U.S. patent application number 09/788103 was filed with the patent office on 2001-09-13 for calcium phosphate microcarriers and microsphers.
This patent application is currently assigned to CaP BIOTECHNOLOGY, INC.. Invention is credited to Starling, L. Brian, Stephan, James E..
Application Number | 20010021389 09/788103 |
Document ID | / |
Family ID | 21922425 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021389 |
Kind Code |
A1 |
Starling, L. Brian ; et
al. |
September 13, 2001 |
Calcium phosphate microcarriers and microsphers
Abstract
The present invention provides calcium phosphate-based (CaP)
microcarriers and their use, for example, in cell culturing
systems, chromatography, and implantable biomedical materials.
Inventors: |
Starling, L. Brian; (Golden,
CO) ; Stephan, James E.; (Arvada, CO) |
Correspondence
Address: |
Gary J. Connell
SHERIDAN ROSS P.C.
Suite 1200
1560 Broadway
Denver
CO
80202-5141
US
|
Assignee: |
CaP BIOTECHNOLOGY, INC.
|
Family ID: |
21922425 |
Appl. No.: |
09/788103 |
Filed: |
February 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09788103 |
Feb 15, 2001 |
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09402308 |
Feb 2, 2000 |
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6210715 |
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09402308 |
Feb 2, 2000 |
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PCT/US98/06456 |
Mar 31, 1998 |
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60042530 |
Apr 1, 1997 |
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Current U.S.
Class: |
424/422 ;
424/489; 424/602 |
Current CPC
Class: |
B01J 20/048 20130101;
A61F 2/30767 20130101; A61F 2002/30593 20130101; A61L 31/026
20130101; B01J 2220/54 20130101; A61F 2002/2817 20130101; C01B
25/32 20130101; B01J 20/2803 20130101; B01J 20/28021 20130101; A61F
2310/00796 20130101; A61F 2002/30242 20130101; A61K 9/1611
20130101; B01J 20/28019 20130101; A61F 2310/00293 20130101; A61F
2230/0071 20130101; B01J 20/28011 20130101; B01J 20/28004 20130101;
B01J 20/282 20130101; A61L 27/12 20130101; A61K 9/501 20130101 |
Class at
Publication: |
424/422 ;
424/489; 424/602 |
International
Class: |
A61K 009/14; A61K
033/42 |
Claims
We claim:
1. A hollow CaP microbead having a density about 1.01 grams/cc to
about 1.12 grams/cc.
2. A hollow microbead according to claim 1 wherein the CaP
comprises 0 to 100% hydroxylapatite (HA), 0 to 100% tricalcium
phosphate (TCP) and/or 0 to 100% other calcium phosphate
materials.
3. A hollow microbead according to claim 2 comprising 0 to 100%
hydroxylapatite (HA) and 0 to 100% tricalcium phosphate (TCP).
4. A hollow microbead according to claim 1 comprising a wall,
wherein the wall is essentially impermeable to aqueous media.
5. A hollow microbead according to claim 1 having an average
diameter in an essentially spherical shape of from about 100
micrometers to about 6 millimeters.
6. A hollow microbead according to claim 1 further comprising a
porous coating.
7. A hollow microbead according to claim 1 further comprising a
biological coating.
8. A hollow microbead according to claim 7 wherein the biological
coating is a growth factor.
9. A hollow CaP microbead having a density from about 1.2 grams/cc
to about 2.0 grams/cc.
10. A hollow microbead according to claim 9 comprising a porous
coating.
11. A hollow microbead according to claim 9 comprising a biological
coating.
12. A hollow microbead according to claim 11 wherein the biological
coating is a growth factor.
13. A hollow microbead according to claim 9 comprising a porous
coating and a biological coating.
14. A hollow microbead according to claim 13 wherein the biological
coating is a growth factor.
15. A biomedical implant comprising a microbead according to claims
1-14.
16. A biomedical implant according to claim 15 further comprising a
biological material or pharmaceutical agent.
17. A biomedical implant according to claim 15 having a density
from about 25% to about 75% of the material's theoretical
density.
18. A biomedical implant according to claim 15 wherein the
microbead comprises a wall which is essentially impermeable to
aqueous media.
19. A biomedical implant according to claim 15 wherein the
microbead comprises a wall which is porous.
20. A biomedical implant according to claim 15 wherein the
microbead comprises holes.
21. A chromatographic column comprising a hollow microbead
according to claim 9.
22. An aggregate comprising the hollow microbeads from claims 1
through 13.
23. A biomedical implant comprising an aggregate according to claim
22.
24. A chromatographic column comprising an aggregate according to
claim 22.
25. Hollow and solid glass or polymer microbeads formed with or
coated with particulate HA, TCP or other CaPs.
26. Hollow and solid microbeads comprising composites of HA, TCP,
other CaPs and ceramic or polymeric materials.
27. A microbead according to claim 26 wherein the surface of said
bead is abraded.
28. An aggregate comprising the hollow microbeads of claim 25 or
27.
29. A biomedical implant comprising the aggregate of claim 28.
30. A chromatographic column comprising the aggregate according to
claim 28.
31. Suspendable and non-suspendable aggregates comprising
microbeads comprising closed and/or open pores, foamed structures
of ceramic, including glass, and/or polymeric composite
materials.
32. An aggregate according to claim 28 or 31 comprising HA and TCP
coatings, porous coatings, or biological coatings including growth
factors.
33. A biomedical implant comprising an aggregate according to 28,
31 or 32.
34. A method of augmenting tissue comprising implanting the
biomedical implant of claim 33.
35. A biomedical implant according to claim 33, further comprising
a biologically active agent.
36. A biomedical implant according to claim 33 having a density
from about 25% to about 75% of the material's theoretical
density.
37. A chromatographic column comprising and aggregate according to
claim 28 or 31
38. An aggregate according to claim 22 that is bonded by
cementatious agents.
Description
FIELD OF THE INVENTION
[0001] The invention disclosed herein relates to calcium phosphate
(CaP) microcarriers and microspheres and their use, for example, in
cell culturing systems, chromatography analysis and processing, and
implantable materials useful for biomedical implants.
BACKGROUND
[0002] Revolutionary advances in biotechnology and genetic
engineering have created enormous potential for marketing cellular
by-products, including for example, proteins, including protein
pharmaceuticals such as interferon, monoclonal antibodies, TPA
(Tissue Plasminogen Activator), growth factors, insulin, and cells
for transplantation. The demand for these products has grown
tremendously and will continue to do so, creating a need for
efficient methods of producing industrial quantities of
cell-derived pharmaceuticals and other products. Further, the
demand for efficient methods of analyzing and isolating biological
products through chromatographic technology, and the need to
improve bio-implantables continues to grow.
[0003] Research and study of cell structure and morphology are
fundamental to continued progress in the diagnosis and treatment of
human diseases. Numerous cell products are of vital importance
therapeutically and commercially, including, for example, hormones,
enzymes, viral products, vaccines, and nucleic acids. The
production of these products requires large scale cell culture
systems for their production.
[0004] Mammalian cells can be grown and maintained in vitro, but
are generally anchorage-dependent, i.e., they require a solid
surface or substrate for growth. The solid substrate is covered by
or immersed in a nutrient medium particular to the cell type to be
cultured. The nutrient medium and solid substrates are contained in
a vessel and provided with an adequate supply of oxygen and carbon
dioxide to support cell growth and maintenance. Cell cultures may
be batch systems, in which nutrients are not replenished during
cultivation although oxygen is added as required; fed batch
systems, in which nutrient and oxygen are monitored and replenished
as necessary; and perfusion systems, in which nutrient and waste
products are monitored and controlled (Lubiniecki, Large Scale
Mammalian Cell Culture Technology, Marcel Dekker, Inc., New York,
1990).
[0005] The primary commercial systems used for mammalian cell
culture use solid matrix perfusion and microcarrier bead systems
(Lubineicke, supra). The solid matrix perfusion systems utilize
glass columns packed with glass beads or helices, which form a
matrix as the solid substrate for cell growth. Once cells have
attached to the matrix, medium is continuously recycled from a
storage vessel for support of cell growth and maintenance. A
similar perfusion system uses hollow fibers as the solid matrix
instead of beads.
[0006] In microcarrier systems, small spheres are fabricated, for
example, from an ion exchange gel, dextran, polystyrene,
polyacrylamide, or collagen-based material. These materials have
been selected for compatibility with cells, durability to agitation
and specific gravities that will maintain suspension of the
microcarriers in growth mediums. Microcarriers are generally kept
in suspension in a growth medium by gently stirring them in a
vessel. Microcarrier systems are currently regarded as the most
suitable systems for large-scale cell culture because they have the
highest surface to volume ratio and enable better monitoring and
control. Nevertheless, current microcarrier culture systems have a
number of serious disadvantages: small microcarrier cultures cannot
be used to inoculate larger microcarrier cultures; therefore, a
production facility must use other culture systems for this
purpose; the cost of microcarriers is high, which can necessitate
reprocessing of the microcarriers for reuse with the attendant
costs; and the oxygen transfer characteristics of existing
microcarrier systems are rather poor.
