U.S. patent application number 10/851773 was filed with the patent office on 2004-12-23 for compositions and methods for production and use of an injectable naturally secreted extracellular matrix.
Invention is credited to Naughton, Gail K..
Application Number | 20040259190 10/851773 |
Document ID | / |
Family ID | 27042969 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259190 |
Kind Code |
A1 |
Naughton, Gail K. |
December 23, 2004 |
Compositions and methods for production and use of an injectable
naturally secreted extracellular matrix
Abstract
The present invention discloses compositions containing natural
human extracellular matrices and methods for the use thereof. More
particularly, the present invention provides compositions and
methods for the repair of skin defects using natural human
extracellular matrix by injection.
Inventors: |
Naughton, Gail K.; (La
Jolla, CA) |
Correspondence
Address: |
KILPATRICK STOCKTON LLP
Attn: John S. Pratt
Suite 2800
1100 Peachtree Street
Atlanta
GA
30309-4530
US
|
Family ID: |
27042969 |
Appl. No.: |
10/851773 |
Filed: |
May 21, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10851773 |
May 21, 2004 |
|
|
|
10805774 |
Mar 22, 2004 |
|
|
|
10805774 |
Mar 22, 2004 |
|
|
|
09948379 |
Sep 7, 2001 |
|
|
|
09948379 |
Sep 7, 2001 |
|
|
|
08660787 |
Jun 6, 1996 |
|
|
|
08660787 |
Jun 6, 1996 |
|
|
|
08470101 |
Jun 6, 1995 |
|
|
|
5830708 |
|
|
|
|
Current U.S.
Class: |
435/41 ;
424/423 |
Current CPC
Class: |
Y10T 442/2525 20150401;
A61L 27/3633 20130101; A61F 2/105 20130101; Y10S 514/801 20130101;
A61L 27/3683 20130101; A61L 27/3839 20130101; A61Q 19/08 20130101;
A61F 2/0059 20130101; A61L 27/3804 20130101; A61L 27/60
20130101 |
Class at
Publication: |
435/041 ;
424/423 |
International
Class: |
C12P 001/00; A61F
002/00 |
Claims
1-29. (cancelled).
30. A composition comprising a three-dimensional framework, said
framework coated with a naturally secreted extracellular matrix
material composed of human proteins.
31. The composition according to claim 30, wherein the framework is
composed of a biodegradable material.
32. The composition according to claim 31, wherein the
biodegradable material is cotton, polyglycolic acid, cat gut
sutures, cellulose, gelatin, or dextran.
33. The composition according to claim 30, wherein the framework is
composed of a non-biodegradable material.
34. The composition according to claim 33, wherein the
nonbiodegradable material is a polyamide, a polyester, a
polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a
polycarbonate, a polytetrafluorethylene, or a nitrocellulose
compound.
35. The composition according to claim 30, wherein the
three-dimensional framework has pore spaces of about 150 .mu.m to
about 220 .mu.m.
36. The composition according to claim 30, wherein the
extracellular matrix is secreted by human stromal cells.
37. The composition according to claim 36, wherein the stromal
cells are fibroblasts.
38. The composition according to claim 36, wherein the stromal
cells are found in loose connective tissue or bone marrow.
39. The composition according to claim 38, wherein the stromal
cells are endothelial cells, pericytes, macrophages, monocytes,
leukocytes, plasma cells, mast cells or adipocytes.
40. An injectable formulation for the treatment of a skin or tissue
defect, comprising a cell-free, injectable formulation of naturally
secreted human extracellular matrix components synthesized by cells
in vitro and a pharmaceutically acceptable carrier formulated for
in vivo administration by injection via a syringe.
41. The injectable formulation according to claim 40, in which the
matrix is produced by a method comprising: (a) providing a living
stromal tissue prepared in vitro comprising human stromal cells and
connective tissue proteins naturally secreted by the stromal cells
attached to and substantially enveloping a framework, said
framework composed of a biocompatible, non-living material formed
into a three-dimensional structure having interstitial spaces
bridged by the stromal cells; (b) killing the cells in the living
stromal tissue; (c) removing the killed cells and any cellular
contents from the framework; (d) collecting the connective tissue
proteins naturally secreted by the stromal cells attached to the
framework; and (e) processing the collected connective tissue
proteins of step (d) with a pharmaceutically acceptable carrier
into a formulation that is suitable for in vivo administration by
injection via a syringe.
42. The injectable formulation according to claim 41, in which the
collected connective tissue proteins of step (d) are processed by
homogenizing, cross-linking, or suspending the collected connective
tissue proteins in a physiological acceptable carrier prior to step
(e).
43. The injectable formulation according to claim 41 in which the
collected connective tissue proteins of step (d) are processed by
adjusting ratios of collagen types I-V, respective to each other,
prior to step (e).
44. The injectable formulation according to claim 41, in which the
stromal cells of the living stromal tissue are fibroblasts.
45. The injectable formulation according to claim 41 in which the
stromal cells of the living stromal tissue are cells found in loose
connective tissue or bone marrow.
46. The injectable formulation according to claim 45 in which the
stromal cells of the living stromal tissue are endothelial cells,
pericytes, macrophages, monocytes, leukocytes, plasma cells, mast
cells, chondrocytes or adipocytes.
47. The injectable formulation according to claim 41, in which the
framework is composed of a biodegradable material.
48. The injectable formulation according to claim 47, in which the
biodegradable material is cotton, polyglycolic acid, cat gut
sutures, cellulose, gelatin, or dextran.
49. The injectable formulation according to claim 41, in which the
framework is composed of a non-biodegradable material.
50. The injectable formulation according to claim 49, in which the
non-biodegradable material is a polyamide, a polyester, a
polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a
polycarbonate, a polytetrafluorethylene, or a nitrocellulose
compound.
51. The injectable formulation according to claim 41, in which the
framework is a mesh
52. The injectable formulation according to claim 51 in which the
mesh has pore spaces of about 150 .mu.m to about 220 .mu.m.
53. The injectable formulation according to claim 41, in which the
pharmaceutically acceptable carrier contains an anesthetic agent, a
lubricating agent, a tissue growth factor or combinations
thereof.
54. The injectable formulation according to claim 40 produced by a
method comprising: (a) providing a living stromal tissue prepared
in vitro comprising human stromal cells and connective tissue
proteins naturally secreted by the stromal cells attached to and
substantially enveloping a framework, said framework composed of a
biocompatible, non-living biodegradable material formed into a
three-dimensional structure having interstitial spaces bridged by
the stromal cells; (b) killing the cells in the living stromal
tissue; (c) removing the killed cells and any cellular contents
from the framework; and (d) processing the connective tissue
proteins naturally secreted by the stromal cells attached to the
framework and the biodegradable framework with a pharmaceutically
acceptable carrier into a formulation suitable for in vivo
administration by injection via a syringe.
55. The injectable formulation according to claim 41, in which the
stromal cells recombinantly express a gene product.
56. The injectable formulation according to claim 55, in which
expression of the gene product is under the control of an inducible
promoter.
57. The injectable formulation according to claim 41, in which
steps (b) and (c) are carried out concurrently.
58. The injectable formulation according to claim 54, in which
steps (b) and (c) are carried out concurrently.
59. The injectable formulation according to claim 30 in which the
cells are grown in vitro on a three-dimensional framework.
Description
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. 08/470,101 filed Jun. 6, 1995,
which is incorporated by reference herein in its entirety.
1. INTRODUCTION
[0002] The present invention relates to compositions and methods
for the treatment and repair of soft tissue and skin defects such
as wrinkles and scars. More particularly, the invention relates to
an injectable composition of human extracellular matrix components
and methods of preparing and using same. The injectable preparation
is obtained from three-dimensional living stromal tissues that are
prepared in vitro.
2. BACKGROUND OF THE INVENTION
[0003] The idea of using an injectable material for soft tissue
augmentation and repair developed soon after the invention of the
hypodermic needle. Various products have been injected into the
human body for correction of soft tissue and skin defects including
paraffin, petrolatum, vegetable oils, lanolin, bees wax, and
silicone. Injectable liquid silicone has been used extensively,
however, due to long term side effects, such as nodules, recurring
cellulitis and skin ulcers which are now being followed more
closely, the use of injectable silicone is on the decline. Further,
in the State of Nevada it is a felony to use injectable silicone in
a human. Orange, Skin and Allergy News (1992) Vol.23, No.6, pg. 1.
