U.S. patent application number 15/584706 was filed with the patent office on 2017-11-23 for tissue regeneration membrane.
The applicant listed for this patent is HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD., YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LTD. Invention is credited to Michael Friedman, Ada Grin, Rami Mosheioff, Jacob Rachmilewitz, Yoel Sasson.
Application Number | 20170333603 15/584706 |
Document ID | / |
Family ID | 41809163 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170333603 |
Kind Code |
A1 |
Friedman; Michael ; et
al. |
November 23, 2017 |
TISSUE REGENERATION MEMBRANE
Abstract
The present invention relates to a membrane comprising at least
one positively charged, synthetic, hydrophobic polymer, at least
one hydrophilic polymer and at least one plasticizer; wherein said
membrane is flexible and is capable of supporting at least one of
cell adherence, cell proliferation or cell differentiation. The
invention further relates to use of a membrane of the invention in
the preparation of an implantable devices including cell delivery
systems, cell growing surfaces and scaffolds. The invention further
provides methods for promoting tissue regeneration in a defected
tissue region applying membranes of the invention.
Inventors: |
Friedman; Michael;
(Jerusalem, IL) ; Sasson; Yoel; (Rehovot, IL)
; Grin; Ada; (Rehovot, IL) ; Mosheioff; Rami;
(Jerusalem, IL) ; Rachmilewitz; Jacob; (Modi'in,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM LTD
HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD. |
JERUSALEM
JERUSALEM |
|
IL
IL |
|
|
Family ID: |
41809163 |
Appl. No.: |
15/584706 |
Filed: |
May 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13144074 |
Feb 24, 2012 |
9669136 |
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PCT/IL2010/000028 |
Jan 12, 2010 |
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15584706 |
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61193947 |
Jan 12, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/502 20130101;
A61P 19/10 20180101; A61L 31/141 20130101; A61L 27/26 20130101;
A61L 27/3834 20130101; A61P 43/00 20180101; A61P 19/08 20180101;
A61L 31/041 20130101; A61L 31/005 20130101 |
International
Class: |
A61L 31/04 20060101
A61L031/04; A61L 27/50 20060101 A61L027/50; A61L 27/26 20060101
A61L027/26; A61L 27/38 20060101 A61L027/38; A61L 31/14 20060101
A61L031/14; A61L 31/00 20060101 A61L031/00 |
Claims
1-28. (canceled)
29. A method for promoting tissue regeneration in a defected tissue
region comprising the steps of: providing a membrane comprising at
least one positively charged synthetic, hydrophobic polymer, at
least one hydrophilic polymer, and at least one plasticizer, and
wherein said membrane is flexible and is capable of supporting at
least one of cell adherence, cell proliferation, or cell
differentiation; and implanting said membrane in the proximity of
said tissue defect region.
30. A method according to claim 29, wherein said defected tissue
results from a condition selected from the group consisting of
non-union fracture, osteoporosis, periodontal disease or condition,
osteolytic bone disease, post-plastic surgery, post-orthopedic
implantation, post neurosurgical surgery, alveolar bone
augmentation procedures, spine fusion, and vertebral fractures.
31. A method according to claim 29, wherein said tissue is selected
from the group consisting of ligament, tendon, cartilage,
intervertebral disc, teeth and bone.
32. A method according to claim 29, wherein said plasticizer is
polyethylene glycol ranging from 300-20,000.
33. A method according to claim 29, wherein said positively charged
synthetic, hydrophobic polymer is ammonia methacrylate copolymer
type A NF (AMCA).
34. A method according to claim 29, wherein both said hydrophilic
polymer and said plasticizer are polyethylene glycol ranging from
300-20,000.
35. A method according to claim 29, wherein both said hydrophilic
polymer and said plasticizer are polyethylene glycol 400.
36. A method according to claim 35, wherein said polyethylene
glycol 400 is present at a concentration of 5-25% w/w.
37. A method according to claim 29, wherein said at least one
hydrophilic polymer is present in a concentration of between about
1% (w/w) to 30% (w/w) of the membrane.
38. A method according to claim 29, wherein said membrane further
comprises at least one type of cell.
39. A method according to claim 38, wherein said cells are selected
from the group consisting of adult stem cells, embryonic stem
cells, pluripotent stem cells, mesenchymal stem cells, umbilical
cord blood cells, osteoblasts, chondroblasts and CD105+ cells.
40. A method according to claim 39, wherein said adult stem cells
are autologous adult stem cells.
41. A method according to claim 29, further comprising at least one
active agent, wherein said at least one active agent is selected
from the group consisting of cytokine, hormone, bisphosphnate,
cannabinoid, beta blocker, bone inducing agent, growth factor,
HMG-CoA reductase inhibitor, drug and antibiotic.
42. A method according to claim 41, wherein said active agent is
selected from the group consisting of statin, estrogen, androgen,
propranolol, transforming growth factor (TGF), bone morphogenetic
protein (BMP), insulin like growth hormone, fibroblast growth
factor (FGF), alendronate, risendronate and parathyroid
hormone.
43. A method according to claim 42, wherein said active agent is
simvastatin or lovastatin.
44. A method according to claim 29, wherein said membrane is
capable of being porous upon hydration, and wherein the pore size
is between about 0.1 to about 5 microns.
45. A membrane comprising at least one positively charged,
synthetic, hydrophobic polymer, at least one hydrophilic polymer
and at least one plasticizer; wherein said membrane is flexible and
is capable of supporting at least one of cell adherence, cell
proliferation or cell differentiation.
46. A three dimensional hollow implant comprising at least one
membrane according to claim 45.
47. A three dimensional hollow implant according to claim 46,
wherein the membrane defines the surface of the implant.
48. A cell delivery system comprising a membrane according to claim
45, wherein said membrane further includes at least one type of
cell.
49. A cell growing surface comprising a membrane according to claim
45.
50. A scaffold substantially coated by a membrane according to
claim 45.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of tissue regeneration,
specifically bone regeneration, using a polymer based membrane or
construct capable of supporting cell adhesion, proliferation and
differentiation.
BACKGROUND OF THE INVENTION
[0002] Non union of the fracture is the condition of cessation of
all reparative processes of healing of fracture without bone union
[1-3]. Non union can also be described as the absence of
progressive repair that has not been observed radiographically
between the 3rd and the 6th month following the fracture [2, 4].
