, pH 5-6. A potential step of -3V (all potentials
are referred to a double junction Ag/AgCl/KCl reference electrode,
197 mV against SHE) was applied over the sample for 5 minutes
resulting in a continuous hydrogen evolution. After the reduction
process the samples were rinsed in MQ water in an ultrasonic bath
for 2 minutes before drying and sterilization. The presence and
distribution of lithium was identified by Time-of-Flight Secondary
Ion Mass Spectrometry (TOF-SIMS) (FIG. 1).
Anodizing in an Alkaline Solution
[0169] Titanium samples were anodized in 0.1M LiOH, pH>11 by LSV
(linear sweep voltametry) from OC (open circuit) to 7V (all
potentials are referred to a double junction Ag/AgCl/KCl reference
electrode, 197 mV against SHE), at a scan rate of 2 and/or 5 mV/s
and a rotation speed of 900 rpm. After the anodization process the
samples were rinsed in MQ water in an ultrasonic bath for 2 minutes
before drying and sterilization. The presence of lithium on the
surface of a sterilized titanium sample is illustrated by the mass
spectrum in FIG. 2a (peak 7), and compared to a reference sample of
an oxalic acid treated sterilized titanium sample (FIG. 2b).
[0170] The presence and distribution of lithium was identified by
TOF-SIMS (FIG. 3).
Example 2
Release of Lithium from a Titanium Implant Surface
[0171] Titanium samples were reduced by potential step technique in
0.1M LiNO.sub.3, pH 5-6. A potential step more negative than -4V
(all potentials are referred to a double junction Ag/AgCl/KCl
reference electrode, 197 mV against SHE) was applied over the
sample for 5 minutes resulting in vigorous hydrogen evolution.
After the reduction process the samples were rinsed in water and
dried.
[0172] The release of lithium was identified by ISE (Ion Selective
Electrode) and ICP (Inductively Coupled Plasma). Four coin shaped
samples, prepared according to the above, were placed in a beaker
with 15 ml MQ water. The water was acidified to pH 4 using 20 mM
HNO.sub.3 and thereafter left for 90 minutes with moderate
agitation before analysis. The solution was analysed using ISE and
ICP, both analyses giving the same result of 0.4 .mu.g lithium /ml
sample.
Reference Example 1
Culturing of MG-63 Cells
[0173] MG-63 is a human cell line (ATCC No CRL-1427, U.S.)
conventionally used in the art for in vitro studies of osteoblasts.
MG-63 origins from a human osteosarcoma and exhibits numerous trait
characteristics of human osteoblasts, such as alkaline phosphatase
(ALP), and osteoprotegerin (OPG).
[0174] In this study, MG-63 cells were obtained from frozen cells
in second passage and further cultured in Dulbecco's modified
Eagle's medium (DMEM) containing FCS 5%, PEST 1% , Gibco, UK) in
37.degree. C. in an atmosphere of 5% CO.sub.2 and 100% humidity.
When the cells reached confluence they were subcultured by the use
of 0.05% Trypsin-EDTA (Gibco, UK) until the amount of cells were
obtained.
[0175] Cell viability was high in all experiments (>98%) and was
checked by the use of trypan blue where the uptake of the stain by
dead cells was checked in a Burkerchamber in a light microscope
(LM).
Reference Example 2
Proliferation of MG-63 Cells and Production of Alkaline Phosphatase
(ALP)
[0176] In order to study the effect of lithium on the proliferation
of MG-63 cells and the production of alkaline phosphatase (ALP),
MG-63 cells prepared in reference example 1 were subcultured into
24 well plates at a plating density of 10 000 cells/cm.sup.2, in
total 20 000 cells/well. Sterile filtered LiNO.sub.3 at final
concentrations of 10 mM, 5 mM, 3 mM, and 1 mM respectively (pH 5.2)
were added to the respective wells of the plate. Untreated cells
were used as controls. Cells were cultured for 3, 7 and 14 days at
a temperature of 37.degree. C. in an atmosphere of 5% CO.sub.2 and
100% humidity.
[0177] The total number of cells in each well (.times.10.sup.5)
after each time period was determined by the NucleoCassette method
by the NucleoCounter (ChemoMetec NS Denmark).
[0178] The number of cells was investigated by lysis of the cells
in "Reagent A" having a pH of 1.25 following stabilization by
"Reagent B". In the NucleoCassette, propidium iodide was
incorporated which targets the amount of released DNA. The cassette
was placed in the NucleoCounter and the amount of measured
fluorochrome corresponded to the amount of DNA. The instructions
from the manufacturer were followed (Chemometec A/S, Denmark).
