U.S. patent application number 15/502633 was filed with the patent office on 2017-08-17 for a method for making a porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal.
This patent application is currently assigned to THE PROVOST, FELLOWS, FOUNDATION SCHOLARS, & THE OTHER MEMBERS IF BOARD, OF THE COLLEGE OF THE HOLY. The applicant listed for this patent is THE PROVOST, FELLOWS, FOUNDATION SCHOLARS, & THE OTHER MEMBERS OF BOARD, OF THE COLLEGE OF THE HOLY. Invention is credited to Henrique ALMEIDA, David BROWE, Conor BUCKLEY, Grainne CUNNIFFE, Pedro DIAZ PEYNO, Rajalakshmanan ESWARAMOORTHY, Daniel John KELLY.
Application Number | 20170232144 15/502633 |
Document ID | / |
Family ID | 51355463 |
Filed Date | 2017-08-17 |
United States Patent
Application |
20170232144 |
Kind Code |
A1 |
KELLY; Daniel John ; et
al. |
August 17, 2017 |
A METHOD FOR MAKING A POROUS SCAFFOLD SUITABLE FOR USE IN REPAIR OF
OSSEOUS, CHONDRAL, OR OSTEOCHONDRAL DEFECTS IN A MAMMAL
Abstract
A method for making a porous devitalised scaffold suitable for
use in repair of osseous, chondral, or osteochondral defects in a
mammal comprises the steps of providing micronized extracellular
matrix (ECM) tissue, mixing the micronized extracellular matrix
with a liquid to provide a slurry, and freeze-drying the slurry to
provide the porous scaffold. A porous scaffold suitable for use in
repair of osseous, chondral, or osteochondral defects in a mammal
and comprising a porous freeze-dried matrix formed from micronised
decellularised extracellular matrix tissue is also described.
Inventors: |
KELLY; Daniel John; (Dublin,
IE) ; CUNNIFFE; Grainne; (Dublin, IE) ;
ALMEIDA; Henrique; (Dublin, IE) ; ESWARAMOORTHY;
Rajalakshmanan; (Dublin, IE) ; BUCKLEY; Conor;
(Dublin, IE) ; DIAZ PEYNO; Pedro; (Dublin, IE)
; BROWE; David; (Dublin, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE PROVOST, FELLOWS, FOUNDATION SCHOLARS, & THE OTHER MEMBERS
OF BOARD, OF THE COLLEGE OF THE HOLY |
Dublin |
|
IE |
|
|
Assignee: |
THE PROVOST, FELLOWS, FOUNDATION
SCHOLARS, & THE OTHER MEMBERS IF BOARD, OF THE COLLEGE OF THE
HOLY
Dublin
IE
|
Family ID: |
51355463 |
Appl. No.: |
15/502633 |
Filed: |
August 17, 2015 |
PCT Filed: |
August 17, 2015 |
PCT NO: |
PCT/EP2015/068855 |
371 Date: |
February 8, 2017 |
Current U.S.
Class: |
424/423 |
Current CPC
Class: |
A61L 27/365 20130101;
A61L 27/3852 20130101; A61L 27/54 20130101; A61L 27/3612 20130101;
A61L 27/3817 20130101; A61L 27/3821 20130101; A61L 27/3847
20130101; A61L 2430/40 20130101; A61L 27/56 20130101; A61L 2430/02
20130101; A61L 27/3683 20130101; A61L 2300/414 20130101; A61L
2430/06 20130101; A61L 27/44 20130101; A61L 2300/64 20130101; A61L
27/3633 20130101; A61L 27/52 20130101; A61L 27/3654 20130101; A61L
27/3695 20130101; A61L 27/3691 20130101 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61L 27/56 20060101 A61L027/56; A61L 27/38 20060101
A61L027/38; A61L 27/54 20060101 A61L027/54 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 15, 2014 |
EP |
14181154.7 |
Claims
1.-65. (canceled)
66. A method for making a porous scaffold suitable for use in
repair of osseous, chondral, or osteochondral defects in a mammal,
the method comprising the step of: providing micronized
extracellular matrix (ECM) tissue; mixing the micronized
extracellular matrix with a liquid to provide a slurry; and
freeze-drying the slurry to provide the porous scaffold, wherein
the extracellular matrix is treated to reduce the GAG content to
less than 90% of the GAG content of untreated ECM, and wherein the
ECM tissue is cartilage ECM.
67. A method according to claim 66 in which the micronized
cartilage extracellular matrix tissue has a mean particle size of
10-200 microns.
68. A method according to claim 66 in which the slurry comprises
100-400 mg/ml micronized cartilage ECM tissue.
69. A method according to claim 66 in which the micronized
cartilage extracellular matrix tissue is cryomilled cartilage
extracellular matrix tissue.
70. A method according to claim 66 in which the porous scaffold is
cross-linked.
71. A method according to claim 66 in which the cartilage
extracellular matrix is hyaline cartilage ECM or growth plate
ECM.
72. A method according to claim 66 in which the cartilage
extracellular matrix is decellularised before or after
micronizing.
73. A method according to claim 66 in which the method of the
invention includes an additional step of seeding the scaffold with
a biological material selected from cells or a biological growth
factor.
74. A method according to claim 73 in which: the cells are selected
from the group consisting of stem cells, chondrocytes, mesenchymal
cells and osteoblasts; and/or in which the biological growth factor
is selected from the group consisting of one or more of the
TGF-.beta. superfamily or cannabinoids.
75. A method of making a multilayer scaffold comprising the steps
of making a first layer comprising a porous scaffold according to a
method of claim 66, making a second layer comprising a porous
scaffold according to a method of claim 66, wherein the first layer
is attached to the second layer.
76. A method according to claim 75 in which the process includes a
step of attaching the first layer to the second layer to form the
multilayer scaffold, in which the first layer comprises hyaline
cartilage ECM and the second layer comprises growth plate ECM.
