Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BONE PRECURSOR COMPOSITIONS
BACKGROUND OF THE INVENTION
Bone is a biological composite having a calcium phosphate mineral phase
within a collagen biopolymer matrix phase. Bone has an average modulus of
elasticity
s of about 20 GN/m2, compressive strength of 170 to 220 MN/m2, tensile
strength of
180 MN/m2 and bending strength of approximately 220 to 270 MN/m2. As a
composite, bone differs from other composite materials by possessing an
orderly
intimate combination of a calcium phosphate mineral phase within a collagen
biopolymer matrix phase. Collagen is deposited by cells which organize the
~o composite structure. The calcium phosphate appears to self-assemble at gaps
in the
collagen phase to create mineral-polymer composite fibers. These mineralized
collagen fibers are bonded together in an orderly manner by further calcium
phosphate cementation.
In many situations bone is broken, destroyed, degraded, or becomes too
brittle.
15 Alternatively, bone can be traumatized by various stressors, it can have
naturally
occurring gaps and/or defects. Various materials have been investigated which
act as
a support, substitute, or an interface for repairing or replacing naturally
occurring
bone structure. Bone replacement structures frequently do not bond to the
affected
bone, thereby providing a weak juncture which is subject to failure due to
stresses
2o associated with normal movement and use of the bone structure. For example,
replacement materials, such as cobalt-chromium or titanium prostheses require
that
the interface between the bone and the prosthesis have a strong bond so that
the
prosthetic device is securely attached to the bone structure. To achieve this,
a bone
cement is generally used in conjunction with prosthetic implants.
25 The standard bone cement currently used in orthopedic surgery is
poly(methylmethacrylate) (PMMA). A common complication associated with
implants cemented with PMMA is that the implant loosens over time due to
everyday
stresses placed upon the implant/cement/bone interface. Further complications
can
be associated with the breakdown of PMMA as a result of mechanical fatigue and
so subsequent degradation in the physiological environment. Additionally, when
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PMMA is used to fill large bone areas, the heat of polymerization often
results in
temperatures high enough to cause deep necrosis of the surrounding bone
tissue.
Additionally, the initial toxicity of the methyl methacrylate monomer and the
non-
resorbability of PMMA preclude its use for bone grafting.
Cement-like biomaterials offer considerable advantages over these standard
bone cements since they can be shaped and hardened in situ, thereby affording
the
best possible fit with the surrounding bone tissue. Various calcium phosphate
formulations have been proposed as resolvable biomaterials. These formulations
typically consist of aqueous mixtures of calcium phosphates, such as
monocalcium
o phosphate monohydrate, dicalcium phosphate anhydrous, dicalcium phosphate
dihydrate, octacalcium phosphate, alpha-tricalciurn phosphate, beta-tricalcium
phosphate, tetra-calcium phosphate monoxide and calcium carbonate. The feature
common to these formulations is that they combine a relatively basic calcium
phosphate with a more acidic material thereby forming a phosphate of
intermediate
~5 acidity. However, the compositions of these cements results in cements with
several
deficiencies which limit their practical use. For example, some cements set
very
rapidly (in less than 30 seconds), making it difficult or impossible for a
surgeon to
inject it into or at the desired location. In contrast, some cements set too
slowly. In
addition, the final diametral strength of some of these cements is rather low
(less than
20 1 MPa) and decreases upon prolonged aging at physiological conditions.
SUMMARY OF THE INVENTION
The present invention provides new cement formulations which are injectable,
have setting times which enable their manipulation in vivo and which maintain
their
strength in physiological environments.
25 The invention is based, at least in part, on the discovery that bone
precursor
compositions of the invention can be prepared with the advantageous properties
of
being injectable, have set times which are between about 1 to about 15
minutes,
preferably between about 5 to about 10 minutes, and/or are biodegradable and
biocompatible and have high diametral strength which does not decrease upon
aging
so at physiological conditions. The bone precursor compositions of the
invention can be
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further modified with cells, reinforcing materials, with pore generating
materials,
extracellular particulates or fibrillar collagen, for example, to further
improve the
compatibility of the cement with the surrounding tissue into or onto which it
is
injected.
s Accordingly, the invention pertains to a bone precursor composition
including
a calcium cement which is suitable for injection, wherein the calcium cement
includes
monobasic calcium phosphate monohydrate and beta-tricalcium phosphate. In a
preferred embodiment, the bone precursor composition further includes calcium
pyrophosphate and alpha-calcium sulfate hemihydrate wherein the ratio by
weight of
~o monobasic calcium phosphate monohydrate to beta-tricalcium phosphate is
between
about 1:2 to about 1:3.75, mare preferably about 1:3.5 and most preferably
about
1:3.05. In one embodiment the reacted and hardened calcium cement is in the
form of
granules with a diameter of between about 1 to 500 Vim, preferably 50 to about
500 ~m
inclusive, preferably between about 100 to about 400 ~m inclusive, most
preferably
15 between about 250 ~m and about 350 wm inclusive. These granules can be
formed by
mechanical action such as grinding and sifting or sorting by size. In a
preferred
embodiment, fibrillar collagen is included in the bone precursor composition.
In
another preferred embodiment, the composition comprises unassembled liquid
collagen.
zo Advantageously, the bone precursor compositions of the invention are
injectable and have selected setting times and compression strengths which
render
them suitable for use as bone precursor compositions. In a preferred
embodiment, the
bone precursor composition includes or is conditioned with cells, such as
those
described infra. Bone precursor compositions of the invention can further
include
25 therapeutic agents or biopolymer fibers, e.g., collagen, such as porcine
collagen.
The bone precursor compositions of the invention can include cells or can be
conditioned with cells prior for use in vitro or in vivo, for example, to
render the
compositions suitable for use in vivo as prosthetic implants, or injectable
compositions
for replacement of damaged or diseased bone or to provide scaffolds which,
when
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occupied by cells, e.g., host cells, are remodeled to become functional tissue
such as
bone. These compositions can be used for in vitro development of bone, to be
implanted as a complete living replacement. This development may require
mechanical or electrical conditioning to stimulate strengthening and tissue
s organization of the product to authentic magnitudes. Optionally, these bone
precursor compositions can be used as model systems for research. In either
case, the
bone precursor compositions and constructs can be seeded with cells, e.g.,
mammalian
cells, e.g., human cells, of the same type as those of that tissue which the
bone
precursor composition or connective tissue is used to repair or reconstruct.
Examples
0 of cells which can seeded onto the bone precursor compositions and
constructs
described herein include tissue cells or mesenchymal cells such as connective
tissue or
bone cells. Suitable examples of soft connective tissue cells include ligament
cells,
tendon cells and chondrocytes. Suitable examples of bone cells include bone
marrow
stem cells, osteocytes, osteoblasts and osteoclasts. In one embodiment, the
bone
~5 precursor compositions and constructs seeded with tissue specific cells are
introduced
into a recipient, e.g., a mammal, such as a human. Typically, the cells
included in the
bone precursor compositions, or the cells which are used to condition the bone
precursor compositions, are connective tissue cells such as mammalian
connective
tissue cells, e.g., fibroblastic ligament cells and chondrocytes, and/or bone
cells such
2o as bone marrow stem cells, osteocytes, osteoblasts and osteoclasts.
In another aspect, the invention pertains to bone precursor compositions which
include a calcium cement and a biopolymer structure, e.g., a foam or matt. The
biopolymer foam can be a single density biopolymer foam or a double density
biopolymer foam. In a preferred embodiment, either or both the calcium cement
and
z5 the biopolymer foam or matt includes or is conditioned with cells.
In yet another aspect, the invention pertains to bone precursor compositions
which include a calcium cement and acid or pepsin extracted collagen. The acid
or
pepsin extracted collagen can be in the form of lyophilized collagen or
microfibrillar
collagen, e.g., microfibrillar collagen in the form of a semisolid pellet. In
a preferred
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embodiment, the collagen in the bone precursor compositions is between about
0.1 to
2.5 dry weight percent of the composition.
In still another aspect, the invention pertains to bone precursor compositions
which include a calcium cement and macromolecules necessary for cell growth,
s morphogenesis, differentiation and tissue building, particularly in the form
of
extracelluiar matrix particulates. The extracellular matrix particulates can
be between
about 0.05 to 20 weight percent of the composition and the ratio by weight of
monobasic calcium phosphate monohydrate to beta-tricalcium phosphate is
between
about 1:2 to between about 1:3.75, more preferably between about 1:3.5 and
most
o preferably about 1:3.05. Alternatively, the reacted hardened calcium cement
is in the
form of granules with a diameter of between about 1 to 500 Vim, preferably 50
to about
500 ~m inclusive, preferably between about 100 to about 400 ~.m, most
preferably
between about 250 ~m to about 350 ~,m. Additionally, the compositions can be
conditioned with cells and/ or growth differentiation or morphogenesis
factors.
15 In a still further aspect, the invention pertains to a method for preparing
a bone
precursor composition suitable for injection. This method includes combining
calcium
pyrophosphate, alpha-calcium sulfate hemihydrate, monobasic calcium phosphate
monohydrate and beta-tricalcium phosphate such that a bone precursor
composition
is prepared. In a preferred embodiment, the bane precursor composition
includes
2o calcium pyrophosphate and alpha-calcium sulfate hemihydrate wherein the
ratio by
weight of monobasic calcium phosphate monohydrate to beta-tricalcium phosphate
is
about 1:2 to about 1:3, preferably about 1:3.5, more preferably about 1:3.75
and most
preferably about 1:3.05. In one embodiment the composition is in the form of
granules
with a diameter of between about 1 to 500 um, preferably 50 to about 500 ~m
2s inclusive, preferably between about 100 to about 400 pm, most preferably
between
about 250 ~m to about 350 ~.m. In a particularly preferred embodiment,
fibrillar
collagen is included in the bone precursor composition.