More recently, bovine collagen has gained widespread use as an
injectable material for soft tissue augmentation. Collagen is the
principal extracellular structural protein of the animal body. At
least fourteen types of mammalian collagen have been described. The
common characteristic amongst them is a three stranded helix,
consisting of three polypeptide chains, called alpha-chains. All
alpha-chains have the same configuration, but differ in the
composition and sequence of their amino acids. Although this leads
to different types of alpha-chains, however, they all have glycine
at every third position in the amino acid sequence. The glycine at
every third position allows for the helical structure of the
alpha-chains. Type I collagen is composed of two alpha.sub.1-chains
and one alpha.sub.2-chain and is the principal extracellular
material of skin, tendon and bone. When clinicians mention
"collagen", they are usually referring to type I collagen. See
Table I, infra, for a detailed listing of collagen types I-V and in
which tissues they are found.
[0004] Collagen has been used as an implant material to replace or
augment hard or soft connective tissue, such as skin, tendon,
cartilage, bone and interstitium. Additionally, collagen implants
have been used for cosmetic purposes for a number of years since
collagen can help cellular ingrowth at the placement site. Early
collagen implants were often solid collagen masses which were
cross-linked with chemical agents, radiation or other means to
improve mechanical properties, decrease immunogenicity and/or
increase resistance to resorption. The-collagen utilized was in a
variety of forms, including cross-linked and non-cross-linked
fibrillar collagens, gelatins, and the like and sometimes was
combined with various other components, such as lubricants,
osteogenic factors and the like, depending on use. A major
disadvantage of solid cross-linked collagen implants is the
requirement for surgical implantation by means of incision. In
addition, lack of deformability and flexibility are other
disadvantages of solid collagen implants.
[0005] Oliver et al., Clinical Orthopaedics & Related Research
(1976) 115:291-302; Br. J. Exp. Path. (1980) 61:544-549; and Conn.
Tissue Res. (1981) 9:59-62 describe implants made by treating skin
with trypsin followed by cross-linking with an aldehyde. The
resulting solid collagen implants were reported to maintain their
original mass after prolonged implantation. A main problem with
such solid implants is that they must be implanted surgically.
Other disadvantages are that they are not as deformable as
injectable implants and residual glutaraldehyde may cause the
implant to lose its flexibility due to continuing cross-linking in
situ.
[0006] Schechter, et al., Br. J. Plas. Surg. (1975) 28:198-202
disclose glutaraldehyde cross-linked skin that was soaked in
L-alanine after cross-linking. The article postulates that the
exposure of the skin to L-alanine blocked residual reactive groups
of the aldehyde, thereby preventing the release of toxic molecules
generated by such groups.
[0007] An alternative to surgically implanted solid collagen
material is disclosed in U.S. Pat. No. 3,949,073. U.S. Pat. No.
3,949,073 describes the use of atelopeptide solutions of bovine
collagen as an injectable implant material for augmenting soft
tissue. According to the patent, the bovine collagen is
reconstituted before implantation and forms a fibrous mass of
tissue when implanted. The patent suggests adding particles of
insoluble bovine collagen microfibrils to control the shrinkage of
the fibrous mass formed at the augmentation site. The commercial
embodiment of the material described in the patent is composed of
reconstituted atelopeptide bovine collagen in saline that contains
a small amount of local anesthetic. While effective, the implant
shrinks in volume after implantation due primarily to absorption of
its fluid component by the body. Thus, if volume consistency is
essential, an additional injection or injections of supplemental
implant material is required. This specific composition has many
serious drawbacks, e.g., the collagen is from a bovine source, not
human, and the preparation process is not only lengthy and
expensive but also requires the addition of microfibrils.
[0008] U.S. Pat. No. 4,424,208 describes an injectable dispersion
of cross-linked atelopeptide bovine collagen and reconstituted
atelopeptide bovine collagen fibers in an aqueous carrier which
exhibited improved volume consistency over the material of U.S.
Pat. No. 3,949,073.
[0009] U.S. Pat. No. 4,582,640 discloses an improved injectable
implant over U.S. Pat. Nos. 3,949,073 and 4,424,208 in which the
improvement consists of improved volume consistency and resistance
to physical deformation, improved injectability as compared to the
dispersion of U.S. Pat. No. 4,424,208 and that the bovine collagen
contains only a single physical form of collagen as compared to the
two physical forms found in U.S. Pat. No. 4,424,208.
[0010] U.S. Pat. No. 4,803,075 describes bovine collagen
compositions including a lubricant material to enhance
injectability through narrow diameter needles for soft tissue
repair.
[0011] Despite the advantages and overall usefulness of the
injectable collagen implant materials disclosed above, problems
associated with producing and injecting the materials have been
encountered. For example, for soft tissue repair, suspensions of
fibrillar collagen have often been used by injecting the
composition to a treatment site through a fine gauge needle. The
use of fibrillar collagen as the primary matrix material in
injectable soft and hard tissue implant compositions has several
limitations. The preparation of fibrillar collagen suitable for
human use is relatively time consuming and expensive. In
particular, the complete removal of contaminating and potentially
immunogenic substances to produce atelocollagen is a relatively
complex and expensive procedure. Moreover, the persistence, shape
retention, cohesiveness, stability, elasticity, toughness-and
intrudability of the fibrillar collagen compositions are not
optimal.
[0012] In addition to the problems associated with producing and
injecting the collagen implant materials, problems with the actual
use of the above mentioned patented injectable implants are also
abundant. For instance, since the above patented injectables derive
collagen from xenogeneic sources, usually bovine collagen, the
collagen must be modified to reduce its immunogenicity. Even with
modified collagen, the implant material is still quite immunogenic
to which some people are either already naturally allergic or
develop an allergic reaction over time to the bovine collagen. Due
to these allergic reactions the injectable collagen implants
described above cannot be given to many people and others are
limited to receiving only three injections per year. Severe
allergic reactions include symptoms of rheumatoid arthritis, while
less severe reactions include redness and swelling at the site-of
injection which may lead to permanent scarring. Because of these
severe side effects, the above described collagen injectables are
no longer used for lip augmentation. Further, the problems
associated with injecting xenogeneic collagen seem so intractable
that rather than injecting collagen, biocompatible ceramic matrices
have been injected to achieve similar results as described in U.S.
Pat. No. 5,204,382.
[0013] In summary, due to the shortcomings of the above-described
injectable compositions for the repair of soft tissue defects, such
as the lack of persistence, the need for repeated injections and
serious concern over adverse reactions, newer injectable materials
for soft tissue augmentation are needed.
3. SUMMARY OF THE INVENTION
[0014] The present invention relates to injectable materials for
soft tissue augmentation and methods for use and manufacture of the
same, which overcome the shortcomings of bovine injectable collagen
and other injectable materials, including silicone, of the prior
art. The injectable materials used in accordance with the present
invention comprise naturally secreted extracellular matrix
preparations as well as preparations derived from naturally
secreted extracellular matrix. These preparations are
biocompatible, biodegradable and are capable of promoting
connective tissue deposition, angiogenesis, reepithilialization and
fibroplasia, which is useful in the repair of skin and other tissue
defects. These extracellular matrix preparations may be used to
repair tissue defects by injection at the site of the defect.
[0015] The injectable preparations of the present invention have
many advantages over conventional injectable collagen preparations
used for the repair of skin defects. The extracellular matrix
preparations of the present invention contain only human proteins,
therefore, there is a reduced risk of an immune response due to
foreign proteins or peptides, especially the type of immune
response seen with bovine collagen found in conventional injectable
collagen preparations. Additionally, the injected preparations of
the present invention should persist longer and even if multiple
injections are required, the injections should not be subject to
the "no more than three injections per year" rule of bovine
collagen-based preparations due to the lack of immunogenicity.
Another advantage provided by the present invention is that the
preparations of native extracellular matrix contain a mixture of
extracellular matrix proteins which closely mimics the compositions
of physiologically normal conditions, for example, in an
extracellular matrix derived from dermal cells, type I and III
collagens, hyaluronic acid as well as various glycosaminoglycans
and natural growth factors are present. Many of these extracellular
matrix proteins and growth factors have been studied extensively
and have been shown to be critical for wound healing and tissue
restoration.
