U.S. patent application number 10/106007 was filed with the patent office on 2003-09-25 for channeled biomedical foams and method for producing same.
Invention is credited to Brown, Kelly R., Chun, Iksoo, Melican, Mora C..
Application Number | 20030181978 10/106007 |
Document ID | / |
Family ID | 28040901 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181978 |
Kind Code |
A1 |
Brown, Kelly R. ; et
al. |
September 25, 2003 |
Channeled biomedical foams and method for producing same
Abstract
The present invention provides a biomedical, biocompatible,
polymeric foam scaffold suitable for use in the repair and
regeneration of tissue and which contains located therein a network
of, branched channels that are effective to encourage and
facilitate vascularization and tissue growth within the scaffold
and to methods for making such biomedical scaffolds.
Inventors: |
Brown, Kelly R.;
(Hillsborough, NJ) ; Melican, Mora C.;
(Bridgewater, NJ) ; Chun, Iksoo; (Flemngton,
NJ) |
Correspondence
Address: |
AUDLEY A. CIAMPORCERO JR.
JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
28040901 |
Appl. No.: |
10/106007 |
Filed: |
March 25, 2002 |
Current U.S.
Class: |
623/11.11 ;
264/28; 264/41 |
Current CPC
Class: |
A61F 2002/30787
20130101; A61L 27/18 20130101; C08L 67/04 20130101; A61F 2/0077
20130101; A61F 2/30767 20130101; A61F 2210/0004 20130101; C08L
67/04 20130101; A61F 2/28 20130101; A61L 27/56 20130101; A61L 31/06
20130101; A61F 2002/3093 20130101; A61F 2/30756 20130101; A61F
2002/30677 20130101; A61F 2002/4648 20130101; A61L 31/146 20130101;
A61F 2002/30762 20130101; A61F 2230/0063 20130101; A61F 2002/0086
20130101; A61F 2002/30199 20130101; A61F 2250/0067 20130101; A61F
2002/2817 20130101; A61L 27/18 20130101; A61F 2002/30062 20130101;
A61F 2002/30766 20130101; A61F 2002/30785 20130101; A61L 31/06
20130101 |
Class at
Publication: |
623/11.11 ;
264/28; 264/41 |
International
Class: |
A61F 002/02; B29C
065/00 |
Claims
We claim:
1. A biomedical, biocompatible scaffold suitable for use in the
repair and regeneration of tissue, comprising a polymeric foam,
said foam comprising a network of branched channels effective to
encourage and facilitate vascularization and tissue growth in said
scaffold.
2. The biomedical scaffold of claim 1 wherein said scaffold is
bioabsorbable.
3. The biomedical scaffold of claim 1 wherein said scaffold
comprises a bioabsorbable polymer selected from the group
consisting of aliphatic polyesters, poly(amino acids),
polyalkylenes oxalates, polyamides, tyrosine derived
polycarbonates, polyorthoesters, polyoxaesters, and
poly(anhydrides).
4. The biomedical scaffold of claim 3 wherein said polymer
comprises an aliphatic polyester selected from the group consisting
of homopolymers and copolymers of lactide, lactic acid, glycolide,
glycolic acid, .epsilon.-caprolactone, p-dioxanone, trimethylene
carbonate, alkyl derivatives of trimethylene carbonate,
.delta.-valerolactone, .beta.-butyrolactone, .gamma.-butyrolactone,
.epsilon.-decalactone, hydroxybutyrate, hydroxyvalerate,
1,4-dioxepan-2-one, 1,5,8,12-tetraoxacyclotetradecane-7,14-dione,
1,5-dioxepan-2-one, and 6,6-dimethyl-1,4-dioxan-2-one.
5. The biomedical scaffold of claim 4 wherein said aliphatic
polyester comprises an elastomer selected from the group consisting
of copolymers of lactide and .epsilon.-caprolactone, and lactide
and glycolide.
6. The biomedical scaffold of claim 5 wherein said elastomer has an
inherent viscosity in the range of about 1 to 2 deciliters per gram
as determined at 25.degree. C. in a 0.1 gram per deciliter solution
of polymer in hexafluoroisopropanol.
7. The biomedical scaffold of claim 6 wherein said copolymer of
lactide and .epsilon.-caprolactone comprises a mole ratio of
lactide to .epsilon.-caprolactone from about 30/70 to about
50/50.
8. The biomedical scaffold of claim 6 wherein said copolymer of
lactide and glycolide comprises a mole ratio of lactide to
glycolide from about 75/25 to about 95/5.
9. The biomedical scaffold of claim 1 comprising a continuous
polymer phase comprising interconnected pores of between about 50
and about 500 microns in diameter.
