U.S. patent application number 17/185545 was filed with the patent office on 2021-11-18 for microcapillary network based scaffold.
The applicant listed for this patent is BONUS THERAPEUTICS LTD., HEALTH CORPORATION OF GALILEE MEDICAL CENTER. Invention is credited to Dror BEN-DAVID, Shai MERETZKI, Samer SROUJI.
Application Number | 20210353828 17/185545 |
Document ID | / |
Family ID | 1000005740394 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210353828 |
Kind Code |
A1 |
SROUJI; Samer ; et
al. |
November 18, 2021 |
MICROCAPILLARY NETWORK BASED SCAFFOLD
Abstract
A scaffold is provided, the scaffold comprising: at least one
inlet tube; at least one outlet tube; and a plurality of porous
elongated microtubes, wherein each one of said porous elongated
microtube has an inner diameter of 5-100 micrometers, wherein said
plurality of elongated microtubes extend from said at least one
inlet tube to said at least one outlet tube and is in fluid
communication thereto, Further provided is a method for producing
and using the scaffold, such a s for tissue engineering.
Inventors: |
SROUJI; Samer; (Haifa,
IL) ; MERETZKI; Shai; (Haifa, IL) ; BEN-DAVID;
Dror; (Kiryat Motzkin, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BONUS THERAPEUTICS LTD.
HEALTH CORPORATION OF GALILEE MEDICAL CENTER |
Haifa
NAHARIYA |
|
IL
IL |
|
|
Family ID: |
1000005740394 |
Appl. No.: |
17/185545 |
Filed: |
February 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15759940 |
Mar 14, 2018 |
10940238 |
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PCT/IL2016/051032 |
Sep 18, 2016 |
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17185545 |
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62220129 |
Sep 17, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0652 20130101;
A61L 2430/02 20130101; A61L 27/54 20130101; A61L 27/46 20130101;
A61L 27/58 20130101; A61L 27/3804 20130101; A61L 27/56 20130101;
A61L 27/446 20130101; A61F 2/06 20130101 |
International
Class: |
A61L 27/38 20060101
A61L027/38; A61L 27/46 20060101 A61L027/46; A61L 27/56 20060101
A61L027/56; A61F 2/06 20060101 A61F002/06; A61L 27/44 20060101
A61L027/44; A61L 27/54 20060101 A61L027/54; A61L 27/58 20060101
A61L027/58; C12N 5/077 20060101 C12N005/077 |
Claims
1. A method of producing a tissue, the method comprising: providing
a scaffold comprising: at least one inlet tube; at least one outlet
tube; a plurality of porous elongated microtubes, wherein each one
of said porous elongated microtube has an inner diameter of 5-100
micrometers, wherein said plurality of elongated microtubes extend
from said at least one inlet tube to said at least one outlet tube
and is in fluid communication thereto; and a plurality of fibers
having a diameter range of 0.5-10 micrometers, wherein said
plurality of fibers is dispersed upon a portion of each of said
plurality of porous elongated microtubes; seeding cells on said
plurality of porous elongated microtubes of said scaffold; and
providing liquid containing nutrients through said inlet of said
scaffold, so as to provide nutrients from pores of said plurality
of porous elongated microtubes to said cells; thereby producing
said tissue.
2. The method of claim 1, wherein cells are seeded on and/or within
said plurality of fibers.
3. The method of claim 1, wherein said tissue is suitable for being
implanted into a subject in need thereof.
4. The method of claim 1, wherein said inlet and said outlet of
said scaffold is suitable for being surgically connected to a
vascular system of a subject in need thereof, thereby providing
fluid communication between the subject's vascular system and said
scaffold.
5. The method of claim 1, wherein said cells are selected from the
group consisting of: adipose-derived stem cells, mesenchymal cells,
mesenchymal stem cells, vascular smooth muscle cells, adipogenic
cells, osteoprogenitors cells, osteoblasts, osteocytes,
chondroblasts, chondrocytes and osteoclasts, endothelial progenitor
cells, hematopoietic progenitor cells, micro vascular endothelial
cells and macro vascular endothelial cells, beta cells, hepatocytes
and a combination thereof.
6. The method of claim 1, wherein said scaffold further comprising
plurality of bioactive particles embedded in between said plurality
of fibers.
7. The method of claim 6, wherein said bioactive particles have a
range of 200-1500 micrometers in diameter.
8. The method of claim 6, wherein said plurality of bioactive
particles are one or more type of osteoconductive particles.
9. The method of claim 6, wherein the one or more types of the
osteoconductive particles are selected from the group consisting
of: calcium carbonate, hydroxyapatite (HA), demineralized bone
material, morselized bone graft, cortical cancellous allograft,
cortical cancellous autograft, cortical cancellous xenograft,
tricalcium phosphate, corraline mineral and calcium sulfate.
10. The method of claim 1, wherein a portion of said scaffold is
printed, molded, casted, polymerized, or electrospun.
11. The method of claim 1, wherein at least one of said inlet tube,
said outlet tube and said porous elongated microtubes are
electrospun tubes.
12. The method of claim 11, wherein said electrospun tubes comprise
a polymer selected from the group consisting of: biodegradable
polymers, non-biodegradable polymers and a combination thereof.
13. The method of claim 12, wherein said polymer is selected from
the group consisting of: polycaprolactone (PCL), polylactic acid
(PLA), polyglycolic acid (PGA), and poly(Lactide-co-Glycolide)
(PLGA), poly(orthoester), a poly(phosphazene), poly(or
polycaprolactone, polyamide, polysaccharide, albumine and
collagen.
14. The method of claim 1, wherein said inlet tube and said outlet
tube have a wall thickness range of 50-2,000 micrometers.
15. The method of claim 1, wherein said plurality of porous
elongated microtubes has a wall thickness range of 0.5-50
micrometers.
16. The method of claim 1, wherein said inlet tube and said outlet
tube have an inner diameter of range of 2,000-10,000
micrometers.
17. The method of claim 1, wherein an average diameter of a pore of
said plurality of porous elongated microtubes is 0.1-5
micrometers.
18. The method of claim 1, further comprising providing the
scaffold at least one agent for promoting cell adhesion,
colonization, proliferation and/or differentiation.
19. The method of claim 18, wherein the at least one agent for
promoting cell adhesion is selected from the group consisting of:
gelatin, fibrin, fibronectin and collagen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. Ser. No.
15/759,940 filed Mar. 14, 2018, which is a National Phase of PCT
Patent Application No. PCT/IL2016/051032 having International
filing date of Sep. 18, 2016, which claims the benefit of priority
U.S. Provisional Patent Application No. 62/220,129 filed on Sep.
17, 2015 entitled MICROCAPILLARY NETWORK BASED SCAFFOLD. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
FIELD OF INVENTION
[0002] The present invention relates to the field of tissue
engineering, and more particularly to the use of synthetic scaffold
for the preparation of prosthetic implants.
BACKGROUND OF THE INVENTION
[0003] Tissue engineering techniques generally require the use of a
scaffold as a three-dimensional template for initial cell
attachment and subsequent tissue formation. As such, scaffold
design is one of the most important aspects of tissue engineering.
Appropriate selection of a scaffold is a key factor in the process
of producing a viable and clinically relevant engineered
tissue.
[0004] A scaffold is expected to possess the following
characteristics for use in tissue engineering: (i) a
three-dimensional porous structure that allows cell/tissue growth
and flow transport of nutrients and metabolic waste; (ii)
biodegradable or bioresorbable with a controllable degradation and
resorption rate to match cell/tissue growth in vitro and/or in
vivo; (iii) conducive surface chemistry for cell attachment,
proliferation, and differentiation; (iv) mechanical properties to
match those of the tissues at the site of implantation; and (v)
process ability to form a variety of shapes and sizes for various
applications.
[0005] In addition, in order to improve the survival and function
of the implanted engineered tissue, adequate vascularization within
the scaffold is crucial. Tissue vascularization is essential for
delivery of nutrients (amino acids, glucose and oxygen) to the
tissue and clearance of the metabolism byproducts from the tissue.
Numerous techniques for development of vascularized tissue have
recently emerged and are classified into two major categories: (a)
vasculogenesis and angiogenesis-based techniques and (b)
prevascularization-based techniques. The former are characterized
by the ingrowth of newly formed blood vessels from the host
microvasculature into the implanted engineered tissue and includes
(1) micropatterning of vascular morphogenesis, (2) use of
functionalized biomaterials to promote vasculogenisis and
angiogenesis, (3) use of growth factor gradients and (4) co-culture
of multiple cell types and control of cell-cell interactions. These
techniques can be utilized to promote the formation of vascular
networks in 3D engineered constructs in a regulated manner, but
their central drawback lies in the time-consuming process of
promoting vasculogenesis and angiogenesis, at a critical time when
survival rates of implanted scaffolds are determined. The
prevascularization-based techniques are founded on generating of
preformed microvascular networks within tissue constructs prior to
their implantation, which later further develop and interconnect
with host blood vessels at the implantation site. The key advantage
of these techniques is the capacity for immediate blood perfusion
within the constructs upon implantation, which boosts the
proliferation and growth of the cells. However, despite these
advanced techniques, clinical use of engineered tissues and tissue
substitutes is still largely restricted to avascular or thin
tissues, and the marked progress achieved in small-scale tissue
engineering applications in vitro was ultimately stalled due to the
lack of vascular perfusion when scaled up to a sizes relevant for
implantation and disease treatment.
[0006] Therefore, engineering a complex bulk tissue that can
maintain its viability in vivo by transporting essential growth
factors throughout the entire volume of the scaffold, remains an
unmet need in the field of tissue engineering in general, and bone
tissue engineering in particular.
SUMMARY OF THE INVENTION
[0007] According to a first aspect the invention provides a
scaffold comprising:
[0008] at least one inlet tube;
[0009] at least one outlet tube; and
[0010] a plurality of porous elongated microtubes, wherein each one
of said porous elongated microtube has an inner diameter of 5-100
micrometers, [0011] wherein said plurality of elongated microtubes
extend from said at least one inlet tube to said at least one
outlet tube and is in fluid communication thereto.