[0007] Specific forms of calcium phosphate ceramic have been
identified for use in microcarriers to support anchorage-dependent
cells in suspension. These specialized ceramics provide a material
which is biomimetic, i.e., it is composed of mineral species found
in mammalian tissues, and which can be further applied to a variety
of in vitro biological applications of commercial interest. A
number of common cell lines used in industrial applications require
attachment in order to propagate and need substrate materials such
as microcarriers for large scale cultivation. U.S. Pat. No.
4,757,017 (Herman Cheung) teaches the use of solid substrates of
mitogenic calcium compounds, such as hydroxylapatite (HA) and
tricalcium phosphate (TCP) for use in in vitro cell culture systems
for anchorage-dependent mammalian cells. The unique features of
this technology include the growth of cells in layers many cells
thick, growth of cells that maintain their phenotype and the
ability to culture cells for extended periods of time. Cheung
demonstrated the application of this technology for culturing red
blood cells. A current limitation of this technology is that the
microcarriers are only available in a non-suspendable granular
form. The density of these microcarriers further limits the ability
to scale-up this technology for large bioreactors, which require a
suspendable microbead carrier.
[0008] A complementary system using an aragonite (CaCO.sub.3) is
disclosed in U.S. Pat. No. 5,480,827 (G. Guillemin et al). Although
this patent also teaches the importance of calcium in a support
system for mammalian cell culture, the calcium compound was not in
a suspendable form.
[0009] The concept of fabricating a suspendable microcarrier bead
with a minor component of glass was discussed by A. Lubiniecki in
Large-Scale Mammalian Cell Culture Technology in which a minimal
glass coating was applied to a polymer bead substrate by a chemical
vapor deposition process or low temperature process. This approach
also was disclosed in U.S. Pat. No. 4,448,884 by T. Henderson (see
also U.S. Pat. Nos. 4,564,532 and 4,661,407). However, this
approach primarily used the polymer bead substrate to maintain
suspendability.
[0010] An example of the use of non-suspendable or porous ceramic
particles for cell culture is taught by U.S. Pat. No. 5,262,320 (G.
Stephanopoulos) which describes a packed bed of ceramic particles
around and through which oxygen and growth media are circulated to
encourage growth of cells. U.S. Pat. No. 4,987,068 (W. Trosch et
al.) also teaches the use of porous inorganic (glass) spheres in
fixed bed or fluidized bed bioreactors. The pores of the particles
act as sites for the culture of cells. Conversely, Richard Peindhl,
in U.S. Pat. No. 5,538,887, describes a smooth surface cell culture
apparatus which would limit cell attachment to chemical adhesion
and prevent mechanical interlocking.
[0011] Macroporous glass beads also have been reported by D. Looby
and J. Griffiths, "Immobilization of Cells In Porous Carrier
Culture", Trends in Biotechnology, 8: 204-209, 1990, and magnesium
aluminate porous systems by Park and Stephanopolous, "Packed Bed
Reactor With Porous Ceramic Beads for Animal Cell Culture,
Biotechnology Bioengineering, 41: 25-34, 1993. Fused alumina foams
have been reported by Lee et al, "High Intensity Growth of Adherent
Cells On a Porous Ceramic Matrix. Production of Biologicals from
Animal Cells in Culture, editors, R. E. et al,
Butterworth-Heinemann pp. 400-405, 1991, and polyurethane foam by
Matsushita et al, "High Density Culture of Anchorage Dependent
Animal Cells by Polyurethane Foam Packed Bed Culture Systems",
Applied Microbiology Biotechnology, 35: 159-64,1991.
[0012] Fluidized bed reactors have been used for cell culture as
reported by J. M. Davis (editor), Basic Cell Culture, (Cartwright
and Shah), Oxford University Press, New York, 1994, but require
carrier systems with densities between 1.3 and 1.6 g/cc. According
to Cartwright (J. M. Davis, supra.), generally, in fluidized beds,
cells do not grow on the exterior surface of carriers where they
would be dislodged by inter-particle abrasion. Instead, as with
macroporous microcarriers, they colonize the interior pores where
they proliferate in a protected microenvironment. As examples,
(Cartwright, supra, p. 78) cell carriers used in fluidized beds
include glass beads (Siran by Schott Glass), and collagen
microspheres produced by Verax. Cartwright also disclosed other
conventional microcarriers weighted with TiO.sub.2 (Percell
Biolytica products) and IAM-carrier polyethylene beads weighted
with silica.
SUMMARY OF THE INVENTION
[0013] Examples of the microcarriers of the present invention are
set forth in FIG. 1.
[0014] The present invention provides hollow microbeads having a
density of about 1.01 grams/cc to about 1.12 grams/cc. More
specifically, the hollow microbeads comprise 0 to 100%
hydroxylapatite (HA), 0 to 100% tricalcium phosphate (TCP) and/or 0
to 100% other calcium phosphate compounds. The hollow microbeads
comprise a wall, wherein the wall may be impermeable to aqueous
media. The essentially spherical hollow microbeads can have a
diameter of from about 100 micrometers to about 6 millimeters. In
another embodiment, the hollow microbead can further comprise a
porous coating and/or a biological coating.
[0015] The present invention also provides hollow microbeads having
a density from about 1.2 grams/cc to 2.0 grams/cc. These hollow
microbeads can further comprise a porous coating and/or a
biological coating.
[0016] Also provided are biomedical implants comprising the
above-described microbeads. The biomedical implants can further
comprise a biological material or pharmaceutical agent. More
specifically, the biomedical implants have a density from about 25%
to about 75% of the material's theoretical density. (By
"theoretical density" is meant the density of a microbead having no
pores.) The biomedical implant may comprise a microbead wherein the
microbead comprises a wall that is essentially impermeable or
porous to aqueous media. The biomedical implant may also comprise
microbeads comprising holes, i.e., portals or channels.
[0017] Also provided are chromatographic columns comprising the
hollow microbeads as set forth above.
[0018] Also provided are aggregates comprising the hollow
microbeads as set forth above. Such aggregates can be used as
biomedical implants and chromatographic columns. The aggregates may
be bonded by cementations agents.
[0019] The invention further provides hollow and solid glass or
polymer microbeads formed with or coated with particulate HA, TCP
and other CaPs.
[0020] Also provided are hollow and solid microbeads comprising
composites of HA, TCP, other CaPs and ceramic, including glass and
polymeric materials. These microbeads may have abraded surfaces and
aggregates may be made from them. The aggregates may be used in
biomedical implants and in chromatographic columns.
[0021] Also provided are suspendable and non-suspendable aggregates
comprising closed and/or open pores, foamed structures of ceramic,
including glass, and/or polymeric composite materials. These
aggregates can comprise HA, TCP and other CaP coatings, porous
coatings, or biological coatings including growth factors.
Biomedical implants can be made comprising these aggregates. Also
provided are methods of augmenting tissue comprising implanting
these biomedical implants. The biomedical implants may further
comprise a biologically active agent and have a density from about
25% to about 75% of the material's theoretical density.
Chromatographic columns also can be made from such aggregates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows examples of microcarriers (microbeads) of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] CaP Microbead Processing Method for Producing a Suspendable
Microcarrier Substrate
[0024] The present invention relates to suspendable microbeads
(also referred to herein as microcarriers and microspheres). These
microcarriers (FIG. 1) can be used for mammalian cell culturing
applications requiring anchorage-dependent cells in laboratory and
commercial bioreactors. The microspheres may be produced by
specialized slurry processing and subsequent use of a coaxial
blowing nozzle, to create the enclosed porosity structure of the
microspheres for suspension in aqueous-based cell culturing media.
More specifically, the CaP microsphere is hollow and comprised of
suitable mixtures of 0 to 100% HA, and 0 to 100% TCP, and/or other
calcium phosphate compounds including any mixture thereof. The
preferred process for the CaP microcarrier ceramic slurry process
uses a nozzle-reactor system with slurry droplet blowing agent, to
produce hollow microspheres in a size range of about 0.5 millimeter
to about 6 millimeter diameter and specific wall thicknesses that
sinter to an aqueous impermeable state sufficient to maintain
suspendability in culture medium. As examples, the microcarrier
wall thicknesses are about 20 to about 40 micrometers for 0.5 mm
diameter, about 50 to about 80 micrometers for 1.0 mm diameter, and
about 170 to about 230 micrometers for 3.0 mm diameter
microcarriers to maintain suspendability. More specifically, the
density of the microspheres is preferably in the range from about
1.01 gms/cc to about 1.12 gms/cc. Preferably, the density range is
controlled from about 1.01 gms/cc to about 1.06 gms/cc. Final
hollow microsphere dimensions are adjusted to compensate for
shrinkage normally encountered during sintering from the formed
state, which in the case of CaP mixtures of HA and TCP is typically
in the range of about 15-25% linear shrinkage.
[0025] Furthermore, using variations in processing conditions or in
post-microsphere fabrication processing, the hollow microspheres
can have either rough (e.g., textured or abraded) or smooth
surfaces for tailoring the surface condition to either enhance the
attachment of cells for increasing the production of cell
by-products, or to enhance the release of grown cells in culture
for applications requiring the proliferation of cells. The surfaces
of the hollow microspheres may also comprise other substances,
including, for example, biological coatings, including growth
factors, selected proteins, amino acids, collagen, and other such
materials.
[0026] Additionally, the density of the hollow microsphere can be
adjusted to about 1.2 gms/cc to about 2.0 gms/cc for fluidized or
fixed bed applications. The microsphere wall thickness may be
increased for any given suspendable size to achieve this range.