In a preferred embodiment, the method further includes the step of contacting,
e.g., immersing or soaking, the reacted, hardened bone precursor composition
with a
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f SS-UI7P~
neutralizing solution such that a neutralized bone precursor composition is
prepared.
The neutralizing solution is selected from the group consisting of CAPS,
triethanolamine, i'pS, tri,rine, HEPFS, glycine, phosphate buffet solution,
leis tris
propane, TAPS, A.'viP and xRLS, preferably tribasic sodium phosphate. The bone
s precursor composition can then be implanted or can be seeded with cells-
In stilj yet another aspect, the invention pertains to methods far producing
or
repairing corulective tissue in a subject, comprising adnvnistering a bone
grerursox
composition to the subject, wherein the bone precursor composition includes
calcium
pyrophosphate, calcium sulfate herxuhydrate, monobasic calcium phosphate
monohydrate and beta-txicalciun't phosphate and, optionally, fibrillax
collagen.
In addition, the injectable bone precursor compvsitiaits of the invention can
include ph,armaceuticaIly acceptable injection vehicles, such as
methylcellulose, saline,
etc. Examples of othex suitable injection vehicles include microfibrillar
collagen or
fibrillax collagen. lNhen injectable calcium cement is used as a vehi~.e it
typically
~s comprises, by weight, between about l and 5 percent, preferably about 1
percent,
calcium p~~ophosnhate, between about ~ and 7.5 percent, preferably about 10
percent,
alpha-calcium su1~ate hemihydrate, between about ~ and 25 perce~wt, preferably
about
22 percent, monnbasic calcium phosphate monohydxate and between about a5 and
percent, preferably about 57 percent, beta-tricalcium phosphate. In one
embodiment,
2o the calcium cement (includes semisolid microfibrillar collagen in an amount
of
about 20'-50 .°~ atdditlonal wet weight, more preferably about 30-5~0~,
and in another
etnbodirnent, at least about 3 ~ ~ .
The bone precursor compositions and Constructs, with or yvithout in vitro
development, with or without Cells Ur extracellular matrix particulates can be
used, far
zs example, as orthopedic implants, max,illofacial irnpl~nts, dental implants,
connective
issue implants, e_g., cartilage implants, bone replacement implants-
Particulaxly, the
bone precursor compositions and cQr~structs which are used as orthopedic and/
or
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SUBSTITE1TE PA~B
AMENDED SHEET
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dental implants include a calcium cement, e.g., a mixture of monobasic calcium
phosphate monohydrate and beta-tricalcium phosphate, and optionally, calcium
sulfate, calcium pyrophosphate or collagen. An example of such an implant is
an
alveolar ridge builder or a bone void filler pellet.
DETAILED DESCRIPTION OF THE INVENTION
The present invention pertains to bone precursor compositions which include a
calcium cement which is suitable for injection. The injectable calcium cement
includes
monobasic calcium phosphate monohydrate and beta-tricalciurn phosphate. One
embodiment further includes calcium pyrophosphate and alpha-calcium sulfate
hemihydrate, preferably the ratio of dry weight of monobasic calcium phosphate
rnonohydrate to beta-tricalcium phosphate is between about 1:2 to about
1:3.75, more
preferably about 1:3.5 and most preferably about 1:3.05. In a preferred
embodiment,
the reacted and hardened calcium cement is in the form of granules with a
diameter of
between about 1 to 500 ~.m, preferably 50 to about 500 ~.m inclusive,
preferably
15 between about 100 to about 400 Vim, most preferably between about 250 um to
about
350 Vim. In a particularly preferred embodiment, collagen, e.g., fibrillar
collagen, is
included in the bone precursor composition. In an even more preferred
embodiment,
the bone precursor composition as either the mixture or in reacted and
hardened form
can include or can be conditioned for cell growth, conditioned with cells, or
treated
2o with macromolecules necessary for cell growth, morphogenesis,
differentiation and
tissue building, particularly in the form of extracellular matrix
particulates. The bone
precursor composition to be conditioned can be in the form of hardened pellets
or a
unitary structure formed before implantation, e.g., an implant.
The language "bone precursor composition" is intended to include those
25 materials, such as the calcium cement compositions described herein, which
can be
used to form, repair, or replace damaged connective tissue, e.g., such as bone
tissue.
In a preferred embodiment, the bone precursor composition is bioabsorbable and
biocompatible. Preferably the base precursor composition is suitable for
injection.
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"Bioabsorbable," as that term is used herein, means materials which are
degraded in response to contact with body fluid or cells while implanted or
injected in
vivo. Examples of bioabsorption processes include hydrolysis, enzymatic
action,
oxidation and reduction. Suitable conditions for hydrolysis, for example,
include
s exposure of the bioabsorbable materials, e.g., calcium cements of the
invention, to
water at a temperature and a pH of body fluids. Bioabsorption of cements of
the
present invention can be enhanced in low pH regions of the mammalian body,
e.g. an
inflamed area. Additionally, the cements of the invention will be remodeled by
host
cells over time and will disappear as it is replaced by new bone.
to "Biocompatible," as that term is used herein, means exhibition of
essentially no
cytotoxicity while in contact with body fluids. Both the material and its
degradation
products are nontoxic. "Biocompatibility" also includes essentially no adverse
interactions with recognition proteins, e.g., naturally occurring antibodies,
cell
proteins, cells and other components of biological systems. However,
substances and
~s functional groups specifically intended to cause the above effects, e.g.,
drugs and
prodrugs which may be added, are required to be biocompatible. The
biocompatible
cement compositions of the invention will not cause adverse tissue reactions
such as
an immune rejection or persistent inflammatory or foreign body response.
The term "calcium cement" is art recognized and is intended to include a
2o material which when combined with a liquid initiator undergoes a chemical
reaction
and/or a crystal rearrangement which results in a cured, e.g., hard, solid.
Via this
setting reaction, the calcium cement can be used as a joiner, or filler for
the assembling
of connective tissue surfaces e.g., bone tissue, which are not in direct
contact, and to
bond bone tissue to metallic or synthetic prosthetic devices. Calcium cements
can
2s include an initiator for the setting reaction. A physiologically acceptable
aqueous
initiator, e.g., water or an aqueous buffer, can be used, such as aqueous
solution,
which can further include additional ingredients such as methylcellulose or
collagen,
e.g., microfibrillar collagen. The water which is used will be substantially
pure, such
as double-distilled or deionized or an equivalent thereof. Other hydroxyl
containing
so materials e.g., methylcellulose, which are water miscible,
pharmacologically
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a'SS-017PC ~ ~y5 P ~ U S 009917871
acceptable and do mot interfere with bone precursor formation, also find value
as
lubricants ox injection vehicles.
The language Hsuitable for injection" is intended to include those bone
pxerursor compositions and calcium cements which have physical characteristics
s which render the materials suitable for passage as a homogenous paste
through a
syringe needle, e_g., typically a 14-?2 gauge needle without clogging the
needle or
separating into uquid axed solid phases.
The term "monobasic calcium. phosphate monohydrate~ is arc recognized and is
intended to include the compound defined as CaH4(PO~Z/ Hz0 and has a calcium
to
phospharoia.s ratio aE Q.S.
The term "beta-tricalciurm phosphate" is art recognized and is intended to
include the compour<d having the chemical formula of Ca3(POg)2 and has a
calcium to
phosphorous ratio of 1.5.
The term. "calcium pyrophosphate" is art recognized and is represented by the
t5 Formula Ca2P2~ and has a calcium to ptiosphoxous ratio of ~..
The term "alpha-calcium sulfate hemihydxate" is art recognized and is
represented by the formula (CaS~J~)/ 0.5 H~~.
The language "i.~cludes or is conditioned with cells" is intended to include
borne
precursor compositions which have cells attached or adhered to the calcium
cement
zo and can attach and grow for a sufficient period of time for deposition of
informational
macrornalecules onto the cement. For example, cells contemplated by the
invention
include tissue cells ox rnesenchymal cells which include com~ctive tissue
cells or bone
cells. Connective tissue ceps further include ligament cells, tendon cells and
chondrocytes. Bone cells are selected from bone marrow stem cells, osteacytes,
25 osteoblasts and osteoclasts.
The term "mesenchymal cell" is art recognized and is iniended to include
uxidifFerentiated cells found in meseruchymal tissue, e.g., ux~lif#erentiated
tissue
composed of branching cells embedded in a rluid matrix which is responsible
for the
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AMENDED SHEET
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production of connective tissue, blood vessels, blood, lymphatic system and
differentiates into various specialized connective tissues.
The term "connective tissue" is art recognized and is intended to include
primary tissue, which is distinguished by an abundance of fibrillar and non-
fibrillar
s extracellular components and cells organized to support or surround other
specialized
tissue.
The term "bone cells" is art recognized and is intended to include
osteoblasts,
osteoclasts and osteocytes.
The term "fibroblast" is art recognized and is intended to include cells found
in
connective tissues.