Non-union may occur either as a result of poor mechanical or
biological environment on the fracture area or as a combination of
the two [2]. This and other situations require manipulation or
augmentation of natural healing mechanisms to regenerate large
quantities of new bone than would naturally occur to achieve
surgical goals [5-7]. Therefore, new bone for the repair or the
restoration of the function of traumatized, damaged, or lost bone
is a major clinical need, and bone tissue engineering has been
heralded as an alternative strategy for regenerating bone [8].
[0003] Tissue engineering, as it applies to bone, focuses on
restoration of large segments of skeleton including weight bearing
bones. Bone can be regenerated through the following strategies:
Osteogenesis--the transfer of cells; Osteoinduction--the induction
of cells to become bone; Osteoconduction--providing a scaffold for
bone forming cells; or Osteopromotion--the promotion of bone
healing and regeneration by encouraging the biologic or mechanical
environment of the healing or regenerating tissues.
[0004] A polymeric poly (L-lactide) tubular membrane spanning a
mechanically stable, large segmental bone defect was shown to
promote woven bone formation and reconstruction of the bone defect
[9]. Mosheiff et al. developed a critical size defect model in
rabbit for bone loss treatment testing. In this model the rabbit
forearm is to produce critical size defect. A critical size defect
is defined as the smallest intraosseus wound that is not bridged by
the skeleton in normal circumstances [10, 11]. Using this model,
our group has successfully employed membranes for guided bone
regeneration (GBR), by osteoconduction [10, 12].
[0005] Gugala et al. demonstrated homogenous growth of mesenchymal
stem cells (MSC) on porous membranes, forming a three-dimensional
fibrillar network [19].
[0006] WO 2005/107826 discloses moldable bone implants comprising
biocompatible granules (e.g. bioceramics), a biocompatible polymer
and a plasticizer. The implant may form an open porous scaffolding
or composite matrix or may be administered as a liquid or
plastically deformable implant.
[0007] WO 2004/084968 discloses a porous matrix suitable for use as
a tissue scaffold or an injectable formulation, preferably prepared
from a degradable cross-linked polymer.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the finding that a novel
polymer-based membrane, comprising both hydrophobic and a
hydrophilic polymers and which further comprises a plasticizer, is
capable of supporting cell adhesion, proliferation and
differentiation, and thus can be used to augment tissue
regeneration, for example in the treatment of large segmental bone
defects. The membranes of the invention operate both as a delivery
system for cells and as a device for guided bone regeneration,
optionally together with active agents that promote cell growth,
adhesion, differentiation, and/or proliferation.
[0009] Specifically, the invention is based on the finding that a
polymer-based membrane which further comprises polyethyleneglycol
(PEG) supported mesenchymal stem cell (MSC) adhesion,
differentiation, and proliferation.
[0010] Accordingly, in one of its aspects the invention provides a
membrane comprising at least one positively charged, synthetic,
hydrophobic polymer, at least one hydrophilic polymer and at least
one plasticizer; wherein said membrane is flexible and is capable
of supporting at least one of cell adherence, cell proliferation or
cell differentiation.
[0011] As used herein the term "membrane" concerns a thin (roughly
two dimensional) continuous homogenous construct having a thickness
of between 30-200 .mu.na, typically constructed by casting at least
one positively charged synthetic, hydrophobic polymer, at least one
hydrophilic polymer and at least one plasticizer into molds.
[0012] The term "positively charged synthetic, hydrophobic polymer"
relates to a synthetically produced polymer which is insoluble in
water, having an overall average positive (surface) charge
resulting from positively charged or partially positively charged
monomer groups (or substituents on said monomers) of the
polymer.
[0013] In some embodiments, said at least one hydrophobic polymer
is an acrylic polymer. In other embodiments, said at least one
hydrophobic polymer is a methacrylate Copolymer substituted by at
least one amine group. The term "amine group" is meant to encompass
any amine group such as for example --NH.sub.3, a secondary amine,
tertiary amine and an ammonium group. In a specific embodiment said
polymer is Ammonic Methacrylate Copolymer, and more preferably
Ammonic Methacrylate Copolymer type A (for example, AMCA
EUDRAGIT.RTM. RL, Degussa Germany).
[0014] It should be understood that a hydrophobic polymer in
accordance with the invention also encompasses co-polymers, or
mixture of hydrophobic, positively charged polymers with
hydrophobic non-positively charged polymers, for example, wherein
at least 30% of the hydrophobic polymer has a positive charge. In
such embodiments, the mixture of hydrophobic polymers may further
include polyethylene, polymethacrylate, polyamide-nylon,
polyethylene vinyl acetate, cellulose nitrate, silicones,
ethylcellulose and any combination thereof.
[0015] As used herein the term "polymer having an amine group and a
methacrylic group", concerns polymers as well as copolymers having
as monomers methacrylic groups substituted with amine groups.
Example of such polymers can be found, for example, in Aggeliki et
al [20].
[0016] The term "hydrophilic polymer" refers to polymers (including
co-polymers and mixtures of polymers) that dissolve in aqueous
media such as in bodily fluids (e.g. extracellular fluid,
interstitial fluid, plasma, blood, or saliva). It is noted that
such polymers generate pores in the membrane upon exposure to
aqueous media.
[0017] In some embodiments, said at least one hydrophilic polymer
is selected from the group consisting of
hydroxylpropylmethylcellulose, hydroxylpropylcellulose,
carboxymethylcellulose, hydroxyethylcellulose, polyvinyl alcohol,
polysaccharides, sodium alginate, polyvinylpyrrolidone, modified
starch, polyethylene glycol, polyethylene oxide and gelatin.
[0018] The term "plasticizer" relates to a compound capable of
endowing the membrane with flexibility. A plasticizer used in
accordance with the present invention should be non-toxic to cells.
In some specific embodiments said plasticizer is nontoxic to stem
cells.
[0019] In some embodiments, said at least one plasticizer is
selected from the group consisting of polyethylene glycol,
polyethylene oxide, triethyl citrate, acetyl triethyl citrate,
tributyl citrate, acetyl tributyl citrate, triacetin, dibutyl
sebacate, diethyl phthalate, propylene glycol, methoxyethylene
glycol and gelatin.
[0020] In some embodiments, the membrane of the invention comprises
at least one plasticizer that is water soluble. Non-limiting list
of water soluble plasticizers includes: PEG, triethyl citrate,
acetyl triethyl citrate, tributyl citrate, acetyl tributyl citrate,
triacetin, dibutyl sebacate, diethyl phthalate, propylene glycol,
methoxyethylene glycol and gelatin.
[0021] In some embodiments said at least one plasticizer and at
least one hydrophilic polymer are compatible with stem cells.