Also, for fixed bed applications, the density of the microcarrier
may be greater than 1.6 gms/cc.
[0027] Alternate Method for Fabricating Hollow CaP Microcarriers
with Diameters Less Than 500 Micrometers (Sol Gel Type System)
[0028] In this aspect of the invention, a reaction precipitation
method for producing submicron CaP microspheres (FIG. 1, 1.2) is
used in conjunction with an oleyl alcohol condensing solution to
fabricate hollow microspheres. The CaP precipitated solution is
nozzle-sprayed onto the oleyl alcohol using nozzle size, pressure,
and spray distance to control the size of the microsphere. This
method utilizes a low solids to liquid ratio (typically less than
about 20%) in conjunction with hot oleyl alcohol for concentrating
the CaP solids into a shell for forming the microspheres. After the
fabricating step, the microspheres are dried, sintered and
classified to size and density by traditional sieving and air
classification methods. The microspheres also may be classified to
desired densities for buoyancy by liquid density separation
methods. The degree of sintering also is used to control the
porosity or permeability of the microsphere. Therefore, a
suspendable or non-suspendable (porous/non-porous) microcarrier may
be produced by this method. More specifically, for fixed bed or
fluidized bed reactors, higher density microcarriers are produced
to meet the requirements for these non-suspendable
applications.
[0029] Alternate Method for Fabricating Hollow CaP Microspheres by
Coating on Wax or Other Organic Microbeads (FIG. 1, 1.2)
[0030] Slurries or powders of calcium phosphate compounds are
applied to the surfaces of wax or other organic microbeads. The
organic microbead is removed by thermal decomposition, solvent
extraction, or a combination thereof. The slurries or powders may
incorporate binder to increase the strength of the shell formed on
the wax or organic microbead, and may further aid in maintaining
the formed CaP microsphere during removal of the wax or organic
substrate. If required, the slurry solids content can be adjusted
to increase or decrease the density of the CaP microsphere.
Likewise, powder can be compacted around the wax or organic
microbead to increase the density of the resulting CaP microsphere.
After the fabrication step, the microspheres are dried, sintered
and classified to size and density by traditional sieving and air
classification methods. The microspheres also may be classified to
desired densities for buoyancy by liquid density separation
methods. The degree of sintering also is used to control the
porosity or permeability of the microsphere. Therefore, a
suspendable or non-suspendable (porous/non-porous) microcarrier may
be produced by this method. More specifically, for fixed bed or
fluidized bed reactors, higher density microcarriers are produced
to meet the requirements for these non-suspendable
applications.
[0031] Porous CaP Coatings for Bonding to the Substrate Surfaces of
Hollow or Solid Beads or Microbeads Comprised of CaP, Glass, Other
Oxide Ceramics or Polymers, Proteinaceous Materials or Composite
Materials (FIG. 1, 1.1 and 1.3)
[0032] A further object of this invention relates to porous CaP
coatings that are comprised of suitable mixtures of particulate HA,
TCP and/or other calcium phosphate compounds, and varying amounts
of porosity for cell culturing applications. The benefits of CaP
coatings include enhanced cell attachment, the protection of cells
during culturing, and increased surface area for cell proliferation
on a variety of substrates, including dense CaP and other dense or
porous substrate materials. The purpose of CaP coatings on non-CaP
substrates is to enhance the beneficial bioactivity of the
substrate surface in cell culturing applications.
[0033] More specifically, two size ranges of porosity have been
identified which include pore sizes less than about 30 micrometers
for increased chemical activity of the substrate, and about 30-80
micrometers for in-growth by cells, protection of cells and
enhanced chemical activity of the substrate. For either pore size
range, the amount of porosity is typically greater than about 10%
but less than about 60% to ensure mechanical integrity of the
coating. More specifically, the amount of porosity to ensure
interconnectivity should be greater than about 20%. Appropriate
methods for applying CaP coatings include a slurry coating
technique and/or applying adherent powder/particulates. The
coatings may comprise open and/or closed porosity. Specific "pore
formers" which produce the desired pore size range include various
organic materials, which are varied in amount to produce porous CaP
coatings for use in cell culturing applications. During the thermal
processing cycle for sintering, the pore formers decompose leaving
channels of interconnected porosity for pore volumes typically
greater than about 20%.
[0034] Microbead substrate materials to be coated include, for
example, dense CaP, and other oxide ceramics such as alumina,
mullite, porcelain or glass. These substrate materials may be used
to produce hollow or solid microspheres for cell culturing
applications. The CaP coatings are applied to either unsintered or
pre-sintered microspheres, and subsequently re-sintered for
bonding. The substrate also may be reheated to soften the surface
in the case of a glass substrate. The CaP coating can be applied to
porous substrates and then co-sintered to enhance bonding and
further densify the substrate layer. For polymeric and
proteinaceous substrates, the preferred method for bonding is
reheating the substrate material to soften the surface or applying
a secondary bio-adhesive material to provide a bonding layer.
[0035] Aggregate Suspendable Microcarriers with Open Porosity (FIG.
1, 1.4)
[0036] CaP microcarrier spheres are prepared as described above
using a nozzle method from either a modified sol gel process or
powder slurry process. The preferred embodiment of this aspect of
the invention requires hollow microspheres with densities less than
1 gm/cc which are subsequently bonded with CaP-prepared powder
slurry, calcium phosphate/sulfate cements, or sol gel-modified
slurries in the unfired state for producing aggregates. Lower
initial microsphere densities are required to offset the additional
weight of the bonding slurries for achieving final suspendability
in the range of about 1.00 gms/cc to about 1.12 gms/cc. After
sintering, aggregates are sized by well known ceramic
granulating/grinding and sieving methods. The sized aggregates are
then sorted for density by liquid density separation methods.
Liquid densities are prepared in the desired range for buoyancy and
aggregates are separated based on the suspending properties. The
starting sphere size can be used to control the open pore size and
pore size distribution. One advantage of this method is that the
open pores are used as sites for cell attachment and growth,
thereby providing protection to the cultured cells and additional
surface area for enhancing growth of cells. As stated above, the
aggregate density is increased by addition of bonding slurry and/or
additionally increasing the wall thickness of starting microspheres
for use in fixed bed or fluidized bed reactors as examples of
non-suspendable applications.
[0037] Aggregate Suspendable Microcarriers with Closed Porosity
(FIG. 1, 1.5)
[0038] As an alternative to a large, single void hollow
microsphere, the closed porosity of the microcarrier can be
distributed in multiple, isolated pores to reduce the density of
the CaP in meeting the suspendability requirement. Closed porosity
refers to microcarriers wherein voids are separated by dense
material not open to the external surface. The preferred approach
for creating closed porosity within the aggregate consists of the
use of a closed-cell organic pore former material which produces
about 60% to 70% closed porosity within the aggregate. After
sintering, aggregates are sized by well known ceramic
granulating/grinding and sieving methods. The sized aggregates may
then be sorted for density by liquid density separation methods.
Liquid densities may be prepared in the desired range for
suspendability and aggregates are separated based on the suspending
property. As stated above, the aggregate density is increased for
use in fixed bed or fluidized bed reactors.
[0039] Composite Suspendable or Non-Suspendable Microcarriers (FIG.
1, 1.0 and 1.6)
[0040] The preferred embodiment of this aspect of the invention
relates to the incorporation of substantial volume fraction of CaP
powder or particulate filler in a hollow or solid natural or
synthetic polymer (e.g., polyethylene, polystyrene, dextran,
gelatin) and/or glass microsphere. More specifically, the void
volume in the hollow microsphere structure of a polymer or glass
reduces the bulk density. As a result, the microcarrier can
accommodate a higher CaP filler solids content and still meet the
suspendability requirement. These microcarriers employ a variety of
hollow glass or polymer microsphere processing methods.
Subsequently, a CaP filler is incorporated to increase density to
the desired suspendability.
[0041] As an alternative to the hollow polymer or glass
microsphere, substantial amounts of "closed porosity" in the
polymer or glass also allows for higher CaP filler additions due to
the lower initial bulk density of the polymer or glass component in
the composite. The closed porosity in the polymer or glass can be
created, for example, by a foaming agent or control of sintering
parameters to produce closed porosity. After the composite
microsphere having a size of about 100 to about 500 micrometers is
made, the surface can be modified by abrading to increase exposure
of the CaP filler for enhanced cell attachment and growth activity.
The density of the microcarrier composite also can be increased
using higher levels of CaP filler in either a hollow microsphere or
solid form for fixed bed or fluidized bed reactor applications.
Additionally, non-suspendable or suspendable microcarriers may be
fabricated from monolithic (continuous solid) forms of combined CaP
filler and polymer and/or glass materials (without or with
porosity) which are subsequently granulated, ground or chopped into
desired sizes and shapes.
[0042] CaP Non-Suspendable Microcarrier with Open Porosity (FIGS.
1, 1.2, 1.7 and 1.8)
[0043] CaP non-suspendable microcarrier spheres with open porosity
are prepared as described above using a conventional spray drying
method or pelletizing method well known in the art from either a
modified sol gel process or powder slurry process. Also the method
taught by Martin (U.S. Pat. No. 3,875,273, supra.) can be used to
form open porous microcarriers. The preferred shape of the
individual microcarrier produced by the methods described above is
sphere-like with a continuous porous phase (FIGS. 1, 1.8). An
alternative shape of microcarrier is a hollow microsphere having a
continuous porous wall that connects the central microsphere void
to the outer surface (FIGS. 1, 1.2). This form of microcarrier can
be produced by the reactor nozzle method taught by Torobin (U.S.
