U.S. patent application number 15/351295 was filed with the patent office on 2017-11-09 for systems and methods for producing gastrointestinal tissues at an anstomosis or other physiological location.
The applicant listed for this patent is Biostage, Inc.. Invention is credited to Saverio La Francesca, David C. Rice, Sherif Soliman, Ara A. Vaporciyan.
Application Number | 20170319325 15/351295 |
Document ID | / |
Family ID | 58695564 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170319325 |
Kind Code |
A1 |
La Francesca; Saverio ; et
al. |
November 9, 2017 |
Systems and Methods for Producing Gastrointestinal Tissues at an
Anstomosis or Other Physiological Location
Abstract
Aspects of the disclosure relate methods and synthetic scaffolds
for regenerating gastrointestinal tissue (e.g., esophageal
tissue).
Inventors: |
La Francesca; Saverio;
(Houston, TX) ; Soliman; Sherif; (Holliston,
MA) ; Rice; David C.; (Houston, TX) ;
Vaporciyan; Ara A.; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biostage, Inc. |
Holliston |
MA |
US |
|
|
Family ID: |
58695564 |
Appl. No.: |
15/351295 |
Filed: |
November 14, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62276715 |
Jan 8, 2016 |
|
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62254700 |
Nov 12, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61F 2210/0076 20130101;
C12M 25/14 20130101; A61L 27/18 20130101; A61F 2230/0069 20130101;
A61L 27/18 20130101; C12M 21/08 20130101; A61F 2/82 20130101; A61L
27/58 20130101; A61L 27/3834 20130101; A61F 2002/045 20130101; C08L
67/02 20130101; A61F 2/0077 20130101; A61F 2/90 20130101; A61L
27/04 20130101; C08L 75/04 20130101; A61F 2/07 20130101; A61F
2002/043 20130101; A61F 2002/046 20130101; A61F 2002/0086 20130101;
A61F 2002/044 20130101; A61F 2210/0004 20130101; A61F 2/04
20130101; A61L 2430/22 20130101; A61L 27/56 20130101; A61L 27/18
20130101; A61F 2250/0067 20130101 |
International
Class: |
A61F 2/04 20130101
A61F002/04; A61L 27/56 20060101 A61L027/56; A61F 2/82 20130101
A61F002/82; A61L 27/18 20060101 A61L027/18; A61L 27/58 20060101
A61L027/58; A61L 27/38 20060101 A61L027/38 |
Claims
1. A method, comprising the steps of: resecting a portion of a
tubular organ in a subject, the tubular organ being proximate to
the stomach region of the subject, the resection step producing a
resected organ portion located primarily in the esophageal region,
the resected organ portion remaining in the subject; implanting a
synthetic scaffold at the site of resection, the synthetic scaffold
including a body member, the body member having a tubular region
having a first end and a second end opposed to the first end and an
outwardly flared region contiguously connected to the tubular
region of the body member at a location proximate to one of the
first end or second end, an outer polymeric surface and a
cellularized sheath layer overlying at least a portion of the outer
polymeric surface, wherein the flared region is connected to the
stomach and the tubular region is connected to the resected organ
portion; maintaining the synthetic scaffold at the resection site
for a period of time sufficient to achieve guided tissue growth
along the synthetic scaffold, the guided tissue growth derived from
and in contact with the tissue present in the resected organ
portion remaining in the subject; and after achieving guided tissue
growth, removing the synthetic scaffold from the implantation site,
the removing step occurring in a manner such that the guided tissue
growth remains in the contact with the resected portion of the
tubular organ remaining in the subject.
2. The method of claim 1 further comprising: imparting cellular
material onto the polymeric surface of the synthetic scaffold; and
allowing the cellular material to grow to form the cellular sheath
layer, the imparting and allowing steps occurring prior to the
resecting step.
3. The method of claim 2 wherein the synthetic scaffold further
comprises a tubular member that is coaxially disposed with the
tubular region, the tubular member extending through an interior
region defined in the flared region of the synthetic scaffold and
the tubular member projects into a central region defined in the
stomach.
4. The method of claim 3 wherein the tubular region is a tubular
member and the outer surface includes spun polymeric fibers.
5. The method of claim 3 wherein the cellularized sheath layer
spans at least a portion outwardly positioned electrospun
fibers.
6. The method of claim 1 wherein the cellularized sheath layer is
composed of cellular material, the cellular material including at
least one of mesenchymal cells, stem cells, pluripotent cells.