[0016] In another aspect of the invention, the preparations can be
used in highly improved systems for in vitro tissue culture. In
this embodiment, naturally secreted extracellular matrix coated
three-dimensional frameworks can be used to culture cells which
require attachment to a support in order to grow but do not attach
to conventional tissue culture vessels. In addition to culturing
cells on a coated framework, the extracellular matrix secreted by
the cells onto the framework can be collected and used to coat
vessels for use in tissue culture. The extracellular matrix, acting
as a base substrate, may allow cells normally unable to attach to
conventional tissue culture dish base substrates to attach and
subsequently grow.
[0017] Yet another embodiment of the present invention is directed
to a novel method for determining the ability for cellular taxis of
a particular cell. The method involves inoculating one end of a
native extracellular matrix coated three-dimensional framework with
the cell type in question and over time measure the distance
traversed across the framework by the cell. Because the
extracellular matrix is secreted naturally by the cells onto the
framework, it is an excellent in vitro equivalent of extracellular
matrix found in the body. Such an assay, for example, may inform
whether isolated tumor cells are metastatic or whether certain
immune cells can migrate across or even chemotact across the
framework, thus, indicating that the cell has such cellular taxis
ability.
3.1. DEFINITIONS AND ABBREVIATIONS
[0018] The following terms used herein shall have the meanings
indicated:
[0019] Adherent Layer:
[0020] cells attached directly to the three-dimensional framework
or connected indirectly by attachment to cells that are themselves
attached directly to the matrix.
[0021] Pharmaceutically Acceptable Carrier:
[0022] an aqueous medium at physiological isotonicity and pH and
may contain other elements such as local anesthetics and/or fluid
lubricants.
[0023] Stromal Cells:
[0024] fibroblasts with or without other cells and/or elements
found in loose connective tissue, including but not limited to,
endothelial cells, pericytes, macrophages, monocytes, plasma cells,
mast cells, adipocytes, chondrocytes, etc.
[0025] Three-Dimensional Framework:
[0026] a three dimensional support composed of any material and/or
shape that (a) allows cells to attach to it (or can be modified to
allow cells to attach to it); and (b) allows cells to grow in more
than one layer. This support is inoculated with stromal cells to
form the living stromal matrix.
[0027] Living Stromal Tissue:
[0028] a three dimensional framework which has been inoculated with
stromal cells. Whether confluent or subconfluent, stromal cells
according to the invention continue to grow and divide. The living
stromal tissue prepared in vitro is the source of the extracellular
matrix proteins used in the injectable formulations of the
invention.
[0029] The following abbreviations shall have the meanings
indicated:
[0030] EDTA ethylene diamine tetraacetic acid
[0031] FBS fetal bovine serum
[0032] HBSS Hank's balanced salt solution
[0033] HS horse serum
[0034] MEM minimal essential medium
[0035] PBS phosphate buffered saline
[0036] RPMI 1640 Roswell Park Memorial Institute Medium No. 1640
(GIBCO, Inc., Grand Island, N.Y.)
[0037] SEM scanning electron microscopy
[0038] The present invention may be more fully understood by
reference to the following detailed description, examples of
specific embodiments and appended figures which are offered for
purposes of illustration only and not by way of limitation.
4. BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1. FIG. 1 is a scanning electron micrograph depicting
fibroblast attachment to the three-dimensional matrix and extension
of cellular processes across the mesh opening. Fibroblasts are
actively secreting matrix proteins and are at the appropriate stage
of subconfluency which should be obtained prior to inoculation with
tissue-specific cells.
[0040] FIG. 2A-D. FIGS. 2A-D are transmission electron micrographs
of collagen isolated from extracellular matrix prepared from dermal
tissue grown in vitro (FIG. 2A-B) or from a normal adult human
dermal sample (FIG. 2C-D).
5. DETAILED DESCRIPTION OF THE INVENTION
[0041] One embodiment of the present invention involves the
preparation and use of an injectable extracellular matrix
composition for the treatment of skin defects. The extracellular
matrix proteins are derived from a living stromal tissue prepared
in vitro by growing stromal cells on a three-dimensional framework
resulting in a multi-layer cell culture system. In conventional
tissue culture systems, the cells were grown in a monolayer. Cells
grown on a three-dimensional framework support, in accordance with
the present invention, grow in multiple layers, forming a cellular
matrix. This matrix system approaches physiologic conditions found
in vivo to a greater degree than previously described monolayer
tissue culture systems. The three-dimensional cell culture system
is applicable to the proliferation of different types of stromal
cells and formation of a number of different stromal tissues,
including but not limited to dermis, bone marrow stroma, glial
tissue, cartilage, to name but a few.
[0042] In accordance with the present invention, the
pre-established living stromal tissue comprises stromal cells grown
on a three-dimensional framework or network. The stromal cells can
comprise fibroblasts with or without additional cells and/or
elements described more fully herein. The fibroblasts and other
cells and/or elements that comprise the stroma can be fetal or
adult in origin, and can be derived from convenient sources such as
skin, liver, pancreas, etc. Such tissues and/or organs can be
obtained by appropriate biopsy or upon autopsy. In fact, cadaver
organs may be used to provide a generous supply of stromal cells
and elements.
[0043] Once inoculated onto the three-dimensional framework, the
stromal cells will proliferate on the framework, and elaborate
growth factors, regulatory factors and extracellular matrix
proteins that are deposited on the support. The living stromal
tissue will sustain active proliferation of the culture for long
periods of time. Growth and regulatory factors can be added to the
culture, but are not necessary since they are elaborated by the
stromal support matrix.
[0044] The naturally secreted extracellular matrix is collected
from the three-dimensional framework and is processed further with
a pharmaceutically acceptable aqueous carrier and placed in a
syringe for precise placement of the biomaterial into tissues, such
as the facial dermis.
[0045] The present invention is based, in part, on the discovery
that during the growth of human stromal cells on a biodegradable or
non-biodegradable three-dimensional support framework, the cells
synthesize and deposit on the three-dimensional support framework a
human extracellular matrix as produced in normal human tissue. The
extracellular matrix is secreted locally by cells and not only
binds cells and tissues together but also influences the
development and behavior of the cells it contacts. The
extracellular matrix contains various fiber-forming proteins
interwoven in a hydrated gel composed of a network of
glycosaminoglycan chains. The glycosaminoglyeans are a
heterogeneous group of long, negatively charged polysaccharide
chains, which (except for hyaluronic acid) are covalently linked to
protein to form proteoglycan molecules.
[0046] The fiber-forming proteins are of two functional types:
mainly structural (collagens and elastin) and mainly adhesive (such
as fibronectin and laminin). The fibrillar collagens (types I, II,
and III) are rope-like, triple-stranded helical molecules that
aggregate into long cable-like fibrils in the extracellular space;
these in turn can assemble into a variety of highly ordered arrays.
Type IV collagen molecules assemble into a sheetlike meshwork that
forms the core of all basal laminae. Elastin molecules form an
extensive cross-linked network of fibers and sheets that can
stretch and recoil, imparting elasticity to the matrix. Fibronectin
and laminin are examples of large adhesive glycoproteins in the
matrix; fibronectin is widely distributed in connective tissues,
whereas laminin is found mainly in basal laminae. By means of their
multiple binding domains, such proteins help cells adhere to and
become organized by the extracellular matrix.
[0047] As an example, a naturally secreted human dermal
extracellular matrix contains type I and type III collagens,
fibronectin, tenascin, glycosaminoglycans, acidic and basic FGF,
TGF-.alpha. and TGF-.beta., KGF, decorin and various other secreted
human dermal matrix proteins. As naturally secreted products, the
various extracellular matrix proteins are produced in the
quantities and ratios similar to that existing in vivo. Moreover,
growth of the stromal cells in three dimensions will sustain active
proliferation of cells in culture for much longer time periods than
will monolayer systems. Further, the three-dimensional system
supports the maturation, differentiation, and segregation of cells
in culture in vitro to form components of adult tissues analogous
to counterparts found in vivo. Thus, the extracellular matrix
created by the cells in culture is more analogous to native
tissues.