77. A porous scaffold suitable for use in repair of osseous,
chondral, or osteochondral defects in a mammal and comprising a
porous freeze-dried matrix formed from micronised decellularised
extracellular matrix tissue, wherein the extracellular matrix
tissue comprises less than 90% of the GAG content of natural ECM,
and wherein the extracellular matrix tissue is cartilage
extracellular matrix tissue.
78. A porous scaffold according to claim 77 in which the porous
scaffold is cross-linked.
79. A porous scaffold according to claim 77 in which the cartilage
extracellular matrix is hyaline cartilage extracellular matrix or
growth plate extracellular matrix.
80. A multilayer scaffold suitable for repair of osteochondral
defects in a mammal and having a first layer comprising a porous
scaffold according to claim 77 in which the cartilage extracellular
matrix is hyaline cartilage extracellular matrix and a second layer
comprising a porous scaffold according to claim 77 in which the
cartilage extracellular matrix is growth plate extracellular
matrix, in which the first layer is attached to the second layer.
Description
INTRODUCTION
[0001] The invention relates to a method for making a porous
scaffold suitable for use in repair of osseous, chondral, or
osteochondral defects in a mammal. The invention also relates to a
porous scaffold suitable for use in repair of osseous, chondral, or
osteochondral defects in a mammal, and a multilayer scaffold
suitable for use in repair of osteochondral defects in a
mammal.
[0002] In humans, 95% of defects to the articular surface of
synovial joints involve cartilage without affecting the subchondral
bone (Hjelle et al., 2002). Such defects fail to heal
spontaneously. An estimated 5.4 million patients in the US alone
will require joint and cartilage procedures to treat such defects
and other degenerative changes by 2019. Bone marrow stimulation
techniques such as microfracture are the most readily available
clinical repair strategies for articular cartilage (Getgood et al.,
2009). By surgically penetrating the subchondral bone, progenitor
cells from the bone marrow can migrate into the defect and form a
repair tissue. In general, a mechanically inferior
fibro-cartilaginous tissue is produced which provides only
temporary symptomatic relief. Alternative cell based therapies such
as autologous chondrocytes implantation (ACI) are available,
however these approaches require two hospital stays and are very
expensive (.about.35,000), which may explain their relatively
limited clinical uptake compared to marrow stimulation techniques.
There is therefore a significant commercial opportunity for a cost
effective `single-stage` or `in-theatre` therapy (such as the
proposed scaffold) for regenerating damaged articular
cartilage.
[0003] Scaffolds fabricated using decellularized extracellular
matrix (ECM) have shown great promise for the regeneration of
damaged tissues. This approach has been used to develop different
tissue-specific (e.g. heart valves, blood vessels, skin and
cartilage) scaffolds. In the case of articular cartilage, numerous
studies have demonstrated that scaffolds derived from devitalized
cartilage are chondroinductive and show great promise for
regenerating damaged joints. There are, however, a number of
limitations associated with current ECM derived scaffolds,
including inhomogeneous pore size with current cartilage ECM
derived scaffolds (which limits cellular infiltration into the
scaffold and leads to inhomogenous deposition of matrix within the
scaffold), failure to generate hyaline cartilage within the
scaffold or a defect treated with the scaffold, poor control over
scaffold pore size, inefficient decellularization of ECM prior to
scaffold fabrication, variability in scaffold composition which
impacts commercial production, and poor control of the release of
exogenous growth factors loaded onto ECM derived scaffolds.
Furthermore, it remains unclear how ECM derived scaffolds can be
used to treat defects that effect multiple different tissues such
as osteochondral defects".
[0004] It is an object of the invention to overcome at least one of
the above-referenced problems.
STATEMENTS OF INVENTION
[0005] The Applicant has discovered that freeze-drying a slurry of
micronized extracellular matrix (ECM) tissue derived from hyaline
cartilage (preferably articular cartilage) or growth plate tissue
provides scaffolds having a homogenous pore size (FIG. 1) that
demonstrate a high level of homogenous stem cell infiltration
in-vitro (FIG. 2) and a high level of deposition of cartilage-like
extracellular matrix (FIG. 3). The Applicant has successfully
employed decellularization techniques in order to sufficiently
reduce xenogeneic DNA from the scaffolds (FIG. 8).
[0006] Cartilage extracellular matrix consists primarily of
glycosaminoglycans (GAG) and type II collagen. The Applicant has
discovered that by reducing the glycosaminoglycan content of the
cartilage ECM using a detergent or similar (FIG. 8), and hence
increasing the ratio of collagen to GAG within the treated ECM,
improves the resultant capacity of the scaffold to induce robust
chondrogenesis after they have been seeded with mesenchymal stem
cells (FIGS. 9 and 10). The Applicant has also discovered that
crosslinking the scaffolds of the invention slows growth factor
release from the scaffolds (FIG. 4), and that reducing the
glycosaminoglycan content of the scaffolds of the invention also
slows growth factor release from the scaffold (FIG. 7). The
Applicant has also discovered that scaffolds formed from micronized
growth plate ECM generates extensive mineralisation of cranial and
femoral defects (FIGS. 23 and 24) and enhanced bone tissue
formation (FIG. 25), and that growth plate ECM derived scaffolds
that are seeded with mesenchymal stem cells support endochondral
bone formation in chondrogenic conditions in vitro (FIGS.
19-21).
[0007] The Applicant has also discovered that treated or native ECM
can be solubilised by solubilisation of the ECM. After
solubilisation the ECM can then be cross-linked and freeze-dried to
create scaffolds. The solubilisation process employed removes the
vast majority of GAG and residual xenogeneic DNA from the resulting
scaffold. (FIG. 25).