[0012] In some embodiments, the scaffold further comprises a
plurality of fibers having a diameter range of 0.5-10 micrometers.
In some embodiments, the plurality of fibers is dispersed upon a
portion of each of said plurality of porous elongated
microtubes.
[0013] In some embodiments, the scaffold further comprises a
plurality of bioactive particles embedded in between said plurality
of fibers. In some embodiments, the scaffold further comprises a
plurality of bioactive particles embedded in between said plurality
of fibers and at least a portion of said porous elongated
microtubes. In some embodiments, the bioactive particles have a
range of 200-1500 micrometers in diameter.
[0014] In some embodiments, the plurality of bioactive particles
comprises one or more type of osteoconductive particles. In some
embodiments, the one or more types of the osteoconductive particles
are selected from the group consisting of: calcium carbonate,
hydroxyapatite (HA), demineralized bone material, morselized bone
graft, cortical cancellous allograft, cortical cancellous
autograft, cortical cancellous xenograft, tricalcium phosphate,
corraline mineral and calcium sulfate. In some embodiments, the
particles comprise hydroxylapatite (HA) and calcium carbonate.
[0015] In some embodiments, at least a portion of the scaffold is
printed, molded, casted, polymerized, or electrospun. In some
embodiments, at least one of said inlet tube, said outlet tube and
said porous elongated microtubes are electrospun tubes. In some
embodiments, said plurality of fibers are electrospun fibers. In
some embodiments, the electrospun tubes or fibers comprise a
polymer (or are formed from a polymeric solution) selected from the
group consisting of: biodegradable polymers, non-biodegradable
polymers and a combination thereof. In some embodiments, the
polymer is selected from the group consisting of: polycaprolactone
(PCL), polylactic acid (PLA), polyglycolic acid (PGA), and
poly(Lactide-co-Glycolide) (PLGA), poly(orthoester), a
poly(phosphazene), poly(or polycaprolactone, polyamide,
polysaccharide, albumine and collagen.
[0016] In some embodiments, the inlet tube and the outlet tube
have, independently, an inner diameter of range of 2,000-10,000
micrometers.
[0017] In some embodiments, the inlet tube and the outlet tube
have, independently, a wall thickness range of 50-2,000
micrometers.
[0018] In some embodiments, the plurality of porous elongated
microtubes have a wall thickness range of 0.5-50 micrometers.
[0019] In some embodiments, an average diameter of a pore of the
plurality of porous elongated microtubes is 0.1-5 micrometers.
[0020] In some embodiments, the scaffold further comprises at least
one agent for promoting cell adhesion, colonization, proliferation
and/or differentiation. In some embodiments, the scaffold further
comprises at least one agent for promoting cell adhesion selected
from the group consisting of: gelatin, fibrin, fibronectin and
collagen.
[0021] In some embodiments, the scaffold further comprises a
plurality of cells. In some embodiments, the scaffold further
comprises a plurality of cells seeded on and/or within the
plurality of fibers. In some embodiments, the scaffold is adapted
for cellular growth.
[0022] In some embodiments, the plurality of cells is selected from
the group consisting of: adipose-derived stem cells, mesenchymal
cells, mesenchymal stem cells, vascular smooth muscle cells,
adipogenic cells, osteoprogenitors cells, osteoblasts, osteocytes,
chondroblasts, chondrocytes and osteoclasts, endothelial progenitor
cells, hematopoietic progenitor cells, micro vascular endothelial
cells and macro vascular endothelial cells, beta cells, hepatocytes
and a combination thereof
[0023] According to another aspect, the invention provides a method
of producing a tissue, the method comprising:
[0024] providing a scaffold comprising: [0025] at least one inlet
tube; [0026] at least one outlet tube; and [0027] a plurality of
porous elongated microtubes, wherein each one of said porous
elongated microtube has an inner diameter of 5-100 micrometers,
[0028] wherein said plurality of elongated microtubes extend from
said at least one inlet tube to said at least one outlet tube and
is in fluid communication thereto; [0029] seeding cells on said
plurality of porous elongated microtubes of said scaffold; and
[0030] providing fluid (e.g., liquid) containing nutrients through
said inlet of said scaffold, so as to provide nutrients from pores
of said plurality of porous elongated microtubes to said cells;
[0031] thereby producing said tissue.
[0032] In some embodiments, the scaffold further comprises a
plurality of fibers dispersed upon each of said plurality of porous
elongated microtubes. In some embodiments, the cells are seeded on
and/or within said plurality of fibers.
[0033] In some embodiments, the tissue is suitable for being
implanted into a subject in need thereof.
[0034] In some embodiments, the inlet and the outlet of the
scaffold is suitable for being surgically connected to a vascular
system of a subject in need thereof, thereby providing fluid
communication between the subject's vascular system and said
scaffold.
[0035] In some embodiments, the cells are selected from the group
consisting of: adipose-derived stem cells, mesenchymal cells,
mesenchymal stem cells, vascular smooth muscle cells, adipogenic
cells, osteoprogenitors cells, osteoblasts, osteocytes,
chondroblasts, chondrocytes and osteoclasts, endothelial progenitor
cells, hematopoietic progenitor cells, micro vascular endothelial
cells and macro vascular endothelial cells, beta cells, hepatocytes
and a combination thereof.
[0036] In some embodiments of the disclosed method, the scaffold
further comprises plurality of bioactive particles embedded in
between said plurality of fibers.
[0037] In addition to the exemplary aspects and embodiments
described above, further aspects and embodiments will become
apparent by reference to the figures and by study of the following
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A-D are schematic presentations of the microcapillary
based scaffold;
[0039] FIGS. 2A-D are optical images of (A) an electrospun
capillary, (B-D) PCL electrospun fibers (9 wt. %).
[0040] FIG. 3 is a schematic illustration of a permeation testing
setup of an electrospun tube connected to inlet/outlet;
[0041] FIGS. 4A-D present images of the complex scaffold: (A) the
complex scaffold: Pro-Osteons dispersed on and in between the
PCL-electrospun tubes. The indicated region is the region that is
seeded with cells, (B) the vasculature-like system: made of
PCL-electrospun tubes, (C-D) a cross-sectional view of the
vasculature-embedded scaffold, showing three PCL tubes surrounded
with layers of Pro-Osteon particles and PCL fiber;
[0042] FIGS. 5A-C present SEM images of the complex scaffold: (A)
cross section of the complex scaffold showing the PCL fibers and
the Pro-Osteon particles dispersed on and in between the
PCL-electrospun tubes, (B) the scaffold bulk including Pro-Osteon
particles and electrospun PCL fibers, (C) electrospun PCL tube
(different magnifications (.times.50, .times.40 & .times.150)
are shown);
[0043] FIG. 6 shows the complex scaffold connected to a medium-flow
bioreactor;
[0044] FIGS. 7A-C present gel electrophoresis results: (A) the
proteins that passed through the PCL tubes walls and into the PBS
bath, (B) quantification of the protein bands observed on the gel,
(C) increase of protein concentrations in the inner PBS bath;
[0045] FIG. 8 is a graph showing cells proliferation when cultured
on complex scaffolds under static conditions. The Alamar blue-based
viability assay showed increase of proliferation rates of MSCs in
correlation to the culture days. Error bars are the standard
deviation (SD) collected from three samples;
[0046] FIGS. 9A-D show H&E-stained histological sections of the
MSC-seeded complex scaffolds. Complex scaffolds were seeded with
MSCs, and cultured under static conditions for one week and then in
dynamic conditions for an additional week before being fixed and
stained. Different magnifications are shown: (A, C) .times.4, (B,
D) .times.10. The PCL tubes are indicated by asterisks.
[0047] FIGS. 10A-C show SEM images of the MSC-seeded complex
scaffold grown under dynamic conditions: (A) cross-section of a PCL
tube showing the integration of cells along the tube walls, (B-C)
cells were distributed on and in between the PCL fibers and
Pro-Osteon particles (different magnifications (.times.450,
.times.2680 and .times.415) are shown); and
[0048] FIGS. 11A-I show H&E- and Trichrome-stained histological
sections of the implanted MSC-embedded complex scaffold in animal
models. Cell-seeded scaffolds were implanted in an ectopic animal
model in order to examine their biocompatibility. Eight weeks
later, the implanted scaffolds were extracted and histological
sections were prepared and stained with H&E staining and
Trichrome staining. PCL tubes, PCL fibers and Pro-Osteon particles
are indicated by black asterisks, arrows and crosses, respectively.
Blood vessels, muscle tissue and collagen accumulation are noted by
arrows, different magnifications are shown: (A, C, E, F and H)
.times.4, (B, D, G and I) .times.10;
[0049] FIGS. 12A-B demonstrate a Hollow Fiber Reactor (HFR) system
FIG. 12A is a photograph of a Hollow Fiber Reactor (HFR) system;
and FIG. 12B is a schematic illustration of the Hollow Fiber
Reactor (HFR) system (1280) of FIG. 12A;
[0050] FIGS. 13A-B show photographs following Giemsa staining of
Coral particles from the inner side (A) and the outer side (B) of
the HFR system;
[0051] FIGS. 14A-B show photographs following Hematoxylin&
Eosin staining of HFR construct after one week of cultivation,
magnification .times.10, these photographs (A and B) demonstrate
that the cells between the mineral particles were embedded within
the PCL fibers, and generated organized connective tissue around
and between the mineral particle;
[0052] FIGS. 15A-B show photograph following MTT staining of
Mineral particles with live cells (A), and mineral particles
without cells used as control (B);
[0053] FIGS. 16A-B are graphs showing FACS results for the
osteogenic marker ALP from MSCs grown in a Static HFR system (A)
and dynamic HFR system (B).