[0179] At harvest, the cell culture medium was analysed with
respect to the ALP content. The intracellular ALP was measured by
cell lysis according to the instructions of the manufacturer
(SenzoLyte.TM. pNPP Alkaline Phosphatase Assay Kit Colorimetric
BioSite, Sweden). The absorption was set at 405 nm by an automatic
platereader (Vmax, Molecular Device, UK). By comparing the optical
density of the samples with the standard provided with the kit, the
ALP concentrations could be determined. The instructions from the
manufacturer were followed (BioSite, Sweden).
[0180] Referring to FIG. 4, an increased proliferation of MG-63
cells was achieved at all the three time points with 5, 3 and 1 mM
lithium. The highest detected cell number was observed after 7
days.
[0181] The production of ALP was initially slow with all lithium
concentrations compared to control (unstimulated cells). A small
increase was detected after 7 days compared to the control.
However, increased ALP levels were detected after 14 days with all
lithium concentrations; 1 mM, 3 mM, 5 mM and 10 mM. The highest
amount of ALP was observed with 1 mM lithium after 14 days. The
results are illustrated in FIG. 5.
Reference Example 3
Preparation of Reference Surfaces
[0182] Titanium samples having the shape of a coin were cleaned,
and then immersed in an 1 M aqueous solution of oxalic acid and
left at 80.degree. C. for 30 minutes under vigorous agitation.
After 30 minutes the samples were removed from the oxalic acid
solution and rinsed in water followed by rinsing in water in an
ultrasonic bath for 2 minutes. The resulting surface is referred to
as "reference surface 1".
[0183] Some of the samples were subjected to a secondary
hydrofluoric acid treatment. Approximately 10 minutes after
rinsing, the samples were immersed in 0.1 M aqueous solution of HF
at room temperature and agitation until the start of active
dissolution, followed by an additional active treatment time of 40
s. Next, the samples were removed from the HF solution and rinsed
in water followed by rinsing in water in an ultrasonic bath for 2
minutes. The samples were dried in air at room temperature for
about 60 minutes before sterilization. The resulting surface is
referred to as "reference surface 2"
Reference Example 4
MG-63 Proliferation on Reference Surfaces
[0184] Sterilized (.beta.-radiation) Ti coins with reference
surfaces 1 and 2, respectively were placed in 24 well plates. MG-63
cells were subcultured onto the coins in the 24 well plates at a
plating density of 10 000 cells/cm.sup.2, in total 20 000
cells/well. Sterile filtered LiNO.sub.3 at final concentrations of
5 mM and 1 mM (pH 5.2) were added to the respective wells.
Untreated cells were used as controls. Cells were cultured for 7
days at a temperature of 37.degree. C. in an atmosphere of 5%
CO.sub.2 and 100% humidity.
[0185] The reference surface 1 comprising lithium in a
concentration of 1 mM induced an increased proliferation of MG-63
cells after 7 days of culture. The reference surface 2 comprising
lithium in a concentration of 1 mM induced a high cell
proliferation after 7 days of cell culture compared to the
unstimulated reference surface 2. The final concentration of 5 mM
lithium induced a modest increased proliferation. The results are
illustrated in FIG. 6.
Reference Example 5
ALP production on Reference Surface 1
[0186] The production of ALP on reference surface 1 comprising a
concentration of 1 mM lithium was compared with unstimulated
reference surface 1 comprising no lithium.
[0187] As is illustrated in FIG. 7, the ALP production increased
with 1 mM lithium after 7 days of cell culture.
Reference Example 6
Morphology
[0188] Titanium samples having (i) reference surface 1 (ii)
reference surface 2, and (iii) reference surface 1+lithium were
prepared for Scanning Electron Microscopy (SEM). The samples were
fixated by glutaraldehyde at +4.degree. C. (Kanowsky's), followed
by osmium tetroxid, dehydration and finally gold sputtered
according to the standard techniques.
[0189] The FIGS. 8, 9, and 10 illustrate the morphology of the
respective surface after 36 h. The reference surface 1 comprising
lithium has a larger amount of cells in proliferative stage (FIG.
10) (round, non-spread cells) as well as a large amount of adhesive
cells compared to cells on the reference surface 1 and 2 comprising
no lithium. On both the reference surface 1 (FIG. 8) and reference
surface 2 (FIG. 9) the cells are thinner, flatter and more spread
out.
[0190] The space between the cells on the reference surfaces 1 and
2 is probably due to fixation difficulties and is often seen when
cells are very thin and spread out.
[0191] The morphology of the cells cultured on the reference
surface 1 comprising lithium shows that the cells are well spread
out, indicating high activity, proliferate, and form matrix and
pseudopodia on the surfaces. This indicates that such surfaces are
osteoconductive as well as osteoinductive and in favour for cell
adhesion, proliferation and matrix formation.