7. The method of claim 1 wherein the removal step is achieved
intrascopically.
8. A method comprising: resecting a portion of a tubular organ
proximate to the stomach in a subject, the resection step producing
a resected organ portion, the resected organ portion remaining in
the subject and having at least one resection edge, wherein the
resected tubular organ includes a tubular organ resection edge and
the stomach includes a resected edge, implanting a synthetic
scaffold at the site of resection, the synthetic scaffold having a
body member, the body member having a tubular region having a first
end and a second end opposed to the first end and an outwardly
flared region contiguously connected to the tubular region of the
body member at a location proximate to one of the first end or
second end, an outer polymeric surface positioned between the first
end and the second end and a cellularized sheath layer overlying at
least a portion of the outer polymeric surface, wherein at least a
portion to the celluralized sheath layer is proximate to at least
one resected, maintaining contact between the synthetic scaffold
and the at least one resected edge for an intervals sufficient to
achieve guided tissue growth along the synthetic scaffold, wherein
at least a portion of the synthetic scaffold is absorbed at the
site of resection within a period of time sufficient to achieve
guided tissue growth along the synthetic scaffold.
9. The method of claim 8 further comprising: imparting cellular
material onto the polymeric surface of the synthetic scaffold; and
allowing the cellular material to grow into the cellular layer, the
imparting and allowing steps occurring prior to the resecting
step.
10. The method of claim 9 wherein the synthetic scaffold comprises
a tubular member that is coaxially disposed with the tubular region
and wherein the outer surface includes electrospun polymeric fibers
and wherein the cellularized sheath layer spans at least a portion
outwardly positioned electrospun fibers.
11. The method of claim 10 wherein the cellular material includes
one of mesenchymal cells, stem cells, pluripotent cells, the
cellular material derived from the subject.
12. The method of claim 9, wherein the tubular organ is an
esophagus and the organ proximate to the esophagus is the stomach
gastrointestinal organ.
13. The method of claim 9, wherein the subject is a mammal.
14. The method of claim 13, wherein the mammal is a human.
15. The method of claim 9 wherein the synthetic scaffold is
completely absorbed.
16. The method of claim 10, further comprising monitoring tissue
regeneration endoscopically.
17. A synthetic scaffold comprising: a body section, the body
section having a first end and a second end opposed to the first
end, the body section further having a least one portion configured
as a tubular member and at least one portion configured as a flared
member, the flared member contiguously connected to the tubular
member and located proximate to either the first end or the second
end of the body section, the body section comprising an outwardly
oriented surface, the outwardly oriented surface having at least
one region composed of spun polymeric fibers, the spun polymeric
fibers having an average fiber diameter between 15 nm and 10
microns, at least a portion of the spun polymeric fibers
interlinked to form pores having an average diameter less than 50
microns.
18. The synthetic scaffold of claim 17 wherein the synthetic
scaffold further comprises a tubular member region the tubular
member region being coaxially positioned relative to the tubular
member and extends with an interior region defined by the flared
member.
19. The synthetic scaffold of claim 19 wherein the spun polymeric
fibers are electropsun, are interconnected and form an outer layer
of the body section and the body section further comprises at least
one inner layer, the inner layer composed of at least one of a
polymeric mesh, a polymeric braided support material, a solid
polymeric member, an electrospun layer, the outer layer in
overlying contact with the inner layer.
20. The synthetic scaffold of claim 19 wherein the electrospun
material has an average fiber diameter of 3 to 10 micrometers and
is composed of at least one of one of the following polymeric
materials: polyvinylidene fluoride, syndiotactic polystyrene,
copolymer of vinylidene fluoride and hexafluoropropylene, polyvinyl
alcohol, polyvinyl acetate, poly(acrylonitrile), copolymers of
polyacrylonitrile and acylic acid, copolymers of polyacrylonitrile
and methacrylates, polystyrene, poly(vinyl chloride), coploymeris
of poly(vinyl chloride), poly(methyl methacrylate), copolymers of
poly(methyl methacrylate), polyethylene terephthalate,
polyurethane.
21. The synthetic scaffold of claim 20 wherein at least one layer
is a polymeric material containing polyethylene terepthalate,
polyurethane, blends of polyethylene terepthlatae and
polyurethane.
