U.S. patent application number 12/195255 was filed with the patent office on 2016-07-21 for method for improvement of differentiation of mesenchymal stem cells using a double-structured tissue implant.
This patent application is currently assigned to HISTOGENICS CORPORATION. The applicant listed for this patent is Hans P.I. Claesson, Joseph Khoury, Sonya Shortkroff, Robert Lane Smith, Laurence J.B. Tarrant. Invention is credited to Hans P.I. Claesson, Joseph Khoury, Sonya Shortkroff, Robert Lane Smith, Laurence J.B. Tarrant.
Application Number | 20160206787 12/195255 |
Document ID | / |
Family ID | 40432742 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206787 |
Kind Code |
A9 |
Shortkroff; Sonya ; et
al. |
July 21, 2016 |
Method For Improvement Of Differentiation Of Mesenchymal Stem Cells
Using A Double-Structured Tissue Implant
Abstract
A double-structured tissue implant (DSTI) and a method for
preparation and use thereof for implantation into tissue defects.
The double-structured tissue implant for differentiation, growth
and transformation of cells, stem cells, mesenchymal stem cells and
bone marrow stem cells. DSTI comprising a primary scaffold and a
secondary scaffold consisting of a soluble collagen solution in
combination with a non-ionic surfactant generated and positioned
within the primary scaffold.
Inventors: |
Shortkroff; Sonya;
(Braintree, MA) ; Khoury; Joseph; (Dedham, MA)
; Tarrant; Laurence J.B.; (Northhampton, MA) ;
Claesson; Hans P.I.; (Wayland, MA) ; Smith; Robert
Lane; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shortkroff; Sonya
Khoury; Joseph
Tarrant; Laurence J.B.
Claesson; Hans P.I.
Smith; Robert Lane |
Braintree
Dedham
Northhampton
Wayland
Palo Alto |
MA
MA
MA
MA
CA |
US
US
US
US
US |
|
|
Assignee: |
HISTOGENICS CORPORATION
Waltham
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20090069903 A1 |
March 12, 2009 |
|
|
Family ID: |
40432742 |
Appl. No.: |
12/195255 |
Filed: |
August 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11894124 |
Aug 20, 2007 |
8685107 |
|
|
12195255 |
|
|
|
|
60967886 |
Sep 6, 2007 |
|
|
|
60958401 |
Jul 3, 2007 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/3834 20130101;
A61L 27/58 20130101; A61F 2/30756 20130101; A61F 2002/30766
20130101; A61L 27/3817 20130101; A61L 27/3852 20130101; A61L
2430/32 20130101; A61L 27/26 20130101; A61L 2430/30 20130101; A61F
2/02 20130101; A61L 27/3804 20130101; A61L 2430/34 20130101; A61L
27/3839 20130101; A61L 27/56 20130101; A61L 27/3878 20130101; A61L
27/54 20130101; A61L 27/386 20130101; A61L 27/52 20130101; A61F
2240/001 20130101; A61L 27/24 20130101; A61F 2/28 20130101; A61L
2430/20 20130101; A61L 27/3873 20130101; A61F 2/08 20130101; A61L
27/3847 20130101; A61L 2430/06 20130101 |
International
Class: |
A61F 2/02 20060101
A61F002/02; A61L 27/56 20060101 A61L027/56; A61F 2/30 20060101
A61F002/30; A61L 27/52 20060101 A61L027/52; A61L 27/26 20060101
A61L027/26; A61L 27/54 20060101 A61L027/54; A61L 27/24 20060101
A61L027/24; A61B 17/08 20060101 A61B017/08; A61L 27/38 20060101
A61L027/38 |
Claims
1. A double-structured tissue implant (DSTI) for treatment of a
tissue defect, said implant comprising: a) a primary scaffold
wherein said primary scaffold is a porous structure prepared from
collagen or a collagen-containing material, said porous structure
comprising randomly or non-randomly organized pores, said primary
scaffold providing a structural support for the secondary scaffold
incorporated therein; and b) a secondary scaffold integrated into
said first scaffold; wherein said DSTI comprises cells, stem cells,
mesenchymal stem cells or bone marrow stem cells.
2. The DSTI of claim 1 wherein said collagen or collagen-containing
material for preparation of the primary scaffold is selected from
the group consisting of Type I collagen, Type II collagen, Type II
collagen, Type IV collagen, Type V collagen, gelatin,
collagen-containing agarose, collagen-containing hyaluronan,
collagen-containing proteoglycan, collagen-containing
glycosaminoglycan, collagen-containing glucosamine,
collagen-containing galactosamine, collagen-containing
glycoprotein, collagen-containing fibronectin, collagen-containing
laminin, collagen-containing bioactive peptide, collagen-containing
growth factor, collagen-containing cytokine, collagen-containing
elastin, collagen-containing fibrin, collagen-containing polylactic
acid, collagen-containing polyglycolic acid, collagen-containing
polyamino acid, collagen-containing polycaprolactone,
collagen-containing polypeptide, a copolymer thereof, a precursor
thereof and a combination thereof, wherein said precursor is
selected from the group consisting of alpha 1 (Type I) peptide,
alpha 2 (Type I) peptide, 2 (alpha 1, Type I) peptide, 1 (alpha 2,
Type I) peptide, 3(alpha 1, Type II), and a combination
thereof.
3. The DSTI of claim 2 wherein said secondary scaffold is
integrated into said primary scaffold by introducing a composition
comprising a soluble collagen or collagen-containing compound in
combination with a non-ionic surfactant into said pores of said
primary scaffold, stabilizing said composition within pores of said
primary scaffold by precipitation or gelling and subjecting a
resulting composite to at least lyophilization and dehydrothermal
treatment.
4. The DSTI of claim 3 wherein said soluble collagen or
collagen-containing compound used for preparation of the
composition for the secondary scaffold is Type I collagen, Type II
collagen, methylated collagen, gelatin or methylated gelatin.
5. The DSTI of claim 4 wherein said non-ionic surfactant used for
preparation of the composition for the secondary scaffold is a
PLURONIC.RTM.-type or a TRITON7-type surfactant comprising
polyethylene oxide with terminal oxide groups.
6. The DSTI of claim 5 wherein said surfactant is a derivatized
polyethylene glycol or a block co-polymer of polyoxyethylene (PEO)
and polyoxypropylene (PPO) having the generic organization of
polymeric blocks PEG-PPO-PEG or PPO-PEG-PPO.
7. The DSTI of claim 6 wherein said surfactant is TRITON.RTM. X100,
namely polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl
ether, or PLURONIC.RTM. F127, namely a polymer of polyoxyethylene
(PEO) and polyoxypropylene (PPO) with two 96-unit hydrophilic PEO
chains surrounding one 69-unit hydrophobic PPO chain.
8. The DSTI of claim 7 wherein said integration of the secondary
scaffold into the primary scaffold results in the double-structured
tissue implant comprising two structurally and functionally
distinct sections, wherein one or both sections may separately be
seeded or rehydrated with cells or incorporated with a cell
modulator.
9. The DSTI of claim 8 wherein said DSTI is implanted as a dry or
rehydrated acellular DSTI or a dry or rehydrated DSTI seeded with
cells.
10. The DSTI of claim 9 wherein said cells, stem cells, mesenchymal
stem cells or bone marrow stem cells migrate into said DSTI from a
native surrounding tissue.
11. The DSTI of claim 10 wherein said migration occurs following a
subchondral microfracture.
12. The DSTI of claim 9 wherein said cells, stem cells, mesenchymal
stem cells or bone marrow stem cells are added to said dry DSTI
before said implantation of said DSTI.
13. The DSTI of claim 12 wherein said chemoattractant property of
said DSTI is enhanced with addition of a growth factor or a
morphogenic factor.
14. The DSTI of claim 13 wherein said growth factor is a
transforming growth factor-beta (TGF.beta.), and wherein said
morphogenic factor is bone morphogenic protein BMP-2, bone
morphogenic protein BMP-7 or repulsive guidance molecule
(RGMA).
15. The DSTI of claim 1 wherein said primary or secondary scaffold
is independently incorporated with a pharmaceutical agent, growth
modulator, growth hormone, mediator, enzyme promoting cell
incorporation, enzyme promoting cell proliferation, enzyme
promoting cell division, a pharmaceutically acceptable excipient,
additive or buffer.
16. The DSTI of claim 15 wherein said growth and morphogenic factor
is a transforming growth factor, insulin-like growth factor 1,
platelet-derived growth factor or a bone morphogenic protein;
wherein said cytokine is interleukin, chemokine, macrophage,
chemoattractant factor, cytokine-induced neutrophil chemoattractant
(gro-1), integral membrane protein, integrin or growth factor
receptors; wherein said membrane associated factor is a repulsive
guidance molecules; wherein said cell attachment or adhesion
protein is fibronectin or chondronectin; wherein said hormone is a
growth hormone, insulin or thyroxine; wherein said pericellular
matrix molecule is perlecan, syndecan, leucine-rich proteoglycan or
fibromodulin; wherein said nutrient is glucose or glucosamine;
wherein said nucleic acid is RNA or DNA; wherein said
anti-neoplastic agent is methotrexate or aminopterin; wherein said
vitamin is ascorbic acid or retinoic acid; wherein said
anti-inflammatory agent is naproxen sodium, salicylic acid,
diclofenac or ibuprofen; wherein said enzyme is phosphorylase,
sulfatase or kinase; and wherein said metabolic inhibitor is RNAi,
cycloheximide or s steroid.
17. The DSTI of claim 1 that is biodegradable and
biocompatible.
18. The DSTI of claim 1 wherein said cells, stem cells, mesenchymal
stem cells and bone marrow stem cells are attached to said primary
or secondary scaffold and proliferate, differentiate and are
transformed into a functionally determined cell, tissue or
organ.
19. The DSTI of claim 18 wherein said functionally determined cell
is a cartilage chondrocyte, blood vessel endothelial cell, cardiac
muscle cell, nerve cell or pancreatic islet cell.
20. The DSTI of claim 1 wherein said DSTI has chemoattractant, cell
adhesion, wettability, shape-memory and structural stability
properties for cells, stem cells, mesenchymal stem cells and bone
marrow stem cells.
21. A method for treatment of a cartilage, bone, tendon, skin,
meniscus, ligament, skeletal, muscle, cardiac muscle or nervous
tissue defect using an implantable double-structured tissue implant
(DSTI), said method comprising steps: a) obtaining or preparing the
DSTI comprising a primary scaffold and a secondary scaffold
integrated into said primary scaffold wherein said primary scaffold
is a porous structure prepared from collagen or a
collagen-containing material, said porous structure comprising
randomly or non-randomly organized pores, said primary scaffold
providing a structural support for the secondary scaffold
incorporated therein and wherein said secondary scaffold comprises
collagen or collagen-containing material in combination with a
non-ionic PLURONIC.RTM.-type surfactant; b) debriding or debriding
and microfracturing said tissue defect for implantation of said
DSTI; c) cutting or trimming the DSTI into a size of the tissue
defect; d) rehydrating said DSTI with a physiologically acceptable
solution with or without cells and with or without a cell
modulator; e) implanting said DSTI into said defect; and f)
covering said implanted DSTI with a tissue adhesive.
Description
[0001] This application claims priority of the U.S. application
Ser. No. 11/894,124 filed on Aug. 20, 2007 and of the Provisional
application Ser. No. 60/967,886 filed Sep. 6, 2007.
BACKGROUND OF THE INVENTION
FIELD OF INVENTION
[0002] The current invention concerns a method for improvement of
differentiation and transformation of cells, stem cells,
mesenchymal stem cells, bone marrow mesenchymal cells and
chondrocytes cultured in a double-structured tissue implant (DSTI)
and a method for determination of a rate of differentiation,
transformation or migration of such cells. The invention further
concerns use of differentiated and transformed cells migrated and
cultured in the DSTI for implantation into tissue defects.
[0003] In particular, the invention concerns a double-structured
tissue implant (DSTI) comprising a primary scaffold and a secondary
scaffold generated and positioned within the primary scaffold. The
primary scaffold is a porous collagen-comprising material having
randomly or non-randomly oriented pores of substantially
homogeneous defined diameter. Under the most favorable conditions,
the pores are vertically oriented and represent a high percentage
of the porosity of the scaffold. The secondary scaffold is
generated within the primary scaffold by introducing a composition
comprising a soluble collagen solution in combination with a
non-ionic surfactant into the pores of the primary scaffold and
solidifying said composition within said pores using a process
comprising lyophilization and dehydrothermal treatment.
[0004] Each, the primary and secondary scaffold, independently, or
together, provides a structural support for cells and/or each
separately may contain cells, progenitor cells, pharmaceutical
agents or growth modulators.
[0005] The DSTI has improved stability, resistance to shrinkage,
swelling, dissolution, wetting, storageability, an increased
surface area for cell adhesion, growth, differentiation and/or
transformation, chemotactic properties for cells or progenitor
cells as well as properties conducive to cell differentiation and
transformation.
[0006] The chemotactic propeties of DSTI toward mesenchymal stem
cells and bone marrow stem cells are enhanced with addition of
morphogenic growth factors that initiate gene expression of
mesenchymal stem cell (MSC) and bone marrow mesenchymal stem cells
(BMSC). Newly expressed mesenchymal stem cell and bone marrow
mesenchymal stem cells cultured in the DSTI differentiate to the
chondrocyte phenotype and ultimately into chondrocytes that are
producing extracellular matrix leading to production of a new
hyaline cartilage.
BACKGROUND AND RELATED DISCLOSURES
[0007] Articular (hyaline) cartilage is a highly organized
avascular tissue composed of chondrocytes embedded within an
extracellular matrix of collagens, proteoglycans, noncollagenous
proteins and glycoproteins which make up about 20-40% of the wet
weight of the tissue. The chemical bonding between the water and
the matrix molecules provides for the smooth articulation of joint
surfaces, and acts as a cushion in response to compressive, tensile
and shearing forces. Because cartilage is avascular, it has little
capacity for self-repair and untreated lesions will result in
continued damage to the joint which eventually leads to
osteoarthritis.
[0008] Current treatment options for osteoarthritis or articular
cartilage injuries include microfracture, autologous chondrocyte
implantation and osteochondral autograft or allograft
transplantation (OATS). Each of these treatments has certain
disadvantages. Microfracture leads to the development of
fibrocartilage which is inferior to a hyaline cartilage because it
lacks the biomechanical strength to support the compressive loads.
Autologous chondrocyte implantation requires cartilage biopsy and,
therefore, a second surgery, and is thus a technically challenging
procedure. Osteochondral autograft transplantation has limitations
as to host tissue availability for large lesions as well as limited
availability of allograft material.
[0009] It would therefore be advantageous to have available a
method and means to treat the injured or osteoarthritic cartilage
that would be more practical and did not require multiple
surgeries.
[0010] Collagen matrices for use as an implant for repair of
cartilage defects and injuries are known in the art. Of a
particular interest is a honeycomb structure developed by Koken
Company, Ltd., Tokyo, Japan, under the trade name Honeycomb Sponge,
described in the Japanese patent JP3170693, hereby incorporated by
reference. Other patents related to the current subject disclose
collagen-based substrates for tissue engineering (U.S. Pat. No.