[0048] Although the applicants are under no duty or obligation to
explain the mechanism by which the invention works, a number of
factors inherent in the three-dimensional culture system may
contribute to these features of the three dimensional culture
system:
[0049] (a) The three-dimensional framework provides a greater
surface area for protein attachment, and consequently, for the
adherence of stromal cells.
[0050] (b) Because of the three-dimensionality of the framework,
stromal cells continue to actively grow in contrast to cells in
monolayer cultures, which grow to confluence, exhibit contact
inhibition, and cease to grow and divide. The elaboration of growth
and regulatory factors by replicating stromal cells may be
partially responsible for stimulating proliferation and regulating
differentiation of cells in culture.
[0051] (c) The three-dimensional framework allows for a spatial
distribution of cellular elements which is more analogous to that
found in the counterpart tissue in vivo.
[0052] (d) The increase in potential volume for cell growth in the
three-dimensional system may allow the establishment of localized
microenvironments analogous to native counterparts found in
vivo.
[0053] (e) The three-dimensional matrix maximizes cell-cell
interactions by allowing greater potential for movement of
migratory cells, such as macrophages, monocytes and possibly
lymphocytes in the adherent layer.
[0054] (f) It has been recognized that maintenance of a
differentiated cellular phenotype requires not only
growth/differentiation factors but also the appropriate cellular
interactions. The present invention effectively recreates the
stromal tissue microenvironment.
[0055] The three-dimensional stromal support, the culture system
itself, and its maintenance, as well as various uses of the
three-dimensional cultures and of the naturally secreted
extracellular matrix are described in greater detail in the
subsections below. Solely for ease of explanation, the detailed
description of the invention is divided into the three sections,
(i) growth of the three-dimensional stromal cell culture, (ii)
isolation of the naturally secreted human extracellular matrix, and
(iii) formulation of the isolated extracellular matrix into
preparations for injection at the site of soft tissue defects.
5.1. Preparing the Living Stromal Tissue In Vitro
[0056] The three-dimensional support used to culture stromal tissue
may be of any material and/or shape that:
[0057] (a) allows cells to attach to it (or can be modified to
allow cells to attach to it); and
[0058] (b) allows cells to grow in more than one layer.
[0059] A number of different materials may be used to form the
framework, such as non-biodegradable or biodegradable materials.
For example, non-biodegradable materials include but are not
limited to: nylon (polyamides), dacron (polyesters), polystyrene,
polypropylene, polyacrylates, polyvinyl compounds (e.g.,
polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene
(PTFE; teflon), thermanox (TPX), etc. Additionally, biodegradable
material may also be utilized, including but not limited to:
nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures,
cellulose, gelatin, dextran, collagen, chitosan, hyaluronic acid,
etc. Any of these materials, bio- or non-biodegradable, can be
woven into a mesh to form a three-dimensional framework.
Alternatively, the materials can be used to form other types of
three-dimensional frameworks, for example, sponges, such as
collagen sponges.
[0060] Certain materials, such as nylon, polystyrene, etc., are
poor substrates for cellular attachment. When these materials are
used as the three-dimensional support framework, it is advisable to
pre-treat the framework prior to inoculation of stromal cells in
order to enhance the attachment of stromal cells to the framework.
For example, prior to inoculation with stromal cells, nylon
frameworks can be treated with 0.1 M acetic acid, and incubated in
polylysine, FBS, and/or collagen to coat the nylon. Polystyrene can
be similarly treated using sulfuric acid. A convenient nylon mesh
which can be used in accordance with the invention is Nitex, a
nylon filtration mesh having an average pore size of 210 .mu.m and
an average nylon fiber diameter of 90 .mu.m (#3-210/36, Tetko,
Inc., N.Y.).
[0061] Stromal cells comprising fibroblasts derived from adult or
fetal tissue, with or without other cells and elements described
below, are inoculated onto the framework. These fibroblasts may be
derived from organs, such as skin, liver, pancreas, etc. which can
be obtained by biopsy, where appropriate, or upon autopsy. In fact,
fibroblasts can be obtained in quantity rather conveniently from
any appropriate cadaver organ. In a preferred embodiment, fetal
fibroblasts can be obtained in high quantity from foreskin.
[0062] Fibroblasts may be readily isolated by disaggregating an
appropriate organ or tissue which is to serve as the source of the
fibroblasts. This can be readily accomplished using techniques
known to those skilled in the art. For example, the tissue or organ
can be disaggregated mechanically and/or treated with digestive
enzymes and/or chelating agents that-weaken the connections between
neighboring cells making it possible to disperse the tissue into a
suspension of individual cells without appreciable cell breakage.
Enzymatic dissociation can be accomplished by mincing the tissue
and treating the minced tissue with any of a number of digestive
enzymes either alone or in combination. These include but are not
limited to trypsin, chymotrypsin, collagenase, elastase,
hyaluronidase, DNase, pronase, and/or dispase etc. Mechanical
disruption can also be accomplished by a number of methods
including, but not limited to the use of grinders, blenders,
sieves, homogenizers, pressure cells, or insonators to name but a
few. For a review of tissue disaggregation techniques, see
Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d
Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.
[0063] Once the tissue has been reduced to a suspension of
individual cells, the suspension can be fractionated into
subpopulations from which the fibroblasts and/or other stromal
cells and/or elements can be obtained. This also may be
accomplished using standard techniques for cell separation
including, but not limited to, cloning and selection of specific
cell types, selective destruction of unwanted cells (negative
selection), separation based upon differential cell agglutinability
in the mixed population, freeze-thaw procedures, differential
adherence properties of the cells in the mixed population,
filtration, conventional and zonal centrifugation, centrifugal
elutriation (counter-streaming centrifugation), unit gravity
separation, countercurrent distribution, electrophoresis and
fluorescence-activated cell sorting. For a review of clonal
selection and cell separation techniques, see Freshney, Culture of
Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss,
Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.
[0064] The isolation of fibroblasts, for example, can be carried
out as follows: fresh tissue samples are thoroughly washed and
minced in Hanks' balanced salt solution (HBSS) in order to remove
serum. The minced tissue is incubated from 1-12 hours in a freshly
prepared solution of a dissociating enzyme such as trypsin. After
such incubation, the dissociated cells are suspended, pelleted by
centrifugation and plated onto culture dishes. All fibroblasts will
attach before other cells, therefore, appropriate stromal cells can
be selectively isolated and grown. The isolated fibroblasts can
then be grown to confluency, lifted from the confluent culture and
inoculated onto the three-dimensional framework, see Naughton et
al., 1987, J. Med. 18(3&4):219-250. Inoculation of the
three-dimensional framework with a high concentration of stromal
cells, e.g., approximately 10.sup.6 to 5.times.10.sup.7 cells/ml,
will result in the establishment of the three-dimensional stromal
support in shorter periods of time.
[0065] In addition to fibroblasts, other cells can be added to form
the three-dimensional stromal cell culture-producing extracellular
matrix. For example, other cells found in loose connective tissue
may be inoculated onto the three-dimensional support framework
along with fibroblasts. Such cells include, but are not limited to,
endothelial cells, pericytes, macrophages, monocytes, plasma cells,
mast cells, adipocytes, chondrocytes, etc. These stromal cells can
be readily derived from appropriate organs such as skin, liver,
etc., using methods known, such as those discussed above.
[0066] In one embodiment of the present invention, stromal cells
which are specialized for the particular tissue to be cultured can
be added to the fibroblast stroma for the production of a tissue
type specific extracellular matrix. For example, dermal fibroblasts
can be used to form the three-dimensional subconfluent stroma for
the production of skin-specific extracellular matrix in vitro.
Alternatively, stromal cells of hematopoietic tissue including, but
not limited to, fibroblast endothelial cells,
macrophages/monocytes, adipocytes and reticular cells, can be used
to form the three-dimensional subconfluent stroma for the
production of a bone marrow-specific extracellular matrix in vitro,
see infra. Hematopoietic stromal cells can be readily obtained from
the "buffy coat" formed in bone marrow suspensions by
centrifugation at low forces, e.g., 3000.times.g. Stromal cells of
liver may include fibroblasts, Kupffer cells, and vascular and bile
duct endothelial cells. Similarly, glial cells can be used as the
stroma to support the proliferation of neurological cells and
tissues. Glial cells for this purpose can be obtained by
trypsinization or collagenase digestion of embryonic or adult
brain. Ponten and Westermark, 1980, In Federof, S. Hertz, L., eds,
"Advances in Cellular Neurobiology," Vol.1, New York, Academic
Press, pp.209-227.
