Note: Descriptions are shown in the official language in which they were submitted.
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USE OF BIOACTIVE GLASS COMPOSITIONS
TO STIMULATE OSTEOBLAST PRODUCTION
FIELD OF THE INVENTION
The present invention is generally in the area of methods for repair and
reconstruction of bone, cartilage and enhancement of healing of other tissues.
BACKGROUND OF THE INVENTION
Bone is a dense network of collagen protein fibers arranged in layers with
crystals
of hydrated and carbonated calcium phosphate between the fibers, where about
25% of the
weight is calcium. Living cells called osteocytes are arranged in lacunae
throughout the
bone. Very small blood vessels extend throughout the bone and supply the
osteocytes with
oxygen and nutrients. The natural process for repairing bone defects involves
having
osteoclasts remove damaged bone, and then having osteoblast cells lay down new
bone.
The osteoblasts repeatedly form layers, each consisting of a network of
collagen ftbers,
which produce enzymes resulting in calcium and phosphorus deposition as
crystalline
hydroxy carbonate apatite until the defect is repaired.
Relatively small bone defects can be repaired using bone cements, pins, screws
and
other devices for mechanical stabilization. Relatively large defects typically
require that
the missing bone be replaced with a biocompatible material that provides
support and
which can be immobilized. Bone grafts are often necessary when bone fails to
repair itself
or when bone loss occurs through fracture or tumor. Bone grafts have to
provide
mechanical stability and be a source of osteogenesis. Bone grafting is
described, for
example, in Friedlaender, G. E., "Current Concepts Review: Bone Grafts,"
Journal of Bone
and Joint Surgery, 69A(5), 786-790 (1987). Osteoinduction and osteoconduction
are two
mechanisms by which a graft may stimulate the growth of new bone. In
osteoinduction,
inductive signals lead to the phenotypic conversion of progenitor cells to
bone cells. In
osteoconduction, the implant provides a scaffold for bony ingrowth. The bone
remodeling
cycle is a continuous event involving the resorption of pre-existing bone by
osteoclasts and
the formation of new bone by the work of osteoblasts.
Bony defects are commonly treated using grafts of organic and synthetic
construction, typically autografts, allografts, and xenografts. An autograft
is tissue
transplanted from one site to another in the patient. The benefits of using
the patient's
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tissue are that the graft will not evoke a strong immune response and that the
material may
or may not be vascularized, which allows for speedy incorporation. However,
using an
autograft requires a second surgery, which increases the risk of infection and
introduces
additional weakness at the harvest site. Further, bone available for grafting
may be
removed from a limited number of sites, for example the fibula, ribs and iliac
crest. An
allograft is tissue taken from a different organism of the same species, and a
xenograft from
an organism of a different species. The latter types of tissue are readily
available in larger
quantities than autografts, but genetic differences between the donor and
recipient may lead
to graft rejection.
Synthetic materials have also been used, for example titanium and steel
alloys,
particularly those having a porous structure to allow cellular ingrowth to
stabilize the
implant, bone cements, alone or mixed with cells, sterilized bone, and
polymeric or
polymeric/hydroxyapatite implants. All have advantages and disadvantages, yet
none
provides a perfect replacement for the missing bone.
Large defects are particularly difficult to treat. One approach involves using
tissue
engineering to stimulate production of osteoblasts or bone tissue. It would be
advantageous
to provide new compositions and methods for stimulating osteoblast production.
The
present invention provides such compositions and methods.
SUMMARY OF THE INVENTION
Compositions comprising bioactive glass compositions or extracts thereof,
which
include ions in an appropriate concentration and ratio that they enhance
osteoblast
production, and methods of preparation and use thereof, are disclosed.
The compositions can be included in implantable devices that are capable of
inducing tissue formation at the implant site, for example as coatings and/or
matrix
materials. Examples of such devices include prosthetic implants, sutures,
stems, screws,
plates, tubes, and the like.
Aqueous extracts of the bioactive glass compositions, which extracts are
capable of
stimulating osteoblast production, are also disclosed. Such extracts can be
formed by
placing bioactive glass in an aqueous solution, allowing the glass to dissolve
over a suitable
period of time, for example one day or more, and filtering out the undissolved
glass
particles. The solvent can also be evaporated to provide a solid material with
osteoblast-
stimulating properties. Alternatively, the solutions can be prepared by mixing
the correct
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ions in an appropriate concentration rather than by extraction from bioactive
glass.
The compositions can be used, for example, to induce local tissue formation
from a
progenitor cell in a mammal, for accelerating allograft repair in a mammal,
for promoting
in vivo integration of an implantable prosthetic device to enhance the bond
strength
between the prosthesis and the existing target tissue at the joining site, and
for treating
tissue degenerative conditions in a mammal.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Biocompatible compositions and methods for enhancing osteoblast production
using the compositions are disclosed. The compositions include an osteoblast-
stimulating
bioactive glass or extract thereof with a ratio and/or concentration of ions
that stimulates
osteoblast proliferation, differentiation and/or function. A major function of
osteoblasts is
the formation of new bone or other tissues, such as those involved in the
process of
membranous or endochondral bone formation. While not wishing to be bound to a
particular theory, it is believed that exposure of human osteoblast cells to
the ions results in
up-regulation of certain cytokines, proteoglycans and/or other proteins such
as growth
factors that are implicated in the growth, differentiation and control of bone
formation in
humans. Genes whose expression is enhanced by exposure to the bioactive glass
solutions
include c-jun and c-myc genes, which are implicated in the early events of
cell proliferation
and differentiation. In some cases, up-regulation is observed even after 48
hours post-
exposure.
In general, the genes shown to be upregulated by exposure to the bioactive
glass or
bioactive glass extract compositions of the invention are involved in:
a) signaling to produce proteins responsible for cell binding,
b) up-regulation of the osteoblast cell cycle, thus stimulating new cell
development,
c) enhancing collagen synthesis, and
d) controlling apoptosis, thereby increasing the rate of the cell cycle.
Exposure to the compositions also increases expression of insulin-like growth
factor-II (IGF-II), an abundant mitogenic molecule found in bone which
stimulates
chondrocyte activity and osteoblast proliferation and differentiation. It is
believed to
appear earlier in the bone regeneration cycle than bone morphogenic proteins
(BMPs).
The term "biocompatible" refers to a material that does not elicit detrimental
effects
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associated with the body's various protective systems, such as cell and
humoral-associated
immune responses, e.g., inflammatory responses and foreign body fibrotic
responses. The
term biocompatible also implies that no specific undesirable cytotoxic or
systemic effects
are caused by the material when it is implanted into the patient.
The terms "morphogenic activity," "inducing activity" and "tissue inductive
activity" alternatively refer to the ability of an agent to stimulate a target
cell to undergo
one or more cell divisions (proliferation) that can optionally lead to cell
differentiation.
Such target cells are referred to generically herein as progenitor cells. Cell
proliferation is
typically characterized by changes in cell cycle regulation and can be
detected by a number
of means which include measuring DNA synthesis or cellular growth. Early
stages of cell
differentiation are typically characterized by changes in gene expression
patterns relative to
those of the progenitor cell, which can be indicative of a commitment towards
a particular
cell fate or cell type. Later stages of cell differentiation can be
characterized by changes in
gene expression patterns, cell physiology and morphology. Any reproducible
change in
gene expression, cell physiology or morphology can be used to assess the
initiation and
extent of cell differentiation induced by the compositions described herein.