[0022] As will be demonstrated in the examples below PEG was shown
to be non toxic to stem cells. Therefore, in a specific embodiment
wherein the membrane includes stem cells the preferred plasticizer
and/or hydrophilic polymer is PEG.
[0023] The term "polyethylene glycol (PEG)" refers to polyethylene
glycol polymers that dissolve in aqueous media. In some embodiments
PEG in the range of 300-20,000 is used in a membrane of the
invention. In other embodiments PEG 400 is used in a membrane of
the invention.
[0024] In some embodiments said hydrophilic polymer is polyethylene
glycol ranging from 300-20,000. In other embodiments said
plasticizer is polyethylene glycol ranging from 300-20,000. In yet
further embodiments both said hydrophilic polymer and said
plasticizer are polyethylene glycol ranging from 300-20,000.
[0025] Without wishing to be bound by theory, in such cases PEG
appears to carry out a dual function in the membrane; it
contributes flexibility to the membrane and it causes pore creation
upon dissolution. As demonstrated in the Examples below, porosity
(number of pores in the membrane) increases according to PEG
concentrations within the membrane. Furthermore, an increased
porosity correlates with increased cell adhesion.
[0026] In other embodiments of the invention, said at least one
hydrophilic polymer is present in a concentration of between about
0.5% weight/weight (w/w) to 30% (w/w) of the membrane. In other
embodiments, said at least one hydrophilic polymer is present in a
concentration of between about 10% (w/w) to about 25% (w/w) of the
membrane.
[0027] In a specific embodiment, said at least one hydrophilic
polymer is selected from the group consisting of
hydroxylpropylmethylcellulose, hydroxylpropylcellulose,
carboxymethylcellulose, hydroxyethylcellulose, being in a
concentration of between about 0.5% (w/w) to 20% (w/w) of the
membrane.
[0028] In another specific embodiment, said at least one
hydrophilic polymer is PEG, being in a concentration of between
about 1% (w/w) to 30% (w/w), or between about 10% (w/w) to about
25% (w/w), or about 15% (w/w) of the membrane.
[0029] In a further embodiment a membrane of the invention further
comprises at least one type of cell. In some embodiments, said
cells are selected from the group consisting of adult stem cells,
embryonic stem cells, pluripotent stem cells, mesenchymal stem
cells, umbilical cord blood cells, osteoblasts, chondroblasts and
CD105+ cells. In other embodiments, said stem cells are autologous
adult stem cells.
[0030] Pluripotent mesenchymal stem cells have the capacity to
undergo commitment to several cell lineages, including osteoblasts,
adipocytes, chondrocytes, and myocytes.
[0031] In one embodiment a membrane of the invention comprises
AMCA, polyethylene glycol ranging from 300-20,000 (in an amount
sufficient to be both a plasticizer and a hydrophilic polymer
capable of forming pores upon contact with aqueous fluids in situ),
and adult stem cells.
[0032] As used herein the term "cell adhesion" or "cell adherence"
refers to the binding/attachment of a cell to a surface while
maintaining viability.
[0033] As used herein the term "cell proliferation" or "cell
growth" refers to reproduction and increase in cell number, i.e.
cell division.
[0034] As used herein the term "cell differentiation" refers to a
process by which a less specialized cell becomes a more specialized
cell type. For example, adult stem cells divide and create
fully-differentiated daughter cells during tissue repair and during
normal cell turnover, e.g. mesenchymal stem cells may differentiate
into osteoblasts.
[0035] The membranes of the invention are capable of supporting
cell adherence, cell proliferation and/or cell differentiation.
[0036] Membranes according to the present invention exhibit
qualities such as the ability to (i) develop direct adhesion and
bonding with existing tissue; (ii) promote cellular function; (iii)
provide a scaffold or template for the formation of new tissue; and
(iv) promote tissue regeneration and act as a carrier for bioactive
materials.
[0037] In some embodiments, a membrane of the invention further
comprises at least one active agent capable of promoting tissue
reproduction and/or deliver pharmaceutical benefits needed in the
site of implantation of said membrane.
[0038] In other embodiments, a membrane of the invention further
comprises at least one active agent, wherein said at least one
active agent is selected from the group consisting of cytokine,
hormone, bisphosphonate, cannabinoid, beta blocker, bone inducing
agent, growth factor, HMG-CoA reductase inhibitor (such as
statins), drug and antibiotic, and any combinations thereof.
[0039] In some embodiments said active agent is selected from the
group consisting of statin, estrogen, androgen, propranolol,
transforming growth factor (TGF), bone morphogenetic protein (BMP,
such as for example BMP-2 and BMP-7), insulin like growth hormone,
fibroblast growth factor (FGF), alendronate, risendronate and
parathyroid hormone. In yet further embodiments, said active agent
is simvastatin or lovastatin.
[0040] Simvastatin is a member of the statin family of
3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, which
are widely used as cholesterol-lowering drugs. Statins may increase
bone mass by anabolic and anticatabolic (antiresorptive)
mechanisms.
[0041] In some embodiments said agent is in a controlled release
formulation.
[0042] In another specific embodiment, the membrane comprises AMCA,
PEG and at least one active agent (e.g. simvastatin).
[0043] In some embodiments a membrane of the invention is capable
of being porous upon hydration (for example hydration achieved upon
contact with bodily fluids in the implantation site), and wherein
the pore size is between about 0.1 to about 5 microns.
[0044] In some embodiments a membrane of the invention may have at
least one pore due to hydration of said water soluble plasticizer
in a size of less than 5 microns. In other embodiments said pore
size is less than 2 microns. In yet other embodiments said pore
size is between about 0.1 to about 5 microns.
[0045] As used herein the term "hydration" refers to exposure of
the membrane of the invention to aqueous solution or a body fluid,
e.g. interstitial fluid, blood, plasma, saliva, which results in
dissolution of the water soluble plasticizer.
[0046] In another aspect, the invention provides a use of a
membrane of the invention, for the preparation of a three
dimensional hollow implant for tissue regeneration of a defected
tissue region.
[0047] Due to the flexibility of a membrane of the invention it is
possible to form a three dimensional implant using said membrane,
without the need to pre-casting or re-molding the membrane or
exposing it to heat. The three dimensional structure constructed
from said membrane may be any type of structure suitable for the
site of implantation, and may further be adjusted at the site of
implantation in accordance with the area wherein said implant is to
be used.
[0048] In some embodiments, said three dimensional hollow implant
is selected from the group consisting of a tubular implant, a
cylindrical implant, a conical implant or a planar implant.