The term "tendon cell" is art recognized and is intended to include those
cells
which when organized into a tendon connect a muscle to bone and permit
concentration of muscle force into a small area.
The term "chondrocytes" is art recognized and is intended to include cartilage
cells.
The term "bone marrow stem cells" is art recognized and is intended to include
cells which can differentiate into mature blood and lymphatic cells or
cartilage or bone
cells.
The term "osteocytes" is art recognized and is intended to include those cells
2o found within the lacunae, which are osteoblasts that have matured and have
become
incorporated within the bone matrix.
The term "osteoblasts" is art recognized and is intended to include those
cells
found most abundantly along bone-forming surfaces and have receptors for
parathyroid hormone and are involved with the synthesis of osteocalcin,
collagen I,
2s alkaline phosphatase, osteonectin and assist in bone mineralization.
The term "osteoclasts" is art recognized and is intended to include monocyte-
macrophage cells which are multinucleated cells found along the cortical
endosteal
surface and the trabeculae in scalloped bays (Howship's lacunae) where
mineralized
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bone is being actively resorbed. These cells contain tartrate-resistant acid
phosphatase, collagenases, dehydrogenases, proteases, and carbonic anhydrase.
Signals from osteoblasts appear to be involved in activation of osteoclastic
bone
resorption.
s The bone precursor composition can be pre-cast into a form, e.g., an
implant, or
pellets, e.g., particles, or a calcium cement which is suitable for injection.
The
injectable composition can further include a pharmaceutically acceptable
vehicle, or
preferably, microfibrillar collagen. The injectable composition noninvasively
fills
voids and hardens there as a resilient bone replacement or prevents the motion
of
small bone fragments during healing. The pellets can be placed and contained
in open
voids to augment the repair of large or irregular defects.
The phrase "pharmaceutically acceptable vehicle" is art recognized and
includes
a pharmaceutically acceptable material, composition or carrier, suitable for
administering bone precursor compositions of the invention to mammals by
injection.
~s The vehicles include liquid or solid filler, diluent, excipient, solvent or
encapsulating
material, involved in carrying or transporting the bone precursor composition
from a
syringe to the cavity in need thereof. Each carrier must be "acceptable" in
the sense of
being compatible with the other ingredients of the formulation and not
injurious to
the patient. Some examples of materials which can serve as pharmaceutically
2o acceptable vehicles, include: sugars, such as lactose, glucose and sucrose;
starches such
as cornstarch and potato starch; cellulose and its derivatives, such as sodium
carboxy
methylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;
malt;
gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils
such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil;
2s glycol such as propylene glycol; polyols such as glycerin, sorbitol,
manitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffering
agents
such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free
water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and
other non-toxic compatible substances employed in pharmaceutical formulations.
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Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, stabilizers, preservatives or
antioxidants can also be present in the compositions.
Methods of preparing these formulations or compositions include the step of
s bringing into association the calcium salts of the present invention with an
initiator
which can include a carrier and, optionally, one or more accessory
ingredients. In
general, the formulations are prepared by uniformly and intimately bringing
into
association the calcium salts of the present invention with liquid initiators
which can
include carriers or finely divided solid additives, or both, and then, if
necessary,
o shaping the product.
In one embodiment, the bone precursor composition further includes solid
additives ("pore-generating particles') which bioabsorb more quickly than the
calcium salts of the composition, thereby causing the bone precursor
composition to
become porous. For example, bioabsorbable particles having a diameter of
between
s about 20 to about 250 ~m inclusive can be added to the bone precursor
compositions.
Suitable bioabsorbable particles or pore-generating particles include gelatin,
hardened
calcium sulfate, salt or sugars, generally in a 5 to 70% range by dry weight
to bone
precursor composition. Porous bone precursor compositions provide the
advantage
of being suitable for osteoconduction.
2o Liquid dosage forms suitable for administration of the bone precursor
compositions of the invention include pharmaceutically acceptable emulsions
and
microemulsions, solutions, suspensions, syrups and elixirs. In addition to the
active
ingredients, e.g. calcium salts, the liquid dosage form can contain inert
diluents
commonly used in the art, such as, for example, water or other solvents,
solubilizing
25 agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl
acetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol,
oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol,
tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters, sorbitan
and
mixtures thereof.
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The bone precursor compositions can also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing agents.
Prevention
of the action of microorganisms may be insured by the inclusion of various
anti-
bacterial and anti-fungal agents, for example, paraben, chlorobutanol, phenol
sorbic
acid, and the like. It may also be desirable to include isotonic agents,
sugars, sodium
chloride and the like into the compositions. In addition, prolonged absorption
of the
injectable bone precursor compositions can be brought about by the inclusion
of
agents which allay absorption such as aluminum monostearate and gelatin e.g.,
collagen.
o A preferred vehicle is microfibrillar collagen. The collagen used in the
compositions and foams of the invention can be in the form of collagen
microfibrils.
In another preferred embodiment, the vehicle is unassembled liquid collagen.
The language "collagen microfibril" is art recognized and is intended to
include
collagen in the form described in Williams, B.R. et al. (1978) J. Biol. Chem.
253
(18):6578-6585 and U.S. Patent Appln. No. 08/910,853, filed August 13,1997,
entitled
"Compositions, Devices, and Methods fvr Coagulating Blood" by Eugene Bell and
Tracy M. Sioussat, the contents of which are incorporated herein by reference.
In a
preferred embodiment, the collagen microfibrils are prepared as a semisolid
(viscous)
pellet of collagen microfibrils resulting from centrifugation of a neutralized
solution of
2o collagen. For example, the collagen can be neutralized by liquid 0.01-2.0 N
NaOH, 0.1-
10% ammonium hydroxide, or other known neutralizing solutions, before spinning
in
a centrifuge to yield a microfibrillar collagen pellet mass. The liquid
content of the
microfibrillar collagen pellet mass can be manipulated by the relative
centrifugal force
employed. For example, the stronger the centrifugal force, the less liquid and
the
higher the resulting concentration of microfibrillar collagen (e.g., from
about 10 to
about 100 mg/ ml). The resultant semisolid pellet of neutralized
microfibrillar
collagen can be manipulated like a fluid such that it can be propelled from,
for
example, a device of the present invention, onto or into a site of bleeding.
Since the
microfibrillar collagen is already neutral, no gelling is required. However,
the density
of this form of collagen allows it to remain in place at the desired site of
bleeding. The
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structure of the microfibrillar collagen provides the surface to initiate the
clotting
cascade at the site of bleeding.
Methods for purifying collagen so it can form microfibrils typically include
the
steps of extracting proteins from, for example, the skin of an animal, e.g.,
chicken,
mammal, e.g., a marine mammal, a cow, goat, sheep, or, preferably, a pig,
e.g., a fetal
or newborn pig. This extraction step involves the use of organic acid such as
formic or
acetic acid. The collagen is then precipitated from the extract by salt (e.g.,
sodium
chloride up to 3.OM or ammonium sulfate up to 50%) and collected by
centrifugation.
The collagen is then redissolved in organic acid and concentrated. The
collagen can
o then be used or subjected to as many rounds, e.g., two rounds, of salt
precipitation
and centrifugation as desired before concentrating and using in the present
invention.
A preferred collagen concentration used to make microfibrillar collagen is 4.0
to 10.0
mg/ml. An alternative method for purifying collagen includes a method in which
pepsin is included in the extraction acid solution, with all other steps the
same as
described above, with the additional updated steps described below.
For further details on the methods for purifying collagen, see U.S. Patent No.
5,562,946 (hereinafter the "'946 patent"), the corresponding PCT application
of which
was published on 17 May 1996 and assigned International Publication No. WO
96/ 14452, the contents of both of which are incorporated herein by reference.
This
2o purification method is described at columns 6-8 of the '946 patent has been
updated as
follows: at Lines 57-bl, of column 7, rather than dialysis bags for dialysis,
hollow fiber
membranes are used with a 0.1 ~m cutoff (or 100,000 MW for pepsin collagen).
Thus,
the centrifugation step at lines 62-64 of column 7, occurs before the dialysis
step and
the concentration step described at 66-67 of column 7 and lines 1-4 of column
8 occurs
2s at the same time as the dialysis in the same hollow fiber.
In certain embodiments of the invention, calcium cement in the form of
granules can be admixed with an injection vehicle which includes
microfibrillar
collagen, unassembled liquid collagen (e.g., at a concentration of about 5
mg/ml to
about 40 mg/ml) or a calcium cement of calcium pyrophosphate, alpha-calcium
so sulfate hemihydrate, monobasic calcium phosphate monohydrate, beta-tri-
calcium
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phosphate and, optionally, a wetting agent. In one embodiment, the mixture can
be
conditioned with cells. These particles can be injected into places where they
can
disperse and infiltrate into multiple small cavities to initiate bone regrowth
throughout the interior of bones weakened by osteoporosis.
The addition of unassembled liquid collagen to the injection vehicle gives the
cement the capability of forming a semi-solid unit with the cement particles
trapped in
a collagen gel. While the unassembled collagen is a liquid in the pH 3 to 6
suspension
prior to injection, after injection and contact with neighboring neutral pH
tissue of the
patient, the collagen can assemble into a gel of sufficient structure to hold
the particles
in their three dimensional suspended positions. This injectable composition,
which
results in a gelled suspension of the cement particulates is useful as a
highly
osteoconductive material for filling larger interior voids than practical with
microfibrillar collagen injection vehicle.