[0054] FIGS. 17A-D are bar graphs showing Real-Time PCR results of
DLX5 expression at day 0 and day 9 in MSCs grown in a static system
(A) or a dynamic system (B), and SP7 expression at day 0 and day 9
in MSCs grown in a static system (C) or a dynamic system (D);
[0055] FIGS. 18A-B are photographs of Giemsa staining (A) and
AC-LDL staining (B) of human adipose microvascular endothelial
cells (HAMEC) grown in microcapillary reactor system;
[0056] FIGS. 19A-B are a photograph (A) and a schematic
illustration (B) of a microcapillary system for testing
permeability to human plasma proteins;
[0057] FIGS. 20A-C are bar graphs demonstrating concentrations of
human serum albumin (A), IgG (B) and lysozyme (C) during the plasma
circulation into the microcapillary construct;
[0058] FIGS. 21A-H are photographs demonstrating "End to End"
anastomosis of vasculature-like system in SD rat using microsurgery
technique: FIG. 21A shows exposure of femoral artery and vain
before the microsurgery procedure, FIG. 21B shows "End to End"
anastomosis of the microcapillary graft to the artery and vain
vessels, FIG. 21C is a magnification of FIG. 21B, FIG. 21D is a
magnification of FIG. 21C with focus on the connected
microcapillary graft, FIG. 21E shows the microcapillary graft
following the "end to end" anastomosis while the microcapillary
graft is already connected and enables the blood circulation, FIG.
21F is a magnification of FIG. 21E, and FIG. 21G is a magnification
of FIG. 21F, both focused on the "end to end" anastomosis area, and
FIG. 21H shows the whole animal following stitching and closure of
the surgery area;
[0059] FIGS. 22A-B are photographs demonstrating the measurement of
blood flow from artery to vein in the anastomosis site (A) and the
measured value for blood flow in the artery as displayed on the
laser flow meter (B);
[0060] FIGS. 23A-E are photographs demonstrating opening of the
stitches (A) and exposure of the anastomosis site (B) performed
one-day post transplantation to allow measurement of blood flow
from artery to vein by laser Doppler (C), and the measured value
for blood flow in the artery (D) and vein (E) as displayed on the
laser flow meters;
[0061] FIG. 24 vasculature-like system extracted 14 days following
the transplantation.
[0062] FIG. 25A-D are histological images magnified by.times.X10
(A) .times.40 (B) .times.60 (C) and .times.100 (D) of the
transplanted vasculature-like system extracted from the rats 14
days after the transplantation and stained by Hematoxylin &
Eosin staining;
[0063] FIGS. 26A-D are photographs demonstrating the anastomosis of
heparin-soaked scaffold to the femoral artery from one side (A) and
the scaffold connected to femoral artery from one side and the
femoral vein from the other side with no apparent leakage from the
connection sites (B), FIGS. 26C-D are photographs demonstrating the
measurement of blood flow through the transplant in anesthetized
rats (C) and the measured value as displayed on the laser flow
meter (D);
[0064] FIG. 27 is a graph plotting flow rate (milliliters/second)
versus pump rate (RPM); and
[0065] FIG. 28 is a bar graph showing fluid loss from the scaffold
(presented as % of fluid flow) in capillary like tubes of human
(diameter of 0.5 mm, width of 237 micrometers) and vein like tubes
of rats (diameter of 1.5 mm, width of 390 micrometers) under a
fluid flow rate of 0.005 cm.sup.3/sec inside the scaffold compared
to a fluid flow rate of 0.038 cm.sup.3/sec inside the scaffold.
DETAILED DESCRIPTION OF THE INVENTION
[0066] The present invention, in some embodiments, provides a
3-dimensional (3D) scaffold that supports growth of cells in a
tissue construct, the scaffold comprises a porous tubular system
that serves as a built-in vascular system. In some embodiments, the
invention provides a scaffold comprising at least one inlet tube;
at least one outlet tube; and a plurality of porous elongated
microtubes, wherein each one of said porous elongated microtube has
an inner diameter of 5-100 micrometers, and wherein said plurality
of elongated microtubes extend from said at least one inlet tube to
said at least one outlet tube and is in fluid communication
thereto.
[0067] In some embodiments, the scaffold provides therein a tubular
system facilitating transport of nutrients, gases and metabolites
from liquid flux within the tubular system through the pores to
cells attached thereto. In some embodiments, the scaffold's tubular
system facilitates transport of by-products of metabolites from the
cells.
[0068] The present invention is based in part on the finding that
the scaffold of the invention serves as an adequate extracellular
matrix (ECM) for cell integration, attachment, proliferation,
growth and differentiation as a result of its structure.
[0069] In embodiments wherein the designated tissue is a bone, the
scaffold may bear features required for effective bone-tissue
engineered constructs and is characterized by osteoconductiveness
that induces mesenchymal stem cells (MSC) differentiation into
bone-forming cells. In some embodiments, the scaffold can serve as
a bioreactor system providing appropriate growth conditions for MSC
proliferation and differentiation into bone-forming cells in vitro
and for their subsequent development into bone structure in vivo
after anastomosis with host blood vessels.
[0070] Reference is now made to FIG. 1A, which is a schematic
illustration of a scaffold 100 according to an embodiment of the
invention. Scaffold 100 comprises an inlet tube 102 which is
fluidly-connected to plurality of porous elongated microtubes 104
which extend in fluid communication to an outlet tube 106.
Optionally, inlet tube 102 may consist of plurality of inlet tubes
such as inlet tubes 102a, 102b, 102c. Optionally outlet tube 106
may consist of plurality of inlet tubes such as inlet tubes 106a,
106b, 106c. Reference is now made to FIG. 1B, which is an enlarged
view of a section of porous elongated microtubes 104. A plurality
of fibers 108 that may serve as a release system of
angiogenic/growth factors are dispersed upon porous elongated
microtubes 104. Optionally, bioactive particles 110 (e.g.,
osteoconductive particles) may be embedded within plurality of
fibers 108. Optionally, a plurality of cells 112 may be seeded
within plurality of fibers 108. For a non-limiting example,
plurality of cells may include bone forming cells. The wide arrow
depicts the flow of blood within porous elongated microtubes 104
and narrow arrows depict the flow of nutrients and oxygen (O.sub.2)
from porous elongated microtubes 104 to plurality of cells 112.
FIGS. 1C-D illustrates forming of cell tissue upon porous elongated
microtubes 104.
Scaffold
[0071] According to another aspect, the present invention provides
a scaffold comprising: at least one inlet tube; at least one outlet
tube; and a plurality of porous elongated microtubes, wherein each
one of said porous elongated microtube has an inner diameter of
5-100 micrometers, and wherein said plurality of elongated
microtubes extend from said at least one inlet tube to said at
least one outlet tube and is in fluid communication thereto.
[0072] In some embodiments, the diameters of the inlet and/or
outlet tubes is 2-1000, or 200-1000, or 2-100 folds greater than
the diameter of the porous elongated microtube. In some
embodiments, the inlet tube and the outlet tube have an inner
diameter range of 2,000-10,000 micrometers. In some embodiments the
inlet tube and the outlet tube have a wall thickness range of
50-2,000 micrometers. In some embodiments the plurality of porous
elongated microtubes has a wall thickness range of 0.5-50
micrometers. In some embodiments the ratio between the number of
porous elongated microtubes and any one of the inlet and outlet
tubes is in the range of 1:1-10:1, or alternatively 1:1-5:1, or
alternatively 1:1-3:1, or alternatively 1:1-50:1, or alternatively
1:1-100:1.
[0073] The term "porous" as used herein relates to a plurality of
openings, pores, or holes that may be filled (permeated) by water,
air or other materials. In some embodiments, pores are not
permeable to cells such as mammalian cells. In some embodiments, a
diameter of a pore is less than 10 micrometers. In some
embodiments, a diameter of a pore is between 0.1-5 micrometers. In
some embodiments, a diameter of a pore is between 0.1-10
micrometers, between 0.5-5 micrometers, or alternatively between
1-10 micrometers.
[0074] As used herein throughout, the term "fluid communication"
means fluidically interconnected, and refers to the existence of a
continuous coherent flow path from one of the components of the
system to the other if there is, or can be established, liquid
and/or gas flow through and between the ports, when desired, to
impede fluid flow therebetween.
[0075] In some embodiments, the scaffold further comprises a
plurality of fibers having a diameter range of 0.5-10 micrometers,
or 0.1-20 micrometers, or 1-5 micrometers. In some embodiments, the
fibers, or at least some of the fibers may be hollow. In some
embodiments, a distance between adjacent fibers ranges between 20
micrometers and 300 micrometers. In some embodiments the fibers are
arranged in a mesh. In some embodiments, the mesh comprises
openings defined between adjacent fibers. In some embodiments, the
openings have a diameter range of 20 micrometers and 300
micrometers. In some embodiments the plurality of fibers and/or the
mesh is dispersed upon at least a portion of the scaffold. In some
embodiments, the plurality of fibers and/or the mesh is dispersed
upon a portion of each of said plurality of porous elongated
microtubes.
[0076] The term "scaffold" as used herein refers to a porous,
artificial, three-dimensional structure comprising biocompatible
material that provides a surface suitable for adherence and
proliferation of cells. Biocompatible, as used herein, is intended
to describe materials that, are non-toxic to cells in vitro and
upon administration in vivo, do not induce undesirable long-term
effects. As used herein the term "in vitro" refers to any process
that occurs outside a living organism. As used herein the term
"in-vivo" refers to any process that occurs inside a living
organism.
[0077] As used herein, the term "diameter" refers to the largest
linear distance between two points on the surface of a described
element (e.g., tube, fiber, openings). The term "diameter", as used
herein, encompasses diameters of spherical elements as well as of
non-spherical elements.
[0078] In some embodiment, the scaffold is biodegradable.
Biodegradable, as used herein, is intended to describe materials
that are biologically degraded in vivo.
[0079] Scaffold of the present invention, or a portion thereof may
be printed, molded, casted, polymerized, or electrospun.
[0080] In some embodiments, the scaffold contains or consists of
electrospun material (e.g. macro micro or nanofibers).
