U.S. patent application number 10/052121 was filed with the patent office on 2002-10-24 for biocompatible, biodegradable polymer-based, lighter than or light as water scaffolds for tissue engineering and methods for preparation and use thereof.
Invention is credited to Botchwey, Edward, Khan, Mohammed Yusuf, Laurencin, Cato T., Levine, Elliot, Lu, Helen H., Pollack, Solomon R..
Application Number | 20020155559 10/052121 |
Document ID | / |
Family ID | 26730212 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155559 |
Kind Code |
A1 |
Laurencin, Cato T. ; et
al. |
October 24, 2002 |
Biocompatible, biodegradable polymer-based, lighter than or light
as water scaffolds for tissue engineering and methods for
preparation and use thereof
Abstract
Scaffolds for tissue engineering prepared from biocompatible,
biodegradable polymer-based, lighter than or light as water
microcarriers and designed for cell culturing in vitro in a
rotating bioreactor are provided. Methods for preparation and use
of these scaffolds as tissue engineering devices are also
provided.
Inventors: |
Laurencin, Cato T.; (Elkins
Park, PA) ; Pollack, Solomon R.; (North Wales,
PA) ; Levine, Elliot; (Cherry Hill, NJ) ;
Botchwey, Edward; (Philadelphia, PA) ; Lu, Helen
H.; (New York, NY) ; Khan, Mohammed Yusuf;
(Philadelphia, PA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 East Main Street
Marlton
NJ
08053
US
|
Family ID: |
26730212 |
Appl. No.: |
10/052121 |
Filed: |
January 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60262128 |
Jan 17, 2001 |
|
|
|
Current U.S.
Class: |
435/174 ;
435/289.1 |
Current CPC
Class: |
C12N 5/0075 20130101;
A61K 35/12 20130101; A61L 27/3847 20130101; C12N 2533/40 20130101;
A61L 27/3895 20130101 |
Class at
Publication: |
435/174 ;
435/289.1 |
International
Class: |
C12N 005/00; C12N
011/00; C12M 003/00 |
Claims
What is claimed is:
1. A scaffold for tissue engineering comprising biocompatible,
biodegradable polymer-based, lighter than water or light as water
microcarriers.
2. The scaffold of claim 1 which is seeded with cells via culturing
in vitro in a rotating bioreactor.
3. The scaffold of claim 2 wherein the seed cells comprise
osteoblast and osteoblast-like cells, endocrine cells, fibroblasts,
endothelial cells, genitourinary cells, lymphatic vessel cells,
pancreatic islet cells, hepatocytes, muscle cells, intestinal
cells, kidney cells, blood vessel cells, thyroid cells, parathyroid
cells, cells of the adrenal-hypothalamic pituitary axis, bile duct
cells, ovarian or testicular cells, salivary secretory cells, renal
cells, chondrocytes, epithelial cells, nerve cells or progenitor
cells.
4. A method for producing scaffolds for tissue engineering
comprising: (a) preparing biocompatible, biodegradable
polymer-based microcarriers which are lighter than water; (b)
bonding the biocompatible, biodegradable polymer-based
microcarriers into a scaffold; and (c) seeding the scaffold with
cells via culturing in vitro in a rotating bioreactor.
5. A method for regenerating a selected tissue comprising seeding
the scaffold of claim 1 with cells which generate the selected
tissue and culturing the scaffold and seeded cells in a rotating
bioreactor.
6. The method of claim 5 wherein the seed cells comprise seed cells
comprise osteoblast and osteoblast-like cells, endocrine cells,
fibroblasts, endothelial cells, genitourinary cells, lymphatic
vessel cells, pancreatic islet cells, hepatocytes, muscle cells,
intestinal cells, kidney cells, blood vessel cells, thyroid cells,
parathyroid cells, cells of the adrenal-hypothalamic pituitary
axis, bile duct cells, ovarian or testicular cells, salivary
secretory cells, renal cells, chondrocytes, epithelial cells, nerve
cells or progenitor cells.
Description
INTRODUCTION
[0001] This invention was supported in part by funds from the U.S.
government (NASA Grant No. NAG 9-832 and NIH Grant No. AR07132-23)
and the U.S. government may therefore have certain rights in the
invention.
FIELD OF THE INVENTION
[0002] The present invention relates to scaffolds for tissue
engineering specifically designed for cell culture in vitro in a
rotating bioreactor. Scaffolds of the present invention comprise
biocompatible, biodegradable polymer-based microcarriers which are
lighter than and/or light as water. In a preferred embodiment, the
biocompatible, biodegradable, lighter than or light as water
microcarriers are bonded into a scaffold which is then cultured
with cells in a rotating bioreactor. Methods for preparation and
use of these scaffolds as tissue engineering devices are also
provided.