[0067] For certain uses in vivo it is preferable to obtain the
stromal cells from the patient's own tissues. The growth of cells
in the presence of the three-dimensional stromal support framework
can be further enhanced by adding to the framework, or coating the
framework support with proteins, e.g., collagens, elastic fibers,
reticular fibers, glycoproteins; glycosaminoglycans, e.g., heparin
sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan
sulfate, keratan sulfate, etc.; a cellular matrix, and/or other
materials.
[0068] After inoculation of the stromal cells, the
three-dimensional framework is incubated in an appropriate nutrient
medium under physiologic conditions favorable for cell growth,
i.e., promoting mitosis, i.e., cell division. Many commercially
available media such as RPMI 1640, Fisher's, Iscove's, McCoy's, and
the like may be suitable for use. It is important that the
three-dimensional stromal culture be suspended or floated in the
medium during the incubation period in order to maximize
proliferative activity. In addition, the culture should be "fed"
periodically to remove the spent media, depopulate released cells,
and to add fresh media.
[0069] During the incubation period, the stromal cells will grow
linearly along and envelop the three-dimensional framework before
beginning to grow into the openings of the framework. The cells are
grown to an appropriate degree to allow for adequate deposition of
extracellular matrix proteins.
[0070] The openings of the framework should be of an appropriate
size to allow the stromal cells to stretch across the openings.
Maintaining actively growing stromal cells which stretch across the
framework enhances the production of growth factors which are
elaborated by the stromal cells, and hence, will support long term
cultures. For example, if the openings are too small, the stromal
cells may rapidly achieve confluence but be unable to easily exit
from the mesh. Trapped cells can exhibit contact inhibition and
cease production of the appropriate factors necessary to support
proliferation and maintain long term cultures. If the openings are
too large, the stromal cells are unable to stretch across the
opening. This will also decrease stromal cell production of the
appropriate factors necessary to support proliferation and maintain
long term cultures. When using a mesh type of matrix, as
exemplified herein, we have found that openings ranging from about
150 .mu.m to about 220 .mu.m will work satisfactorily. However,
depending upon the three-dimensional structure and intricacy of the
framework, other sizes may work equally well. In fact, any shape or
structure that allows the stromal cells to stretch and continue to
replicate and grow for lengthy time periods will work in accordance
with the present invention.
[0071] Different proportions of the various types of collagen
deposited on the framework can be achieved by inoculating the
framework with different tissue-specific cells. For example, for
hematopoietic cells, the matrix should preferably contain collagen
types III, IV and I in an approximate ratio of 6:3:1 in the initial
matrix. For skin, collagen types I and III are preferably deposited
in the initial matrix. The proportions of collagen types deposited
can be manipulated or enhanced by selecting fibroblasts which
elaborate the appropriate extracellular matrix proteins. This can
be accomplished using monoclonal antibodies of an appropriate
isotype or subclass which are capable of activating complement, and
which define particular collagen types. These antibodies in
combination with complement can be used to negatively select the
fibroblasts which express the desired collagen type. Alternatively,
the stroma used to inoculate the framework can be a mixture of
cells which synthesize the appropriate collagen types desired. The
distribution and origins of the five types of collagen is shown in
Table I.
1TABLE I DISTRIBUTIONS AND ORIGINS OF THE FIVE TYPES OF COLLAGEN
Collagen Principal Tissue Type Distribution Cells of Origin I Loose
and dense ordinary Fibroblasts and connective tissue; collagen
reticular cells; fibers smooth muscle cells Fibrocartilage Bone
Osteoblast Dentin Odontoblasts II Hyaline and elastic Chondrocytes
cartilage Retinal Cells Vitreous body of eye III Loose connective
tissue; Fibroblasts and reticular fibers reticular cells Papillary
layer of dermis Smooth muscle cells; Blood vessels endothelial
cells IV Basement membranes Epithelial and endothelial cells Lens
capsule of eye Lens fibers V Fetal membranes; placenta Fibroblasts
Basement membranes Bone Smooth muscle Smooth muscle cells
[0072] Thus, depending upon the collagen types desired, the
appropriate stromal cell(s) can be selected to inoculate the
three-dimensional framework.
[0073] The three-dimensional extracellular matrix producing culture
of the present invention affords a vehicle for introducing gene
products in vivo. In certain situations, it may be desirable to
prepare an extracellular matrix containing a foreign gene product,
growth factor, regulatory factor, etc. In such cases, the cells may
be genetically engineered to express the gene product, or altered
forms of the gene product that are immobilized in the extracellular
matrix laid down by the stromal cells. For example, using
recombinant DNA techniques, a gene of interest can be placed under
the control of an inducible promoter. The recombinant DNA construct
containing the gene can be used to transform or transfect a host
cell which is cloned and then clonally expanded in the
three-dimensional culture system. The use of the-three-dimensional
culture in this regard has a number of advantages. First, since the
culture comprises eukaryotic cells, the gene product will be
properly expressed and processed in culture to form an active
product. Second, the number of transfected cells can be
substantially enhanced to be of clinical value, relevance, and
utility. The three-dimensional cultures of the present invention
allow for expansion of the number of transfected cells and
amplification (via cell division) of transfected cells.
[0074] Preferably, the expression control elements used should
allow for the regulated expression of the gene so that the product
can be over synthesized in culture. The transcriptional promoter
chosen, generally, and promoter elements specifically, depends, in
part, upon the type of tissue and cells cultured. Cells and tissues
which are capable of secreting proteins (e.g., those characterized
by abundant rough endoplasmic reticulum and golgi complex) are
preferable.
[0075] During incubation of the three-dimensional culture,
proliferating cells are released from the framework. These released
cells can stick to the walls of the culture vessel where they can
continue to proliferate and form a confluent monolayer. This should
be prevented or minimized, for example, by removal of the released
cells during feeding, or by transferring the three-dimensional
framework to a new culture vessel. The presence of a confluent
monolayer in the vessel will "shut down" the growth of cells in the
three-dimensional framework and/or culture. Removal of the
confluent monolayer or transfer of the stromal culture to fresh
media in a new vessel will restore proliferative activity of the
three-dimensional culture system. Such removal or transfers should
be done in any culture vessel which has a stromal monolayer
exceeding 25% confluency. Alternatively, the culture system can be
agitated to prevent the released cells from sticking, or instead of
periodically feeding the cultures, the culture system could be set
up so that fresh media continuously flows through the system. The
flow rate can be adjusted to both maximize proliferation within the
three-dimensional culture, and to wash out and remove cells
released from the matrix, so that they will not stick to the walls
of the vessel and grow to confluence. In any case, the released
stromal cells can be collected and crypreserved for future use.
[0076] Once inoculated onto the three-dimensional framework,
adherence of the fibroblasts is seen quickly (e.g., within hours)
and the fibroblasts begin to stretch across the framework openings
within days. These fibroblasts are metabolically active, secrete
extracellular matrix and rapidly form a dermal equivalent
consisting of active fibroblasts and collagen.
[0077] FIG. 1 illustrates the ability of the fibroblasts to arrange
themselves into parallel layers between the naturally-secreted
collagen bundles. These fibroblasts exhibit a rapid rate of cell
division and protein secretion.
5.2. Removal of the Extracellular Matrix from the Framework
[0078] After the cells have been inoculated onto the framework and
cultured under conditions favoring cellular growth, such that a
desired amount of extracellular matrix is secreted on to the
three-dimensional framework, the cells are killed and the naturally
secreted extracellular matrix is processed further.