[0081] In some embodiments the scaffold may consists of, or
include, one or more polymers selected from the group consisting
of: biodegradable polymers and non-biodegradable polymers. In some
embodiments the scaffold may consists of or include any of the
following materials: polycaprolactone (PCL), polylactic acid (PLA),
polyglycolic acid (PGA), poly(Lactide-co-Glycolide) (PLGA),
poly(orthoester), a poly(phosphazene), a polyamide, a
polysaccharide, albumin, collagen (e.g., collagen I or IV), fibrin,
hyaluronic acid, poly(vinyl alcohol) (PVA), Polyhydroxybutyrate
(PHB), poly(ethylene oxide) (PEO), fibrin, polydioxanone (PDO),
trimethylene carbonate (TMC), polyethyleneglycol (PEG), alginate,
chitosan copolymers or mixtures thereof.
[0082] In some embodiments, the scaffold further comprises
bioactive agents. As used herein, the terms "bioactive" is used to
refer to any effect on, interaction with, or response from living
cells and/or tissue. The term "bioactive agent" refers to a
molecule that exerts an effect on a cell or tissue.
[0083] Representative examples of types of bioactive agents include
therapeutics, vitamins, electrolytes, amino acids, peptides,
polypeptides, proteins, carbohydrates, lipids, polysaccharides,
nucleic acids, nucleotides, polynucleotides, glycoproteins,
lipoproteins, glycolipids, glycosaminoglycans, proteoglycans,
growth factors, differentiation factors, hormones,
neurotransmitters, prostaglandins, immunoglobulins, cytokines, and
antigens. Various combinations of these molecules can be used.
Examples of cytokines include macrophage derived chemokines,
macrophage inflammatory proteins, interleukins, tumor necrosis
factors. Examples of proteins include fibrous proteins (e.g.,
collagen, elastin) and adhesion proteins (e.g., actin, fibrin,
fibrinogen, fibronectin, vitronectin, laminin, cadherins,
selectins, intracellular adhesion molecules, and integrins). In
various cases, the bioactive agent may be selected from
fibronectin, laminin, thrombospondin, tenascin C, leptin, leukemia
inhibitory factors, RGD peptides, anti-TNFs, endostatin,
angiostatin, thrombospondin, osteogenic protein-1, bone morphogenic
proteins, osteonectin, somatomedin-like peptide, osteocalcin,
interferons, and interleukins. In some embodiments, the bioactive
agent includes a growth factor, differentiation factor, or a
combination thereof
[0084] As used herein, the term "growth factor" refers to a
bioactive agent that promotes the proliferation of a cell or
tissue. Representative examples of growth factors that may be
useful include transforming growth factor-.alpha. (TGF-.alpha.),
transforming growth factor-.beta. (TGF-.beta.), platelet-derived
growth factors (PDGF), fibroblast growth factors (FGF), nerve
growth factors (NGF) including NGF 2.5s, NGF 7.0s and beta NGF and
neurotrophins, brain derived neurotrophic factor, cartilage derived
factor, bone growth factors (BGF), basic fibroblast growth factor,
insulin-like growth factor (IGF), vascular endothelial growth
factor (VEGF), EG-VEGF, VEGF-related protein, Bv8, VEGF-E,
granulocyte colony stimulating factor (G-CSF), insulin like growth
factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic
growth factor (GDNF), stem cell factor (SCF), keratinocyte growth
factor (KGF), transforming growth factors (TGF), (e.g., TGFs
.alpha., .beta., .beta.1, .beta.2, and .beta.3), any of the bone
morphogenic proteins, skeletal growth factor, bone matrix derived
growth factors, and bone derived growth factors and mixtures
thereof.
[0085] As used herein the term "differentiation factor" refers to a
bioactive agent that promotes the differentiation of cells.
Representative examples include neurotrophins, colony stimulating
factors (CSF), and transforming growth factors. Some growth factors
may also promote differentiation of a cell or tissue. Some
differentiation factors also may promote the growth of a cell or
tissue. For example, TGF may promote growth and/or differentiation
of cells. In some embodiments, the scaffold comprises at least one
bioactive agent for promoting cell adhesion, colonization,
proliferation and/or differentiation. In some embodiments, the at
least one agent for promoting cell adhesion is selected from the
group consisting of: gelatin, fibrin, fibronectin and collagen.
[0086] The bioactive agent may be incorporated into the scaffold in
a variety of different ways. In one embodiment, the bioactive agent
is located and/or formulated for controlled release to affect the
cells or tissues in or around the oriented nanofiber structures.
For instance, it may be dispersed in a controlled release matrix
material. In one embodiment, the bioactive agent is provided in
lipid microtubules or nanoparticles selected to modulate the
release kinetics of the bioactive agent. Such particles may be
dispersed among the nanofibers, or provided within the scaffold. In
another embodiment, the bioactive agent is actually integrated
into, forms part of, the tubes, microtubes and/or fibers
themselves. This may be done, for example, by adding the bioactive
agent to a polymer solution prior to electrospinning the solution
to form the tubes, microtubes and/or fibers themselves. Release of
the bioactive agent may be controlled, at least in part, by
selection of the type and amounts of biodegradable matrix materials
in the nanoparticles or nanofibers.
[0087] In some embodiments the scaffold further comprises cells
such as endothelial cells attached thereto within one or more the
tubes or microtubes of the scaffold. One skilled in the art will
appreciate that incorporation of endothelial cells within tubes of
the scaffold may enhance vascular tissue formation, e.g., so as to
replace the scaffold's synthetic vascular tubes/microtubes which
are degraded in vivo.
[0088] In some embodiments, the scaffold further comprises
plurality of bioactive particles having a range of 200-1500
micrometers in diameter. In some embodiments, particles of the
invention are larger than 1 micrometer in diameter. Particles of
the invention may have any shape or form (e.g., spherical,
triangular, rectangular, etc.). In some embodiments, the particles
are embedded within the scaffold. In some embodiments, the
particles are embedded in between the plurality of fibers dispersed
upon the scaffold or a portion thereof
[0089] In some embodiments, the bioactive particles are ceramic
particles. As used herein, the term "ceramic" is intended to refer
to an inorganic, nonmetallic material, typically crystalline in
nature, though it could be amorphous as well. Ceramics generally
may be compounds formed between metallic and nonmetallic elements,
such as, for example, aluminum and oxygen (e.g.,
alumina--Al.sub.2O.sub.3), calcium and oxygen (e.g., calcia--CaO),
silicon and oxygen (e.g., silica--SiO.sub.2) and other analogous
oxides, nitrides, borides, sulfides, and carbides as well as carbon
matrices. However, "ceramic", as used herein, should not be unduly
construed as being limited to a ceramic body in the classical
sense, that is, in the sense that it consists entirely of inorganic
materials, but rather refers to a body which is predominantly
ceramic with respect to either composition or dominant
properties.
[0090] In some embodiments, the plurality of particles comprises
one or more type of osteoconductive particles. As used in here
"osteoconductive" refers to the ability of a substance to serve as
a suitable template or substance along which bone may grow. In one
embodiment, the one or more types of the osteoconductive particles
are osteoconductive ceramic particles selected from the group
consisting of: calcium carbonate, hydroxyapatite (HA),
demineralized bone material, morselized bone graft, cortical
cancellous allograft, cortical cancellous autograft, cortical
cancellous xenograft, tricalcium phosphate, coralline mineral and
calcium sulfate. In some embodiments, the bioactive particles
comprise hydroxylapatite (HA) and calcium carbonate.
Applications
[0091] A scaffold according to the present invention can be used
for a wide variety of applications. Embodiments of the scaffolds
disclosed herein are suitable for uses such as for cell culture and
cell transplantation. In some embodiments the scaffold may serve as
a bioreactor system providing appropriate growth conditions for
cells in vitro. In some embodiments, the scaffold may serve as
perfusion bioreactor that provides for immediate supply of
nutrients and gases to cells grown in a tissue culture.
[0092] Reference is now made to FIG. 12B which is a schematic
illustration of a hollow fiber reactor (HFR) system 1270 comprising
a scaffold (also referred to as microcapillary system) 1273,
according to an embodiment of the invention. Scaffold 1273 is
connected to an inlet tube 1275 and an outlet tube 1276.
Optionally, inlet tube may be equipped with a peristaltic pump 1277
to facilitate constant circulation of a growth medium. Optionally,
the circulation of growth medium facilitates a turbulence movement
of the growth medium. Scaffold 1273 is enclosed within a HFR vessel
1274. The HFR system may further include a medium reservoir 1271.
Optionally, medium reservoir 1271 is equipped with a cap 1272.
Optionally, inlet tube 1275 and outlet tube 1276 which are
connected to scaffold 1270 contact medium reservoir 1271 via ports
(not shown) in cap 1272. Optionally, waste removal is done using a
waste outlet 1279 via a port (not shown) in cap 1272. Optionally,
growth medium replenishment is done using a feed inlet 1280 via a
port (not shown) in cap 1272. Optionally, system 1270 is
continuously aerated through aeration inlet 1281 with filtered
air/CO.sub.2 (95%/5%, respectively) gas mixture bubbling out
through a sparger 1282 dipped into the growth medium. Optionally,
pressure is release through an output 1283. Optionally, cell
seeding is conducted through an HFR inoculation port 1278.
Following cell seeding onto scaffold 1273, inoculation port 1278 is
closed and during closed system is maintained during the 3D growth
phase of the cells. In some embodiment, the flow rate of the growth
medium is regulated. In some embodiments, the required flow rate of
growth medium is determined according to the characteristics of the
scaffold (e.g. diameter). In some embodiment, the RPM of pump 1277
motor is used as the main control of the flow rate. For a
non-limiting example, when using a microcapillary system including
a capillary tube, which has a diameter of 0.5 mm, connected at each
side to a one of two vein like tubes having a diameter of 0.86 mm
the required RPM is between 10 and 50. As exemplified in example 10
the required flow rates and RPM may be calculated (i.e. by applying
equations 2-4) for specific applications.