10. The biomedical scaffold of claim 1 further comprising
cells.
11. The biomedical scaffold of claim 1 further comprising a
bioactive substance.
12. A process for making biomedical, biocompatible scaffolds
suitable for use in the repair and regeneration of tissue,
comprising: preparing a homogenous mixture comprising a synthetic,
biocompatible polymer, a solvent in which said polymer is soluble,
and a non-solvent in which said polymer is not soluble, wherein
said solvent and said non-solvent are miscible, and wherein the
freezing point of said non-solvent is higher than the freezing
point of said solvent, placing said homogenous mixture in a mold or
other device suitable for preparing foam scaffolds suit-able for
use in repair and regeneration of tissue, cooling said homogenous
mixture to a first temperature effective to freeze said
non-solvent, maintaining said first temperature for a time
effective to allow phase separation of said non-solvent from said
homogenous mixture, cooling said homogenous mixture to a second
temperature sufficient to form a solid, and removing said solvent
and said non-solvent from said solid to provide a biocompatible,
porous foam scaffold which comprises a network of branched
channels.
13. The process of claim 12 wherein said polymer is
bioabsorbable.
14. The process of claim 13 wherein said bioabsorbable polymer is
selected from the group consisting of aliphatic polyesters,
poly(amino acids), polyalkylenes oxalates, polyamides, tyrosine
derived polycarbonates, polyorthoesters, polyoxaesters and
poly(anhydrides).
15. The process of claim 14 wherein said polymer comprises an
aliphatic polyester selected from the group consisting of
homopolymers and copolymers of lactide, lactic acid, glycolide,
glycolic acid, .epsilon.-caprolactone, p-dioxanone, trimethylene
carbonate, alkyl derivatives of trimethylene carbonate,
.delta.-valerolactone, .beta.-butyrolactone, .delta.-butyrolactone,
.epsilon.-decalactone, hydroxybutyrate, hydroxyvalerate,
1,4-dioxepan-2-one, 1,5,8,12-tetraoxacyclotetradecane-7,14-dione,
1,5-dioxepan-2-one, and 6,6-dimethyl-1,4-dioxan-2-one.
16. The process of claim 15 wherein said aliphatic polyester
comprises an elastomer selected from the group consisting of
copolymers of lactide and .epsilon.-caprolactone, lactide and
glycolide, and blends thereof.
17. The process of claim 16 wherein said copolymer of lactide and
.epsilon.-caprolactone comprises a mole ratio of lactide to
.epsilon.-caprolactone from about 30/70 to about 50/50.
18. The process of claim 16 wherein said copolymer of lactide and
glycolide comprises a mole ratio of lactide to glycolide from about
75/25 to about 95/5.
19. The process of claim 14 wherein said solvent is selected from
the group consisting of dimethyl carbonate, 1,4-dioxane and diethyl
carbonate.
20. The process of claim 19 wherein said non-solvent is selected
from the group consisting of t-butanol, tert-amyl alcohol, 3,3
dimethyl-2 butanol, octanol, nonanol, decanol, n-decanol and
dodecanol.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to biomedical porous polymeric
foam scaffolds useful for tissue repair and regeneration and
methods for preparing same.
BACKGROUND OF THE INVENTION
[0002] Over the past two decades, the field of tissue engineering
has focused on the repair and reconstruction of tissue utilizing
scaffolds, both as a means to culture cells in vitro for subsequent
implantation in vivo and as an acellular implant to encourage
tissue ingrowth and incorporation. Scaffolds seeded and cultured
with cells are utilized to deliver and/or direct cells to desired
sites in the body, to define a potential space for engineered
tissue, and to guide the process of tissue development. In the case
of cell culture, cell transplantation, on or from scaffolds, has
been explored for the regeneration of skin, nerve, liver, pancreas,
cartilage, adipose and bone tissue, using various biological and
synthetic materials.
[0003] Acellular scaffolds have also been developed for promoting
the attachment and migration of cells from the surrounding living
tissue to the surface and interior of the scaffold. In these cases,
bioabsorbable materials are useful in order to provide a substrate
for incipient tissue growth and subsequent degradation and
elimination from the area leaving behind newly regenerated tissue.
Examples of such materials include-poly(lactic acid) (PLA),
poly(caprolactone) (PCL), poly(glycolic acid) (PGA),
poly(dioxanone) (PDO), poly(trimethylene carbonate) (TMC), and
their copolymers and blends.