[0079] This involves first killing the cells and removing the
killed cells and any cellular debris from the three-dimensional
framework. This process is carried out in a number of different
ways. For example, the cells can be killed by flash-freezing the
living stromal tissue prepared in vitro in liquid nitrogen without
a cryopreservative. Another way to kill the cells is to irrigate
the inoculated three-dimensional framework with sterile water, such
that the cells burst in response to osmotic pressure. Once the
cells have been killed, one can, for example, disrupt the cellular
membranes and remove the cellular debris by a mild detergent rinse,
such as EDTA, CHAPS or a zwitterionic detergent, followed by
treatment with a cryoprotectant such as DMSO, propylene glycol,
butanediol, raffinose, polyvinyl pyrrolidone, dextran or sucrose
and vitrified in liquid nitrogen. Alternatively, the framework can
be subjected to enzymatic digestion and/or extracting with reagents
that break down the cellular membranes and allow removal of cell
contents. Examples of detergents include non-ionic detergents (for
example, TRITON X-100, octylphenoxy polyethoxyethanol, (Rohm and
Haas); BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical
Co.), TWEEN 20, a polyethoxyethanol sorbitan monolaureate (Rohm and
Haas), LUBROL-PX, or polyethylene lauryl ether (Rohm and Haas));
and ionic detergents (for example, sodium dodecyl sulphate,
sulfated higher aliphatic alcohol, sulfonated alkane and sulfonated
alkylarene containing 7 to 22 carbon atoms in a branched or
unbranched chain). Enzymes can be used also and can include
nucleases (for example, deoxyribonuclease and ribonuclease),
phospholipases and lipases. An advantage to using a mild detergent
rinse is that it will solubilize membrane-bound proteins, which are
often highly antigenic.
[0080] Once the cells have been killed and the cellular debris has
been removed, the collection of the naturally secreted human
extracellular matrix can be accomplished in a variety of ways which
depends on whether the three-dimensional framework is composed of
material that is biodegradable or non-biodegradable. For example,
if the framework is composed of non-biodegradable material, one can
remove the extracellular matrix from a non-biodegradable support by
subjecting the three-dimensional framework to sonication and/or to
high pressure water jets and/or to mechanical scraping and/or to a
mild treatment with detergents and/or enzymes to remove the
attached extracellular matrix from the framework.
[0081] If the extracellular matrix is deposited on a biodegradable
three-dimensional framework, after killing and removing the cells
and cellular debris, the extracellular matrix can be recovered, for
example, by simply allowing the framework to degrade in solution,
i.e., allow the framework to dissolve, thus freeing the
extracellular matrix. Additionally, if the biodegradable support is
composed of a material which can be injected, like the
extracellular matrix itself, one can process the entire
extracellular matrix coated framework into syringes for injection.
Further, if the extracellular matrix is deposited on a
biodegradable support, the matrix can be removed by the same
methods as if the matrix had been deposited on a non-biodegradable
support, i.e., by subjecting the three-dimensional framework to
sonication and/or to high pressure water jets and/or to mechanical
scraping and/or to a mild treatment with detergents and/or enzymes
to remove the attached extracellular matrix from the framework.
None of the removal processes are designed to damage and/or
denature the naturally secreted human extracellular matrix produced
by the cells.
5.3. Formulation and Use of Injectable Preparations
[0082] Once the naturally secreted extracellular matrix has been
collected, it is processed further. The extracellular matrix can be
homogenized to fine particles, such that it can pass through a
surgical needle. Homogenization is well known in the art, for
example, by sonication. Further, the extracellular matrix can be
cross-linked by gamma irradiation without the use of chemical
cross-linking agents, such as glutaraldehyde, which are toxic. The
gamma irradiation should be a minimum of 20 M rads to sterilize the
material since all bacteria, fungi, and viruses are destroyed at
0.2 M rads. Preferably, the extracellular matrix can be irradiated
from 0.25 to 2 M rads to sterilize and cross-link the extracellular
matrix.
[0083] Further, the amounts and/or ratios of the collagens and
other proteins may be adjusted by mixing extracellular matrices
secreted by other cell types prior to placing the material in a
syringe. For example, biologically active substances, such as
proteins and drugs, can be incorporated in the compositions of the
present invention for release or controlled release of these active
substances after injection of the composition. Exemplary
biologically active substances can include tissue growth factors,
such as TGF-.beta., and the like which promote healing and tissue
repair at the site of the injection.
[0084] Final formulation of the aqueous suspension of naturally
secreted human extracellular matrix will typically involve
adjusting the ionic strength of the suspension to isotonicity
(i.e., about 0.1 to 0.2) and to physiological pH (i.e., about pH
6.8 to 7.5) and adding a local anesthetic, such as lidocaine,
(usually at a concentration of about 0.3% by weight) to reduce
local pain upon injection. The final formulation will also
typically contain a fluid lubricant, such as maltose, which must be
tolerated by the body. Exemplary lubricant components include
glycerol, glycogen, maltose and the like organic polymer base
materials, such as polyethylene glycol and hyaluronic acid as well
as non-fibrillar collagen, preferably succinylated collagen, can
also act as lubricants. Such lubricants are generally used to
improve the injectability, intrudability and dispersion of the
injected biomaterial at the site of injection and to decrease the
amount of spiking by modifying the viscosity of the compositions.
This final formulation is by definition the processed extracellular
matrix in a pharmaceutically acceptable carrier.
[0085] The matrix is subsequently placed in a syringe or other
injection apparatus for precise placement of the matrix at the site
of the tissue defect. In the case of formulations for dermal
augmentation, the term "injectable" means the formulation can be
dispensed from syringes having a gauge as low as 25 under normal
conditions under normal pressure without substantial spiking.
Spiking can cause the composition to ooze from the syringe rather
than be injected into the tissue. For this precise placement,
needles as fine as 27 gauge (200.mu. I.D.) or even 30 gauge
(150.mu. I.D.) are desirable. The maximum particle size that can be
extruded through such needles will be a complex function of at
least the following: particle maximum dimension, particle aspect
ratio (length:width), particle rigidity, surface roughness of
particles and related factors affecting particle:particle adhesion,
the viscoelastic properties of the suspending fluid, and the rate
of flow through the needle. Rigid spherical beads suspended in a
Newtonian fluid represent the simplest case, while fibrous or
branched particles in a viscoelastic fluid are likely to be more
complex.
[0086] The above described steps in the process for preparing
injectable naturally secreted human extracellular matrix are
preferably carried out under sterile conditions using sterile
materials. The processed extracellular matrix in a pharmaceutically
acceptable carrier can be injected intradermally or subcutaneously
to augment soft tissue, to repair or correct congenital anomalies,
acquired defects or cosmetic defects. Examples of such conditions
are congenital anomalies as hemifacial microsomia, malar and
zygomatic hypoplasia, unilateral mammary hypoplasia, pectus
excavatum, pectoralis agenesis (Poland's anomaly) and
velopharyngeal incompetence secondary to cleft palate repair or
submucous cleft palate (as a retropharyngeal implant); acquired
defects (post-traumatic, post-surgical, post-infectious) such as
depressed scars, subcutaneous atrophy (e.g., secondary to discoid
lupis erythematosus), keratotic lesions, enophthalmos in the
unucleated eye (also superior sulcus syndrome), acne pitting of the
face, linear scleroderma with subcutaneous atrophy, saddle-nose
deformity, Romberg's disease and unilateral vocal cord paralysis;
and cosmetic defects such as glabellar frown lines, deep nasolabial
creases, circum-oral geographical wrinkles, sunken cheeks and
mammary hypoplasia. The compositions of the present invention can
also be injected into internal tissues, such as the tissues
defining body sphincters to augment such tissues.
[0087] Various sample embodiments of the invention are described in
the sections below. For purposes of description only, and not by
way of limitation, the three-dimensional culture system of the
invention is described based upon the type of tissue and cells used
in various systems. These descriptions specifically include but are
not limited to bone marrow, skin, epithelial cells, and cartilage
but it is expressly understood that the three-dimensional culture
system can be used with other types of cells and tissues. The
invention is also illustrated by way of examples, which demonstrate
characteristic data generated for each system described.
EXAMPLES
6. Example
Three-Dimensional Skin Stromal Culture System
[0088] The subsections below describe the three-dimensional culture
system of the invention for culturing different stromal cells in
vitro. Briefly, cultures of fibroblasts were established on nylon
mesh which had been previously sterilized. Within 6-9 days of
incubation, adherent fibroblasts began to grow into the meshwork
openings and deposited parallel bundles of collagen. Indirect
immunofluorescence using monoclonal antibodies showed predominantly
type I collagen with some type III as well.