[0093] The term "bioreactor" as used herein means any apparatus,
which provides biologically active, protected environment suitable
for cultivation of cells. The term "perfusion bioreactor" as used
herein means a fluidized-bed reactor for cell culture designed for
continuous operation as a perfusion system, i.e., a system in which
fresh medium is fed to the bioreactor at the same rate as spent
medium is removed. In some embodiments, the scaffold is
implantable, and may be surgically connected to a subject's blood
vessels. In some embodiments the scaffold may serve as a bioreactor
system providing appropriate growth conditions for cells in vitro
and for their subsequent development into bone structure in vivo
after anastomosis with host blood vessels. As used herein, the term
"anastomosis" refers to the joining together of two hollow
structures, for a non-limiting example, two arteries or veins, to
achieve continuity. An anastomosis can be end-to-end, side-to-side,
or end-to-side depending on the circumstances of the required
reconstruction or bypass procedure.
[0094] In some embodiments, the scaffold is used to produce a
tissue. In some embodiments the method for producing a tissue
comprises: providing the scaffold of the invention, seeding cells
on said plurality of porous elongated microtubes of said scaffold;
and providing liquid containing nutrients through said inlet of
said scaffold, so as to provide nutrients from pores of said
plurality of porous elongated microtubes to said cells; thereby
producing the tissue.
[0095] In some embodiments, the method comprises a preliminary step
of determining a desired flow rate of said liquid containing
nutrients through said scaffold. In some embodiments, the desired
flow rate is suitable for producing a tissue. In some embodiments,
the desired flow rate is suitable for the step of cells seeding. In
some embodiments, the desired flow rate is suitable for the step of
cells culturing. In some embodiments, the flow rate is controlled
by a pump. In some embodiments, an RPM range of the pump is
determined according to a desired flow rate.
[0096] The term "subject" as used herein refers to an animal, more
particularly to non-human mammals and human organism. Non-human
animal subjects may also include prenatal forms of animals, such
as, e.g., embryos or fetuses. Non-limiting examples of non-human
animals include: horse, cow, camel, goat, sheep, dog, cat,
non-human primate, mouse, rat, rabbit, hamster, guinea pig, pig. In
one embodiment, the subject is a human. Human subjects may also
include fetuses. In one embodiment, a subject in need thereof is a
subject afflicted with a fractured bone, a bone injury, diminished
bone mass and/or bone abnormality.
[0097] The terms "liquid", "fluid" and "media" as used
interchangeably herein refer to water or a solution based primarily
on water such as phosphate buffered saline (PBS), or water
containing a salt dissolved therein. The term aqueous medium can
also refer to a cell culture medium. The term "cell culture medium"
refers to any liquid medium which enables cells proliferation.
Growth media are known in the art and can be selected depending of
the type of cell to be grown. For example, a growth medium for use
in growing mammalian cells is Dulbecco's Modified Eagle Medium
(DMEM) which can be supplemented with heat inactivated fetal bovine
serum.
[0098] The term "nutrients" may include but is not limited to fats,
glucose, mono- or oligo-saccharides, minerals, trace elements
and/or vitamins. Nutrients may further include one or more gaseous
components such as primarily oxygen and carbon dioxide. Nutrients
may further include one or more metabolite.
[0099] The term "metabolite" or "metabolites" as used herein
designates compounds that are naturally produced by an organism
(such as a plant or animal) and that are directly involved in the
normal growth, development or reproduction of the organism. This
includes, but is not limited to, any compound produced by plant or
animal cells, or genetically modified plant or animal cells, such
as proteins or other types of chemical compounds.
[0100] The terms "cell" and "cells" as used herein, refer to
isolated cells, cell lines (including cells engineered in vitro),
any preparation of living tissue, including primary tissue explants
and preparations thereof. Any type of cell can be added to the
scaffold for culturing and possible implantation, including cells
of the muscular and skeletal systems, such as chondrocytes,
fibroblasts, muscle cells and osteocytes, parenchymal cells such as
hepatocytes, pancreatic cells (including Islet cells), cells of
intestinal origin, and other cells such as nerve cells and skin
cells, either as obtained from donors, from established cell
culture lines, or even before or after genetic engineering. Pieces
of tissue can also be used, which may provide a number of different
cell types in the same structure. The scaffold can also be used as
a three-dimensional in vitro culture system for
attachment-dependent cells, e.g., hepatocytes in a 3D micro
environment which mimics the physiological micro environment more
closely. In some embodiments, cells are selected from the group
consisting of: adipose-derived stem cells, mesenchymal cells,
mesenchymal stem cells, vascular smooth muscle cells, adipogenic
cells, osteoprogenitors cells, osteoblasts, osteocytes,
chondroblasts, chondrocytes and osteoclasts, endothelial progenitor
cells, hematopoietic progenitor cells and a combination
thereof.
[0101] Scaffolds or portions thereof described herein can be used
to generate synthetic organs or tissues or portions thereof,
including but not limited to, respiratory tissues (e.g., tracheal,
bronchial, esophageal, alveolar, and other pulmonary or respiratory
tissues), circulatory tissues (e.g., arterial, venous, capillary,
and other cardiovascular tissue, for example, heart chambers of
other heart regions or heart or cardiac valves or valve
structures), renal tissue (for example renal pyramids of the
kidney), liver tissue, cartilaginous tissue (e.g. nasal or
auricular), bone tissue, skin tissue, and any other tissue or organ
or portion thereof that is being engineered on a synthetic
scaffold.
[0102] In some embodiments, the cells may be allowed to proliferate
on the scaffold for a time period, in which the cells can grow to
form colonies, after which the colonies can fuse to form a network
of cells, and subsequently forming a tissue. Generally, the time
for proliferation can range from a few hours or days to a few
weeks, such as about 1 day to about 4 weeks, or about 1 day to
about 2 weeks, or about 1 day to about 1 week, or about 1 day to
about 4 days. The time for proliferation can also depend on the
cultivation conditions for the cells. Parameters of the cultivation
condition can include, for example, temperature, pH, amount of
water, pressure, nutrients present, and type of cell. Cultivation
conditions of cells are known in the art and can therefore be
adapted by a person skilled in the art depending on the desired
cell type and application.
[0103] The term "tissue" refers to a structure formed by related
cells joined together, wherein the cells work together to
accomplish specific functions. An organ refers to a differentiated
structure of an organism composed of various cells or tissues and
adapted for a specific function. Therefore, one or more species of
living cells can be added into the mixture to form a specific
organ. For a non-limiting example, the heart which is an organ
contains muscle tissue that contracts to pump blood, fibrous tissue
that makes up the heart valves and special cells that maintain the
rate and rhythm of heartbeats.
[0104] As used herein, the term "seeding" refers to plating,
placing and/or dropping the cells of the present invention into the
electrospun scaffold of the present invention. It will be
appreciated that the concentration of cells which are seeded on or
within the electrospun scaffold depends on the type of cells used
and the composition of the scaffold.
Electrospun Scaffold
[0105] In some embodiments, at least a portion of the scaffold is
produced by electrospinning. In some embodiments, portions produced
by electrospinning may be connected such as by epoxy glue.
[0106] As used herein, the term "electrospinning" refers to a
technology which produces electrospun fibers (e.g. nanofibers) from
a polymer solution. During this process, one or more polymers are
liquefied (i.e. melted or dissolved) and placed in a dispenser. An
electrostatic field is employed to generate a positively charged
jet from the dispenser to the collector. Thus, a dispenser (e.g., a
syringe with metallic needle) is typically connected to a source of
high voltage, preferably of positive polarity, while the collector
is grounded, thus forming an electrostatic field between the
dispenser and the collector. Alternatively, the dispenser can be
grounded while the collector is connected to a source of high
voltage, preferably with negative polarity. As will be appreciated
by one ordinarily skilled in the art, any of the above
configurations establishes motion of positively charged jet from
the dispenser to the collector. Reverse polarity for establishing
motions of a negatively charged jet from the dispenser to the
collector is also contemplated. At the critical voltage, the charge
repulsion begins to overcome the surface tension of the liquid
drop. The charged jets depart from the dispenser and travel within
the electrostatic field towards the collector. Moving with high
velocity in the inter-electrode space, the jet stretches and the
solvent therein evaporates, thus forming fibers which are collected
on the collector forming the electrospun scaffold.
[0107] In some embodiments, the inner diameter and wall thickness
of the tubes are adjusted by changing the collecting mandrels and
controlling the deposition time of electrospinning, respectively.
In some embodiments, the porosity of a porous tube correlates with
fiber diameter and polymer weight concentration, which enable
manipulation of the scaffold porosity by changing the polymer
weight concentration. In some embodiments, a porous scaffold
produced by electrospinning exhibits permeability within the
permeability range for human trabecular bone as exemplified below
(permeability constant K=10-10-10-12 [m2]). In some embodiments,
Cell adherence is supported by the high surface area-to-volume
ratios of the electrospun nanofibers, whose nanoscale architectures
expose the cells to more binding sites compared with micro- and
macro-scale architecture, and by that lead to a better adherence of
every cell by allowing its attachment to multiple nanofibers.
[0108] The descriptions of the various embodiments of the present
invention have been presented for purposes of illustration, but are
not intended to be exhaustive or limited to the embodiments
disclosed. Many modifications and variations will be apparent to
those of ordinary skill in the art without departing from the scope
and spirit of the described embodiments. The terminology used
herein was chosen to best explain the principles of the
embodiments, the practical application or technical improvement
over technologies found in the marketplace, or to enable others of
ordinary skill in the art to understand the embodiments disclosed
herein.
[0109] In the discussion unless otherwise stated, adjectives such
as "substantially" and "about" modifying a condition or
relationship characteristic of a feature or features of an
embodiment of the invention, are understood to mean that the
condition or characteristic is defined to within tolerances that
are acceptable for operation of the embodiment for an application
for which it is intended. Unless otherwise indicated, the word "or"
in the specification and claims is considered to be the inclusive
"or" rather than the exclusive or, and indicates at least one of,
or any combination of items it conjoins.
[0110] It should be understood that the terms "a" and "an" as used
above and elsewhere herein refer to "one or more" of the enumerated
components. It will be clear to one of ordinary skill in the art
that the use of the singular includes the plural unless
specifically stated otherwise. Therefore, the terms "a," "an" and
"at least one" are used interchangeably in this application.