[0004] Scaffolds, whether acellular or seeded, have certain
requirements with regards to the penetration of the scaffold by
cells and the nutrient flow to cells. Scaffolds with pores of
diameters up to 500 microns provide sufficient open space for the
formation of functional tissue, but lack the means necessary to
provide sufficient infiltration of cells, diffusion of nutrients
and oxygen to the cells, removal of metabolic waste away from the
cells, and to guide the cells and fluids.
[0005] Several attempts to provide scaffolds with architectures to
improve the diffusion of nutrients through the scaffold have been
made in the recent past. These include bimodal porous structures
that enhance the available surface area and internal volume of the
scaffold. These structures were created using leachable particles
incorporated into either a polymer or a polymer solution. In the
case of the polymer solution, freeze drying was used to create a
polymer foam embedded with leachable particles. The foam was then
subjected to a subsequent step in which the particles were leached
out of the system to create a second set of pores.
[0006] Alternatively, biocompatible porous polymer membranes were
prepared by dispersing salt particles in a biocompatible polymer
solution. The solvent was evaporated and the salt particles were
leached out of the membrane by immersing the membrane in a solvent
for the salt particles. A three-dimensional porous structure was
then manufactured by laminating the membranes together to form the
desired shape.
[0007] Others have circumvented the use of leachable particles to
form porous membranes of various pore diameters by casting a layer
of polymer solution on a substrate and submerging the
layer/substrate in a non-solvent for the polymer. This created a
porous polymer structure. The cast layers were laminated to achieve
gradients in porosity in the three-dimensional structure.
[0008] Still others have used a rigid-coil/flexible-coil block
copolymer mixed with a solvent that selectively solubilized one of
the blocks. The other block of the copolymer was permitted to
self-assemble into organized mesostructures. The solvent was then
evaporated, leaving the structure mesoporous.
[0009] The field of tissue engineering to repair and reconstruct
tissue has utilized scaffolds to encourage tissue ingrowth and
incorporation, scaffolds in the form of porous polymer foams. The
morphology of foams has progressed from random to controlled
formation, but the controlled morphology has resulted either in a
monomodal, isotropic distribution of pores through spinodal
decomposition of polymer solvent mixtures or in the production of
uniaxial channels in the foam. There remains a need for
biodegradable porous polymer scaffolds for tissue engineering that
have an architecture providing for the effective and thorough
distribution of fluids and nutrients necessary for tissue growth.
In addition, it would be advantageous to be able to produce this
scaffold by way of a method that does not require any manipulation
of the material post-processing.
SUMMARY OF THE INVENTION
[0010] The present invention provides a biomedical, biocompatible,
foam scaffold suitable for use in the repair and regeneration of
tissue that comprises a network of branched, channels effective to
encourage and facilitate vascularization and tissue growth therein
and a process for making the biomedical scaffolds. The process
comprises preparing a homogenous mixture of a synthetic,
biocompatible polymer, a solvent in which the polymer is soluble
and a non-solvent in which the polymer is not soluble. The solvent
and non-solvent are miscible and the freezing point of the
non-solvent is higher than the freezing point of the solvent. The
homogeneous mixture is placed in a mold and cooled to a temperature
effective to freeze the non-solvent. This temperature is maintained
for a time effective to allow the non-solvent to phase-separate
from the mixture. The mixture is then cooled to a temperature
effective to form a solid, and the solvent and non-solvent are
removed from the solid to provide a biocompatible, porous scaffold
suitable for use in the repair and regeneration of tissue
comprising a network of branched channels. This network of channels
provide a high degree of interconnectivity that aids in
transferring nutrients to the center of the scaffold, thus
encouraging and facilitating vascularization and, ultimately,
tissue growth within the scaffold structure.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a sectional view of a foam scaffold according to
the present invention.
[0012] FIG. 2 is a scanning electron micrograph of a cross-section
of a foam scaffold according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present invention provides a biocompatible foam scaffold
for use in the repair and regeneration of tissue and a method of
producing a scaffold in which a network of branched channels is
embedded. The process involves a combination of phase separation
and lyophilization in order to achieve this novel internal
architecture.
[0014] According to one aspect of the present invention, a
biocompatible porous scaffold is provided having a substantially
continuous polymeric foam phase with a highly interconnected
distribution of pores between about 50 and about 500 microns in
diameter, in which is embedded a network of branched channels. The
presence of the branched, channeled network in the porous scaffold
provides passageways between the pores for the distribution of
nutrients and the removal of waste. The resulting foams have a
porosity of about 90%.
[0015] The branched channels provide interconnectivity that is
useful for transmitting cell-to-cell, signaling molecules across
the scaffold and allowing for the diffusion of nutrients through
the scaffold. The channeled network also provides a patterned
surface that is useful for guiding cell growth. In addition, the
large surface area in the overall foam is ideal for cell seeding,
cell growth and the production of extracellular matrices. Finally,
the existence of channels in a three-dimensional structure
encourages cell growth in the pores and further provides a means of
infiltration into the interior of the scaffold by way of the
channels.