BACKGROUND OF THE INVENTION
[0003] In 1993, Langer and Vacanti et al. estimated the number of
bone repair procedures performed in the United States at over
800,000 per year (Science 1993 260(5110):920-926). Today, skeletal
reconstruction has become an increasingly common and important
procedure for the orthopaedic surgeon. Conventional approaches in
bone repair have involved biological grafts such as autogenous bone
or autografts, allogenic bone or allografts and xenografts
(Burwell, R. G. History of bone grafting and bone substitutes with
special reference to osteogenic induction, in Bon Grafts,
Derivatives and Substitutes., M. R. Urist and R. G. Burwell,
Editors. 1994, Butterworth-Heinemann Ltd.: Oxford. p.3). Currently,
autograft is the preferred biological graft most often utilized in
the clinical setting, having success rates as high as 80-90% and no
risk of immune rejection or disease transfer (Cook et al. J. Bone
Joint Surg. Am. 1994 76(6):827). However, due to limited
availability of autografts and risks of donor site morbidity,
alternative approaches to bone repair have been sought.
[0004] Numerous tissue engineering solutions have been proposed to
address the need for new bone graft substitutes.
[0005] One potentially successful repair solution seeks to mimic
the success of autografts by removing cells from the patient by
biopsy and growing sufficient quantities of mineralized tissue in
vitro on implantable, three-dimensional scaffolds for use as a
functionally equivalent autogenous bone tissue. In this way, an
ideal bony repair environment is created by reproducing the
intrinsic properties of autogenous bone material, which
include:
[0006] a porous, three-dimensional architecture allowing
osteoblast, osteoprogenitor cell migration and graft
re-vascularization;
[0007] the ability to be incorporated into the surrounding host
bone and to continue the normal bone remodeling processes;
[0008] and the delivery of bone forming cells and osteogenic growth
factors to accelerate healing and differentiation of local
osteoprogenitor cells (Burwell, R. G.. History of bone grafting and
bone substitutes with special reference to osteogenic induction, in
Bon Grafts, Derivatives and Substitutes., M. R. Urist and R. G.
Burwell, Editors. 1994, Butterworth-Heinemann Ltd.: Oxford. p.3;
Gadzag et al. J. Amer. Acad. Ortho. Surg. 1995 3(1):1).
[0009] Biodegradable scaffolds for in vitro bone engineering, which
possess a suitable three-dimensional environment for the cell
function together with the capacity for gradual resorption and
replacement by host bone tissue have also been described. See, e.g.
Cassebette et al. Calcified Tissue International 1990 46(1):46-56;
Masi et al. Calcified Tissue International 1992 51(3):202-212;
Rattner et al. In Vitro Cellular & Developmental Biology-Animal
1997 33(10):757-762; Mizuno et al. Bon 1997 20(2):101-107;
Elghannam et al. J. Biomed. Mater. Res. 1995 29(3):359-370;
Ducheyne et al. J. Cell. Biochem. 1994 56(2):162-167; Ishuag et al.
J. Biomed. Mater. Res. 1997 36(1):17-28; Ishuag-Riley et al.
Biomaterials 1998 19(15):1405-1412; Goldstein et al. Tissue
Engineering 1999 5(5):421-433; Devin et al. J. Biomater.
Science-Polymer Edition 1996 7(8):661-669; Laurencin et al. Bone
1996 19(1):S93-S99; Thomson et al. Biomaterials 1998
19(21):1935-1943; and Laurencin et al. J. Biomed. Mater. Res. 1996
30(2):133-138. This three-dimensional matrix milieu provides the
necessary microenvironment for cell-cell and cell-matrix
interaction, and is sufficient for the production of limited
amounts of mineralized bone matrix in static culture. To
demonstrate clinical feasibility of tissue engineered bone and to
sufficiently match the intrinsic properties of autogenous bone
graft material, however, rapid mineralization of osteoid tissue
grown in vitro must be achieved. In the above-described
three-dimensional matrices, nonhomogeneous cell seeding confines
cell density to the near surface of the scaffold and mineralized
tissue formation is limited by inadequate diffusion of oxygen,
nutrients, and waste.
[0010] Using porous polylactic glycolic acid (PLAGA) foams with
pore sizes ranging from 150 to 710 .mu.m, Ishaug-Riley et al.
(Biomaterials 1998 19(15):1405-1412) have observed a limit to
osseous tissue ingrowth and mineralization in a static culture
environment of about 200 .mu.m. While it is possible that
structures with larger pores would facilitate greater diffusion,
important cell-cell interactions and scaffold mechanical integrity
could be compromised.
[0011] Formation of three-dimensional assemblies for culturing of
various cell types in a rotating bioreactor have been described.
See e.g. Goldstein et al. Tissue Engineering 1999 5(5):421-433;
Granet et al. Medical & Biological Engineering and Computing
1998 36(4):513-519; Klement et al. J. Cellular Biochem. 1993
51(3):252-256; Qui et al. Tissue Engineering 1998 4(1):19-34; Lewis
et al. J. Cellular Biochem. 1993 51(3):265-273; Becker et al. J.
