Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
Partially degradable scaffolds for biomedical applications
Cross-reference to related applications
The present invention claims priority of U.S. provisional application serial
no. 60/939,607 filed May 22, 2007, the entire disclosure of which is
incorporated
herein by reference.
Field of the invention
The present invention is directed to a scaffold for tissue engineering,
comprising a support structure having an outer surface, at least a part of the
outer
surface being covered by a bioactive material layer coating that allows cell
attachment, wherein the bioactive material layer is at least partially
degradable in an
environment of use, to allow a detachment of the cells from the support
structure by
degradation of the bioactive material layer. The invention is further directed
to the
use of an at least partially degradable bioactive material layer on a scaffold
for tissue
engineering, for allowing detachment of the cells or grown tissue by
degradation of
the bioactive material layer.
Backaound of the invention
Tissue engineering is an emerging discipline and field of application.
Techniques are being developed to add, modify, repair or replace cells,
organized
cells, tissue, parts of organs or complete organs within the body of living
animals or
human beings, or to provide such biological constructs outside of the living
organisms. While surgical procedures for replacement or repair of tissues or
organs
may be based on artificial non-biologic substitutes, tissue engineering
potentially
could provide solutions that avoid known issues of surgically used substitutes
such
as, e. g., incompatibility of materials, foreign body reactions, wear, debris,
fatigue or
fracturing. Examples for artificial surgical substitutes include heart valves,
joint
implants, breast implants and the like.
Tissue engineering techniques can require the growth, induction of growth,
differentiation of, conduction of and organization of cells, organized cells,
tissue, and
parts of organs or complete organs of the same or different organisms.
Conventional
techniques can be based on providing a support structure, sometimes referred
to as a
scaffold, for organization. Those scaffolds can, e.g., imitate structures for
ordered
cell organization. Scaffolds can be made synthetically from biodegradable
organic
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-2-
polymer materials such as, e. g., poly(glycolic acid) (PGA), poly(lactic acid)
(PLA)
and their co-polymers.
Scaffolds are used to support the biologically active construct for adding,
modifying, repairing or replacing cells, organized cells, tissue, parts of
organs, but it
may also be desirable to use a scaffold that can be completely absorbed.
Alternatively, cells may require external and/or mechanical, structural,
electrical,
chemical or other biologic signals. Deficient, insufficient or lacking signals
can cause
improper differentiation or dedifferentiation of cells and/or result in
inappropriate or
defective organization or function. For example, growth factors may be
involved in
cell differentiation and development, for example for controlling the
migration of
cells, morphogenesis from one cell type to another or mitogenesis. The mode of
action of growth factors can include, e. g., autocrine, on neighbouring cells,
on cells
far away by travelling through the bloodstream or by inducing a single cell to
pass a
signal onto a neighbouring cell by direct cell-cell interaction. Biologically
active
agents such as growth factors may not only be present and used during the
growth of
cells, tissue or similar constructs, but also can be relevant and active for
remodelling,
repair or healing of injuries.
In some applications, it can be advantageous to use porous scaffolds, for
example, to allow the osteoconductive growth of bone-forming cells. Also,
porous
scaffolds can facilitate their seeding with cells, for example to allow the
diffusion
and/or distribution of cells into the scaffold and potentially allow a more
even
distribution. Replacement of bone is one application where an increasing
demand
may require appropriate solutions. Bone replacement can be indicated due to,
e. g.,
trauma, infections, cancer or muscular-skeletal diseases.
A range of bone grafting materials have been established in clinical use, such
as demineralised human bone matrix, bovine collagen mineral composites and
processed coralline hydroxyapatite, calcium sulphate scaffolds, bioactive
glass
scaffolds and calcium phosphate scaffolds. Such orthopedic scaffolds can be
used as
both temporary and permanent conduits for bone. These exemplary materials can
be
used to facilitate and direct the growth of bone or cartilage tissue across
sites of
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-3-
fractures or to re-grow them in defective, damaged or infected bone. The
provision of
appropriate scaffolds also requires considering the structure of bone that has
to be
treated. Cortical and cancellous bone are structurally different, although the
material
composition is very similar. Cancellous bone comprises a thin interstitium
lattice
interconnected by pores of 500-600 micron width with a spongy and open-spaced
structure, whereby the interstitium can be substituted by a scaffolding
material.
Cortical bone comprises neurovascular "Haversian" canals of about 50-100
micron
width within a hard or compact interstitium. Any suitable scaffold may allow
at least
osteoconduction or osteoinduction. Osteoinductive materials can actively
trigger and
facilitate bone growth, for example by recruiting and promoting the
differentiation of
mesenchymal stem cells into osteoblasts. Osteoconductive materials induce bone
to
grow in areas where it would not normally grow, also called "ectopic" bone
growth,
usually by biochemical and/or physical processes. Osteogenic materials may
contain
cells that can form bone or can differentiate into osteoblasts.
Different manufacturing techniques for scaffolds and porous scaffolds have
been developed and used. One conventional method of making solid scaffolds is
solvent casting, ct., e. g., Mikos, et al., Polymer, 1994, 35:1068-77; de
Groot, et al.,
Colloid Polym. ScL, 1991, 268:1073-81; Laurencin, et al, J. Biomed. Mater.
Res.,
1996, 30:133-8. Others can include solvent casting and particulate leaching,
melt
molding, fiber bonding, gas foaming or membrane lamination. For ceramic based
systems also different conventional techniques such as hydrothermal conversion
and
bum-out of dispersed polymer phase may be used.
Conventional scaffolds have many shortcomings which may limit the use and
application of tissue engineering techniques For example, it may be desirable
to
match the mechanical properties to the tissue that shall be replaced or
regenerated, to
match the degradation rate appropriately, to provide a scaffold that comprises
a
structure and geometry that allows growing, conducting and inducting different
entities of cells, organized cells, tissue, parts of organs or organs. Other
disadvantages can include that the scaffolds either can not be used for ex-
vivo
growth or, if so, then only limited for in-vivo use. Moreover, particularly
for
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-4-
scaffolds, the accurate design and distribution of pores, including the
combination of
different porous structures and fine-structures is significantly limited. For
example,
porous ceramic systems may suffer from poor control over pore size
distribution, and
may also exhibit limited moldability compared to polymers. Polymers, either
biodegradable or not, may be degraded to toxic metabolites, cause inflammation
or
allergic reactions or adversely interact with blood, such as triggering
thrombosis.
A specific disadvantage of conventional scaffolds for ex-vivo use, e.g. in a
cell cultivation system, can be that harvesting the grown tissue may be
problematic
because of difficulties to separate the grown tissue from the scaffold
material without
substantially damaging cells.
Further disadvantages can include that the function of the scaffolds is only
inductive or only conductive but not both. From a clinically relevant
perspective, a
major drawback can be that in-vivo used scaffolds are typically difficult to
be
detected by imaging methods during or after implantation.
Objects and summary of the invention
One exemplary object of the present invention is to provide a tissue
engineering scaffold that can be used ex-vivo in a cell cultivation system or
bioreactor for tissue engineering, and which may additionally optionally also
be used
in-vivo as an implantable scaffold or seed implant. Another object of the
present
invention is to provide a class of scaffolds that can be used as complex
structures for
ex-vivo and/or in-vivo growth of cells, organized cells, tissue or organs or
parts
thereof. A further object of the present invention is to provide scaffolds for
ex-vivo
use, e.g. for use in a cell cultivation system, that allow harvesting or
separation of the
grown tissue from the scaffold material without the use of enzymes and/or
essentially
without substantially damaging cells. A still further object of the present
invention is
to provide scaffolds for replacement or repair of tissues, parts of organs or
organs in-
vivo or ex-vivo. Another object of the present invention is to provide
scaffolds
wherein the mechanical, chemical, biological and physical properties such as
electrical conductivity, optical or other suitable properties can be tailored
appropriately to the intended use. One further object of the present invention
is to
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-5-
provide scaffolds that can be used for ex-vivo perfused systems, such as
assisted
systems, to partially or completely replace organ functions. Another object is
to
provide a manufacturing process for the scaffolds as described herein.
According to an exemplary embodiment of the present invention, a scaffold
for tissue engineering is provided, comprising a support structure having an
outer
surface, at least a part of the outer surface being covered by a bioactive
material layer
that allows cell attachment, wherein the bioactive material layer is at least
partially
degradable in an environment of use, to allow a detachment of the cells by
degradation of the bioactive material layer.
According to another exemplary embodiment of the present invention, a
scaffold as described above for tissue engineering is provided, which may be
used in
ex-vivo perfused systems, such as assisted systems, to partially or completely
replace
organ functions.
According to another exemplary embodiment of the present invention the use
of an at least partially degradable bioactive material layer on the surface of
a support
structure of a scaffold for tissue engineering for separating tissue grown on
the
scaffold by degrading the bioactive material layer in an environment of use is
provided.
According to a further exemplary embodiment of the present invention a
method for tissue engineering is provided, comprising the steps of providing,
in a cell
culture system or bioreactor, a scaffold comprising a support structure having
an
outer surface, at least a part of the outer surface being covered with a
bioactive
material layer that allows cell attachment, wherein the bioactive material is
at least
partially degradable in an environment of use to allow a detachment of the
cells from
the support structure by degradation of the bioactive material layer;
inoculating the
scaffold with cells or living tissue; cultivating the inoculated scaffold in a
suitable
environment to grow tissue; and harvesting the grown tissue after and/or by
optionally actively induced degradation of the bioactive layer.
For example, the environment of use may be an ex-vivo environment which
may include e.g. a liquid medium, such as a cell culture medium or a
nutritional
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-6-
medium, a solvent or solvent system, or even an at least partially gaseous
medium.
Alternatively or additionally, the environment of use may include an in-vivo
environment inside the human or animal body which is exposed to body fluids
such
as blood, blood plasma, lymph, bile, urine. In particular, such environments
may
include extra-cellular fluids such as organ-specific plasma, interstitial and
transcellular fluids. Transcellular fluids for example include the digestive
secretions
(within glands and ducti), cerebrospinal, intraocular, pleural, pericardial,
peritoneal,
seminal and synovial fluids, cochlear endolymph and the secretions of glands.