Pat. No. 5,397,759). The open porosity of microspheres is created
by sintering at a lower temperature of about 1100.degree. C., which
is less than that typically required to densify the material, or by
adding a pore former as previously discussed, followed by
sintering. The preferred embodiment of this invention requires
microspheres with densities greater than 1.12 gm/cc for packed or
fluidized bed bioreactors.
[0044] The above-described microspheres can be subsequently bonded
with CaP-prepared powder slurry or sol gel-modified slurries in the
unfired state for producing aggregates. The aggregate form is
created by sintering the agglomerates in a fabricated form. After
sintering, aggregates are sized by well known ceramic
granulating/grinding and sieving methods. An alternative bonding
method comprises the use of CaP or calcium sulfate cement or other
cement for bonding microspheres. One advantage of the aggregate
method for fluidized bed applications is that the open pores of the
bonded aggregate are used as sites for cell attachment and growth,
thereby providing protection to the cultured cells and additional
surface area for enhancing growth of cells. The starting sphere
size can be used to control the open pore size and pore size
distribution for optimizing cell growth of different cell types.
The density of these microcarriers will, in general, be greater
than 1.12 gms/cc in either individual microcarrier or aggregate
form.
[0045] CaP Microspheres for Chromatographic Applications
[0046] The processing methods as set forth for CaP microsphere
fabrication (e.g., CaP Microbead Processing Method for Producing a
Suspendable Microcarrier Substrate and Alternate Method for
Fabricating Hollow CaP Microcarriers with Diameters Less Than 500
Micrometers (Sol Gel Type System) described above) can be used to
produce microspheres for use in chromatographic applications. In
this application, a preferred embodiment for the microspheres is in
the size range of about 10 to 100 micrometers diameter with open
porosity in the range of about 20% to about 60% and a pore size
range from about 0.01 to about 0.5 micrometers. Microsphere sizes
larger than 100 micrometers, up to about 2 millimeters diameter in
either the hollow or non-hollow sphere form, improve permeability
by minimizing the resistance to flow in the chromatographic column
while maintaining the ability to separate and purify proteins,
enzymes, nucleic acids, viruses, and other macromolecules. In
addition, the thin wall of the hollow porous microsphere improves
permeability for greater efficiency of separation and
purification.
[0047] Implantable CaP Microspheres and Aggregates of Bonded
Microspheres
[0048] The processing methods as set forth for CaP microsphere
fabrication (e.g., CaP Microbead Processing Method for Producing a
Suspendable Microcarrier Substrate and Alternate Method for
Fabricating Hollow CaP Microcarriers with Diameters Less Than 500
Micrometers (Sol Gel Type System) described above) also can be used
to produce microspheres for use as a biomedical implant. In this
application, a preferred embodiment for the microspheres is in the
size range of about 500 micrometers to about 1,000 micrometers
diameter. Compacts of microspheres in this size range produce an
interstitial open porosity of about 60% with a pore size range of
about 350 micrometers to about 500 micrometers. For other tissue
in-growth applications, such as epithelial tissue, the microcarrier
diameter size range can be adjusted to provide an open pore size
range from about 50 micrometers to about 150 micrometers to
facilitate tissue in-growth.
[0049] As stated previously, aggregates can be formed by bonding
microspheres using the CaP slurry and cement methods (e.g., CaP
Non-Suspendable Microcarrier with Open Porosity) and can be used
for biomedical implant applications. Cemented aggregates offer the
advantage of conformation to the implant site without subsequent
sintering. In addition, these microspheres can be used with
collagen to form a composite implantable material. The open pore
size within the microsphere-bonded aggregates can be adjusted for
specific tissue in-growth as stated above.
[0050] The microspheres and aggregates of microspheres discussed
above can be used as carriers of biological growth factors and
other pharmaceutical agents including anti-inflammatory and
anti-tumor agents. Open porosity within the microsphere can be made
by sintering at a temperature less than that required to fully
densify the material, or by adding a pore former to the material.
This open porosity can be adjusted to facilitate delivery of
specific biological growth factors and pharmaceutical agents. The
tissue growth factors or pharmaceutical agents are incorporated
either as a coating on microspheres or aggregate, or are
impregnated within the open porosity of the microsphere or
aggregate. The size of the open porosity between individual
microspheres and within aggregates of microspheres can be adjusted
by changing the size of the microspheres. Also, these microspheres
and aggregates can be used to culture tissues which may be
subsequently implanted to augment tissue defects.
[0051] Hollow microspheres and hollow microspheres bonded in
aggregates provide a central cavity as a reservoir for growth
factors or other pharmaceutical agents. The hollow microsphere
provides compressive strength due to its geometry as a basic sphere
in distributing mechanical stress within its wall. In addition, the
thin wall of the hollow microsphere can be replaced by tissue
in-growth as the material within the wall resorbs. The degree of
resorbability may be adjusted by changing the wall thickness, the
amount and size of porosity within the wall, and the amount of HA,
TCP, and/or other CaP phases. Generally, an increased amount of TCP
increases the resorbability of the microsphere.
[0052] Aggregates of the material can be shaped by hand carving or
mechanized grinding. The degree of carvability can be adjusted by
changing the strength of the bond between the microspheres. This
bond strength can be changed by adjusting the sintering temperature
of the bonding slurry or by adjusting the bonding cement chemistry.
As previously stated, aggregates formed with cements can also be
molded and allowed to set and conform to the implant site, thereby
reducing the need for grinding or carving to shape.
[0053] A typical application for microspheres and bonded aggregates
of microspheres is the repair and augmentation of bony defects.
Also, microspheres of smaller sizes can be used to augment soft
tissue, e.g., cartilage defects.
[0054] An object of this invention is to provide appropriate forms
of calcium phosphate ceramic materials to be fabricated in specific
shapes and sizes for anchorage-dependent mammalian cell culturing
applications. Fabricated forms of the calcium phosphate ceramic to
be used as microcarriers in, for example, mammalian cell culture
applications include hollow microspheres, solid spheres, aggregates
of microspheres, multi-pore aggregates, polymeric and glass CaP
composites. A variety of coatings that can be made highly porous or
combined with organic or polymeric materials, including growth
factors, to form composite structures also can be used in
conjunction with these fabricated forms. Combinations of the
aforementioned fabricated forms also can be used to make the
microcarriers of the present invention. To achieve the objects of
this invention, appropriate mixtures of hydroxylapatite, tricalcium
phosphate, and/or other CaP compounds and, in certain cases, an
open pore phase are used to enhance cellular growth through the
higher surface area of the porous structure. Closed porosity is
used to maintain buoyancy in growth media. Although more limited in
application, calcium carbonate can be used in granular form, as a
coating on a substrate carrier for cell culturing applications or
as the Ca phase in polymeric composites. A major advantage of the
CaP ceramic microcarriers is that the finished material can be
heated to as high as 1,000.degree. C. for decomposing organic cell
culture components to recycle the microcarrier after its use in
culture. This heating step can not be done with the polymeric/CaP
composite microcarriers described herein. Other advantages of the
CaP substrate as a microcarrier for cell culturing applications, as
compared to polymeric materials, are that the CaP substrate is
dimensionally stable, due to non-swelling, by absorption of
media.
[0055] For bioreactor applications which require a suspendable form
of microcarrier, the preferred form of the calcium phosphate
substrate comprises a hollow microcarrier of about 0.2 to about 6
mm diameter that has been sintered to a sufficiently dense,
impervious or impermeable state. For anchorage-dependent mammalian
cells, the hollow microcarrier is suspended in the growth medium of
the bioreactor. Therefore, a degree of buoyancy for the
microcarrier is required. The preferred microcarrier density is in
the range of about 1.00 to about 1.12 gms/cc. The density is more
preferably in the range of about 1.00 to about 1.06 gms/cc. The
hollow microspheres are fabricated from a CaP ceramic based on
mixtures of HA and TCP, and/or other CaP compounds. The microsphere
wall is of a controlled thickness in the unsintered state that
varies depending on sphere size (see Table 1). The microsphere wall
is sintered to a sufficiently dense state of about 3.0 gms/cc, or
greater for HA/TCP mixtures to maintain an impervious state for
suspendability. Alternatively, the open porosity structure can be
sealed with a polymeric or other organic film former to achieve an
impervious state.
[0056] U.S. Pat. No. 5,397,759 by Leonard Torobin teaches a process
for fabricating porous ceramic hollow microspheres of uniform
diameter and uniform wall thickness in sizes ranging from 1-4 mm in
diameter. U.S. Pat. No. 5,225,123 by Leonard Torobin also teaches a
process for fabricating hollow ceramic microspheres with closed
porosity. The present invention uses aspects of this technology to
produce a suspendabie calcium phosphate ceramic for use in cell
culture applications. An alternative technology for fabricating
porous ceramic hollow spheres is described in U.S. Pat. No.