22. The synthetic scaffold of claim 20 polymeric braided support
material is composed of at least one of polyethylene terepthalate,
polyurethane, nitinol and mixtures thereof.
23. The synthetic scaffold of claim 20 further comprising at least
one sheath layer, the sheath layer composed of cellular material,
the cellular material composed of mesenchymal cells and stem cells
present in a defined layer the defined layer being between 1 and
100 celled thick.
24. The synthetic scaffold of claim 23 wherein the sheath layer of
cellular material overlays the electrospun fibers present on the
outer surface such that the cellular material is contained on the
outer surface and spans pores defined therein.
25. The synthetic scaffold of claim 19 further comprising at least
one hole, indent, protrusion, or a combination thereof defined
proximate to at least one of the first or second ends that is
adapted to assist in at least one of the following: retrieval of
the scaffold from a subject after tissue regeneration has occurred
around the scaffold at the site of implantation in the subject or
implanting the synthetic scaffold in a location in the body of a
subject.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims priority to and the benefit of U.S.
Provisional Application Patent Ser. No. 62/254,700 filed Nov. 12,
2015, and U.S. Provisional Application Ser. No. 62/276,715 filed
Jan. 8, 2016, the entire disclosure of both is hereby incorporated
by reference.
TECHNICAL FIELD
[0002] This disclosure relates to engineered tissues that are
useful for replacement or repair of damage tissues.
BACKGROUND
[0003] Engineered biological tissues that are useful for
replacement or repair of damaged tissues are often produced by
seeding cells on synthetic scaffolds and exposing the cells to
conditions that permit them to synthesize and secrete extracellular
matrix components on the scaffold. Different techniques have been
used for producing synthetic scaffolds, including nanofiber
assembly, casting, printing, physical spraying (e.g., using pumps
and syringes), electrospinning, electrospraying and other
techniques for depositing one or more natural or synthetic polymers
or fibers to form a scaffold having a suitable shape and size for
transplanting into a subject (e.g., a human subject, for example,
in need of an organ or region of engineered tissue).
[0004] It is estimated that over 500.000 individuals worldwide are
diagnosed with esophageal malignancy each year. Congenital
malformations of the esophagus, such as esophageal atresia, have an
average prevalence of 2.44 per 10,000 births. Chronic esophageal
stricture after esophageal injury is also common. While there have
been advances in minimization of the extent of esophageal resection
for early stage malignant disease, such as endoscopic mucosal
resection, the mainstay of treatment for many esophageal disorders
is surgical esophagectomy. Traditionally, autologous conduits such
as stomach, small bowel, or colon are harvested and rerouted into
the chest to restore gastrointestinal continuity. Many children
with esophageal atresia or patients affected by either trauma or
caustic injury to the esophagus ultimately undergo similar
reconstruction. These treatment modalities are associated with high
morbidity and mortality.
[0005] Autologous conduits are traditionally used because of the
complex structure of the esophagus. Comprised of stratified
squamous epithelium, submucosa and outer circular and longitudinal
muscle layers, these multiple layers of the esophagus provide a
barrier to contain oral intake and contamination from escape
outside of the gastrointestinal tract. Furthermore, the combined
layers provide a physiological mechanism for propulsion, and
management of stresses during passage of the bolus either during
swallowing or emesis.
[0006] It would be desirable to provide structure as well as a
method of making a structure that can support tissue
regeneration.
SUMMARY
[0007] Disclosed herein are implementations that pertain to
synthetic scaffolds and related systems that enable production of
gastrointestinal tissues (e.g., tissues of the esophagus, stomach,
intestine, colon, or other hollow gastrointestinal tissue). In some
embodiments, scaffolds provide guides for gastrointestinal (e.g.,
esophageal) tissue growth and regeneration in a subject. In some
embodiments, the regenerated gastrointestinal tissue comprises
muscle tissue, nervous system tissue, or muscle tissue and nervous
system tissue. In some embodiments, gastrointestinal (e.g.,
esophageal) tissue is regenerated surrounding a scaffold. In some
embodiments, the scaffold is not incorporated into the final
regenerated tissue (e.g., the new esophageal tissue does not
incorporate the scaffold into the regenerated esophageal walls).
Accordingly, aspects of the disclosure relate to guided tissue
regeneration where a scaffold provides support and/or signals that
promote host tissue regeneration without the scaffold needing to be
incorporated into the regenerated tissue (e.g., without the
scaffold providing structural or functional support in the final
regenerated tissue).