[0016] In another aspect of the present invention, the polymer
phase is bioabsorbable. Here, the scaffold undergoes biodegradation
as the tissue grows and becomes incorporated in the site of the
implantation.
[0017] Referring to FIGS. 1 and 2, scaffold 10 includes a polymeric
foam component 12 including pores 14 with open cell pore structure.
Continuous branched channels 16 are embedded in foam component 12.
These branched channels 16 have primary branches 18 as well as
secondary branches 20. The branched channels 16 in the
three-dimensional scaffold 10 structure encourages and facilitates
cell growth in pores 14 and further provides a means for
transferring nutrients to the center of scaffold 10, thus
encouraging and facilitating vascularization into scaffold 10.
[0018] Biomedical polymers are suitable for use in the present
invention. These types of polymers are biocompatible at the time of
implant, causing no harm to living tissue. Preferably, the polymers
should be biodegradable, where the polymer degradation products are
biocompatible, non-toxic and physiologically compatible, and may
also be bioabsorbable, or resorbed into living tissue. Additional
parameters that play an important role include the mechanical
properties of the material, especially its mechanical rigidity.
High rigidity is advantageous where cells growing within the
scaffold exert forces. It is also important that the biodegradation
kinetics of the polymer match the rate of the healing process.
Finally, from a processing standpoint, the thermal properties of
the polymer are important to allow the polymer to retain mechanical
integrity post-processing, e.g. a sufficiently high glass
transition temperature to avoid pore/channel collapse upon solvent
removal.
[0019] Polymers that can be used for the preparation of scaffolds
for use in the repair and regeneration of tissue according to the
present invention include polymers selected from the group
consisting of aliphatic polyesters, poly(amino acids),
polyalkylenes oxalates, polyamides, tyrosine derived
polycarbonates, polyorthoesters, polyoxaesters, poly(anhydrides),
and blends thereof. For the purpose of this invention aliphatic
polyesters include, but are not limited to, homopolymers and
copolymers of lactide (which includes lactic acid, D-,L- and meso
lactide), glycolide (including glycolic acid),
.epsilon.-caprolactone, p-dioxanone (1,4-dioxan-2-one),
trimethylene carbonate (1,3-dioxan-2-one), alkyl derivatives of
trimethylene carbonate, .delta.-valerolactone,
.beta.-butyrolactone, .gamma.-butyrolactone, .epsilon.-decalactone,
hydroxybutyrate (repeating units), hydroxyvalerate (repeating
units), 1,4-dioxepan-2-one (including its dimer
1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,
and 6,6-dimethyl-1,4-dioxan-2-one and blends thereof.
[0020] Elastomeric copolymers are also particularly useful in the
present invention. These elastomeric copolymers will have an
inherent viscosity in the range of about 1.0 dL/g to 4 dL/g, more
preferably about 1.0 dL/g to 2.0 dL/g and most preferably about 1.0
dL/g to 1.7 dL/g as determined at 25.degree. C. in a 0.1 gram per
deciliter (g/dL) solution of polymer in hexafluoroisopropanol
(HFIP). For the purpose of this invention, an "elastomeric
copolymer" is defined as a polymer which, at room temperature, can
be stretched repeatedly to at least about twice its original length
and which, upon immediate release of stress, will return to
approximately its original length.
[0021] Exemplary bioabsorbable, biocompatible elastomers include,
but are not limited to, elastomeric copolymers of lactide
(including L-lactide, D-lactide, blends thereof, and lactic acid
polymers and copolymers) and .epsilon.-caprolactone where the mole
ratio of lactide to .epsilon.-caprolactone is from about 30/70 to
about 65/35, and more preferably from about 30/70 to about 50/50;
elastomeric copolymers of lactide (including L-lactide, D-lactide,
blends thereof, and lactic acid polymers and copolymers) and
glycolide (including polyglycolic acid) where the mole ratio of
lactide to glycolide is from about 75/25 to about 95/5.
[0022] The scaffolds of the present invention are foams prepared by
the lyophilization of a polymer dissolved in a homogeneous mixture
of components that act as solvents and non-solvents for the
polymer. The process according to the present invention employs
thermally induced phase separation to fabricate highly porous foam
scaffolds comprising a network of branched channels embedded
therein for the optimization of properties necessary to encourage
and facilitate vascularization. Phase separation will occur by
liquid-liquid demixing and crystallization based on the
thermodynamics of the system. After lyophilization, the resulting
foam contains an embedded network of branched channels resulting
from the phase separation and crystallization of the non-solvent
from the mixture during processing.