6,790,454) collagen/polysaccharide bi-layer matrix (U.S. Pat. No.
6,773,723), collagen/polysaccharide bi-layer matrix (U.S. Pat. No.
6,896,904), matrix for tissue engineering formed of hyaluronic acid
and hydrolyzed collagen (U.S. Pat. No. 6,737,072), method for
making a porous matrix particle (U.S. Pat. No. 5,629,191) method
for making porous biodegradable polymers (U.S. Pat. No. 6,673,286),
process for growing tissue in a macroporous polymer scaffold (U.S.
Pat. No. 6,875,442), method for preserving porosity in porous
materials (U.S. Pat. No. 4,522,753), method for preparation of
collagen-glycosaminoglycan composite materials (U.S. Pat. No.
4,448,718), procedures for preparing composite materials from
collagen and glycosaminoglycan (U.S. Pat. No. 4,350,629) and a
crosslinked collagen-mucopolysaccharide composite materials (U.S.
Pat. No. 4,280,954).
[0011] However, many of the above disclosed structures have
uncontrolled and unpredictable parameters such as an uneven and
uncontrolled porosity, uneven density of pores, uneven sizes of the
pores and random distribution of pores within the support matrix.
Such uncontrolled parameters lead to an uneven distribution of
cells and to uneven distribution of extracellular matrix produced
by the cells because the actual usable pore structure represents
only a small percentage of the total implant. Additionally, when
introduced into tissue defects or cartilage lesions during the
surgery, these structures are difficult to handle as they are
unstable and do not have appropriate wetting properties in that
they may shrink or swell and, therefore, are not easily manipulated
by a surgeon.
[0012] For a tissue implant to be suitable for implantation,
particularly for implantation into the cartilage lesion, the
implant needs to be stable, easily manipulated, easily stored in
sterile form and have a long shelf-life.
[0013] In order to provide a more uniform and sterically stable
support structure for implantation into a tissue defect or
cartilage lesion, inventors previously developed a collagen matrix
having narrowly defined size and density of pores wherein the pores
are uniformly distributed, mostly vertically oriented and
non-randomly organized. This matrix is disclosed in the co-pending
patent application Ser. No. 11/523,833, filed on Sep. 19, 2006,
hereby incorporated by reference in its entirety. Additionally, the
acellular matrix suitable to be used as the primary scaffold is
described by inventors in the U.S. Pat. No. 7,217,294, on May 15,
2007, hereby incorporated in its entirety.
[0014] However, even with the above-described improvements, a
solution to problems faced by the surgeon during surgery is still
lacking. A practicality needed for routine use of the tissue
implants, such as, for example, the articular cartilage implants by
the orthopedic surgeons, where the implant needs to be readily
available, manipulatable, wettable, stable, sterile and able to be
rapidly prepared and used for implantation, is still not achieved.
All the previously described and prepared matrices or scaffolds
require multiple steps before they are fully implantable.
[0015] Thus, it would be advantageous to have available an implant
that would be easily manufactured and packaged, would be stable for
extended shelf-life, would be easily manipulatable and rapidly
wettable upon introduction into the lesion, could provide a support
for cell migration and seeding, for cell differentiation and
transformation and that could have, additionally, pre-incorporated
drug or modulator in at least one compartment of the implant.
[0016] The implant should also allow the surgeon to introduce a
drug or modulator during the surgical procedure.
[0017] It would also be an advantage to provide an implant with an
increased area of internal membranes which, while not interfering
with cell migration and nutrient exchange, nevertheless, would
provide a substrate favorable to cell adhesion, growth and
migration as well as cell differentiation, transformation and
formation of hyaline cartilage.
[0018] It is, therefore, a primary object of this invention to
provide an implant that would have a double-structure comprising of
a primary scaffold and a secondary scaffold compartments where each
compartment of the implant can assume a different function, be
incorporated with cells, different drugs or modulators and/or be
selectively chosen for performing the different functions following
the implantation.
[0019] The current invention provides such double-structured tissue
implant and/or a method for use and fabrication thereof by
providing the double-structured tissue implant (DSTI) comprising a
first scaffold providing a sterically stable and biocompatible
support structure, preferably made of Type I collagen, having
defined pore sizes and density with said pores organized mostly
vertically, and a second scaffold wherein said second scaffold is
formed within said pores of said first scaffold. The
double-structured scaffold of the invention is stable, resistant to
shrinkage, swelling and dissolution, rapidly wettable, prepared in
the sterile storageable form having a long-shelf life that can be
easily manipulated and surgically implanted.
[0020] All patents, patent applications and publications cited
herein are hereby incorporated by reference in their entirety.
SUMMARY
[0021] One aspect of the current invention is a method for
improvement of differentiation and transformation of cells, stem
cells, mesenchymal stem cells, bone marrow mesenchymal stem cells
or chondrocytes by introducing and culturing such cells in a
double-structured tissue implant (DSTI).
[0022] Another aspect of the current invention is a
double-structured tissue implant, process for preparation thereof
and a method for use thereof for differentiation and transformation
of cells, stem cells, mesenchymal stem cells, bone marrow
mesenchymal stem cells or chondrocytes.
[0023] Yet another aspect of the current invention is a DSTI
comprising migrated and cultured cells, stem cells, mesenchymal
stem cells, bone marrow mesenchymal stem cells or chondrocytes
suitable for implantation into tissue defects.
[0024] Still another aspect of the current invention is a method
for determination of a rate of differentiation and transformation
of cells, stem cells, mesenchymal stem cells, bone marrow
mesenchymal stem cells or chondrocytes.
[0025] Still yet another aspect of the current invention is a
double-structured tissue implant having two distinct qualitatively
different compartments wherein each of the compartments may be
independently seeded with cells, stem cells, mesenchymal stem
cells, bone marrow mesenchymal stem cells or chondrocytes or
wherein one or both compartments of the implant may comprise a
pharmaceutical agent or growth modulator.
[0026] Another aspect of the current invention is a process for
preparation of a double-structured implant by providing a primary
porous scaffold prepared from a biocompatible collagen material
wherein said scaffold has a substantially homogenous defined
porosity and uniformly distributed randomly and non-randomly
organized pores of substantially the same size of defined diameter
of about 300.+-.100 .mu.m, wherein said primary scaffold is brought
in contact with a soluble collagen based solution comprising at
least one non-ionic surfactant (Basic Solution), wherein such
solution is introduced into said pores of said primary scaffold,
stabilized therein by precipitation or gelling, dehydrated,
lyophilized and dehydrothermally processed to form a distinctly
structurally and functionally different second scaffold within said
pores of said primary scaffold.
[0027] Still yet another aspect of the current invention is a DSTI
having chemotactic properties toward stem cells, mesenchymal stem
cells and bone marrow stem cells that make these cells migrate into
such DSTI, differentiate and/or be transformed within the DSTI
wherein such differentiation and transformation is enhanced with
addition of morhogenetic growth factors.
[0028] Yet another aspect of the current invention is a method for
determination of a rate of differentiation and transformation of
stem cells, mesenchymal stem cells and bone marrow stem cells
seeded within a DSTI.
[0029] Still yet another aspect of the current invention is a
method for implantation of the DSTI containing mesenchymal stem
cells, bone marrow stem cells or bone marrow slurry in a tissue
defect.
BRIEF DESCRIPTION OF FIGURES
[0030] FIG. 1 shows a scanning electron microscopic view of a
hydrated double-structured tissue implant (DSTI).
[0031] FIG. 2 is a photomicrograph of a DSTI showing pores filled
with human chondrocytes.
[0032] FIG. 3 is a graph illustrating compatibility of cells with
DSTIs by determining production and accumulation of S-GAG/DNA in 21
days by chondrocytes embedded in DSTIs (DSTI-1- DSTI-3) and in a
collagen matrix (control) that is not lyophilized or treated
dehydrothermally.
[0033] FIG. 4 is a graph showing gene expression of Type I collagen
(COL 1A1), Type II collagen (COL 2A1) and aggrecan (AGC 1) by bone
marrow stem cells (BMSC) and by human articular chondrocytes (hAC)
seeded in the DSTI and cultured for two (2 WK) and four weeks (4
WK).
[0034] FIG. 5 is a graph illustrating migration of bone marrow stem
cells (BMSC migrated) and human articular chondrocytes (hAC
migrated) into the DSTI and production of S-GAG/DNA by these cells
after four weeks of culture compared to BMSC and hAC controls
without the DSTI.
[0035] FIG. 6 is a graph illustrating gene expression,
up-regulation of Type II collagen (COL 2A1) and down-regulation of
Type I collagen (COL 1A1) mRNA production by bone marrow stem cells
(BMSC) that migrated into DSTI compared to mRNA produced by human
articular chondrocytes (hAC).
[0036] FIG. 7 is a graph illustrating chemoattractant properties of
DSTI for bone marrow stem cells. The FIG. 7 shows a chemotactic
activity of BMSC in response to various culture media formulations
in the presence or absence of DSTI. Bovine serum albumin (BSA),
chondrogenic (CCM) and growth (Growth) media were used with or
without DSTI to determine chemoattractant property of the DSTI.
Chemoattractant property of DSTI is expressed as an average
increase in cell count per field.
[0037] FIG. 8 is a graph illustrating the chemoattractant property
of DSTI that can be enhanced by the addition of a growth factor,
namely repulsive guidance molecule (RGMA). Fetal bovine serum (FBS)
medium was used as a control against the DSTI containing FBS medium
and DSTI containing FBS medium and RGMA.
[0038] FIG. 9 is a graph illustrating up-regulation of cartilage
specific genes determined by the levels of mRNA for protein
aggrecan (AGC1), collagen Type I (COL 1A1), collagen Type II (COL
2A1), cartilage oligomeric matrix protein (COMP), and the
chondrogenic transcription factor SOX9 by bone marrow mesenchymal
stem cell (BMSC) migration toward DSTI containing the chondrocyte
growth medium and growth factors RGMA, BMP-2 and BMP-7 compared to
DSTI containing the chondrocyte growth medium without the growth
factor (DSTI) and to the chondrocyte growth medium only without
DSTI and growth factor (Empty).
[0039] FIG. 10 is a graph illustrating up-regulation of cartilage
specific genes determined by the levels of mRNA for protein
aggrecan (AGC1), collagen Type I (COL 1A1), collagen Type II (COL
2A1), cartilage oligomeric matrix protein (COMP), and the
chondrogenic transcription factor SOX9 by human articular
chondrocytes migrated toward DSTI containing the chondrocyte growth
medium and growth factors RGMA, BMP-2 and BMP-7 compared to DSTI
containing the chondrocyte growth medium without the growth factor
(DSTI) and to the chondrocyte growth medium only without DSTI and
growth factor (Empty).
[0040] FIG. 11 is a graph showing an effect of hydrostatic pressure
on production of mRNA for Type I collagen (COL 1A1), Type II
collagen (COL 2A1) and aggrecan (AGC1) for human articular
chondrocytes (hAC) and bone marrow stem cells (BMSC). The effect of
the hydrostatic pressure is expressed as increase in mRNA over
levels observed at static pressure.
[0041] FIGS. 12A-12F are schematic illustrations of treatment
protocols for implantation of the DSTI into cartilage lesions using
double-structured tissue implants containing stem cells,
mesenchymal stem cells or bone marrow stem cells. FIG. 12A is a
schematic illustration of a method for implantation of DSTI into
the tissue lesion or defect where the DSTI is rehydrated with
non-differentiated or pre-differentiated stem cells or mesenchymal
stem cells. The stem or mesenchymal stem cells are first dissolved
in a physiologically acceptable solution and such solution is
applied to the dehydrated DSTI previously trimmed to a size and
shape of the defect. Rehydrated DSTI is implanted into the defect
and sealed with an adhesive. FIG. 12B illustrates a method for
implantation of the DSTI with microfracture of the subchondral
plate. The subchondral plate is penetrated and the rehydrated DSTI
is placed in the defect as in FIG. 12A. After sealing the defect
containing rehydrated DSTI with the adhesive, the marrow components
are able to enter the DSTI through the microfracture. FIG. 12C
illustrates a method for implantation of DSTI with microfracture as
seen in FIG. 12B except that the adhesive is also applied to the
bottom of the lesion in between the microfracture penetrations. The
FIG. 12D shows the implantation of the DSTI into the tissue lesion
where the DSTI is rehydrated with mesenchymal stem cells. The DSTI
rehydrated with MSC is then implanted and the defect is sealed with
adhesive. FIG. 12E show implantation of the DSTI seeded with bone
marrow stem cells that were cultured and optionally could also be
activated by applying a hydrostatic/constant pressure regimen. The
DSTI seeded with these cultured and/or activated cells is placed
into the lesion or defect and the defect is sealed with adhesive
placed over the implant. FIG. 12F illustrates implantation of the
DSTI into the tissue defect where the DSTI is rehydrated with
freshly aspirated bone marrow that is either undiluted or diluted
with saline or another physiologically acceptable solution.
DEFINITIONS
[0042] As used herein:
[0043] "Double-structured tissue implant" or "DSTI" means a tissue
implant prepared according to a process of the invention wherein
the primary scaffold is loaded with a Basic Solution thereby
forming a composite that is subsequently subjected to
precipitation, dehydration and lyophilization to obtain a
Lyophilized Composite that is subsequently treated with
dehydrothermal (DHT) treatment to result in a stable
double-structured tissue implant.
[0044] "Primary scaffold" means a porous honeycomb, sponge, lattice
or another structure made of collagen or collagen based material
having randomly or non-randomly oriented pores of substantially
homogenous defined diameter. Under the most favorable conditions,
the pores are vertically oriented and represent a high percentage
of the porosity of the scaffold.
[0045] "Secondary scaffold" means a collagen based structure
prepared from a collagen or collagen-based compound in the presence
of a non-ionic surfactant. The secondary scaffold is generated
within the primary scaffold by introducing a composition comprising
a soluble collagen solution in combination with a non-ionic
surfactant (Basic Solution) into the pores of the primary scaffold
and solidifying said composition within said pores using a novel
process of the invention.
[0046] "Basic Solution" means a solution comprising a collagen in
admixture with a surfactant, preferably PLURONIC.RTM.-Type
surfactant, neutralized to the pH of about 7.4. Basic Solution is
used for preparation of the secondary scaffold.
[0047] "Composite" means a primary scaffold loaded with a
composition comprising a precipitated or gelled soluble collagen in
combination with a non-ionic surfactant (Basic Solution). The
composite is in a hydrated form because the Basic Solution is added
in a fluid form as a gel, suspension or solution.
[0048] "Lyophilized composite" means the hydrated "Composite", as
defined above, that is subsequently subjected to a freezing and
lyophilization step.
[0049] "Collagen matrix" means a collagen support matrix that is
not lyophilized or dehydrothermally treated.
[0050] "Surfactant" means a non-ionic or ionic surfactant polymer.
Suitable surfactants, such as PLURONIC.RTM.-Type polymers or
TRITON.RTM.-Type polymers, are non-ionic co-polymer surfactants
consisting of polyethylene and polypropylene oxide blocks.