[0008] Accordingly, in a first aspect, the invention provides a
method for making a porous scaffold suitable for use in repair of
osseous, chondral, or osteochondral defects in a mammal, the method
comprising the step of: [0009] providing a slurry of micronized
extracellular matrix (ECM) tissue or a gel comprising solubilised
and crosslinked extracellular matrix (ECM) tissue; and [0010]
freeze-drying the slurry or gel to provide the porous scaffold.
[0011] Thus, the ECM material that is freeze-dried may be a slurry
formed from micronized ECM or it may be a gel formed by
solubilisation of ECM (optionally micronized ECM) that is
cross-linked, typically chemically cross-linked, to form a gel
prior to freeze-drying. Preferably, the solution of enzymatically
digested ECM is cross-linked prior to freeze-drying.
[0012] In one embodiment, the ECM is solubilised by enzymatic
digestion. In one embodiment, the ECM is micronized prior to
solubilisation.
[0013] Preferably, the slurry comprises 100-400 mg/ml micronised
ECM tissue, ideally 200-300 mg/ml micronised ECM tissue.
[0014] Preferably, the micronized extracellular matrix tissue has a
mean particle size of 10-200 microns, ideally 20-70 microns.
[0015] Typically, the micronized extracellular matrix tissue is
cryomilled extracellular matrix tissue.
[0016] Suitably, extracellular matrix is treated to reduce the GAG
content. Preferably, the extracellular matrix is treated to reduce
the GAG content after the extracellular matrix is micronized.
[0017] Typically, the porous scaffold is cross-linked.
[0018] Preferably, the extracellular matrix is hyaline cartilage
(preferably articular cartilage) ECM or growth plate ECM.
[0019] Preferably, the extracellular matrix is decellularised
before or after micronizing, ideally after micronisation.
[0020] Suitably, the method of the invention includes an additional
step of seeding the scaffold with a biological material, for
example cells, preferably mesenchymal cells, or a biological
molecule, for example a growth factor. This could be achieved by,
for example, soaking the prepared scaffold in a solution containing
the growth factor or cells of interest. Suitably, the biological
material or molecule (biologic) is selected from the groups of:
cells; and biological growth factors. Typically, the biological
growth factors are selected from the group consisting of one or
more of the TGF-.beta. superfamily, (IFG, FGF, BMP, PDGF, EGF) or
cannabinoids. These growth factors can also be included during the
production process as opposed to post-fabrication soaking of the
scaffolds.
[0021] In a preferred embodiment, the invention provides a method
for making a porous devitalised scaffold suitable for use in repair
of osseous, chondral, or osteochondral defects in a mammal, the
method comprising the step of: [0022] providing micronized
extracellular matrix (ECM) tissue having a mean particle size of
30-70 microns; [0023] mixing the micronized extracellular matrix
with a liquid to provide a slurry comprising 200-300 mg/ml
micronized ECM; and [0024] freeze-drying the slurry to provide the
porous scaffold.
[0025] The invention also relates to a method of making a
multilayer scaffold comprising the steps of making a first layer
comprising a porous scaffold according to a method of the
invention, making a second layer comprising a porous scaffold
according to a method of the invention, wherein the first layer is
attached to the second layer.
[0026] Preferably, the process includes a step of attaching the
first layer to the second layer to form the multilayer
scaffold.
[0027] In one embodiment, the first layer comprises ECM from a
first source and the second layer comprises ECM from a different
source to the first source. Preferably, the first source of ECM is
hyaline cartilage ECM and the second source of ECM is growth plate
ECM. The latter type of multilayer scaffolds are suitable for
repair or treatment of osteochondral defects.
[0028] Suitably, the layers are attached together by means of
freeze-drying. Thus, for example, the layers may be freeze-dried
independently, and then placed in a mould and freeze-dried
together. Alternatively, one layer may be freeze-dried and then
placed in a mould with a slurry and freeze-dried to form the
layered scaffold. Other methods of attaching the two layers include
use of adhesives, stitching and intermediate bonding layers.
[0029] In a preferred embodiment, the invention also to a method of
making a multilayer scaffold comprising the steps of making a first
layer comprising a porous scaffold according to a method of the
invention in which the ECM is cartilage ECM, making a second layer
comprising a porous scaffold according to a method of the invention
in which the ECM is growth plate ECM, wherein the first layer is
attached to the second layer
[0030] The invention also relates to a porous scaffold formed
according to a method of the invention.
[0031] The invention also relates to a porous multilayer scaffold
formed according to a method of the invention.
[0032] The invention also provides a porous scaffold typically
suitable for use in repair of osseous, chondral, or osteochondral
defects in a mammal and comprising a porous freeze-dried matrix
formed from micronised decellularised extracellular matrix or
solubilised and crosslinked extracellular matrix.
[0033] Preferably, the micronized extracellular matrix tissue has a
mean particle size of 10-200 microns, ideally 20-70 microns.
[0034] Typically, the micronized extracellular matrix tissue is
cryomilled extracellular matrix tissue.
[0035] Suitably, extracellular matrix comprises reduced GAG
content.
[0036] Typically, the porous scaffold is cross-linked.
[0037] Preferably, the extracellular matrix is hyaline cartilage
ECM (ideally articular cartilage ECM) or growth plate ECM.
[0038] Preferably, the micronised extracellular matrix is
decellularised.
[0039] Suitably, the porous scaffold is seeded with a biological
material, for example cells, preferably mesenchymal stem cells, or
a biological molecule. Preferably, the ECM is growth plate or
hyaline cartilage ECM, and the porous scaffold is seeded with
cells, preferably mesenchymal stem cells.
[0040] The invention also provides a porous scaffold according to
the invention suitable for use in repair of chondral defects in a
mammal, in which the extracellular matrix is hyaline (ideally
articular) cartilage extracellular matrix.
[0041] The invention also provides a porous scaffold according to
the invention suitable for use in repair of osseous defects in a
mammal, in which the extracellular matrix is growth plate tissue
extracellular matrix.