[0008] In some embodiments, a gastrointestinal (e.g., esophageal)
scaffold includes biodegradable and/or bioresorbable material that
is resorbed after gastrointestinal (e.g., esophageal) tissue
regeneration is initiated (e.g., after functional esophageal tissue
is regenerated).
[0009] In some embodiments, a gastrointestinal (e.g., esophageal)
scaffold includes one or more structures that can be used to assist
in removing the scaffold after gastrointestinal (e.g., esophageal)
tissue regeneration is initiated (e.g., after functional esophageal
tissue is regenerated).
[0010] In some embodiments, the scaffold comprises an end having an
enlarged inner diameter relative to the inner diameter of the
opposite end of the scaffold.
[0011] In some embodiments, the scaffold comprises an anti-reflux
system. In some embodiments, the anti-reflux system comprises a
valve. In some embodiments, the anti-reflux system comprises a
hollow structure (e.g., a tubular structure) extending beyond the
edge of an end having an enlarged inner diameter of the scaffold.
In some embodiments, the hollow structure (e.g., a tubular
structure) of the reflux system is configured to extend within the
stomach.
[0012] In some embodiments, a scaffold is cellularized with one or
more cell types prior to implantation. In some embodiments, the
cells are autologous cells. In some embodiments, the cells are
progenitor or stems cells. In some embodiments, the cells are
obtained from bone marrow, adipogenic tissue, esophageal tissue, or
other suitable tissue. In some embodiments, the cells can be
obtained from various allogenic sources, including but not limited
to sources such as amniotic fluid, cord bold and the like. In some
embodiments, the cells are mesenchymal stem cells (MSCs)
[0013] In some embodiments, a scaffold is implanted at a site that
provides a sufficient stem cell niche (e.g., an esophageal or other
gastrointestinal site that provides a stem cell niche) for
regenerating tissue in the subject. In some embodiments, without
wishing to be bound by theory, the scaffold and/or cells that are
provided on the scaffold help promote growth and/or regeneration of
gastrointestinal tissue from host stem cells present at the site of
scaffold implantation.
[0014] In some aspects, the disclosure relates to the discovery
that growth of esophageal tissues can be promoted or enhanced by
the presence of synthetic scaffolds that are engineered to replace
or repair natural structural patterns and/or functional properties
of diseased or injured tissues or organs, without the scaffolds
becoming fully integrated into the final regenerated tissue. Thus,
in some aspects, the disclosure provides a method for promoting or
enhancing growth of gastrointestinal (e.g., esophageal) tissue, the
method comprising: delivering to a gastrointestinal (e.g.,
esophageal) region of a subject a synthetic scaffold, wherein
delivery of the synthetic scaffold results in growth of new
gastrointestinal (e.g., esophageal) tissue in that region of the
subject. In some embodiments, the diseased or injured
gastrointestinal region is removed (e.g., surgically) prior to
implanting the scaffold. In some embodiments, the scaffold is an
approximately tubular structure that is implanted (e.g., sutured to
the ends of the remaining gastrointestinal tissue after removal of
the diseased or damaged tissue). In some embodiments, the implanted
scaffold is shorter than the tissue that was removed (e.g., 5-50%
shorter). In some embodiments, the remaining gastrointestinal
tissue near the site of the implant is stretched when the tissue is
attached (e.g., sutured) to the both ends of the scaffold. In some
embodiments, new gastrointestinal (e.g., esophageal) tissue is
regenerated over the implanted scaffold without being fully
integrated with the scaffold. In some embodiments, the walls of the
regenerated tissue do not incorporate the walls of the scaffold
even though the scaffold can be retained within the lumen of the
regenerated tissue. In some embodiments, the scaffold can be
removed from the lumen formed by the regenerated tissue at a
suitable point in the tissue regeneration process.
[0015] In some embodiments, the growth of new gastrointestinal
(e.g., esophageal) tissue results in the formation of functional
tissue (e.g., a functional esophagus) that does not require the
continued presence of the scaffold for function.
[0016] In some embodiments, the synthetic scaffold is resorbable or
dissolvable under physiological conditions. In some embodiments,
the synthetic scaffold is removed from the gastrointestinal (e.g.,
esophageal) region of the subject after the formation of a
functional esophagus.
[0017] In some embodiments, methods and compositions described
herein also can be used for tracheal and/or bronchial tissue
regeneration.