[0111] For purposes of better understanding the present teachings
and in no way limiting the scope of the teachings, unless otherwise
indicated, all numbers expressing quantities, percentages or
proportions, and other numerical values used in the specification
and claims, are to be understood as being modified in all instances
by the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the following specification
and attached claims are approximations that may vary depending upon
the desired properties sought to be obtained. At the very least,
each numerical parameter should at least be construed in light of
the number of reported significant digits and by applying ordinary
rounding techniques.
[0112] In the description and claims of the present application,
each of the verbs, "comprise," "include" and "have" and conjugates
thereof, are used to indicate that the object or objects of the
verb are not necessarily a complete listing of components, elements
or parts of the subject or subjects of the verb. Other terms as
used herein are meant to be defined by their well-known meanings in
the art.
[0113] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0114] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
herein above and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0115] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Maryland
(1989); Perbal, "A Practical Guide to Molecular Cloning", John
Wiley & Sons, New York (1988); Watson et al., "Recombinant
DNA", Scientific American Books, New York; Birren et al. (eds)
"Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold
Spring Harbor Laboratory Press, New York (1998); methodologies as
set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;
5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook",
Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal
Cells--A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y.
(1994), Third Edition; "Current Protocols in Immunology" Volumes
I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and
Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk,
CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference. Other general
references are provided throughout this document.
Materials and Methods
Electrospinning of Fibrous Tubes
[0116] A 9% solution of polycaprolactone (PCL, Mw 80,000 Da;
Sigma-Aldrich) in a 80:20 (w/w) mixture of chloroform (Frutarom)
and dimethylformamide (DMF, Frutarom) was electrospun using the
following parameters: an applied voltage of 12 kV, a flow rate of
2.5 ml/hour and a tip-collector distance of 12-17 cm. PCL fibrous
tubes were collected using a rotating aluminum wire with a diameter
of either 0.5 mm or 2.8 mm, at 350 rpm. The collected tubes were
then dried in a vacuum, at a pressure of .about.10-3 atm, and then
stored in a desiccator at relative humidity of .about.30%.
Capillary Design
[0117] Tubular scaffolds with an inner diameter of 2 mm, wall
thickness of 0.2 mm.+-.0.02 mm and a length of 25 mm, were
successfully constructed (FIG. 2a). Inner diameter and wall
thickness of electrospun tubular scaffolds can be adjusted by using
mandrels with different external diameters and by controlling the
electrospinning deposition time, respectively. The design may be
adjusted according to a desired application. SEM images of the
electrospun scaffolds are shown in FIGS. 2b-d. All scaffolds showed
randomly oriented fibers and interconnected pore structure
throughout the scaffold. However, fiber diameter and pore size were
distinct. To quantify the permeability of the scaffolds, different
air pressure drop through 10.times.10.times.0.1 mm.sup.3 layers of
scaffolds was applied and the total pressure drop (dP) was measured
using a commercially available water manometer. Permeability was
calculated using Equation 1.
v = - k .mu. a dP dx Equation .times. .times. 1 ##EQU00001##
where .nu. is the flux (discharge per unit area, with units of
length per time[m/s]), dP/dx is the pressure gradient vector
(Pa/m), and .mu..sub.a is the air viscosity (Pas).
Fabrication of the Complex Scaffold
[0118] The complex scaffold is composed of a vasculature-like
system embedded in a bulk scaffold. The vasculature-like system is
comprised of electrospun PCL tubes, whereas the bulk surrounding
scaffold includes bone-inducing material (Pro-Osteon particles of
0.5-1 mm diameter, Bepex) and electrospun PCL fibers (FIGS. 4 &
5a). Using a wet brush, the Pro-Osteon particles are dispersed in
between and on the PCL tubes and are kept in place by the PCL
fibers that also are dispersed around the tubes and Pro-Osteon
(FIGS. 4a & 5b). Each vasculature structure consists of three
or more small electrospun PCL tubes (inner diameter. 0.5 mm, FIG.
5c), connected together from both sides by another electrospun PCL
tube with a diameter of 2.8 mm (FIG. 4b). The latter tubes resemble
inlet and outlet grafts for media infusion into the construct. The
small tubes tips are inserted into the large one and connected to
it using an epoxy adhesive that forms a liquid-tight adherence. The
radius of the entire scaffold system is adjusted to .about.1 cm.
All experiments were performed 3 or 4 times at least, and more, if
necessary. All data are expressed as mean .+-.standard deviation
(SD).
Example 1
Preliminary Capillary Design
[0119] As mentioned in the method section, inner diameter and wall
thickness of electrospun tubular scaffolds can be adjusted. The
average fiber diameter and pore size increased as a function of the
PCL weight concentration in the electrospun polymer solution. More
specifically, the mean diameter of fibers electrospun from the low
concentration (8 wt. %) PCL solution was 200 nm.+-.65 nm, while for
the higher PCL solution concentration (11 wt. %) mean fiber
diameter was 550 nm.+-.45 nm. As expected, the porosity of the
electrospun scaffold is correlated with the fiber diameter, namely,
higher porosity was observed in scaffolds with fibers of larger
diameter. Additionally, the permeability of the scaffolds was
quantified using Darcy equation, and the Darcy constant k was found
to be in the range of 10.sup.(-10)-10.sup.(-12) [m.sup.2].
Example 2
Preliminary Flow and Permeability Study Using Homemade
Bioreactor
[0120] Flow and tube wall permeability were characterized by tube
assembly in a homemade bioreactor. The tube was connected to an
inlet and outlet (FIG. 3) and immersed in a PBS bath (Invitrogen).
A 10% solution of fetal calf serum (FCS) serum in PBS (Invitrogen)
was pushed through the tube using a peristaltic pump, while the
longitudinal pressure was regulated, in the range of 1-2 N/m2, with
a valve close to the outlet. The serum that passed through the PCL
wall tubes (the permeate) entered the surrounding bath and the
excess fluid (concentrate) exited and collected in the feed
reservoir (FIG. 3). The permeate was collected during the
experiment and protein concentration was measured using a native
10% gel electrophoresis system.
[0121] The PCL tubes proved permeable, as demonstrated from the
increasing protein concentrations measured over time in the PBS
bath in which perfused tubes were immersed (FIGS. 7A-C). The system
reached saturation within 120 minutes of perfusion. The tubes were
permeable to all protein sizes present in the serum.
Example 3
Preliminary Adjustment of Culture Conditions
[0122] Human adipose-derived mesenchymal stem cells (MSCs) were
isolated from human adipose tissue explants (1-2 mm.sup.3)
extracted from abdominal area. This study was approved by the
Rambam Health Care Campus Helsinki Committee (#0370-12-RMB). The
explants were placed in fibronectin-coated flasks and incubated in
a basic growth medium, containing Dulbecco's Modified Eagle Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 1%
L-glutamine and 1% Pen-strep antibiotics (all from Biological
Industries) for 5-7 days. After one week, adipose tissue explants
were washed and MSCs were trypsinized. MSCs were cultured in a
basic growth medium, which was changed once in 3 days. Cells were
expanded up to passage 3 or 4 in growth medium and were then used
in in vitro and in vivo studies.
[0123] Inductive conditions were achieved by culturing MSCs in
inductive medium comprised of DMEM, supplemented with 10% FBS,
10.sup.-8 M dexamethasone and 100 .mu.g/ml L-ascorbic acid
2-phosphate sesquimagnesium (both from Sigma Aldrich). Moreover,
osteogenic differentiation was induced by culturing the cell-seeded
scaffolds in osteoconductive medium comprised of inductive medium
further supplemented with 10 mM .beta.-glycerophosphate (Sigma
Aldrich).
[0124] MSCs were only seeded onto scaffold regions containing
Pro-Osteons (FIG. 2a). The seeding region dimensions were
approximately 2.2 cm long and 1 cm wide. Prior to seeding, all
scaffold samples were soaked in 70% ethanol for sterilization and
washed several times with phosphate buffered saline (PBS). One
million of trypsin-released cells were counted and re-suspended in
50 .mu.l growth or inductive medium and were seeded onto the
scaffold. The cell-seeded samples were then incubated for 70 min
with slow rotation, and then re-suspended in growth or inductive
medium, respectively.
[0125] Proliferation of MSCs cells in a static culture: To evaluate
the proliferation rates and growth viability of the isolated MSCs,
the Alamar blue-based viability assay was used. Cells were seeded
on the scaffolds and cultured in growth medium for 28 days. Cell
viability was assessed on days 1, 3, 7, 14, 21 and 28 post-seeding.
At each time point, the cell-seeded scaffold was washed twice in
PBS and incubated for 2 hours in medium containing 10% Alamar blue
reagent (Serotec, UK). The fluorescence of Alamar blue reagent was
recorded by FLUOstar galaxy fluorescence reader (BMG Labtech,
Germany) at 540 nm excitation and 580 nm emission.
[0126] Histological analysis of cell-seeded scaffolds cultured
under dynamic conditions: MSCs were seeded on the complex scaffolds
(.about.1 million cells per scaffold) and cultured for two weeks in
a medium flow bioreactor system (FIG. 6). During the first week,
the cells were cultured in inductive medium, before being
transferred to osteoconductive conditions. For histological
staining, cell-seeded scaffolds were fixed in 10% neutral buffered
formalin (NBF) for 48 hours, and then decalcified in 10%
ethylenediaminetetraacetic acid (EDTA) solution for 1 week. The
constructs were then embedded in paraffin after undergoing standard
fixation. Transverse, 5 .mu.m-thick sections were placed on
silanized slides for hematoxylin & eosin (H&E) or Masson's
Trichrome staining.
[0127] Morphological analysis of cell-seeded complex scaffolds
cultured under dynamic conditions: MSCs were seeded on complex
scaffolds, which were then cultured in growth medium for
approximately 1 month in a medium flow bioreactor system. Samples
were then fixed in 10% NBF, dehydrated in graded ethanol solutions
and soaked in hexamethyldisilazane for 15 minutes. The samples were
then sputter-coated with gold and characterized using a Phenom
desktop scanning electron microscope (SEM) (5 kV accelerating
voltage, FEI Company).