6.1. Establishment of the Three-Dimensional Stroma of Skin
Fibroblasts
[0089] Skin fibroblasts were isolated by mincing dermal tissue,
trypsinization for 2 hours, and separation of cells into a
suspension by physical means. Fibroblasts were grown to confluency
in 25 cm.sup.2 Falcon tissue culture dishes and fed with RPMI 1640
(Sigma, MO) supplemented with 10% fetal bovine serum (FBS),
fungizone, gentamicin, and penicillin/streptomycin. Fibroblasts
were lifted by mild trypsinization and cells were plated onto nylon
filtration mesh, the fibers of which are approximately 90 .mu.m in
diameter and are assembled into a square weave with a mesh opening
of 210 .mu.m (Tetko, Inc., NY). The mesh was pretreated with a mild
acid wash and incubated in polylysine and FBS. Adherence of the
fibroblasts was seen within 3 hours, and fibroblasts began to
stretch across the mesh openings within 5-7 days of initial
inoculation. These fibroblasts were metabolically active, secreted
an extracellular matrix, and rapidly formed a dermal equivalent
consisting of active fibroblasts and collagen. FIG. 1 is a scanning
electron micrograph depicting fibroblast attachment and extension
of cellular processes across the mesh opening.
6.2 Establishment of the Three-Dimensional Bone Marrow Stromal
Cultures
[0090] Bone marrow was aspirated from multiple sites on the
posterior iliac crest of hematologically normal adult volunteers
after informed consent was obtained. Specimens were collected into
heparinized tubes and suspended in 8 ml of RPMI 1640 medium which
was conditioned with 10% FBS and 5-10% HS and supplemented with
hydrocortisone, fungizone, and streptomycin. The cell clumps were
disaggregated and divided into aliquots of 5.times.10.sup.6
nucleated cells.
[0091] Nylon filtration screen (#3-210/36, Tetko Inc., NY) was used
as a three-dimensional framework to support all stromal cell
cultures described in the examples below. The screen consisted of
fibers, which were 90 .mu.m in diameter, assembled into a square
weave pattern with sieve openings of 210 .mu.m. Stromal cells were
inoculated using the protocols described in Section 6.1. Adherence
and subsequent growth of the stromal elements was monitored using
inverted phase contrast microscopy and scanning electron microscopy
(SEM).
6.3 Preparation of the Three-Dimensional Oral Mucosal Epithelial
Stromal Matrix
[0092] Samples of oral mucosal tissue were obtained from
orthodontic surgical specimens. Tissue was washed three times with
fresh MEM containing antibiotics (2 ml of antibiotic antimycotic
solution from GIBCO, Cat. #600-5240 AG; and 0.01 ml of gentamicin
solution from GIBCO Cat. #600-5710 AD per 100 cc MEM), cut into
small pieces, then washed with 0.02% EDTA (w/v). 0.25% trypsin (in
PBS without Ca.sup.++ or Mg.sup.++) was added; after a few seconds,
the tissue pieces were removed and placed in fresh trypsin (in PBS
without Ca.sup.++ or Mg.sup.++) and refrigerated at 4.degree. C.
overnight. Tissues were removed and placed in fresh trypsin
solution, and gently agitated until cell appeared to form a
single-cell suspension. The single-cell suspension was then diluted
in MEM containing 10% heat inactivated fetal bovine serum and
centrifuged at 1400.times.g for 7 minutes. The supernatant was
decanted and the pellet containing mucosal epithelial cells was
placed into seeding medium. Medium consisted of DMEM with 2%
Ultrosen G, 1.times.L-glutamine, 1.times.non-essential amino acids,
penicillin and streptomycin. The cells were seeded onto a
three-dimensional framework. The three-dimensional stromal culture
was generated using oral fibroblasts and 8 mm.times.45 mm pieces of
nylon filtration screen (#3-210/36, Tetko Inc., NY). The mesh was
soaked in 0.1 M acetic acid for 30 minutes and treated with 10 mM
polylysine suspension for 1 hour. The meshes were placed in a
sterile petri dish and inoculated with 1.times.10.sup.6 oral
fibroblasts collected as described above in DMEM complete medium.
After 1-2 hours of incubation at 5% CO.sub.2 the meshes were placed
in a Corning 25 cm.sup.2 tissue culture flask, floated with an
additional 5 ml of medium, and allowed to reach subconfluence,
being fed at 3 day intervals. Cultures were maintained in DMEM
complete medium at 37.degree. C. and 5% CO.sub.2 in a humidified
atmosphere and were fed with fresh medium every 3 days.
6.4 Establishment of the Three Dimensional Small Vessel Endothelial
Stromal Cell Culture
[0093] Small vessel endothelial cells isolated from the brain
according to the method of Larson et al., 1987, Microvasc. Res.
34:184 were cultured in vitro using T-75 tissue culture flasks. The
cells were maintained in Dulbecco's Modified Eagle Medium/Hams-F-12
medium combination (the solution is available as a 1:1 mixture).
The medium was supplemented with 20% heat-inactivated fetal calf
serum (FCS), glutamine, and antibiotics. The cells were seeded at a
concentration of 1.times.10.sup.6 cells per flask, and reached a
confluent state within one week. The cells were passaged once a
week, and, in addition, were fed once a week with DMEM/Hams-F-12
containing FCS, glutamine, and antibiotics as described. To passage
the cells, flasks were rinsed twice with 5 ml of PBS (without
Ca.sup.++ or Mg.sup.++) and trypsinized with 3 ml of 0.05% Trypsin
and 0.53 mM EDTA. The cells were pelleted, resuspended, and tested
for viability by trypan blue exclusion, seeded and fed with 25 ml
of the above mentioned DMEM/Hams-F-12 supplemented medium. A factor
VIII related antigen assay, Grulnick et al., 1977, Ann. Int. Med.
86:598-616, is used to positively identify endothelial cells, and
silver staining was used to identify tight junctional complexes,
specific to only small vessel endothelium.
[0094] Nylon filtration screen mesh (#3-210/36, Tetko, Inc., NY)
was prepared essentially as described above. The mesh was soaked in
an acetic acid solution (1 ml glacial acetic acid plus 99 ml
distilled H.sub.2O) for thirty minutes, was rinsed with copious
amounts of distilled water and then autoclaved. Meshes were coated
with 6 ml fetal bovine serum per 8.times.8 cm mesh and incubated
overnight. The meshes were then stacked, three high, and
3.times.10.sup.7 small vessel endothelial cells (cultured as
described) were seeded onto the stack, and incubated for three
hours at 37.degree. C. under 5% CO.sub.2 in a humidified
atmosphere. The inoculated meshes were fed with 10 ml of
DME/Hams-F-12 medium every 3-4 days until complete confluence was
reached (in approximately two weeks).
6.5 Establishment of the Three Dimensional Chondrocyte Stromal Cell
Culture
[0095] Cartilage was harvested from articular surfaces of human
joints. The cartilage pieces were digested with collagenase (0.2%
w/v) in complete medium (DMEM with 10% fetal bovine serum,
glutamine, non-essential amino acids, sodium pyruvate, 50 .mu.g/ml
ascorbate and 35 .mu.g/ml gentamicin) for 20 hours at 37.degree. C.
Liberated chondrocytes were spun, resuspended in complete medium,
counted and plated at 1.times.10.sup.6 cells per T-150 flask. Cells
were routinely passed at confluence (every 5-7 days).
[0096] Polyglycolic acid mesh (1 mm diameter.times.2 mm thick) was
sterilized by ethylene oxide or electron beam treatment and
presoaked overnight in complete medium. The mesh was seeded in 6
well plates with 3-4.times.10.sup.6 cells per mesh in a total
volume of 10 .mu.l and incubated for 3-4 hours at 37.degree. C. in
a tissue culture incubator. At this time, 1.5 ml of media was
added. The seeded mesh was incubated overnight. 5 ml of media was
added the next day. Media was changed three times per week until
confluence is reached.
7. Example Extracellular Matrix Composition
[0097] The extracellular matrix has been characterized by a number
of analytic methods to determine its content of matrix proteins,
each value is the average of at least two independent
determinations. The matrix contained type I and type III collagens,
fibronectin, tenascin, sulfated glycosaminoglycans, decorin and
various other secreted human extracellular matrix proteins.