A most preferred embodiment of cement includes by dry weight,1 % calcium
pyrophosphate,10% alpha-calcium sulfate hemihydrate, 22% monobasic calcium
phosphate monohydrate and 67% beta-tricalcium phosphate and, optionally,
between
about 0.1 to about 2.5% collagen by dry weight.
The use of the injectable cement injection vehicle does provide the
microparticulates with a structural binder. The cement binder and the collagen
binder
2o have some important differences, such as the degree of porosity, the degree
of
structural strength and the rate of remodeling. The cement binder will result
in a
structure of higher strength, lower porosity, and slower remodeling rate than
the gel
binder. A person of skill in the art will be able to apply expertise in
deciding the
appropriate binder for the situation. Self assembling molecules, such as
fibrinogen
and certain synthetic polymer precursors are also suitable agents for
inclusion in the
injection vehicle for the purpose of binding the cement particles in
suspension after
injection. However, fibrinogen already is present at the injection site in the
blood
supply and, through natural clotting mechanisms, may form a fibrin gel clot in
conjunction with or adjacent to the collagen gel or microfibrils in any
injected
so composition of cement particles. Synthetic polymer precursors form
materials of less
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- instructive value than collagen and are not actively remodeled by host
tissue while
they degrade by hydrolytic mechanisms. Cells and liquids can transverse the
collagen
gel and cells can bind to it. Bound cells can remodel the collagen into
structures they
need or they can associate into tissues using the collagen filament framework
and
s rebuild the bone at the injected site, with the cement microparticulates
giving a jump-
start of calcification. In a further embodiment, the injection vehicle may
also,
advantageously, include materials which increase the viscosity of the
composition
such as, for example, microfibrillar collagen, collagen foam, collagen fiber
particles, or
0.1 to 15%, more preferably 0.5 to 10%, methyl cellulose. The injection
vehicle may
also comprise a pharmaceutically acceptable carrier as mentioned supra.
Bone precursor compositions can include therapeutic agents. For example,
therapeutic agents include antibiotics, such as gentamycin, penicillin,
streptomycin,
anti inflammatory agents, such as cyclosporin, and/or agents such as
cytokines,
growth factors, or macromolecules necessary for growth, morphogenesis,
~s differentiation, or tissue building, or extracellular matrix particulates.
As indicated above, a bone precursor composition of the invention can be
fabricated with biopolymer fibers. For example, a biopolymer fiber, a mufti-
fiber
element, or a biopolymer fabric comprising fibers can be embedded in or about
cement. The cement can serve as an anchor for fibers embedded in the cement,
for
2o example, in a ligament replacement where the cement anchors the ligament
precursor
fibers in the bone at the site of ligament attachment Alternatively, the
calcium cement
can be deposited into these fibers in the form of a coating or in granulated
form.
Methods and apparatus for fabricating biopolymer fibers are known to those
with
ordinary skill in the art as disclosed in U.S. Patent No. 5,562,946, entitled
"Apparatus
2s and Method for Spinning and Processing Collagen Fiber," issued October
8,1996 and
herein incorporated by reference. Preferably, the biopolymer fiber is formed
of
collagen, most preferably from fetal porcine collagen.
The term "biopolymer" as used herein, is intended to include naturally
occurring polymers or man-made polymers from naturally-occurring components
3o which are suitable for introduction into a living organ, e.g. a mammal,
e.g., a human.
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Preferably, the biopolymer is non-toxic and bioabsorbable when introduced into
a
living organism and any degradation products of the biopolymer are also non-
toxic to
the organism. Biopolymers of the invention can be formed into structures such
as
biocompatible foams, e.g. single or double density foams, composite foams, and
s biocompatible constructs within or attached to bone precursor composition
which
include biopolymer fibers, e.g., collagen fibers, biopolymer fabrics, e.g.,
collagen
fabrics, and/or extraceilular matrix particulates. Examples of molecules which
can
form biopolymers which can be used in the present invention include collagen,
thrombospondin, gelatin, polysaccharides, poly-1-amino acids, elastin,
laminin,
heparin sulfate proteoglycan, fibronectin and fibrinogen and combinations
thereof.
For example, a combination of collagen with a calcium cement can form a bone
precursor composition.
Preferred sources of molecules which form biopolymers include mammals such
as pigs, e.g., near-term fetal pigs, sheep, and cows. Other sources of
molecules which
~s can form the biopolymers include both land and marine vertebrates and
invertebrates.
In one embodiment, the collagen can be obtained from skins of near-term,
domestic
porcine fetuses which are harvested intact, enclosed in their amniotic
membranes.
Collagen or combinations of collagen types can be used in the foams, fibers,
and foam
compositions described herein. Examples of collagen or combinations of
collagen
2o types include collagen type I, collagen type II, collagen type III,
collagen type IV,
collagen type V, collagen type VI, collagen type VII, collagen type VIII,
collagen type
IX, collagen type X, collagen type XI, collagen type XII, collagen type XIII,
and
collagen type XIV. A preferred combination of collagen types includes collagen
type I,
collagen type III, and collagen type IV. Preferred mammalian tissues from
which to
2s extract the molecules which can form biopolymer include entire mammalian
fetuses,
e.g., porcine fetuses, dermis, tendon, muscle and connective tissue. As a
source of
collagen, fetal tissues are advantageous because the collagen in the fetal
tissues is not
as heavily cross linked as in adult tissues. Thus, when the collagen is
extracted using
acid extraction, a greater percentage of intact collagen molecules is obtained
from fetal
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i SS-Q 17PC
tissues in comparison to adult tissues. Fetal tissues also ixLCIude various
mt~lecular
factors which are present in normal tissue at different stages of animal
development.
The biopolymers can be u~ to create sponges or foams, e.g., single or double
density foams, which can be in any form or shaQe, e.g., strips. sheets, tubes,
etc. In
s addition, the biopolymers cart be used to create foams which are then
combined with
cem~xtt carnpositior< to forrn implants, such as a cement overlaid with a
single density
foam to produce an osteochondxal replacement to repair an. articular joint-
The forms and shapes ire which the foams and foam compositions are made can
mimic those of tissues or body parts to be replaced and thus can be used as
prostheses
~o or grafts which tissue cells remodel to promote regeneration of a
replacement tissue itt
the recipient. Single density or double density foam compositions which are
useful
with the cement co~.npos-itions of the invention axe described in U.S. Patent
No.
5,891,558, the contents of which are expressly incorporated herein by
referexuee. Fxtra-
cellular matrix particulates andl or v~iabie cells can also be added to the
biopalymers to
~ s further promote cell its growth and tissue development and organization
within the
foams.
For example, single dexsity foams can be cast into the inner portion of a
preformed cement tube. This composite can replace bane segments, with the
cement
providing the replaceable srtructural bone cortex and the foam core allowing
the
2o regrowth of the bone marrow. Alternatively, a singe or double density foam
can be
cast onto a hardened centerit form, or preferably adding a foam to a cement
cornposifion which is still hardening and includes collagen within the cemeslt
form.
Reacted, haxdexied cement particulates can be mixed with collagen and cast
into a
foam with the particles suspended in the foam matrix. Particle density ranges
from
z5 0.5 to 4096 particle weight to wet collagen volume. The foam and/ or the
cement can
be further treated ~n~ith extraceiluiar matrix pazticulates and/ or viable
cells as
described above. This material provides acv immediately osteoconductive sway
to fill
voids with a precast cohesive material, for applications such as filling post-
extraction
tooth sockets or filling other open bore cavities.
_ lg _
SU85'r'lTu't"E PwGE
AMENDED SHEET
CA 02353949 2001-06-05
WO 00/07639 PCT/US99/17$71
In another embodiment, a matt can be cast onto a cement composition of the
invention. Alternatively, cement particulates (granules) can be cast within or
bonded
onto a matt, as described below. Particle density ranges from 0.1 to 5%
particle weight
to wet collagen fibrillar pellet volume. The matt-cement composites can be
used to
s repair cortical bone defects where the periosteum was removed or destroyed.
The
cement provides the replacement for the lost bone and the matt provides the
replacement for the lost periosteum. The term matt is art recognized and is
intended
to include those matts described in pending U.S. Patent Application No.
09/042,549,
entitled "Biopolymer Matt for Use in Tissue Repair and Reconstruction," filed
March
0 17,1998, the contents of which are expressly incorporated herein by
reference.
As used herein, the term "matt" refers to a biopolymer scaffold comprising a
densely packed random array of biopolymer fibrils or bundles of fibrils or
particles,
e.g., collagen fibrils. Malts which have been dried, as discussed previously,
possess a
wet tensile strength of at least 0.02 MPa with a preferred strength of greater
than 1
~5 MPa and have a collagenase resistance of at least 20 min per mg of collagen
at a
collagenase concentration of 10 units per 1 cm2 of product. Typically the
fibrils or
bundles of fibrils are between about 0.01 ~m and 50 um in diameter and between
about 0.0002 and 5.0 mm in length, preferably 0.1 um to 20 um wide and 0.01 mm
to 3
mm long. Matts, whether dried or not, possess the following characteristics:
(1)
2o physically stable in aqueous solutions; (2) nontoxic to living organisms;
(3) can serve
as a substrate for cell attachment and growth; (4) approximately 0.01 mm to 20
mm
thick, preferably 0.1 to 5.0 mm thick. In a preferred embodiment, the
biopolymer
matt, matt composite, or matt composition is a collagen matt, collagen matt
composite,
or collagen matt composition prepared from collagen solution as previously
2s described.