Cellular Biochem. 1993 51(3):283-289; and Prewett et al. J. Tissue
Culture Methods 1993 15:29-36. Using such assemblies, it has been
shown that osteoblast-like MC3T3 cells form cell aggregates when
grown on non-degradable microspheres and produce collagen fibrils
in the matrix between microspheres (Klement et al. J. Cellular
Biochem. 1993 51(3):252-256). Also, rat stromal cells cultured for
2 weeks on cytodex-3 beads formed aggregates, began synthesizing
mineralized matrix and showed elevated expression of type I
collagen and osteopontin (Qui et al. Tissue Engineering 1998
4(1):19-34). However, when microspheres with greater density than
the surrounding medium are placed in a rotating bioreactor,
centrifugal force induces heavier-than-water microspheres to move
outward and collide with the bioreactor wall. These collisions
induce cell damage and are a confounding variable in tissue
engineering.
[0012] In the present invention, lighter than or light as water,
biocompatible, biodegradable microcarriers and scaffolds comprising
these microcarriers are used in a three-dimensional culturing
method for the growth of mineralized tissues in vitro in a rotating
bioreactor. The combination of three-dimensionality and fluid flow
of the present invention circumvents limitations associated with
static three-dimensional culturing methods, eliminates confounding
wall collisions, and increases the rate and extent of mineralized
tissue formation in the rotating bioreactor. Scaffolds prepared in
accordance with the present invention exhibit controllable and
quantifiable motion in a bioreactor environment, thereby enhancing
fluid transport throughout the scaffold. As demonstrated herein,
scaffolds produced in accordance with the present invention support
cell attachment, growth, and phenotypic expression over short-term
culture ultimately resulting in enhanced synthesis of mineralized
bone graft quality tissue.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide scaffolds
for tissue engineering comprising biocompatible, biodegradable
polymer-based, lighter than or light as water microcarriers. In a
preferred embodiment, the scaffolds are seeded with cells via
culturing in vitro in a rotating bioreactor.
[0014] Another object of the present invention is to provide a
method of producing scaffolds for tissue engineering which
comprises preparing biocompatible, biodegradable polymer-based
microcarriers which are lighter than or light as water; bonding the
biocompatible, biodegradable polymer-based microcarriers into a
scaffold and seeding the scaffold with cell via culturing in vitro
in a rotating bioreactor.
[0015] Another object of the present invention is to provide
methods for using scaffolds comprising biocompatible, biodegradable
polymer-based, lighter than or light as water microcarriers seeded
with cells via culturing in vitro in a rotating bioreactor as
tissue engineering devices. Scaffolds of the present invention can
be seeded with cells including, but not limited to, osteoblast and
osteoblast-like cells, endocrine cells, fibroblasts, endothelial
cells, genitourinary cells, lymphatic vessel cells, pancreatic
islet cells, hepatocytes, muscle cells, intestinal cells, kidney
cells, blood vessel cells, thyroid cells, parathyroid cells, cells
of the adrenal-hypothalamic pituitary axis, bile duct cells,
ovarian or testicular cells, salivary secretory cells, renal cells,
chondrocytes, epithelial cells, nerve cells and progenitor cells
such as myoblast or stem cells, particularly pluripotent stem
cells, and used in the regeneration of tissues derived from such
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to tissue engineering
scaffolds and methods for production of tissue engineering scaffold
which promote growth in vitro of mineralized bone tissue in a
rotating bioreactor. To produce these scaffolds, polymer
microencapsulation methods were adapted for the formation of
hollow, lighter than or light as water microcarriers of
biocompatible, biodegradable polymers. Scaffolds were then
fabricated by sintering together the lighter than or light as water
microcarriers into a fully interconnected, three dimensional
network. The microcarriers and scaffolds of the present invention
move within the fluid medium of the rotating bioreactor in a near
circular trajectory while avoiding collision with the bioreactor
wall. Cell culture studies on the scaffolds of the present
invention show that cells readily attach to microcarrier scaffolds.
In addition, cells cultured in vitro in a rotating bioreactor on
these lighter-than-water scaffolds retained their phenotype and
showed significant increases in alkaline phosphatase expression and
alizarin red staining by day 7 as compared to statically cultured
controls.
[0017] By "lighter than or light as water microcarriers" it is
meant microcarriers with a density equal to or less than water.
[0018] It has been shown previously that when osteoblast cells are
co-inoculated with microcarriers in a rotating bioreactor a random
aggregation occurs generated by the adherence of cells to
microcarrier beads and the formation of cellular bridges between
adjacent microcarriers (Granet et al. Medical & Biological
Engineering & Computing 1998 36(4):513-519; Qiu et al. Tissue
Engineering 1998 4(1):19-34; and Watts et al. Critical Reviews in
Therapeutic Drug Carrier Systems 1990 7(3):235-259). However, this
random aggregation that occurs in the rotating bioreactor is not
conducive to strict quantitative comparison, because the size and
shape of cell-bead aggregates as well as the degree of aggregation
varies greatly. Such a limitation is overcome by the present
invention via the sintered pre-assembly of microcarriers into
dimensionally reproducible cell scaffolds prior to culture in the
bioreactor. Furthermore, the microcarrier sintering method of the
present invention is not limited by the adverse effects associated
with the particulate leaching and consequently no unwanted
degradation of the scaffold occurs during fabrication.