[0018] These and other aspects are described in more detail
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The disclosure is best understood from the following
detailed description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity.
[0020] FIG. 1A is a perspective view of an embodiment of a
synthetic scaffold as disclosed herein with a portion being
rendered in partial cross-section;
[0021] FIG. 1B is a photomicrograph of a surface of a tubing
surface of an embodiment of the synthetic scaffold as disclosed
herein;
[0022] FIG. 1C side perspective view of a second embodiment of a
synthetic scaffold as disclosed herein;
[0023] FIG. 2 is a non-limiting depiction of the biological layers
of an esophagus; and
[0024] FIG. 3 is a side-by-side illustration that illustrates a
non-limiting example of regenerated esophageal tissue in comparison
with corresponding native tissue;
[0025] FIG. 4 A is a SEM photomicrograph of an outer surface region
of an embodiment of the synthetic scaffold as disclosed herein
showing cellular growth after seven days of bioreaction taken at
5000.times.;
[0026] FIG. 4B is a photomicrograph of an outer surface region of
an embodiment of the synthetic scaffold as disclosed herein showing
cellular growth after seven days of bioreaction;
[0027] FIG. 5 is a process diagram of an embodiment an embodiment
of the regeneration method as disclosed herein;
[0028] FIG. 6 is an overall study flow for an embodiment of the
process as disclosed herein including generation of a cellularized
scaffold and subsequent implantation
[0029] FIG. 7A are SEMs of samples of an electrospun scaffold
according to an embodiment as disclosed herein taken at
1000.times., 2000.times. and 5000.times. respectively;
[0030] FIG. 7B is a graphic depiction of representative uniaxial
mechanical testing loading of pre-implantation and
post-implantation electrospun scaffolds according to an embodiment
as disclosed herein;
[0031] FIG. 7C is a Table directed to the uniaxial mechanical
properties of pre- and post-implantations scaffolds prepared
according to an embodiment as disclosed herein;
[0032] FIG. 8 is a diagrammatic representation of flow cytometry of
MSCs isolated and propagated from adipose tissue for up to 5
passages;
[0033] FIG. 9 is an overview of implantation surgery according to
an embodiment as disclosed herein;
[0034] FIG. 10 is a representation of a timeline according to an
embodiment of the process as disclosed herein;
[0035] FIG. 11A is a photograph of regenerated esophageal tissue
that is located on the interior of the esophagus of a first test
subject at an esophageal resection site of the first test subject
taken after removal of an embodiment of the scaffold device as
disclosed herein at 3 to 4 weeks post-surgery;
[0036] FIG. 11B is a photograph of regenerated tissue that is
located on the interior of the esophagus at the esophageal
resection site of FIG. 11A at an intermediate interval after
removal of the scaffold device showing tissue growth;
[0037] FIG. 11C is a photograph of regenerated tissue that is
located on the interior of the esophagus at the esophageal
resection site of FIG. 11A at an interval subsequent to the
intermediate interval of FIG. 11B showing tissue growth;
[0038] FIG. 12A is a photograph of regenerated esophageal tissue
that is located on the interior of the esophagus at an esophageal
resection site of a second test subject taken after removal of an
embodiment of the scaffold device as disclosed herein at 3 to 4
weeks post-surgery;
[0039] FIG. 12B is a photograph of regenerated tissue that is
located on the interior of the esophagus at the esophageal
resection site of FIG. 12A at an intermediate interval after
removal of the scaffold device showing tissue growth;
[0040] FIG. 12C is a photograph of regenerated tissue that is
located on the interior of the esophagus at the esophageal
resection site of FIG. 12A at an intermediate interval after
removal of the scaffold device showing tissue growth subsequent to
the tissue growth depicted in FIG. 12B;
[0041] FIG. 12D is a photograph of regenerated tissue that is
located on the interior of the esophagus at the esophageal
resection site of FIG. 12A at an intermediate interval after
removal of the scaffold device showing tissue growth subsequent to
the tissue growth depicted in FIG. 12C;
[0042] FIG. 12E is a photograph of regenerated tissue that is
located on the interior of the esophagus at the esophageal
resection site of FIG. 12A at an intermediate interval after
removal of the scaffold device showing tissue growth subsequent to
the tissue growth depicted in FIG. 12D;
[0043] FIG. 13A is a photograph of tissues from a representative
test animal esophagus at 2.5 month post implantation including the
surgical site and adjacent distal and proximal tissues excised for
histological analysis;
[0044] FIG. 13B is a photograph of a magnified cross-sectional
sample of mucosa tissue taken from proximal section 1 of FIG.