The biopolymers can be used to create matts, matt composites, or matt
compositions which can be in any form or shape, e.g., strips, sheets, tubes,
etc. In
addition, the biopolymers can be used to create matts which can be supported
by
polymer mesh, e.g., a Teflon~ mesh, or used with tissue culture inserts for
multiwell
3o plates which can be used as molds in which matt, matt composites, and matt
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compositions of the invention can be #orrned on the polycarbonate membrane of
the
insert. Polymer meshes used with the matt, matt composites, and matt
compositions
of the invention can expose cells, such as chondrocytes, contained on arid
within the
matt, matt composites, and mart compositions tv body tissues and fluids, far
example,
s where the matt, matt composites, and matt compositions are used as support
to
stimulate formation of bone. Both the meshes and culture uzserts have the
advantage
of providing a means for handling the matt, matt composites, and matt
compositions
without requiring actual eons-pct with the matt, matt composites, or matt
compositions. The forms anal shapes in which the matt, matt coutposites, and
matt
so Compositions are invade can mimic those of tissues or body parts to be
replaced and
thus can be used as prostheses or grafts which tissue cells remodel to
groznote
regeneration of a replacement tissue in thz recipient.
Selected reinforcing material can be added to the calcium cement ox to
biopolymer solutions incorporated into the calcium cements of the invention.
The
~ reinforcing material should be added to the cement prior to hardening. Such
reinforcing materials include biopolyxner fibers, threads, e_g., woven or
braided
threads, and/ or fabrics, e.g., non woven fabrics, prepared, for example, by
textile
methods. $iopolyxr'.er thFeads, e.g., collagen threads, can be prepared by
extruding
the biopolymer in solution into a coagulation bath and transferring the
biopolymer to
zo a bath containing ethanol or acetone or another dehydratixig solution.
Alternatively,
the thread can be dehydrated by subjection to vacuum-drying. The biopolymer
thread can thenhe cross linked by, far example, methods described herein. Ars
example of an apparatus for spinning and processing a biopolyrnez fiber, e.g.,
collagen
fiber, is described in LJ.S. Patent No. 5,562.,94, the contents of which is
incorporated
2s herein by reference in its entirety. The threads can then be dried,
spooled, for
example, by pulling the moving thread over more rollers, stretching and drying
it and
then winding it onto spools. Textile implements can be employed to weave or
braid
the threads into fabric or other complex forms or constructs for use as
described
herein-
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CA 02353949 2001-06-05
WO 00/07639 PCT/US99/17871
Biopolymer fabrics, e.g., non woven biopolymer fabrics, are typically composed
of a mat of entangled biopolymer fibers of a selected size and density.
Typically, the
non woven biopolymer fabrics are produced by spooling dry biopolymer fiber
onto a
drum of circumference equal to that of the length of an individual fiber
element.
s Spooling is continued until the number of wraps of fiber on the drum equals
the
number of pieces of fiber required for the fabric. A cut is then made across
the wound
fiber in a direction parallel to the drum axis and the fibers are removed from
the
drum. The fiber can then be cross linked if it has not been previously cross
linked.
The fiber is then dispersed in a volume of a buffer solution for a period of
time to
o stabilize its pH and soften the fiber. The fiber is transferred to a volume
of water and
agitated mechanically to produce entanglement of the fiber strands. The
entangled
fiber strands are sieved from the water onto a collection screen until they
coat the
screen in a mat of uniform density. The mat is then dried on the screen or
after
transfer to another surface, screen, or cell culture device. If desired, the
non woven
~5 fabric can be cut or punched into smaller shapes after drying.
Macromolecules necessary for cell growth, morphogenesis, differentiation, and
tissue building can also be added to the biopolymer molecules or to the
biopolymer
fibrils or to the cement composition of the invention to further promote cell
ingrowth
and tissue development and organization on or within the cement composition or
2o biopolymer construct. The phrase "macromolecules necessary for cell growth,
morphogenesis, differentiation, and tissue building" refers to those
molecules, e.g.,
macromolecules such as proteins, which participate in the development of
tissue.
Such molecules contain biological, physiological, and structural information
for
development or regeneration of the tissue structure and function. Examples of
these
2s macromolecules include, but are not limited to, growth factors,
extracellular matrix
proteins, proteoglycans, glycosaminoglycans and polysaccharides.
Alternatively, the
biopolymer matts, matt composites, and matt compositions of the invention can
include extracellular matrix macromolecules in particulate form or
extracellular
matrix molecules deposited by cells or viable cells.
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The term "growth factors" is art recognized and is intended to include, but is
not limited to, one or more of platelet derived growth factors (PDGF), e.g.,
PDGF AA,
PDGF BB; insulin-like growth factors (IGF), e.g., IGF-I, IGF-II; fibroblast
growth
factors (FGF), e.g., acidic FGF, basic FGF, ~i-endothelial cell growth factor,
FGF 4, FGF
s 5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF), e.g.,
TGF-(31,
TGF-(31.2, TGF-(32, TGF-~i3, TGF-(35; bone morphogenic proteins (BMP), e.g.,
BMP 1,
BMP 2, BMP 3, BMP 4; vascular endothelial growth factors (VEGF), e.g., VEGF,
placenta growth factor; epidermal growth factors (EGF), e.g., EGF,
amphiregulin,
betacellulin, heparin binding EGF; interleukins, e.g., IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-
0 7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14; colony stimulating factors
(CSF), e.g., CSF-
G, CSF-GM, CSF-M; nerve growth factor (NGF); stem cell factor; hepatocyte
growth
factor, and ciliary neurotrophic factor. Adams et al., "Regulation of
Development and
Differentiation by the Extracellular Matrix" Development Vol.117, p.1183-1198
(1993)
(hereinafter "Adams et al.") and Kreis et al. editors of the book entitled
"Guidebook to
15 the Extracellular Matrix and Adhesion Proteins," Oxford University Press
(1993)
(hereinafter "Kreis et al.") describe extracellular matrix components that
regulate
differentiation and development. Further, Adams et al. disclose examples of
association of growth factors with extracellular matrix proteins and that the
extracellular matrix is an important part of the micro-environment and, in
2o collaboration with growth factors, plays a central role in regulating
differentiation and
development. The teachings of Adams et al. and Kreis et al. are incorporated
herein
by reference. The term encompasses presently unknown growth factors that may
be
discovered in the future, since their characterization as a growth factor will
be readily
determinable by persons skilled in the art.
zs The term "extracellular matrix proteins" is art recognized and is intended
to
include one or more of fibronectin, laminin, vitronectin, tenascin, entactin,
thrombospondin, elastin, gelatin, collagens, fibrillin, merosin, anchorin,
chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin,
epinectin,
hyaluronectin, undulin, epiligrin, and kalinin. The term encompasses presently
so unknown extracellular matrix proteins that may be discovered in the future,
since
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their characterization as an extracellular matrix protein will be readily
determinable
by persons skilled in the art.
The term "proteoglycan" is art recognized and is intended to include one or
more of decorin and dermatan sulfate proteoglycans, keratin or keratan sulfate
s proteoglycans, aggrecan or chondroitin sulfate proteoglycans, heparan
sulfate
proteogiycans, biglycan, syndecan, perlecan, or serglycin. The term
encompasses
presently unknown proteoglycans that may be discovered in the future, since
their
characterization as a proteoglycan will be readily determinable by persons
skilled in
the art.
The term "glycosaminoglycan" is art recognized and is intended to include one
or more of heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan
sulfate,
hyaluronic acid. The term encompasses presently unknown glycosaminoglycans
that
may be discovered in the future, since their characterization as a
glycosaminoglycan
will be readily determinable by persons skilled in the art.
15 The term "polysaccharide" is art recognized and is intended to include one
or
more of heparin, dextran sulfate, chitin, alginic acid, pectin, and xylan. The
term
encompasses presently unknown polysaccharides that may be discovered in the
future, since their characterization as a polysaccharide will be readily
determinable by
persons skilled in the art.
2o Suitable living cells include, but are not limited to, epithelial cells,
e.g.,
keratinocytes, adipocytes, hepatocytes, neurons, glial cells, astrocytes,
podocytes,
mammary epithelial cells, islet cells; endothelial cells, e.g., aortic,
capillary and vein
endothelial cells; and mesenchymal cells, e.g., dermal fibroblasts,
mesothelial cells,
stem cells, osteoblasts, smooth muscle cells, striated muscle cells, ligament
fibroblasts,
25 tendon fibroblasts, chondrocytes, and fibroblasts.
Extracellular matrix particulates or extracellular matrix particulates
dispersed
or suspended in a vehicle can also be mixed with the calcium cements of the
invention
and/or supports detailed above, thereby forming a bone precursor composition
having extracellular matrix particulates. As used herein, the language
"extracellular
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matrix particulate" xefars to a fragment of an extracellular matrix derived
from a tissue
source forrnexly having living cells but which has been processed to remove
the cells
and to retain noncellular extraceijulaz matrix factors such as, for example,
growth
factors necessary far cell growth, moxphogenesis, and diff et~entiation-
Methods for
farming extracellular matrix particulates for producing graft tissue are
disclosed in
L.S. Patent Nos. 5,893,8$8 and 5,~~,5~~. the contents of which are
incorporated herein
by reference.