[0049] In a further embodiment, said tissue to be regenerated upon
use of a membrane of the invention or a three dimensional hollow
implant made thereof, is selected from ligament, tendon, cartilage,
intervertebral disc, dental tissue (including teeth, enamel,
dentin, cementum, pulp, periodontal ligaments, alveolar bone,
gingiva tissue) and bone.
[0050] In a further aspect the invention provides a three
dimensional hollow implant comprising at least one membrane of the
invention. In one embodiment, a membrane of the invention comprised
within a three dimensional hollow implant of the invention defines
the surface of the implant.
[0051] In another aspect the invention provides a cell delivery
system comprising a membrane of the invention.
[0052] In yet a further aspect the invention provides a cell
growing surface comprising a membrane of the invention.
[0053] In another one of its aspects the invention provides a
scaffold substantially coated by a membrane of the invention.
[0054] The invention also provides granular material substantially
coated by a membrane of the invention. In certain embodiments, the
granular material is composed of bone compatible ceramics e.g.
calcium-based minerals.
[0055] The invention further provides a method for promoting tissue
regeneration in a defected tissue region comprising the steps of:
providing a membrane of the invention; and implanting said membrane
in the proximity of said tissue defect region. In some embodiments
said defected tissue results from a condition selected from
non-union fracture, osteoporosis, periodontal disease or condition,
osteolytic bone disease, post-plastic surgery, post-orthopedic
implantation, post neurosurgical surgery, alveolar bone
augmentation procedures, spine fusion, vertebral fractures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] In order to understand the invention and to see how it may
be carried out in practice, embodiments will now be described, by
way of non-limiting example only, with reference to the
accompanying drawings, in which:
[0057] FIGS. 1A-1F shows micrographs of hMSC adherence on
polystyrene tissue culture dish and on AMCA membrane. CFSE-labeled
hMSC were seeded either on polystyrene tissue culture dish (A-C) or
on membrane (D-F). After 24 h CFSE-labeled cells were visualized by
confocal microscopy. Three representative images for each culturing
condition are shown.
[0058] FIGS. 2A-2F shows scanning electron micrographs of the hMSC
adherence on AMCA/PEG membranes. hMSC were seeded on AMCA membrane
with PEG 400. After 24 h in culture cells were fixed as described
in Materials and Methods and analysis was performed using scanning
electron microscopy (SEM). Magnifications of images from left to
right are: upper panel--.times.200 (FIG. 2A), .times.1000 (FIG.
2B), .times.2000 (FIG. 2C); .times.4000 (FIG. 2D), .times.5000
(FIG. 2E), .times.6000 (FIG. 2F).
[0059] FIGS. 3A-3B shows flow cytometric analysis of CFSE labeled
hMSC. 1.times.10.sup.5 CF SE-loaded hMSC were cultured on either
AMCA membrane with 15% PEG 400 (FIG. 3A) or polystyrene tissue
culture dish (FIG. 3B) for 24 h, 48 h, 96 h and 144 h. At the
indicated time points cells were harvested and flow cytometric
analysis was performed. Loss of CFSE reflects cellular
division.
[0060] FIG. 4 is a graph showing hMSC proliferation on AMCA
membranes prepared with various PEG 400 concentrations. CFSE-loaded
hMSC were cultured and analyzed as in FIG. 3. Data are presented as
Mean Fluorescent Intensity (MFI) of CFSE over time for AMCA
membranes containing different concentrations of PEG 400 vs
polystyrene tissue culture dish (control). Decrease in MFI
represent rate of MSC proliferation over time.
[0061] FIGS. 5A-5C show scanning electron micrographs of the AMCA
membranes: Membranes were prepared using solvent casting technique.
The membranes were then analyzed using SEM or immersed in
phosphate-buffered saline (PBS) for 24 h and then analyzed by SEM.
(FIG. 5A): 15% PEG 400 membrane before immersion in PBS--showing no
porosity; (FIG. 5B): membrane with 5% PEG after immersion in
PBS--slightly porous; (FIG. 5C): Membrane with 15% PEG after
immersion in PBS--porous. Magnification: .times.5000.
[0062] FIGS. 6A-6B shows hMSC differentiation on AMCA membranes.
1.times.10.sup.5 hMSC were cultured on either membrane or
polystyrene tissue culture dish. After 3 days when cells reached
confluency differentiation medium containing culture medium with
ascorbic acid (50 .mu.gimp, dexamethasone (10.sup.-8 M) and
.beta.-glycerophosphate (10 mM) was added and the cells were fed
with fresh differentiation medium twice a week. On the 17th day of
culture cells were fixed with 70% ethanol and alizarin red staining
was performed. (FIG. 6A): Control polystyrene dish; (FIG. 6B): AMCA
membrane with 15% PEG 400.
[0063] FIG. 7 is a graph showing bone regeneration by mean callus
area. An increased mean callus area was measured in bones implanted
with AMCA membranes as compared to untreated controls.
[0064] FIGS. 8A-8B is a graph showing quantitative analysis of
relative callus density changes over time (FIG. 8A) and callus area
changes over time (FIG. 8B) observed for ethyl cellulose membrane
and ethyl cellulose membrane controllably releasing simvastatine in
6 male New Zealand rabbits for which critical size defect (10 mm)
was created in both forelimbs. Forelimb was inserted with Ethyl
Cellulose (EC) membrane which contained simvastatin; Contralateral
limb was inserted with EC membrane. Calibration was done using
Osirix software.
[0065] FIG. 9 shows a microCT of bone regeneration with EC
membrane. In this experiment bone defect was left untreated. The
bone defect was in a non union state. Arrows mark the bone defect
area.
[0066] FIG. 10 shows microCT (computed tomography) of bone
regeneration with EC membrane containing simvastatin. In this
experiment bone defect was treated and successful bridging of the
defect was shown. Arrows mark the bone defect area.
[0067] FIGS. 11A-11B is a graph showing mean callus area growth for
AMCA membrane of the invention, with and without addition of
controlled release simvastatine. A--Shows results obtained 4 weeks
after implantation of the AMCA membrane+simvastatin. B--Shows
results obtained 8 weeks after implantation of the AMCA
membrane+simvastatin
[0068] FIGS. 12A-12D is a graph showing various parameter effects
on simvastatine release from membrane of the invention (measured in
vitro): effect of simvastatin concentration on simvastatin release
rate (FIG. 12A); effect of membrane width on simvastatin release
rate (FIG. 12B); effect of plasticizer on simvastatin release rate
(FIG. 12C); effect of plasticizer type on simvastatine release rate
(FIG. 12D).