[0023] As mentioned above, the homogeneous mixture is comprised of
several components, each of which has a particular relationship
with the polymer. Specifically, the mixture comprises at least a
first component in which the polymer is soluble, referred
heretofore as the solvent, and a second component in which the
polymer is not soluble, referred heretofore as the non-solvent.
[0024] Typically, the two components are liquids at, or slightly
above, room temperature, and must also conform to certain criteria
relative to one another. First, the solvent and non-solvent must be
miscible; i.e. the solvent solubilizes both the polymer and the
non-solvent. Second, the melting point of the non-solvent must be
above that of the solvent. The higher melting point of the
non-solvent allows phase separation of the non-solvent from the
polymer mixture upon cooling. This is necessary to create the
channels in the polymer foam. Finally, the freezing points of the
solvent and non-solvent must be sufficiently disparate in order
that the solid phase of the non-solvent is favorable, while the
solid phase of the solvent is unfavorable, so that complete phase
separation may occur in a given temperature range between the two
freezing points. In this temperature range, phase separation occurs
as the non-solvent crystallizes from the mixture. Due to
colligative properties of the mixture, the solvent and non-solvent
are expected to undergo a freezing point depression, the magnitudes
of which are dependent on the properties of the pure solvent and
non-solvent. As a result of this freezing point depression, a
spread in the freezing points is also useful to allow the complete
phase separation and crystallization of the non-solvent from the
homogeneous mixture.
[0025] In processes for making the scaffolds of the present
invention, a homogeneous mixture is made by combining polymer,
solvent, and non-solvent. The mixture is then poured into a mold
and placed in a lyophilizer. The formation of the channeled
scaffold is a one-step process that relies on the properties of the
respective components of the mixture and the lyophilization cycle.
The first segment of the cooling cycle involves a ramping-down to a
temperature below the depressed freezing point of the non-solvent,
but above that of the solvent. The temperature is held while
crystals of the non-solvent begin to nucleate and grow. This phase
separation may occur in the form of dendrites of the non-solvent
that grow in the mixture, creating a branch-like structure within
the mixture. Since the polymer is not soluble in the non-solvent,
the dendrites of non-solvent do not contain polymer. The dendrites
of non-solvent growing in the polymer solution act as placeholders
for the branched channel network structure that will result upon
the sublimation of the non-solvent.
[0026] Once the non-solvent has crystallized, the temperature is
decreased below the freezing point of the solvent. At this point,
the polymer/solvent mixture solidifies. The degree of crystallinity
of this solid God phase depends on the rate of temperature
decrease. Whether amorphous, crystalline, or some combination
thereof, this solidified polymer/solvent mixture is responsible for
foam formation.
[0027] The solid mixture sublimes by way of the lyophilization
cycle as vacuum is applied to the frozen sample, leaving behind a
polymer foam with pores forming as the solvent sublimes and
branched channels forming in the foam as the non-solvent
sublimes.
[0028] Non-solvent crystallization during liquid-liquid demixing
occurs with the correct selection of solvents and processing
conditions. A mixture of at least one component which is a solvent
and at least one component which is a non-solvent for the polymer
is employed. As mentioned above, the solvent and non-solvent must
be miscible. Further, the proportions and mixing of the two
components are chosen so as to retain solubility of the polymer in
the solvent despite its insolubility in the non-solvent. This
yields a uniform, homogenous mixture.
[0029] Solvents useful in the present invention include, but are
not limited to, dimethyl carbonate (DMC; m.p. 5.degree. C.),
1,4-dioxane (m.p. 12.degree. C.) and diethyl carbonate (m.p.
-43.degree. C.). Non-solvents in which the polymer is insoluble
that are suitable for use include, but not limited to, alcohols
such as t-butanol (m.p. 25.degree. C.), tert-amyl alcohol (m.p.
-12.degree. C.), 3,3 dimethyl-2 butanol (m.p. -5.degree. C.),
octanol (m.p. -15.degree. C.), nonanol (m.p. -8.degree. C.),
decanol (m.p. 7.degree. C.), n-decanol (m.p. 11.degree. C.), and
dodecanol (m.p. 22-26.degree. C.). It is critical that the polymer
be soluble in the overall solvent/non-solvent mixture.