3,875,273 by Robert M. Martin. There also are other processes for
fabricating hollow microspheres from ceramic materials as described
by David Wilcox in Hollow and Solid Spheres and Microspheres:
Science and Technology Associated With Their Fabrication and
Application, Materials Research Society Symposium Proceedings,
Volume 372, 1995. These fabrication methods include sacrificial
cores, nozzle-reactor systems, emulsion/phase separation techniques
(including sol gel processing), and mechanical attrition. Although
these approaches do not specifically address calcium phosphate
materials or cell culture microcarrier system applications, the
described processes could be modified to produce the media
suspendable calcium phosphate microcarrier systems of the present
invention based on teachings herein. However, the aforementioned
technologies must be combined with either commercial sources of
calcium phosphate materials or be allied to the chemical
formulation of hydroxylapatite and/or other calcium phosphate
compounds such as tricalcium phosphate (tribasic calcium phosphate)
in order to produce the carriers of the present invention. These
materials are of primary interest, since they can be fabricated in
dense non-permeable forms as taught by A. Tampieri in the Journal
of Material Science: Materials in Medicine, Vol. 8, pp. 29-37,
1997.
[0057] The efficacy of cell culture microcarriers for
anchorage-dependent cells can also be greatly improved by the use
of coatings that enhance cell attachment. Cartwright and Shah in
Basic Cell Culture (J. M Davis, supra.) indicate collagen,
fibronectin, laminin, and Pronectin (synthetic fibronectin
promoting better attachment) are coatings currently used to promote
cell attachment. Cheung (Cheung, supra.) and others have also
reported poly-lysine as a coating that promotes cell attachment and
proliferation.
[0058] Other applications of hydroxylapatite bead materials
(non-suspendable) for biotechnology include, for example, use for
chromatographic filtration/separation columns as taught by Louis
Lange in U.S. Pat. No. 5,492,822 for the isolation of human
pancreatic cholesterol. The HA form of CaP is also used for the
separation and purification of proteins, enzymes, nucleic acids,
viruses, and other macromolecules. According to Bio-Rad in Bulletin
No. 1115, HA has unique separation properties and high selectivity
and resolution. Applications of HA chromatography include
separation of monoclonal and polyclonal antibodies of different
classes, antibodies which differ in light chain composition,
antibody fragments, isozyme, supercoiled DNA from linear duplexes,
and single-stranded from double-stranded DNA.
[0059] For biomedical implant applications, HA has been used in
particulate, monolithic and coating forms for tissue augmentation,
primarily bone. HA has a number of advantages as an implant
material including biological attachment to bone tissue,
outstanding biocompatibility, elastic modulus close to that of
bone, other reasonable mechanical properties such as strength, and
support of new bone growth.
[0060] HA also may be used as a host material for delivery of
biological materials, including growth factors, and referred to
herein as biological coatings, and other pharmaceutical agents
including anti-inflammatory and anti-tumor agents. Hideki Aoki in
Medical Applications of Hydroxyapatite discusses the properties and
uses of HA for biomedical applications. U.S. Pat. No. 5,422,340
teaches the use of calcium phosphate particles as carriers for bone
growth factors. For bone in-growth applications of HA, Kenna in
U.S. Pat. No. 5,192,324 teaches that a particle size of +30 to -20
U.S. Standard mesh size provides pore sizes of 350 micrometers to
500 micrometers to facilitate bone in-growth.
[0061] Hydroxylapatite can be produced by a variety of methods
including: 1) preparation from calcium nitrate and ammonium
phosphate (E. Hayek and H. Newesely in Inorganic Synthesis, Jacob
Kleinberg, editor, McGraw-Hill, New York, Vol. 7, pp 63-65, 1963,
or S. Larsen et al in Experimentia, Vol. 27, No. 40, pp. 483-485,
1971, 2) synthesis from calcium hydroxide and phosphoric acid as
described by J. Tagai et al in Adv. Biomaterials 2, pp 477-488,
1980, 3) sol gel processing as described by A. Deptula in the
Journal of Non Crystalline Solids 2, pp. 477-488, 1980, and 4)
hydrolysis of CaHPO.sub.4 under heat and pressure (hydrothermal
bomb) as described by A. Posner et al, Acta Cryst., 11, p 308,
1958. Likewise, variations of these methods, well know in the art,
are also used to produce hydroxylapatite.
[0062] Tricalcium phosphate can be produced by the partial
decomposition of hydroxylapatite upon calcining at >1200.degree.
C. (A. Tampieri, supra.) and/or the reaction of a stoichiometric
mixture of hydroxylapatite with CaHPO.sub.4 with subsequent thermal
processing at or above 900.degree. C. as described by C. Rey
(Biomaterials 11, p. 13 (1990).
EXAMPLE 1
Process for Producing CaP Hollow Microspheres for Mammalian Cell
Culturing Applications
[0063] CaP hollow ceramic spheres in the range of about 0.2 mm to
about 6 mm in the unsintered state are produced from ceramic
slurries using either a commercial source of starting CaP raw
material (calcium phosphate tribasic) or a precipitated form of CaP
based on the nitrate solution process as taught by Jacob Kleinberg,
editor, Inorganic Synthesis, McGraw-Hill, New York, Vol. 7, pp.
63-65 (Hayek and Newesley), 1963. Using the commercial source of
CaP raw material (FMC, Lawrence, Kans.) slurry processing
properties are controlled by optimizing several variables,
including slurry density-related to solids content, viscosity,
particle size distribution, film stabilizing agents, and dispersing
agents. The characteristic spheres are formed from slurries at
around several thousand per minute. After forming the spherical
geometry from the nozzle, the spheres are subsequently dried in
free flight by loss of solvent with the aid of a high vapor
pressure organic as the dispersing liquid. The dried spheres in the
green state are polymer-bonded shells which are then conventionally
fired to sinter the walls.
[0064] In more detail, the ceramic slurry is fed through the outer
orifice of the nozzle, and the pressurized forming gas that
produces the microsphere void is fed through a center orifice with
the outer orifice acting as a metering area for size control. The
basic coaxial nozzle process is set forth in U.S. Pat. Nos.
5,225,123 and 5,397,759 by Torobin. The forming gas, described
above, acts as a blowing agent within the slurry droplet and
expands during flight from the drop tower. This method of hollow
microsphere formation is set forth in Wilcox, Hollow and Solid
Spheres and Microspheres: Science and Technology Associated With
Their Fabrication and Application, Materials Research Society
Symposium Proceedings, Volume 372, 1995.
[0065] The advantage of the nozzle-reactor system is that it has
been demonstrated, for a variety of ceramic materials, to meet
exacting specifications on sphericity, size and wall thickness
control. Well-designed nozzle systems dispense individual CaP
slurry droplets, with precise control of dimensions, into a reactor
for forming the microsphere geometry. In the reactor, the slurry
droplet is converted to a firm hollow microsphere. The reactor in
drop tower configuration is the preferred method for fabricating
CaP hollow microspheres for use as suspendable microcarriers for
anchorage-dependent cells grown in cultures.
[0066] Sintering of the fabricated hollow microspheres is conducted
in the temperature range of about 1100.degree. C. to 1350.degree.
C., for time periods of about 0.1 to 6 hours to densify the outer
wall of the microsphere to a sufficient level of impermeability to
ensure the suspendability required in typical bioreactor
applications using aqueous media. The product that results from
this process comprises hollow microspheres with the preferred
sphere densities of about 1.00 to about 1.06 gms./cc. For typical
sphere diameters in the range of about 1 to 3 millimeters, sphere
wall thicknesses must be controlled to size limits within the range
of about 75 to about 250 micrometers to achieve sphere densities
slightly greater than 1.00 gms/cc for optimizing suspendability.
Table 1 sets forth the relationship of sphere density as a function
of sphere wall thickness for typical 0.5 mm, 1 mm, 2 mm, and 3 mm
diameter microspheres based on a typical calcium phosphate density
of 3.0 gms./cc.
1TABLE 1 CaP Sphere Diameter, Wall Thickness, Sphere Density Tables
Based on CaP Density of 3.0 Grams Per Cubic Centimeter Sphere Dia.
(mm) Wall Thk. (microns) Sphere Density (gms/cc) 0.5 25 0.81 0.5 30
0.96 0.5 35 1.09 0.5 40 1.22 0.5 50 1.46 0.5 65 1.78 0.5 75 1.97
0.5 100 2.35 1.0 25 0.43 1.0 50 0.81 1.0 60 0.96 1.0 65 1.02 1.0 75
1.16 1.0 100 1.46 1.0 125 1.73 2.0 75 0.63 2.0 100 0.81 2.0 125
0.99 2.0 150 1.16 2.0 175 1.32 2.0 200 1.46 2.0 225 1.60 2.0 250
1.73 3.0 100 0.56 3.0 125 0.69 3.0 150 0.81 3.0 175 0.93 3.0 190
1.00 3.0 200 1.05 3.0 210 1.09 3.0 225 1.16 3.0 250 1.26
[0067] An alternative method for fabricating ceramic hollow spheres
is set forth in U.S. Pat. No. 3,875,273 by Martin and can be used
to manufacture the CaP microspheres of the present invention.
However, it is not as readily scaled for high volume production
needs. The Martin method produces porous ceramic microbeads which
subsequently require sealing of the outer microsphere surface with
a polymeric film to attain a suspendable microcarrier.