13A;
[0045] FIG. 13C is a photograph of a magnified cross-sectional
sample of mucosa tissue taken from proximal section 2 of FIG.
13A;
[0046] FIG. 13D is a photograph of a magnified cross-sectional
sample of submucosa tissue taken from proximal section 1 of FIG.
13A;
[0047] FIG. 13E is a photograph of a magnified cross-sectional
sample of submucosa tissue taken from proximal section 2 of FIG.
13A;
[0048] FIG. 13F is a photograph of a magnified cross-sectional
sample of mucosa tissue taken from distal section 3 of FIG.
13A;
[0049] FIG. 13I is a photograph of a magnified cross-sectional
sample of mucosa tissue taken from distal section 4 of FIG.
13A;
[0050] FIG. 13J a photograph of a magnified cross-sectional sample
of mucosa tissue taken from distal section 4 of FIG. 13A;
[0051] FIG. 13K a photograph of a magnified cross-sectional sample
of submucosa tissue taken from distal section 4 of FIG. 13A;
[0052] FIG. 14A is a photograph of tissues of pig esophagus for
histological analysis at 2.5 months post implantation with an
embodiment of the scaffold as disclosed herein;
[0053] FIG. 14B is photograph of a magnified cross-sectional sample
taken a section B of FIG. 14A illustrating the presence of mucosal
tissue;
[0054] FIG. 14C is photograph of a magnified cross-sectional sample
taken a section C of FIG. 14A illustrating the presence of mucosal
tissue;
[0055] FIG. 14D is photograph of a magnified cross-sectional sample
taken a section D of FIG. 14A illustrating the presence of mucosal
and submucosa tissue and muscular layers;
[0056] FIG. 14E is photograph of a magnified cross-sectional sample
taken a section E of FIG. 14A illustrating the presence of mucosal
and submucosa tissue and muscular layers;
[0057] FIG. 14F is a photograph of a cross-sectional sample of
esophageal tissue of FIG. 14A used for Ki67 immunoreactivity
analysis;
[0058] FIG. 14G is a photograph of a cross-sectional sample of
esophageal tissue of FIG. 14A used for CD31 immunoreactivity
analysis;
[0059] FIG. 14H is a photograph of a cross-sectional sample of
esophageal tissue of FIG. 14A used for CD3.epsilon.
immunoreactivity analysis;
[0060] FIG. 14I is a photograph of a cross-sectional sample of
esophageal tissue of FIG. 14A used for .alpha.SMA immunoreactivity
analysis;
[0061] FIG. 14J is a photograph of a cross-sectional sample of
esophageal tissue of FIG. 14A used for Transgelin/SMA22.alpha.
immunoreactivity analysis;
[0062] FIG. 14K is a photograph a cross-sectional sample of
esophageal tissue of FIG. 14A used for striated myosin analysis
[0063] FIG. 15A is a perspective view of a first alternate
embodiment of a synthetic tubular scaffold as disclosed herein
including an enlarged end and anti-reflux system;
[0064] FIG. 15A'' is a perspective view of the synthetic tubular
scaffold of FIG. 15A;
[0065] FIG. 15B is a perspective view of a second alternate
embodiment of a synthetic tubular scaffold as disclosed herein
including an enlarged end and anti-reflux system;
[0066] FIG. 15B' is a perspective view of the synthetic tubular
scaffold of FIG. 15B;
[0067] FIG. 16A is a front perspective view of the synthetic
tubular scaffold of FIG. 14 A in position and implanted at a
gastroesopageal junction;
[0068] FIG. 16B is a side perspective view of FIG. 16A;
[0069] FIG. 16C is a rear perspective view of FIG. 16A;
[0070] FIG. 17 A is a bottom perspective of the implanted synthetic
tubular scaffold of FIG. 16A;
[0071] FIG. 17B is a bottom perspective of the implanted synthetic
tubular scaffold of FIG. 16A;
[0072] FIG. 17C is a bottom perspective of the implanted synthetic
tubular scaffold of FIG. 16A; and
[0073] FIG. 17D is a bottom view of FIG. 16A.