[0042] A multilayer scaffold typically suitable for use in repair
of osteochondral defects in a mammal and having a first layer
comprising a porous scaffold of the invention and a second layer
comprising a porous scaffold of the invention, in which the first
layer is attached to the second layer.
[0043] In one embodiment, the first layer of porous scaffold
comprises ECM from a first source and the second layer of porous
scaffold comprises ECM from a different source to the first source.
Preferably, the first source of ECM is hyaline cartilage ECM and
the second source of ECM is growth plate ECM. The latter type of
multilayer scaffolds are suitable for repair or treatment of
osteochondral defects.
[0044] Suitably, the layers are seamlessly attached by means of,
for example, freeze-drying. Thus, for example, the layers may be
freeze-dried independently, and then placed in a mould and
freeze-dried together. Alternatively, one layer may be freeze-dried
and then placed in a mould with a slurry and freeze-dried to form
the layered scaffold. Other methods of attaching the two layers
include use of adhesives, stitching and intermediate bonding
layers.
[0045] The invention also relates to a porous scaffold or
multilayer scaffold of the invention for use in a method of
treating osseous, chondral, or osteochondral defects in a mammal,
in which the porous scaffold is inserted into the defect.
[0046] The invention also relates to a porous scaffold of the
invention for use in a method of treating chondral defects in a
mammal, in which the porous scaffold comprises cartilage ECM and in
which the porous scaffold is inserted into the chondral defect.
[0047] The invention also relates to a porous scaffold of the
invention for use in treating osseous defects in a mammal, in which
the porous scaffold comprises growth plate ECM and in which the
porous scaffold is inserted into the osseous defect. The porous
scaffold comprises cells, ideally mesenchymal stem cells, although
the scaffold may also be cell-free.
[0048] The invention also relates to a multilayer scaffold of the
invention for use in treating osteochondral defects in a mammal, in
which the multilayer scaffold comprises a first layer of porous
scaffold comprising hyaline cartilage ECM and a second layer of
porous scaffold comprising growth plate ECM, and in which the
multilayer scaffold is inserted into the osteochondral defect. The
first and/or second layer of porous scaffold may comprise cells,
ideally mesenchymal stem cells, although the scaffold is preferably
cell-free.
[0049] In another aspect, the invention relates to a gel suitable
for use in repairing osseous, chondral, or osteochondral defects in
a mammal and comprising a gel base and micronized ECM homogenously
distributed throughout the gel base.
[0050] Typically, the gel base comprises fibrin.
[0051] Ideally, the gel is injectable.
[0052] The invention also relates to a method of making a gel
suitable for use in repairing osseous, chondral, or osteochondral
defects in a mammal and comprising the steps of mixing micronised
ECM with a gel base. Typically, the process comprises a step of
mixing micronised ECM with a liquid gel base precursor, and then
adding to the mixture an activator capable of converting the liquid
gel base precursor to a gel base. Suitably, the gel base precursor
is fibrinogen, the activator is thrombin.
[0053] The invention also relates to a method of making a porous
devitalised ECM-based scaffold comprising the step of mixing
ECM-producing cells within a hydrogel base, culturing the mixture
in-vitro such that the ECM-producing cells deposit ECM within the
mixture, and then freeze-drying the mixture to provide the porous
devitalised ECM-based scaffold.
[0054] Typically, the ECM-producing cells are selected from
chondrocytes and osteoblasts.
[0055] The invention also relates to a porous ECM-based scaffold
formed according to the method of the invention.
[0056] The invention also relates to a porous ECM-based scaffold
formed according to the method of the invention in a micronized
form.
[0057] The invention also relates to micronized growth plate ECM, a
slurry comprising micronized ECM, or a freeze-dried scaffold formed
from a slurry of micronized growth plate ECM.
[0058] The invention also relates to a porous scaffold formed from
micronized, freeze-dried, growth plate ECM.
[0059] The invention also relates to a porous freeze-dried scaffold
comprising cryomilled ECM.
[0060] The invention also relates to a porous scaffold formed from
solubilised and crosslinked, freeze-dried, growth plate ECM.
[0061] The invention also relates to a porous multilayer scaffold
comprising at least first and second layers, the first layer
comprising a porous freeze-dried scaffold formed from micronized
hyaline cartilage ECM and the second layer comprising a porous
freeze-dried scaffold formed from micronized growth plate cartilage
ECM.
[0062] The invention also relates to a porous growth plate
ECM-based scaffold formed according to the method of the invention
in a solubilised form.
BRIEF DESCRIPTION OF THE FIGURES
[0063] FIG. 1: Concentration modulates scaffold morphology. Helium
ion (HIM) micrographs showed different architecture in scaffolds
when cartilage ECM slurry concentration was altered: (A) 250 mg/ml;
(B) 500 mg/ml; (C) 1000 mg/ml. Mean pore size decreased with
increased concentration of ECM.
[0064] FIG. 2: Distribution of live cells. Confocal microscopy at
day 1 and day 28 of human infrapatellar fat pad derived stem cells
seeded in ECM derived scaffolds: (A) 250 mg/ml, (B) 500 mg/ml and
1000 mg/ml. Picture represents cross-section of ECM derived
scaffolds. Poor cellular penetration at day 1 was observed in the
(B) 500 mg/ml and (C) 1000 mg/ml scaffolds, which contrasts with
the 250 mg/ml scaffold where a homogeneous cellular infiltration
was observed (A). At day 28, scaffold with 1000 mg/ml of ECM
continued to show poor stem cells infiltration (F).
[0065] FIG. 3: sGAG deposition for day 0, 7, 14 and 28.
Histological images of glycosaminoglycans (GAG) (alcian blue) and
cell nuclei (nuclear fast red) staining for ECM derived
scaffolds--250, 500 and 1000 mg/ml--at day 0, 7, 14 and 28 of
culture (A). In (A) it is possible to observe strong GAG deposition
and cell distribution for the 250 mg/ml scaffold. With high
magnification it is possible to observe the superior GAG deposition
for 250 (B), followed by 500 (C) and 1000 mg/ml scaffold (D).