[0019] Microcarriers of the present invention which are light than
or light as water exhibit buoyancy after immersion in deionized
water, phosphate buffer solution and tissue culture medium. In a
preferred embodiment, microcarriers of the present invention are
fabricated to produce lighter than water densities from about 0.6
to about 0.99 g/cc as estimated using a density gradient column
(ASTM D-1505). Microcarriers with densities as light as water or
1.0 g/cc can also be used. Using PLAGA to produce microcarriers of
the present, the majority of lighter than or light as water
microcarriers (47%) were within the range of 500 to 860 .mu.m in
diameter, with 19% from 300-500 .mu.m, 8% at 100-300 .mu.m and 2%
less than 100 .mu.m. Though 29% of microcarriers were greater than
860 .mu.m in diameter, it is preferred that only microcarriers 860
.mu.m and below are used for scaffold fabrication. For bone tissue
engineering devices, it is preferred that microcarriers in the size
range of 500-860 .mu.m be used for scaffold fabrication, as they
form structures with an expected pore size range of 113 to 356
.mu.m shown to be suitable for osteoblast adherence and migration
(Ishaug-Riley et al. Biomaterials 1998 19(15):1405-1412; Laurencin
et al. Bone 1996 19(1):S93-S99). PLAGA microcarriers of this size
range, when sintered, produce an interconnected network with an
average pore size of 187 .mu.m and aggregate density of 0.65
g/cc.
[0020] While PLAGA has been used as the exemplary microcarrier, as
will be understood by those of skill in the art upon this
disclosure, other biocompatible, biodegradable polymers can be used
in the production of scaffolds of the present invention. Examples
of such polymers include, but are not limited to, lactic acid
polymers such as poly(L-lactic acid (PLLA), poly(DL-lactic acid
(PLA), and poly(DL-lactic-co-glycolic acid)(PLGA). Blends of PLLA
with PLGA, can also be used for these scaffolds. Other exemplary
biodegradable polymers useful in the scaffolds of the present
invention include, but are not limited to, polyorthoesters,
polyanhydrides, polyphosphazenes, polycaprolactones,
polyhydroxybutyrates, degradable polyurethanes,
polyanhydrideco-imides, polypropylene fumarates, and
polydiaxonane.
[0021] The hollow microcarriers are then fabricated into scaffolds,
preferably via sintering in a mold for tissue engineering devices
at a temperature which promotes bonding of the microcarriers but is
below the melting temperature of the polymer. For example, PLAGA
microcarriers were fabricated into 4 mm.times.2.5 mm cylindrical
scaffolds by sintering at 60.degree. C. At this temperature,
amorphous polymer chains of adjacent microcarriers move past one
another and inter-lock forming a mechanical bond. Because this
temperature is well below the melting temperature, however,
collapse of individual microcarriers is avoided, thereby preserving
their hollow, spherical geometry and the lighter-than-water density
of the aggregate structure. Porosity is a result of the imperfect
packing of spherical microcarriers inside the mold, and thus
geometry dictates that there are no isolated spaces (pores) within
the structure and that the network of pores in the scaffold is
fully interconnected. The effect of sintering on the connectivity
of microspheres was evident from SEM linkages showing two or more
microspheres fused together at the contact regions. Assuming the
spheres approach a close packed configuration in the mold, the
diameter of the scaffold pores can be represented as interstitial
voids in the structure. Again, geometry dictates that the pore of
the structure is given by 0.225R in the case of a tetrahedral site
(a void surrounded by 4 spheres in the shape of regular
tetrahedron) or 0.414R in an octahedral, site (a void surrounded by
6 spheres in the shape of an octagon), where R is the radius of the
surrounding spheres.
[0022] The porosity and pore size distribution of typical
microcarrier scaffolds was measured using mercury porosimetry.
Although the broad distribution of microcarrier size likely
decreases the resulting pore diameter and increases packing
efficiency, the measured porosity of 31% slightly exceeds that of
close packing (26%). The average pore size distribution of 12
microcarrier scaffolds where the median pore is 187 .mu.m is well
within the theoretical expectation for close packed spheres.
Although the value median pore diameter exceeds the minimum
requirement for cell ingrowth and migration (Ishaug et al. J.
Biomed. Mater. Res. 1997 36(1):17-28; Ishaug-Riley et al.
Biomaterials 1998 19(15):1405-1412; Goldstein et al. Tissue
Engineering 1999 5(5):421-433; Laurencin et al. Bone 1996
19(1):S93-S99), the level of total pore volume or porosity of
microcarrier scaffolds is 50-60% less than that of similar
polymeric matrices proposed for bone repair (Ishaug et al. J.
Biomed. Mater. Res. 1997 36(1):17-28; Ishaug-Riley et al.
Biomaterials 1998 19(15):1405-1412; Goldstein et al. Tissue
Engineering 1999 5(5):421-433).
[0023] The motion of microcarrier scaffolds constructed primarily
from 500 to 860 .mu.m lighter than or light as water microcarriers
and fashioned into 4.times.2.5 mm cylindrical discs within the
rotating bioreactor was assessed. Particle tracking analysis
revealed an instantaneous velocity of 98 mm/second and a trajectory
completely absent of wall collisions once equilibrium motion was
reached.