[0051] "TRITON.RTM.-Type surfactants are commercially available
derivatized polyethylene oxides, such as for example, polyethylene
oxide p-(1,1,3,3-tetramethylbutyl)-phenyl ether, known under its
trade name as TRITON.RTM.-X100. Other TRITON.RTM.-Type surfactants
that may be suitable for use in the instant invention are
TRITON.RTM. X-15, TRITON.RTM. X-35, TRITON.RTM. X-45, TRITON.RTM.
X-114 and TRITON.RTM. X-102. TRITON.RTM. surfactant are
commercially available from, for example, Union Carbide, Inc.
PLURONIC.RTM.-Type surfactants are commercially available block
co-polymers of polyoxyethylene (PEO) and polyoxypropylene (PPO)
having the following generic organization of polymeric blocks:
PEO-PPO-PEO (Pluronic) or PPO-PEO-PPO (Pluronic R). Exemplary
PLURONIC.RTM.-Type surfactants are PLURONIC.RTM. F68, PLURONIC.RTM.
F127, PLURONIC.RTM. F108, PLURONIC.RTM. F98, PLURONIC.RTM. F88,
PLURONIC.RTM. F87, PLURONIC.RTM. F77, PLURONIC.RTM. F68,
PLURONIC.RTM. 17R8 and PLURONIC.RTM. 10R8.
[0052] "The porosity" means a pore size defined by the diameter of
holes within the primary scaffold as well as density of the pore
distribution as a function of cross-sectional area in millimeters.
Porosity is defined as a total volume of pores relative to the
implant.
[0053] "Substantially homogeneous" means at least 85-99%
homogeneity. Preferable homogeneity is between 95% and 99%.
[0054] "Substantially homogeneous porosity" means that a pore size
and diameter is within pore size range of about 200-300.+-.100
.mu.m, preferably 300.+-.50 .mu.m, in diameter.
[0055] "Wettability" means an ability to quickly absorb a fluid
into the DSTI without changes in the size and shape of the
implant.
[0056] "Shrinkage" means a volumetric reduction in surface area in
all dimensions of a double-structured tissue implant.
[0057] "Swelling" means a volumetric increase of a surface area in
all dimensions of a double-structured tissue implant.
[0058] "Dissolution" means the act of a solid matter being
solubilized by a solvent.
[0059] "Rehydration" means the act of hydrating, wetting or
rewetting a dehydrated composite, lyophilized composite, stand
alone secondary scaffold or double-structured tissue implant.
[0060] "Shape-memory" means the capacity of the rehydrated DSTI to
maintain its shape and size after deformation.
[0061] "Dehydrothermal treatment" means removing water at low
pressure and at high temperature for cross-linking of polymers.
[0062] "Top surface" means an apical or synovial side of the matrix
turned toward the joint.
[0063] "Bottom surface" means basal, closest to bone surface of the
matrix.
[0064] "Chondrocytes" means the cells naturally residing in
articular cartilage.
[0065] "Stem cells" or "SC" means the human body's master cells,
with the ability to grow into any one of the body's more than 200
cell types. Stem cells are the cells that can differentiate into
many different cell types when subjected to the right biochemical
signals.
[0066] "Mesenchymal stem cells" or "MSC" means multipotent stem
cells that can proliferate and differentiate into a variety of
cells types. Cell types that MSC have been shown to differentiate
into include osteoblasts, chondrocytes, myocytes or adipocytes.
MSCs can encompass multipotent cells derived from bone marrow or
from other non-marrow tissues, such as adult muscle side-population
cells or the Wharton's jelly present in the umbilical cord. MSCs
have a large capacity for self-renewal while maintaining their
multipotence. However, the capacity of cells to proliferate and
differentiate is known to decrease with the age of the donor, as
well as the time in culture. Further, no unique surface markers
have been identified for MSCs.
[0067] "Bone marrow" means the soft, fatty, vascular tissue that
fills most bone cavities and is the source of red blood cells and
many white blood cells.
[0068] "Bone marrow stem cells", "bone marrow mesenchymal stem
cells" or "BMSC" means the most primitive cells in the bone marrow
or bloodstream or umbilical cord cells that after entering the
bloodstream travel to the bone marrow.
[0069] "Stromal cells" means a highly heterogenous cell population
consisting of multiple cell Types with different potentials for
proliferation and differentiation.
[0070] "Human articular chondrocytes" or "hAC" means chondrocytes
isolated from human articular cartilage.
[0071] "S-GAG" means sulfated glycosaminoglycan.
[0072] "AGC1" means aggrecan.
[0073] "COL 1A1" means Type I collagen gene.
[0074] "COL 2A1" means Type II collagen gene.
[0075] "RGMA" means repulsive guidance molecule.
[0076] "SOX9" means the chondrogenic transcription factor.
[0077] "COMP" means cartilage oligomeric matrix protein.
[0078] "RT-PCR" means reverse transcription-polymerase chain
reaction used for quantification of gene expression through
detection of messenger RNA (mRNA) levels.
[0079] "Cell modulator" means a pharmaceutical agent, drug, growth
factor, growth hormone, mediator, enzyme promoting cell
incorporation, enzyme promoting cell proliferation, enzyme
promoting cell division, a pharmaceutically acceptable excipient,
additive or buffer.
DETAILED DESCRIPTION OF THE INVENTION
[0080] The current invention concerns a method for treatment tissue
defects by using a novel double-structured tissue implant (DSTI)
that has properties resulting in chemotactic chemoattraction of
cells, stem cells, mesenchymal stem cells, bone marrow mesenchymal
cells and chondrocytes and in improvement of their differentiation
and transformation when these cells are cultured in said DSTI. The
invention further concerns a method for determination of a rate of
differentiation, transformation or migration of such cells. The
invention additionally concerns use of differentiated and
transformed cells migrated or seeded into and cultured in the DSTI
for implantation into tissue defects ultimately leading to
production of hyaline cartilage or another replacement tissue.
[0081] The DSTI has improved stability, resistance to shrinkage,
swelling, dissolution, wetting, storageability, an increased
surface area for cell adhesion, growth, differentiation and/or
transformation, as well as chemotactic properties for cells or
progenitor cells. The chemotactic properties of DSTI toward
mesenchymal stem cells and bone marrow stem cells are
advantageously enhanced with addition of morphogenetic growth
factors that initiate gene expression of mesenchymal stem cell
(MSC) and bone marrow mesenchymal stem cells (BMSC) and their
differentiation to the chondrocyte phenotype and extracellular
matrix production leading to and resulting in production of a new
hyaline cartilage.
[0082] Finally, the invention is directed to a double-structured
tissue implant suitable for implantation into a tissue lesion, and
to the method for use of said implant in a clinical setting for
repair of tissue lesions. The DSTI is particularly suitable for
implantation into articular cartilage defects where it facilitates
regeneration and formation of new hyaline cartilage. The secondary
scaffold facilitates better cell attachment while at the same time
maintaining a porous character of the DSTI. The DSTI may be
advantageously hydrated before or during surgery with a
physiologically acceptable solution optionally containing cells,
stem cells, mesenchymal stem cells or bone marrow stem cells or
bone marrow slurry. The double-structured implant has improved
properties compared to a single structured implant and provides for
variability in use.
[0083] A scanning electron micrograph of a rehydrated
double-structured tissue implant (DSTI) is seen in FIG. 1. FIG. 1
is a cross-section of the DSTI showing the multiple flat
feather-like surfaces spanning the pores as well as struts and
flaps throughout and within the implant's pores. These surfaces
generated by combination of the primary and secondary scaffold are
responsible for improved cell attachment. The DSTI is produced by a
process disclosed in Section III.
[0084] I. Double-Structured Tissue Implant
[0085] Double-structured tissue implant (DSTI) is a composite
structure comprising a primary scaffold and a secondary scaffold
generated and positioned within the primary scaffold. The primary
scaffold is a porous collagen-comprising material having randomly
or non-randomly, preferably vertically, oriented pores of
substantially homogeneous defined diameter. The primary scaffold
represents a high percentage of the DSTI. The secondary scaffold is
generated within the primary scaffold by introducing a composition
comprising a soluble collagen solution in combination with a
non-ionic surfactant into the pores of the primary scaffold and
solidifying said composition within said pores using a specific
multi-step process described in Section III.
[0086] The DSTI thus comprises two separate compartments, namely
the primary scaffold that provides a structural support for the
secondary scaffold incorporated within the primary scaffold. Each,
the primary and secondary scaffold independently, or together,
provides a structural support for cells and/or each separately may
contain cells, progenitor cells, pharmaceutical agents or growth
modulators.
[0087] A. The Primary Scaffold
[0088] The primary scaffold is a collagen-based matrix prepared as
a honeycomb, lattice, sponge or any other similar structure made of
a biocompatible and/or biodegradable collagen containing material
of defined density and porosity that is stable, pliable,
storageable and, most importantly, highly porous.
[0089] Typically, the primary scaffold is prepared from compounds
influencing fibrillar organization, such as collagen,
collagen-containing composition or collagen containing a polymer.
Representative compounds suitable for preparation of the primary
scaffold are a Type I collagen, Type II collagen, Type III
collagen, Type IV collagen, Type VI collagen, gelatin, collagen
containing agarose, collagen containing hyaluronan, collagen
containing proteoglycan, collagen containing glycosaminoglycan,
collagen containing glycoprotein, collagen containing glucosamine,
collagen containing galactosamine, collagen containing fibronectin,
collagen containing laminin, collagen containing growth factor,
collagen containing cytokine, collagen containing elastin, collagen
containing fibrin, collagen containing polylactic acid, collagen
containing polyglycolic acid, collagen containing polyamino acid,
collagen containing polycaprolactone, collagen containing
polypeptide, a copolymer thereof, each alone or in combination.
Additionally, the primary scaffold may be prepared from the
collagen precursors such as, for example, peptide monomers, such as
alpha 1 (Type I), and alpha 2 (Type I) collagen peptide or alpha 1
(Type I) alpha 2 (Type I) peptides, alone or in combination, or
from a combination of precursors, such as 2 (alpha 1, Type I)
peptide and 1 (alpha 2, Type I) peptide together with collagens,
such as Type IX or Type XI collagen.
[0090] The collagen containing material used for preparation of the
primary scaffold may further be supplemented with other compounds,
such as pharmaceutically acceptable excipients, surfactants,
buffers, additives and other biocompatible components.
[0091] Preferably, the primary scaffold of the invention is
prepared from collagen and most preferably from Type I collagen or
from a composition containing Type I collagen.
[0092] In one embodiment, the primary scaffold is a structure
containing a plurality of narrowly defined randomly or non-randomly
organized pores having a substantially homogeneous narrowly defined
size and diameter that are uniformly distributed through the
scaffold, dividing the scaffold space into columns or pore network.
The exemplary primary scaffold is described in the co-pending
application Ser. No. 11/523,833, filed on Sep. 19, 2006, herein
incorporated by reference in its entirety.
[0093] In another embodiment, the primary scaffold may be the Type
I collagen-based support matrix that is a porous honeycomb, sponge,
lattice or scaffold having randomly or non-randomly organized pores
of variable pore diameters such as described in, for example, the
U.S. Pat. No. 7,217,294 on May 15, 2007, herein incorporated by
reference.
[0094] In yet another embodiment the primary scaffold is a
honeycomb collagen matrix developed by Koken Company, Ltd., Tokyo,
Japan, under the trade name Honeycomb Sponge, described in the
Japanese patent JP3170693, hereby incorporated by reference.
[0095] The primary scaffold according to the invention has,
preferably, a substantially defined pore size in diameter and pore
density in randomly or non-randomly organized manner that creates
an apical (top) or basal (bottom) surface to the implant where the
sizes and diameters of the pores on both the apical or basal
surface are substantially the same. When used as a primary scaffold
only, the scaffold provides conditions for a sterically-enhanced
enablement of cells. Chondrocytes seeded in the primary scaffold,
for example, have been shown to produce within said implant an
extracellular matrix comprising glycosaminoglycan and Type II
collagen in ratios characteristic for a normal healthy articular
hyaline cartilage.
[0096] A secondary scaffold structure is generated within the pores
of the primary scaffold. To that end, the primary scaffold is
loaded with a composition suitable for preparation of the secondary
scaffold (Basic Solution). Such composition comprises a soluble
collagen, collagen-containing or collagen-like mixture, typically
of Type I collagen, in combination with a non-ionic surfactant.
[0097] B. The Secondary Scaffold
[0098] The secondary scaffold is created or generated within the
pores of the primary scaffold. The secondary scaffold is a
qualitatively different structure formed within the confines of the
first scaffold.
[0099] The secondary scaffold is generated by a process comprising
preparing a soluble collagen-based composition as described below,
further comprising a suitable non-ionic or ionic surfactant (Basic
Solution).
[0100] The secondary scaffold comprises a collagen, methylated
collagen, gelatin or methylated gelatin, collagen-containing and
collagen-like mixtures, said collagen being typically of Type I or
Type II, each alone, in admixture, or in combination, and further
in combination with a surfactant, preferably a non-ionic
surfactant. The suitable non-ionic surfactant is preferably a
polymeric compound such as a PLURONIC.RTM.-Type polymer.
[0101] In preparation of the DSTI, said composition suitable for
generation of the secondary scaffold within the primary scaffold is
then brought into contact with a primary scaffold structure by
absorbing, wicking, soaking or by using a pressure, vacuum, pumping
or electrophoresis, etc., to introduce said composition for the
secondary scaffold into the pores of the primary scaffold. In
alternative, the primary scaffold may be immersed into the
composition for the secondary scaffold. The primary scaffold
containing basic solution is then processed according to Section
III.
[0102] C. Surfactants
[0103] Improved properties of the DSTI, such as its rapid
wettability and resistance to shrinkage, swelling and dissolution,
are due to a presence of a secondary scaffold as a distinct
functional entity.
[0104] The secondary scaffold prepared according to the process of
the invention requires, as an essential part, a presence of a
surfactant, preferably a non-ionic or, in some instances, even an
ionic surfactant. The surfactant, preferably the non-ionic
surfactant of Type such as TRITON.RTM. or PLURONIC.RTM., preferably
PLURONIC.RTM. F127, is an essential component of a composition used
for preparation of the secondary scaffold, or micellar substrate
bound to the implant. The presence of the surfactant improves
stability and particularly wettability and rehydration properties
of the implant without causing its shrinkage or swelling.
[0105] Suitable surfactants, such as PLURONIC.RTM.-Type polymers or
TRITON.RTM.-Type polymers, are non-ionic co-polymer surfactants
consisting of polyethylene and polypropylene oxide blocks.
[0106] TRITON.RTM.-Type surfactants are commercially available
derivatized polyethylene oxides, such as for example, polyethylene
oxide p-(1,1,3,3-tetramethylbutyl)-phenyl ether, known under its
trade name as TRITON.RTM.-X100. Other TRITON.RTM.-Type surfactants
that may be suitable for use in the instant invention are
TRITON.RTM. X-15, TRITON.RTM. X-35, TRITON(.RTM. X-45, TRITON.RTM.