[0066] FIG. 4: TGF-.beta.3 release profile for constructs with or
without EDAC crosslinking. ELISA results for TGF-.beta.3 release
into the media from TGF-.beta.3 loaded ECM derived scaffold
indicates slower release rate for scaffolds with EDAC crosslinking,
with significant difference at day 4 (n=6, *p<0.05).
[0067] FIG. 5: Tailored sGAG scaffold morphology. Helium ion (HIM)
micrographs showed altered pore size and architecture in tailoring
GAG concentration of scaffolds: (A) 5% GAG; (B) 50% GAG; (C) 100%
GAG. Mean pore size increased with decreasing GAG
concentration.
[0068] FIG. 6: sGAG Histology. GAG staining on decellularized
tailored GAG cartilage explants. micrographs showed in tailoring
GAG concentration: (A) 5% GAG; (B) 50% GAG; (C) 100% GAG.
[0069] FIG. 7: TGF-.beta.3 release profile from tailored GAG
cartilage ECM scaffolds. ELISA results for TGF-.beta.3 release into
the media from TGF-.beta.3 loaded ECM derived scaffold 4 (n=4). By
removing sGAGs from the ECM prior to scaffold fabrication, it is
possible to slow the release of growth factors from the
construct.
[0070] FIG. 8: Biochemical assay for DNA and GAG content of
tailored GAG scaffold.
[0071] FIG. 9: Biochemical assays performed on cartilage tissues
engineered in vitro using tailored GAG scaffolds seeded with human
stem cells
[0072] FIG. 10: Histological staining for sGAG (Alcian blue) and
Collagen (Picrosirius red) of cartilage tissues engineered in vitro
using ECM derived scaffolds seeded with human stem cells.
[0073] FIG. 11: Gross appearance of Fibrin-ECM particle constructs
post-gelation
[0074] FIG. 12: TGF-.beta.3 release profile for Fibrin hydrogel
loaded with ECM particles. ELISA results for TGF-.beta.3 release
into the media from TGF-.beta.3 loaded ECM derived particles
(n=4).
[0075] FIG. 13: GAG content of cartilage tissues engineered in
vitro using ECM particle loaded hydrogels. Fibrin hydrogels
containing ECM particles loaded with TGF-.beta.3 showed higher GAG
accumulation than constructs where TGF-.beta.3 was either added
directly to the media or added to control gelatine microparticles
(MPs).
[0076] FIG. 14: Histology. GAG and Collagen staining of cartilage
tissues engineered in vitro within fibrin hydrogels loaded with ECM
derived particles
[0077] FIG. 15: Gross morphology after of tissues generated in vivo
using proposed injectable construct.
[0078] FIG. 16: Histology. GAG and Collagen staining for tissues
generated in vivo.
[0079] FIG. 17. A representative PDMS mould used to control
freeze-drying of growth plate ECM to specific dimensions
[0080] FIG. 18: Schematic outlining the steps involved in
endochondral ossification, whereby a cartilage template is
converted to a mature bone
[0081] FIG. 19: Histological analysis of constructs at day 0 and
following 28 days of culture in either chondrogenic or osteogenic
medium, demonstrating the deposition of the main constituents of
cartilage, sGAG and collagen.
[0082] FIG. 20: MSC-seeded growth plate ECM constructs with
positive collagen type II and collagen type X staining
[0083] FIG. 21: Mineral deposition was observed in MSC-seeded
growth plate ECM scaffolds cultured in either chondrogenic or
osteogenic medium, in comparison to MSCs seeded on a cartilage ECM
construct which only mineralised in osteogenic culture
conditions.
[0084] FIG. 22: An image of a bi-layered construct containing
cartilage ECM in the top layer and growth plate ECM in the bottom
layer and histological analysis of tissue deposited by MSCs seeded
onto osteochondral scaffolds after 28 days in culture.
[0085] FIG. 23: Reconstructed images of growth plate scaffold
treated and untreated cranial defects at 4 and 8 weeks showing the
level of mineralisation achieved
[0086] FIG. 24: Reconstructed images of growth plate scaffold
treated and untreated femoral defects at 4 and 8 weeks showing the
level of mineralisation achieved
[0087] FIG. 25: Histological analysis of repair tissue formed
across the cranial defect after 4 and 8 weeks either (a) untreated
or (b) treated with the growth plate scaffold. (c) Higher power
images of the repair tissue, demonstrating de novo bone forming
both upon and within the original growth plate ECM tissue.
[0088] FIG. 26: Biochemical assays for DNA and GAG content of
solubilised ECM scaffold (a). Scaffolds were generated using
micronized ECM (High GAG) or solubilised ECM. Macroscopic images of
wet and dry solubilised ECM scaffolds (b).
[0089] FIG. 27: Biochemical assays performed on cartilage tissues
engineered in vitro using solubilised or micronized ECM scaffolds
seeded with human stem cells (a). Scaffolds were generated using
micronized ECM (High GAG) or solubilised ECM. Gross morphology of
tissues generated using scaffolds (b).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0090] In this specification, the term "porous" as applied to a
scaffold should be understood to mean having a porosity of at least
90% as determined using the method of Gleeson et al (J. P. Gleeson,
N. A. Plunkett, F. J. O'Brien--Addition of hydroxyapatite improves
stiffness, interconnectivity and osteogenic potential of a highly
porous collagen-based scaffold for bone tissue regeneration--Eur
Cell Mater, 20 (2010), pp. 218-223) In one embodiment, the scaffold
(or each layer in the scaffold) has a porosity of at least 91%,
92%, 93%, 94%, 95%, 96%, 97% or 98%. Ideally, the scaffold has a
porosity of at least 98%, ideally at least 98.5%.