Additionally, the secreted matrix proteins were found throughout
the three-dimensional support framework. The extracellular matrix
contained a total protein amount of 292 mg/cm.sup.2.+-.0.06;
fibronectin was present at 3.4 mg/cm.sup.2.+-.1.2; and tenascin at
1.7 mg/cm.sup.2.+-.0.6. Both fibronectin and tenascin showed the
expected molecular weight distributions on immunoblots.
7.1. Collagen Content of the Extracellar Matrix
[0098] Collagen content of the extracellular matrix was determined
using the Sirius Red assay. The binding of Sirius Red F3BA in
saturated picric acid solution has been used widely to estimate
fibrotic collagen deposition. Bedossa et al., 1989, Digestion
44(1):7-13; Finkelstein et al., 1990, Br. J. Ophthalmol.
74(5):280-282; James et al., 1990, Liver 10(1):1-5. The specificity
of Sirius Red binding to collagen is based largely on its use as a
histological stain. In rat liver with various degrees of
cholestatic fibrosis, collagen content measured by Sirius Red
binding shows strong correlation with hydroxyproline content. Walsh
et al., 1992, Analyt. Biochem. 203:187-190. In addition,
histological staining with Sirius Red is birefringent, indicating
directional binding related to the orientation of the collagen
strands. Sirius Red is known to bind to proteins other than the
classical collagens that contain collagen-like triple helices, such
as the complement component C1. Some minor binding to serum albumin
has also been found, although control experiments using bovine
serum albumin standard showed no interference with the assay. The
interference is estimated to represent less than 2% of the collagen
signal in the extracellular matrix and the use of Sirius Red assay
gives a reproducible method for measuring collagens. The
extracellular matrix contained a collagen content of 0.61
mg/cm.sup.2.+-.0.09. Further, collagens I and III showed the
expected molecular weight distributions on immunoblots
7.2. Collagen Fibers Visualized Via Electron Microscopy
[0099] Collagen derived from the dermal tissue grown in vitro and
collagen derived from a normal adult human dermal sample were
processed and visualized by transmission electron microscopy (TEM).
Briefly, the respective collagens were weighed and placed in a
sterile 50 ml centrifuge tube with 30 ml 0.05 M Tris buffer, pH
8.0. After mixing for two hours on a wrist shaker, the Tris buffer
was removed and the specimen placed in a homogenization cylinder
along with 30 ml fresh 0.05 M Tris buffer. The sample was
homogenized for 30 seconds in buffer alone and then for two 30
second bursts following the addition of a dispersing agent as
described in U.S. Pat. Nos. 4,969,912 and 5,332,802. The
temperature was maintained at 5-10.degree. C. during the mechanical
disruption process. The dispersing agent was added at a
concentration of 0.05% (wet weight of the collagen). The
homogenized preparation was centrifuged at 3500 rpm for 6 minutes
to separate the dispersed collagenous material from the yet
undispersed material. The undispersed residue was again treated
with dispersing agent at 0.05% (wet weight of the collagen) and
homogenized for two 30 second bursts. The dispersion was again
centrifuged to recover dispersed collagenous material which was
added to the first recovery.
[0100] The collagenous dispersion was filtered through a 100 micron
filter, centrifuged at 3500 rpm and the pellet was washed 3 times
with 0.004 M phosphate buffer, pH 7.4. The last centrifugation was
conducted at 10,000 rpm to pack the collagenous pellet. Samples
were then collected for TEM. As shown in FIGS. 2A-D, the collagen
fibers isolated from either the extracellular matrix prepared from
dermal tissue grown in vitro (FIGS. 2A-B) or from normal adult
human dermis (FIGS. 2C-D) appeared identical in that intact
collagen fibers with typical collagen banding and normal
periodicity in both preparations.
7.3. Glycosaminoglycans Present in the Extracellular Matrix
[0101] Glycosaminoglycans have been shown to play a variety of
structural and functional roles in the body and their presence in
the secreted extracellular matrix is important. Table II lists a
number of examples of glycosaminoglycans which have been determined
to be found in the extracellular matrix as well as their functional
importance in normal dermis.
2TABLE II NAME LOCATION GLYCAN FUNCTION MECHANISM Versican Matrix
12-15 Structural Binds hyaluronic Chondroitin sulfate acid and
collagen Decorin Matrix 1 Chondroitin/dermatan Binding TGF-.beta.
Inactivates sulfate and other growth factors growth factors; binds
to collagen Betaglycan Cell 1-4 TGF-.beta. Type III Adjunct
receptor membrane Chondroitin/heparan receptor for TGF.beta.
sulfate Syndecan Cell 1-3 Chondroitin Growth factor membrane
sulfate, 1-2 heparan binding sulfate
[0102] Further, the extracellular matrix was found to contain a
total of 2.8 mg/cm.sup.2.+-.0.1 sulfated glycosaminoglycans.
7.4. Growth Factors Present in the Extracellular Matrix
[0103] The cells producing and depositing the extracellular matrix
also expressed a number of different growth factors. Growth factors
are important in the extracellular matrix for two reasons. During
the growth of and deposition of the extracellular matrix, naturally
seeded growth factors help to control cell proliferation and
activity. Further, growth factors remain attached to the
extracellular matrix. A variety of growth factors have been
determined to be expressed during the deposition of the matrix.
[0104] The expression of growth factors has been examined by
polymerase chain reaction of reverse transcripts (RT-PCR) of total
RNA. Briefly, RNA was extracted from the growing cells by an SDS
precipitation and organic solvent partition procedure. The RNA was
transcribed using superscript reverse transcriptase and random
hexamer primers. The same batch of reverse transcript was used for
detection of all the growth factors. PCR was performed under
standard conditions, using 4 .mu.l reverse transcript,
corresponding to 200 ng RNA in a total volume of 20 .mu.l.
[0105] Based on this assay, acidic and basic FGF, TGF-.alpha. and
TGF-.beta., and KGF mRNA transcripts were present as were several
others as shown in Table III, including PDGF, amphiregulin, HBEGF,
IGF, SPARC and VEGF. Of these, PDGF and TGF-.beta.3 are thought to
be involved in regulation of cell proliferation and matrix
deposition in culture, while TGF-.beta.1, HBEGF, KGF, SPARC, VEGF
and decorin are deposited in the matrix. Amphiregulin, IGF-1, IGF-2
and IL-1 were not expressed at the sensitivity used in these
experiments.
3TABLE III Messenger RNA Full Name Function Expression PDGF-A Chain
Platelet- Mitogen for ++ derived growth fibroblasts, factor, A
granulation chain tissue, chemotactic PDGF-B Chain Platelet-
Mitogen for 0 - (+)* derived growth fibroblasts, factor, B
granulation chain tissue, chemotactic IGF-1 Insulin-like Mitogen
for 0 growth factor-1 fibroblasts IGF-2 Insulin-like Mitogen for
(+) growth factor-2 fibroblasts TGF-.alpha. Transforming Mitogen
for + growth factor-.alpha. fibroblasts, keratinocytes Amphiregulin
Amphiregulin Mitogen for 0 fibroblasts, keratinocytes KGF
Keratinocyte Mitogen for ++ growth factor keratinocytes HBEGF
Heparin- Mitogen for +++ binding fibroblasts, epidermal
keratinocytes growth factor- like growth factor TGF-.beta.1
Transforming Stimulates + growth factor- matrix .beta.1 deposition
TGF-.beta.3 Transforming Stimulates ++ - ++ growth factor- matrix
.beta.3 deposition VEGF Vascular Angiogenic ++ endothelial factor
growth factor SPARC Secreted Complex anti- ++++ protein acidic
angiogenic, and rich in angiogenic cysteine ICAM-1 Intercellular
Lymphocyte + adhesion adhesion, molecule-1 mobility VCAM Vascular
Lymphocyte +++ cellular adhesion, adhesion mobility molecule GAPDh
Glyceraldehyde Glycolytic +++ 3-phosphate housekeeping
dehydrogenase gene .beta.2- .beta.2- Antigen +++ microglobulin
microglobulin presentation *A small amount of PDGF B chain is seen
in some preparations.
[0106] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed since
these embodiments are intended as illustrations of several aspects
of the invention. Any equivalent embodiments are intended to be
within the scope of this invention. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims.
[0107] A number of references are cited herein, the entire
disclosures of which are incorporated herein, in their entirety, by
reference.
* * * * *