X-114 and TRITON.RTM. X-102. TRITON.RTM. surfactant are
commercially available from, for example, Union Carbide, Inc.
[0107] PLURONIC.RTM.-Type surfactants are commercially available
block co-polymers of polyoxyethylene (PEO) and polyoxypropylene
(PPO) having the following generic organization of polymeric
blocks: PEO-PPO-PEO (Pluronic) or PPO-PEO-PPO (Pluronic R).
Exemplary PLURONIC.RTM.-Type surfactants are PLURONIC.RTM. F68,
PLURONIC.RTM. F127, PLURONIC.RTM. F108, PLURONIC.RTM. F98,
PLURONIC.RTM. F88, PLURONIC.RTM. F87, PLURONIC.RTM. F77,
PLURONIC.RTM. F68, PLURONIC.RTM. 17R8 and PLURONIC.RTM. 10R8.
[0108] The most preferred non-ionic surfactant of
PLURONIC.RTM.-Type suitable for use in the invention is a block
co-polymer of polyoxyethylene (PEO) and polyoxypropylene (PPO) with
two 96-unit hydrophilic PEO blocks surrounding one 69-unit
hydrophobic PPO block, known under its trade name as PLURONIC.RTM.
F127. PLURONIC.RTM. surfactants are commercially available from
BASF Corp.
[0109] D. Double-Structured Tissue Implant
[0110] The double structured tissue implant (DSTI) is prepared by
treating the primary scaffold loaded with a combination of the
soluble collagen and non-ionic surfactant and processes according
to the process for preparation of the DSTI described below in
Section III, Scheme 1.
[0111] Briefly, the primary scaffold is loaded with the
collagen/surfactant combination, precipitated or gelled, washed,
lyophilized and dehydrothermally treated to solidify and stabilize
the secondary scaffold within the pores of the primary
scaffold.
[0112] The double-structured tissue implant can be seeded with
cells, loaded with pharmaceutical agents, drugs or growth
modulators. Additionally and preferably, the two of its distinct
compartments, namely the primary scaffold and the secondary
scaffold, can each be independently loaded with living cells, cell
suspension, with a pharmaceutically effective agent or agents or
with a growth modulator. These may be loaded into the implant
individually or in any possible combination, such as, for example,
where the cells may be introduced into one component and the drug
into the second component, or the drug into one component and the
modulator into the second component and/or any variation
thereof.
[0113] The DSTI loaded with chondrocytes is shown in FIG. 2 wherein
chondrocytes are seen to form clusters within the pores of the DSTI
and retain their rounded morphology. The cells are seen to be well
distributed within the pores of the DSTI and attached to the core
(primary) scaffold or to the flaps and struts of the secondary
scaffold.
[0114] The DSTI provides a biocompatible environment for culturing,
growth, proliferation, differentiation and transformation of cell,
particularly chondrocytes, stem cells, mesenchymal stem cells, bone
marrow cells and bone marrow mesenchymal cells.
[0115] II. Properties of the Double-Structured Tissue Implant
[0116] The DSTI of the invention has distinctly improved properties
when compared to the primary scaffold alone or to a composite
loaded with a composition for preparation of the secondary scaffold
(Composite), unprocessed, or to the Composite that has been frozen
and lyophilized (Lyophilized Composite) but not dehydrothermally
treated.
[0117] Typically, a tissue implant is implanted into a tissue
defect during a surgery. Also typically, such surgery has a
time-limit on implantation that has preferably about one hour
window when the implant is placed into the defect. For these
reasons, it is important that a specification for an implantable
double-structured tissue implant provides stability, resistance to
change in shape, size and shrinkage or swelling, resistance to
dissolution, consistency with respect to pore size permitting an
ingrowth of cells from the native tissue into the implant and
conditions for synthesis and formation of extracellular matrix
within the implant. The DSTI appears to have all the above
properties.
[0118] Furthermore, the presence of the secondary scaffold improves
the function of the DSTI by providing a multitude of small
membranous attachment sites which can provide cell anchorage and
phenotype stability while preserving the through porosity of the
overall implant, thereby allowing nutrients and growth factors and
migratory cells to permeate the implant.
[0119] A Stability of the Double-Structured Tissue Implant
[0120] From the point of view of the implantability, stability of
the implant is one of the major requirements. The implant stability
depends on several factors. There must be low or, preferably, no
initial dissolution of collagen from the implant into the
physiological fluids and there must be low or preferably no change
in size and shape of the implant following rehydration or wetting
before, during or after surgery prior to biodegradation in
situ.
[0121] 1. Collagen Retention and Resistance to Dissolution
[0122] One of the most important requirements for the implant is
its resistance to dissolution of its components upon wetting and
rehydration of said implant during implantation, during preparation
of the implant for implantation and subsequently also after
implantation. A minimally low dissolution or, preferably, no
dissolution of the collagen component from the implant into the
physiologic solution immediately after or before placement of the
implant into the tissue defect and into an interstitial fluid,
plasma or blood following the surgery, under normal physiological
conditions ensures continued functionality of the implant following
its implantation into the tissue defect, such as, for example into
the cartilage lesion. Low or minimal dissolution of collagen from
the implant means that the DSTI has the high retention of the
collagen within the implant during the initial, most important
implantation period.
[0123] In order to determine the stability of the implant subjected
to transport and handling, an additional study was performed with
and without agitation and the dissolution of collagen from the
DSTI. These conditions were compared to the dissolution of collagen
from the non-lyophilized composite (Composite). Results are not
shown. These studies confirmed that even with agitation, there is a
relatively small change in the accumulated release of collagen into
the solution over a period of eight days but particularly during
the first hour following the rehydration.
[0124] The DSTI has been shown to have almost complete retention of
collagen and high resistance to dissolution during the implantation
and immediately after implantation.
[0125] 2. Resistance to Change in Size and Shape
[0126] Another important feature of the DSTI is its shape-memory,
that is its resistance to change in size and shape. This feature is
very important for implant efficacy as any change in the size and
shape by shrinking or swelling can negatively effect the outcome of
the implantation surgery. An implant that would get smaller by
shrinking will not fill the defect, will not provide a structural
support for migration of cells from the surrounding tissue or cell
integration into the surrounding tissue and may also be dislodged
from the defect. Swelling of the implant could, on the other hand,
cause the implant to swell within the defect, decrease the
structural support for cells and be rejected or ejected from the
defect because of its larger size.
[0127] The resistance to change in shape and size means that for
implantation into a defect of discernable size, the functional
construct must not swell or shrink extensively upon rehydration
during time of preparation before surgery or after placement of the
implant into the defect.
[0128] The DSTI has been shown to have a high resistance to change
in size and shape of the implant consistent with shape memory after
hydration.
[0129] B. Viability of Cells Cultured in DSTI
[0130] Another important feature of the tissue implant is to
provide support and conditions for cell migration from surrounding
tissue into the implant or for the cell integration into
surrounding tissue in a case when the cells are seeded into the
DSTI before implanting. This feature is determined by cell
viability within the DSTI and provides another criteria for
determining functionality and usefulness of the DSTI.
[0131] In order for an implant to be functionally viable, the
implant must provide a structural support for cells as well as
provide or permit conditions to be provided for cell seeding into
the implant, cell growth within the implant and/or cell migration
into or from the surrounding tissue.
[0132] Conditions for cell seeding, their growth within the
implant, their nutritional and metabolic needs are designed based
on the type of cells that the implant is supposed to deliver and
support. For example, if the implant is designed for repair of a
skin defect, the cells and their requirement will be different than
if the implant is designed for repair of a chondral or bone lesion.
Conditions for structural support and conditions for promotion of
cell growth, their migration and/or integration into the
surrounding tissue will be adjusted based on the tissue where the
DSTI will be implanted and the function the implant will assume in
repair of the tissue defect.
[0133] While the DSTI of the invention is preferably suitable for
use in treatment and repair of chondral, subchondral or bone
lesions, the DSTI, as such, is suitable to be used for repair of
any other tissue or tissue defect.
[0134] The successful implant, such as, for example, DSTI implanted
into the cartilage lesion, must provide conditions allowing cells
to form, generate and/or synthesize a new extracellular matrix
(ECM). In this regard, the implant porous structure must allow
cells to migrate, be attached or aggregate into and within the
pores and function similarly to their normal function in the
healthy tissue.
[0135] Consequently, the pore size of the implant and the
consistency with respect to pore size for the ingrowth of cells is
important both for cell adhesion, extracellular matrix production
and cell to cell contact and communication. Depending on the tissue
to be repaired, the pore size of the primary and/or secondary
scaffold will vary. For example, cartilage scaffolds would have an
optimal pore size of between 200-300.+-.100 .mu.m and bone would
have a pore size in the range of 300 to 350.+-.100 .mu.m.
[0136] A significant advantage of having a double-structured tissue
scaffold arises from the increase in mechanical integrity relative
to a primary porous collagenous material because the polymerization
creates fiber-like structure between the primary and secondary
scaffold that serves as a reinforcing network for cells.
[0137] In addition, due to inclusion of the secondary scaffold
there is an increase in overall surface area within the DSTI that
permits cells spreading and migration throughout the interstices of
the DSTI. At the same time, the secondary scaffold must be designed
such that it is not of such high density that it becomes a blocking
agent that acts as a steric hindrance for cell ingrowth and tissue
repair.
[0138] DSTIs according to the invention have all the above
properties.
[0139] Chondrocytes seeded in the DSTI are viable and proliferate
as evidenced from testing for viability. Viability testing was
performed as described in Example 5. In this study, the total
number of cells and the percent of live cells that were retained in
the DSTIs (DSTI-1-DSTI-3) over the course of 21 days in culture
were determined. Results are seen in Table 1.
TABLE-US-00001 TABLE 1 Percent Viability of Chondrocytes Seeded in
DSTI Group Initial 21 Days DSTI-1 98 .+-. 1% 97 .+-. 2% DSTI-2 97
.+-. 2% 100 .+-. 1% DSTI-3 97 .+-. 2% I 98 .+-. 1%
[0140] Results summarized in Table 1 demonstrate that all three
DSTIs had initially at least 97% viability and such viablity has
not significantly changed after 3 weeks of culturing the DSTI
seeded with chondrocytes.
[0141] Chondrocytes maintained the chondrocyte cartilage phenotype.
Testing for chondrocyte phenotype included measurement of synthesis
of chondrocyte specific extracellular matrix, namely production of
S-GAG/DNA, by chondrocytes seeded in DSTIs. The testing was
performed according to Example 6. An increase in S-GAG/DNA levels
over time indicated that the environment is conducive to
chondrocyte synthesis and deposition of ECM. Results are seen in
FIG. 3.
[0142] In the study illustrated in FIG. 3, three DSTI discs and one
collagen matrix that is not lyophilized and dehydrothermally
treated (Control), seeded with chondrocytes were tested and
compared initially and after 21 days in culture for production of
S-GAG/DNA (.mu.g/.mu.g).
[0143] As seen in FIG. 3, all three DSTIs had significantly,
between 2 and 5 fold, increased S-GAG deposition after 21 days in
culture, when compared to the initial 24 hours reading. The levels
of two of three DSTIs were not significantly different from the
levels seen in non-treated collagen matrix used as a positive
control, leading to the conclusion that seeding the cells within
DSTI did not alter cell viability.
[0144] Chondrocytes seeded in the DSTI remained viable,
proliferated and synthesized proteoglycan, as determined by
increased synthesis of S-GAG. Proteoglycan is an extracellular
matrix (ECM) constituent of cartilage. Moreover, over time, the
S-GAG accumulated within the DSTIs indicating deposition of the ECM
within DSTI. Results obtained in this study show that the DSTI
provides a biocompatible environment for chondrocytes and other
cells.
[0145] C. Chemotactic Properties of DSTI toward Chondrocytes MSC
and BMSC
[0146] DSTI have been found to have chemoattraction for cells in
general and particularly for MSC and BMSC. These cells were found
to migrate into the DSTI. Such migration was proven to be important
for treatment of tissue defects.
[0147] When DSTI is implanted into the tissue defect, its
chemotactic properties attract and/or increase migration of stem
cells and particularly bone marrow stem cells into the DSTI where
they differentiate and/or are transformed into the appropriate
differentiated cells that will begin to synthesize the
extracellular matrix ultimately generating the hyaline cartilage or
another target tissue.
[0148] D. Migration
[0149] The ability of multipotent, undifferentiated bone marrow
stem cells to migrate toward and enter into the DSTI, to attach,
grow, and differentiate into chondrocytes was studied using a
procedure according to Example 7.
[0150] Following two or four weeks of culturing of DSTIs seeded
with BMSC, individual DSTIs were removed and analyzed for DNA and
S-GAG content. RT-PCR was used for determination of RNA expression
of Type I collagen (COL 1A1), Type II collagen (COL 2A1), and
aggrecan (AGC1 ).
[0151] Quantification of DNA within a cell-seeded DSTI showed that
in a chondrogenic medium hAC proliferation was increased 2.6 fold
and bone marrow stem cells fproliferation increased 1.5 fold when
compared to non-chondrogenic medium.
[0152] Measurement of S-GAG levels showed that in chondrogenic
medium hAC produced 3.1 fold more S-GAG and bone marrow stem cells
produced 1.6 fold more S-GAG compared to non-chondrogenic medium.
On a per cell basis, the bone marrow stem cells produced more S-GAG
per amount of DNA, consistent with being metabolically active.
[0153] The DSTI was both supporting proliferation and ECM
production in a different culture medium without showing negative
effects on differentiation.
[0154] E. Gene Expression
[0155] Cells migrated into the DSTI show increased gene expression
for collagen Type I, collagen Type II and aggrecan.
[0156] Human articular chondrocytes (hAC) in the DSTI exhibit an
increase in Type I collagen and a decrease in Type II collagen and
aggrecan production at 2 and 4 weeks. Such increase is possibly
associated with the chondrocyte proliferation in chondrogenic
medium. Results are seen in FIG. 4.
[0157] FIG. 4 shows gene expression of Type II collagen by bone
marrow stem cells in DSTI. Such expression is greatly increased
after two and four weeks in culture. Specifically, FIG. 4 shows
results of RT-PCR of bone marrow stem cells (BMSC). RT-PCR shows
bone marrow stem cells differentiating into chondrogenic phenotype:
collagen Type I (COL 1A1), collagen Type II (COL 2A1) and aggrecan
(AGC1). For bone marrow stem cell, there was substantial increase
in production of Type II collagen at four weeks of culture compared
to the amount seen at two weeks of culture.
[0158] Results seen in FIG. 4 clearly show that bone marrow stem
cells seeded in DSTI and grown in chondrogenic medium produce
glycosaminoglycans and display differentiation markers indicative
of the chondrocyte phenotype. By 4 weeks in culture, Type II
collagen gene expression in the stem cell seeded in DSTI is
dramatically up-regulated and the bone marrow stem cells are
producing more S-GAG/cell than the articular chondrocytes.
[0159] The above studies clearly indicate that the DSTI supports
gene expression of migrated cells and also that the culture medium
has an important function in differentiation and migration of cells
into DSTI.