The at least partially degradable bioactive material layer is typically
provided
in the form of a coating which covers the outer surface of the scaffold's
supporting
structure at least partially, or completely. The at least partially degradable
bioactive
material layer allows a simple and efficient separation of the tissue grown on
the
scaffold, since the bioactive layer can be degraded over time in the
environment of
use, so that the tissue grown may be harvested easily in one homogeneous part
or
layer without damages after a certain time essentially determined by the
composition
of the layer and/or the environment of use. For example, the tissue may be
incrementally or gradually loosened from the underlying support structure or
an
interconnecting cell layer. Alternatively, the bioactive material layer can be
made of
a material that at least partially degrades after degradation has been
actively induced,
for example by applying an electrical current, a pH-change in the medium or
the like.
Thus, in accordance with exemplary embodiments of the present invention,
problems
conventionally associated with separating a grown tissue from e.g. complex
shaped
supports without substantially damaging the tissue can be successfully
overcome.
Also, the use of expensive enzymes to separate tissue from the support can be
avoided.
The scaffolds may be used for growing biologic constructs made of all types
of cells and organs, including, for example, but not limited to, hepatocytes,
pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, exocrine
cells, cells of
intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of
the adrenal-
hypothalamic-pituitary axis, heart muscle cells, kidney epithelial cells,
kidney
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-7-
tubular cells, kidney basement membrane cells, nerve and neural cells, blood
vessel
cells, cells forming bone and cartilage, smooth muscle cells, skeletal muscle
cells,
ocular cells, integumentary cells, keratinocytes or stem cells of mammal, in
particular human origin. The biologic constructs can include cells and
compounds
from any desired species or any combination thereof, including genetically
modified
biologic material.
A scaffold providing in accordance with exemplary embodiments can be
tailored to have conductive or inductive or combined properties for growing
cells and
tissues. Such scaffold may also comprise rationally designed structures to
allow
engraftment, ingrowth, induction or conduction of attached cells or tissues or
any
combination thereof. For example, as desired for a particular application, the
support
may have a complex structure, e.g. osteoconductive, structure while it is
still possible
to easily detach the grown tissue from the support by inducing degradation of
the
bioactive material layer covering the support and forming the physical link
between
the support and the adherent cells or tissue grown there upon.
A further aspect is that the present invention comprises a class of scaffolds
that can incorporate and/or release or absorb beneficial agents useful for
growth,
induction or conduction, differentiation or dedifferentiation of cells and
living tissue.
Definitions
The terms "biodegradable" or "degradable" as used herein refer to any
material which can be removed in-vivo or ex-vivo in an environment of use,
e.g. by
(bio)corrosion or (bio)degradation. Thus, any material, e.g. a metal or
organic
polymer that can be degraded, absorbed, metabolized, or which is resorbable in
the
human or animal body, in a cell culture system or bioreactor may be used in
the
embodiments of the present invention. Also, as used in this description, the
terms
"biodegradable", "bioabsorbable", "resorbable", and "biocorrodible" refer to
materials that are broken down and may be gradually absorbed or eliminated,
regardless whether these processes are due to hydrolysis, metabolic processes,
bulk
or surface erosion.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-8-
The term "biological construct" as used herein refers to and can be used as a
synonym for cells, any agglomeration of cells or tissue which may be grown on
the
scaffold and harvested from it, specifically living cells, organized cells,
living tissue,
and parts of organs or complete organs of the same entity or different
entities,
particularly of mammals, animals and human origin, also including cells and
tissue
having been genetically modified.
The terms "active ingredient", "active agent" or "beneficial agent" as used
herein can include any material or substance which may be used to add a
function to
the scaffold. Examples of such active ingredients include biologically,
therapeutically or pharmacologically active agents such as drugs or
medicaments,
diagnostic agents such as markers, or absorptive agents. The active
ingredients may
be a part of the support structure or the bioactive material layer, such as
incorporated
into the scaffold or being coated on at least a part of the scaffold.
Biologically or
therapeutically active agents comprise substances being capable of providing a
direct
or indirect therapeutic, physiologic and/or pharmacologic effect in a human or
animal organism. A therapeutically active agent may include a drug, pro-drug
or
even a targeting group or a drug comprising a targeting group. An "active
ingredient" may further include a material or substance which may be activated
physically, e.g. by radiation, or chemically, e.g. by metabolic processes.
The term "porous" as used herein designates a property of a material, which
is determined by the presence of a plurality of interconnected pores. The
volume of
the pores can be assessed by measuring the porosity of the material as
conventionally
known, e. g. by N2-adsoption methods such as BET and further defined herein.
"Porous" does not include holes such as boreholes or the like.
The term "support structure" is used to designate the bulk structure of the
scaffold, i.e. the device body. To the contrary, a coating cannot be a part of
a support
structure.
Exemplary embodiments of the present invention will now be described in
greater detail. The following description makes reference to numerous specific
details in order to provide a thorough understanding of the present invention.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-9-
However, each and every specific detail needs not to be employed to practice
the
present invention. Also, the features of all exemplary embodiments are
principally
combinable with each other, if not expressly stated otherwise.
Detailed description of preferred embodiments
In an exemplary embodiment of the invention a scaffold is provided which
includes a support structure which is at least partially covered on its outer
and/or
inner surfaces with a bioactive and at least partially degradable material
layer. The
degradable bioactive material layer can allow a separation of the tissue grown
on the
scaffold while it is degraded over time in the environment of use, or its
degradation
can be induced by a stimulus, so that the tissue grown may be harvested easily
in one
homogeneous part or layer without significant damages, by reducing or
eliminating
the physical contact between the tissue and the scaffolds support structure or
the
bioactive material layer on its outer surface. Such exemplary embodiments of
the
present invention can overcome the problems conventionally associated with
separating the grown tissue from e.g. complex shaped supports essentially
without
damaging the tissue. Also, the use of expensive enzymes to separate tissue
from the
support can be avoided.
The support structure may have any desired shape or form, depending on the
specific application, suitable for growing cells or tissue on it. For example,
the
support structure may have a honeycomb, mesh or tubular structure for ex-vivo
cell
culturing systems, or may be in the form of a "must-fit" implant for
replacement of
bone or cartilage, which may be implanted into the human or animal body after
tissue
has been grown on the scaffold in an ex-vivo culturing system. Basically, the
scaffold can be made from one part or from an assembly of multiple parts as
desired.
Support structure
The support structure may consist of or include materials such as, e.g.
inorganic, organic or mixed inorganic/organic hybrid materials, including
materials
as conventionally used for cell culture supports or tissue engineering
scaffolds,
which may be porous or non-porous, and may be structured or designed as
desired
for the intended application. The support structure can be made from the same
or a
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-10-
different material than the bioactive material layer, typically from a
different
material, so that the materials discussed below in the context of the support
structure
may also be used in the bioactive material layer and vice versa, with the
prerequisite
that the material of the bioactive material layer is at least partially
degradable in an
environment of use as described herein. Also, the support structure material
and the
bioactive material layer may be both degradable, preferably having different
degradation rates/properties. In exemplary embodiments, the support structure
is
made of a substantially non-degradable material.
For example, the support structure can be made from at least one of an
inorganic material, an organic material, an inorganic-organic hybrid material,
a
carbon material, a polymer material, a ceramic material, a metal or metal
alloy
material, or any composites or combinations thereof.
In an exemplary embodiment of the present invention the support structure
consists of or includes organic materials. Such materials can include
biocompatible
polymers, oligomers, or pre-polymerized forms as well as polymer composites
which
may include at least partially degradable materials. The polymers used may be
thermosets, thermoplastics, synthetic rubbers, extricable polymers, injection
molding
polymers, moldable polymers, spinnable, weavable and knittable polymers,
oligomers or pre-polymerizes forms and the like or mixtures thereof. In
exemplary
embodiments, the material of the support structure can include organic
materials
being biodegradable per se, such as, but not limited to, collagen, albumin,
gelatin,
hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, carboxymethylcellulose-phtalate); furthermore
casein, dextrane, polysaccharide, fibrinogen, poly(D,L lactide), poly(D,L-
lactide-Co-
glycolide), poly(glycolide), poly/hydroxybutylate), poly(alkylcarbonate),
poly(orthoester), polyester, poly(hydroxyvaleric acid), polydioxanone,
poly(ethylene
terephtalate), poly(maleic acid), poly(tartaric acid), polyanhydride,
polyphosphohazene, poly(amino acids), and/or copolymers or mixtures thereof.
In another exemplary embodiment the support structure is based on inorganic
composites, organic composites or hybrid inorganic/organic composites.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-11-
In one exemplary embodiment, the support structure of the scaffold includes
or consists of glassy carbon or vitreous carbon, e.g., a non-graphitizing type
of
inorganic carbon material, which combines glassy and ceramic properties with
those
of graphite. Important properties of this material are, e.g. its
biocompatibility, bio-
inertness, and resistance to chemical attack. Glassy carbon is a conventional
material,
widely used, e.g., as an electrode material in electrochemistry, and may be
produced
from organic precursor materials such as polymers or phenolic resins at
temperatures
up to 3000 C by carbonization, and may further be widely varied in its
physical
properties.
The structure of glassy carbon can be 100% sp2 -hybridized carbon, e.g., a
graphite or fullerene like structure. Certain molecular models assumed that
both sp2
and sp3 -bonded atoms may be present. A later model was based on the
assumption
that the molecular orientation of the polymeric precursor material can be
"memorized" to some extent after carbonization. Thus, the structure may bear
some
resemblance to that of a polymer, in which the "fibrils" can be very narrow
curved
and twisted ribbons of graphitic, and thus inorganic, carbon. However, more
recent
research has suggested that glassy carbon has a fullerene-related structure.
For
example, glassy or vitreous carbon can include two-dimensional structural
elements
(sp2-C) and does not exhibit `dangling' bonds, such as e.g. amorphous carbon
does.
The support structure may include amorphous carbon, e.g., a glassy carbon
material that essentially does not have any crystalline structure, but can
include a
certain amount of sp3-carbon structural elements. Amorphous carbon can reveal
some short-range order, but there may be no long-range pattern of atomic
positions.
In further exemplary embodiments, the support structure may include
diamond-like carbon (DLC) which is also an amorphous carbon material that can
display some of the properties of diamond. Such materials may contain
significant
amounts, for example, up to 100 %, of sp3 hybridized carbon atoms, wherein the
carbon atoms may be arranged in a cubic lattice or a hexagonal lattice, or
mixtures
thereof. Furthermore, mixtures of amorphous, diamond-like, vitreous, glassy,
or
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-12-
other carbon materials may be used for preparing the support structure of
exemplary
scaffolds of the present invention.