[0091] In this specification, the term "osseous defect" should be
understood to mean any defect within bony tissue.
[0092] In this specification, the term "chondral defect" should be
understood to mean any defect within the articular surface of a
joint that does not penetrate through the subchondral bone.
[0093] In this specification, the term "osteochondral defect"
should be understood to mean a defect to the articular surface that
affects both the articular cartilage and the underlying bone.
[0094] In this specification, the term "extracellular matrix
tissue" or "extracellular matrix" or "ECM" should be understood to
mean a collection of extracellular molecules secreted by cells that
provides structural and biochemical support to the surrounding
cells. The ECM may be obtained from a mammal, for example a human
or a non-human mammal, or it may be engineered in-vitro using
published techniques, for example Vinardell et al (Vinardell, T.,
Sheehy, E., Buckley, C. T., Kelly, D. J. A comparison of the
functionality and in vivo phenotypic stability of cartilaginous
tissues engineered from different stem cells sources. Tissue
Engineering Part A, 18(11-12), 1161-1170, 2012) and Buckley et al
(Buckley, C. T., Vinardell, T., Kelly, D. J. Oxygen Tension
Differentially Regulates the Functional Properties of Cartilaginous
Tissues Engineered from Infrapatellar Fat Pad Derived MSCs and
Articular Chondrocytes. Osteoarthritis and Cartilage, 18 (10),
1345-1354, 2010). Examples of extracellular matrix for the purpose
of the present invention include cartilage ECM (obtained from
porcine articular cartilage tissue) and growth plate ECM (typically
obtained from the epiphysial plate of porcine tibia or femora).
[0095] In this specification, the term "hyaline cartilage ECM"
should be understood to mean ECM obtained from hyaline cartilage
which is a tissue found, for example, in the ear and nose and on
joint surfaces. It is mostly composed of type II collagen and
chondroitin sulphate.
[0096] In this specification, the term "articular cartilage ECM"
should be understood to mean ECM obtained from articular cartilage,
which is a form of hyaline cartilage found at the articular end of
joints.
[0097] In this specification, the term "growth plate ECM" or
"growth plate tissue ECM" should be understood to mean ECM obtained
from growth plate tissue of developing bones, typically developing
long bones. This could include the epiphyseal plate in the
metaphysis of a long bone, or articular cartilage from skeletally
immature joints as this tissue is also known to act as a surface
growth plate during development and skeletal maturation.
[0098] In this specification, the term "micronised" as applied to
ECM should be understood to mean provided in a particulate form, in
which the particles of ECM have a mean particle size of less than
200 microns as determined using routine light microscopy.
Preferably, the micronised ECM has a mean particle size of less
than 150 or 100 microns. Ideally, the micronized ECM has a mean
particle size between 20 and 200 microns, 20 and 150 microns, 20
and 100 microns, 20 and 70 microns, 30 and 70 microns, 30 and 60
microns, 40 and 60 microns, and ideally about 50 microns. Methods
of micronisation include milling, cryomilling,
[0099] In this specification, the term "cryomilled" should be
understood to mean a process in which a material is cryogenically
frozen and then milled. Examples of cryomilling machines include
the RETCH CRYOMILL.TM..
[0100] In this specification, the term "solubilised" should be
understood to mean a process by which ECM tissue is digested,
ideally enzymatically digested, to become soluble in an aqueous
solvent. Suitably solubilising agents will be known to the person
skilled in the art, and include enzymes and denaturing agents such
as urea. An example of an enzyme that can be used to digest ECM
tissue to become soluble is a protease, for example pepsin, or a
collagense. Preferably, the solubilised ECM will be a purified
collagen with substantial removal of GAG and xenogeneic DNA.
Ideally, the solubilised ECM will have greater than 50%, 60%, 70%,
80% or 90% removal of GAG and DNA when compared to native ECM
tissue.
[0101] In this specification, the term "freeze-drying" as applied
to a slurry refers to a process in which the slurry is frozen,
typically to a final freezing temperature of from -10.degree. C. to
-70.degree. C. and then sublimated under pressure. In one
embodiment, the desired final freezing temperature is between
-10.degree. C. and -70.degree. C. Suitably, the desired final
freezing temperature is between -30.degree. C. and -50.degree. C.
Typically, the desired final freezing temperature is between
-35.degree. C. and -45.degree. C., ideally about -40.degree. C. In
one embodiment of the invention, freezing or freeze-drying is
carried out at a constant cooling rate. This means that the rate of
cooling does not vary by more than +/-10% of the target cooling
rate, i.e. if the desired rate of cooling is 1.0.degree. C./min,
and the actual rate of cooling varied between 0.9.degree. C./min
and 1.1.degree. C./min, this would nonetheless still be considered
to be a constant cooling rate. Typically, the constant cooling rate
is between 0.1.degree. C./min to 10.degree. C./min. Preferably,
freeze-drying is carried out at a constant cooling rate of between
0.5.degree. C./min to 1.5.degree. C./min. More preferably, freezing
or freeze-drying is carried out at a constant cooling rate of
between 0.8.degree. C./min to 1.1.degree. C./min. Typically,
freezing or freeze-drying is carried at a constant cooling rate of
about 0.9.degree. C./min. The temperature of the freeze-drying
chamber at a start of the freeze-drying process (i.e. when the
slurry is placed in the chamber) is usually greater than 0.degree.
C., preferably at about ambient temperature. The sublimation step
is generally carried out after the final freezing temperature is
reached. This step involves heating the freeze-drying chamber to a
sublimation temperature (generally about 0.degree. C.), preferably
at a constant heating rate. The process typically includes a final
sublimation step where an ice phase in the formed scaffold is
sublimated under vacuum for a suitable period of time.
[0102] In this specification, the term "slurry" should be
understood to mean a suspension of micronized ECM in a solvent,
suitably an aqueous solvent, for example water. Typically, the
slurry comprises less than 500, 400, 300 mg/ml micronized ECM.