EXAMPLE 1A
Alternative Method for Producing CaP Hollow Microspheres with
Diameters in the Size Range From 0.2 mm to 6.0 mm by Coating Wax or
Other Organic Microbeads with CaP Compounds
[0068] Hollow ceramic microspheres may also be fabricated by
coating ceramic powders or slurries onto microbeads of wax or other
organic materials and subsequently removing the wax microbead
through thermal decomposition and/or solvent extraction. The
resulting hollow ceramic microspheres are sintered to the desired
density.
[0069] More specifically, a microbead of polyethylene wax or other
wax or organic material is formed by spraying from a melt and
re-solidifying at a lower temperature. Size of the microbeads is
determined by the size of the spraying orifice and the pressure
under which the organic material or wax is sprayed. Wax or other
organic microbeads also can be produced, for example, by compaction
of wax powders by rolling in heated ball mills or pan pelletizers
or by rolling the powders and gradually adding a solvent to the
powders to consolidate them in the form of beads. The size of the
beads is controlled by the particle size of the starting powder,
heat of the ball mill or pan pelletizer, speed of rotation of the
ball mill or pan pelletizer, size of the ball mill or pan
pelletizer, length of rolling time, and amount and speed of
addition of an organic solvent system. The desired size of bead is
obtained by screening. This screening process also removes the
unconsolidated powders from the powder consolidation method.
[0070] In the case of hollow ceramic (CaP) microspheres prepared
from slurries, powders with a broad particle size distribution are
prepared by ball milling dry powders. These powders are further
reduced in particle size by wet milling. By this means, a slurry
with a high solids content can be prepared as is well known in the
art of preparation of ceramic powders and materials. Likewise,
dispersants and binders can be milled with the slurry to produce a
higher solids content, promote adherence to the wax/organic
microbead and to promote stronger resulting ceramic microspheres.
The solids content of the slurry controls the final
density/porosity of fabricated ceramic microbead in the unsintered
state. The prepared slurry is mixed with the previously described
wax or organic microbeads of the desired size. The mixture is
sprayed under pressure through an orifice of sufficient size to
allow passage of the wax/organic beads with a coating of slurry.
The slurry coating of the wax/organic microbeads may also be
accomplished by converging the slurry mixture with a liquid mixture
of the wax/organic microbeads such that the two streams of
materials converge causing the coating of the ceramic slurry onto
the wax/organic microbeads. The slurry-coated wax/organic
microbeads are allowed to dry during falling in air or by drying in
heated air sufficient to cause drying of the coating, but not
melting or decomposing of the wax/organic microbead.
[0071] The coated microbeads are further classified to the desired
sizes by screening through screens of the desired mesh sizes. The
wax is removed from the ceramic-coated wax beads by heating the
coated beads to melt and decompose the wax/organic substrate. The
porosity of the unsintered ceramic-coated shell allows for the
removal of the wax/organic substrate by melting and thermal
decomposition. Likewise, the porosity of the unsintered
ceramic-coated shell allows for removal of the wax/organic by
solvent extraction or a combination of solvent extraction and/or
thermal decomposition. After the fabricating step, the microspheres
are further dried, sintered and classified to size and density by
traditional sieving and air classification methods. The
microspheres also may be classified to desired densities for
buoyancy by liquid density separation methods.
[0072] In the case of preparation by compaction of ceramic powders
onto wax/organic beads, wax/organic beads are prepared as
previously described in this example. A fine ceramic powder
distribution is obtained by numerous methods well known in the art.
An example of such a method is dry ball milling and subsequent wet
ball milling. The wet milled powder is subsequently dried and
further ball milled or air jet milled to break up agglomerates. The
resulting powder and wax/organic microbeads of the desired size are
placed in a ball mill, pan pelletizer or other container and rolled
or vibrated to compact the powders onto the wax/organic microbead.
The use of a dense micro-media may also be added to a ball mill or
other container to further compact the powders onto the wax/organic
microbeads. Furthermore, the resulting shell thickness and density
of the ceramic coating is controlled by the energy imparted to the
fabricated bead. The amount of energy is controlled by the amount
of time of compaction, and speed of rotation or vibration, and/or
addition of liquid to promote the agglomeration of powders onto the
wax/organic microbeads. Excess or unconsolidated powders are
removed from the coated microbeads by sieving through screens of
sufficient size to retain the coated microbeads and allow excess
powders and compacting media to pass through. The wax/organic is
removed as previously described and the ceramic microspheres are
classified to size by methods previously described in this example,
and sintered to the desired density. The above-mentioned methods
are applicable to the formation of CaP-coated wax/organic
microbeads.
EXAMPLE 2
Method for Fabricating Hollow CaP Microcarriers with Diameters Less
Than 500 Micrometers (Modified Sol Gel Type System)
[0073] Using the method set forth by Hayek and Newesley in Jacob
Kleinberg, editor, Inorganic Synthesis, McGraw-Hill, New York, Vol.
7, pp. 63-65, 1963 for the synthesis of hydroxylapatite, basic
solutions of calcium nitrate and ammonium phosphate are prepared
and adjusted to a pH above 9 to promote the precipitation of
hydroxylapatite upon addition of the ammonium phosphate to the
calcium nitrate. Upon completion of the reaction, the solids are
allowed to partially settle and are then decanted from the reacting
solution. The decanting process is then repeated three times with
distilled H.sub.2O. Care must be taken not to wash away the
hydroxylapatite precipitate. Based upon initial reaction
calculations, the precipitate is diluted to a solid concentration
of approximately 15-20%. A dispersant such as Pluronic (BASF,
Parsippany, N.J.) may be titrated into this precipitate until a
fluid "water-like" consistency is obtained. Using a method as set
forth by Kyung Moh for non-CaP ceramics ("Sol Gel Derived Ceramic
Bubbles", Hollow and Solid Microspheres, Material Research Society
Proceedings, #372, ed. by D. L. Wilcox, 1995), a solids content
consisting of 100 grams of CaP, 16 grams of acetone and 0.5 grams
of methyl cellulose (Dow Chemical, Midland, Mich.) is added and
stirred into an even consistency. The mix is covered with Parafilm
wax (VWR Scientific, Chicago, Ill.) to prevent evaporation. This
solution is subsequently sprayed into hot oleyl alcohol (at
95.degree. C.). The nozzle size, pressure, and distance from the
oleyl alcohol can be adjusted to give the desired microsphere size.
The mixture of droplets and oleyl alcohol is stirred for
approximately 20 minutes. The gelled bubbles are subsequently
filtered from the oleyl alcohol and placed on a refractory dish or
plate, dried at .about.100.degree. C. for 1 hour and fired at a
rate of 100.degree. C./hr to 1100-1300.degree. C. for approximately
1 hour to obtain dense or semi-permeable hollow microspheres
depending on the desired permeability. The microspheres can be
separated into desired classifications by well known sieving, air
classification methods, and/or buoyancy classification in solutions
of desired densities.
EXAMPLE 3
Porous CaP Coatings for Bonding to the Substrate Surfaces of Hollow
or Solid Beads or Microbead Materials Comprised of CaP, Glass,
Other Oxide Ceramics, Polymers, Proteinaceous Materials or
Composite Materials
[0074] The surface of the hollow microsphere can also be altered by
applying a porous layer of suitable particulate calcium phosphate
ceramic which (1) will increase the chemical activity of the
material due to the higher surface area of the material and (2)
through larger interconnecting pore sizes, can also provide porous
channels to accommodate cell and tissue in-growth.
[0075] The porous calcium phosphate coating composition is
comprised of a commercial source of tricalcium phosphate powder
(FMC, Lawrence, Kans.) which is either used as-received, or
preferably, is calcined in the temperature range of 1100.degree. C.
to 1250.degree. C. to slightly coarsen the material for making
porous coatings. The tribasic calcium phosphate ceramic powder is
readily processed in an aqueous medium or other solvent if desired.
The aqueous vehicle can be used in a process that readily
accommodates a dispersant for increasing solids while minimizing
shrinkage during drying. It also accommodates an organic-based pore
former that is the preferred method for generating porosity within
the calcium phosphate structure after sintering. Typically, the
pore former is added in sufficient quantity (>30 volume percent)
to create a continuous porous phase when higher levels of porosity
in the coating are desired. The aqueous tribasic calcium phosphate
slurry is cast onto the surface of the microcarrier substrate and
allowed to dry. The inclusion of a small amount (.about.1%) of an
acrylic emulsion binder aids in providing higher green strength to
the unfired microcarrier for improved handling, and offers
resistance to cracking during the drying process.
[0076] For calcium phosphate hollow microspheres and other solid
forms of calcium phosphate to ultimately be sintered to an
impervious state, the preferred method for coating the microbead or
other substrate surface is to pre-sinter the microbead to a
temperature of about 1100-1300.degree. C. This provides
sufficiently high strength for slurry coating such that the coating
and microcarrier can be co-sintered to bond the coating to the
microcarrier substrate. The microcarrier substrate is densified
during final sintering. Final sintering can be done at a higher
temperature typically in the range of 1150-1400.degree. C. This
sinters the calcium phosphate microcarrier wall with the coating
having residual porosity derived from the pore formers that are
incorporated into the coating formulation. The resulting material
is at least a two-layer calcium phosphate structure (the substrate
is one of the layers) with a dense wall supporting a tailored
porous coating layer for enhancing the chemical activity and
in-growth potential for cells and tissues grown in culture. The
amount and size of porosity can be altered based on changes in
coating formulation. The amount and type of pore former is the
primary material variable that controls the porous phase.