Optionally, the carbon material, such as amorphous, diamond-like, vitreous or
glassy carbon material may be mixed with other materials such as metals,
alloys,
ceramic, polymers, or the like, preferably in minor amounts, e.g. less than 30
% by
weight, preferably less than 10 % by weight. According to exemplary
embodiments
of the present invention the optionally porous scaffolds may have a carbon
content of
at least about 20% by weight, preferably sp2 carbon or, in specific
embodiments, sp3
carbon or any mixture thereof. Inorganic materials with sp2 carbon or sp3
carbon
contacted with physiologic fluids or living cells or tissue exhibit bioinert
or bioactive
properties and can be superior to other materials in terms of cytotoxicity,
haemocompatibility, inflammation or engraftment and respective tissue or cell
adhesion.
Different materials may also be used, e. g., to provide or form different
sections or parts of the scaffold support structures. According to an
exemplary
embodiment of the invention, the support structure includes a composite
material
comprising inorganic carbon as described above, and a further inorganic
material
selected from, e.g., at least one of a metal, a metal alloy, or a metal
compound.
According to an exemplary embodiment, an optionally porous support
structure can consist of or include a metal compound, a metal or metal alloys,
e.g.
metals and metal alloys selected from main group metals of the periodic
system,
transition metals such as copper, gold and silver, titanium, zirconium,
hafiiium,
vanadium, niobium, tantalum, chromium, molybdenum, tungsten, manganese,
rhenium, iron, cobalt, nickel, ruthenium, rhodium, palladium, osmium, iridium
or
platinum, or from rare earth metals. For example, the metal compound may
include
zero-valent metals, metal oxides, metal carbides, metal nitrides, metal
oxynitrides,
metal carbonitrides, metal oxycarbides, metal oxynitrides, metal
oxycarbonitrides
and the like, and any mixtures thereof. The metals or metal oxides or alloys
used may
also be magnetic. Examples include iron, cobalt, nickel, manganese and
mixtures
thereof, for example iron, platinum mixtures or alloys, or for example,
magnetic
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
- 13 -
metal oxides such as iron oxide and ferrite. Semi-conducting materials or
alloys may
also be used, for example semi-conductors from Groups II to VI, Groups III to
V,
and Group IV. Suitable Group II to VI semi-conductors include, for example,
MgS,
MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe,
ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, or mixtures thereof. Examples for
suitable Group III to V semi-conductors are GaAs, GaN, GaP, GaSb, InGaAs, InP,
InN, InSb, InAs, AlAs, AIP, AISb, AIS and mixtures thereof. Examples for Group
IV semi-conductors include germanium, lead and silicon. The semi-conductors
may
also comprise mixtures of semi-conductors from more than one group and all the
groups mentioned above are included.
Metal compounds which may be used can include metals or metal-oxides or
alloys that comprise MRI visibility or radiopacity, preferably implants made
from
ferrite, tantalum, tungsten, gold, silver or any other suitable metal, metal
oxide or
alloy, such as platinum-based radiopaque steel alloys, so-called PERSS
(platinum-
enhanced radiopaque stainless steel alloys), cobalt alloys or any mixture
thereof. In
such embodiments, the scaffold can be detectable by non-invasive diagnostic
methods, e.g., if used in-vivo.
Exemplary, support structure may also include a combination or composite of
carbon materials such as those described herein, together with a metal or
metal alloy
as described above.
In certain embodiments, the material of the support structure can be carbon-
based, i.e. having a carbon content of at least 50% or more. For example, such
carbon-based material can be made by using discrete carbon particles. Such
particles
can include tubes, fibers, fibrous materials or wires or spherical or
dendritic or any
regular or irregular particle form and the preferred particle sizes are in,
but not
limited to, a range of lnm up to 1000 m. Suitable particles can include carbon
species such as fullerenes, in particular C36, C60, C70, C76, C80, C86, C112
etc., or
any mixtures thereof, , nanotubes such as MWNT, SWNT, DWNT, random-oriented
nanotubes, so-called fullerene onions or metallo-fullerenes, also graphite
fibers, or
particles or diamond particles. The material can be a composite, for example a
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-14-
carbon material combined with a polymer, metal or metal alloy, ceramic or bio-
ceramic, mineral or any mixture thereof.
In an exemplary embodiment, the scaffold can be formed from a
biodegradable metal, alloy or metal composite as described in further detail
below
with respect to the bioactive material layer, for example, an alloy based on
magnesium or calcium. Such scaffold can be primarily degraded to hydroxyl
apatite
within a medium or living body. This property can be especially advantageous
for
scaffolds with a temporary function. By alloying the aforesaid metals it is
possible to
control the physiologic or ex-vivo degradation rate from a few days up to 20
years.
Moreover, by introducing precious metals, either within the alloy, or as a
part of the
scaffold, either ex-vivo or in-vivo, or alternatively by applying a current or
voltage,
for example, with an appropriate electrode or similar device, the degradation
can be
substantially altered. Using a metal also can allow to utilize the mechanical
strength
of these compounds and to provide a tailored scaffold that can both satisfy
any
mechanical requirements as well as being biodegradable.
In an exemplary embodiment, the composition of the materials for the support
structure as described above is selected such that the degradation rate of the
support
structure is lower, e.g., by about 10 %, 20 %, 50% or even 100 % lower than
the
degradation rate of the bioactive material layer, to allow a detachment of the
grown
tissue before the support is completely degraded.
In other embodiments the scaffold can be formed using a composite
comprising at least about 10% of a degradable metal composition together with
a
polymer or polymer mixture that can be degradable or not, or with a ceramic,
also
degradable or not, or any mixture thereof.
The scaffold can also be made using an inorganic or an organic material, such
as, e. g., a metal, a ceramic material, or a composite such as an inorganic-
organic
hybrid material, as further defined herein below, either degradable or not.
The scaffold material can also include materials that are capable of absorbing
specific compounds or of exchanging ions, or diagnostic markers and the like,
as
further described herein below.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
- 15 -
Bioactive material layer
A scaffold of exemplary embodiments of the present invention includes at
least one bioactive surface that allows cell attachment. The bioactive
material layer
can cover the surface of the support partially or completely. To facilitate
detachment
of grown tissue, at least a portion of the bioactive surface of the scaffold
includes a
bioactive material layer that is at least partially degradable in an
environment of use,
e.g. a cell culture medium or a body fluid, as described above. The bioactive
material
layer can comprise a patch-like composite of degradable and non-degradable
structures and materials. Degradation is useful, e.g. if the biologic
construct has to be
detached without using enzymes that affect the vitality or the structure of
such
biologic constructs or tissue. For example, controlled detachment of biologic
constructs by applying enzymes in conventional complex formed or shaped
scaffolds
with porous structures can be difficult.
In further exemplary embodiments, the bioactive material layer consists of or
includes, attached to the support structure, an at least partially degradable
or
corrodible layer of an inorganic material such as a solid metal or metal
alloy, or
optionally metal or metal alloy particles embedded in a matrix material, e.g.
an
alkoxide-based gel or a polymer. For example, the at least partially
degradable layer
may consist of or include biodegradable metals or metal oxides, carbides,
nitrides
and mixed forms thereof, or metal alloys, including alkaline or alkaline earth
metals,
Fe, Zn or Al, such as Mg, Fe or Zn, and optionally alloyed with or combined
with
other metals selected from Mn, Co, Ni, Cr, Cu, Cd, Pb, Sn, Th, Zr, Ag, Au, Pd,
Pt,
Si, Ca, Li, Al, Zn and/or Fe. Other suitable materials include, e.g., alkaline
earth
metal oxides or hydroxides such as magnesium oxide, magnesium hydroxide,
calcium oxide, and calcium hydroxide or mixtures thereof.
In exemplary embodiments, the degradable bioactive material layer can
include biodegradable or biocorrosive metals or alloys based on at least one
of
magnesium or zinc, or an alloy comprising at least one of Mg, Ca, Fe, Zn, Al,
W, Ln,
Si, or Y. Furthermore, the bioactive material layer may be substantially
completely
or at least partially degradable in-vivo or ex-vivo as described herein.
Examples for
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-16-
suitable biodegradable alloys include e.g. magnesium alloys comprising more
than
90 % of Mg, about 4-5 % of Y, and about 1.5-4 % of other rare earth metals
such as
neodymium and optionally minor amounts of Zr; or biocorrosive alloys
comprising
as a major component tungsten, rhenium, osmium or molybdenum, for example
alloyed with cerium, an actinide, iron, tantalum, platinum, gold, gadolinium,
yttrium
or scandium.
The biodegradable metal or metal alloy may include in an exemplary
embodiment
(i) About 10-98 wt.-%, about 35-75 wt.-% of Mg, and about 0-70 wt.-%, or
about 30-40% of Li and about 0-l2wt.-% of other metals, or
(ii) About 60-99wt.-% of Fe, about 0.05-6wt.-% Cr, about 0.05-7wt.-% Ni
and up to about l Owt.-% of other metals; or
(iii) About 60-96wt.-% Fe, about 1-lOwt.-% Cr, about 0.05-3wt.-% Ni and
about 0-l5wt.-% of other metals, wherein component percentages selected for a
particular alloy add up to 100 %.
In further exemplary embodiments, the bioactive material layer may include
particles of the above mentioned biodegradable metallic materials embedded in
e.g. a
polymeric matrix or a sol/gel derived matrix which may itself be degradable or
not.
Degradation of the metallic particles in the bioactive layer allows a partial
degradation of the layer where the particles are exposed to the medium and/or
biological cells, resulting in the loss of "anchoring sites" for the cells or
tissue, thus
leading to a detachment of the tissue from the support.