Suitably, the slurry comprises 100-500, 100-400, 200-300, 230-270,
and ideally about 250 mg/ml micronized ECM.
[0103] In this specification, the term "cross-linked" should be
understood to mean treated to introduce cross-links between
different polymeric molecules in the ECM. The ECM may be micronised
ECM or solubilised ECM. Crosslinking may be performed on the
solubilised ECM or on the formed freeze-dried scaffold. Typically,
the scaffold is cross-linked by one or more of the means selected
from the group comprising: dehydrothermal (DHT) cross-linking; and
chemical cross-linking. When crosslinking is be performed on the
solubilised ECM, the crosslinking agent is typically a chemically
crosslinking agent. Suitable chemical cross-linking agents and
methods will be well known to those skilled in the art and include
a glyoxal, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride (EDAC) or Glutaraldehyde. Ideally, the scaffold is
cross-linked using DHT and EDAC cross-linking. Cross-linking can be
carried out at any stage of the fabrication process. In a preferred
embodiment, scaffold pore symmetry can be controlled by varying the
degree of cross-linking within each respective layer using cross
linking methods familiar to one skilled in the art. Similarly, in
another embodiment, scaffold permeability or flow conductivity can
be varied by varying the mechanical properties of the scaffold
using either cross linking or other stiffness improvement
methodologies known to one skilled in the art.
[0104] In this specification, the term "GAG" should be understood
to mean glycosaminoglycan, particularly sulphated
glycosaminoglycans.
[0105] In this specification, the term "reduced GAG content" as
applied to ECM from a given source should be understood to mean a
GAG content that is reduced compared to natural ECM from the same
source, for example less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,
20%, 10% or 5% GAG content of natural ECM. Methods of reducing GAG
content include the use of buffers, detergents (such as Sodium
dodecyl sulfate or Triton-X or Sodium deoxycholate) or other
chemicals (e.g. chondroitinase ABC) known to reduce the sGAG
content of tissues.
[0106] In this specification, the term "decellularised" or
"devitalised" as a applied to a material (for example ECM, a
scaffold, or a gel) should be understood to mean that the cellular
content of the material is reduced partially or preferably
completely. Method of decellularising a material include chemical
nucleic acid digestion, possibly following partial or total removal
of matrix components from the ECM.
[0107] In this specification, the term "seeding" as applied to a
scaffold should be understood to mean incorporating a biological
material into a scaffold. Method of seeding a scaffold include
soaking the scaffold in a solution containing the biological
material for a sufficient time to allow the biological material
infiltrate the pores of the scaffold.
[0108] In this specification, the term "cells" should be understood
to mean any type of cell, particularly stem cells, chondrocytes,
and osteoblasts. Preferably, the cells are mesenchymal stem
cells.
[0109] In this specification, the term "biological material" should
be understood to mean proteins, peptides, nucleic acid, nucleic
acid constructs, nucleic acid vectors, or chemical molecules having
biological activity. Preferably, the biological material comprises
a biological growth factor, for example one or more of the
TGF-.beta. superfamily, (IFG, FGF, BMP, PDGF, EGF) or
cannabinoids.
[0110] In this specification, the term "cannabinoids" should be
understood to mean a biological compound which can be naturally or
synthetically derived and that acts on the cannabinoid receptor
types 1 and/or 2 (CB.sub.1 and CB.sub.2), for example
.DELTA.9-tetrahydrocannabinol (.DELTA.9-THC).
[0111] In this specification, the term "gel base" should be
understood to mean a matrix having both solid and liquid
properties. An exemplary gel base is an agarose gel.
[0112] In this specification, the term "injectable" should be
understood to mean that the gel is sufficiently deformable to
enable it to be injected into a defect in cartilage or bone.
EXPERIMENTAL
[0113] Development of Decellularized ECM Derived Scaffolds with a
Uniform Pore Size.
[0114] Cartilage used in the fabrication of ECM derived scaffolds
was harvested, in sterile conditions, from the femoral condyles of
female pigs (3 months old) shortly after sacrifice. The cartilage
was first broken up into small pieces using a scalpel. Cartilage
particles were then broken up using a cryogenic mill (6770
Freezer/Mill, SPEX, UK). These small pieces of cartilage where then
homogenized in distilled water (dH2O) using a homogenizer (IKAT10,
IKA Works Inc, NC, USA) to create a cartilage slurry (250 mg/ml).
The slurry was transferred to custom made moulds (containing wells
5 mm in diameter and 3 mm in height) and freeze-dried (FreeZone
Triad, Labconco, KC, USA) to produce porous scaffolds. Briefly, the
slurry was frozen to -30.degree. C. (1.degree. C./min) and kept at
that temperature for one hour. The temperature was then increased
to -10.degree. C. (1.degree. C./min), followed by a hold of 24
hours and then finally increased to room temperature (0.5.degree.
C./min). Next, two different crosslinking techniques were applied
to the scaffolds. The scaffolds underwent DHT and
1-Ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking
The DHT process was performed in a vacuum oven (VD23, Binder,
Germany), at 115.degree. C., in 2 mbar for 24 hours. The EDAC
(Sigma-Aldrich, Germany) crosslinking consisted of chemical
exposure for 2 hours at a concentration of 6 mM in the presence of
N-Hydroxysuccinimide (NHS) (Sigma-Aldrich, Germany). A molar ratio
of 2.5 M EDAC/M N-Hydroxysuccinimide was used. After EDAC
crosslinking the scaffolds were washed twice in sterile PBS
(Sigma-Aldrich, Germany).
Development of Decellularized ECM Derived Scaffolds with Controlled
Pore Size and Tailored Growth Factor Release Rates.
[0115] Articular cartilage was harvested from femoral condyles of
female 4 months old pigs under sterile condition shortly after
sacrifice. All steps of the decellularization and tailoring GAG
protocol were performed in 2 mL working volume at room temperature.