Typically, the amount of porosity in the coating will be in the
range of about 20-60% with the pore size controlled in either a
fine distribution or coarser distribution, depending on the
application. The fine pore size distribution is comprised of a
majority of pores less than about 30 micrometers. The coarser pore
size distribution will have pore channels in about the 30 to 80
micrometer range, but the pore size distribution can be further
altered to produce an even coarser and/or finer pore size
distribution for certain applications.
[0077] As an alternative to the slurry method of coating, a powder
or particulate agglomerate of CaP may be directly bonded to the
surface of the solid CaP, or other suitable substrate, using a
coating process that simulates a spray granulation (or disk
pelletizing) processing method that is well known in the art as
taught by J. Reed, Principles of Ceramics Processing, Second
Edition, John Wiley & Sons, Inc. New York, 1995. Any form of
the solid substrate, including a hollow CaP microsphere, is
introduced in a contained system with a liquid or binder-liquid
(such as an aqueous-based acrylic emulsion) and sprayed onto the
surface to promote adhesion of the loose powder or particulate onto
the solid substrate surface and subsequently sintered. The
particulate may be an agglomerate of CaP powder that contains open
porosity in order to increase surface area of the CaP material for
greater activity in cell culturing applications.
[0078] A CaP coating, typical of the type described above, is not
limited in application to the surface of hollow microspheres.
Calcium phosphate coatings, as formulated and tailored to specific
property needs, can also be applied to a variety of other substrate
materials including, but not limited to, alumina, mullite,
cordierite, or other ceramic materials all of which are examples of
oxide materials. The porous coating can be applied to either a
hollow or a solid microbead. The microbead is a substrate for
bonding the porous calcium phosphate coating in an appropriate
sintering cycle. In addition to the variety of oxide ceramic
substrate materials that are available in either a hollow or solid
microbead or both, other materials may be used in providing a
substrate surface for bonding the porous calcium phosphate coating.
Glass beads, with or without BioGlass (L. L. Hench, Univ. of
Florida), as an example, offer the advantage of softening over a
broad temperature range. The softening characteristic of glass is
useful for bonding the calcium phosphate coating. Similarly, a
polymeric substrate material, particularly one that has an
amorphous phase in its structure, also may exhibit a softening
effect with temperature or solvent treatment of the surface. This
softening makes it easier to bond a calcium phosphate coating
because of the softening and wetting of the particulate calcium
phosphate coating material. Examples of polymeric/organic materials
that have utility as a substrate in either a hollow or solid
microsphere form include, but are not limited to, dextran,
polyethylene, polypropylene, polystyrene, polyurethane, and
collagen.
[0079] The porous structure of the calcium phosphate coating
provides a large surface area interface that is well suited for
culturing anchorage-dependent cells. In certain applications,
growth enhancing materials, such as biological coatings, including
growth factors which may be added separately in the porous
structure or coated onto the surface of the coating, are useful in
promoting cell and tissue growth with the calcium phosphate
substrate providing additional mechanical and anchorage-dependent
functionality. Collagen and other bio-adhesives (coated or added
separately) provide special utility in promoting cell adhesion and
are well suited for adapting to the coarse porosity in the CaP
coating. Collagen also has the potential for forming "bridges"
between individual CaP coated hollow or solid microbeads.
EXAMPLE 4
Porous Calcium Phosphate Coating for Application on Calcium
Phosphate, Other Ceramic, Glass (BioGlass.RTM.), and Polymer Hollow
Microspheres and Spheres
[0080] A typical example of a porous calcium phosphate coating,
having a fine pore size distribution, typically less than about 30
micrometers, is comprised of the following components:
2 Volume Percent As-received or Calcined Tribasic Calcium Phosphate
Powder 50-80 Corn Starch Pore Former or Expancel 461-DE 20 20-50
Total Calcium Phosphate and Fine Pore Former Components 100 Aqueous
Formulation Total Calcium Phosphate/Fine Pore Former Components
25-40 Distilled Water 60-75 Ammonium Polyacrylate Dispersant
0.5-1.0
[0081] A typical example of a porous calcium phosphate coating
having a coarse pore size distribution with the majority of pore
sizes in the range of 30-60 micrometers is comprised of the
following components:
3 Volume Percent As-received or Calcined Tribasic Calcium Phosphate
Powder 50-80 Potato Starch Pore Former or Expancel 091-DE 80 20-50
Total Calcium Phosphate and Coarse Pore Former Components 100
Aqueous Formulation Total Calcium Phosphate/Coarse Pore Former
Components 25-40 Distilled Water 60-75 Ammonium Polyacrylate
Dispersant 0.5-1.0
[0082] Material Sources:
[0083] Tribasic Calcium Phosphate: FMC, Lawrence, Kans.
[0084] Corn Starch: Argo Brand, CPC Int., Inc., Englewood Cliffs,
N.J.
[0085] Expancel Polymers: Expancel, Inc., Duluth, Ga.
[0086] Potato Starch: Western Polymer Corp., Moses Lake, Wash.
[0087] Polyacrylate Dispersant (Duramax D-3021): Rohm & Haas,
Montgomeryville, Pa.
[0088] To reduce the surface area of the as-received tribasic
calcium phosphate powder for ease of slurry processing at higher
solids content, the material is calcined in the 1100-1250.degree.
C. temperature range prior to mixing with the pore former, water
and dispersant components. The preceding porous coating
formulations are sintered in the temperature range of about
1000.degree. C. to 1300.degree. C. for bonding to the surface of
CaP or other ceramic spheres. For glass or polymer spheres, the
coatings are bonded to the surface at a temperature near the
softening point of the glass or polymer composition.
EXAMPLE 5
Aggregate Suspendable Microcarriers with Open Porosity
[0089] Microcarrier spheres are prepared as set forth above by the
modified sol gel, the Torobin spray nozzle method or other methods
adapted from procedures as set forth in Hollow and Solid
Microspheres (D. Wilcox, supra.) The density of the hollow
microspheres is adjusted to less than 1.0 gms/cc to offset the
additional fired weight of the bonding CaP medium to be added. A
slurry of CaP, made either from commercially available powders such
as FMC (Lawrence, Kans.) or sol-prepared reacted sources (see Hayek
and Newesley in Jacob Kleinberg, supra.) is prepared in H.sub.2O
with a solids content of 15-35% by weight. Approximately 1% of
methylcellulose (Dow Chemical, Midland, Mich.) and 6 drops of
Pluronic (BASF, Parsippany, N.J.) per 20 gms of CaP solids is added
to improve processing and green strength of formed aggregates. The
slurry and microspheres are mixed together in a container until the
slurry covers the microspheres with a thin coating. The coated
spheres are subsequently placed in a form for containment during
sintering. The form used for containment can either be thermally
decomposed (paper container) during sintering or separated from the
aggregate after sintering in a ceramic crucible. The green
aggregate is dried at 100.degree. C. for one hour and subsequently
fired at a rate of about 100.degree. C./hr to 1100-1300.degree. C.
for 1 hour to obtain composites of spheres with interconnecting
open porosity. The fired aggregate can subsequently be pulverized,
sieved and classified by liquid gravity separation into the desired
size/density of aggregate which would allow for the growth of
cells, during cell culture, within protected open pores, in
addition to the outer surface of the aggregates.
EXAMPLE 6
Aggregate Suspendable Microcarriers with Closed Porosity
[0090] For a CaP hollow microsphere, the amount of enclosed
porosity required to achieve suspendability is the range of about
60% to 70%. For other than a hollow microsphere, this amount of
porosity is redistributed in an aggregate form having multiple
enclosed pores. Microcarriers are fabricated by addition of foaming
agents to CaP slurries, and subsequently drying these slurries,
such that the enclosed bubbles formed during foaming remain in
place to provide a closed multi-pore structure. A typical foaming
agent is an organic wetting agent which in a slurry, exhibits low
surface tension and is capable of creating a foamed structure with
the mechanical shear of the slurry. Such foaming agents include,
for example, a Triton X-100 surfactant (Spectrum Chemical Mfg.
Corp., Gardena, Calif.). Another example of a foamed microcarrier
is one created by a chemical reaction which produces gas that
coalesces as bubbles within the CaP slurry (e.g., oxidation of
hydrogen peroxide) and is subsequently dried and thermally
processed.
[0091] In both cases, the foamed aggregates are sintered to
approximately 1200 to 1350.degree. C. such that interstitial CaP
material between the voids is impermeable to liquid media
penetration. These aggregates are made to have neutral buoyancy in
liquid media by adjusting the CaP solids to void content of the
monolith. These materials are subsequently ground by standard
granulation techniques and classified by screening or air
classification to the desired size needed in cell culture.
EXAMPLE 7
Composite Suspendable or Non-Suspendable Microcarriers
[0092] In forming suspendable composites, the amount of CaP filler
additive that can be used relative to a polymer or glass material
is directly related to the density of the bulk polymer or glass
material. However, the solid form polymer or glass can be made to
contain a significant amount of closed porosity phase to reduce its
bulk density. For polystyrene or dextran as examples of polymers
exhibiting utility as a cell culture material, the presence of
closed porosity created by a foaming agent which lowers the bulk
density, will allow for the addition of more CaP filler in meeting
the suspendability requirement due to its higher density.