Comparative Example 1
MG-63 Proliferation on Reference Surfaces Comprising Lithium,
Calcium and Magnesium, Respectively
[0192] Sterilized (.beta. radiation) Ti coins with reference
surfaces 1 and 2 were compared with Ti coins with reference surface
1 comprising lithium, calcium and magnesium, respectively, and the
coins were placed in 24 well plates. MG-63 cells were subcultured
onto the coins in the 24 well plates at a plating density of 10 000
cells/cm.sup.2, in total 20 000 cells/well. Cells were cultured for
7 days at a temperature of 37.degree. C. in an atmosphere of 5%
CO.sub.2 and 100% humidity.
[0193] The total number of cells in each well (.times.10.sup.5)
after each time period was determined by the NucleoCassette method
by the NucleoCounter (ChemoMetec A/S Denmark).
[0194] The number of cells was investigated by lysis of the cells
in "Reagent A" having a pH of 1.25 following stabilization by
"Reagent B". In the NucleoCassette, propidium iodide was
incorporated which targets the amount of released DNA. The cassette
was placed in the NucleoCounter and the amount of measured
fluorochrome corresponded to the amount of DNA. The instructions
from the manufacturer were followed (Chemometec A/S, Denmark).
[0195] Referring to FIG. 11, the proliferation with the reference
surface 1 comprising lithium showed the highest MG-63 cell
proliferation after 7 days of cell culture. The cell proliferation
was markedly higher than the reference surfaces comprising calcium
and magnesium.
Comparative Example 2
Production of Osteoprotegerin on Reference Surface 1
[0196] The production of osteoprotegerin (OPG) was compared between
Ti coins with reference surface 1 comprising lithium, calcium and
magnesium, respectively.
[0197] The amount OPG was determined by DuoSet ELISA human
OPG/TNFRSF11B (R&D Systems, UK). The supernatant from each well
was centrifuged free of cells (5 min in 400 g) and further used for
investigation. Sample OPG concentrations were determined by
correlating the amounts to the standard curve provided by the
manufacturer. The sensitivity of the test (MDD, minimum detectable
dose) was 50 pg/ml. The instructions from the manufacturer were
followed (R&D Systems, UK).
[0198] As is illustrated in FIG. 12, the highest production of OPG
was observed with reference surface 1 comprising lithium after 7
and 14 days of cell culture.
Comparative Example 3
Bone Tissue Response In Vivo
[0199] Integration of implants according to the invention was
tested in a rabbit model. The objective was to qualitatively and
quantitatively study the in vivo bone tissue response of implant
surface modifications according to the invention compared to a
commercially available control implant.
Implants for Removal Torque Study
[0200] Torque fixtures (square headed removal torque design,
3.5.times.8.2 mm) with reference surface 1 comprising lithium were
compared with control torque fixtures (3.5.times.8.2 mm) with the
commercially available Osseospeed.TM. surface.
Implant Insertion
[0201] Twelve mature male New Zealand white rabbits were scheduled
for surgery. Two rabbits died during initial anaesthesia (#9, 10).
The surgery went uneventful. Low speed drilling (1500 rpg for
drilling the holes and 20 rpm for implant insertion) was done with
continuous NaCl cooling. The first drill was a small round burr and
thus used as a marker for the coming larger spiral drills
(altogether 6 drills having diameters in the range of from 1.2 to
3.35 mm).
[0202] Three implants ("square headed removal torque design";
3.5.times.8.2 mm) were inserted in each tuburositas tibia. The
tibia implants were scheduled for removal torque tests.
Removal Torque Results
[0203] After six weeks the study was terminated and the rabbits
were sacrificed. The implants and surrounding tissue were examined.
The tibia rtq implants were easy to locate and all of them showed
signs of periosteal bone tissue up-growth. The biomechanical test
of the implant-bone interface was performed with the removal torque
test (RTQ). The RTQ instrument is an electronic equipment (Detektor
AB, Goteborg, Sweden) involving a strain gauge transducer used for
testing the implant stability (the peak loosening torque in Ncm) in
the bone bed and can thus be regarded as a three dimensional test
roughly reflecting the interfacial shear strength between bone
tissue and the implant (Johansson C. B., Albrektsson T. Clin Oral
implants Res 1991; 2:24-9). A linear increasing torque was applied
on the same axis of the implant until failure of integration was
obtained and the peak value was noted.
[0204] As is illustrated in FIG. 13, the removal torque value for
the implant comprising lithium according to the invention was
improved compared to the commercially available Osseospeed.TM.
surface.
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