[0030] The melting point of the non-solvent must be higher than
that of the solvent. In addition, the disparity in melting points
between the two solvents should be sufficiently large to allow
thorough crystallization of the non-solvent during liquid/liquid
demixing step of the process. Preferably, the disparity in melting
points is greater than about 20.degree. C. A preferred
solvent/non-solvent pair meeting the above requirements are
dimethyl carbonate (DMC; m.p. 5.degree. C.) which is a solvent for
the disclosed polymers, and t-butanol (m.p. 25.degree. C.), a
non-solvent for the disclosed polymers.
[0031] Combining the polymer, solvent and non-solvent can be
accomplished in two ways. The polymer may first be completely
dissolved in the solvent followed by the addition of the
non-solvent. Alternatively, the solvent and non-solvent may be
mixed followed by the addition of polymer. If the non-solvent is
added to the polymer solution, then it is added into a constantly
agitated polymer solution at a rate effective to avoid localized
precipitation of the polymer in the homogeneous mixture. The weight
ratio of the non-solvent to the total volume of the solvent and
non-solvent is preferably between about 1 to about 50 weight
percent and more preferable between about 15 to about 30 weight
percent. The polymer concentration in the solvent mixture is
preferably between about 0.5 and about 25 weight percent and more
preferably between about 2.5 and about 10 weight percent.
[0032] Alternatively, the method of the present invention can also
be used to make a porous scaffold of a first polymer with branched
channels of a second porous polymer embedded within the structure.
This structure is created if the second polymer is insoluble in the
solvent, and soluble in the non-solvent. This mixture creates dual
solid-liquid phase transformation processes occurring within the
same system. Again, the two components must be miscible, but the
two polymers used in the system must only be soluble in one of the
components, i.e. its respective solvent. Upon cooling the system,
the temperature is held for a sufficient time to allow the first
component to crystallize, thus forcing a solid-liquid phase
transformation and precipitation of the first polymer in a
dendritic branched fashion. After complete crystallization of the
first component, the temperature is then lowered until the second
solid-liquid phase transformation occurs. Sublimation of the system
leaves behind a branched foam structure of one polymer embedded
within a second foam structure of another polymer.
[0033] The porous polymer scaffolds can be molded or cut to shape
for tissue engineering and tissue guided regeneration applications.
Cellular pre-seeding can be used prior to implantation or the
scaffold can be used in an acellular fashion due to the structure
of the scaffold that allows generous cellular ingrowth. The
scaffold serves both as a physical support and an adhesive
substrate for isolated cells during in vitro culture and subsequent
implantation. As the transplanted cell populations grow, the cells
function normally and begin to secrete their own extracellular
matrices (ECM) which allows the scaffold to mimic the ECM of an
organ. The porous polymer scaffold may, therefore, be used as an
external scaffolding for the support of in vitro culturing of cells
for the creation of external support organs. In all cases, the
scaffold polymer is selected to degrade as the need for the
artificial support diminishes.
[0034] In applications where the tissue shape is integral to tissue
function, the polymer scaffold may be molded to have the
appropriate dimensions. Any crevices, apertures or refinements
desired in the three-dimensional structure can be created by
fashioning the matrix with scissors, a scalpel, a laser beam or any
other cutting instrument. Scaffold applications include the
regeneration of tissues such as adipose, pancreatic, cartilaginous,
osseous, musculoskeletal, nervous, tendenous, hepatic, ocular,
integumeary, arteriovenous, urinary or any other tissue forming
solid or hollow organs.
[0035] The scaffold may also be used in transplantation as a matrix
for dissociated cell types. These include fibrochondrocytes,
adipocytes, pancreatic Islet cells, osteocytes, osteoblasts,
myeloid cells, chondrocytes, hepatocytes, exocrine cells, cells of
intestinal origin, bile duct cells, parathyroid cells, nucleus
pulposus cells, annulus fibrosis cells, thyroid cells, endothelial
cells, smooth muscle cells, fibroblasts, meniscal cells, sertolli
cells, cells of the adrenal-hypothalamic-pituitary axis, cardiac
muscle cells, kidney epithelial cells, kidney tubular cells, kidney
basement membrane cells, nerve cells, blood vessel cells, cells
forming bone and cartilage, smooth muscle cells, skeletal muscle
cells, ocular cells, integumentary cells, keratinocytes, peripheral
blood progenitor cells, fat-derived progenitor cells, glial cells,
macrophages, mesenchymal stem cells, embryonic stem cells, stem
cells isolated from adult tissue, genetically engineered cells, and
combinations thereof. Pieces of tissue can also be used, which may
provide a number of different cell types in the same structure.