[0093] A glass or polymer hollow microsphere is the preferred
composite pre-cursor. A composite structure is formed using CaP
powder or particulate as filler material. The final desired
density, for achieving buoyancy as a suspendable microcarrier, will
take into account the polymer or glass phase, CaP filler phase and
a substantial pore phase that results from the hollow
microsphere-forming process. Process methods for forming hollow
microsphere diameters in the size range of about 100 micrometers to
about 6 millimeters are useful in this invention. A method that
allows for a substantial void (e.g., about 30% to about 60% of the
overall bulk volume) in the microcarrier provides the means for a
high loading of CaP filler in forming the composite structure.
Polymeric materials that can be used include polystyrene, dextran,
polyethylene, and others available in hollow microsphere form. The
example below illustrates the degree of CaP loading for such a
polymeric hollow microsphere. Although higher in density, a hollow
glass microsphere is also capable of accepting CaP filler loading
for achieving suspendability.
EXAMPLE 8
CaP Filler Loading in Hollow Polymeric Microsphere
[0094] For a polymer with a 1.1 gms/cc density in a hollow
microsphere form with 60% void volume, the bulk density is 0.44
gms/cc. Therefore, the following equation can be written:
x(0.44 gms/cc)+(1-x)(3.0 gms/cc)=1.05 gms/cc as example of density
for buoyancy for the composite
[0095] where
[0096] x=volume fraction of polymer in hollow microsphere form
[0097] 1-x=volume fraction of CaP filler in the composite
[0098] with x=0.76 as the volume fraction of hollow polymer
microsphere, the corresponding volume fraction of CaP filler is
0.24 or 24%.
[0099] Non-suspendable composites of CaP can be prepared by the
addition of CaP powders and/or particulates to conventional
materials used in cell culture, such as polystyrene, collagen,
dextran, gelatin, or glass as examples. Similar materials such as
polyethylene, polyurethane, and silicones or the like could also be
used. In the case of thermoplastic polymers, sufficient CaP could
be added to the melted polymer to bond the powders or particulates
into a coherent mass of sufficient strength to resist crumbling
during cell culture. CaP particle loading ranges can be from about
10 to 80 volume percent and are adjustable to the requirements of
the culture of specific cells. Likewise, the CaP
powders/particulates can be added to liquid/collagen or gelatin
slurries in equivalent previously cited ranges of CaP volume
percent sufficient to maintain mechanical integrity in cell culture
environments. In the aforementioned cases, the materials can be
either dried under heat or frozen and subsequently broken up by
standard means of granulation to the desired particle sizes, or
atomized by spray drying or other conventional polymer processing
techniques, as previously described, and further classified by
standard sieving methods. A similar process could also be used in
the case of CaP additions to glass, although the CaP material would
have to be added to the melted glass preferably below 1300.degree.
C. The melted glass is subsequently cooled to room temperature and
ground/classified to size or atomized from the melt to produce
particulates, which are classified to the desired sizes. All of
these composites could be used as cell culture substrates or in
discrete particulate forms in cell culture-packed beds or fluidized
beds that require higher density as previously described. Likewise,
the activity of the CaP filler can be further enhanced by abrading
the surface of the composite structures.
EXAMPLE 9
CaP Non-Suspendable Microcarrier with Open Porosity
[0100] CaP ceramic microspheres in the range of about 0.5 mm to
about 6 mm in the unsintered state are produced from ceramic
slurries using either a commercial source of starting CaP raw
material (calcium phosphate tribasic) or a precipitated form of CaP
based on the nitrate solution process as taught by Jacob Kleinberg,
editor, Inorganic Synthesis, McGraw-Hill, New York, Vol. 7, pp.
63-65 (Hayek and Newesley), 1963. Using the commercial source of
CaP raw material (FMC, Lawrence, Kans.), slurry processing
properties are controlled by optimizing several variables,
including slurry density-related to solids content, viscosity,
particle size distribution, organic binders, including pore
formers, and dispersing agents. Slurry processing is followed by
spray drying which is performed using conventional methods well
known in the art as taught by K. Masters, Spray Drying, Leonard
Hill Books, London, England, 1972. The preferred method for
producing microspheres with diameters greater than one (1)
millimeter is by disk pelletizing which is well known in the art of
ceramic processing. Methods for producing open porosity include the
previously cited organic pore formers, and/or by sintering the
microspheres to a temperature less than that required to completely
density the material, e.g., about 1100.degree. C. for the HA form
of CaP. The coaxial nozzle process as set forth in Example 1 above
can also be used to produced hollow microspheres with open
porosity. Open porosity is obtained by lower temperature sintering
as described above. An alternative method for fabricating ceramic
hollow spheres is set forth in U.S. Pat. No. 3,875,273 by Martin
and can be used to manufacture the CaP microspheres of the present
invention.
EXAMPLE 10
Aggregate CaP Non-Suspendable Microcarrier with Open Porosity
[0101] Microcarrier spheres are prepared as set forth above in
Example 9. A slurry of CaP, made either from commercially available
powders such as from FMC (Lawrence, Kans.) or sol-prepared reacted
sources (see Hayek and Newesley in Jacob Kleinberg, supra.), is
prepared in H.sub.2O with a solids content of about 10-20% by
weight. Approximately 1% of methylcellulose (Dow Chemical, Midland,
Mich.) and 6 drops of Pluronic (BASF, Parsippany, N.J.) per 20 gms
of CaP solids is added to improve processing and green strength of
formed aggregates. The slurry and microspheres are mixed together
in a container until the slurry covers the microspheres with a thin
coating. The coated spheres are subsequently placed in a form for
containment during sintering. The form used for containment can
either be thermally decomposed during sintering or separated from
the aggregate after sintering. The green aggregate is dried at
100.degree. C. for one hour and subsequently fired at a rate of
about 100.degree. C./hr to about 1100.degree. C./hr to obtain
bonded microspheres with interconnecting open porosity. An
alternative method for bonding microspheres to make aggregates uses
a CaP cement as taught by L. Chow and S. Takagi in U.S. Pat. No.
5,525,148. The fired or cement-bonded aggregate can subsequently be
pulverized, sieved and classified by liquid gravity separation into
the desired size/density of aggregate which would allow for the
growth of cells, during cell culture, within protected open pores,
in addition to the outer surface of the aggregates.
[0102] For Examples 1-10, the CaP microcarriers can be used in
conventional cell culturing systems. For example, in cell cultures
comprising the BHK 21 (baby hamster kidney) cell line, BHK 21 cells
are anchored to the surface of the CaP microcarrier. This cell line
may be used to produce IL2 (Interleukin 2 Factor) by secretion
(Cartwright, supra).
EXAMPLE 11
CaP Microspheres for Chromatographic Applications
[0103] Microspheres for this application are produced by the
processing methods set forth in Examples 1, 1A, and 2 with the
exception that the microspheres are sintered to less than full
density to make open interconnected porosity in the range of about
20% to 50% and a pore size range from about 0.01 to 0.5
micrometers. This open porosity is produced by sintering the
microspheres at a temperature in the range of about 1100.degree. C.
to 1200.degree. C. An example of an application is the separation
of single- and double-stranded DNA in a HA column as taught by K.
Sundaram and L. Loane, "Liquid Chromatographic Assay For The
Separation of Single- and Double-Stranded DNA By Using UV and UV
Diode-Array Detectors and Hydroxylapatite Column", Journal of
Liquid Chromatography, 18(5), 925-939 (1995).
EXAMPLE 12
Implantable CaP Hollow Microspheres
[0104] Hollow microspheres with a diameter of about 500 micrometers
are produced by methods set forth in Examples 1, 1A, and 2 with the
exception that the microspheres are sintered to less than full
density to make open interconnected porosity in the range of about
20% to 30% and a pore size range from about 0.1 to 1.0 micrometers.
These microspheres are placed in a solution containing transforming
growth factor--beta to infiltrate and coat the microspheres with
the growth factor for the repair of bone defects as taught by A.
Ammann et al. in U.S. Pat. No. 5,422,340. The amount of growth
factor in the microsphere can be increased by infiltrating under
vacuum. The microspheres are suspended in saline in a syringe and
subsequently delivered to the bone defect site. An alternative
method for delivering the microspheres for implantation is to mix
the microspheres with CaP cement at the time of implantation. The
cement as taught by L. Chow and S. Takagi in U.S. Pat. No.
5,525,148 can be used for this method.
EXAMPLE 13
Implantable CaP Aggregate with Bonded Hollow Microspheres
[0105] An aggregate for this application is produced by methods set
forth in Example 10 with the exception that the aggregate is left
in a monolithic form and is not ground or pulverized. The aggregate
is subsequently infiltrated with the transforming growth
factor--beta as described in Example 12. During implantation, the
monolith is carved to the desired implant shape and inserted in the
defect site.
[0106] The foregoing description of the invention is only exemplary
for purposes of illustration. Without departing from the spirit and
scope of the invention, one skilled in the art can make changes and
modifications to the invention to adapt it to various uses and
conditions. Such changes and modifications are within the scope of
the disclosed invention.
[0107] All documents referred to herein are incorporated by
reference.
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* * * * *