[0024] Cell attachment to microcarrier scaffolds during rotating
culture was estimated from cell concentration profiles taken at
times 4, 8, 12, and 24 hours following co-inoculation with lighter
than or light as water scaffolds. Cells used in these experiments
were osteoblast-like cells. As will be understood by those of skill
in the art upon reading this disclosure, however, the scaffolds of
the present invention can actually be seeded with any cell type
which exhibits attachment and ingrowth and is suitable for the
intended purpose of the scaffold. Some exemplary cell types which
can be seeded into these scaffolds include, but are not limited to,
osteoblast and osteoblast-like cells, endocrine cells, fibroblasts,
endothelial cells, genitourinary cells, lymphatic vessel cells,
pancreatic islet cells, hepatocytes, muscle cells, intestinal
cells, kidney cells, blood vessel cells, thyroid cells, parathyroid
cells, cells of the adrenal-hypothalamic pituitary axis, bile duct
cells, ovarian or testicular cells, salivary secretory cells, renal
cells, chondrocytes, epithelial cells, nerve cells and progenitor
cells such as myoblast or stem cells, particularly pluripotent stem
cells.
[0025] In experiments with osteoblast-like cells, cell density in
the bioreactor medium decreased about 60%. The decreased
concentration of suspended cells during culture is assumed to
reflect the attachment of these cells to the scaffolds. By dividing
the estimated quantity of attached cells by the total number of
scaffolds present in culture, cell seeding was estimated to be
approximately 1.4.times.10.sup.5 cells/scaffold. After 24 hours of
dynamic seeding, a sampling of 6 to 10 cell-scaffolds was used to
measure directly the number of cells attached to scaffolds using
fluorometric DNA analysis. Measurements of attached cell were in
excellent agreement with cell concentration estimates with an
average value of 1.3.times.10.sup.5 cells per scaffold and standard
deviation of 2.0.times.10.sup.4 cells. The average surface area per
scaffold was calculated to be approximately 2 cm.sup.2 resulting in
a cell seeding density of approximately 6.5.times.10.sup.4
cells/cm.sup.2.
[0026] Cell proliferation was examined on lighter than or light as
water microcarrier scaffolds over a period of 7 days with cell
numbers measured immediately following cell seeding and at days 3
and 7. Cells cultured on lighter than or light as water scaffolds
in the rotating bioreactor show evidence of a lower rate and extent
of proliferation than those cultured on non-rotating controls.
Significant differences in cell numbers could be detected by day 7
(p<0.05). The presence of cells within the pores of the scaffold
that nearly cover the entire surface of the internal microcarriers
was verified by SEM. By progressively focusing the microscope down
the pore of the structure, it was estimated that cells had
penetrated as deep as 800 .mu.m.
[0027] The retention of osteoblastic phenotype was evaluated by ALP
histochemical staining and calorimetric analysis. Cells were
stained for ALP expression on lighter than or light as water
scaffolds in the rotating bioreactor and on the non-rotating
three-dimensional controls at days 3 and 7. Positive ALP staining
is evident at each time point and for each culture condition. At
each time point, more cells per unit area are present on scaffolds
cultured under non-rotating three-dimensional conditions than those
cultured in the rotating bioreactor, which is consistent with
fluorometric DNA analysis described herein. Calorimetric analysis
was also performed at 24 hours and at day 7. These results were
normalized by the actual number of cells present in each scaffold.
It was found that by day 7 the actual amount of ALP expressed per
cell is significantly higher for cells cultured in the rotating
bioreactor than on non-rotating three-dimensional controls
(p<0.05).
[0028] The production of calcified matrix was analyzed by alizarin
red histochemical staining. Scaffolds cultured in the rotating
bioreactor showed substantially greater alizarin positive
extracellular matrix material by day 7 as compared to
three-dimensional controls (p<0.05). To quantify the amount of
early stage calcified matrix formation, alizarin red staining
techniques were adapted for calorimetric analysis by solubilizing
the red matrix precipitate with cetyl pyridinium chloride to yield
a purple solution suitable for optical density measurements at 562
nm. Quantities of ALZ stained matrix were expressed as a molar
equivalent CaCl.sub.2 concentration and normalized by the average
number of cells per scaffold as determined in companion
proliferation studies. Significant increases in the quantity of ALZ
stained matrix produced on lighter than or light as water scaffolds
under rotating conditions at days 3 and 7 as compared to
non-rotating controls were observed.
[0029] Thus, as demonstrated herein, lighter than or light as water
polymer-based microcapsules are excellent cell microcarriers
providing a low shear, non-turbulent flow environment for attached
cells that avoids damaging collisions with the bioreactor wall.