In other preferred embodiments, detachment is achieved by bulk degradation
of the bioactive material layer or parts of the bioactive layer. Specifically,
the
bioactive layer can be partially or completely made from magnesium, magnesium
compounds or magnesium alloys. In one example, the bioactive layer is a
composite
material of an inorganic or organic polymer or sol/gel derived matrix
comprising
magnesium or magnesium alloy particles, preferably in a particle size range of
100nm to 2000 m, more referred from 200nm to 10 m and most preferred from
200nm to 1 m. In physiologic fluids or suitably selected liquid media, the
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-17-
magnesium or magnesium alloys will be degraded to degradation products such as
hydroxyl apatite and hydrogen gas. If partially or completely used in or for
the
bioactive material layer, the degradation of the magnesium-based materials or
other
degradable metal materials over time will result in a loss of integrity of the
bioactive
layer and allow mechanical removal of previously attached cells or biological
constructs. In specifically preferred embodiments the magnesium, magnesium
compounds or magnesium alloys are used as particulates within a bioactive
layer,
preferably with a volume content of 10 to 90 %, more preferred from 20% to 90%
and most preferred from 30% to 60% of degradable metallic or metal-based
particles,
e.g. magnesium particles, in a polymeric or sol/gel-derived matrix. Adhesion
of cells
and biological constructs will then be affected over time due to a loss of
connective
surface. Preferred materials for use as the matrix for such composite
bioactive layers
are biocompatible polymers such as poly(lactic) acid (PLA) or PGLA, and the
other
biocompatible and/or biodegradable polymers mentioned herein below. In further
embodiments the bioactive material layer is completely made out of magnesium,
magnesium compounds or magnesium alloys, for example using thin metallic foils
at
least partially covering the support.
The bioactive material layer may also consist of or comprise, for example as a
matrix of a composite material, an organic material such as a polymer that can
be
degraded ex-vivo or in-vivo, and such layer can be used to detach the biologic
construct ex-vivo. Useful polymers can include pH-sensitive polymers, shape
memory polymers and the like. The polymers used should be biocompatible. In
exemplary embodiments, it can be particularly preferred to select polymers
from pH-
sensitive polymers, such as: homopolymers such as poly(amino carboxylic acid),
poly(acrylic acid), poly(methyl acrylic acid), and their copolymers. This
applies
likewise for polysaccharides such as celluloseacetatephthalate,
hydroxylpropylmethylcellulose-phthalate,hydroxypropylmethylcellulosesuccinate,
celluloseacetate trimellitate and chitosan. Further polymers may be selected
from
temperature sensitive polymers, such as, for example, however not exclusively:
poly(N-isopropylacrylamide-co-sodium-acrylate-co-n-N-alkylacrylamide),poly(N-
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-18-
methyl-N-n-propylacrylamide), poly(N-methyl-N-isopropylacrylamide), poly(N-N-
propylmethacrylamide), poly(N-isopropylacrylamide), poly(N,N-
diethylacrylamide),
poly(N-isopropylmethacrylamide), poly(N-cyclopropylacrylamide), poly(N-
ethylacrylamide), poly(N-ethylmethylacrylamide), poly(N-methyl-N-
ethylacrylamide), poly(N-cyclopropylacrylamide). Other polymers suitable to be
used as polymers with thermogel characteristics are hydroxypropylcellulose,
methylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose and
pluronics such as F-127, L-122, L-92, L-81, L-61. Preferred polymers include
also,
however not exclusively, functionalized styrene, such as amino styrene,
functionalized dextrane and polyamino acids. Furthermore, polyamino acids,
(poly-
D-amino acids as well as poly-L-amino acids), for example polylysine, and
polymers
which contain lysine or other suitable amino acids may be used. Other useful
polyamino acids are polyglutamic acids, polyaspartic acid, copolymers of
lysine and
glutamine or aspartic acid, copolymers of lysine with alanine, tyrosine,
phenylalanine, serine, tryptophan and/or proline.
In exemplary embodiments, the material for the bioactive material layer can
include organic materials being biodegradable per se, for example collagen,
albumin,
gelatin, hyaluronic acid, starch, cellulose (methylcellulose,
hydroxypropylcellulose,
hydroxypropylmethylcellulose, carboxymethylcellulose-phtalate); furthermore
casein, dextrane, polysaccharide, fibrinogen, poly(D,L lactide), poly(D,L-
lactide-Co-
glycolide), poly(glycolide), poly/hydroxybutylate), poly(alkylcarbonate),
poly(orthoester), polyester, poly(hydroxyvaleric acid), polydioxanone,
poly(ethylene,
terephtalate), poly(maleic acid), poly(tartaric acid), polyanhydride,
polyphosphohazene, poly(amino acids), and all of the copolymers and any
mixtures
thereof. Such polymeric materials may also be used in the bioactive material
layer as
a matrix material, e.g., to embed biodegradable metallic or other particles as
described above, thus providing a composite bioactive material layer.
In further exemplary embodiments, the bioactive material layer may include
particles of the above mentioned biodegradable polymer materials embedded in
e.g. a
metallic matrix or a sol/gel derived matrix or combined with metallic
particles, which
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-19-
may all itself be degradable or not. Degradation of the polymeric particles in
the
bioactive layer allows a partial degradation thereof, resulting in the loss of
"achoring
sites" for the cells or tissue, thus leading to a detachment of the tissue
from the
support.
In further exemplary embodiments, the bioactive material layer may comprise
or consist of an inorganic-organic hybrid material or a ceramic material. For
example, the hybrid material can be obtained using a sol-gel processing
technique
and may include a gel obtained by hydrolysis of a reaction mixture comprising
a
silicon alkoxide compound. For example, the reaction mixture may further
include a
carbon material selected from at least one of fullerenes, for example, C36,
C60, C70,
C76, C80, C86, Cl12 etc., or any mixtures thereof, carbon nanotubes such as
MWNT, SWNT, DWNT, random-oriented nanotubes, as well as so-called fullerene
onions or metallo-fullerenes, soot, lamp black and the like. Carbon fibers,
diamond
particles or graphite particles may also be used.
Biocompatibility of such carbon-based materials may be used to promote cell
adhesion to the bioactive material layer, and the typically non-degradable
carbon
species can be embedded in a material that is at least partially degradable.
Sol/gel technology allows for the production of highly biocompatible, in
some instances bioerodible or biodegradable materials at low temperatures.
Sol/gel-
process derived materials may be used to form at least partially degradable
bioactive
material layers, or these materials may be used to form suitable matrices for
degradable particles as described above, suitable for providing the bioactive
material
layer. Additionally, sol/gel-derived materials may be easily applied to a
support in
the form of a liquid sol coating, which is then cured to form the bioactive
layer.
The sol/gel-process technology can be widely applied to build up different
types of networks. The linkage of the components under formation of the sol or
gel
can take place in several ways, e.g. via conventional hydrolytic or non-
hydrolytic
sol/gel-processing, and may be used to produce, e.g., aerogels or xerogels.
A "sol" is a dispersion of colloidal particles in a liquid, and the term "gel"
connotes an interconnected, rigid network of pores of typically sub micrometer
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-20-
dimensions and polymeric chains whose average length is typically greater than
a
micrometer. For example, the sol/gel-process may involve mixing of the
precursors,
e.g. a sol/gel forming components into a sol, adding further additives or
materials,
casting the mixture in a mould or applying the sol onto a substrate in the
form of a
coating, gelation of the mixture, whereby the colloidal particles are linked
together to
become a porous three-dimensional network, aging of the gel to increase its
strength;
converting the gel into a solid material by drying from liquid and/or
dehydration or
chemical stabilisation of the pore network, and densification of the material
to
produce structures with ranges of physical properties. Such processes are
described,
for example, in Henge and West, The SoUGel-Process, 90 Chem. Ref. 33 (1990).
The term "sol/gel" as used within this specification may mean either a sol or
a
gel. The sol can be converted into a gel as mentioned above, e.g. by aging,
curing,
raising of pH, evaporation of solvent or any other conventional methods, which
can
result in inorganic-organic hybrid materials or ceramic materials.
The term semi-solid refers to materials having a gel-like consistency, i.e.
being sbstantially dimensionally stable at room temperature, but have a
certain
elasticity and flexibility, typically due to a residual solvent content.
The bioactive material layer may be produced from a sol which is
subsequently being converted into a solid or semi-solid material layer. The
sol can
be prepared from any type of sol/gel forming components in a conventional
manner.
The skilled person will -depending on the desired properties and requirements
of the
material to be produced - select the suitable components / sols for preparing
the
bioactive material layer based on his professional knowledge.
The sol/gel forming components can include, e. g., alkoxides, oxides,
acetates, nitrates of various metals, e.g. silicon, aluminum, boron,
magnesium,
zirconium, titanium, alkaline metals, alkaline earth metals, or transition
metals, and
from platinum, molybdenum, iridium, tantalum, bismuth, tungsten, vanadium,
cobalt,
hafinium, niobium, chromium, manganese, rhenium, iron, gold, silver, copper,
ruthenium, rhodium, palladium, osmium, lanthanum and lanthanides, as well as
combinations thereof.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-21 -
In some exemplary embodiments of the present invention, the sol/gel forming
components can be selected from metal oxides, metal carbides, metal nitrides,
metaloxynitrides, metalcarbonitrides, metaloxycarbides, metaloxynitrides, and
metaloxycarbonitrides of the above mentioned metals, or any combinations
thereof.
These compounds, which may be in the form of colloidal particles, can be
reacted
with oxygen containing compounds, e.g. alkoxides to form a sol/gel, or may be
added as fillers if not in colloidal form.
In other exemplary embodiments of the present invention, the sols may be
derived from at least one soUgel forming component selected from alkoxides,
metal
alkoxides, colloidal particles, particularly metal oxides and the like. The
metal
alkoxides that may be used as sol/gel forming components may be conventional
chemical compounds that may be used in a variety of applications. These
compounds
have the general formula M(OR)X wherein M is any metal from a metal alkoxide
which e.g. may hydrolyze and polymerize in the presence of water. R is an
alkyl
radical of 1 to 30 carbon atoms, which may be straight chained or branched,
and x
has a value equivalent to the metal ion valence. Metal alkoxides such as
Si(OR)4,
Ti(OR)4, Al(OR)3, Zr(OR)3 and Sn(OR)4 may be used. Specifically, R can be the
methyl, ethyl, propyl or butyl radical. Further examples of suitable metal
alkoxides
can include Ti(isopropoxy)4, Al(isopropoxy)3, Al(sec-butoxy)3, Zr(n-butoxy)4
and
Zr(n-propoxy)4.