[0069] FIGS. 13A-13B is a graph showing bone regeneration
parameters achieved with AMCA membrane carrying hMSC in rabbit
critical size defect model. FIG. 13A shows callus area growth. FIG.
13B shows a histological evaluation of defects after 8 weeks from
implantation of membrane.
DETAILED DESCRIPTION OF EMBODIMENTS
[0070] The present invention provides a membrane composed of at
least three elements, the first being a synthetic, hydrophobic
polymer having a positive surface charge, which is
non-biodegradable under physiologic conditions, at least one
hydrophilic polymer which is biodegradable under physiological
conditions and at least one plasticizer. Without wishing to be
bound by theory, the combination of these elements generates a
membrane which is flexible enough to be able to generate three
dimensional structures suitable for various therapeutic
applications, for example, a hollow tube. Moreover, upon exposure
to fluid (in vivo or ex vivo), the hydrophilic polymer at least
partially disintegrates and the membrane becomes porous, thus
enabling the adhesion of cells.
[0071] Cells may be seeded on a membrane of the invention as will
be further described below in detail.
[0072] In further embodiments, a membrane of the invention may
further include an active agent, as further detailed below.
[0073] A membrane of the invention can serve as an infrastructure
to allow guided tissue repair as well as a cell delivery system. A
membrane of the invention may also serve as a barrier membrane for
eliminating infiltration of unwanted cells, blood vessels and
soft/scar tissue into the treated area, and for isolating the cells
delivered in said membrane from the surrounding tissue, and
preventing the leakage of cells and factors from the space inside
the membrane to the surrounding tissue.
[0074] A membrane of the invention may be used as such, for
example, by covering a region into which the cells are delivered,
however, in certain embodiments it may be used to form a three
dimensional device (for example a hollow tubular device) which
holds the cells to be deliver, or may coat a tissue engineering
scaffold containing the cells to be delivered.
[0075] In some embodiments, a membrane of the invention is folded
into a desired three dimensional structure, e.g. a tubular device.
The tubular device can be used as an infrastructure to allow guided
tissue repair as well as to deliver cells into a tubular region of
defect, such as a bone defect, and the membrane can be used to hold
the delivered cells and components in the device and prevent
infiltration of cells, extracellular matrix and blood vessels from
the surrounding tissue into the space surrounded by the device.
[0076] In other embodiments, a membrane of the invention is used
for coating a tissue engineering scaffold. Such a membrane-coated
scaffold can hold cells to be delivered into a site in the body.
The membrane coating isolates the cells delivered in the scaffold
from the surrounding tissue and prevents the leakage of cells, and
soluble factors from the space inside the scaffold into the
surrounding tissue. Coating of the scaffold with a membrane of the
invention may allow better cell adhesion and higher doses of cells
to be delivered to the target site.
[0077] In yet other embodiments, a bone defected area can be
wrapped after implantation of a scaffold with a membrane of the
invention in order to prevent leakage of cells and soluble factors
and to prevent growth of soft tissue into the scaffold.
[0078] The term "cell delivery" refers to introduction of cells
into a desired site in the body of an individual for therapeutic
purposes.
[0079] A membrane of the invention is suitable for seeding of any
type of cells for example stem cells (both adult and embryonic stem
cells). In other embodiments cell type may be selected from the
following non-limiting list: mesenchymal (stromal) stem cells,
umbilical cord blood cells, osteoblasts, chondroblasts, or CD105+
cells. The invention also encompasses seeding of pluripotent stem
cells of embryonic origin as well as adult cells that have been
reprogrammed to become pluripotent. The cells may be autologous,
allogenic or xenogenic.
[0080] In some embodiments, the cells are autologous adult stem
cells, obtained, for example, from bone marrow or adipose
tissue.
[0081] Cell seeding is performed in some embodiments ex vivo. The
cells may be placed on the membranes (for example formed as a
hollow tubular device) or placed in a tissue engineering matrix
(also termed herein "scaffold") coated by the membrane of the
invention. Examples of tissue engineering matrix are those
fabricated from either biological materials or synthetic
polymers.
[0082] In certain embodiments, a membrane of the invention, a
tubular implant of the invention or a coated scaffold of the
invention, with or without ex vivo seeded cells are placed at a
desired location in the body. This location is typically a location
where it is desired to generate new tissue which has been damaged
by trauma, surgical interventions, genetic or disease
processes.
[0083] In some embodiments a desired site is a site where tissue
should be generated from adult stem cells; is some embodiments such
a site is ligament, tendon, cartilage, intervertebral disc, dental
tissue or bone tissue, most preferably bone tissue.
[0084] Generation of bone tissue is required in conditions such as
non-union fractures, osteoporosis, periodontal disease or teeth
implantation, osteolytic bone disease, post-plastic surgery,
post-orthopedic implantation, post neurosurgical surgery that
involves calvaria bone removal, in alveolar bone augmentation
procedures, for spine fusion and in vertebral fractures.
[0085] Generation of tendon/ligament tissue is required for example
following tissue tear due to trauma or inflammatory conditions.
[0086] Generation of cartilage tissue is required in conditions
such as Rheumatoid Arthritis, Osteoarthritis, trauma, cancer
surgery or cosmetic surgery.
[0087] Generation of intervertebral disc tissues including nucleous
pulposus and annulus fibrosus, is required in conditions such as
nucleons pulposus degeneration, annulus fibrosus tears, or
following nucleotomy or discectomy.
[0088] Typically the membrane, for example in the form of a hollow
tube is placed at the desired site by implantation.
[0089] In certain embodiments, the membrane of the invention
comprises a synthetic, hydrophobic positively charged polymer, a
hydrophilic polymer, a plasticizer and an active agent and is
further seeded with cells.
[0090] In a specific embodiment the membrane of the invention
comprises a synthetic, hydrophobic positively charged polymer and
PEG and is further seeded with stem cells.
[0091] As used herein the term "cell-growing surface" refers to any
artificial surface suitable for cell growth for example a slide,
vessel or cell/tissue culture dish. The membrane coated cell
growing surface in accordance with the invention thereby gains
properties suitable for cell adhesion, proliferation and/or
differentiation.
[0092] The present invention provides a flexible membrane capable
of supporting MSC adherence, proliferation and differentiation.