DETAILED DESCRIPTION
[0074] Aspects of the disclosure relate in part to the remarkable
discovery that inserting a synthetic scaffold into the esophageal
region of a subject can promote or enhance the regeneration of new
esophageal tissue (e.g., a complete and functional esophagus) in
the subject without fully incorporating the scaffold into the
regenerated tissue. Thus, in some embodiments, the disclosure
provides a method for promoting or enhancing growth of
gastrointestinal (e.g., esophageal) tissue, the method comprising:
delivering to the gastrointestinal (e.g., esophageal) region of a
subject a synthetic scaffold, wherein delivery of the synthetic
scaffold results in growth of new gastrointestinal (e.g.,
esophageal) tissue in that region of the subject.
[0075] Tissue that is regenerated using methods described herein
can be any gastrointestinal tissue, such as tissues of the
esophagus, stomach, intestine, colon, rectum, or other hollow
gastrointestinal tissue. In some aspects, the disclosure is based,
in part, on the surprising discovery that methods described herein
result in the regeneration of gastrointestinal tissue comprising
muscle tissue, nervous system tissue, or muscle tissue and nervous
system tissue.
[0076] In some embodiments, the synthetic scaffold is resorbable or
dissolvable under physiological conditions (e.g., within a time
period corresponding approximately to the time required for tissue
regeneration). In some embodiments, at least a portion of the
synthetic scaffold is resorbable or dissolvable under suitable
physiological conditions.
[0077] In some embodiments, the synthetic scaffold is removed from
the subject after the formation of a regenerated functional tissue
(e.g., esophagus or portion thereof).
[0078] In some embodiments, a scaffold is designed to be readily
retrievable by having a) one or more reversible attachments that
can be easier to remove than a suture, for example to help
disconnect the scaffold from the surrounding tissue (e.g.,
esophagus) after tissue regeneration, and/or b) one or more
features that can be used to help retrieve the scaffold, for
example after it has been disconnected from the surrounding tissue
(e.g., adjacent esophageal tissue).
[0079] Non-limiting examples of reversible attachments include
mechanical mechanisms (for example hooks and loops, connectors such
as stents, or other mechanical attachments that can be
disconnected) and/or chemical mechanisms (for example biodegradable
or absorbable attachments and/or attachments that can be
selectively removed by chemical or enzymatic means). In some
embodiments, absorbable staples can be used. In some embodiments,
absorbable staples comprise a co-polymer of
polylactide-polyglycolide for example, or any other absorbable
blend of material.
[0080] In some embodiments, surgical implantation and/or retrieval
of a scaffold can be performed with thoracoscopic assistance.
[0081] Non-limiting examples of structural features that can assist
in the retrieval or removal of a scaffold (e.g., after it is
disconnected from the surrounding gastrointestinal tissue) include
holes, indents, protrusions, or other structural features, or any
combination thereof these structural features are located only on
the outer surface of the scaffold. One or more of these structural
features can be used to help grip or hold a tool (e.g. a grasper)
that is being used to retrieve the scaffold. In some embodiments,
one or more of these structural features can be located at only one
end of the scaffold (e.g., the end that is proximal to the mouth of
the subject). In some embodiments, one or more of these structural
features can be located at both ends, or throughout the length of
the scaffold. In some embodiments, one or more of these structural
features are located only on the outer surface of the scaffold. In
some embodiments, one or more of these structural features are
located only on the inner surface of the scaffold. In some
embodiments, one or more of these structural features are located
on both the outer and inner surfaces of the scaffold. In some
embodiments, a scaffold is reinforced (e.g., is thicker and/or
includes stronger material) at or around the location of one or
more structural features that are used to retrieve the
scaffold.
[0082] In some embodiments, a disconnected scaffold can be removed
endoscopically via the lumen of the airway leading to the
esophagus. In some embodiments, a disconnected scaffold can be
removed surgically
[0083] In some embodiments, the subject has diseased (e.g.,
cancerous) or injured gastrointestinal tissue that needs to be
replaced. In some embodiments, the subject is a human (e.g., a
human patient).
[0084] In some embodiments, the disclosure provides engineered
scaffolds that can be used to replace or repair an esophagus or a
portion thereof. In some embodiments, esophageal scaffolds
described herein may be used for promoting tissue regeneration
(e.g., a regenerated esophagus or portion thereof) to replace a
tissue in a subject (e.g., a human). For example, subjects (e.g., a
human) having certain cancers (e.g., esophageal cancer) may benefit
from replacement of a tissue or organ affected by the cancer.