In exemplary embodiments, the sols are based on silanes or alkoxide
compounds, e.g.. being made from silicon alkoxides such as tetraalkoxysilanes,
wherein the alkoxy may be branched or straight chained and may contain 1 to 25
carbon atoms, e.g. tetramethoxysilane (TMOS), tetraethoxysilane (TEOS) or
tetra-n-
propoxysilane, as well as oligomeric forms thereof. Also suitable are
alkylalkoxy-
silanes, wherein alkoxy is defined as above and alkyl may be a substituted or
unsubstituted, branched or straight chain alkyl having about 1 to 25 carbon
atoms,
e.g., methyltrimethoxysilane (MTMOS), methyltriethoxysilane,
ethyltriethoxysilane,
ethyltrimethoxysilane, methyltripropoxysilane, methyltributoxysilane,
propyltri-
methoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, isobutyltri-
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-22-
methoxysilane, octyltriethoxysilane, octyltrimethoxysilane, which is
commercially
available from Degussa AG, Germany, methacryloxydecyltrimethoxysilane
(MDTMS); aryltrialkoxysilanes such as phenyltrimethoxysilane (PTMOS), phenyl-
triethoxysilane, which is commercially available from Degussa AG, Germany;
phenyltripropoxysilane, and phenyltributoxysilane, phenyl-tri-(3-glycidyloxy)-
silane-
oxide (TGPSO), 3-aminopropyltrimethoxysilane, 3-aminopropyl-triethoxysilane,
2-aminoethyl-3-aminopropyltrimethoxysilane, triaminofunctional propyltri-
methoxysilane (Dynasylan TRIAMO, available from Degussa AG, Germany), N-
(n-butyl)-3-aminopropyltrimethoxysilane, 3-aminopropylmethyl-diethoxysilane, 3-
glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropyltriethoxy-silane, vinyl-
trimethoxysilane, vinyltriethoxysilane, 3-mercaptopropyltrimethoxy-silane,
Bisphenol-A-glycidylsilanes; (meth)acrylsilanes, phenylsilanes, oligomeric or
polymeric silanes, epoxysilanes; fluoroalkylsilanes such as
fluoroalkyltrimethoxy-
silanes, fluoroalkyltriethoxysilanes with a partially or fully fluorinated,
straight chain
or branched fluoroalkyl residue of about 1 to 20 carbon atoms, e.g.
tridecafluoro-
1,1,2,2-tetrahydrooctyltriethoxysilane and modified reactive
flouroalkylsiloxanes
which are available from Degussa AG under the trademarks Dynasylan F8800 and
F8815; as well as any mixtures of the foregoing. Such sols may be easily
converted
into solid porous aerogels by drying.
In another exemplary embodiment of the present invention, the sol may be
prepared from carbon-based nano-particles and organic alkaline or earth
alkaline
metal salts, e.g. their formiates, acetates, propionates, malates, maleates,
oxalates,
tartrates, citrates, benzoates, salicylates, phtalates, stearates, phenolates,
sulfonates,
and amines, as well as acids, such as phosphorous acids, pentoxides,
phosphates, or
organo phosphorous compounds such as alkyl phosphonic acids. Further
substances
that may be used to form sols for e.g. bioerodible or degradable bioactive
material
layers include sols made from magnesium acetate, calcium acetate, phosphorous
acid, P205 as well as triethyl phosphite as a sol in ethanol or ethanediol,
whereby
biodegradable composites can be prepared from physiologically acceptable
organic
or inorganic components. For example, by varying the stoichiometric Ca/P-
ratio, the
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
- 23 -
degeneration rate of such composites can be adjusted. A molar ratio of Ca to P
can be
about 0.1 to 10, or preferably about 1 to 3.
In some exemplary embodiments of the present invention, the sols can be
prepared from colloidal solutions, which may comprise carbon-based
nanoparticles,
preferably in solution, dispersion or suspension in polar or nonpolar
solvents,
including aqueous solvents as well as cationically or anionically
polymerizable
polymers as precursors, such as alginate. By addition of suitable coagulators,
e.g.
inorganic or organic acids or bases, including acetates and diacetates, carbon
containing composite materials can be produced by precipitation or gel
formation.
Optionally, further additives can be added to adjust the properties of the
resultant
drug delivery material.
The sol/gel components used in the sols may also comprise colloidal metal
oxides, preferably those colloidal metal oxides which are stable long enough
to be
able to combine them with the other sol/gel components and the polymer-
encapsulated active agents. Such colloidal metal oxides may include, but are
not
limited to, Si0z, A1203, MgO, Zr02, Ti02, Sn0z, ZrSiO4, B203, La203, Sbz05 and
ZrO(N03)2. Si0z, A1203, ZrSiO4 and Zr02 may be preferably selected. Further
examples of the at least one sol/gel forming component include
aluminumhydroxide
sols or -gels, aluminumtri-sec-butylat, A100H-gels and the like.
Such colloidal sols may be acidic in the sol form and, therefore, when used
during hydrolysis, it may not be necessary to add additional acid to the
hydrolysis
medium. These colloidal sols can also be prepared by a variety of methods. For
example, titania sols having a particle size in the range of about 5 to 150 nm
can be
prepared by the acidic hydrolysis of titanium tetrachloride, by peptizing
hydrous
Ti02 with tartaric acid and, by peptizing ammonia washed Ti(S04)z with
hydrochloric acid. Such processes are described, for example, by Weiser in
Inorganic
Colloidal Chemistry, Vol. 2, p. 281 (1935). To preclude the incorporation of
contaminants in the sols, the alkyl orthoesters of the metals can be
hydrolyzed in an
acid pH range of about 1 to 3, in the presence of a water miscible solvent,
wherein
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-24-
the colloid is present in the dispersion in an amount of about 0.1 to 10
weight
percent.
Where the sol is formed by a hydrolytic sol/gel-process, the molar ratio of
the
added water and the sol/gel forming components, such as alkoxides, oxides,
acetates,
nitrides or combinations thereof may be in the range of about 0.001 to 100,
preferably from about 0.1 to 80, more preferred from about 0.2 to 30.
In a typical hydrolytic sol/gel processing procedure which can be used in
exemplary embodiments of the invention, the sol/gel components are optionally
blended with (optionally chemically modified) fillers such as biodegradable
particles
in the presence of water, applied to the surface of the support structure and
cured.
Optionally, further solvents or mixtures thereof, and/or further additives may
be
added, such as surfactants, fillers and the like. The solvent may contain
salts, buffers
such as PBS buffer or the like to adjust the pH value, the ionic strength etc.
Further
additives such as cross linkers may be added, as well as catalysts for
controlling the
hydrolysis rate of the sol or for controlling the cross linking rate. Non-
hydrolytic
sols may be similarly made, but likely essentially in the absence of water.
When the sol is formed by a non-hydrolytic sol/gel-process or by chemically
linking the components with a linker, the molar ratio of the halide and the
oxygen-
containing compound may be in the range of about 0.001 to 100, or preferably
from
about 0.1 to 140, even more preferably from about 0.1 to 100, particularly
preferably
from about 0.2 to 80.
In non-hydrolytic soUgel processes, the use of metal alkoxides and carboxylic
acids and their derivatives may also be suitable. Suitable carboxylic acids
include
acetic acid, acetoacetic acid, formic acid, maleic acid, crotonic acid,
succinic acid,
their anhydrids, esters and the like.
Non-hydrolytic sol/gel processing in the absence of water may be
accomplished by reacting alkylsilanes or metal alkoxides with anhydrous
organic
acids, acid anhydrides or acid esters, or the like. Acids and their
derivatives may be
suitable as sol/gel components and/or for modifying/functionalizing the
encapsulated
active agents.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
- 25 -
In certain exemplary embodiments of the present invention, the sol may also
be formed from at least one soUgel forming component in a non-hydrous sol/gel
processing, and the reactants can be selected from anhydrous organic acids,
acid
anhydrides or acid esters such as formic acid, acetic acid, acetoacetic acid,
succinic
acid, maleic acid, crotonic acid, acrylic acid, methacrylic acid, partially or
fully
fluorinated carboxylic acids, their anhydrides and esters, e.g. methyl- or
ethylesters,
and any mixtures of the foregoing. It is often preferred to use acid
anhydrides in
admixture with anhydrous alcohols, wherein the molar ratio of these components
determines the amount of residual acetoxy groups at the silicon atom of the
alkylsilane employed.
Typically, according to the degree of cross linking desired in the resulting
sol,
either acidic or basic catalysts may be applied, particularly in hydrolytic
sol/gel
processes. Suitable inorganic acids include, for example, hydrochloric acid,
sulfuric
acid, phosphoric acid, nitric acid as well as diluted hydrofluoric acid.
Suitable bases
include, for example, sodium hydroxide, ammonia and carbonate as well as
organic
amines. Suitable catalysts in non-hydrolytic sol/gel processes include
anhydrous
halide compounds, for example BC13, NH3, A1C13, TiC13 or mixtures thereof.
In certain exemplary embodiments of the present invention, the sol may be
further modified by the addition of at least one cross linking agent to the
sol, the
encapsulated active agent or the combination. The cross linking agent may
comprise, for example, isocyanates, silanes, diols, di-carboxylic acids,
(meth)acrylates, for example such as 2-hydroxyethyl methacrylate,
propyltrimethoxysilane, 3-(trimethylsilyl)propyl methacrylate, isophorone
diisocyanate, polyols, glycerine and the like. Biocompatible cross linkers
such as
glycerine, diethylene triamino isocyanate and 1,6-diisocyanato hexane may be
preferably used.
Fillers can be used to modify the pore sizes and the degree of porosity, if
desired. Some preferred fillers include inorganic metal salts, such as salts
from
alkaline and/or alkaline earth metals, preferably alkaline or alkaline earth
metal
carbonates, -sulfates, -sulfites, -nitrates, -nitrites, -phosphates, -
phosphites, -halides, -
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-26-
sulfides, -oxides, as well as mixtures thereof. Further suitable fillers
include organic
metal salts, e.g. alkaline or alkaline earth and/or transition metal salts,
such as
formiates, acetates, propionates, malates, maleates, oxalates, tartrates,
citrates,
benzoates, salicylates, phtalates, stearates, phenolates, sulfonates, and
amines as well
as mixtures thereof.