These scaffolds adopt a particle trajectory absent of confounding
wall collisions, while maintaining a three-dimensional geometry
open to mass transport of nutrients and waste products. In
particular, the hydrodynamic flow environment produced by the
motion of lighter than or light as water scaffolds in the rotating
bioreactor enhances O.sub.2 and nutrient transport to cells at the
near surface (external) of the scaffold and possibly those in the
scaffold interior (internal). This may act to advance phenotype
development and tissue formation in the system of the present
invention. Further, cell seeding of the scaffolds in the rotating
bioreactor, as opposed to static seeding methods for these
scaffolds, enhances cell migration to the interior of the scaffold
and promotes homogeneity of initial cell seeding from one scaffold
to another. Accordingly, the scaffolds of the present invention
provide a combination of three-dimensionality and fluid transport
in the absence of damaging wall collisions that appears to be a
closer approximation of the in vivo environment of the cell thereby
expanding capacity for ex vivo tissue synthesis.
[0030] Scaffolds of the present invention are expected to
particularly useful in developing bone graft quality tissue.
However, as will be understood by one of skill in the art upon
reading this disclosure, the method of scaffold fabrication
disclosed herein can be used to generate a variety of microcarrier
scaffolds of different component size ranges, associated
three-dimensional architecture, and density useful in a variety of
tissue engineering applications. Scaffolds seeded with cells
including, but not limited to, osteoblast and osteoblast-like
cells, endocrine cells, fibroblasts, endothelial cells,
genitourinary cells, lymphatic vessel cells, pancreatic islet
cells, hepatocytes, muscle cells, intestinal cells, kidney cells,
blood vessel cells, thyroid cells, parathyroid cells, cells of the
adrenal-hypothalamic pituitary axis, bile duct cells, ovarian or
testicular cells, salivary secretory cells, renal cells,
chondrocytes, epithelial cells, nerve cells and progenitor cells
such as myoblast or stem cells, particularly pluripotent stem
cells, are useful in the regeneration of tissues derived from such
cells.
[0031] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
[0032] Example 1: Buoyant Microcarrier Fabrication
[0033] A conventional microsphere fabrication technique was adapted
for the formation of hollow, lighter than or light as water
microcarriers of bioerodible poly(d,l-lactic-co-glycolide)
copolymer. For this technique, a 25% w/v polymer solution of 50:50
PLAGA (molecular weight approximately 30,000) was dissolved in
methylene chloride, and poured slowly into a 1000 ml beaker
containing 0.1% PVA (Polysciences, Lot #413322, molecular weight
25,000). The solution was stirred continuously (Caframo, Model
BDCISSO) at 1000 rpm for 4 to 6 hours to allow for solvent
evaporation. Buoyant microcarriers were harvested by vacuum
filtration (Whatman, 54 .mu.m), washed with deionized water, and
lyophilized (Lyph-lock 12, Labconco Corp.) for 24 hours. Size
distribution was determined by mechanically sifting the
microcarriers using a series of stainless steel sieves with
selected mesh sizes. Microcarriers were freeze fractured and
analyzed with scanning electron microscopy to confirm that the
carriers were indeed hollow. In vitro buoyancy was verified over 7
days by immersion inside a water-tight container maintained at
37.degree. C. in an oscillating (60 opm) water bath.
[0034] Example 2: Scaffold Fabrication and Characterization
[0035] Microcapsules of a selected size range and weight were
poured into a stainless steel mold and heated in an oven (Precision
Gravity Convection Incubator) for 1 hour at 60.degree. C., several
degrees above the glass transition temperature for the PLAGA 50:50
(T.sub.g=45-50.degree. C). Microcarriers bonded to each other while
maintaining their hollow, spherical geometry. Scaffolds used for
bioreactor culture were exposed to ultraviolet irradiation for 30
minutes on each side in an effort to minimize bacterial
contamination. Microcarrier scaffolds were characterized using a
low field emission scanning electron microscope (SEM, JEOL 6300).
For SEM, specimens were coated with gold and examined for pore
inter-connectivity, degree of microcarrier bonding, and deformation
of microcarriers. The porosity of the structure was measured by
porosimetry using the Micromeritics Autopore HI porosimeter.
Specifically, cylindrical polymer scaffolds, 4 mm in diameter and
approximately 2.5 mm in length were placed in a 5cc penetrometer,
subjected to a vacuum of 50 .mu.m Hg, and infused with mercury.
Porosity is determined by measuring the volume of the mercury
infused. In addition to an overall percentage of porosity for the
polymer scaffold, porosimetry will also give an approximate
distribution of pore sizes within the polymer scaffold, allowing
for more accurate characterization of the scaffold geometry.