Observed Effect in Cell Culture
The bioactive glass or bioactive glass extract compositions described herein,
when
added to cells in culture, were observed to have the following effects:
The population of cells in primary human osteoblast cultures that are capable
of dividing and proliferating increased;
The population of cells in primary human osteoblast cultures that are not
dividing, proliferating, or differentiating, or producing extra-cellular
matrices
undergo rapid apoptosis;
The cells in primary human osteoblast cultures that are capable of dividing
and
proliferating showed a more rapid differentiation from an osteoblast precursor
towards a mature phenotype characteristic of osteocytes;
Mineralized bone nodules were rapidly formed in primary human osteoblast
' cultures;
The cells in mouse embryonic cell cultures underwent rapid selection and
differentiation into cells of the osteoblast lineage;
Rapid mineralization of the femora was observed in mouse fetal femoras in
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culture, even under micro-gravity conditions where mineralization does not
occur in the absence of the compositions;
Enhanced mineralization of the metatarsals was observed in mouse fetal
metatarsals in culture, even under simulated hyper-gravity conditions; and
A series of genes which influence growth and formation of new bone was up-
regulated in human osteoblast cultures. These genes include IGF-II, IGFBP3,
MMP2, MMP14, TIMP1, TIMP2, procollagen a2, Decorin, c jun, c-myc,
calcium proteinase (calpain) and DAD 1 (defender against cell death). The
most significant up-regulation was observed with IGF-II, MMP2, MMP14,
TIMP1, calpain and DAD 1.
The effect varied depending on the concentration of the ions in solution when
aqueous extracts of bioactive glass were used. For example, for extracts
derived from 4555
Bioglass, when the concentration was about 10 g/1, the effect was optimized,
and either
below 2 g/1 or above 40 g/1 in culture, was not significantly observed. For
extracts derived
from other bioactive glass compositions, the concentrations will be expected
to be different.
An effective amount of bioactive glass or bioactive glass extract 'for
stimulation of
osteoblast production, or osteoblast proliferation, differentiation, function
or a combination
thereof, will be an amount which will provide at least one of the above-listed
effects.
I. Bioactive Glass Compositions
The compositions include osteoblast-stimulating bioactive glass, preferably in
the
form of fibers, particles, preferably non-interlinked particles, extracts
derived from the
bioactive glass, and sols, gels or solids derived from the extracts. The
compositions can
optionally include other therapeutic agents.
As used herein, the terms "bioactive glass" or "biologically active glass"
mean an
inorganic glass material having an oxide of silicon as its major component and
which is
capable of bonding with growing tissue when reacted with physiological fluids.
The term
"osteoblast-stimulating" refers to bioactive glasses and aqueous extracts
thereof with
particular ratios and/or concentrations of ions which stimulate osteoblast
proliferation,
differentiation and/or function.
Bioactive glasses are well known to those skilled in the art, and are
disclosed, for
example, in An Introduction to Bioceramics, L. Hench and J. Wilson, eds. World
Scientific,
New Jersey (1993).
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The glass includes a composition by approximate weight percent of between
about
42 and 52% by weight of silicon dioxide (Si02), between about 1 S and 2S% by
weight of
sodium oxide (NazO), between about 1S and 2S% by weight calcium oxide (Ca0),
and
between about 1 and 9% by weight phosphorus oxide (P205), when the glass is
melt-
S derived. The glass includes between about SS and 80% by weight of silicon
dioxide (SiOz),
between about 0 and 9% by weight of sodium oxide (NazO), between about 10 and
40% by
weight calcium oxide (Ca0), and between about 3 and 8% by weight phosphorus
oxide
(P205), when the glass is sol gel-derived. The oxides can be present as solid
solutions or
mixed oxides, or as mixtures of oxides. The currently most preferred glass is
4SSS
bioglass, which has a composition by weight percentage of approximately 4S%
SiOz,
24.5% CaO, 24.5% Na20 and 6% PZOS.
CaF2, B203, A1203, MgO, AgzO, Zn0 and I~20 can be included in the composition
in addition to silicon, sodium, phosphorus and calcium oxides. The preferred
range for
BZOg 15 between 0 and 10% by weight. The preferred range for Kz0 is between 0
and 8%
1S by weight. The preferred range for Mg0 is between 0 and S% by weight. The
preferred
range for A1203 is between 0 and 1.S% by weight. The preferred range for CaF2
is between
0 and 12.5 % by weight. The preferred range for Ag20 and Zn0 is between 0 and
2% by
weight.
Particulate, non-interlinked bioactive glass is preferred. That is, the glass
is in the
form of small, discrete particles, rather than a fused matrix of particles or
a mesh or fabric
(woven or non-woven) of glass fibers. Note that under some conditions the
discrete
particles of the present invention can tend to cling together because of
electrostatic or other
forces but are still considered to be non-interlinked. Useful ranges of
particle sizes are less
than about 1200 microns, typically between 1 and 1000 microns. For direct
implantation,
2S the particle size range depends on the intended application. In one
embodiment, the size
range of the particles is about 100 to about 800 microns. In a preferred
aspect of the
invention, the size range of the particles is about 300 to about 700 microns.
To produce
extracts, the particle size is preferably less than about 90 microns; more
preferably, less
than about 20 microns; even more preferably, less than about S microns, and
ideally, less
than about 3 microns, as measured by SEM or laser light scattering techniques.
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Highly porous bioactive glass can also be used, particularly in tissue
engineering
applications where the high porosity can be useful in matrix materials for
cell culture.
Highly porous bioactive glass has a relatively fast degradation rate and high
surface area, in
comparison to non-porous bioactive glass compositions. When highly porous
bioactive
glass is used in place or in addition to small particles of bioactive glass,
the pore size is
between about 0 and 500 ~,m, preferably between about 50 and 500 Vim, more
preferably
between 100 and 400 pm. The degree of porosity of the glass is between about 0
and 85 %,
preferably between about 30 and 80 %, and more preferably between about 40 and
60 %.
Porous bioactive glass can be prepared, for example, by incorporating a
teachable
substance into the bioactive glass composition, and leaching the substance out
of the glass.
Suitable teachable substances are well known to those of skill in the art and
include, for
example, sodium chloride and other water-soluble salts. The particle size of
the teachable
substance is roughly the size of the resulting pore. The relative amount and
size of the
teachable substance gives rise to the degree of porosity. Also, as described
herein, porosity
can be achieved using sintering and/or by controlling the treatment cycle of
glass gels to
control the pores and interpores of the material.
The glass composition can be prepared in several ways, to provide melt-derived
glass, sot-gel derived glass, and sintered glass particles. The sintered
particles can be in
sot-gel derived, or pre-reacted melt derived form. Sol-gel derived glass is
generally
prepared by synthesizing an inorganic network by mixing metal alkoxides in
solution,
followed by hydrolysis, gelation, and low temperature (around 200-900 °
C) firing to
produce a glass. Sol-gel derived glasses produced this way are known to have
an initial
high specific surface area compared with either melt-derived glass or porous
melt-derived
glass. Melt derived glass is generally prepared by mixing grains of oxides or
carbonates,
melting and homogenizing the mixtures at high temperatures, typically between
about 1250
and 1400°C. The molten glass can be fritted and milled to produce a
small particulate
material.
The glass composition is preferably melt-derived. In each preparation, it is
preferred to use reagent grade glass and/or chemicals, especially since the
glass and/or
chemicals are used to prepare materials which ultimately can be administered
to a patient.
A. Melt Derived Glass
A melt-derived glass composition can be prepared, for example, by preparing an
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admixture of the individual metal oxides and other components used to prepare
the glass
composition, blending the admixture, melting the admixture, and cooling the
mixture. The
melting temperature is determined in large part by the glass composition, and
ranges, for
example, from about 900-1500°C, preferably between about 1250 and
1450°C. The melt is
preferably mixed, for example, by oxygen bubbling, to ensure a thorough
homogenation of
the individual components.