This protocol consists of three phases. In Phase I, the 50 and 5%
GAG groups were incubated in basic buffer (10 mM Tris-HCl (pH 8.0))
containing 100 mM DTT, 2 mM MgCl2, and 10 mM KCl for 24 hrs; and
anatomically adjacent pieces of cartilage subjected to 1 min
incubations for 100% GAG group. The 5% GAG groups were additionally
subjected to 0.5% SDS treatment with basic buffer containing 100 mM
DTT, 2 mM MgCl2, and 10 mM KCl for 24 hrs. Following sGAG removal,
nucleic acid digestion (2.5 Kunitz units/mL deoxyribonuclease I,
7.5 Kunitz units/mL ribonuclease A, 0.15 M NaCl, 2 mM MgCl.sub.2
(H2O) in 10 mM Tris-HCl (pH 7.6)) was performed for 24 h and
washout (10 mM Tris-buffered saline (pH 7.5)) for 48 h. In phase
II, the cartilage tailored GAG-ECM scaffolds were prepared by
cryo-milling followed by DHT+EDAC crosslinking as described in
section 1 above.
Development of Solubilised ECM Derived Scaffolds
[0116] Cartilage used in the fabrication of ECM derived scaffolds
was harvested, in sterile conditions, from the femoral condyles or
growth plates of female pigs (3 months old) shortly after
sacrifice. The cartilage was first broken up into small pieces
using a scalpel. ECM tissue was then transfer to sterile
containers. ECM tissue was then pre-treated with 0.2M NaOH for 24
hours at 4.degree. C. After washing and removal of pre-treatment
solution, the ECM tissue was then digested with pepsin in 0.5 M
Acetic Acid. Pepsin is added at a concentration of .about.1500
units pepsin per 50 mg ECM tissue. The ECM was then incubated in
the pepsin solution for 24 hours at <20.degree. C. with rotation
at a speed of 4 rpm. Salt precipitation was then performed to
extract purified collagen using concentration of NaCl between
0.1M-5M. In order to remove any remaining salt, acid or pepsin,
dialysis can be performed on the solubilised collagen. Dialysis was
performed against 0.02 M Na.sub.2HPO.sub.4 (pH 9.4) for 24 h at
4.degree. C. The solubilised collagen can then be freeze-dried. To
generate scaffolds, the freeze dried collagen was rehydrated in an
aqueous solution at a concentration range of 1 mg/ml to 200 mg/ml
preferably, 20 mg/ml. Once rehydrated the collagen can then be
cross-linked to form a gel with Glyoxal at a concentration between
1 mM and 50 mM preferably, 10 mM. The solution is then incubated
for 30 minutes at 37.degree. C. to allow cross-linking to take
place. After incubation the gel can then be transferred to moulds
and freeze-dried to create scaffolds.
Development of Injectable Decellularized ECM Derived Particles as
Growth Factor Delivery Systems.
[0117] Particulated cartilage ECM is fabricated as described in 1
or 2 above. Instead of freeze-drying these particles to produce a
porous scaffold, it is also possible to combine these particles
with a hydrogel to develop an injectable chondroinductive composite
biomaterial that also acts as a growth factor delivery system.
[0118] One manifestation of this invention would be to combine ECM
particles with a fibrin hydrogel. The particulated cartilaginous
material is incorporated into the hydrogel by mixing directly with
the fibrinogen, with the desired ratio. Gelation occurs by adding
thrombin to the fibrinogen/ECM-particles slurry. Appropriate mixing
ensures a homogeneous distribution of bioactive cartilage
ECM-derived micro-particles within the hydrogel.
Development of Decellularized Growth Plate ECM Derived
Scaffolds.
[0119] Growth plate used in the fabrication of ECM derived
scaffolds was harvested, in sterile conditions, from the femur,
fibula and tibia of female pigs (3 months old) shortly after
sacrifice. The growth plate was first broken up into small pieces
using a scalpel, and then broken up using a cryogenic mill (6770
Freezer/Mill, SPEX, UK). These small pieces of growth plate were
then homogenized in distilled water (dH.sub.2O) using a homogenizer
(IKAT10, IKA Works Inc, NC, USA) to create a slurry (250 mg/ml).
The slurry was transferred to custom made moulds and freeze-dried
(FreeZone Triad, Labconco, KC, USA) to produce porous scaffolds.
Briefly, the slurry was frozen to -30.degree. C. (1.degree. C./min)
and kept at that temperature for one hour. The temperature was then
increased to -10.degree. C. (1.degree. C./min), followed by a hold
of 24 hours and then finally increased to room temperature
(0.5.degree. C./min). Next, two different crosslinking techniques
were applied to the scaffolds. The scaffolds underwent DHT and
1-Ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking.
The DHT process was performed in a vacuum oven (VD23, Binder,
Germany), at 115.degree. C., in 2 mbar for 24 hours. The EDAC
(Sigma-Aldrich, Germany) crosslinking consisted of chemical
exposure for 2 hours at a concentration of 6 mM in the presence of
N-Hydroxysuccinimide (NHS) (Sigma-Aldrich, Germany). A molar ratio
of 2.5 M EDAC/M N-Hydroxysuccinimide was used. After EDAC
crosslinking the scaffolds were washed twice in sterile PBS
(Sigma-Aldrich, Germany).
[0120] Results obtained from both in vitro and in vivo
characterisation of the growth plate scaffold will be presented
below, and demonstrate its potential for use in bone tissue
regeneration. Also, we will display the ability of the growth plate
scaffold layer to be combined with a cartilage ECM layer to
generate an osteochondral graft which can be potentially applied to
repair both bone (osteo) and cartilage (chondral) layers
simultaneously.
[0121] The invention is not limited to the embodiments hereinbefore
described which may be varied in construction and detail without
departing from the spirit of the invention.
* * * * *