[0036] Example 3: Numerical Model Simulation and Particle Motion
Analysis
[0037] The equations of motion governing microcarrier motion in the
rotating bioreactor are as follows. For the particle position (x,
y) and velocity (v.sub.x, v.sub.y), microcapsule motion relative to
the rotating fluid is governed by equations: 1 x t = v x v x t = -
1 part V part [ p S C d v x + ( part - fluid ) V part 2 x + 2 (
part - fluid ) V part x - ( part - fluid ) V part g sin ( t ) ] y t
= v y v y t = - 1 part V part [ p S C d v y + ( part - fluid ) V
part 2 y - 2 ( part - fluid ) V part y - ( part - fluid ) V part g
cos ( t ) ] S = R part 2 C d 24 Re + 6.0 1.0 + Re + 0.4 ( 1 )
[0038] where (.rho..sub.sphere-.rho..sub.fluid) is the difference
between the density of the microcapsule and surrounding fluid, Re
is the Reynolds number, V.sub.part is the microcarrier volume,
C.sub.d is the drag coefficient at Re<2.times.10.sup.5, p is the
stagnation pressure, S is the microcapsule planar surface area, and
Z is the axis of rotation. A numerical solution to these equations
was obtained by way of a fourth order Runga Kutta integration
scheme run on a local workstation, using an adaptive stepwise
control algorithm to ensure convergence through the integration
period and assuming a specific starting position (x,y) within the
bioreactor. Using this numerical model, scaffold parameters (e.g.
density and drag coefficient) have been identified which yield
particle trajectories without any confounding wall collisions.
Scaffolds were then fabricated from component microcarriers which
meet these design criteria.
[0039] A particle tracking system built for the rotating bioreactor
was used to compare resulting scaffold motion in the rotating
bioreactor relative to the culture medium. The particle tracking
system is comprised of a rotating CCD camera (Cohu, Inc.) that is
in synchrony with a rotating High Aspect Ratio Vessel (HARV).
Particle motions are videotaped (Sony SVO-9500 MD) and digitally
re-recorded using a Sony Frame Code Generator and frame grabber
(Media Cydernetics). Image analysis is carried out using Image Pro
(Phase 3 Imaging, Inc.). Lighter-than-water PLAGA microcarriers and
microcarrier scaffolds were incubated in distilled water at room
temperature for 24 hours in a non-rotating bioreactor vessel and
their trajectories recorded during bioreactor rotation using the
tracking apparatus. A temporal description of scaffold trajectory
was measured over consecutive frames from which particle velocities
were computed. From these velocity measurements and based on the
geometry of the scaffolds (and diameter of isolated microcarriers),
maximum fluid shear stress is estimated by assuming uniform flow
past a single microcarrier and using the stokes equation: 2 = - 3 U
2 a ( 2 )
[0040] where .sigma. is shear stress, .mu. is viscosity, U is flow
velocity and .alpha. is the diameter of the microcarrier.
[0041] Example 4: Cell Seeding and Culture The human SaOS-2 line
(ATCC A HTB-85), which exhibits homogeneous and reproducible
expression of cellular alkaline phosphatase over an infinite life
span was used. For all experiments, cells were maintained in M199
(Gibco) culture medium supplemented with 10% fetal bovine serum
(Sigma), 2.5 mM L-glutamine and 3 mM b-glycerol phosphate. SaOS-2
cells were grown to confluency and digested in 0.01% trypsin in
0.04% EDTA (Gibco) for 10 minutes. Cells were then resuspended in a
minimal amount of media, their numbers determined with a Coulter
Counter, and diluted to an appropriate cell density. Prior to cell
seeding, PLAGA scaffolds (n=36) were washed in phosphate buffered
saline (PBS), and placed inside a single bioreactor vessel
(Synthecon) filled with 55 ml of complete medium containing no
cells. After 10 minutes, the bioreactor vessel was inoculated with
8.times.10.sup.6 cells and mounted onto a multi-HARV rotating unit
turning at 25 rpm. Cell attachment to microcarrier scaffolds in the
rotating vessel was estimated from the decrease of cell density in
the supernatant fluid observed over 24 hours. At time intervals 4,
8, and 12 hours, 0.5 ml of the cell suspension was removed from the
bioreactor, re-suspended in trypsin solution to dissociate cell
aggregates and cell numbers were determined using a coulter
counter. At 24 hours, the entire cell suspension was removed and
the cell number determined.
[0042] Example 5: Cell Counting Immediately following the seeding
of the cells for an experiment, a random sampling (n=6 to 10) of
selected scaffolds was removed and the initial number of attached
cells were determined by means of a fluorometric DNA assay as
described by Labarca and Paigen (Anal. Biochem. 1980 102:344-352).
The remaining scaffolds were washed with PBS and divided equally
into two experimental groups. Each group of scaffolds was placed,
respectively, into two new bioreactor vessels and re-fed with 55 ml
of fresh culture medium. To determine the effect of culture vessel
rotation on cell function, one vessel was mounted onto a multi-HARV
unit and rotated at 25 rpm and the other was cultured statically
(no-rotation) as a control. Each vessel was cultured at 37.degree.
C. and 5% CO.sub.2 for 7 days. At days 3 and 7, additional
scaffolds were removed for DNA quantification. Scaffolds used for
DNA analysis were washed 3 times in PBS, combined with 3 ml of
additional PBS containing 2 mM EDTA, and pulverized using a tissue
homogenizer (PowerGen 35, Fisher) with a 10 mm diameter saw-tooth
generator for 1 minute. Cells were ruptured by 2 minutes of further
homogenization at 30,000 rpm with a 5 mm diameter flat bottom
generator. Homogenates were frozen at -70.degree. C. until the day
of analysis. On the day of analysis, 1 ml of scaffold homogenate
was combined with 7 .mu.l of a 200 .mu.g/ml solution of
bisbenzimide H33258 dye (Calbiochem) and vortexed vigorously.