The methods for farming extracellular matrix particulates Include freezing a
tissue source, e.g., a connective tissue source, having living cells, whereby
the living
o cells are disrupted to form cell remnants consisting of, for example,
cytoplasmic and
nuclear components. The tissue source is then processed, e.g., by grinding,
washing
and sieving, to xemove the cytoplasmic and nuclear components without removing
extracellular matrix including factors necc~sary fox cell growth, migration,
differ-
entiation, and m.orphogenesis. The extracellular matrix is freeze-dried and
~5 fragmented, e.g., cryomilled to produce particulates of defined sizes, to
prndvee
extracellular matrix particulates.
The extracellular matrix particulates can include extraceliular matrix
proteins.
For cxarnple, extracellular matrix particulates obtained fror~n skin include
transforming growth factor ~1, platelet-derived growth factor, basic
fibxoblast growth
Zo factor, epidermal growth factor, syndecztn 1, decorixt" fibronectin,
collagens, Iaminin,
teiiascin, and dexrnatan sulfate. Extracellular matrix particulates froal lung
include
syrsdecan~l, fzbronectix~, i.aminir~, and tenascin. The extracellular matrix
particul.ates
can also include cytokixxes, e.g., growth factors necessary for tissue
development. The
term "cytakixle" includes but is not limited tv growth factors, interleukins,
iriterferons
25 and colony stimulating factors. These factors are present in normal tissue
at different
stages of tissue development, marked by cell division, morphogenesis and
differentiation. Aznoatg these factors are stimulatory molecules that provide
the
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AMENDED SHEET
CA 02353949 2001-06-05
WO 00/07639 PCT/US99/17871
signals needed for in vivo tissue repair. These cytokines can stimulate
conversion of
an implant into a functional substitute for the tissue being replaced. . This
conversion
can occur by mobilizing tissue cells from similar contiguous tissues, e.g.,
from the
circulation and from stem cell reservoirs. Cells can attach to the prostheses
which are
s bioabsorbable and can remodel them into replacement tissues.
Extracellular matrix particulates can be obtained from specific tissues. The
particulates have two kinds of informational properties. The first is their
molecular
diversity, and the second is their micro-architecture, both of which are
preserved in
the preparation of the microparticuiates. The preferred associations among the
o different molecules of the extracellular matrix are also preserved in the
preparation of
the microparticulates.
The extracellular matrix plays an instructive role, guiding the activity of
cells
which are surrounded by it or which are organized on it. Since the execution
of cell
programs for cell division, morphogenesis, differentiation, tissue building
and
is regeneration depend upon signals emanating from the extracellular matrix,
three-
dimensional scaffolds, such as collagen foams, are enriched with actual matrix
constituents, which exhibit the molecular diversity and the microarchitecture
of a
generic extracellular matrix, and of extracellular matrices from specific
tissues.
To provide further cellular and molecular binding sites on the surfaces of the
2o bone precursor compositions and calcium cements to replace, for example,
binding
sites which have been compromised as a result of the setting process, a
coating process
can precede or accompany the application of extracellular matrix particulates
to these
materials. In addition, artificial microstructures, typically having a size in
the range of
between about 5 and 500 ~.m, composed of a matrix polymer, such as collagen,
2s combined with other proteins, proteoglycans, glycosaminoglycans,
extracellular
matrix enzymes, cytokines (including growth factors), and glycosides can be
created
in the form of wet or dry particulates that can be applied with the coating
solution to
the surfaces of the bone precursor composition and calcium cement. The
selected
components can be chemically or electrostatically bound to the bone precursor
so composition and calcium cement or can be contained in the microparticulate
lattice or
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CA 02353949 2001-06-05
WO 00/07639 PCT/US99/17871
in a dehydrated form of the lattice. Thus, the invention also pertains to
methods for
preparing collagen-coated bone precursor compositions and calcium cements and
extracellular matrix particulate-coated bone precursor compositions and
calcium
cements. These methods typically include forming the selected type of bone
precursor
s composition or calcium cement as described herein and applying a collagen
solution
or an extracelluiar matrix particulate solution to the bone precursor
composition or
calcium cement, thereby forming the collagen-coated or extracellular matrix
particulate-coated bone precursor composition or calcium cement. The coated
bone
precursor compositions and calcium cements can be further freeze-dried. In one
~o embodiment, the collagen solution also includes extracellular matrix
particulates.
Preferably, bone precursor compositions and calcium cements of the present
invention
include extracellular matrix particulates in amounts between about 0.05 to
about 20
dry weight percent of the compositions.
In one preferred method, the hardened bone precursor composition, in pellet or
~s granular form is contacted with a neutralizing solution such that a
neutralized bone
precursor composition is prepared. The term "neutralizing solution" is art
recognized
as intended to include suitable chemical, biochemical, enzymatic or other
components
which alter the pH of calcium containing materials. For example, neutralizing
solutions are selected from CAPS {3-[cyclohexylaminoJ-1-propanesulfonic acid),
2o triethanolamine, TES(N-tris[hydroxymethylJmethyl-2-aminoethanesulfonic
acid),
tricine, HEPES (N-2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acidJ)
glycine,
phosphate buffer solution, bis tris propane, TAPS (N-tris[hydroxymethylJmethyl-
3-
aminopropane sulfonic acid), AMP (2-amino-2-methyl-1-propanol) and TRIS
(tris[hydroxymethyl]aminomethane). A preferred neutralizing solution is
tribasic
2s sodium phosphate. Bone precursor materials and calcium cements prepared in
this
manner can include or can be conditioned with cells described supra.
The bone precursor compositions and calcium cements of the present invention
can be used as substrates for cell growth In vitro and in vivo, e.g., for
establishing
research model systems. For example, in one embodiment, the bone precursor
so composition calcium cement can be seeded with abnormal cells to study
disease states
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including cancer. In another embodiment, the bone precursor composition or
calcium
cement and can serve as diagnostic test models for determining
chemotherapeutic
strategies by selecting for agents capable of killing cancer cells cultivated
in or on the
cements. In yet another embodiment, the bone precursor compositions or calcium
s cements can be used to test the toxicity of various substances to which
cells in or on
the cements are exposed.
The bone precursor composition or calcium cement can also be used as
prostheses which can be introduced or grafted into recipients, e.g., such as
mammalian recipients, e.g., humans. For example, the bone precursor
composition or
o calcium cement can be used as a prosthesis or to reconstitute, for example,
the
following types of tissue: connective tissue such as bone or cartilage, and to
anchor
tissue such as ligament and tendon. Tissue cells seeded into these bone
precursor
compositions or calcium cements can be obtained from a mammal, e.g., a human.
Tissue cells axe delivered to the bone precursor composition or calcium cement
by first
~s suspending the cells in small volumes of tissue culture medium. The tissue
culture
medium which contains the cells can then be applied in drops to the bone
precursor
composition or calcium cement. Alternatively, the bone precursor composition
or
calcium cement can be placed in a vessel which contains the tissue culture
medium
and cells in suspension and which shakes such that the tissue culture medium
2o containing the cells is distributed throughout the bone precursor
composition or
calcium cement. In another embodiment, tissue cells can be suspended in a
biopolymer solution e.g., a collagen solution, at low concentrations, at a
temperature
of about 4°C to 10°C, and at a pH of about 7Ø The solution
containing the cells can
then be delivered to the bone precursor composition or calcium cement. As bone
2s precursor composition or calcium cement is warmed to 37°C, the
biopolymer solution,
e.g., collagen solution, forms a gel on the bone precursor composition or
fragmented
calcium cement. As used herein, the term "gel" refers a network or mesh or
biopolymer filaments together with an aqueous solution trapped within the
network
or mesh of biopolymer filaments. An alginate gel for use as a delivery vehicle
of cells
3o to the bone precursor composition or fragmented calcium cement of the
invention can
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be produced by addition of calcium which causes polymerization at room
temperature
and at a neutral pH. Selected epidermal, endodermal, mesenchymal-derived,
epithelial, endothelial, or mesothelial cells can then be seeded onto the
surface of the
gel-coated bone precursor composition or calcium cement.
s The bone precursor composition or calcium cement and other forms of
biopolymers described herein can be conditioned, e.g., made tissue-ready or
established with pre-tissue elements by cells. For example, the bone precursor
composition or calcium cement with or without other forms of biopolymers can
be
seeded with a selected cell type or selected cell types. The cells can then be
allowed to
grow, proliferate, and secrete factors, e.g., extracellular matrix factors,
which attach or
adhere to the cement and/or biopolymers that support, for example, cell
growth,
differentiation, morphogenesis. The cell conditioning of the bone precursor
composition or calcium cement and other biopolymer forms described herein
serves at
least two functions. First, the cells provide chemical conditioning of the
bone
15 precursor composition or calcium cement, i.e., the cells secrete
extracellular matrix
components which attract ingrowth of cells into the bone precursor composition
or
calcium cement and biopolymer forms and support the growth and differentiation
of
the cells in the foams. Second, the cells provide structural conditioning of
the bone
precursor composition or calcium cement and biopolymer forms, i.e., the cells
remodel
2o the bone precursor composition or calcium cement and biopolymer forms to
form a
scaffold which provides the appropriate physical structure for the type of
cells in the
tissue which the bone precursor composition or calcium cement is to replace or
reconstruct, e.g., the cells arrange themselves in the lacunae. The cell-
cement scaffold
can be further treated by mechanical and/ or electrical conditioning to
stimulate
2s further remodeling and strengthening of the material into a bone as cells
respond to
the applied forces. The bone precursor composition or calcium cement and/ or
biopolymer forms containing viable cells can be introduced into a recipient
subject.