Static Culture Conditions
[0128] The metabolic activity rate and proliferation capacities of
MSCs cultured on complex scaffolds under static conditions
increased in direct correlation with the duration of culture, as
presented in FIG. 8. This result is a preliminary indication of the
effective biological support provided by the complex scaffold.
Dynamic Culture Conditions
[0129] After examining the biocompatibility of the scaffold in a
static culture, it was tested in a dynamic culture. An MSC-embedded
complex scaffold was first cultured for one week under static
conditions and then transferred for an additional week in an
inductive medium, delivered via a medium flow bioreactor system
(FIG. 6). The scaffold was then fixed, decalcified, embedded in
paraffin and stained. The H&E-stained histological sections
(FIGS. 9A-D of the seeded scaffold, illustrate the complex
structure of the scaffold that consisted of three PCL tubes
embedded within a bulk tissue (asterisks indicate the tubes). The
images also demonstrated the extensive MSC proliferation and the
expansion within the scaffold. It was also noted that MSCs did not
penetrate the PCL tubes, but rather, settled on the wall of the
tubes.
[0130] The SEM analysis corroborated these findings (FIGS. 10A-C),
as well as excellent integration of the cells within the scaffold.
Cells were distributed along and in between the osteoconductive
particles, depositing their extracellular matrix components
throughout the bulk part of the scaffold. In addition, cells were
observed along the external walls of the embedded tubes. It is
important to state that in contrast to histological sections,
wherein the ceramics are dissolved through a decalcification
process, the Pro-Osteon particles can still be seen in the SEM
analysis.
Example 4
Preliminary Implantation of MSC-Seeded Scaffolds in Ectopic
Models
[0131] In order to examine the biocompatibility of the complex
scaffolds in vivo, they were seeded with induced cells and then
implanted in ectopic animal models.
[0132] On the implantation day, complex scaffolds were seeded with
cells that were previously cultured for at least 10 days in
inductive medium. Prior to implantation, a fibrin clot, composed of
1:1 rat fibrinogen: rat thrombin (Sigma Aldrich), was added to the
cell-seeded constructs to stabilize the sample.
[0133] All surgical processes described below were performed
following the protocols approved by the Institutional Animal Care
and Use Committee. Three groups of 6-week-old, nude female mice
(n=5 per group, Harlan Laboratories) were anesthetized using a
0.5:0.5:9 ketamine:xylazine:PBS cocktail at a dose of 400 .mu.L/20
g, delivered with a 25-gauge needle. Cell-seeded complex constructs
were subcutaneously implanted to the dorsum of the anesthetized
mice. In parallel, unseeded scaffolds and seeded Pro-Osteons
particles were subcutaneously implanted as negative and positive
controls, respectively. Tissue samples of the construct area were
extracted for histological analysis 8 weeks post-implantation.
[0134] Histological sections of the implanted scaffolds extracted 8
weeks after implantation, showed the new formed tissues around the
Pro-Osteons (crosses represent their position before
decalcification; FIGS. 11C, E and H) and the vasculature-like
system within the implanted scaffold, represented by three PCL
electrospun-tubes (asterisks; FIGS. 11A, F and H). Moreover, the
PCL fibers that constitute the tubes wall (black arrows; FIGS. 11D
and I), MSC integration within the scaffold (black dots examining
the cells' nuclei), formation of blood vessels (red arrows; FIGS.
11B, F, G and H) and muscle tissue (yellow arrows; FIGS. 11A and F)
and accumulation of collagen into the scaffold (blue arrows; FIGS.
11B, F and G) were observed. Furthermore, maintained structural
integrity of vasculature-like scaffold was apparent and no
inflammatory reaction toward the graft was detected, indicating its
biocompatibility with the host.
Example 5
A Custom Designed Hollow Fiber Reactor (HFR) System for Dynamic
Culturing
[0135] A hollow fiber reactor (HFR) (1270) was developed for use
with the microcapillary system to enable cell growth and
differentiation prior to transplantation. The bioreactor system
(FIGS. 12A and 12B) is based on 500 ml Erlenmeyer flask used as a
medium reservoir (1271); it has a specially designed cap (1272)
that contains inlets and outlets for feed, waste and aeration. The
microcapillary system (1273) is placed into the HFR vessel (1274),
connected to an inlet tube (1275) and an outlet tube (1276) and
then the growth medium is constantly circulated throughout the
system. Cell seeding is conducted using the HFR inoculation port
(1278). Following cell seeding onto microcapillary system (1273),
it is closed and during all of the 3D growth phase maintains closed
system parameters.
[0136] Seeding conditions: 8-12.times.10.sup.6 MSCs in 300 ul
growth medium were injected onto the construct (using the
inoculation port of the HFR) in several points on the construct and
incubated at static conditions for adequate cell adherence.
Following 20 minutes, 2 ml growth medium was added and incubation
continued at static conditions (37.degree. C., 5% CO2) for total of
120 minutes. After the incubation, the HFR vessel was connected to
the system tubing and the growth medium (total volume of 150 ml)
circulation through the microcapillary construct began (at 18 RPM
based on preliminary studies). The circulating medium was aerated
by air+5% CO2 mix into the medium reservoir using a sparger
(aeration rate is 15 ml/hour). Medium change (50 ml) was performed
twice a week using the waste and the feed ports on the cap. The
bioreactor system was very stable and was able to run during a long
period (one month was successfully tested). Osteogenic induction
was performed for two days by adding BMP2 (150 ng/ml final conc.)
to the growth medium.
Example 6
Cells Characterization Following Culturing of MSCs on the
Microcapillary Scaffold in the HFR Dynamic System Compared to a
Static System
a. Cell Attachment and Growth on the Microcapillary Scaffold
[0137] Since cell counting on the microcapillary scaffold is
challenging, cells growth and construct coverage is shown using
Giemsa staining, which demonstrates the MSCs growth onto the
microcapillary system (FIGS. 13A and B, magnification
.times.1).
[0138] Histological evaluation (Hematoxylin & Eosin staining)
after one week of culturing in the HFR system supports MSCs growth
onto the scaffolds construct. As can be seen in the images (FIGS.
14A and B), the cells between the mineral particles, embedded
within the PCL fibers, have generated organized connective tissue
around and between the mineral particles.
[0139] Cells viability was demonstrated by MTT staining (FIGS. 15A
and B). The live cells are violet colored (15A) and the control
mineral particles (without cells) are not colored (15B). This
method demonstrates cells coverage as well as viability.
b. Osteogenic Potential of MSCs Following Osteogenic Induction by
BMP-2, in Static and Dynamic Growth Systems
[0140] In order to evaluate the MSCs osteogenic potential following
cultivation in the HFR system experiments ending with osteogenic
induction of the cultured cells were performed. The experiments
compared static and dynamic (HFR) growth systems. The static system
is composed of a petri dish or in an Erlenmeyer in which cells are
cultured on the microcapillary scaffold and medium is manually
replaced. The dynamic system is composed of a fully closed
bioreactor which enables medium aeration and flow along and within
the microcapillary graft, as well as an automatic medium
replacement by specific ports.
[0141] Osteogenic induction was evaluated by the osteogenic marker
ALP, using FACS (FIGS. 16 A-B). Results show that cells cultivated
in the dynamic HFR system had a higher level of ALP positive cells
(33.23%, 16B) compared to the level of ALP positive cells (18.65%)
of cells cultivated in the static system (16A).
[0142] Osteogenic differentiation was also evaluated using
Real-Time PCR (FIGS. 17A-C). The RNA was extracted using the
PureLink.RTM. RNA Mini Kit (Life technologies). TaqMan osteogenic
primers (DLX5 and Osterix (SP7)) were used for Real-Time reactions
preparation. The osteogenic genes expression, following 2 days of
osteogenic induction (day 9 of cultivation), was compared to the
osteogenic genes expression at HFR seeding day (day 0). Both
dynamic and static growth conditions were evaluated. Real time PCR
results demonstrated DLX5 and SP7 gene expression are elevated post
osteogenic induction (day 9) relative to day 0, indicating for
osteogenic induction in both systems, with higher expression in
dynamic HFR system compared to static system.
c. Endothelial Cells (ECs) Growth on the Microcapillary Scaffold in
Dynamic and Static Culturing Systems
[0143] The ability of the microcapillary system to support Seeding
and growth of Human Adipose Microvascular Endothelial Cells (HAMEC)
inside the microcapillary tubes was tested as following: the growth
system was built as was described before, but with single PCL tube.
Before cell seeding, the tube was coated with fibronectin to allow
adherence of endothelial cells onto the inner surface of the tube
lumen. The sterile PCL tube was aseptically filled with fibronectin
solution and rotated for one hour at 37.degree. C. incubator to
allow even coating with fibronectin. Subsequently, suspension of
endothelial cells was prepared in Endothelial Cell Medium (ECM)
(from ScienCell). To seed cells, the PCL tube was drained out of
the fibronectin solution, and filled instead with the prepared
suspension of endothelial cells. Cells were seeded at a density of
5.times.10.sup.5 cells/cm.sup.2, and the tube was filled in the
appropriate volume of medium. ECM medium was also used to submerge
the PCL tube to cover the outside surfaces and the tube was rotated
for two hours at 37.degree. C. incubator to allow even seeding of
cells onto the inner surface of the tube. After two
hour-cell-seeding, the PCL tube was opened from both sides and was
connected to the dynamic system including peristaltic pump and
aerated growth medium reservoir. The growth medium was circulated
constantly as previously described. The medium flow was kept for 48
hours. Next, the tube was taken out of the incubator and washed
twice with PBS and stained with Giemsa and visualized (FIG. 18A).
Adequate cell coverage of the inner walls of the tubes is seen with
characteristic cobble stone morphology. Moreover, the HAMECs
retained their AC-LDL uptake capabilities (FIG. 18B) which points
out that their biological functionality is intact.