Preferably, porosity in the resultant bioactive material layers can be
produced
by treatment processes such as those described in German Patent publication
DE 10335131 and in PCT Application No. PCT/EP04/00077. Further additives may
include, e.g., drying-control chemical additives such as glycerol, DMF, DMSO
or
any other suitable high boiling point or viscous liquids that can be suitable
for
controlling the conversion of the sols to gels and solid or semi-solid
materials.
Sol-gels can be cured into a solid or semi-solid bioactive material layers
Curing into a gel, preferably an aerogel or xerogel, may be accomplished by,
e.g.,
aging, raising of pH, evaporation of solvent or any other conventional method.
The
sol may be preferably cured at room temperature, particularly where the
materials
used result in polymeric glassy composites, aerogels or xerogels.
Curing can be achieved by drying including a thermal treatment of the
sol/combination or gel, in the range of about -200 C to +200 C, such as in the
range
of about -100 C to 100 C, or in the range of about -50 C to 100 C, about 0
C to
90 C, or even from about 10 C to 80 C or at about room temperature. Drying
or
aging may also be performed at any of the above temperatures under reduced
pressure or in vacuo.
Typically, many sol/gel derived materials obtained by hydrolysis reactions
are biodegradable in physiologic fluids or cell culturing media themselves.
Additionally, biodegradable particles such as metals or polymers as described
above
may be embedded in sol-gel-derived materials in some exemplary embodiments, to
provide the bioactive material layer. In such embodiments, degradation of the
particles in the bioactive layer allows a partial degradation thereof,
resulting in the
loss of "anchoring sites" for the cells or tissue, thus leading to a
detachment of the
tissue from the support.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-27-
If particles are provided in the bioactive material layer, the particles can
be
selected from tubes, fibers, fibrous materials or wires or spherical or
dendritic or any
regular or irregular particle form and the particle sizes can be in, but not
limited to, a
range of about 1 nanometer (nm) up to about 1000 micrometer ( m), such as from
1
nm to 500 gm, or from 1 nm to 10 m.. The degradable layer can be porous.
In further preferred embodiments, the support structure can be made from
magnesium, magnesium compounds or magnesium alloys, and the bioactive layer
comprises a slower degradation rate. In these embodiments, the bioactive layer
including the cell culture or biological construct will be available after
degradation of
the support structure for in-vivo implantation, wherein the layer is the
subsequently
degraded in-vivo.
In other embodiments, the support structure includes pH-sensitive or
temperature sensitive polymers as described above, that change the properties
upon
change of pH or temperature either resulting, e.g., in a shift of surface
charge,
volume and/or surface structure allowing the removal of the bioactive material
layer
together with the biological construct thereon in toto.
Functionalization and further exemplary embodiments
As described herein above, the bioactive material layer may include
biodegradable inorganic, organic or inorganic-organic hybrid materials in a
particulate form. The materials can, e.g., comprise organic or inorganic micro-
or
nano-particles or any mixture thereof, which may also be included in the
support
structure. In the bioactive layer or in the support, the degradable material
particles
may be used to accelerate degradation and detachment of cells, in that
selective
degradation of the particles may mechanically destabilize the structure of the
layer or
support structure.
Furthermore, it may also be preferable to add substantially non-degradable
particles to the bioactive material layer or the support structure, regardless
whether it
already includes degradable particles or not.
For example, the particles added in some exemplary embodiments of the
present invention can include materials enhancing diagnostic properties or
visibility
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
- 28 -
in diagnostic methods, such as at least one of zero-valent metals, metal
oxides, metal
carbides, metal nitrides, metal oxynitrides, metal carbonitrides, metal
oxycarbides,
metal oxynitrides, metal oxycarbonitrides and the like. The particles may also
be
magnetic, e.g. to allow attracting, by magnetic forces, cell material or
tissue adhering
to the magnetic particles floating in a medium after detachment from the
support.
Examples for magnetic particles are - without excluding others - iron, cobalt,
nickel,
manganese and mixtures thereof, for example iron, platinum mixtures or alloys,
or
for example, magnetic metal oxides such as iron oxide and ferrite.
Semi-conducting particles may also be used, e.g. to improve visibility for
diagnostic or monitoring purposes, for example semi-conductors, as mentioned
as
diagnostic agents in WO 2006/069677 and as described herein above.
Particles may also include super paramagnetic, ferromagnetic, ferromagnetic
metal particles. Suitable examples are magnetic metals, alloys, preferably
made of
ferrites such as gamma-iron oxide, magnetite or cobalt-, nickel- or manganese
ferrites, particularly particles as disclosed in WO 83/03920, WO 83/01738, WO
85/02772 and WO 89/03675; and US patent 4,452,773, US patent 4,675,173; and
WO 88/00060 and US patent 4,770,183; WO 90/01295 and WO 90/01899.
Additionally, particles incorporated into the support structure or in the
bioactive material layer of the scaffolds may include carbon particles, for
example
soot, lamp-black, flame soot, furnace soot, gaseous soot, carbon black, and
the like,
furthermore, carbon-containing nano particles and any mixtures thereof.
Particle
sizes especially for carbon-based particles are in the region of about 1 nm to
1,000
m, such as from 1 nm to 300 m, or from 1 nm to 6 m.
Nano-morphous carbon species may also be used, such as fullerenes and the
like as mentioned herein above.
In a further exemplary embodiment, the particles can include polymers,
oligomers or pre-polymeric particles such as beads. Examples of suitable
polymers
for use as particles in the present invention are mentioned herein above.
In certain exemplary embodiments, the particles can include electrically
conducting polymers, preferably from saturated or unsaturated
polyparaphenylene-
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-29-
vinylene, polyparaphenylene, polyaniline, polythiophene,
poly(ethylenedioxythiophene), polydialkylfluorene, polyazine, polyfurane,
polypyrrole, polyselenophene, poly-p-phenylene sulfide, polyacetylene,
monomers
oligomers or polymers thereof or any combinations and mixtures thereof with
other
monomers, oligomers or polymers or copolymers made of the above-mentioned
monomers. Particularly preferred are monomers, oligomers or polymers including
one or several organic, for example, alkyl- or aryl-radicals and the like or
inorganic
radicals, such as, for example, silicon or germanium and the like, or any
mixtures
thereof. Preferred are conductive or semi-conductive polymers having an
electrical
resistance between l0exp(12) and l0exp(12) Ohm=cm. It may particularly be
preferred to select those polymers which comprise complexated metal salts.
In a further exemplary embodiment, cell attachment can be further promoted
by a physical surface design of the bioactive material layer, hereinafter
referred to as
a "texture". Appropriate textures are ultra-micro porous or nano-porous
structures.
Nano-porous surfaces in a range of about 2 nm (nanometer) up to 20 nm
particularly
promote cell attachment. In exemplary embodiments, the attachment efficiency
of
cells may be tuned specifically by increasing or decreasing the size and
density of the
pores, depending on the properties of the cells or tissue to be grown on the
scaffold.
For example, for HUVEC (Human Umbilical Vein Endothelial Cells) it was found
that the optimal attachment efficiency can be realized with a pore size at
about 2 nm,
for osteoblast cells it was found that the optimum pore size is at about 500
nm.
Applicable pore sizes for improving cell attachment in exemplary embodiments
may
therefore be selected in a range from about 2 nm to about 10 m (micrometer),
depending on the cell type and cell entity. It is assumed that the
implementation of
the aforesaid textures is mimicking the known tight and gap junctions of cell-
cell
contacts. These textures may be applied to all materials an material
combinations as
described herein.
In exemplary embodiments it can be preferred to have, as the bioactive
material layer or as an additional layer on top thereof, a degradable
biochemical
active surface on the scaffold. Biochemical active surfaces are capable to
interact
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-30-
with glycoproteins, carbohydrates, proteins, lipids, lipoproteins, glycolipids
and
similar compounds that allow cell attachment, such as membrane or
transmembrane
receptors. Examples for cell adhesion proteins include selectins, integrins as
well as
cadhereins. Those membrane compounds of cells are capable to bind to
absorptive
surfaces or physiologic ions. Presence of calcium, sulfur, magnesium or heavy
metals, preferably as ions, on the surface of scaffolds, can promote the
adhesion of
such cell attachment structures of cells, whereby the selection of the ions
and ions
entities significantly allows attracting or attaching selectively specific
cells and cell
types. An isolated specific membrane receptor or transmembrane receptor can be
included as a biochemical active surface in the bioactive material layer. The
bioactive material layer may comprise at least one of physiologically
acceptable ions
and at least one isolated specific membrane receptor or transmembrane
receptor, or
both.
According to a further exemplary embodiment of the present invention,
physiologic ions or salts may be embedded into the surface of the bioactive
material
layer and/or into the support structure material. Preferably, the material may
include
at least one physiologically acceptable ion, i.e. cation or anion, that is
embedded into
the material structure, e.g. at the surface. Preferably, the ions are selected
from
alkaline or alkaline earth ions such as calcium (Ca2+), magnesium (Mg2+),
sodium
(Na+), potassium (K+), lithium (Li+), cesium (Cs+), aluminum (A13+) a
transition
metal ion such as Zn2+, Ag+, a heavy metal ion, anions such as sulfur,
Chloride(Cl-),
nitrate (N03), sulfate (S042 ), phosphate (P043-) etc, most preferred ions of
Ca and S
and Mg.
Such ions may include any ion having a beneficial effect on the tissue grown.
The ratio between some of the anions and cations can be varied, according to
the
underlying nature of the cell type that selectively shall be attached. A
mixture can be
selected resulting in different patterns of ions and ion concentrations. As it
is widely
accepted, the attachment of cells to physiologic matrices such as
extracellular matrix
(ECM) is depending on the interaction between cell adhesion molecules (CAMs),
such as cadherins, Ig superfamily of CAMs (for example N-CAM, V-CAM) and
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-31 -
integrins. The ECM, for, example is well known to comprise substances such as
proteoglykanes that are chemically containing a core protein and
glykosaminoglykanes. Like ceratane sulphate or heparansulfate, many of those
compounds comprise sulfur groups and the like, e. g., or they have partially
the
function to bind selectively physiologic ions such as, sodium, magnesium or
calcium.
Incorporating a tissue-specific composition of ions within the support
material or the
bioactive material layer may directly correlate with the selective attachment
and
growth of cells.