Alternatively, the bone precursor composition or calcium cement and/or
biopolymer
forms containing the cells can be further processed to kill the cells, e.g.,
freeze-dried to
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remove antigenic determinants but leave the deposited extracellular matrix
macromolecules, and then introduced into a recipient subject.
The invention further includes methods for preparing bone precursor
compositions. The methods include combining calcium monopyrophosphate, alpha
s calcium sulfate hemihydrate, monobasic calcium phosphate monohydrate and
beta-tri
calcium phosphate such that bone precursor compositions are prepared. The
ingredients for the bone precursor composition can be admixed first as dry
components and in the presence of liquid vehicles as described supra to form a
paste
which can be injected or molded into desired shapes and left to cure, e.g.,
harden.. In
to one embodiment, the method includes the step of crushing hardened pellets
then
sifting and washing the particles to produce the bone precursor composition as
granules having a diameter between about 1 to 500 Vim, preferably 50 to 500 ~m
inclusive. If porosity-imparting particles have been included in the cement
composition which can then be molded, the hardened cement composition can be
~5 treated to dissolve the particles to create the pores prior to
implantation, if desired.
Alternatively, in vivo dissolution, e.g., bioabsorption, of the particles will
create pores
within the cement over time.
In yet another embodiment of the present invention, methods for producing or
repairing connective tissue in a subject is disclosed. The methods include
2o administering a bone precursor composition to the subject by injection or
implantation
at the tissue site, wherein the bone precursor composition includes calcium
pyrophosphate, calcium phosphate hemihydrate, monobasic calcium phosphate
monohydrate and beta-tri calcium phosphate. The language "producing or
repairing"
is art recognized and is intended to include the ability to cause, enhance, or
stimulate
2s tissue to grow or begin growth in a subject.
One advantage of the present invention is that the bone precursor composition
includes calcium salts such as calcium pyrophosphate, calcium sulfate
hemihydrate,
monobasic calcium phosphate monohydrate, and beta-tricalcium phosphate in
amounts which help promote the production and/or repair of the connective
tissue in
3o the subject. The bone precursor composition preferably has a ratio by
weight of
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monobasic calcium phosphate monohydrate to beta-tri-calcium phosphate of
between
about 1:2 to about 2:3, preferably about 1:3.5, more preferably about 1:3.75
and most
preferably about 1:3.05. The bone precursor composition can be preferably, in
the
form of granules with a diameter of between about 1 to 500 Vim, preferably 50
to 500
~m inclusive. Furthermore, the bone precursor compositions, which can be
granulated can include or be conditioned with cells described supra.
Alternatively,
the bone precursor composition can further include a pharmaceutically
acceptable
injection vehicle, a biopolymer foam, a therapeutic agent, a biopolymer fiber,
acid or
pepsin extracted collagen or extracellular matrix particulates.
The bone precursor compositions and calcium cements of the present invention
provide advantages over those known in the art. For example, the bone
precursor
compositions and calcium cements of the invention can be admixed such that
setting
times between about one to 15 minutes, preferably about 5 to about IO minutes,
can be
accomplished, thereby providing the practitioner with sufficient time to
formulate the
~ s bone precursor bone composition or cement and yet have the material
solidify in a
relatively short period of time after injection or application to a site in
need thereof.
Setting times, for example, were estimated by the procedure similar to that
used for
conventional cements. Set time was considered to be complete the moment a
cylindrical rod (stainless steel, 0.18 centimeters diameter, loaded with 60
grams) put
2o vertically on to the specimen no longer left any mark on its surface.
Setting time
measurements started at the end of the molding operation. The cement continues
to
cure, e.g., harden, after setting and reaches full compression strength within
48 hours
of preparation of the cement. The compressive strength, C, was calculated by
dividing
the crushing force by the cross-section of a sample, whereas the diametral
tensile
25 strength, T, was calculated from the formula T=2F/~ LD in which F is the
crushing
force, L is length and D is diameter. Values for both T and C are expressed in
MPa.
EXPERIMENTAL
Preparation of Microfibrillar Collagen:
This procedure produces a semisolid pellet of collagen microfibrils results
from
so centrifugation of a neutralized solution of collagen. Collagen, 0.5 to 15
mg/ml,
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preferably 3 to 10 mg/ml, pH about 3.0, was neutralized, by mixing with a
neutralizing solution. This collagen mixture was treated with either a dilute
base or
buffer, in a volume ratio of 85-95% collagen to 5-15% buffer. Suitable bases
were 0.02
to 2.0 M sodium, ammonium or potassium hydroxide, preferably 0.6 N NaOH at
8.5%
s volume to 91.5% collagen volume, or buffer, such as 0.02 to 2.0 M,
preferably 0.44 M,
sodium bicarbonate pH 6 to 14 or 0.02 to 2.0 M, preferably 0.2 M, sodium or
potassium
phosphate pH 6 to 14, or other buffers useful in broad pH ranges, such as tris
or
tricine. The collagen then was incubated for at least 30 min at a temperature
between
37 and 4°C, preferably 15°C. The pH was fine-tuned to between 5
and 10, preferably
pH 6 to 8, using a dilute base, such as 0.02 to 2.0 M sodium, ammonium or
potassium
hydroxide or additional amounts of the buffers mentioned above with pH's of 10
or
above. The neutral collagen formed into fibrils, which after additional
incubation time
were pelleted by centrifugation between 1000 and 30,000 x g. This
centrifugation
yielded a pellet of 3 -100 mg/ml collagen, depending on the starting
concentration of
~s the collagen, the total volume and the time spun, usually starting at 5
mg/ml,
spinning at 2000 x g for 60 min to yield a 10 -15 mg/ml pellet (more
centrifugation
time or a higher speed yielded a higher concentration collagen pellet). The
supernatant was discarded, and after gentle stirring to combine the pellet
layers, the
semisolid pellet was used for the liquid applications listed above (e.g.,
liquid
2o ingredient mixed with inorganic calcium compounds to make a cement, used to
spray
onto bleeding wounds to accelerate clotting, or is used as a vehicle to carry
particles
during an injection), or was overlaid or combined with cells for cell
cultivation or for
seeding implant structures for cell conditioning, or was poured into molds or
onto
hardened cements for freeze drying.
2s The fibrils or fibril bundles generated, as observed under a light
microscope
with 1:10.5% toluidine blue stain, were between 0.01 ~m to 20 ~,m wide and
about 0,01
mm to 3.0 mm long. The collected microfibrillar collagen pellet has a collagen
concentration of at least 7 mg/rnl and the supernatant collagen concentration
is no
more than 1 mg/ml. The collected microfibrillar collagen pellet has an
absorbance at
30 410 nm of at least 1.5, preferably over 2Ø The isolated material from the
microfibrillar
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collagen pellet has no low molecular weight collagen degradation products, as
can be
determined by electrophoretically analyzing denatured and reduced samples on a
10% dodecyl sulfate polyacrylamide gel.
Preparation of Injectable Cements
s Criteria: Injectable=mixed paste able to be loaded into a pharmaceutical
syringe
and injected through a 14 gauge needle. Set time=the time after molding when a
1.5
mm diameter rod weighed down with 60 g no longer leaves an impression on the
surface of the cement.
Key: MCPM=monobasic calcium phosphate monohydrate; ~i-TCP=beta
o tricalcium phosphate; CSH=(alpha) calcium sulfate hemihydrate; CPP=calcium
pyrophosphate; AFC=acid-extracted collagen microfibrils; PFC=pepsin-extracted
collagen microfibrils. The % collagen liquid was calculated as a percent of
the dry
weight of cement and added to the total, i.e. if 1 g total dry ingredients are
used, then
0.33 g liquid collagen was added to mix the cement into a paste for a 33%
collagen
t5 amount.
% (3- % % % Type Set Time,Strength
# MCPM Injectable
TCP ~H CPP Coll Coll min , MPa
1 16 64 15 5 none n.a. no 17 8.6
2 16 64 15 5 33 AFC no 14 8.6
3 16 64 15 5 35 AFC yes 19 11.5
4 16 64 15 5 35 PFC yes 21 7.5
20 73 6 1 35 AFC almost 11 8.3
6 8 72 15 5 35 AFC no 18 7.1
7 24 56 15 5 35 AFC yes 20 9.8
8 22 67 10 1 35 AFC yes 12 10.3
9 22 67 10 1 35 PFC yes 10 11.3
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Cement pastes were packed into a mold and were allowed to harden into a
uniform geometric shape. Injectability was determined prior to molding and
setting
time was determined with the cements in the mold. After allowing at least 48
hours to
s harden, hardened cement pellets from the mold were tested for compressive
strength,
by taking into account the surface area subjected to compression. Recipe (1)
used 33%
distilled water to mix the cement into a paste. Recipes 3 and 4, demonstrated
that
injectability was achieved at the expense of setting time. Recipes 6 and 7
vary the
relative concentration of MCPM and (3-TCP, with the lower amount being
uninjectable
o and the higher amount being injectable but slowly setting. Recipes S, 8 and
9 utilize
high MCPM while manipulating the concentrations of the CPP and CSH. High
MCPM, lower CSH and CPP in 8 and 9 resulted in injectability, quick setting
times,
and high compressive strength. The effect was noted regardless of extraction
method
of the collagen (both AFC and PFC can produce this result). The diametral
tensile
~s strength of these mixtures was measured at 9 to 10 MPa.