The mixture can be cooled, for example by casting the molten admixture into a
suitable liquid such as deionized water, to produce a glass frit. Porosity can
be introduced
by grinding the glass into a powder, admixing the powder with a foaming agent,
and hot
pressing the mixture under vacuum and elevated temperature. The particle size
of the glass
powder is between about 2 and 70 Vim, the vacuum is preferably less than 50
MPa, and the
hot pressing is preferably performed at a temperature above 400°C,
preferably between
about 400 and 500°C. Suitable foaming agents include compounds which
evolve carbon
dioxide and/or water at elevated temperatures, for example metal hydroxides,
metal
carbonates, and peroxides such as hydrogen peroxide. Preferred metal
carbonates are
sodium bicarbonate, sodium carbonate and calcium carbonate. The foaming agents
are
preferably added in a range of between about 1-5, more preferably 2-3 percent
by weight of
the glass powder. The preparation of melt-derived porous glass is described,
for example,
in U.S. Patent No. 5,648,301 to Ducheyne and El Ghannam.
B. Sintered Glass Particles
Glass can be sintered using lrnown methodology. In one embodiment, an aqueous
slurry of the glass powder and a foaming agent with a suitable binder, such as
polyvinyl
alcohol, is formed. The slurry is then poured into a mold, allowed to dry, and
sintered at
high temperatures. These temperature can range, depending on the glass
composition and
foaming agent used, between about 450 and 1000°C, more preferably
between about 550
and 800°C.
C. Leaching of the Porous Material
To aid in preparing glass compositions with high porosity, the glass
composition
can include a material which can be preferably leached out of the glass
composition, and in
doing so, provide the composition with high porosity. For example, minute
particles of a
material capable of being dissolved in a suitable solvent, acid or base can be
mixed with or
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9
melted into the glass, and subsequently leached out. The resulting voids have
roughly the
same size as the particle that was leached out. In the case of a material
which is part of a
melt-derived glass composition, the size of the pores and degree of porosity
depends on the
amount of added material relative to the amount of glass. For example, if the
leached
material constituted about 80% of the glass, then the glass would be
approximately 80%
porous when the material was leached out. When leaching the glass composition,
care
should be taken not to leach out those components which add to the bioactivity
of the glass,
i.e., the calcium, silica and phosphorus oxides.
II. Solutions Derived from Bioactive Glass
Osteoblast-stimulating compositions derived from aqueous or other extracts of
bioactive glass, and/or solutions including the same ions at the same
concentration ranges
can be used in the methods described herein. The extracts can be formed by
placing an
osteoblast-stimulating bioactive glass in an aqueous solution, allowing the
glass to dissolve
over a suitable period of time, and filtering out the un-dissolved glass
particles. The
solvent can be evaporated to provide a sol, gel or solid material with
osteoblast-stimulating
properties. The compositions can be used in situations where osteoblast
production is
desired, for example solutions used for cell culture, and buffer solutions.
The extract may be incorporated into hydrogels or other aqueous based
biocompatible carriers for delivery to specific sites in the body. Those of
skill in the art
will appreciate that the molecular weight and/or water content of polymers or
other
materials utilized as carriers may be used to control the rate of release of
the ionic bioactive
glass extracts.
The concentration of ions in aqueous osteoblast-enhancing solutions is as
follows:
Si - 1 ppm to 100 ppm
Ca 10 ppm to 150 ppm
P 5 ppm to 50 ppm.
Typically, the osteoblast-enhancing solutions will also contain sodium ions.
The
amount will depend on the environment in which the solution is used and the
amount of
time of reaction of the initial glass composition.
The preferred range of ions is:
Si 3 ppm to 40 ppm
Ca 60 ppm to 100 ppm
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P 10 ppm to 40 ppm.
Without being bound to a particular theory, it is believed that there is a
complex
relationship between the type of ion being released from the glass, the amount
of that ion,
the rate at which release occurs, the pH of the solution, and the resulting
osteoblast
stimulating response. This effect is observed with respect to the particles of
bioactive glass
themselves and also in the ionic solutions derived from the glass particles.
Accordingly, in
the uses described below, particles of bioactive glass can be used in place of
or in addition
to the solutions derived from the particles.
Solid Compositions
The aqueous solutions can be dried, for example by spray drying or by drying
ih
vacuo, to provide an antibacterial composition. The compositions can be
incorporated into
other solutions used in cell culture or other tissue engineering applications,
such as cell
culture media.
There are many types of cell culture media, each of which are essentially
isotonic
with the cells to be cultured. These include Dulbecco's minimal essential
media, Hank's
balanced salt solution, and others. The compositions described herein can be
added to any
of these solutions to enhance osteoblast proliferation, differentiation and/or
function in the
cell culture media. The cell culture media including the compositions
described herein are
also useful for other cell types, including fibroblasts, chondroblasts and
other cells with a
phenotype similar to osteoblasts.
III. Formulations Includinu Bioactive Glass
The compositions can be in a variety of forms. These include, for example,
solid,
semi-solid and liquid dosage forms such as tablets, pills, powders, liquid
solutions or
suspensions, suppositories, and injectable and infusible solutions. The
preferred form
depends on the intended mode of administration and therapeutic application and
can be
selected by one skilled in the art. Modes of administration can include oral,
parenteral,
subcutaneous, intravenous, intralesional or topical administration, or direct
injection into a
bony defect or an adjacent tissue locus. In most cases, the pharmaceutical
compositions
will be administered in the vicinity of the treatment site in need of tissue
regeneration or
repair.
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The compositions can, for example, be placed into sterile, isotonic
formulations
with or without co-factors which stimulate uptake or stability. Solutions
including the ions
at appropriate concentrations and/or ratios can be lyophilized, stored under
refrigeration
and reconstituted prior to administration with sterile Water-For-Injection
(USP).
The compositions can include conventional pharmaceutically acceptable carriers
well known in the art (see for example Remington's Pharmaceutical Sciences,
16th Edition,
1980, Mac Publishing Company). Such pharmaceutically acceptable carriers can
include
other medicinal agents, carriers, genetic carriers, adjuvants, excipients,
etc., such as human
serum albumin or plasma preparations. The compositions are preferably in the
form of a
unit dose and will usually be administered as a dose regimen that.,depends on
the particular
tissue treatment.
The pharmaceutical compositions can also be administered, for example, in
microspheres, liposomes, other microparticulate delivery systems, polymers or
sustained
release formulations placed in, near, or otherwise in communication with
affected tissues or
the bloodstream bathing those tissues.
Liposomes containing the compositions described herein can be prepared by
well-known methods (See, e.g., DE 3,218,121; Epstein et al., Proc. Natl. Acad.
Sci. U.S.A.,
82, pp. 3688-92 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77, pp.
4030-34
(1980); U.S. Pat. Nos. 4,485,045 and 4,544,545). Ordinarily, the liposomes are
of the small
(about 200-800 Angstroms) unilamellar type in which the lipid content is
greater than about
mol. % cholesterol. The proportion of cholesterol is selected to control the
optimal rate
of release.
Dosing of the compositions can be via a single dose, sequential dosing, or
continuous release.
Other Therapeutic Agents
In addition to the osteoblast-stimulating bioactive glass and/or extracts
thereof, the
formulations can include other therapeutic agents such as antibiotics,
antivirals, healing
promotion agents, anti-inflammatory agents, immunosuppressants, growth
factors, anti-
metabolites, cell adhesion molecules (CAMS), bone morphogenic proteins (BMPs),
vascularizing agents, anti-coagulants, and topical anesthetics/analgesics.