[0036] Allogeneic or autologous cells may be used and are
dissociated using standard techniques and seeded onto or into the
foam scaffold. If the cells are seeded onto the scaffold, seeding
may take place prior to, or after, the scaffold is implanted. If
the cells are added after implantation, the added benefit is that
cells are placed into the scaffold after it has had an opportunity
to vascularize and be incorporated into the implant site. Methods
and reagents for culturing cells in vitro and implantation of a
tissue scaffold are known to those skilled in the art.
[0037] After fabrication, scaffolds can be further modified to
increase effectiveness of the implant. For example, the scaffolds
can be coated with bioactive substances that function as receptors
or chemoattractors for a desired population of cells. The coating
can be applied through absorption or chemical bonding and may be
designed to deliver therapeutic or medicated additives in a
controlled fashion. In addition, since the lyophilization of the
foam takes place at low temperatures, thermally sensitive additives
can be used without concern of degradation during polymer
processing. The additive may be released by a bioerosion of the
polymer phase or by diffusion from the polymer phase. Alternative
to release, the additive may simply migrate to the polymer surface
of the scaffold structure where it is active.
[0038] Depending on the additive and the nature of the components
used in the system, the additive may be added to the pre-blended
mixture or it may be added first to the component in which it is
most soluble before adding another component. The additive may be
provided in a physiologically acceptable carrier, excipient, or
stabilizer, and may be provided in sustained release or timed
release formulations. The additives may also incorporate biological
agents to facilitate their delivery, such as antibodies, antibody
fragments, growth factors, hormones, demineralized bone matrix, or
other targeting moieties, to which the additives are coupled.
[0039] Acceptable pharmaceutical carriers for therapeutic use are
well known in the pharmaceutical field. Such materials are
non-toxic to recipients at the dosages and concentrations employed,
and include diluents, solubilizers, lubricants, suspending agents,
encapsulating materials, solvents, thickeners and dispersants. Also
acceptable are buffers such as phosphate, citrate, acetate and
other organic acid salts. Anti-oxidants such as ascorbic acid,
preservatives, low molecular weight peptides (less than about 10
residues), such as polyarginine, proteins such as serum albumin,
gelatin or immunoglobulins may also be used. The pharmaceutical
carriers can also include hydrophilic polymers such as
poly(vinylpyrrolindinone), amino acids such as glycine, glutamic
acid, aspartic acid or arginine, monosaccharides, disaccarides, and
other carbohydrates including cellulose or its derivatives,
glusocse, mannose or dextrines. Chelating agents such as EDTA,
sugar alcohols, such as mannitol or sorbitol, counter-ions such as
sodium and/or non-ionic surfactants such as tween, pluronics or PEG
are all acceptable carriers as well.
[0040] As mentioned, the coating can be applied through absorption
or chemical bonding, the latter taking place by covalently binding
the additive to a pendent free carboxylic acid group on the
polymer. For example, moieties having reactive functional groups or
being derivatized to contain active functional groups may be
reacted with polymer pendent free carboxylic acid groups to form a
polymer conjugate. If the additive is active in the conjugate form,
then conjugates that are resistant to hydrolysis are utilized. The
opposite is true if the additive is inactive in the conjugate form
in which case the conjugate used is hydrolyzable.
[0041] The amount of additive incorporated into the porous polymer
scaffold is chosen to provide optimal efficacy to the subject in
need of treatment, typically a mammal. A dose and method of
administration will vary from subject to subject and be dependent
upon such factors as the type, sex, weight, and diet of the mammal
being treated. Other factors include concurrent medication, the
particular compounds employed, overall clinical condition, and
other factors that those skilled in the art will recognize. The
porous polymer scaffolds can be utilized in vitro or in vivo as
tissue engineering and tissue guided regeneration scaffold in
mammals such as primates, including humans, sheep, horses, cattle,
pigs, dogs, cats, rats, and mice. As the polymers used in this
invention are typically suitable for storage at ambient or
refrigerated temperatures, the polymer-drug combinations of this
invention may be prepared for storage under conditions suitable for
the preservation of drug activity. Sterility is also an issue for
polymer scaffolds to be used in tissue engineering and tissue
guided regeneration applications and may be accomplished using
conventional methods such as treatment with gases, heat, or
irradiation.
[0042] Additives suitable for use with the present invention
include biologically or pharmaceutically active compounds. Examples
of biologically active compounds include cell attachment mediators,
such as peptide containing variations of the "RGD" integrin binding
sequence known to affect cellular attachment, biologically active
ligands, and substances that enhance or exclude particular
varieties of cellular or tissue ingrowth. Examples of such
substances include integrin binding sequence, ligands, bone
morphogenic proteins, epidermal growth factor, fibroblast growth
factor, platelet-derived growth factor, IGF-I, IGF-II, TGF-.beta.