[0160] The ability of human articular chondrocytes, bone marrow
stem cells or bone marrow cells to migrate into DSTI and
differentiate into chondrocytes was studied by migration assays
using Boyden chambers, commonly used test to study the chemotaxis
potential of a media/agent and migration ability of the cells. This
study simulated the ability of bone marrow stem cells to migrate
into a defect following microfracture surgery.
[0161] Evaluation of the study using the Boyden chamber assay, as
described in Example 8, showed that human articular chondrocytes
that migrated into DSTI remained in the DSTI. In general, a DNA
content of cells substantiated a 4 fold increase in cell number in
human articular chondrocytes that migrated into the DSTI as
compared to those that were seeded in the DSTI as controls.
[0162] F. Production of Extracellular Matrix
[0163] Production of extracellular matrix by the individual cells
is measured by the ratio of S-GAG to DNA. To determine the amount
of extracellular matrix produced either by human articular
chondrocytes or by bone marrow stem cells, another study was
performed. Results of this study are shown in FIG. 5.
[0164] FIG. 5 shows production of S-GAG/DNA by bone marrow stem
cells (MSC) and human articular chondrocytes (hAC) after four weeks
in culture. The cells were either placed in a Boyden chamber and
allowed to migrate into the DSTI (Migrated) or were seeded directly
into the DSTI (Control). As seen in FIG. 5, the bone marrow stem
cells that migrated into the DSTI (MSC Migrated) produced almost
twice as much S-GAG per cell than MSC seeded in the DSTI (MSC
Control) and almost two and a half times more than human articular
chondrocytes that either migrated into DSTI or were seeded in the
DSTI. There was no observable difference between the amount of
S-GAG production by human articular chondrocyte between the
migrated cells into the DSTI and seeded cells in the DSTI.
[0165] The above results clearly show that the combination of DSTI
with bone marrow stem cells cultured in an appropriate medium leads
to increased differentiation of cells and increased production of
extracellular matrix by the cells migrated into the DSTI.
[0166] Uniqueness of stem cells and particularly bone marrow stem
cells behavior during migration into the DSTI is shown in another
study where the gene expression of bone marrow stem cells migrating
into the DSTI was determined vis-a-vis human articular
chondrocytes. Results are seen in FIG. 6.
[0167] FIG. 6 shows differences between gene expression of bone
marrow stem cells and human articular chondrocytes migrated into
the DSTI. The bone marrow stem cells that migrate into DSTI in the
presence of chondrogenic medium show increases in gene expression
of Type II collagen (COL 2A1) and aggrecan (AGC1) whereas human
articular chondrocytes begin to show signs of dedifferentiation
leading to an increase in Type I collagen (COL 1A1) and decreases
in Type II collagen (COL 2A1) and aggrecan (AGC1). Gene expression
for bone marrow stem cells migrated into the DSTI is approximately
50 times higher that for human articular chondrocytes.
[0168] These data indicate a role of the DSTI in the modulation of
the bone marrow stem cells differentiation as they populate and
adhere within a 3D support structure.
[0169] G. DSTI as Chemoattractant for Bone Marrow Stem Cells
[0170] The DSTI possess a unique chemoattractive property for bone
marrow stem cells that migrate toward and into the DSTI
[0171] The potential for the DSTI to act as a chemoattractant for
bone marrow stem cells was studied. To determine if cells will
migrate across the Boyden Chamber in response to the presence of
the DSTI, cell were seeded in the Boyden chamber and processed
according to Example 9. Three different culture media, namely
bovine serum albumin (BSA), growth (Growth) and chondrogenic (CCM)
medium, were used for this study to explore possibility of
enhancing the chemoattraction by using different culture medium.
Results are seen on FIG. 7.
[0172] FIG. 7 shows chemotactic activity of bone marrow stem cells
in response to various culture media formulations and in the
presence or absence of DSTI. As seen in FIG. 7, when the DSTI
contained 1% BSA medium or the chondrogenic media, there were 2 or
less cells per field. No difference was observed between the empty
chambers and chamber containing DSTI. Number of cells/field
increased somehow in the growth medium without DSTI when compared
to the BSA and chondrogenic medium. However, the presence of DSTI
containing a growth medium showed remarkable increase in the cell
number/field. Specifically, in the presence of growth medium
without DSTIs number of cells/field averaged 17.5 cells compared to
number of cells/field observed in the presence of DSTI containing
the growth medium where the number of cells/field averaged almost
90 cells. As seen in FIG. 7, there was a highly significant
difference between the DSTI containing samples and the empty
samples containing only the media for all three media. The 1% BSA
medium showed a 2.8 fold increase in cells/field in the presence of
DSTI compared to the medium alone without DSTI. The chondrogenic
medium showed a 1.9 fold increase in cells/field for chambers
containing DSTIs. The growth medium showed a 3.5+ fold increase in
cells/field in chambers containing DSTIs.
[0173] This study clearly demonstrated that bone marrow stem cells
are highly attracted to DSTI. Significantly more cells penetrated
the Boyden Chamber membrane in response to the DSTI presence.
Selection of media was able to further accentuate such attraction.
Showing that such attraction may be manipulated by media selection
and that it is particularly enhanced with use of the growth medium
but not with chondrogenic medium indicates that such manipulation
may be advantageously utilized for provoking or suppressing
chemoattraction of DSTI.
[0174] H. The Double-Structured Tissue Implant Containing Cell
Modulator
[0175] The double-structured implant of the invention, as shown
above, has properties never before observed or described in any
other tissue implant. One other important property is its
variability of uses due to its double structure.
[0176] One embodiment of the DSTI use is the DSTI containing a cell
modulator. The cell modulator may be a pharmaceutical agent, drug,
growth modulator, growth hormone, mediator, enzyme promoting cell
incorporation, cell proliferation or cell division,
pharmaceutically acceptable excipient, additive, buffer, etc.,
incorporated into the primary or secondary scaffold, or to both
scaffolds.
[0177] Any of the above mentioned compound may be introduced
separately into the primary scaffold or into the secondary scaffold
at a time of its formation by simply introducing such compound to
the dehydrated dry DSTI. In alternative, such compound may be added
to the DSTI at a time of rehydration before its implantation.
[0178] Suitable pharmaceutical agents, drugs or modulators are
selected from the group consisting of:
[0179] growth and morphogenic factors, such as, for example,
transforming growth factor, insulin-like growth factor 1,
platelet-derived growth factor, bone morphogenetic proteins
(bmps);
[0180] cytokines, such as, for example, interleukins, chemokines,
macrophage, chemoattractant factors, cytokine-induced neutrophil
chemoattractants (gro-1), integral membrane proteins such as
integrins and growth factor receptors;
[0181] membrane associated factors that promote growth and
morphogenesis, such as, for example, repulsive guidance
molecules;
[0182] cell attachment or adhesion proteins, such as, for example,
fibronectin and chondronectin;
[0183] hormones, such as, for example, growth hormone, insulin and
thyroxine;
[0184] pericellular matrix molecules, such as perlecan, syndecan,
small leucine-rich proteoglycans and fibromodulin;
[0185] nutrients, such as, for example, glucose and
glucosamine;
[0186] nucleic acids, such as, for example, RNA and DNA;
[0187] anti-neoplastic agents, such as, for example, methotrexate
and aminopterin;
[0188] vitamins, such as, for example, ascorbate and retinoic
acid;
[0189] anti-inflammatory agents, such as, for example, naproxen
sodium, salicylic acid, diclofenac and ibuprofen;
[0190] enzymes, such as, for example, phosphorylase, sulfatase and
kinase; and
[0191] metabolic inhibitors, such as, for example, RNAi,
cycloheximide and steroids.
[0192] These, and other similar compounds and/or compounds
belonging to the above-identified groups may be added individually
or in combination to a primary scaffold, to a secondary scaffold,
to a composition (Basic Solution) for formation of the secondary
scaffold or to the lyophilized composite or DSTI before, during or
after implantation.
[0193] Addition of agents such as growth factors, cytokines and
chemokines will increase cell migration, cell growth, will maintain
or promote appropriate cell phenotype and will stimulate
extracellular matrix synthesis. Loading the scaffold with
anti-inflammatory agents or other drugs can provide a local
site-specific delivery system.
[0194] The range of concentration of the added drug or compound
depends on the drug or compound and its function and it extends
from picograms to milligrams.
[0195] I. Differentiation of Mesenchymal Stem Cells
[0196] DSTI is a novel implant that has chemoattraction for
mesenchymal stem cells as well as for bone marrow stem cells that
migrate into the DSTI. Such migration further results in
differentiation of these cells and in their transformation into
specifically functioning cells, such as, for example, chondrocytes.
These properties were shown to be enhancible by using selected
media as well as by complementing such media with compounds such as
growth factors, pharmaceutical agents or growth modulators.
[0197] To confirm results obtained above that indicated that by
using DSTI, differentiation of mesenchymal stem cells may be
achieved and further improved by addition of growth factors,
migration of mesenchymal stem cells (MSC) towards DSTI loaded with
a repulsive guidance molecule (RGMA) that alters utilization of
protein morphogenetic protein was studied.
[0198] Purpose of this study was to determine if DSTI loaded with a
growth factor such as RGMA will increase the chemoattraction of the
DSTI. In the above described studies on migration, it was
determined that the DSTI possesses chemotactic properties towards
BMSC and hAC. Although the results obtained above indicated that
the migration of such cells increased in culture containing DSTI
and growth factor containing medium, it was not known if the
addition of a growth factor could also enhance the cell
differentiation.
[0199] For this purpose, cells were tested in the Boyden Chamber
according to a protocol of Example 10 in the presence and absence
of repulsive guidance molecule (RGMA) with or without DSTI in the
culture for 18 hours. Results are seen in FIG. 8.
[0200] FIG. 8 shows average cell count per field is samples of
medium only, DSTI containing medium and DSTI containing medium and
RGMA. Results seen in FIG. 8 show that in 18 hours, the presence of
DSTI signifiantly (p<0.00001) increased migration of BMSC by
approximately 1.7 times in DSTI containing the FBS medium when
compared to samples containing only the FBS medium. The addition
RGMA to DSTI further significantly (p<0.000001) increased cell
count 2.1 times when compared to samples containing only FBS
medium. An increase in average cell count per field also increased
(p<0.002) compared to DSTI containing the medium without
RGMA.
[0201] This study confirmed that DSTI is chemoattractive to MSC and
that this effect can be advantageously enhanced by the addition of
a growth factor such as RGMA.
[0202] Further studies were directed to determination if migration
of MSC towards DSTI loaded with other growth factors also initiates
differentiation gene expression.
[0203] For this purpose, the potential morphogenetic factors RGMA,
BMP-2 and BMP-7 were investigated for their ability to initiate
gene expression relating to MSC differentiation and maintenance of
chondrocytes.
[0204] Initial studies on RGMA have demonstrated not only its
ability to enhance chemoattraction to DSTI but also its ability to
up-regulate BMP-2 gene expression in both chondrocytes and MSCs.
BMP-2 and BMP-7 have been well documented to act as growth factors
that induce cartilage production. This study was designed to
compare these three potential growth factors on their ability to
increase initiation of MSC differentiation and maintenance of
chondrocytes.
[0205] Human bone marrow mesenchymal stem cells (MSC) were
subjected to treatment in Boyden Chamber assay in the absence
(empty) or presence of DSTI (DSTI), DSTI and RGMA (RGMA), DSTI and
BMP-2 (BMP-2) and BMP-7 and DSTI (BMP-7). At intervals of 72 hours
and three weeks, samples of DSTI were tested for RNA production for
RT-PCR (72 hours) and for production of RNA and subjected to
histological analyses.
[0206] Results are seen in FIGS. 9 and 10. FIG. 9 shows results
obtained with mesenchymal stem cells. FIG. 10 shows results
obtained with human articular chondrocytes. RT-PCR analysis of the
72 hour experiments showed that MSC (FIG. 9) migrating towards DSTI
show significantly increased production of aggrecan (AGC 1) and
cartilage oligomeric matrix protein (COMP) expression, whereas hAC
(FIG. 10) did not have similar increases (note a different scale).
RGMA, BMP-7, and BMP-2 displayed increasing expression,
respectively, when compared to DSTI in FIG. 9 for MSC for AGC1, COL
1A1, COMP, and a chondrogenic transcription factor SOX9. hAC showed
similar increases in AGC1 and COL2A1 in response to BMP-2 and BMP-7
only (FIG. 10).
[0207] The results shown in FIGS. 9 and 10 show that migration of
MSC towards DSTI containing growth factors up-regulates cartilage
specific genes, such as COMP and SOX9. Migration of hAC towards
DSTI containing growth factor maintains chondrocyte phenotype.
Migration of MSC (FIG. 9) toward DSTI leads to chondrocytic gene
expression up-regulation. The addition of a growth factor such as
RGMA, BMP-7 or BMP-2 to DSTI enhances this gene up-regulation at 72
hours. Chondrocytes (FIG. 10) maintain their phenotype in response
to migration towards DSTI combined with growth factors.
[0208] These studies showed that BMP-2-stimulated MSC show
increased production of AGC1, COL2A1 and COMP, but that is also had
significantly increased a bone gamma-carboxyglutamic acid protein
(BGLAP) Such increase may be a possible indication of conversion to
osteogenic phenotype. BMP-7 and RGMa show a similar increase in
COL2A1 and COMP, but lack the increase in BGLAP. hAC subjected to
similar analyses show a similar pattern of AGC 11, COL2A1, and
BGLAP increased expression when stimulated with BMP-2, with no
apparent changes in response to RGMa or BMP-7.
[0209] J. The Effects of Hydrostatic Pressure on Chondrocyte
Differentiation in DSTI
[0210] Previously, inventors discovered an effect of hydrostatic
pressure on proliferation of articular chondrocytes in studies
where the adjustment of hydrostatic pressure, culture medium
perfusion and oxygen levels has been used to simulate the articular
cartilage environment. Consequently, a purpose of this study was to
investigate if similar conditions would enhance cells
differentiation and growth in the DSTI. This study investigates the
effect of hydrostatic pressure and low oxygen on bone marrow stem
cells differentiation potential in DSTI.
[0211] Briefly, bone marrow stem cells and human articular
chondrocytes were subjected to culturing conditions under either
the hydrostatic pressure or the static pressure. The tissue
engineering processor (TEP) and chondrogenic medium were used to
investigate the role of hydrostatic pressure on differentiation of
bone marrow stem cells seeded into DSTI. The protocol used for this
study is described in Example 13. Results are seen in FIG. 11.
[0212] FIG. 11 shows that hydrostatic pressure increases production
of Type II collagen and depresses production of Type I collagen
gene expression. Hydrostatic pressure protocol generated by (TEP
leads to stimulation of both cell types compared to static
cultures. Bone marrow stem cells leads to increased expression of
Type II collagen and decrease of Type I collagen.
[0213] Real time RT-PCR data show that human articular chondrocytes
do relatively well in TEP compared to static conditions, where COL
2A1 and AGC1 remained elevated. Bone marrow stem cells showed an
increase in COL 2A1 and a decrease in COL 1A1 with relatively no
change in AGC1 expression.