Suitable growth factors include platelet-derived growth factor (PDGF),
vascular
endothelial growth factor (VEGF), epidermal growth factor (EGF), basic
~broblast growth
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12
factor (FGF), insulin-like growth factors (IGF-I and IGF-II), endothelial
derived growth
supplement (EDGS), keratinocyte growth factor (I~GF), osteogenin, skeletal
growth factor
(SGF), osteoblast-derived(BDGFs), retinoids, growth hormone (GH), bone
morphogenic
proteins (BMPs), tissue growth factor-beta (TGF-(3), CBFA-1 and transferrin.
IV. Devices
Devices can be prepared which include the compositions described herein, for
example, dispersed in an implantable or extracorporeal biocompatible carrier
material that
functions as a suitable delivery or support system for the composition.
Suitable examples
of sustained release carriers include semi-permeable polymer matrices in the
form of
shaped articles such as suppositories or capsules. Implantable or
microcapsular sustained
release matrices include polylactides (U.S. Pat. No. 3,773,319; EP 058 481),
copolymers of
L-glutamic acid and ethyl-L-glutamate (Sidman et al., Biopolymers, 22, pp. 547-
56 (1985));
poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al., J.
BionZed.
Mate. Res., 15, pp. 167-277 (1981); Langer, Chem. Tech., 12, pp. 98-105
(1982)).
In one embodiment, the carrier includes a biocompatible matrix made up of
particles or porous materials. The pores are preferably of a dimension to
permit progenitor
cell migration and subsequent differentiation and proliferation. Various
matrices known in
the art can be employed (see, e.g., U.S. Pat. Nos. 4,975,526; 5,162,114;
5,171,574 and PCT
WO 91/18558).
The matrix can be formed, for example, by close packing particulate material
into a
shape spanning the particular tissue or bone defect to be treated.
Alternatively, a
biocompatible, preferably biodegradable material can be structured to serve as
a temporary
scaffold and substrate for recruiting migratory progenitor cells, and as a
base for their
subsequent anchoring and proliferation.
Useful matrix materials include, for example, collagen; hydrogels;
homopolymers
or copolymers of glycolic acid, lactic acid, and butyric acid, including
derivatives thereof;
and ceramics, such as hydroxyapatite, tricalcium phosphate and other calcium
phosphates.
The bioactive glass or bioactive glass extracts of the invention may be used
with,
incorporated into or encapsulated within matrix carrier materials, such as
hydrogels, to
enable the release of the ions from the glass or extract in a controlled
fashion. This release
of the ions preferably will be controlled over time and may be a sustained
release
formulation. Various therapeutic agents, as described above, can be adsorbed
onto or
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13
dispersed within the Garner material, and will also be released over time at
the implantation
site as the matrix material is slowly absorbed.
Implantable prosthetic devices including the compositions described herein can
also be prepared. Such prosthetic implant can be selected for a particular
treatment by the
skilled practitioner, and can include materials such as metals and/or
ceramics. The
compositions can be moldable or machinable.
Examples of prosthetic devices include hip devices, screws, rods, cages for
spine
fusion, stems, plates, sheets, pins, valves, sutures, tubes and the like.
In one embodiment, the composition is disposed as a coating on prosthetic
implants. For example, a surface region that is implantable adjacent to a
target tissue in a
mammal, preferably, a human, can be coated. The coating is present in an
amount
sufficient to promote enhanced tissue growth into the surface of the implant.
The amount
of the composition sufficient to promote enhanced tissue growth can be
determined
empirically by those of skill in the art using appropriate bioassays.
Preferably, animal
studies are performed to optimize the concentration of the composition
components before
a similar prosthetic device is used in the human patient. Such prosthetic
devices will be
useful for repairing orthopedic defects, injuries or anomalies in the treated
mammal.
In vivo integration of implantable prosthetic devices into target tissue can
be
performed, for example, by providing the composition on a surface of a
prosthetic device,
and implanting the device in a mammal at a locus where the target tissue and
the surface of
the prosthetic device are maintained at least partially in contact for a time
sufficient to
permit enhanced tissue growth between the target tissue and the device.
V. Methods for Using the Compositions
The compositions and devices disclosed herein will permit the physician to
treat a
variety of tissue injuries, tissue degenerative or disease conditions and
disorders that can be
ameliorated or remedied by localized, stimulated tissue regeneration or
repair. For
example, the compositions and devices of the invention may be used to treat
osteoblast-
related tissue degenerative conditions.
The devices can be used to induce local tissue formation from a progenitor
cell in a
mammal by implanting the device at a locus accessible to at least one
progenitor cell of the
mammal. The devices can be used alone or in combination with other therapies
for tissue
repair and regeneration.
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14
The devices can also be implanted in or surrounding a joint for use in
cartilage and
soft tissue repair, or in or surrounding nervous system-associated tissue for
use in neural
regeneration and repair.
The tissue specificity of the particular composition will determine the cell
types or
tissues that will be amenable to such treatments and can be selected by one
skilled in the
art. The ability to enhance tissue regeneration by administering the
compositions described
herein is thus not believed to be limited to any particular cell-type or
tissue. The
compositions and methods disclosed herein can be practiced to enhance new
tissue
inductive functions as they are discovered in the future.
The compositions and devices will permit the physician to obtain predictable
bone
and/or cartilage formation. The compositions and devices can be used to treat
more
efficiently and/or effectively all of the injuries, anomalies and disorders
that have been
described in the prior art of osteogenic devices. These include, for example,
forming local
bone in fractures, non-union fractures, fusions and bony voids such as those
created in
tumor resections or those resulting from cysts; treating acquired and
congenital craniofacial
and other skeletal or dental anomalies (see e.g., Glowacki et al., Laneet, l,
pp. 959-63
(1981)); performing dental and periodontal reconstructions where lost bone
replacement or
bone augmentation is required such as in a jaw bone; and supplementing
alveolar bone loss
resulting from periodontal disease to delay or prevent tooth loss (see e.g.,
Sigurdsson et al.,
J. PeYiodontol., 66, pp. 511-21 (1995)).
In addition to the osteoblast-stimulating bioactive glass and/or extracts
thereof, the
devices can also include a matrix including allogeneic bone. Such devices can
also be
implanted at a site in need of bone replacement to accelerate allograft repair
and
incorporation in a mammal.
The devices can also be used in cartilage repair, for example, following joint
injury
or in osteoarthritis treatment. The ability to enhance cartilage-inducing
activity by
administering the compositions described herein can permit faster or more
extensive tissue
repair and replacement.
The compositions and devices described herein will be useful in treating
certain
congenital diseases and developmental abnormalities of cartilage, bone and
other tissues.
Developmental abnormalities of the bone can affect isolated or multiple
regions of the
skeleton or of a particular supportive or connective tissue type. These
abnormalities often
require complicated bone transplantation procedures and orthopedic devices.
The tissue
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repair and regeneration required after such procedures can occur more quickly
and
completely using the bioactive glasses as described herein.
Examples of heritable conditions, including congenital bone diseases, for
which use
of the morphogenic compositions and devices described herein will be useful
include
5 osteogenesis imperfecta, the Hurler and Marfan syndromes, and several
disorders of
epiphyseal and metaphyseal growth centers such as is presented in
hypophosphatasia, a
deficiency in alkaline phosphatase enzymatic activity.