Fluorescence was read using a Tecan Spectrofluor microplate reader
with an emission wavelength of 465 nm and an excitation wavelength
of 360 nm. Cell standards were used to convert measured
fluorescence to cell numbers, and unseeded but cultured scaffolds
were analyzed to determine any effect of PLAGA
autofluorescence.
[0043] Example 6: Alkaline Phosphatase Activity
[0044] Alkaline Phosphatase (ALP) activity was measured by using
adaptations of standard histochemical (Vaan Belle, H. Biochimica et
Biophysica Acta 1972 289:158-168) and calorimetric (Rattner et al.
In Vitro Cellular & Developmental Biology - Animal 1997
33(10):757-762) methods. At days 3 and 7, scaffolds were removed
from both the rotating and non-rotating bioreactor vessels and
washed two times with PBS. Scaffolds were then incubated for 30
minutes at 37.degree. C. with Napthol AS-BI (Sigma, N-2250)
phosphate salt (0.5 mg/ml; Sigma) and N,N-Dimethyl Formamide (10
.mu.g/ml; Sigma D-8654) in 50 mM Tris buffer (pH 9.0), in the
presence of Fast Red (Sigma, F-2768) violet salt (1.0 mg/ml). After
30 minutes, cells were washed two times with PBS and fixed by
incubation in 2% paraformaldehyde for 30 minutes at 4.degree. C.
ALP staining was viewed by light microscopy. Scaffolds were
fractured into halves in order to visualize cells in the interior
regions of the 3-dimensional structure.
[0045] In addition, ALP expression was quantified in each of the
cell-scaffold homogenates used for fluorometric DNA analysis. For
this analysis, aliquots of cell homogenates were incubated at
37.degree. C. for 30 minutes in 0.1 M Na.sub.2CO.sub.3 buffer
solution (pH 10) containing 2 mM MgCl.sub.2 with disodium
p-nitrophenyl phosphate (pNP-PO.sub.4) as the substrate. Standard
solutions were prepared by serial dilutions of 0.5 mM p-nitrophenol
(pNP) in Na.sub.2CO.sub.3 buffer. Enzymatic activity was expressed
as total mmoles of pNP produced per minute per total cell number
determined by fluorometric DNA analysis. Absorbance was measured at
415 nm using a Tecan Spectrofluor microplate reader.
[0046] Example 7: Alizarin Red Calcium Quantification The
effectiveness of sodium 1,2-dihydroxy anthraquinone-3-sulfonate,
commonly known as Alizarin Red (ALZ), as a chelating compound and
colorometric reagent for spectrophotometric determination of
calcium is well established (Wu, L. and Forsling, W. Acta Chemica
Scandinavica 1992 46:418-422). ALZ spectrophotometric methods were
adapted for the determination of mineralized matrix production
(Stanford et al. J. Biol. Chem. 1995 270:9420-9428) on lighter than
or light as water PLAGA by osteoblast-like cells. Scaffolds were
removed from the bioreactor, washed in ddH.sub.20, and incubated in
40 mM Alizarin red solution (pH 4.2) for 10 minutes at room
temperature. To remove unreacted ALZ, scaffolds were washed 5-10
times in ddH.sub.20 (until water was clear) . Scaffolds were then
incubated in 10% cetyl pyridinium chloride for 15 minutes to
solubilize reacted ALZ and pulverized using a tissue homogenizer
(PowerGen 35, Fisher) with a 10 mm diameter saw-tooth generator.
Serial dilutions of 1 N CaCl.sub.2 were used as standards. ALZ
concentration per cell was calculated as molar equivalent
CaCl.sub.2 divided by the average cell number at each time point as
determined by fluorometric DNA analysis. Absorbance was measured at
570 nm using a Tecan Spectrofluor microplate reader.
[0047] Example 8: Scanning Electron Microscopy
[0048] Before seeding with cells, microcarriers and microcarrier
scaffolds were coated with gold and visualized using a low field
emission electron microscope (JEOL 6300) at 2 keV accelerating
voltage. To evaluate cell attachment and morphology to
lighter-than-water scaffolds cultured in the bioreactor, scaffolds
were cultured as described above and removed at day 7 for SEM
analysis. Attached cells were fixed to scaffold substrates by
washing thoroughly with PBS, then incubation in 1% and 3%
glutaraldehyde for 1 hour and 24 hours, respectively. Following
fixation, cells were washed with PBS, placed through a series of
graded ethanol dehydrations and allowed to air dry. Finally,
cell-scaffolds were coated with carbon and analyzed at 2 keV.
[0049] Example 9: Statistical Analysis
[0050] Statistical analysis was performed using JMP IN 3.2.1
software. One-way ANOVA was performed to determine any
statistically significant relationship between the rotating and
non-rotating conditions with respect to the quantity of reacted
ALZ, ALP expression, and cell number. Statistical significance was
attained at p<0.05. Three scaffolds were analyzed at each time
point and for each quantitative assay.
* * * * *