Conventional techniques may be used for measuring or verifying the surface
structure and composition. One exemplary method is ESCA (Electron Spectroscopy
for Chemical Application), another suitable method is EDX (Energy Dispersive X-
ray Spectroscopy). The enrichment or doping of the material with at least one
physiologic ion results preferably in following content (atom%): 0,1% up to
90%,
preferably 1% to 85%, most preferred 5% to 25%, as measured by EDX.
Ion exchangers which may be included in the scaffolds include those for
binding positively charged ions or cations, which display on their surface
negatively
charged groups; or those for binding negatively charged ions or anions, which
display on their surface positively charged groups. The ion exchanger can be
composed of the solid support material, a liquid or gel, or any combination
thereof,
such as for example a hydrogel or polymer composed for easily hydrated groups
such
as cellulose consisting of polymers of sugar molecules. These materials
consist of
polymeric matrixes to which are attached functional groups. The chemistry of
the
matrix structure is polystyrenic, polyacrylic or phenol-formaldehyde. The
functional
groups are numerous: sulfonic, carboxylic acids, quatemary, tertiary and
secondary
ammonium, chelating (thiol, iminodiacetic, aminophosphonic and the like). The
various types of matrices and their degree of cross linking translate into
different
selectivity for given species and into different mechanical and osmotic
stability. Ion
exchange resins are also characterized by their operating capacities function
of the
process conditions. Ion exchange resins are mostly available in a moist beads
form
(granular or powdered forms are also sometime used, dry form is also available
for
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-32-
applications in a solvent media) with a particle size distribution typically
ranging 0.3
- 1.2 mm (16 - 50 mesh) with a gel or macro porous structure. The ion
exchanger
may be included in at least one part of the scaffold, for example in the
active layer or
in the structural material. Other useful materials are absorbents to absorb at
least one
compound of the cells, organized cells, tissue, organ or biologic construct,
either in-
vivo or ex-vivo.. Suitable absorbers, for example, are used to absorb
proteins. For
protein absorption diethylaminoethyl (DEAE) or carboxymethyl (CM) absorbers
are
appropriate. Since proteins are charged molecules, proteins in the cultivation
system
will interact with the absorber depending on the distribution of charged
molecules on
the surface of the protein, displacing mobile counter ions that are bound to
the resin.
The way that a protein interacts with the absorber material depends on its
overall
charge and on the distribution of that charge over the protein surface. The
net charge
on a given protein can depend on the composition of amino acids in the protein
and
on the pH of the fluid. The charge distribution can further depend on how the
charges
are distributed on the folded protein. An appropriate absorber or combination
of
absorbers and/or the pH of the fluid can be selected based on the protein's
isoelectric
point, for adjusting the absorption properties and function.
In addition to conventional absorbents, further useful absorbents can include
from materials that comprise imidazolium, quatemary ammonium, pyrrolidinium,
pyridinium, or tetra alkylphosphonium as the base for the cation, whereby
possible
anions include hexafluorophosphate [PF6]-, tetrafluoroborate [BF4]-,
bis(trifluoromethylsulfonyl) imide [(CF3S02)2N]-, triflate [CF3SO3]-, acetate
[CH3CO2]-, trifluoroacetate [CF3CO2]-, nitrate [N03] , chloride [Cl]-, bromide
[Br]-,
or iodide [I]-, among many others. Any combination of a absorbing material can
be
selected. Suitable absorbers are also activated carbon or activated carbon-
like
materials, chelating agents such as penicillamine, methylene tetramine
dihydrochloride, ethylenediaminetetraacetic acid (EDTA),
Distearyldimethylamine
(DMSA) or deferoxamine mesylate and the like. The absorber can be provided as
a
liquid solution, gel, solid or any combination thereof. The solid can include
particles,
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-33-
a structured mold or any combination thereof. The absorber can be included in
the
active layer and/or the structural material of the scaffold.
Further, beneficial agents may be added to the bioactive material layer and/or
the support structure. Beneficial agents can be selected from biologically
active
agents, pharmacological active agents, therapeutically active agents,
diagnostic
agents or absorptive agents or any mixture thereof. Beneficial agents can be
incorporated partially or completely into the support or bioactive layer or
into a
further overcoating of the scaffold. Furthermore, it is also one aspect of the
present
invention to optionally or further coat the inventive scaffold with beneficial
agents
partially or completely, e.g. with growth factors, gene-vectors etc.
Examples of beneficial ingredients include biologically, therapeutically or
pharmacologically active agents such as drugs or medicaments, diagnostic
agents
such as markers, ion exchangers or absorptive agents. The active ingredients
may be
incorporated into the scaffold or being coated on at least a part of the
scaffold.
Biologically or therapeutically active agents comprise substances being
capable of
providing a direct or indirect therapeutic, physiologic and/or pharmacologic
effect in
a cell, tissue or a human or animal organism. A therapeutically active agent
may
include a drug, pro-drug or even a targeting group or a drug comprising a
targeting
group. An "active ingredient" may further include a material or substance
which
may be activated physically, e.g. by radiation, or chemically, e.g. by
metabolic
processes.
Suitable therapeutically active agents may be selected from the group of
enzyme inhibitors, hormones, cytokines, growth factors, receptor ligands,
antibodies,
antigens, ion binding agents such as crown ethers and chelating compounds,
substantial complementary nucleic acids, nucleic acid binding proteins
including
transcriptions factors, toxines and the like. Further examples of active
agents,
beneficial agents, absorbents, signal-generating agents and diagnostic agents
or
markers are disclosed in WO 2006/069677, all of which are incorporated herein
by
reference.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-34-
Geometric scaffold structures
The support structure may have any desired shape or form, depending on the
specific application, suitable for growing cells or tissue on it. For example,
the
support structure can have a honeycomb, mesh or tubular structure for ex-vivo
cell
culturing systems, or it may be in the form of a "must-fit" implant for
replacement of
bone or cartilage, which may be implanted into the human or animal body after
tissue
has been grown on the scaffold in an ex-vivo culturing system. Basically, the
scaffold can be made from one part or from an assembly of multiple parts as
desired.
Material used to make the support and/or the bioactive material layer may be
porous, e.g. nanoporous, ultramicroporous, microporous or mesoporous or
macroporous, or may have combined pores or porosities. The materials can be
completely or partially porous within any section or part or at different
sections or
parts. The porous scaffold material can include a gradient of different porous
layers
or sections in any desired geometric or three-dimensional direction. For
example, the
bioactive material layer can have a bulk volume porosity of about 10-90 %,
such as
from about 30% to 80%, or from about 50% to 80%. Also, such pores can have a
dimension suitable for osteoconduction, such as from about 50 to 1000 m.
Porosity
and average pore sizes may be measured by conventional methods such as
adsorptive
methods, e.g. N2- or Hg- porosity measurements. The porous structure can also
be
partially or completely a mesh-like porous structure or a lattice, or it may
comprise a
mesh-like trabecular, regular or irregular or random or pseudo-random
structure, or
any combination thereof.
In an exemplary embodiment, the scaffold can be provided in a shape of a
cylinder having at least one excavation. For example, the excavation may pass
through a mould body connecting one side of the surface with another side. The
excavation may include a flow-channel for inflow or outflow or through-flow of
a
fluid, fluid mixture or components or compounds of a fluid or fluid mixture,
wherein
the flow-channel can be centric or eccentric, linear or non-linear. The
scaffold may
include a Y-like or star-shaped form, at least in one plane, whereby the form
has at
least three parts that intersect at a node and the parts may be formed to
lamellas with
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-35-
a linear profile in the cross-section. The combined shape can be symmetric or
asymmetric, regular or irregular, whereby each individual lamella can have a
different geometry.
The scaffold may also have a honeycomb-like structure. The honeycomb
configuration can be provided, e. g., as a pentagonal, hexagonal, polygonal or
tubular
or rectangular or any other geometric configuration, preferably a symmetric
pattern.
Special embodiments
In an exemplary embodiment, the bioactive material layer includes a
combination of degradable metal particles, e.g. particles based on Mg, Ca,
and/or Zn
or alloys thereof, as described above, with magnetic particles, e.g. iron-
based
particles, both optionally embedded in a degradable or non-degradable polymer
or
sol/gel-derived material.
In a further exemplary embodiment having enhanced biocompatibility or
haemocompatibility, the bioactive material layer includes nanomorphous carbon
species, such as fullerenes, for example, C36, C60, C70, C76, C80, C86, Cl 12
etc.,
or any mixtures thereof, furthermore, nanotubes such as MWNT, SWNT, DWNT,
random-oriented Nanotubes, as well as so-called fullerene onions or metallo-
fullerenes, in a silane based inorganic-organic hybrid material, which may
optionally
be prepared by using sol/gel technology.
Completely degradable scaffolds may provide a degradation rate that
corresponds to the re-growth or repair rate of the tissue. Typical
biodegradation rates
for maintaining the structure or structural integrity of a scaffold can be, e.
g., about 4-
10 weeks for cartilage repair and about 3-8 weeks for bone repair. The
mechanical
requirements of the scaffolds can be highly dependent on the type of tissue
being
replaced. For example, cortical bone has a Youngs Modulus of 15-30 GPa whereby
cancellous (or spongy, trabecular) bone has a Youngs Modulus of 0.01-2GPa.
Cartilage has a Youngs Modulus of less than 0.001 GPa. The materials used for
a
scaffold in any particular case should be selected appropriately to reflect
such
considerations.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-36-
Manufacturin
The scaffolds can, for example, be manufactured using conventional methods,
for example by coating, sputtering, molding or metallizing a template as
disclosed in
US-Patent Applications 12/016,835; 12/030,304; 12/016,519; 12/030,392;
12/030,350; 12/030,315; 12/030,680; 12/098,282; 12/033,238 and 12/016,536.
Other
suitable methods may use an organic precursor or polymer or pre-polymer and
carbonize the material. Such techniques are described, e. g., in WO
2005/021462;
WO 2004/082810; WO 2004/101177; WO 2004/101017; WO 2004/105826; WO
2004/101433; WO 2005/012504; WO 2005/042045; WO 2005/065843; WO
2006/074809; and WO 2006/069677
Techniques for producing porous support structures are disclosed in
W02005/021462 Al, including techniques for introducing porosity into carbon
materials produced by carbonization of organic polymer precursors. The
aforesaid
techniques also permit molding of a scaffold to any desired geometric shape.