Inflammatory joint diseases can also benefit from the compositions and devices
described herein. These include infectious, non-infectious, rheumatoid and
psoriatic
10 arthritis, bursitis, ulcerative colitis, regional enteritis, Whipple's
disease, and ankylosing
spondylitis (also called Marie Strumpell or Bechterew's disease); the so-
called "collagen
diseases" such as systemic lupus erythematosus (SLE), progressive systemic
sclerosis
(scleroderma), polymyositis (dermatomyositis), necrotizing vasculitides,
Sjogren's
syndrome (sicca syndrome), rheumatic fever, amyloidosis, thrombotic
thrombocytopenic
15 purpura and relapsing polychondritis. Heritable disorders of connective
tissue include
Marfan's syndrome, homocystinuria, Ehlers-Danlos syndrome, osteogenesis
imperfecta,
alkaptonuria, pseudoxanthoma elasticum, cutis laxa, Hurler's syndrome, and
myositis
ossiftcans progressiva.
In one embodiment, the compounds are used to fill voids, including voids
created
during medical procedures. For example, during a root canal operation, the
hollowed-out
tooth can be filled with a composition including bioactive glass. This will
help prevent
bacterial infection until the tooth is ultimately ftlled. Also, bioactive
glass-containing
compositions can be used to fill the pockets that can develop between the
teeth and gums.
The compositions can also be used to ftll voids, for example those present in
aneurysms,
and those formed surgically, such as removal of a spleen, ovary, gall bladder,
or tumor.
VI. Bioassays
The utility of the compositions at enhancing bone and/or tissue growth can be
demonstrated using conventional bioassays. Examples of useful bioassays are
described in
U.S. Pat. No. 5,344,654 to Rueger et al.
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Feline and Rabbit Models
The feline and rabbit as established large animal efficacy models for
osteogenic
device testing have been described in detail (See, for example, U.S. Pat. No.
5,354,557
Oppermann et al.).
In the feline model, a femoral osteotomy defect is surgically prepared.
Without
further intervention, the simulated fracture defect would consistently
progress to non-union.
The effects of osteogenic compositions and devices implanted into the created
bone defects
can be evaluated by the following study protocol.
Briefly, the procedure is as follows: Sixteen adult cats each weighing less
than 10
Ibs. undergo unilateral preparation of a 1 cm bone defect in the right femur
through a lateral
surgical approach. In other experiments, a 2 cm bone defect can be created.
The femur is
immediately internally fixed by lateral placement of an 8-hole plate to
preserve the exact
dimensions of the defect.
Three different types of materials can be implanted in the surgically created
cat
femoral defects: group I is a negative control group which undergoes the same
plate
fixation with implants of 4M guanidine-HCl-treated (inactivated) cat
demineralized bone
matrix powder (GuHCI-DBM) (360 mg); group II is a positive control group
implanted
with biologically active demineralized bone matrix powder (DBM) (360 mg); and
groups
III and IV undergo a procedure identical to groups I-II, with the addition of
the
compositions to be evaluated.
All animals are allowed to ambulate ad libiturn within their cages post-
operatively.
All cats are injected with tetracycline (25 mg/kg subcutaneously (SQ) each
week for four
weeks) for bone labeling. All but four group III and four group IV animals are
sacrificed
four months after femoral osteotomy.
In vivo radiomorphometric studies are carried out immediately post-op at 4, 8,
12
and 16 weeks by taking a standardized X-ray of the lightly-anesthetized animal
positioned
in a cushioned X-ray jig designed to consistently produce a true anterio-
posterior view of
the femur and the osteotomy site. All X-rays are taken in exactly the same
fashion and in
exactly the same position on each animal. Bone repair is calculated as a
function of
mineralization by means of random point analysis. A final specimen
radiographic study of
the excised bone is taken in two planes after sacrifice.
At 16 weeks, the percentage of groups III and IV femurs that are united, and
the
average percent bone defect regeneration in groups I-IV are compared. The
group I
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GuHCI-DMB negative-control implants should generally exhibit no bone growth at
four
weeks, less than 10% at eight and 12 weeks, and about 16% (+/-10%) at 16
weeks. The
group II DMB positive-control implants should generally exhibit about 15-20%
repair at
four weeks, 35% at eight weeks, 50% (+/-10%) at 12 weeks and 70% (+/-12%) by
16
weeks.
Excised test and normal femurs can be immediately studied by bone
densitometry,
or wrapped in two layers of saline-soaked towels, placed into sealed plastic
bags, and
stored at -20 °C. until further study. Bone repair strength, load-to-
failure, and
work-to-failure are tested by loading to failure on a specially designed steel
4-point
bending jig attached to an Instron testing machine to quantitate bone
strength, stiffness,
energy absorbed and deformation to failure. The study of test femurs and
normal femurs
yields the bone strength (load) in pounds and work-to-failure in joules.
Normal femurs
exhibit a strength of 96 (+/-12) pounds.
Following biomechanical testing, the bones are immediately sliced into two
longitudinal sections at the defect site, weighed, and the volume measured.
One-half is
fixed for standard calcified bone histomorphometrics with fluorescent stain
incorporation
evaluation, and one-half is fixed for decalcified hemotoxylin/eosin stain
histology
preparation.
Selected specimens from the bone repair site are homogenized in cold 0.15 M
NaCI, 3 mM NaHC03, pH 9.0 by a Spex freezer mill. The alkaline phosphatase
activity of
the supernatant and total calcium content of the acid soluble fraction of
sediment are then
determined.
Rabbit Model Bioassay for Bone Repair
This assay is described in detail in U.S. Pat. No. 5,354,557 to Oppermann et
al. and
Cook et al., J. Bone and Joint Surgery, 76-A, pp. 827-38 (1994). Ulnar non-
union defects
of 1.5 cm are created in mature (less than 10 lbs) New Zealand White rabbits
with
epiphyseal closure documented by X-ray. The experiment can include
implantation of
devices into at least eight rabbits per group as follows: group I negative
control implants of
4M guanidine-HCl-treated (inactivated) demineralized bone matrix powder
(GuHCl-DBM); group II positive control implants with biologically active
demineralized
bone matrix powder (DBM); group III implants with osteogenic protein alone;
group IV
implants with osteogenic protein/MPSF (morphogenic protein stimulatory factor)
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18
combinations, and group V controls receiving no implant. Ulnae defects are
followed for
the full course of the eight week study in each group of rabbits.
In another experiment, the marrow cavity of the 1.5 cm ulnar defect is packed
with
activated osteogenic protein in rabbit bone powder in the presence or absence
of a MPSF.
The bones are allografted in an intercalary fashion. Negative control ulnae
are not healed
by eight weeks and reveal the classic "ivory" appearance.
Tendon/ligament-like tissue formation bioass~
A modified version of the Sampath and Reddi rat ectopic implant assay (see
above)
is disclosed in PCT WO 95116035. The modified assay monitors tendon and
ligament-like
tissue formation. This tendon/ligament-like tissue assay can be used to
identify
compositions that stimulate tendon/ligament-like tissue formation in a
particular treatment
site. The assay can also be used to optimize concentrations and treatment
schedules for
therapeutic tissue repair regimens.
It should be understood that the above experimental procedure can be modified
within the skill of the art in a number of ways to be useful in determining
whether a device
is capable of inducing tendon and/or ligament-like tissue in vivo. It can be
used to test
various ion concentrations and/or ratios, and to produce an in vivo dose
response curve
useful in determining effective relative concentrations and/or ratios of ions
in the bioactive
glasses or extracts thereof.
Histological evaluation
Histological sectioning and staining is preferred to determine the extent of
osteogenesis in implants. Implants are fixed in Bouins Solution, embedded in
paraffin, and
cut into 6-S ~m sections. Staining with toluidine blue or hemotoxylin/eosin
demonstrates
clearly the ultimate development of endochondral bone. Twelve-day implants are
usually
sufficient to determine whether the implants contain newly-induced bone.