[0214] K. Properties of the Double-Structured Tissue Implant
[0215] Double-structured tissue implant of the invention has
chemoattractant, cell adhesion, wettability, shape-memory and
structural stability properties for cells, stem cells, mesenchymal
stem cells and bone marrow stem cells.
[0216] These properties were demonstrated in the above studies. The
double structure of the implant provides structural stability,
wettability and possess a shape-memory when hydrated. In addition,
DSTI acts as a chemoattractant for bone marrow stem cells and for
human articular chondrocytes. The above-described studies
demonstrated that when the DSTI is brought into contact with bone
marrow stems cells and/or human articular chondrocytes, such cells
migrate into the DSTI, adhere to the DSTI double structure,
differentiate and express the chondrocyte genotype and produce
cartilage specific extracellular matrix. Moreover, such cell
behavior may be advantageously modified and amplified by addition
of cell modulators, by different culture media or by applying a
hydrostatic pressure and other culture conditions. By using DSTI
for seeding of cells or as a recipient of the cell migration, the
differentiation of bone marrow stems cells into the chondrocyte
genotype is enhanced.
[0217] DSTI, therefore, is a suitable implant that supports both
human articular chondrocyte and bone marrow mesenchymal stem cell
adhesion as well as their proliferation. Upon implantation of DSTI
in a chondral defect with the addition of bone marrow stem cells,
the joint environment will facilitate differentiation into the
chondrocyte phenotype. The DSTI as an implant is therefore
eminently suitable for repair of chondral or other tissue defects.
Use for repair of the muscle, vascular, cardiac or neural tissue
may be accomplished by directing the stem cells, mesenchymal stem
cells, bone marrow stem cells or neural stem cells to differentiate
into muscle, cardiac, epithelial or nerve cells.
[0218] III. Process for Preparation and Use of the
Double-Structured Tissue Implant
[0219] The secondary scaffold is generated within confines of the
primary scaffold by a process comprising several stages and steps
as set forth in Scheme 1. The process stages comprise pre-loading,
loading, polymerization, treatment of composite double-structured
scaffold, dehydrothermal treatment, packaging and, ultimately, its
surgical delivery.
Scheme 1
Process for Production and Use of a Double-Structured Implant
Stage 1--Pre-Loading
[0220] The pre-loading stage is a preparatory stage where the
primary scaffold is either obtained from commercial sources or is
prepared according to the procedure described in Example 1.
[0221] Step 1
[0222] Step 1 comprises obtaining or preparing a primary scaffold,
typically a collagen containing honeycomb, sponge or lattice
providing a structural support for incorporation of the secondary
scaffold.
[0223] In one embodiment, a bovine Type I collagen matrix with
honeycomb (HC) like structure is obtained, for example, from Koken,
Inc. (Japan) or from other commercial sources and used as primary
scaffold. However, such commercially available honeycomb matrices
have typically randomly distributed pores of irregular shape and
size. The pores of these structures are not always vertically
positioned.
[0224] In another embodiment, and preferably, a primary honeycomb
scaffold is produced according to a process described in Example 1,
wherein said primary scaffold has randomly or non-randomly oriented
pores of substantially the same size and shape.
[0225] The shape and size of the primary scaffold determines a size
of the double-structured tissue implant (DSTI) ultimately delivered
to the surgeon for implantation into the tissue defect.
[0226] Typically the DSTI has a rectangular, circular or oval shape
with dimensions of about 50 mm and a vertical thickness of about 1
to 5 mm, preferable 1-2 mm. Preferred dimensions of the DSTI and,
therefore, the dimensions of the primary scaffold are 50.times.50
mm.times.1.5 mm, with pores oriented substantially vertically, said
pores having a pore size of from about 100 to about 400 .mu.m,
preferably about 200.+-.100 .mu.m and pore length of 1.5 mm.
However, dimensions of the primary scaffold may be any that are
required by the tissue defect to be repaired and that can be
prepared by the process of the invention.
[0227] Step 2
[0228] Step 2 comprises preparing a composition for preparation of
a secondary scaffold (Basic Solution) and comprises neutralization
of a soluble collagen solution having an initial acidic pH of about
pH 1.5-4, preferably between about pH 1.9-2.2, a collagen
concentration from about 0.5 to about 10 mg/ml of collagen,
preferably about 2.9 to about 3.2 mg/ml, a surfactant concentration
from about 0.05 to about 10 mg/ml, preferably about 0.29 to about
0.32 mg/ml and osmolality from about 20 to about 400 mOsm/kg,
preferably about 280 to about 320 mOsm/kg. The soluble collagen
solution is then neutralized with any suitable base and/or buffer
to pH in a range from about pH 7.3 to about pH 7.7 to derive the
Basic Solution. Preferably, the solution is neutralized by
adjusting pH to neutrality 7.4 using a collagen/surfactant,
10.times. Dulbecco phosphate buffered saline (DPBS) and 0.1 M NaOH
in 8:1:1 ratio or using an aqueous solution or ammonia vapor in
concentration sufficient to neutralize acid within the collagen
solution. The final osmolality and pH of the Basic Solution is
about 290 mOsm/kg and pH 7.4, respectively.
[0229] The suitable buffers for solubilization of the Type I
collagen are, for example, a formic acid containing buffer at pH
4.8, acetic acid containing buffer at pH 5.0 or a diluted
hydrochloric acid containing buffer at pH 3.0.
[0230] Neutralization is typically carried out using ammonia
aqueous solution or a vapor of about 0.3%-1% ammonia, or in
concentration sufficient to neutralize the acidic pH over about 12
to about 24 hour period. This factor has also been found to affect
the collagen polymerization and formation of pores having
homogeneous pore size.
Stage 2--Loading and Precipitation
[0231] The primary scaffold is loaded with a Basic Solution for the
secondary scaffold comprising soluble collagen solution containing
a surfactant. This Basic Solution is subsequently precipitated
within pores of the primary scaffold.
[0232] Loading the primary scaffold with the Basic Solution for the
secondary scaffold is performed using any suitable method. Soaking,
wicking, submerging the primary scaffold in the solution,
electrophoresis and any other suitable means. Once the Basic
Solution for the secondary scaffold is introduced into the primary
scaffold, a composite of both is subjected to a process or
treatment that results in formation of the secondary scaffold
inside pores of the primary scaffold.
[0233] Step 3
[0234] The neutralized Basic Solution of step 2 is loaded into the
primary scaffold by placing from about 3.75 to about 7.5 ml
(approximately 1 to 2.times.volume), preferably a volume about 4.9
ml (approx. 1.3.times.volume of the primary scaffold) of the
secondary scaffold Basic Solution on the bottom of a dish and then
placing the primary scaffold in this solution and allowing it to be
soaked up.
Stage 3--Polymerization of the Soluble Collagen within a Primary
Scaffold into a Secondary Scaffold
[0235] The primary scaffold loaded with the neutralized Basic
Solution comprising the soluble collagen and the surfactant is then
subjected to conditions resulting in precipitation of the
neutralized Basic Solution within the pores of the primary scaffold
thereby generating a structurally distinct secondary scaffold.
[0236] Typically, and allowing for variability of the Basic
Solution or composition used for creating of the secondary
scaffold, the composition introduced into the pores of the primary
scaffold is gelled or precipitated within said primary scaffold and
may also be cross-linked using chemicals such as glutaraldehyde or
another multifunctional aldehyde, where the aldehyde reacts with
amino groups of the collagen yielding a Schiff base, which can be
stabilized by a reduction reaction; carbodiimide reagent, such as
carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC)
with or without N-hydroxy-succinimide (NHS) where the HNS is used
to suppress side reactions. Additionally, EDC and NHS can be used
in combination with diamine or diacid compounds to introduce
extended cross-links; acyl azide where the acid are activated and
subsequently reacted with an adjanced amine group; epoxy compounds
such as 1.4-butanediol diglycidyl ether, or cyanamide.
[0237] In addition, irradiation such as short wave length UV
irradiation (254 nm) can introduce cross-links in the collagen.
[0238] Step 4
[0239] The primary scaffold loaded with the neutralized
collagen/surfactant solution in a range from about 1 to about 2
volumes of the primary scaffold is then placed in an incubator at a
temperature from about 25.degree. C. to about 38.degree. C.,
preferably to about 37.degree. C. temperature, typically for from
about 10 minutes to about 18 hours, more typically for about 20 to
about 100 minutes, preferably for about 40 to 60 minutes, and most
preferably for a time when the precipitation of the neutralized
collagen solution into a solid secondary scaffold occurs.
[0240] Step 5
[0241] In order to assure that the vast majority of the salt of the
precipitated collagen solution within the pores of the primary
scaffold is removed, a composite consisting of the primary scaffold
having the secondary scaffold precipitated within is subjected to a
washing step whereby the majority of the salts are removed.
[0242] The composite (Composite) comprising the primary scaffold
and the secondary scaffold precipitated within, is washed by
placing said composite in a volume of from about 20 ml to about 10
liters, preferably about 500 ml, of de-ionized water further
containing a non-ionic surfactant. The surfactant is typically
present in concentration from about 0.05 to about 1.0 mg/ml,
preferably about 0.23 mg/ml. Most preferred surfactant is
PLURONIC.RTM. F127.
[0243] Typically, the washing step takes approximately 30 minutes.
There may be one or several washing step repetitions. All excess
non-precipitated collagen is removed during the extraction from the
composite into the wash solution.
[0244] Polymerizing of the collagen present in the secondary
scaffold solution loaded within the primary scaffold pores results
in formation of a solid double-structured composite, as defined
above, comprising the primary scaffold and the secondary scaffold
precipitated therewithin.
[0245] Following the precipitation or gelling and washing, the
composite is subjected to lyophilization and dehydrothermal
treatment.
Stage 4--Dehydration of Composite Double-Structured Scaffold
[0246] The solid double-structured composite is then dehydrated
using any method suitable for such dehydration. Typically, such
dehydration will be freeze-drying or lyophilization. Freezing is
typically carried out at temperature from about -10.degree. C. to
about -210.degree. C., preferably from about -80.degree. C., over a
period of about 2 to about 60 minutes. The frozen composite is then
lyophilized forming the Lyophilized Composite.
[0247] The gradual nature of the polymerization and slow process f
water removal typically maintains the architectural elements of the
secondary scaffold and achieves the proper orientation and diameter
of the pores.
[0248] Step 6
[0249] Freezing is achieved with freezing the solid
double-structured composite by placing it on the metal shelf of a
freezer and adjusting the temperature to from about -10.degree. C.
to about -210.degree. C., preferably to about -60.degree. C. to
about -90.degree. C., and most preferably for about -80.degree. C.,
for about 2 to about 60 minutes, preferably for about 20-45 minutes
and most preferably for about 30 minutes.
[0250] Step 7
[0251] The frozen solid double-structured composite is then
subjected to lyophilization. The frozen composite is removed from
the freezer and placed into a pre-cooled lyophilization chamber.
Lyophilization typically occurs in about 15-21 hours, depending on
the size and shape of the composite but is typically and preferably
completed in about 18 hours.
Stage 5--Dehydrothermal Treatment
[0252] To further stabilize the composite to provide resistance to
dissolution and to achieve sterility of the final product, the
solid double-structured composite is subjected to dehydrothermal
(DHT) treatment. DHT treatment achieves cross-linking of the
collagen with the surfactant and at higher temperatures also
sterilizes the DSTI.
[0253] Cross-linking step prevents dissolution of the secondary
scaffold upon rehydration before or after implantation.
[0254] Step 8
[0255] This step is performed to sterilize and cross-link the
double structured tissue implant.
[0256] The lyophilized double-structured composite is placed into a
dry glass chamber or container and covered with the glass, aluminum
foil or another suitable material resistant to higher temperatures.
The container with the lyophilized double-structured composite is
placed into the pre-heated dehydrothermal oven and subjected to a
temperature in a range from about 70.degree. C. to about
200.degree. C., preferably from about 130.degree. C. to about
150.degree. C., and most preferably about 135.degree. C., under
vacuum, for about 30 minutes to about 7 days, preferably for about
5-7 hours and most preferably for about 6 hours.
[0257] Such treatment stabilizes the composite, makes it resistant
to collagen dissolution upon wetting, provides for rapid wetting
and assures none or minimal shrinkage or swelling upon wetting with
a physiological solution or buffer, and sterilizes the
double-structured tissue implant.
Stage 6--Packaging and Storage
[0258] The double-structured tissue implant fabricated by the
process described above is then ready for a sterile packaging and
storage. In this form, the DSTI has a long shelf-life.
[0259] Step 9
[0260] The double structured tissue implant is removed from the
dehydrothermal oven and transferred aseptically into sterile
environment, such as a Bio Safety Cabinet (BSC), where it is
packaged under conditions assuring sterility. The double-structured
tissue implant is then ready to be stored at room temperature until
its use.
Stage 7--Delivery by Implantation
[0261] Packaged double-structured tissue implant is delivered or
made available to a surgeon for implantation into a tissue
defect.
[0262] Step 10
[0263] During surgery, surgeon determines an extent of the defect
or lesion to be repaired, opens the packaged product, cuts the DSTI
to size of the defects and places the cut-to-size piece into said
defect. Typically, the implant is placed into the defect in a dry
form and a suitable physiologically acceptable solution is then
added to wet the implant in situ. In alternative, the implant may
be wetted before the implantation and then placed into the
defect.
[0264] Since the implant is very stable, and does not change its
size or shape significantly by shrinking or swelling, the implant
fits tightly into the defect or lesion. To assure that the implant
stays within the defect or lesion, such defect or lesion is first
coated with a suitable tissue adhesive, sealant or glue that keeps
the implant in place. In alternative, the defect or lesion may be
pretreated with microfracture where the tissue underlying the
lesion or defect is microfractured with microchannels to permit the
blood and nutrient supply into the lesion or defect, lining the
defect or lesion but not the microfracture, with the adhesive, glue
or sealant and placing the implant as described above. In both
instances, the implant placed into the lesion or defect may
optionally be covered with another layer of the adhesive, sealant
or glue.
[0265] In some instances, cells, drugs or modulators may be loaded
into the DSTI or attached to the second scaffold before
implantation and wetting, during wetting following the
implantation, or independently provided after the implantation.
[0266] Results obtained for three separate lots containing three
rehydrated DSTIs per each lot, are seen in Table 2. The DSTI is
rehydrated by placing a droplet of phosphate buffer saline
(1.5.times.volume of PBS), on top of the DSTI and the rehydration
time is measured as the time it takes for the DSTI to be completely
hydrated.
TABLE-US-00002 TABLE 2 Number of Sample Results Attribute (n/lot)
Lot #1 Lot #2 Lot #3 Rehydration Time (seconds) 3 <2 <2 <2
Rehydrated pH 3 7.7 .+-. 0.1 7.8 .+-. 0 7.7 .+-. 0.1 Rehydrated
osmolality 3 317 .+-. 6 356 .+-. 4 319 .+-. 1 (mOsm/kg) Size
variation at hydration (%) 3 99.8% .+-. 5.2% 100.6% .+-. 10.0%
99.7% .+-. 2.3% Collagen Retention in PBS (%) 3 99.4% .+-. 0.2%
99.1% .+-. 0.1% 99.2% .+-. 0.2%
[0267] As seen in Table 2, results obtained in three different lots
in three different studies are closely similar confirming the
reproducibility of the process as well as consistency of the
parameters observed after rehydration.