Such a membrane can be used as treatment for bone regeneration
applications. The healing of displaced fractures and regeneration
of bone defects does not result only from proliferation of the
locally present osteoblasts, but involves recruitment,
proliferation, and differentiation of preosteoblastic cells. The
differentiation of multipotent osteoblastic precursors is the main
initial event in bone healing and callus formation, although
preexisting osteoblasts might also be involved. Any failure in the
recruitment, establishment, proliferation, and differentiation of
these progenitor cells can lead to delayed union or nonunion. There
are many difficulties related to the healing of critical-size bone
defects. In general, these difficulties result from the fact that
there is an insufficient number and/or activity of osteogenic cells
of the host to allow for healing.
[0093] A membrane of the invention can guide bone regeneration as
well as prevent unwanted vascularization in the newly formed bone.
The membrane can also protect the area of bone defect from
infiltration by connective and scar tissues, guide the osteogenic
cells and allow storage of osteogenic components in the space
enclosed by the membrane, which may potentially be released from
the bone ends and bone marrow [10, 12]. Furthermore, placing MSC
attached to a membrane at the site of critical size defect model
will provide starting material for a new bone tissue. Therefore,
implanting GBR membrane with expanded ex vivo MSC may greatly
improve the bone repair outcome.
[0094] As demonstrated in the Examples provided below, several
polymers were tested in conjugation with various plasticizers.
[0095] In one embodiment, a membrane constituted from AMCA and 15%
PEG 400 could support good MSC adhesion, proliferation and
differentiation: (I) MSC adhered to AMCA membrane with 15% PEG 400
as determined by light microscopy, fluorescent microscopy and SEM.
(II) MSC maintain their proliferative activity as determined by
CFSE labeling and flow cytometric analysis (III) MSC maintained
their differentiation ability as determined by Alizarin Red
staining.
[0096] AMCA membrane containing 15% PEG 400 supported MSC
differentiation to osteoblasts.
Materials and Methods
[0097] Polymers:
[0098] Ammonio Methacrylate Copolymer type A NF (AMCA,
EUDRAGIT.RTM. RL, Degussa, Germany) and Ethyl Cellulose (EC,
ETHOCEL.RTM. N 100, Hercules Inc., Wilmington, Del.).
[0099] Plasticizers:
[0100] Polyethylenglycol 400 (PEG 400, Merck, Germany), Glyceryl
triacetate (Triacetin, Fluka, Rehovot, Israel), Glycerin (Frutarom,
Israel), Triethyl Citrate (Fluka, Rehovot, Israel), Dibutyl
Sebacate (Fluka, Rehovot, Israel), Dibutyl Phtalate (Fluka,
Rehovot, Israel).
[0101] Polymeric membranes preparation and sterilization--
[0102] Membranes were prepared using solvent casting technique as
disclosed in Friedman M. and Golomb G. J. [13]. Polymeric membranes
were cast from solution consisting of polymer, plasticizer and
Ethanol (Frutarom, Israel) into the TEFLON.RTM. moulds (round
plates, inner diameter 9.6 cm) and the solvent was allowed to
evaporate over night. Membranes width was: 100.+-.5 .mu.M.
[0103] Prior to use in tissue culture, membranes were immersed in
PBS (Biological Industries, Beit Haemek, Israel) for 24 hours to
wash out possible remains of ethanol and then sterilized by UV
irradiation for 2 hr.
[0104] Characterization of Membranes--Scanning Electron Microscopy
(SEM) Photomicrographs--
[0105] AMCA membranes containing 15% PEG 400 were fixed with 2%
glutaraldehyde in cocodylate buffer (0.1 M; pH=7.2) for 2 hours.
The specimens were then processed according to the air drying
method skipping the ethanol dehydration series (Ethanol dissolves
AMCA; therefore it should be excluded from the specimen
preparation). The process was accomplished through 100% Freon 113.
The specimens were vigorously shaken, which allowed rapid
evaporation of the Freon phase. The membranes were mounted in
copper stubs, coated with gold and then examined in FEI quanta 200
at an accelerating voltage of 30 KV.
[0106] Cell Harvesting and Culture--
[0107] hMSCs were obtained from discarded bone tissues from
patients undergoing total hip replacement surgeries, under approval
of Hadassah Medical Center Helsinki Ethics Committee following an
informed consent.
[0108] The hMSCs were separated from other bone marrow-residing
cells by plastic adherence and were then grown under tissue culture
conditions, as described in Krampera M. et al. [14], and Djouad
Fetal [15]. The cells were maintained in a lowglucose Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% heatinactivated
fetal calf serum, 2 mM glutamine, and penicillin/streptomycin
(Biological Industries, Beit-Haemek, Israel). Primary cultures were
usually maintained for 12-16 days, and were then detached by
trypsinization and subcultured (Barry F P. et al. [16]). The medium
was changed every 3-4 days.
[0109] MSC Adhesion to Membrane--
[0110] For MSC labeling, MSC were re-suspended in PBS (10.sup.7
cells/ml) containing 5-carboxyfluorescein diacetoxymethyl ester
(BCECF/AM or CFSE; 5 .mu.g/mL; Calbiochem), incubated at 37.degree.
C. for 10 min, and the cells were then washed three times. Cells
were cultured on a sterilized membrane wetted with PBS,
15.times.10.sup.4 cells in 150 .mu.l medium, and incubated for six
hours at 37.degree. C. Afterwards 3 ml of medium were added. Cells
were examined 24 hours after seeding by fluorescent microscope.
Tissue culture polystyrene dishes were used as a positive control
for membrane in cell adhesion test.
[0111] CFSE-Based Proliferation Assay--
[0112] For cell division studies, MSC were resuspended in PBS
(10.sup.7 cells/nil) containing
3'-O-Acetyl-2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein
diacetoxymethyl ester (BCECF/AM or CFSE; 5 .mu.g/mL; Calbiochem),
incubated at 37.degree. C. for 10 min, and washed three times.
CFSE-labeled cells were then seeded on the membrane or on the
tissue culture dishes as described above. At the indicated time
points cells were harvested and proliferation of cells was
visualized by incremental loss of CFSE fluorescence as analyzed on
a FACSCalibure flow cytometer (Becton Dickinson) using Cell Quest
software.
[0113] MSC Differentiation on Membrane--
[0114] MSC were seeded on the membranes or on the center well organ
culture dishes (Falcon) for control, as described above. As soon as
MSC were confluent, the culture medium was supplemented with
ascorbic acid (50 .mu.gimp, dexamethasone (10.sup.-8 M) and
.beta.-glycerophosphate (10 mM). Medium was changed twice a week
for 17 days, afterwards membranes and dishes were dyed with
Alizarin Red, as described below.