Biological markers
Alkaline phosphatase (AP)activity can be used as a marker for osteogenesis.
The
enzyme activity can be determined spectrophotometrically after homogenization
of the
implant. The activity peaks at 9-10 days in vivo and thereafter slowly
declines. Implants
showing no bone development by histology have little or no alkaline
phosphatase activity
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under these assay conditions. The assay is useful for quantification and
obtaining an
estimate of bone formation quickly after the implants are removed from the
rat.
Alternatively, the amount of bone formation can be determined by measuring the
calcium
content of the implant.
Gene expression patterns that correlate with endochondral bone or other types
of
tissue formation can also be monitored by quantitating mltNA levels using
procedures
known to those of skill in the art such as Northern Blot analysis. Such
developmental gene
expression markers can be used to determine progression through tissue
differentiation
pathways after osteogenic protein/MPSF treatments. These markers include
osteoblastic-related matrix proteins such as procollagen az(I), procollagen a,
(I), procollagen a,
(III), osteonectin, osteopontin, biglycan, and alkaline phosphatase for bone
regeneration
(see e.g., Suva et al., J. Bone Miner. Res., 8, pp. 379-88 (1993); Benayahu et
al., J. Cell.
Biochern., 56, pp. 62-73 (1994)).
The procedures described above can be used to assess the ability of one or
more of
the compositions described herein to enhance bone and/or cartilage
regeneration and repair
in vivo. It is anticipated that the efficacy of any of the compositions
described herein can
be characterized using these assays. Various compositions, dose-response
curves,
naturally-derived or synthetic matrices, and any other desired variations on
the device
components can be tested using the procedures essentially as described.
The following are examples which illustrate the compositions and devices
described herein, and methods used to characterize them. These examples should
not be
construed as limiting; the examples are included for purposes of illustration
and the present
invention is limited only by the claims.
Example 1: The effect of the ionic dissolution products of Bioglass D 4555 on
human
primary osteoblasts
The use of biomaterial resorption as a means to deliver morphogenic stimuli in
cells and tissues was evaluated. Specifically, the effect of the ionic
dissolution products of
Bioglass D 4555 on human primary osteoblasts in vitro was evaluated. Bioglass
4555 is a
bioactive glass ceramic material which resorbs initially by selective leaching
of at least
silicon, calcium and phosphorus ions followed by network dissolution mediated
by surface
re-polymerization.
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The ionic dissolution products of Bioglass 4555 stimulate gene transcription
in
human primary osteoblasts, as demonstrated using cDNA micro-array and real
time PCR
methodologies. The ionic dissolution products of Bioglass 4555 can increase
IGF-II
availability in cells and tissues in two ways: i) by inducing the
transcription of the growth
5 factor and its carrier protein and ii) by regulating the dissociation of
this factor from its
binding protein resulting in an increase of free-active IGF-1 l, as determined
by EIA. Free
IGF-II increases the cell proliferation observed in cultures stimulated with
the ionic
dissolution products of Bioglass 4555. The data demonstrate that the
biomaterials
described herein are useful not only for structural support, but also, through
their
10 resorption, for stimulating the intrinsic cellular pathways for bone
growth, repair and
regeneration.
Materials and methods
Cell culture and stimulation.
15 Osteoblasts were isolated from trabecular bone of femoral heads taken
during total
hip arthroplasty using the method described by Beresford et al (Beresford et
al., Metab.
Bone Dis. and Rel. Res., 5:229-234 (1984)). Cultures were grown in DMEM
(Dulbecco's
modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS), 2 mM
L-
glutamine, 50 U/ml penicillin G, 50 pg/ml streptomycin B and 0.3 pg/ml
amphotericin B
20 (complete medium) at 37°C, in 95% air humidity and 5% CO2.
A solution containing the ionic dissolution products of Bioglass 4555 was
prepared
by incubating 1 g of Bioglass 4555 particulate (710-300 pm in diameter, US
Biomaterials
Corp, USA) in 100 ml DMEM for 24 hours at 37°C. The particulates were
removed by
filtration through a 0.20 ~.m filter (Sartorius, UK) and the collected medium
was
supplemented as described above for the complete medium. The elemental content
of this
solution in calcium (Ca), silicon (Si), phosphorus (P) and sodium (Na) ions
was determined
by ICP analysis.
Human primary osteoblast cells at passages 2-3 Were used. Cultures at
approximately 75% confluence were stimulated with the ionic dissolution
products of
Bioglass. Non-stimulated cells were cultured in complete DMEM. After 48 hours
the cells
were released by trypsin, centrifuged and snap frozen in liquid N2.
RNA Extraction
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Total RNA was extracted using a phenol:chloroform method (Clontech
Laboratories, Inc., Palo Alto, USA), and precipitated with isopropanol by
15000 g
centrifugation at 4°C. The RNA pellet was washed with 80% ethanol, re-
suspended in
diethylpyrocarbonate-treated water. To remove genomic DNA, the RNA samples
were
then treated with DNase (0.10 units/~.1 of DNase 1, in DNAse I buffer,
Clontech
Laboratories, Inc., Palo Alto, USA). The concentration and purity of total RNA
in each
sample was determined by light absorbance at 260 nm and RNA integrity was
assessed by
electrophoresis on a denaturing agarose/formaldehyde/EtBr gel to verify that
the RNA was
intact.
Analysis of gene expression using cDNA microarrays
Gene expression analysis in four different donor primary osteoblast cell lines
was
performed using the ATLAS Platform (Atlas 1.2 Human array, Clontech
Laboratories, Inc.,
Palo Alto, USA) which allows the simultaneous screening of 1172 genes.
Briefly, gene
specific primers were used for cDNA synthesis using Superscript II RNase H-
(Life
Technologies, UI~) in the presence of [32-P]-dATP (Amersham, UK). Labeled cDNA
was
purified from unincorporated nucleotides by gel filtration using CHROMA SPIN-
200
columns. The incorporation of 32P in the probe was determined by scintillation
counting.
Each filter was hybridized with equal amount of radioactive probe.
Prehybridization and
hybridization was done at 68°C for 30 minutes and 16 hours
respectively. Membranes
were washed according to the manufacturers protocol. Arrays were scanned using
a
Molecular Dynamics 445 Sl PhosphorImager. Data analysis was performed using
the
Atlasimage 1.1 software package (Clontech Laboratories, Inc., Palo Alto, USA).
Differential gene expression between stimulated and un-stimulated cells was
normalized
towards the expression of the 'housekeeping genes' 40S Ribosomal Protein S9
and 23 KDa
highly basic protein.
Tlerification of c-DNA microarray data with Real Time Quantitative PCR
IGF-II that has been identified by the microarray analysis was selected for
further
analysis. RT reactions were carried out for each RNA sample using the
Thermoscript RT-
PCR System (Life Technologies, UI~), according to manufacturer's protocol.
Each reaction
tube contained 1 ~,g of DNAse free total RNA in a total volume of 20 ~,1
containing Ix
cDNA Synthesis Buffer, SmM DTT, 40 U RNASEOUT, ImM dNTP Mix, 15U
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THERMOSCRIPT RT and 2.5 ~M oligo (dT),2-,8 primer. RT reaction was carried out
at
50 ° C for 60 min and terminated by incubating at 85 ° C for 5
min. Finally 2U of RNase H
was added to each reaction and the reaction mixture was incubated for a
further 20 min. at
37°C.