[0268] The rehydration time for each lot is less then 2 second
evidencing a very fast wettability of the DSTI products.
[0269] The pH of the rehydrated DSTI products is between 7.7 and
7.8 in all lots.
[0270] Osmolality of the rehydrated DSTI products is between 317
and 356 mOsm/kg in all lots.
[0271] Variation in size of rehydrated DSTI products is negligible
evidencing that there is no shrinkage or swelling upon hydration of
DSTI.
[0272] Collagen retention within the rehydrated DSTI is above 99%,
evidencing a great stability of the DSTI products.
[0273] IV. Method of Use of Double-Structured Tissue Implant
[0274] Double-structured tissue implant of the invention is useful
for treatment and repair of tissue defects of various tissues. Such
treatment is achieved by implanting the DSTI into the defect in
surgical setting with cells, stem cells, mesenchymal stem cells or
bone marrow stem cells either added tot he DSTI by seeding said
cells within the DSTI before or during surgery or by preparing a
surgical site of the defect in such a way that said cells will
naturally migrate into said DSTI due to tis chemoattractant
properties.
[0275] In this regard, the use of DSTI for implantation is
illustrated in FIGS. 12A-12E. These Figures illustrate, in a
schematic way, implantation of DSTI into the articular cartilage
lesion. However, the same or similar process would be used for
implantation of the DSTI into defect of any other tissue.
[0276] FIGS. 12A-12F is a schematic illustration of several
treatment protocols for implantation of the DSTI into a tissue
lesions, exemplarized here with the implantation of the DSTI into
the cartilage lesion, using a double-structured tissue implant
(DSTI) containing stem cells, mesenchymals stem cells or bone
marrow stem cells. These cells may be seeded within said DSTI with
or without previous culture or activation or may migrate into the
DSTI from the surrounding native tissue.
[0277] FIGS. 12A-12F are schematic illustrations of treatment
protocols for implantation of the DSTI into cartilage lesions using
double-structured tissue implants containing mesenchymal stem cells
or bone marrow stem cells. FIG. 12A is a schematic illustration of
a method for implantation of DSTI into the tissue lesion or defect
where the DSTI is provided as a dry DSTI, is precut and trimmed
into the size and shape of the lesion, rehydrated with a
physiologically acceptable solution that may contain
non-differentiated or pre-differentiated stem cells or mesenchymal
stem cells and the DSTI is placed into the lesion. Before
rehydration, the stem or mesenchymal stem cells are first dissolved
in a physiologically acceptable solution and such solution is
applied to the dehydrated, previously trimmed DSTI to a size and
shape of the defect. Rehydrated DSTI is implanted into the defect
and sealed with an adhesive. FIG. 12B illustrates a method for
implantation of the DSTI that is essentially the same as seen in
FIG. 12A except that the subchondral plate is first penetrated with
microfracture penetrations to permit migration of cells or marrow
components from the underlying tissue into the DSTI implanted in
the tissue lesion. In this method, the subchondral plate is
penetrated and the rehydrated DSTI is placed in the defect as in
FIG. 12A. After sealing the defect containing rehydrated DSTI with
the adhesive, the marrow components are able to enter the DSTI
through the microfracture. FIG. 12C illustrates a method for
implantation of DSTI with microfracture as seen in FIG. 12B except
that the adhesive previously applied only on the top of the DSTI
and to seal the lesion is also applied to the bottom of the lesion
in between the microfracture penetrations. Additionally, it can
also be applied to the bottom of the lesion in instances when the
migration of cells from the underlying tissue is undesirable. The
FIG. 12D shows the implantation of the DSTI into the tissue lesion
where the DSTI is rehydrated with mesenchymal stem cells (MSC). The
DSTI rehydrated with MSC is then implanted into the tissue lesion
and the defect is sealed with adhesive as described above. FIG. 12E
show implantation of the DSTI seeded with bone marrow stem cells
that were cultured in the DSTI prior its trimming into the size and
shape of the lesion. In this instance the dry DSTI is rehydrated
with the solution containing the MSC, cultured according to
protocols used for such culturing in the presence of a culture
medium optionally containing cell modulators. Such cell seeded DSTI
could optionally be also activated by applying a
hydrostatic/constant pressure regimen. The DSTI seeded with these
cultured and/or activated cells is placed into the lesion or defect
and the defect is sealed with adhesive placed over the implant.
FIG. 12F illustrates implantation of the DSTI into the tissue
defect where the DSTI is rehydrated with freshly aspirated bone
marrow that is either undiluted or diluted with saline or another
physiologically acceptable solution.
[0278] A. Use of DSTI for Treatment of Chondral Defects
[0279] One example of utility of the DSTI is its use for treatment
of chondral defects.
[0280] To be successful for treatment of articular cartilage, the
DSTI must provide conditions allowing the chondrocytes or
mesenchymal stem cells seeded therein to be able to form and
generate the new extracellular matrix. In this regard, the DSTI
pore structure must allow cells to migrate into the pores and
function similarly to their normal function in the healthy tissue.
The extracellular matrix formed by the cells seeded within the DSTI
then provides means for growing a new hyaline or hyaline-like
cartilage for treatment, replacement or regeneration of the damaged
or injured articular cartilage. Such treatment is currently
difficult because of the unique properties of the articular
cartilage that is not the same as and does not behave as other soft
tissues.
[0281] As amply illustrated above, the DSTI of the invention
provides all conditions necessary for migration or seeding the
chondrocytes, stem cells, mesenchymal stem cells or bone marrow
stem cells into the DSTI. Moreover, it provides conditions for
their differentiation, transformation into chondrocytes, production
of extracellular matrix as ultimately for production of a new
hyaline cartilage. All these conditions may be enhanced with
additions of cell mediators, using different culture conditions or
by intermittently applying the hydrostatic pressure.
[0282] B. Use of DSTI for Treatment of Other Conditions
[0283] In addition to cartilage repair, a number of other acute or
chronic conditions represent instances where the implantation of
the double-structured tissue implant can provide a clinically
important means for tissue repair.
[0284] The DSTI once placed at the site of tissue damage will
provide a support for development of new tissues occurs in
accordance with predefined configuration. For example, ischemic
area in the myocardium may be treated with DSTI containing stem
cells or mesenchymal stem cells differentiated into myocardial
cells. In these applications, similarly to cartilage, the DSTI must
resist, at least initially, the dynamic forces generated by the
surrounding muscle and connective tissues and maintaining its
structure during a necessary period of cellular infiltration,
tissue formation and healing.
[0285] Since every tissue is a subject to metabolic turnover, the
rapidity by which tissue differentiation and structural integrity
are established is subject to various endogenous modulations, the
ability to up-regulate or down-regulate such modulations through
placement of specific signaling factors placed within the primary
and secondary scaffold of the DSTI provides for vast therapeutic
utility of the DSTI. Although the limits by which, for example, new
muscle formation can be derived from progenitor cells suggests that
localization of the mesenchymal cells to the site of damage in
response to homing molecules, such as chemokines and cell receptor
ligands, may be used to accelerate repair of muscle, either cardiac
or skeletal, there must be a means to deliver these progenitor
cells to such site. DSTI have been shown to be able to deliver
these progenitor cells or modulators to the site of damage.
[0286] In another instance, wound healing applications have
remained a primary goal in the use of tissue implants for
cell-based tissue repair. Treatment of acute and chronic wounds is
dependent on a multi-faceted transition by which progenitor cells
encounter soluble mediators, form blood elements, extracellular
matrix macromolecules and parenchymal cells that then serve to
reestablish a body surface barrier through epithelization. In this
instance either the double-structured tissue implant may provide a
novice stromal layer into which blood vessels and progenitor cells
can migrate. From this migration, the progenitor cells may then
undergo differentiation into the fibroblast stromal cell and
generate or recruit epithelial cells to support reestablishment of
dermal and epidermal layers at the time of wound closure.
[0287] C. Basic Requirements for DSTI
[0288] The collagen-based primary and secondary scaffolds of the
DSTI are essential components of the DSTI and are responsible for
capability of DSTI to initiate the repair and induction of repair
of tissue defects.
[0289] The first requirement is that the scaffolds are prepared
from the biocompatible and preferably biodegradable materials that
are the same or similar to those observed in the tissues to be
repaired, hence the instant DSTI are prepared from collagen or
collagen-like materials.
[0290] The second requirement is that the scaffolds have a spatial
organization and orientation similar to that of the tissue to be
repaired. The porous structure of both primary and secondary
scaffold provides such organization.
[0291] The third requirement is that the scaffold have a pore
density permitting the seeding or migration of the cells into said
primary or secondary scaffolds in numbers needed for initiation of
a tissue recovery or formation of new tissue in vivo. The
substantially homogenous pore size and distribution within the
DSTIs allows the cell seeding and assures cell viability.
[0292] The fourth requirement is that the scaffolds have a
sufficient number of pores for the number of cells needed for
initiation of the tissue recovery and repair. The spatial
organization of both scaffolds have optimized number of pores and
such number and pore size may be easily adjusted to the tissue
requirement.
[0293] The fifth requirement is that the pores have substantially
the same size and that such size is substantially the same from the
top apical to the bottom basal surface of the pores, said pores
being organized substantially vertically from the top to the
bottom. The primary scaffold have such organization.
[0294] The sixth requirement is stability of the DSTI. The
double-structured organization of the DSTI provides such stability
during wetting, reconstitution, resistance to dissolution and to
shrinkage or swelling.
[0295] The seventh requirement is that DSTI provides support and
conditions for cell migration from the surrounding tissue, for
integration of seeded cells into the surrounding tissue and
generally that assures the cell viability. The DSTI provides such
conditions and the cells seeded within DSTI have almost 100%
viability.
[0296] V. Adhesives and Tissue Sealants
[0297] As described in the FIGS. 12A-12E, the double-structured
tissue implant is implanted into a tissue defect or cartilage
lesion covered with a biocompatible adhesive, tissue sealant or
glue. Typically, the sealant is deposited over the implant after
the implant is placed into the defect. However, in instances, such
adhesive is also placed at the bottom of the lesion to prevent the
migration of cells from the surrounding tissue or to prevent
effects of endogenously present tissue modulators from interfering
with a therapeutic effect of the DSTI. For example, when he DSTI is
seeded with precultured cells or progenitor cells, a deposition of
the adhesive to the bottom of the lesion is preferred. The adhesive
is almost always used to cover the implanted DSTI.
[0298] Generally, the tissue sealant or adhesive useful for the
purposes of this application has adhesive, or peel strengths at
least 10 N/m and preferably 100 N/cm; has tensile strength in the
range of 0.2 MPa to 3 MPa, but preferably 0.8 to 1.0 MPa. In
so-called "lap shear" bonding tests, values of 0.5 up to 4-6
N/cm.sup.2 are characteristic of strong biological adhesives.
[0299] Such properties can be achieved by a variety of materials,
both natural and synthetic. Examples of suitable sealant include
gelatin and di-aldehyde starch described in PCT WO 97/29715,
4-armed pentaerythritol tetra-thiol and polyethylene glycol
diacrylate described in PCT WO 00/44808, photo-polymerizable
polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromers
described in U.S. Pat. No. 5,410,016, periodate-oxidized gelatin
described in U.S. Pat. No. 5,618,551, serum albumin and
di-functional polyethylene glycol derivatized with maleimidyl,
succinimidyl, phthalimidyl and related active groups described in
PCT WO 96/03159.
[0300] Sealants and adhesives suitable for purposes of this
invention include sealants prepared from gelatin and dialdehyde
starch triggered by mixing aqueous solutions of gelatin and
dialdehyde starch which spontaneously react and/or those made from
a copolymer of polyethylene glycol and polylactide, polyglycolide,
polyhydroxybutyrates or polymers of aromatic organic amino acids
and sometimes further containing acrylate side chains, gelled by
light, in the presence of some activating molecules.
[0301] Another type of the suitable sealant is 4-armed polyethylene
glycol derivatized with succinimidyl ester and thiol plus
methylated collagen in two-part polymer compositions that rapidly
form a matrix where at least one of the compounds is polymeric,
such as polyamino acid, polysaccharide, polyalkylene oxide or
polyethylene glycol and two parts are linked through a covalent
bond, for example a cross-linked derivatized PEG with methylated
collagen, such as methylated collagen-polyethylene glycol.
[0302] Preferable sealants are 4-armed tetra-succinimidyl ester
PEG, tetra-thiol derivatized PEG and PEG derivatized with
methylated collagen, commercially available from Cohesion Inc.,
Palo Alto, Calif. and described in U.S. Pat. Nos. 6,312,725B 1 and
6,624,245B2 and in J. Biomed. Mater. Res., 58:545-555 (2001), J.
Biomed. Mater. Res., 58:308-312 (2001) and The American Surgeon,
68:553-562 (2002), all hereby incorporated by reference.
[0303] Sealants and adhesives described in copending U.S.
application Ser. No. 10/921,389 filed Aug. 18, 2004 and Ser. No.
11/525,782 filed Dec. 22, 2006, are hereby incorporated by
reference.
EXAMPLE 1
Preparation of the Primary Scaffold
[0304] This example describes one exemplary method for preparation
of the collagen-based primary scaffold suitable as a structural
support for preparation of the DSTI.
[0305] Type I collagen is dissolved in a weak hydrochloric acid
solution at pH 3.0 with the collagen concentration and osmolality
of the solution adjusted to about 3.5 mg/ml and 20 mOsm/kg,
respectively. The solution (70 ml) is poured into a 100 ml Petri
dish and the Petri dish containing the collagen solution is
centrifuged at 400.times.g for 30 minutes to remove air bubbles.
Neutralization and subsequent precipitation or gelling is carried
out in a 7 liter chamber containing 10 ml of 15% ammonia solution
over a 45 minutes period. The precipitated collagen is then washed
in a large excess of de-ionized water. The water is changed as many
times as needed over next 3 days to remove formed salts and excess
ammonia.
[0306] The solution is then subjected to unidirectional freezing
over a period of about 4 hours. The Petri dish is placed on a
stainless steel disc which is partially submerged in a cooling
bath. The temperature of the cooling bath is increased from
0.degree. C. to -18.degree. C. at a rate of 0.1.degree. C./minute.
The frozen water is removed by lyophilization over a period of
about 3 days. The lyophilized primary scaffold is then
dehydrothermally (DHT) treated at 135.degree. C. for about 18 hours
before being precut into slices of an appropriate thickness.
[0307] The organization of the newly synthesized cartilage specific
matrix within the porous Type I collagen is visualized and
quantified using histological and image analysis methods.
EXAMPLE 2
Preparation of a Basic Solution for a Secondary Scaffold
[0308] This example describes preparation of the Basic Solution
used for formation of the secondary scaffold.
[0309] The Basic Solution comprises a soluble collagen in admixture
with a PLURONIC.RTM. surfactant. The Basic Solution is incorporated
into the primary scaffold and processes into the double scaffold
tissue implant or processed as a stand alone implant.