Example 7
Demonstration of Human Plasma Protein Permeability via the
Microcapillary Scaffold, in Conditions That Mimic Blood Flow Within
the Scaffold
[0144] The microcapillary construct permeability to human plasma
proteins was tested using the system illustrated in FIGS. 19A-B. As
demonstrated in the schematic illustration of FIG. 19B, a sample of
human plasma (diluted 1:4) 1991 was streamed into the
microcapillary system 1992 in a HFR vessel 1993 using a peristaltic
pump 1994 in a circular manner. The microcapillary system wetted as
planned and liquid droplets formed on the construct outer
perimeter, the droplets were gathered using a collection tube 1995.
Sampling the liquid gathered inside the collection tube was sampled
every 30 min for 5 hr. Human serum albumin, IgG and lysozyme
concentrations were determined in every sample (using IMPLEN's
NanoPhotometer.RTM. P-Class) and were compared to the
concentrations in the plasma before the experiment. Results
demonstrate that the system is permeable to all tested proteins:
human serum albumin (FIG. 20A), IgG (FIG. 20B) and Lysozyme (FIG.
20C). Further a constant wetting rate was observed meaning there is
no plugging of the system with time.
Example 8
Standardization and Stability of the Microcapillary Scaffold
[0145] The electrospinning machine includes syringe that contains
the polymer solution which is 12% polycaprolactone (PCL, Mw 80,000
Da) in an 80:20 (w/w) mixture of chloroform and dimethylformamide
(DMF). The polymer syringe (5 ml BD plastic syringe) is driven by a
syringe pump which is used to control the flow rate (1 ml/hour) of
the polymer being ejected. The electrospinning machine also
includes high voltage power supply which apply a fixed voltage of
15-20 kV to a metallic needle (21 G) connected to the polymer
syringe. The polymer solution (12% PCL) pass through the needle and
is charged by the high voltage that is directly opposite to the
surface tension of the polymer solution, leading to the elongation
of the hemispherical surface of the solution at the tip of the
syringe to form a conical shape known as "Taylor cone". Due to
elongation and solvent evaporation, the charged jet forms randomly
oriented nanofibers that are collected on a grounded metallic
collector made of wire of 0.5 mm diameter, or tubes of 0.86 mm or
1.5 mm diameter.
[0146] Tubes of 0.5 mm diameter are the capillary-like tube and
tubes of 0.86 mm and 1.5 mm diameter are the vein-like tube that is
cut to two tubes and connected to both sides of the capillary-like
tube.
[0147] The vasculature-like system for in vitro experiments is made
of three PCL tubes of 0.5 mm (collection time 8 minutes, flow rate
1 ml/hour) which are connected together from both sides by PCL
tubes of 1.5mm (collection time 40 minutes, flow rate 1 ml/hour),
which resemble an inlet and outlet grafts for media infusion into
the construct. The three tubes are surrounded with pro-osteon
particles that are dispersed in between and onto the PCL tubes and
are kept in place by the PCL fibers.
Example 9
Demonstration of Microsurgery Technique Proof of Concept--for
Transplantation of the Microcapillary Scaffold End to End in a Rat
Model
[0148] To provide compatibility between the diameters of the
vein-like tube (inlet/outlet) and the rat femoral/artery vein, a
vein-like tube having a diameter of 0.86 mm was produced, using a
collector of 0.86 mm by the electrospinning technique. The
adjustment in the vein diameter was done to overcome any
incompatibility between the diameters of the vein-like tube
(inlet/outlet) and the rat femoral/artery vein that can lead to
blood leakage from the anastomosis site the vein-like tube
diameter. In addition, the number of capillary-like tubes connected
to the vein-like tubes was adjusted to two capillary-like PCL tubes
of 0.5 mm. These two capillaries were surrounded by bone-inducing
material (pro-osteon) and electrospun PCL fibers, and connected
together to 0.86 mm vein-like tube in both sides using PCL solution
as glue.
[0149] Next, the system was sterilized and transplanted in Sprague
Dawley (SD) rat model using microsurgery technique. The
vasculature-like system was fixated to the rat muscle tissue, then
anastomosed to the femoral artery in one side and the femoral vein
in the other one by 6-8 stitches in each side done with 10-0
Prolene microsurgical suture via "End to End" anastomosis (FIG.
21A-H). After finishing the anastomosis and releasing the clamps
that held the artery and vein, no blood was leaked from the
connection site. Using laser Doppler, the blood flow in the artery
and vein was measured (FIG. 22A), demonstrating a blood flow
through the vasculature-like system (FIG. 23). Later, the rat skin
was stitched above the transplant and coated with polydine ointment
to prevent any infections. After the surgery, the rat was examined
for a week; and no pain was shown by the rat and it moved freely
which mean that no damage was done to its limbs although it lost
the main blood supply.
[0150] One day later, the rat was anesthetized and the stitches
were cut of the skin in order to measure the blood flow through the
femoral artery and vein. As demonstrated in FIGS. 23A-B the blood
still flowed through the anastomosed vasculature-like system but it
was reduced by 41-47% according to the laser Doppler (FIGS. 23C and
23D), probably as a result of blood clots. Two weeks later, the rat
was sacrificed and the transplant was extracted (FIG. 24).
[0151] Next, the extracted transplants were fixated,
Paraffin-embedded blocks were prepared and stained with Hematoxylin
& Eosin. Histological analysis of the transplant showed that
the scaffold structure is maintained, the fibers of PCL tubes were
not destroyed and there is accumulation of cells into the
capillary-like tubes (FIG. 25A-D).
[0152] In order to prevent clotting inside the vasculature-like
system during the surgery, the scaffold was soaked with phosphate
buffered Saline (PBS), and 2 ml of Heparin were injected (5000
unit/ml) through it to be soaked with heparin. Next, the
heparin-soaked scaffold was anastomosed to the femoral artery from
one side, blood passage through the scaffold was examined by
releasing the blocking clamp for seconds (FIG. 26A). Next, the
other side was connected to the femoral vein. After releasing the
clamps, the blood flowed through the scaffold without any leakage
from the connection site (FIG. 26B). In addition, every rat was
injected subcutaneously with 75 Units/kg of heparin after
completing the surgery. Three weeks later, the rats were
anesthetized; their blood flow through the transplants was measured
(FIG. 26C), and the laser Doppler showed that although much
improved, there is still a slight reduction in the blood flow (FIG.
26D).
[0153] In the following surgeries, in order to overcome any
clotting that may occur in the transplant and reduce the blood flow
through it; a daily dosage of 100 units/kg heparin was inject for a
week after the transplantation. There was no bleeding as a result
of heparin and the rats acted and moved normally.
Example 10
Calibrated Flow Rates
[0154] The flow rates were evaluated using a calibrated peristaltic
pump and relevant blood flow rates obtained from the Literature.
The pressure on the blood vessel wall is influence by the blood
flow rate inside the blood vessels and this pressure in turn is the
driving force for blood penetration throws the walls. As mention
above, in the large blood vessels it is not desirable process,
while in capillary it is necessary process for the cell nourish.
Since the blood flow rate influence by the blood velocity and the
cross section of the blood vessels, penetration tests should be
evaluated in the relevant flow rates.
[0155] The peristaltic pump was calibrated in order to examine the
range of its flow rate. Two pump rate were examined (high 100 rpm
and low 11 rpm) and the coming out fluid volume was measured after
defined time (20-75 minutes). Flow rate (Q) was calculated using
Equation 2 (V--volume, t--time) and the flow velocity was measured
using Equation 3 (v--velocity, A--area=.pi.r.sup.2).
Q .function. ( ml Sec ) = V .function. ( ml ) t .function. ( sec )
Equation .times. .times. 2 v .function. ( cm Sec ) = Q .function. (
ml / sec ) A .function. ( cm 2 ) Equation .times. .times. 3
##EQU00002##
[0156] The results are shown in Table 1, while blood flow velocity
in different types of blood vessels is demonstrated in Table 2.
FIG. 8 shows the flow rate of the peristaltic pump in correlation
with pump rate.
TABLE-US-00001 TABLE 1 Peristaltic pump calibration and flow rate
calculation Volume (ml) Time (min) Flow Rate (ml/hr) Pump Rate
(90/10) = 100 rpm 21 20 63.00 48 40 72.00 62 72 51.67 Q (ml/hr)
Mean 62 SD 10 Q (ml/sec) 0.017 v (cm/sec) 0.152 Pump Rate (10/1) =
11 rpm 5 20 15.00 8 48 10.00 11 63.5 10.39 Q (ml/hr) Mean 12 SD 3 Q
(ml/sec) 0.003 v (cm/sec) 0.029
TABLE-US-00002 TABLE 2 Peristaltic pump calibration and flow rate
calculation Relation between blood flow velocity and total
cross-section area in human .sup.[1] Blood cross- Blood velocity
Vessel section flow Type of in cm/s diameter area rate blood
vessels V (cm/s) D (cm) A (cm.sup.2) Q (cm.sup.3/sec) Aorta 40 2.5
4.91 196 Capillary 0.03.sup.[2] 0.0008 0.000001 0.00000002 Vein 15
0.5 0.20 3 Tube like Vain 0.152 0.38 0.11 0.0172
[0157] The fibers penetration levels of our tubes like vain and
capillary fibers were evaluated based on fiber diameter, fiber
thickness--depending on electrospinning duration--and PBS flow
rate. Electrospun PCL tubes at different diameters and thickness
were connected to the bioreactor system in order to examine their
permeability (FIG. 27). The tube permeability was calculated using
Equation 4.
% .times. .times. permeability = leakage .times. .times. rate
.times. .times. ( ml / sec ) Pump .times. .times. flow .times.
.times. rate .times. .times. ( ml / sec ) 100 .times. % Equation
.times. .times. 4 ##EQU00003##
[0158] As demonstrated in the results below, tubes simulating veins
were almost not penetrable. However, tubes simulating capillaries
were 65% permeable. Moreover, it seems that .about.40 minutes of
electrospinning is sufficient to block almost completely the fiber
wall. (FIG. 28).
* * * * *