PCR primers and TaqMan probes for IGF-II were designed using Primer Express
1.0 Software program (PE Biosystems, UI~). The human IGF-II cDNA sequence was
obtained from GenBank (accession number 577035). The following forward and
reverse
primers were used 5'-GTGCTACCCCCGCCAAGT-3' (located on exon four, anneals
between residues 584 and 601) and 5'-CTGCTTCCAGGTGTCATATTGGA-3' (located on
exon 5, anneals between residues 696 and 674). The TaqMan probe sequence was 5-
CTCCGACCGTGCTTCCGGACAACT-3' (spans exon 4-exon 5 boundary, anneals
between residues 623 and 646) and was labeled with the reporter fluorescent
dye FAM (6-
carboxyfluorescein), at the 5' end and the fluorescent dye quencher TAMRA (6-
carboxy-
tetramethyl-rhodamine) at the 3' end.
0.5 ~1 of each reaction mixture was subjected to PCR in a total volume of 25
~1
containing lx TaqMan Universal Master Mix (PE Biosystems, UK), 300 nM forward
primer, 300 nM reverse primer and 50 nM probe, TaqMan Ix 18s ribosomal RNA
endogenous control reagent (VIC fluorescent labeled probe and appropriate
primers) was
added in each reaction tube and served as internal amplification control. Each
sample was
run in quadruplicate. DNA amplification was carried out on the PE-ABI 7700
sequence
detection system for the test samples, standards and no template controls
using the
sequence detector V 1.6 program. Cycling parameters, were: 50°C for
Smin, 95°C for 10
min followed by 40 cycles of a two-stage temperature profile of 95°C
for 15s and 60°C for
1 min. Data points collected following primer extension were analyzed at the
end of
thermal cycling. A threshold value was determined as 10 S.D. above the mean of
the
background fluorescence emission for all wells between cycles 1 and 15. The
cycle
number at which the fluorescence signal from a positive sample crosses this
threshold was
recorded.
Normalisation of data
Serial dilutions of human primary osteoblast cDNA were analyzed for each
target,
IGF-II and IBS, and threshold Cycle (CT were plotted versus the log of the
initial amount of
cDNA to give a standard curve. CTS for IGF-II and 18S RNA were adjusted using
the
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23
appropriate standard curves. Then IGF-II adjusted CT was normalized to I8S
adjusted CT to
minimize variability in the results due to differences in the RT efficiency
and RNA
integrity among test samples.
Free IGF II Elisa.
Cells were plated on a 24 well plate at a seeding density of 50000 cell/well
and
allowed to attach. Seven different donor osteoblast cell lines were used in
the experiment
(n=7). Two days following seeding cells were stimulated with ionic dissolution
products of
Bioglass~ 4555 and control medium, which were not supplemented with FCS. Free
IGF-II
was assayed in the supernatant of stimulated and non-stimulated cells after
two days in
culture using an IGF-II ELISA Kit (Diagnostic Systems Laboratories, Inc,
Webster, USA)
following the manufacturer's protocol. All samples were assayed in duplicate
and free IGF-
II levels were referred to total protein concentration. Protein concentration
of the cell
lysates was assayed by the Bradford dye binding method using bovine serum
albumin as a
standard (Bradford et al., Anal. Biochem., 72:248-254 (197.6)).
Evaluation of cell proliferation
Cells were plated on a 24 well plate at a seeding density of 50000 cell/well
and
allowed to attach. Five different donor primary osteoblast cell lines were
used in the
experiment (n=5). Two days following seeding cells were stimulated with ionic
dissolution
products of Bioglass~ 4555 and control medium. After four days in culture the
cells were
released by trypsin and counted using a hemocytometer.
Results
ICP analysis
Analysis of the ionic composition of the two solutions used by ICP revealed an
increase in concentration of Ca and most notably Si in the DMEM solution
containing the
ionic dissolution products of Bioglass 4555, relative to control. 'These ions,
along with P
and Na, are constitutive elements of Bioglass 4555 and their reaction kinetics
in
physiological solutions are well characterized chemically. However, their
biological
properties have not been described.
Gene Profiling using cDNA rnicroarrays
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Microarray analysis of gene expression on four different donor cell lines
revealed a
similar pattern of gene expression. Approximately 5-7% of genes represented on
the Atlas
human 1.2 arrays were differentially expressed. These included insulin like
growth factor
II, and its binding protein IGFBP-3. Gene transcription of both these
molecules was
induced. Also induced were proteases (MMP-2 and cathepsin-D) that have been
shown to
cleave IGF-II from their binding proteins and release the active form of the
molecule.
MMP-14, a previously non-described IGFBP cleaving protease, shows a similar
pattern of
induction suggesting possible involvement in the process. Steady state mRNA
transcripts
for the IGF-II receptor was relatively unaffected by the stimulus. The
analysis identified
60 mRNA species that were upregulated greater than twofold in the treated
cultures
compared to the untreated control (Table 1). Only five genes were identified
as down-
regulated, including E-16 amino acid transporter, c jun terminal kinase 2,
polycystin
precursor, Sp2 protein and proteasome inhibitor HP131 subunit.
TABLE I
List of Genes Up-Regulated or Down-Regulated Greater Than Twofold in Human
Osteoblasts Treated with the Ionic Products of Bioactive Glass Dissolution
GeneBank
Accession No. Protein/Gene Ratio Function
M59040 CD44 antigen hematopoietic 7 Cell surface
form precursor receptor
U12779 MAP kinase-activated protein 6 Signal transduction
kinase 2
(MAPI~AAP kinase 2)
X06256 Integrin beta 1; fibronectin 6 Cell surface
receptor beta receptor
subunit
AF04010 RCL growth-related c-myc-responsive5 Growth related
5 gene gene
D15057 Defender against cell death 4.5 Apoptosis
1, (DAD-1)
Y00371 Heat shock cognate 71-kDa protein4.5 Heat shock
protein
X04106 Calpain; calcium-dependent 4.1 Apoptosis
protease small
subunit
X59798 G1/S-specific cyclin D1 4 Cell cycle
regulator
D11428 Peripheral myelin protein 22 4 Cell surface
antigen
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M34079 26S protease regulatory subunit4 Transcription
6A; TAT- factor
binding proteinl
D26512 Matrix metalloproteinase 14 3.6Matrix component
precursor
(NIIVIP 14)
J03075 Protein kinase C substrate 3.5Signal transduction
80-kDa protein
heavy chain
U09579 Cyclin-dependent kinase inhibitor3.4Cell cycle regulator
1
(CDKN1A)
5 L19185 Natural killer cell enhancing 3.3Antioxidant
factor AFB)
M2964S Insulin-like growth factor 3.2Growth factor
II (IGF2)
L11285 Dual specificity mitogen-activated3 Signal transduction
protein
kinase kinase 2
M13194 DNA excision repair protein 3 DNA repair
ERCC1
U07418 Mutt protein homolog 3 DNA repair
10 X69391 60S ribosomal protein L6 3 Transcription
M13667 Major prion protein precursor 3 Cell surface
antigen
M37722 N-sam; fibroblast growth factor3 Cell surface
receptorl receptor
precursor
X79389 Glutathione S-transferase T1 3 Enzyme
L42379 Bone-derived growth factor 3 Growth factor
I (BPGFI)
15 K00065 Cytosolic superoxide dismutase3 Enzyme
1 (SODI)
M26880 Ubiquitin 2.9Enzyme
M17733 Thymosin beta 4; FX 2.9Nuclear protein
J03210 Matrix metalloproteinase 2 2.7Matrix Component
(MMP2)
M37435 Macrophage-specific colony 2.6Growth factor
stimulating
factor (MCSF)