[0115] Alizarin Red Staining--
[0116] A stock solution of 2% alizarin red in distilled water was
adjusted to pH 4.2 with NaOH and passed through a 0.22 .mu.m
filter. Cultures in the center well organ culture dishes were
rinsed with 150 mM NaCl three times, fixed in ice cold 70% ethanol,
rinsed with distilled water and stained at room temperature for 10
min with 500 .mu.l of alizarin red stock per well. Individual wells
were rinsed five times with distilled water; a sixth and final wash
with distilled water was performed for 15 min (Halvorsen Y D. et
al. [17]). Membranes due to their positive charge had a higher
affinity towards alizarin red stain than a negatively charged
center well organ culture dishes, therefore rinsing with distilled
water didn't remove the stain from the membranes well enough. To
reduce background we applied a single rinse with 0.02 M HCl on the
membranes. Photomicrographs were then obtained.
Example 1--Cell Adhesion Using a Membrane of the Invention
[0117] Various membranes were tested for their ability to support
cell attachment and growth. The tested membranes varied in their
polymer and plasticizer types. Several plasticizers were tested,
i.e. glycerin, polyethylene glycol, triethyl citrate, dibutyl
sebacate, dibutyl phtalate, triacetin. The plasticizers tested were
hydrophobic or hydrophilic and were added in order to contribute
flexibility to membrane. MSC were seeded on sterilized membranes as
described hereinabove.
[0118] EC Membranes:
[0119] MSC cells showed little adherence to all formulations of EC
membranes and cell aggregation was slight. The various plasticizers
had no influence on either cell adhesion or cell shape. As control,
poly-1-lysine coated membranes were used. Poly-1-lysine, a highly
positively charged amino acid chain, is commonly used as a coating
agent to promote cell adhesion in culture. Cells adhered in
monolayer spindle shape to EC membranes coated with poly-1-lysine,
hence it was concluded that EC does not support cell adhesion, as
such. However EC was found to be non toxic in the presence of
poly-1-lysin.
[0120] AMCA Membranes:
[0121] Cell adhesion test was performed with Ammonio Methacrylate
Copolymer type A (AMCA, EUDRAGIT.RTM. RL, Degussa, Germany) [85%],
mixed with various plasticizers disclosed herein above [15%].
[0122] MSC adhered well to AMCA membranes prepared with the various
plasticizers (FIG. 1D-F) in spindle monolayer shape. Cell spreading
on the AMCA membranes was similar to spreading on the polystyrene
dishes which were used as a positive control for cell adhesion
(FIG. 1 A-C). The mode of spreading is indicative of the cells'
well being.
[0123] Cell adhesion was further analyzed using SEM. As shown in
FIG. 2 cells on the AMCA membrane, were flat and monolayer spindle
shaped. Furthermore, at higher magnification, cell-membrane
interaction was seen, with a cellular podia attached to the
membrane, (FIG. 2, D-F). Moreover, the release of numerous vacuoles
from the cell surface was observed, demonstrating cell
functionality. Similar results were obtained using both human as
well as rabbit MSC.
Example 2--Cell Proliferation Using a Membrane of the Invention
[0124] Proliferative capacity of MSC was tested using the
fluorescent marker of cell division, CFSE and flow cytometric
analysis. This method is based on the fluorescein related dye CFSE,
which is partitioned with remarkable fidelity between daughter
cells allowing eight to 10 discrete generations to be identified
both in vitro and in vivo. The technique allows complex information
on proliferation kinetics and differentiation to be collected
According to this technology; individual cells are tagged with the
fluorescent CFSE dye that binds irreversibly to cell cytoplasm. As
cells divide, their fluorescence halves sequentially with each
generation, allowing the proliferative history of any single cell
present to be monitored over time (see Lyons A B. Et al [18]).
[0125] MSC proliferated on AMCA and PEG 400 membrane (FIG. 3B) (but
no proliferation was detected with other plasticizers; data not
shown) although at somewhat reduced rates as compared to their
proliferative capacity on tissue culture dishes used as control
(FIG. 3A).
[0126] Subsequently, MSC proliferation rate was tested over time on
membranes containing different concentrations of PEG 400 (10%, 15%,
20% and 25% w/w). The rate of MSC proliferation inversely
correlated to the mean fluorescent intensity value (MFI) (FIG. 4).
This analysis revealed that, membranes containing 15% PEG 400 and
20% PEG 400 were fairly close to the polystyrene control, while
other concentrations of PEG resulted in either higher or lower
proliferation rates.
[0127] In addition, AMCA membrane with 15% and with 5% PEG 400 was
characterized using scanning electron microscopy (SEM). Membranes
were observed before and after immersion in PBS (FIG. 5). It is
noted that membranes were immersed in PBS for 24 hours before each
MSC seeding, in order to wash out residual ethanol. Since PEG 400
is soluble in water and thus porogenic, only after immersion in
PBS, pores were observed on the membrane surface (FIG. 5B-C). In
both concentrations of PEG 400, SEM pictures demonstrated a porous
surface, with average pore size of 0.18 .mu.m. Pore distribution
correlated directly to different PEG 400 concentrations.
Example 3--Cell Differentiation Using a Membrane of the
Invention
[0128] Differentiation medium was added as described hereinabove.
Membranes and dishes were then dyed with Alizarin Red. (FIGS. 6A
and 6B). Alizarin red binds irreversibly to bivalent positive ions
and has especially high affinity towards calcium. Calcium is
secreted from osteoblasts and deposits on the membrane as part of
the creation of an extracellular matrix. Therefore presence of
calcium marks the differentiation from MSC that do not secrete
calcium into osteoblast. FIG. 6 demonstrates that MSC cultured on
both AMCA membrane and polystyrene controls have differentiated to
osteoblast and produced extracellular matrix. This finding confirms
that AMCA membrane with 15% PEG 400 supports MSC differentiation
and that MSC after adhesion to membrane maintain their stem cells
traits.
Example 4--In Vivo Bone Regeneration Study Using a Membrane of the
Invention
[0129] Study Group:
[0130] Five male New Zealand rabbits weighing 3.8-4.4 kg underwent
bilateral midshaft resection of radial bone segment (1 cm in
length) in forelimbs. Tubular AMCA membranes were implanted in the
left forelimb (treated osteotomy) and the right limb served as a
control (untreated osteotomy).
[0131] Evaluation of healing process: radiographic
evaluation--lateral radiographs of forelimbs were obtained 2, 4, 6
and 8 weeks postoperatively. To obtain standardized measurements of
the bone defects during the regenerative healing process, true
lateral radiographs of both forelimbs were performed in standard
conditions (42 kV, 2 mas). Radiographs were examined using OsiriX
medical imaging software to evaluate the area and density of the
new bone.