I-III, growth differentiation factor, parathyroid hormone, vascular
endothelial growth factor, hyaluronic acid, gylcoprotein,
lipoprotein, and the like.
[0043] Examples of pharmaceutically active compounds include
antiinfectives, analgesics, anorexics, antihelmintics,
antiarthritics, antiasthmatics, anticonvulsants, antidepressants,
antidiuretics, antidiarrheals, antihistamines, antiinflammatory
agents, antimigraine preparations, antinauseants, antineoplastics,
antiparkinsonism drugs, antipruritics, antipsychotics,
antipyretics, antispasmodics, anticholinergics, sympathomimetics,
xanthine derivatives, calcium channel blockers, beta-blockers,
antiarrhythmics, antihypertensives, diuretics, vasodilators,
central nervous system stimulants, decongestants, hormones,
steroids, hypnotics, immunosuppressives, muscle relaxants,
parasympatholytics, psychostimulants, sedatives, tranquilizers,
naturally derived or genetically engineered proteins,
polysaccharides, glycoproteins, or lipoproteins, oligonucleotides,
antibodies, antigens, cholinergics, chemotherapeutics, hemostatics,
clot dissolving agents, radioactive agents and cystostatics, and
the like. Therapeutically effective dosages may be determined by in
vitro or in vivo methods. For each particular additive, individual
determinations may be made to determine the optimal dosage
required. The determination of effective dosage levels to achieve
the desired result will be within the realm of one skilled in the
art. The release rate of the additives may also be varied within
the routine skill in the art to determine advantageous profile,
depending on the therapeutic conditions to be treated.
[0044] A typical additive dosage might range from about 0.001 mg/kg
to about 1000 mg/kg, preferably from about 0.01 mg/kg to about 100
mg/kg, and more preferably from about 0.10 mg/kg to about 20 mg/kg.
The additives may be used alone or in combination with other
therapeutic or diagnostic agents.
[0045] The invention will be better understood by reference to the
following non-limiting examples.
EXAMPLE 1
[0046] A mixture to be lyophilized was first prepared. The mixture
was composed of a 60/40 copolymer of PLA/PCL (I.V. of 1.7 dL/g at
25.degree. C. in a 0.1 g/dL solution of HFIP), and dimethyl
carbonate, a solvent for 60/40 PLA/PCL, in a 95/5 weight ratio. The
polymer and solvent were placed into a flask that was then placed
into a water bath and heated to 70.degree. C. The solution was
heated and stirred for 5 hours. Afterwards, the solution was
filtered using an extraction thimble (extra coarse porosity, type
ASTM 170-220 (EC)) and stored in the flask.
[0047] Twenty milliliters of t-butanol was added to 80 ml of the
polymer solution in a dropwise fashion to form an 80/20 volumeric
mixture. The polymer solution was constantly agitated during the
dropwise addition of t-butanol.
[0048] Twenty milliliters of the 80/20 mixture was poured into a
50-ml recrystallization dish. The dish was placed on the shelf of a
pre-cooled (20.degree. C.) laboratory scale lyophilizer (Model
Freeze Mobile G from Virtis Company (Gardiner, N.Y.), and was
subjected to the following freeze dry sequence: cool at 2.5.degree.
C./min to 0.degree. C., hold 40 minutes; cool at 2.5.degree. C./min
to -10.degree. C., hold 120 minutes; cool at 2.5.degree. C./min to
-50.degree. C., hold 15 minutes; hold at -48.degree. C. for an
additional 60 minutes; turn on the condenser; turn on vacuum pump
once condenser reaches -40.degree. C.; hold until vacuum in chamber
is 150 mT and vacuum in foreline is 100 mT, then hold an additional
60 minutes; warm at 2.5.degree. C./min to -30.degree. C., hold 60
minutes; warm at 2.5.degree. C./min to -15.degree. C., hold 60
minutes; warm at 2.5.degree. C./min to 0.degree. C., hold 60
minutes; warm at 2.5.degree. C./min to 15.degree. C., hold 60
minutes; warm at 2.5.degree. C./min to 22.degree. C., hold 60
minutes.
[0049] As the temperature decreased to -10.degree. C., dendritic
crystals grew in the solution as the t-butanol phase separated from
the mixture. The remaining polymer in dimethyl carbonate was frozen
at -50.degree. C. The foam was formed as the dimethyl carbonate
sublimed and the channels were formed as the dendritic crystals of
t-butanol sublimed.
[0050] Scanning electron micrographs (SEMs) showed the average pore
diameter of the foam to be in the range of 50 to 400 microns and
the channels to have an average diameter in the range of 0.5 to 1.0
mm.
* * * * *