Casting Collagen Foams onto Cements
A mold was constructed with a solid base and wells which consisted of several
detachable horizontal layers. The first well layer was assembled onto the bed
and a
calcium cement was mixed and applied to the wells. The cements were allowed to
set
2o for ten minutes and the next layer of the mold was added. Into the next
well Layer,
fibrillar collagen was overlaid on the setting cement surface. The remainder
of the
mold was assembled and the mold was placed in the freeze dryer. The foam
portion
of this construct was seeded with chondrocytes for development of an articular
cartilage prosthesis. Mechanical conditioning of the construct for articular
cartilage, if
2s desired, is achieved by anchoring the chondrocyte-seeded construct in the
apparatus
described US patent #5,882,929.
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250 u.m Cernent Particles and Collagen
A. Injectable 250 ~m cement particles.
Hardened cement pellets (Recipe #8 described above) were ground in a mortar
and pestle. The ground material was sifted to define the size classes.
Particular size
classes were mixed with microfibrillar collagen and tested for injectability
through a
19 ga needle. Particles sifted to a size range of 140 to 250 ~.m were added in
various
proportions to acid-extracted fibrillar collagen (AFC). These particles were
injectable
in the proportion of 1 g particles to 1 g AFC prepared after neutralization
with 8% of
0.6 N NaOH. These particles were injectable in the proportion of 1 g particles
to 0.75 g
o AFC prepared after neutralization with 10% 0.2 M dibasic sodium phosphate.
These
particles were injectable in the proportion of 1 g particles to 0.5 g AFC
prepared after
neutralization with 10% sodium bicarbonate.
B. Cernent particles in single density Foams.
Hardened cement pellets (Recipe #8 described above) are ground in a mortar
~5 and pestle. The ground material was sifted to define the size classes.
Particles with
sizes between 140 ~.m and 250 ~m were collected and 1 g of these particles
were placed
into a sieve with a pore size of 53 Vim. Deionized water was poured in four 25
ml
batches onto the particles in the sieve. With swirling of the buffer, fine
particles were
washed off the larger particles. The particles then were left in the sieve to
dry at room
2o temperature. Particles were mixed with pepsin-extracted fibrillar collagen
(PFC) in
the proportion of 0.5 g particles to 10 ml PFC. The mixtures were dispensed
into
molds, freeze-dried and UV crosslinked. The resulting foams had an even
distribution
of particles throughout and after wetting, could support their own weight
without
disintegrating. The foams then could be implanted to fill in bone cavities or
used for
25 tissue culture or implantation.
A. Preparatio n of Cements including Collagen and Pore-~eneratin~ Particles
I. Calcium sulfate hemihydrate was mixed into a paste with 36% isotonic
saline. Calcium sulfate paste was loaded into pellet molds hardened and dried
for
two days. Calcium sulfate pellets were crushed and ground in a mortar. Pellet
3o particles were placed in a sieve stack and sifted. Particles with sizes
between 140 ~m
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and 400 ~m were collected and 2 g of these particles were placed into a sieve
with a
pore size of 53 Vim. A buffer of 10 mM sodium phosphate, pH 7.4 was poured in
four
25 rnl portions onto the particles in the sieve. The particles and buffer were
swirled,
causing fine particles to wash through the sieve. The remaining particles were
left in
the sieve to dry at room temperature.
II. A 22% MCPM:67% ~i-TCP:10% CSH:1% CPP cement dry mixture was
prepared and then washed. Dried 140-400 ~m calcium sulfate hemihydrate
particles
from step I were added to a proportion of 50% dry weight of the mixture.
Microfibrillar collagen was then added wet to reach a 35% weight of the final
50:50
o mixture. The components were mixed into a paste, loaded into a mold and
allowed to
set, harden and dry. Compression tests at 48 h demonstrated a 3.3 MPa strength
of the
50:50 pellets.
The product of the 22:67:10:1 hardening reaction did not dissolve readily in
deionized water; pellets were placed in deionized water to dissolve the
calcium sulfate
~s particles and produce pores in the pellets, when could then be implanted
into bone
voids. Alternatively, instead of pre-dissolving the calcium sulfate, the
pellets could be
implanted to fill bone voids and allow biological processes to dissolve the
calcium
sulfate particles before dissolving the pellet superstructure to allow pore
formation to
occur gradually during the process of bone ingrowth.
2o B. Preparation of Cements including_Collagen and Pore-generating Particles
The above experiment was conducted generally as above, with changes and
observations in the above procedure noted as follows. In Step I, calcium
sulfate paste
was loaded into pellet molds and allowed to harden and dry for two days.
Calcium
sulfate pellets were then crushed and ground in a mortar and pestle, and
pellet
2s particles were placed in a sieve stack and sifted. Particles with sizes
between 250 ~m
and 400 ~m were collected, and 2 g of these particles were placed into a sieve
with a
pore size of 53 Vim. With swirling of the sodium phosphate buffer added to the
sieve
as above and draining through the sieve, fine particles were washed through
the sieve
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off the larger particles which remained in the sieve. The particles then were
left in the
sieve to dry at room temperature.
Step II. A 22% MCPM:67% ~3-TCP:10% CSH:1% CPP cement dry mixture was
prepared as above, but washed, dried 250-400 ~,m calcium sulfate hernihydrate
s particles from step I were added to a proportion of 30 % dry weight.
Microfibrillar
collagen then was added wet to reach a 40% weight of the final 70:30 mixture.
The
components were mixed into a paste, which was confirmed to be injectable,
loaded
into a mold and allowed to set (a 9 min set time was measured), harden and
dry.
Compression tests at 48 h demonstrated a 16 MPa strength of the 70:30 pellets
compared to 22 MPa of pellets without particles. The standard product of the
22:67:10:1 hardening reaction does not dissolve readily in deionized water.
Therefore,
the pellets with the calcium sulfate particles were placed in deionized water
to
dissolve the calcium sulfate particles and pxoduce pores in the pellets, which
then
could be implanted into bone voids. Particle-containing pellets placed in
deionized
~s water for one day exhibit pores up to 250 ~m diameter and absorb liquid at
a rate 4
times faster than before creating the pores. More pores form with additional
time in
deionized water, so after two days, the liquid absorption rate is 14 times
faster than
before creating the pores. Alternatively, instead of pre-dissolving the
calcium sulfate,
the pellets could be implanted to fill bone voids and allow biological
processes to
2o dissolve the calcium sulfate particles before dissolving the pellet
superstructure to
allow pore formation gradually during the process of bone ingrowth.
Cultivation of mammalian Cells on Cement constructs
The following demonstrative cultivation of cells on cement pellets, on cement
microparticulates or in foams containing cement microparticulates. The cement
25 ingredients were sterilized by methods standard to the art. Cements were
aseptically
measured, mixed, molded into pellets and allowed to harden. If
rnicroparticulates
were used, then the pellets were ground and sifted to desired size classes. If
particulates were embedded in foams, the method of example B was followed. For
small volume culture, cements first conducted buffer conditioning. For
example, 2.4 g
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of cement pellets (Recipe #8 described above) was washed for 6 hours in 30 mL
0.05 M
tribasic sodium phosphate, pH 12, followed by water and phosphate-buffered
saline
rinses prior to equilibrating in culture medium. For larger volume cultures,
rinses and
soaks in culture medium were sufficient to ensure pH compatibility of cement
s constructs with cell culture (the pH of hardened cements is ~5.5, low for
tissue
culture). Suspensions of 1 to 5 x 105 cells per ml were added to cements for
seeding 1
to 2 h while gently agitating. After seeding, excess unattached cells were
removed
with a change of culture medium and cement constructs were returned to the
incubator for further incubation with the loose cement particulates
continuously being
o gently agitated. Cells were cultivated on cements or particulates for as
long as
desired, given sufficient medium changes. Cells were observed during culture
after
staining with fluorescent dye. Metabolic assays were performed with the cells
on the
cements or after cells were released from cements by trypsin. Cements or
particulates
with cells were fixed and prepared for histology and immunohistochemical
staining.
~5 Cement constructs supporting cell growth for sufficient time, 7 days to 3
weeks,
for the cells to deposit extracellular matrix on the cements were treated
further as cell-
conditioned cements. For this process, if desired, the cement construct were
treated
by mild solutions to iyse cells and release intracellular contents. Regardless
of
whether cell washing was undertaken, the construct was washed in dilute
neutral
2o buffer and freeze-dried. These freeze-dried materials were used as cell-
conditioned
products with much of the tissue foundation already deposited on the cement
construct and implanted by methods appropriate for each construct format for
rapid
tissue induction.
EQUI V ALENTS
2s The features and other details of the invention will now be more
particularly
described and pointed out in the claims. It will be understood that the
particular
embodiments of the invention are shown by way of illustration and not as
limitations
of the of the invention. The principal features of this invention can be
employed in
various embodiments without departing from the scope of the invention.
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Those skilled in the art will recognize, or be able to ascertain, using no
more
than routine experimentation, many equivalents to specific embodiments of the
invention described specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
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