[0132] Measured Parameters: [0133] Total area of regenerated bone
tissue (appearing around and within the bone gap defect). To
eliminate possible bias by variability of bone dimensions, data
calibration was made using the diameter of olecranon process at its
narrowest zone as a standard reference. This diameter was defined
as 10 mm in each specimen. [0134] Relative density of the newly
regenerated bone in the gap defect. The segmented area was
outlined, and the density was measured. The bone density in the
center of the olecranon process was measured in each forelimb for a
calibration, as a reference value. The density of olecranon process
was defined as a 100% for each specimen (see Mosheiff R. et al.
[10]).
[0135] Results:
[0136] FIG. 7 shows bone regeneration expressed by mean callus area
(mm.sup.2) throughout the study (weeks 2 to 8). At week 2 of the
study the mean callus area produced in control arm was larger then
that of arm treated with AMCA membrane, possibly due to formation
of hematome or blood clot at the surgery site. When the site was
surrounded by membrane it isolated the area and thus slowed the
degradation of the hematome. However from week four of the study,
mean area of callus generated in the limb treated with AMCA
membrane was slightly bigger than that of the control (144.8
mm.sup.2 vs. 114.5 mm.sup.2). This trend continued at weeks 6 and
8, hand in hand with widening the difference between mean callus
areas of AMCA membrane treated limb and control limb. At week 8,
the difference between mean callus areas produced in two limbs
(treated with AMCA membrane and control) reached its peak and was
143.91 mm.sup.2 (see Table 1 below). However, this difference is
not statistically significant, due to small sample size (n=5) of
this preliminary study and high variability of results, as it often
happens in in vivo studies.
TABLE-US-00001 TABLE 1 Radiographic parameters of the study at week
8 (end point) mean control AMCA membrane sig Difference mean mean
Measured parameter (2-tailed) (mm.sup.2) std (mm.sup.2) std
(mm.sup.2) surface area of callus 0.08 143.91 85.633 129.74 277.203
273.65 relative density of callus 0.68 20.54 39.8 123.33 101.95
143.87 relative density of prox 0.89 31.96 38.35 126.12 107.43
158.08 quad relative density of prox 0.68 55.84 51.93 96.44 126.81
152.28 med quad relative density of distal 0.34 57.89 37.7 106.28
114.28 164.17 med quad relative density of distal 0.5 40.41 41.99
110.07 88.78 150.48 quad
Example 5--In Vivo Bone Regeneration Study Using an EC Membrane of
the Invention Further Comprising Simvastatin
[0137] In 6 male New Zealand rabbits critical size defect (10 mm)
was created in both forelimbs. In one forelimb EC membrane which
contained simvastatin was inserted, in the contralateral limb EC
membrane with no active agent was inserted. Callus density and
Callus area were measured and calibrated using Osirix software.
FIGS. 8A and 8B show the quantitative analysis of the
radiographs.
[0138] FIG. 9 shows the microCT of bone regeneration with EC
membrane. In this experiment bone defect was left untreated. The
bone defect is in non union state. Arrows mark the bone defect
area.
[0139] FIG. 10 shows microCT of bone regeneration with EC membrane
containing simvastatin. Arrows mark the bone defect area.
[0140] In this experiment bone defect was treated and successful
bridging of the defect is evident.
Example 6--In Vivo Bone Regeneration Study Using an AMCA Membrane
of the Invention Further Comprising Simvastatin
[0141] Rabbit model: critical size bone defect of 1 cm in radius
bone were created. 5 rabbits were treated with simvastatin
controlled release AMCA membrane and 5 others with AMCA membrane
without any active ingredient.
Membranes:
[0142] AMCA membrane comprising simvastatine: [0143] Simvastatin
20% w/w-0.36 g [0144] AMCA (EUDRAGIT.RTM. RL) 70% w/w-1.26 g [0145]
PEG 400 10% w/w-0.18 g [0146] Membrane width was 180 micrometer.
Control AMCA membrane: [0147] AMCA (EUDRAGIT.RTM. RL) 90% w/w-1.62
g [0148] PEG400 10% w/w-0.18 g [0149] Membrane width was 180
micrometer. FIG. 11 shows significantly larger callus area formed
at the defect site treated with simvastatin controlled release AMCA
membrane (Wilcoxon summed ranks test), as well as increase in
callus growth rate at 2 first post operation weeks--may be
important from clinical point of view.
Example 7--In Vitro Release Rate of Simvastatin from Different
Membranes of the Invention
[0150] The effects of various parameters on simvastatine release
from membranes of the invention were measured in vitro as
follows:
[0151] The effect of simvastatin concentration on simvastatin
release rate is shown in FIG. 12A; the composition of the tested
membranes was as follows:
TABLE-US-00002 Membrane components Membrane 1 Membrane 2 Membrane 3
Simvastatin 20% 5% 10% PEG 400 10% 10% 10% EUDRAGIT .RTM. RL 70%
85% 80% width (micron) 90 75 87
[0152] The effect of membrane width on simvastatin release rate is
shown in FIG. 12B; the composition of the tested membranes was as
follows:
TABLE-US-00003 Membrane components Membrane 4 Membrane 5
Simvastatin 20% 20% PEG400 10% 10% EUDRAGIT .RTM. RL 70% 70% width
(micron) 90 220
[0153] The effect of plasticizer on simvastatin release rate is
shown in FIG. 12C; the composition of the tested membranes was as
follows:
TABLE-US-00004 Membrane components Membrane 6 Membrane 7 Membrane 8
Simvastatin 20% 20% 20% PEG400 10% 5% 10%-Klucel HF EUDRAGIT .RTM.
RL 70% 75% 70% width (micron) 90 104 87
[0154] The effect of plasticizer type on simvastatine release rate
is shown in FIG. 12D.
Example 8--Bone Regeneration with AMCA Membrane Comprising hMSC
[0155] Rabbit model: critical size bone defect of 1 cm in radius
bone was created. Two rabbits were treated with AMCA membrane
carrying hMSC in one forearm and on another forearm AMCA membrane
without hMSC.
[0156] Properties of the membrane:
[0157] AMCA (EUDRAGIT.RTM. RL) 85% w/w-1.512 g
[0158] PEG 400 15% w/w-0.266 g
[0159] Membrane width was of 180 micrometer.
[0160] FIG. 13A demonstrates the development of the callus area in
the effected bone. As shown in FIG. 13B the histological score of
various parts of the defected bone area at 8 weeks post operation
is higher in bones implanted with an AMCA membrane carrying
hMSC.
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