20 M92843 Tristetraproline 2.5Transcription
factor
D90209 CAMP-dependent transcription 2.3Transcription
factor ATP-4 factor
X69550 rho GDP dissociation-inhibitor2.3Signal transduction
1
M23619 High mobility group protein 2.3Nuclear protein
(HMG-I)
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26 -
X15480 Glutathione S-transferase pi 2.3 Enzyme
X03124 Metalloproteinase inhibitor 2.2 Matrix component
1 precursor
(TINIP 1 )
M14219 Decorin; bone proteoglycan 2.2 Matrix component
II precursor
J05594 TM'-2 2.1 Matrix component
M11233 Cathepsin D precursor 2 Enzyme
X60188 Extracellular signal-regulated2 Signal transduction
kinase 1
(ERKl)
M77234 fte-1 2 Transcription
factor
AF06051 Cyclin K 2 Cell cycle
5 regulator
M36340 ADP-ribosylation factor 1 2 Signal transduction
L35253 Mitogen-activated protein kinase2 Signal transduction
p38 (MAP
kinase p38)
M36429 Guanine nucleotide-binding 2 signal transduction
protein -G-ilG-
s/G-t beta subunit 2
U32944 Cytoplasmic dynein light chain2 Translocation
1
L07541 Replication factor C 38-kDa 2 DNA synthesis
subunit
J00123 Proenkephalin A precursor 2 Cell surface
receptor
M65212 Membrane-bound & soluble catechol-O-2 Enzyme
methyltransferase
U04847 Inil 2 Transcription
factor
L31881 Nuclear factor 1 (NP1) 2 Transcription
factor
M30257 Vascular cell adhesion protein2 Cell adhesion
1 precursor
(V-CAM 1)
D28468 DNA-binding protein TAXREB302 2 Transcription
factor
M62831 Transcription factor ETR101 2 Transcription
factor
J03746 Microsomal glutathione S-transferase2 Enzyme
12
X06985 Herne oxygenase 1 (HO1) 2 Enzyme
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M35977 Vascular endothelial growth 2 Growth factor
factor precursor
(VEGF)
M36717 Ribonuclease/angiogenin inhibitor2 Nuclear protein
(RAI)
D16431 Hepatoma-derived growth factor2 Growth factor
(HDGF)
K03515 Neuroleukin (NLK) 2 Enzyme
M24545 Monocyte chemotactic protein 2 Cytokine
1 precursor
(MCP1)
X04602 Interleukin-6 precursor (1L-6)2 Cytokine
AF07786 E16 amino acid transporter .5 Transporter
6
L31951 a jun N-terminal kinase 2 (JNK2).5 Signal transduction
U24497 Polycystin precursor .5 Cell adhesion
M97190 Sp2 protein .5 Transcription
factor
D88378 Proteasome inhibitor HPI31 .4 Enzyme
subunit
CoYroboration of results with Taqman real time PCR
Taqman real time PCR was used to confirm induction of 1GF-II mRNA expression
demonstrated by eDNA microarray analysis. Expression and induction of IGF-II
followed
the same pattern in all four donor osteoblast cell lines examined.
Free IGF II ELISA
Free IGF-II represents the fraction of the molecule, which is not bound to IGF
binding proteins (IGFBPs) and hence represents the active form of IGFII. The
ionic
dissolution products of Bioglass 4555 were shown to statistically increase the
concentration
of free IGF-II by approximately 70%.
Evaluation of cell pYOliferatioh
Osteoblast proliferation was increased 50.2% (P<0.001) over control, following
four days of stimulation with the ionic dissolution products of Bioglass 4555.
The
stimulatory effect on cell proliferation observed is believed to be mediated
by IGF-II,
which has been described as a potent mitogenic, growth factor for osteoblasts.
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Effects of stimulation of cells by ionic dissolution products
Chemical substances released by the bioactive glass substrate are believed to
account for the observed changes in cellular performance. Bioglass 4555
resorbs initially
by selective leaching of Si, Ca, and P ions followed by network dissolution
mediated by
surface re-polymerization.
Using cDNA microarray methodology, the data show that human primary
osteoblast transcription is directly regulated by the ionic dissolution
products of Bioglass
4555. Among the genes which were found to be up-regulated in human primary
osteoblasts were IGF-II and to a lesser extent its carrier protein IGFBP-3.
IGF-II is an anabolic peptide of the insulin family and constitutes the most
abundant growth factor in bone (Mohan et al., 1988, Bautista et al., 1990). It
is produced
locally by bone cells and is considered to exert mostly paracrine or autocrine
effects.
Nonetheless, differences in IGF-II expression occur and can significantly
impact bone cell
function in various physiological and pathological conditions. In vitro
studies using
osteoblasts of various animal sources have shown that IGF-II is a potent
inducer of
osteoblast proliferation and collagen synthesis.
The majority of IGF-II in vivo is found bound to IGE binding proteins
(IGFBPs).
The latter can inhibit or potentiate its biological activity, form storage
complexes with IGFs
or stabilize IGFs in the circulation for slow release into the peripheral
tissues. Therefore
IGF-II activity appears to be influenced not only by the level of expression
of IGF-Il
polypeptide but also by the type and concentrations of IGFBPs present locally.
Thus
changes in IGFBPs expression by bone cells can well contribute to the
effectiveness of
IGF-II in the tissue.
The induction of IGF-II m-RNA expression represents a true difference in IGF-
II
protein synthesis and IGF-II availability. IGF-Il bioavailability at the local
level is
regulated through IGFBPs limited proteolysis by several proteases resulting in
IGF-II
release in its free 'active' form. These include members of the
metalloproteinase family,
such as M1V11' I and 2 and cathepsin-D (Conover et al., 1994), some of which
were found to
be transcriptionally induced in the system described in this example, This
effect was
correlated with a statistically significant increase of free-active IGF-II in
cells stimulated
with the ionic dissolution products of Bioglass 4555.
The ionic dissolution products of Bioglass 4555 can increase the availability
of
IGF-II in cells and tissues in two ways, (i) by inducing the transcription of
the growth
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factor and its carrier protein and (ii) by regulating the dissociation of this
factor from its
binding protein. One of the direct effects of free IGF-II is the observed
increase in cell
proliferation.
In summary, the ionic dissolution products of Bioglass 4555 induce the
bioavailability of IGF-II, IGFBP3, MMP2, MMP14, TIIVIPl, TIIVVIP2, procollagen
a2,
Decorin, c-jun, c-myc, calcium proteinase (calpain) and DAD 1 in human primary
osteoblasts, and effect bone cell proliferation and differentiation as well as
bone tissue
growth.
Moreover, the ionic dissolution products of Bioglass were found to upregulate
genes, at a rate greater than twofold in human osteoblasts, such as CD44
antigen
hemotopoietic form precursor, MAP kinase-activated protein kinase 2, integrin
beta 1, RCL
growth-related c-myc-responsive gene, defender against cell death 1 (DAD-1),
cyclin D1,
MMP14, CDKNlA, IGF-II, MMP2, TIlVII'1, decorin, TIMP-2, extracellular signal-
regulated kinase 1, cyclin K, ADP-ribosylation factor l, MAP kinase p38,
nuclear factor 1
(NFI), vascular endothelial growth factor precursor (VEGF), among others. It
is believed
that the upregulation of these genes by bioactive glass or glass extracts as
taught herein
contributes, directly or indirectly, to the stimulation of osteoblast
proliferation,
differentiation and/or function.
While the invention has been described in detail and with reference to
specific
embodiments thereof, it will be apparent to one skilled in the art that
various changes and
modifications can be made without departing from the spirit and scope thereof.