Production of sol/gel-based coatings that can be degraded in physiologic
fluids and
which may be applied to at least a portion of the scaffold, are described, e.
g., in WO
2006/077256 or WO 2006/082221.
Tissue en inn method
In an exemplary embodiment of the present invention a method for tissue
engineering is provided, comprising the steps of providing, in a cell culture
system or
bioreactor, a scaffold as described herein, the scaffold comprising a support
structure
having an outer surface, at least a part of the outer surface being covered
with a
bioactive material layer that allows cell attachment, wherein the bioactive
material is
at least partially degradable in an environment of use to allow a detachment
of the
cells from the support structure by degradation of the bioactive material
layer;
inoculating the scaffold with cells or living tissue; cultivating the
inoculated scaffold
in a suitable environment to grow tissue; harvesting the grown tissue after
degradation of the bioactive layer.
The properties of the scaffold or the bioactive material layer may be modified
as described above, e.g. with other substances selected from organic and
inorganic
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-37-
substances or compounds. Examples include compounds or ions of iron, cobalt,
copper, zinc, manganese, potassium, magnesium, calcium, sulphur or phosphorus.
The incorporation of such additional compounds may be used, for example, to
promote the growth of certain tissue or cells on the scaffold. Further,
impregnation or
coating of the scaffold with carbohydrates, lipids, purines, pyromidines,
pyrimidines,
vitamins, proteins, growth factors, amino acids and/or sulfur sources or
nitrogen
sources are also suitable in promoting growth.The following substances may
also be
used to stimulate cell growth: bisphosphonates (e.g., risedronates,
pamidronates,
ibandronates, zoledronic acid, clodronic acid, etidronic acid, alendronic
acid,
tiludronic acid), fluoride (disodium fluorophosphate, sodium fluoride);
calcitonin,
dihydrotachystyrene as well as all growth factors and cytokins (epidermal
growth
factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors
(FGFs), transforming growth factors b (TGFs-b), trans-forming growth factor a
(TGF-a), erythropoietin (Epo), insulin-like growth factor I(IGF-I), insulin-
like
growth factor II (IGF-II), interleukin 1(IL-1), interleukin 2 (IL-2),
interleukin 6 (IL-
6), interleukin 8 (IL-8), tumor necrosis factor a (TNF-a), tumor necrosis
factor b
(TNF-b), interferon g (INF-g), monocyte chemo-tactic protein, fibroblast
stimulating
factor 1, histamine, fibrin or fibrinogen, endothelin 1, angiotensin II,
collagens,
bromocriptine, methysergide, methotrexate, carbon tetrachloride,
thioacetamide,
ethanol.
In an exemplary embodiment of the present invention, the scaffold is loaded
with viable and/or propagable biological material, capable of replication,
such as
single-cell or multi-cell micro organisms, fungi, spores, viruses, plant
cells, cells
culture or tissue or animal or human cells, cell cultures or tissue or
mixtures thereof.
Such loading typically leads to extensive immobilization of the biological
material.
The loading can be performed with tissue-forming or non-tissue-forming
mammalian cells, primary cell cultures such as eukaryotic tissue, e.g., bone,
cartilage, skin, liver, kidney as well as exogenous, allogenic, syngenic or
autologous
cells and cell types and optionally also genetically modified cell lines and
in
particular also nerve tissue.
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-38-
The biological material can be applied to the scaffold using conventional
methods. Examples include, e. g., immersion of the scaffold in a
solution/suspension
of the cell material, spraying the scaffold with cell material solution or
suspension,
inoculating a fluid medium in contact with the scaffold and the like. An
incubation
time may be used after loading to allow the immobilized biological material to
completely permeate the supporting body or substrate.
Such scaffolds are suitable in particular for immobilizing and propagating all
types of tissue cultures, especially cell tissues. In such processes, the
cells can be
supplied with liquid or gaseous nutrients in a bioreactor, while metabolites
may be
removed easily with a fluid flow.
The scaffolds, optionally installed in suitable housings to form cartridge
systems which are optionally loaded with different cell cultures, may be
immersed in
a single culture medium for the sake of reproduction and may be removed from
the
culture medium after a certain period of time, when the bioactive material
layer is
sufficiently degraded to separate the tissue from the support structure.
Suitable ex-vivo bioreactors include e.g., flasks, bottles, in particular cell
culture flasks, roller bottles, spinner bottles, culture tubes, cell culture
chambers, cell
culture dishes, culture plates, pipette caps, snap cover dishes, cryotubes,
agitated
reactors, fixed bed reactors, tubular reactors and the like.
Before, during or after loading with the biological material, the scaffold is
brought in contact with a fluid medium. The fluid medium may optionally be a
different medium before loading than after loading. The term "fluid medium"
includes any fluid, gaseous, solid or liquid, such as water, organic solvents,
inorganic
solvents, supercritical gases, conventional substrate gases, solutions or
suspensions
of solid or gaseous substances, emulsions and the like. The medium is
preferably
selected from liquids or gases, solvents, water, gaseous or liquid or solid
reaction
educts and/or products, liquid culture media for enzymes, cells and tissues,
mixtures
thereof and the like.
Examples of liquid culture media include, for example, RPMI 1640 from Cell
Concepts, PFHM II, hybridoma SFM and/or CD hybridoma from GIBCO, etc. These
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-39-
may be used with or without serum, e.g., fetal bovine serum medium with or
without
amino acids such as L-glutamine. The fluid medium may also be mixed with
biological material, e.g., for inoculating the scaffold.
The contact may be accomplished by complete or partial immersion of the
scaffold or the housing/container holding it into the fluid medium. The
scaffolds may
also be secured in suitable reactors so that fluid medium can flow through
them. An
important criterion here may be the wettability and removability of any
enclosed air
bubbles from the substrate material. Evacuation, degassing and/or flushing
operations may be necessary here and may be used as needed.
The scaffold can be immersed in a solution, emulsion or suspension
containing the biological material for a period for time from about 1 second
up to
about 1000 days or may be inoculated with it, optionally under sterile
conditions, to
give the material an opportunity to diffuse into the porous body and form
colonies
there. The inoculation may also be performed by spray methods or the like.
The fluid medium, e.g., a culture medium, may be moved or agitated to
ensure the most homogeneous possible vital environment and supply of nutrients
to
the cells. This may be accomplished through various methods, e.g., by moving
the
scaffold in the medium or moving the medium through the scaffold. This is
usually
done for a sufficient period of time to permit growth, reproduction or
adequate
metabolic activity of the biological material.
Harvesting of tissue or cells may be done as desired and as further described
herein, after degradation of at least a part of the bioactive material layer.
In an exemplary embodiment of the present invention, the scaffold may also
be used in ex-vivo perfused systems, such as assisted systems, to partially or
completely replace organ functions. For example the scaffold can include
supporting
structure formed from a carbon-based material, preferably made from pyrolytic,
glassy carbon, diamond-like carbon or any other carbon material obtainable by
carbonization or using CVD or PVD methods, for example a porous, spongy or
trabecular carbon body, and a bioactive surface structure that supports cell
attachment. The bioactive surface structure can include a biodegradable
polymer
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-40-
coating such as a poly(lactic acid) coating, or other such polymers as
described
herein above. For long-term cultivation, the bioactive material layer
comprises a
bioactive nano-structure with a porosity in a range of 2nm up to 500nm to
support
cell attachment of hepatocytes or any co-culture comprising hepatocytes or
hepatic
progenitor cells. Preferably, the scaffold has a random or pseudo-random or
geometric mesh-structure in order to trigger a trabecular growth pattern of
the
adherent cell culture and to increase the available biologically active
surface of the
scaffold within the assisted system.
Hence, the scaffold can have any desired superordinate mould structure,
preferably with flow-channels or appropriate shape for gas exchange within a
compartment of the scaffold. In some preferred configurations, the compartment
in
the scaffold is first used to seed, for example, hepatocytes or hepatic
progenitor cells
or any co-culture comprising hepatocytes or hepatic progenitor cells, and to
increase
the cell density until the complete surface of the scaffold has been covered
by the
culture. Such a pre-grown tissue on the scaffold can subsequently be used as a
part
or component of an liver assisting system, taking over at least a part of the
organ
function of liver tissue outside the patient's body. In this embodiment
patient blood
is circulated through the compartment of the scaffold comprising the liver
tissue with
sufficient contact time.
In other embodiments, the scaffold can be made from a carbon hollow-fiber
membrane and the bioactive material layer is comprised by a biodegradable
polymer
coating such as a PLA coating. The layer can provide a nano-structured porous
surface for promoting attachment and growth of, e.g. different entities of
renal cells,
renal progenitor cells or any combination thereof, preferably comprising renal
proximal tubule cells or the like. An exemplary use of this scaffold includes
a first
step of seeding and cultivating the cells on the scaffold and subsequently
providing
the scaffold and respective compartment to a kidney assisting system.
In further embodiments the scaffolds include magnesium-based alloys as the
bioactive material layer. Such scaffolds can preferably be used to seed and
cultivate
CA 02687637 2009-11-18
WO 2008/142129 PCT/EP2008/056299
-41 -
chondroblasts, chondrocytes, osteoblasts or osteocytes or any co-culture
thereof
within a bio-reactor to produce cartilage or bone tissue.
***
Having thus described in detail several exemplary embodiments of the
present invention, it is to be understood that the invention described above
is not to
be limited to particular details set forth in the above description, as many
apparent
variations thereof are possible without departing from the spirit or scope of
the
present invention. The embodiments of the present invention are disclosed
herein or
are obvious from and encompassed by the detailed description. The detailed
description, given by way of example, but not intended to limit the invention
solely
to the specific embodiments described, may best be understood in conjunction
with
the accompanying Figures.
The foregoing applications, and all documents cited therein or during their
prosecution ("appln. cited documents") and all documents cited or referenced
in the
appln. cited documents, and all documents cited or referenced herein ("herein
cited
documents"), and all documents cited or referenced in the herein cited
documents,
together with any manufacturer's instructions, descriptions, product
specifications,
and product sheets for any products mentioned herein or in any document
incorporated by reference herein, are hereby incorporated herein by reference,
and
may be employed in the practice of the invention.
The invention is further characterized by the following claims, which should
not be construed to limit the invention to the feature combinations given
therein.