[0310] Solution for the secondary scaffold is prepared by mixing
PLURONIC.RTM. F127 (2.32 mg, 0.29 mg/ml), obtained commercially
from BASF, Germany, with 8 ml of a solution containing 2.9 mg/ml
bovine Type I collagen dissolved in hydrochloric acid (pH 2.0) at
room temperature. The resulting solution is neutralized with 1 ml
of 10.times. Dulbecco's phosphate buffered saline (DPBS) and 1 ml
of 0.1M NaOH to the final pH of 7.4.
[0311] In the alternative, the neutralization is achieved by
ammonia aqueous solution or ammonia vapor in concentration
sufficient to neutralize acid within the collagen solution.
EXAMPLE 3
Preparation of the Double-Structured Tissue Implant
[0312] This example describes preparation of the double-structured
tissue implant (DSTI). The preparation of DSTI includes
incorporation of the Basic Solution for formation of a secondary
scaffold within the primary scaffold and its further processing
into DSTI.
[0313] 4.9 ml (1.3.times.volume of the primary scaffold) of the
neutralized basic collagen/PLURONIC.RTM. solution prepared in
Example 2, is placed in a dish and a primary scaffold, prepared in
Example 1, precut into a square having 50.times.50.times.1.5 mm
dimensions is then placed into the neutralized Basic Solution for
the secondary scaffold. The basic neutralized solution is absorbed
into the primary scaffold by wicking or soaking.
[0314] The primary scaffold containing the neutralized solution is
then placed in a 37.degree. C. incubator over a period of 50
minutes to precipitate or gel the neutralized collagen solution.
The composite consisting of the primary scaffold with the gelled or
precipitated neutralized solution within is then washed in 500 ml
of de-ionized water over a period of 30 minutes. The washed
composite is placed on metal shelf of a freezer at a temperature
-80.degree. C. over a period of 30 minutes. The frozen composite is
removed from the freezer and lyophilized.
[0315] Lyophilization is performed over a period of 18 hours. The
lyophilized composite is then dehydrothermally treated at
135.degree. C. under vacuum for a period of 6 hours to form the
double-structured tissue implant (DSTI).
[0316] The DSTI is removed from the dehydrothermal oven and
transferred aseptically into a Bio Safety Cabinet (BSC) where it is
packaged.
EXAMPLE 4
Testing of Viability, Growth and Functionality of Cell seeded into
DSTI
[0317] This example describes procedure used for determination of
viability, growth and functionality of cells seeded into the
DSTI.
[0318] Human chondrocytes obtained from NDRI Research Services
depository were dissolved in 30 .mu.l of culture medium and the
medium containing the chondrocytes was placed into culture plates
and the 6 mm DSTI discs were placed into the culture medium
containing the cells. The cells were allowed to penetrate the discs
for about 1 hour at which time 1 ml of medium was added to the
wells. For comparison, human chondrocytes were introduced into 6 mm
discs of a well-characterized non-lyophilized 3-dimensional
honeycomb collagen scaffold. The discs were incubated for 1 or 21
days at 2% O.sub.2 and 37.degree. C.%CO.sub.2.cndot.Medium was
changed twice weekly. On termination 3 discs were taken for
viability testing and 3 for GAG and DNA analysis.
EXAMPLE 5
Testing Viability of Cell Cultured in the DSTI
[0319] This example describes a procedure used for testing of cell
viability.
[0320] Three DSTI discs (DSTI-1, DSTI-2 and DSTI-3) obtained in
Example 4 were used for viability testing. Each DSTI was digested
with collagenase at 37.degree. C. for 1 hour and the digest was
centrifuged to obtain a cell pellet. The pellet was dispersed into
an aliquot of culture medium and a cell count was performed using
trypan blue exclusion test to determine the total number of cells
and the percent of live cells that were retained in the DSTI. over
the course of 21 days in culture.
[0321] Results were described above in Table 1. All three discs
have shown at least 97% viability initially and such viability
increased after 3 weeks in culture.
EXAMPLE 6
S-GAG and DNA Assays
[0322] This example describes procedure used for determination of
S-GAG and DNA.
[0323] The DSTI discs obtained in Example 4 were digested with
papain at 56.degree. C. overnight. An aliquot of the digest was
taken for analysis of S-GAG content by the DMMB assay and an
additional aliquot was used to measure DNA by Hoechst dye.
EXAMPLE 7
Determination of Cell Attachment Growth and Differentiation
[0324] This example describes conditions for determination of the
ability of multipotent, undifferentiated bone marrow stem cells to
attach, grow and differentiate into chondrocytes in DSTI.
[0325] Human bone marrow mesenchymal stem cells (HBMSC) were
purchased from Lonza and maintained in culture with bone marrow
stem cells growth media containing 10% FBS. Human articular
chondrocytes (hAC) were extracted by placing finely minced
cartilage in a collagenase solution overnight. Liberated cells were
then plated on tissue culture plastic in chondrocyte growth media
(DMEM/F12+10% FBS). At confluence, BMSC and hAC were lifted with
0.05% Trypsin+0.01% EDTA and cells were counted. The DSTI was cut
into discs (6.times.1 mm diameter) using a biopsy punch, placed on
Teflon.RTM. dishes and seeded by aliquoting 95,000 cells in 30
.mu.l growth media on top of each DSTI. The cells were allowed to
attach for 1 hour. Seeded DSTIs were then placed in individual 15
ml polypropylene tubes and incubated with either 0.5 ml growth
media or complete chondrogenic medium (Lonza,
serum-free+TGF.beta.3), and placed in 37.degree. C. incubators at
2% O.sub.2, Medium was replaced twice weekly. At 2 and 4 weeks,
individual DSTIs were removed and analyzed for DNA content using
the Hoechst assay; S-GAG using dimethylene blue assay. RT-PCR was
used for RNA expression of Type I collagen (COL1AI), Type II
collagen (COL2A 1), and aggrecan (AGC1) measurements. The
measurements were corrected using 188 and .beta.-actin (ACT.beta.).
All primers were obtained from Applied Biosystems.
EXAMPLE 8
Boyden Chamber Assay
[0326] This example describes conditions used for determination of
migration of cells into DSTI using a Boyden chamber assay.
[0327] A Boyden chamber was placed in a 24 well plate and 50,000
cells of undifferentiated bone marrow stem cells or human articular
chondrocytes were plated on the 8 .mu.m pore size membrane and
allowed to attach for 48 hours in 200 .mu.l growth medium. A DSTI
disc (6 mm diameter) was then placed in the bottom portion of the
chamber and 600 .mu.l growth or chondrogenic induction medium was
placed in the lower chamber and incubated at low O.sub.2. Media was
replaced 2 times per week. Cells begin to migrate into the lower
chamber and into DSTI, and differentiate into chondrocytes.
Following 1, 2, or 3 weeks, DSTI were analyzed for biochemical
determination of S-GAG, DNA, and for gene expression by RT-PCR.
[0328] Certain modifications to a classical Boyden chamber assay
were introduced. Membrane inserts with 8 .mu.m pore size were
placed in individual wells of a 24 well plate. 50,000 human
articular chondrocytes or bone marrow stem cells were plated in the
top chamber in growth media and allowed to attach for 48 hours with
nothing in the bottom chamber. This allowed cells to attach and
form a barrier that leads to decreased permeability to the lower
chamber, allowing a unidirectional effect from top to bottom
chambers. Following 1, 2, and 3 weeks in culture, DSTI were assayed
by DNA/GAG, and RT-PCR.
EXAMPLE 9
Determination of DSTI Chemoattractant Potential
[0329] This example describes procedure used for determination of
chemoattractant potential of the DSTI.
[0330] Approximately 50,000 cells/insert were seeded onto the top
chambers of 8 .mu.m pore Boyden Chambers and filled with DMEM/12
medium supplemented with 1% bovine serum albumin (BSA). The lower
chambers contained 6 mm DSTIs or were left empty and were filled
with one of three medium formulations: the same medium as in the
top chamber (1% BSA medium), growth medium (DMEM/F12 medium
supplemented with 10% fetal bovine serum (FBS)) or chondrogenic
medium (Lonza medium supplemented with TGF.beta.3).
[0331] The movement of cells through the membrane was observed at 4
hours and at 18 hours. Medium was aspirated from the upper chamber
and the membrane swabbed to remove cells still on top. The membrane
was then fixed with methanol and stained by immunofluorescence with
actin and DAPI to visualize and count the cells that penetrated the
membrane. Six high power fields were counted using a fluorescence
microscope and the mean .+-. standard deviation was calculated for
each group at each time point.
EXAMPLE 10
Method of Improvement of Differentiation
[0332] This example describes a protocol used for determination of
cell differentiation.
[0333] Approximately 50,000 MSC per one Boyden Chamber were seeded
onto the top chambers of 8 .mu.m pore Boyden chambers and filled
with Advanced Dulbecco's Minimal Essential Medium (ADMEM) medium
supplemented with 1% BSA. The lower chambers were either empty or
contained DSTI or DSTI loaded with 250 ng RGMA in 30 .mu.l PBS and
allowed to absorb into the scaffold for 60 minutes prior to start
of experiment. 500 .mu.l ADMEM containing 10% FBS was placed in the
lower chamber. After 18 hours, medium was aspirated from the upper
chamber and the membrane swabbed to remove cells on top. The
membranes were then fixed with methanol and stained by
immunofluorescence with 4,6-diamino -6-phenylindole (DAPI) to
visualize and count the cells that penetrated the membrane. Six
high power fields were counted using a fluorescence microscope and
the mean .+-. standard deviation was calculated for each group at
each time point.
EXAMPLE 11
Assay for Determination of Differentiation of Mesenchymal Stem
Cells
[0334] This example describes procedure used for determination of
fo differentiation of mesenchymal stem cells.
[0335] Human bone marrow mesenchymal stem cells (MSC) were
purchased from Lonza and maintained in culture with bone marrow
stem cells growth media containing 10% FBS. Human articular
chondrocytes (hAC) were extracted by placing finely minced
cartilage in a collagenase solution overnight. Liberated cells were
then plated on tissue culture plastic in chondrocyte growth media
(DMEM/F12+10% FBS). At confluence, MSC and hAC were lifted with
0.05% Trypsin+0.01% EDTA and cells were counted. A Boyden Chamber
of 8 .mu.m pore size was placed in a 24 well plate and 50,000 cells
of undifferentiated MSC or hAC were plated on the insert and
allowed to attach for 72 hours in 200 .mu.l growth medium. A DSTI
disc (6 mm diameter.times.10.5 m thickness) was then placed in a 24
chamber and hydrated with 30 .mu.l PBS (control) or 100 ng BMP-2,
100 ng BMP-7, or 250 ng RGMA and allowed to absorb into the DSTI
for 1 hour. 500 .mu.l of growth medium was placed in the lower
chamber together with DSTI preloaded with drug. Medium in the upper
chamber was replaced with 200 .mu.l DMEM/F12+1% BSA and the DSTI is
placed into the medium. Cells were allowed to migrate, proliferate,
and differentiate for 72 hours. Following 72 hours, DSTI in the
lower chamber was placed in TRIzol to extract RNA. In the longer 3
weeks experiments, DSTI were either placed in TRIzol to extract RNA
for RT-PCR or placed in buffered formalin for histological
analyses
EXAMPLE 12
Method for Production of Hyaline Cartilage
[0336] This example describes a protocol for investigation of
production of hyaline cartilage using cultured seeded mesenchymal
stem cells and human articular chondrocytes.
[0337] Human bone marrow mesenchymal stem cells (MSC) were
purchased from Lonza and maintained in culture with bone marrow
stem cells growth media containing 10% FBS. Human articular
chondrocytes (hAC) were extracted by placing finely minced
cartilage in a collagenase solution overnight. Liberated cells were
then plated on tissue culture plastic in chondrocyte growth media
(DMEM/F12+10% FBS). At confluence, MSC and hAC were lifted with
0.05% Trypsin+0.01% EDTA and cells were counted. A Boyden chamber
of 8 .mu.m pore size was placed in a 24 well plate and 50,000 cells
of undifferentiated MSC or hAC were plated on the insert and
allowed to attach for 72 hours in 200 .mu.l growth medium. A DSTI
disc (6 mm diameter.times.10.5 m thickness) was placed in a 24 well
plate and hydrated with 30 .mu.l PBS (control) or 100 ng BMP-2, 100
ng BMP-7, or 250 ng RGMA and allowed to absorb into the DSTI for 1
hour. 500 .mu.l of growth medium was placed in the lower well with
the drug preloaded DSTI. Medium in the upper insert was replaced
with 200 .mu.l DMEM/F12+1% BSA and the DSTI was placed in the
chamber. Cells are allowed to migrate, proliferate, and
differentiate for 3 weeks. Medium is replaced twice weekly; in half
of the growth factor conditions, fresh growth factor is added to
the lower well in the same concentrations as above. After 3 weeks,
DSTI was placed in TRIzol to extract RNA for RT-PCR analyses.
EXAMPLE 13
The Effect of Hydrostatic Pressure on Cell Differentiation in
DSTI
[0338] This example describes a protocol used for determination of
the effect of hydrostatic pressure on cell differentiation using
DSTI.
[0339] Bone marrow stem cells or human articular chondrocytes were
seeded into DSTI and placed in growth medium or chondrogenic
medium. The human articular chondrocytes or bone marrow stem cells
were seeded onto DSTI and allowed to attach for 48 hours. DSTIs
were then either placed in static conditions for 21 days or were
subjected to TEP for 7 days followed by static for 14 days. In both
situations, chondrogenic media was used for the first 7 days
followed by 3D media (DMEM:F12 supplemented with 10% FBS and ITS)
for the following 14 days. DSTIs were analyzed for histology by
Safranin-O, for biochemical determination of S-GAG by DMB, DNA by
Hoechst dye assay, and gene expression by RT-PCR.
EXAMPLE 14
Determination of Retention of Collagen within DSTI
[0340] This example describes a procedure used for determination of
the stability of the double-structured tissue implant in vitro.
[0341] Three lots of DSTIs are prepared as described in Example 4
and cut to a size of 1.5.times.1.5.times.0.15 cm. Cut DSTIs are
placed in 35 mm Petri dishes, rehydrated with 450 m of phosphate
buffered saline and additional 2 ml of phosphate buffered saline
are added to each Petri dish containing the DSTI. The analysis for
each lot consist of three replicates for a total of 9 samples for
the three lots.
[0342] Dishes containing individual DSTIs are placed in the
incubator for the duration of testing. In the predetermined
intervals of zero hour, 1 hour, 3 days, 7 days and 14 days, 1 ml
aliquot of the phosphate buffered saline is removed from each
plate. Each removed 1 ml is replaced with 1 ml of a fresh phosphate
buffer saline. The removed aliquots are subjected to a calorimetric
protein assay for quantification of total collagen released into
the saline.
[0343] Cumulative collagen retention curves are generated by
subtraction of the amount of collagen released into the solution
from the theoretical collagen load estimated at 0.777 mg of
collagen/DSTI sample.
* * * * *