Without wising to be bound by any particular theory, synthetic
scaffolds described herein promote the growth of new tissue (e.g.,
esophageal tissue) in a subject and therefore provide a therapeutic
benefit to the subject.
[0085] In some embodiments, the growth of new esophageal tissue
results in the formation of a functional esophagus in the subject.
In some embodiments, the new esophageal tissue does not incorporate
the scaffold into the regenerated esophageal walls. In some
embodiments, the scaffold is designed and manufactured to be
absorbable and/or readily retrievable after the esophageal tissue
has regenerated. In some embodiments, the scaffold is designed to
be at least partially absorbable.
[0086] In some embodiments, the growth of new gastrointestinal
tissue at other locations in the gastrointestinal tract results in
formation of functional gastrointestinal tissue specific to the
location in question. In some embodiments, the new gastrointestinal
tissue does not incorporate the scaffold into the regenerated
gastrointestinal walls. In some embodiments, the scaffold is
designed and manufactured to be absorbable and/or readily
retrievable after the gastrointestinal tissue has regenerated. In
some embodiments, the scaffold is designed to be at least partially
absorbable.
[0087] In some embodiments, a synthetic scaffold has a size and
shape that approximates the size and shape of a diseased or injured
gastrointestinal (e.g., esophageal) region that is being
replaced.
[0088] In some embodiments, a scaffold will have at least two
layers. The scaffold can have an approximately tubular structure in
certain embodiments. FIG. 1A illustrates a non-limiting embodiment
of a scaffold 10 having an approximately tubular body 12 having an
interiorly oriented surface 14 and an exteriorly oriented surface
16. In some embodiments, a lateral cross-section of the scaffold 10
is approximately circular. In some embodiments, a lateral
cross-section is approximately "D" shaped. However, scaffolds 10
having other cross-sectional shapes can be used. Scaffold 10 can
have any suitable length and diameter depending on the size of the
corresponding tissue being regenerated. In some embodiments, a
scaffold 10 can be from around 1-10 cms in length (for example 3-6
cms, e.g., about 4 cms) in certain embodiments, or 10-20 cms long
in other embodiments. However, it is contemplated that shorter or
longer scaffolds 10 can be used depending on the application, needs
of the patient and/or locations in the gastrointestinal tract
requiring treatment. In some embodiments, a scaffold 10 can have an
inner diameter of 0.5 to 5 cms. However, scaffolds with smaller or
larger inner diameters can be used depending on the application,
needs of the patient and/or locations in the gastrointestinal tract
requiring treatment.
[0089] In some embodiments, the inner diameter of each end (e.g.,
the end proximal to the mouth of the subject and the end distal to
the mouth of the subject) of an approximately tubular scaffold is
not equal. For example, in some embodiments, the inner diameter of
an approximately tubular scaffold increases along the length of the
scaffold (e.g., having one end with an enlarged diameter, also
referred to as an "enlarged end"), resulting in a "bell shape" or a
"trumpet shape" scaffold. However, it should be appreciated that
the end of an approximately tubular scaffold having an enlarged
diameter can be any shape, including but not limited to
cylindrical, elliptical, pyramidal, cuboid, bell-shaped, and
trumpet-shaped. FIG. 4 provides a non-limiting example of an
approximately tubular scaffold increases along the length of the
scaffold. Either end of a scaffold (e.g. proximal or distal in
relation to the mouth of a subject), may have an enlarged
diameter.
[0090] In some embodiments, the inner diameter of the enlarged end
of a tubular scaffold is between about 0.1 cm and 2 cm larger than
the non-enlarged end of the scaffold. In some embodiments, the
inner diameter of the enlarged end of a tubular scaffold is about
0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9
cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm,
1.8 cm, 1.9 cm, or 2.0 cm larger than the non-enlarged end of the
scaffold. In some embodiments, the inner diameter of the enlarged
end of a tubular scaffold ranges from about 2 cm to about 5 cm. In
some embodiments, the inner diameter of a scaffold is about 2 cm,
about 2.5 cm, about 3.0 cm, about 3.5 cm, about 4.0 cm, about 4.5
cm, or about 5 cm. An enlarged end can be fabricated at the same
time as the synthetic scaffold (e.g., electrospun as part of the
synthetic scaffold) or fabricated separately and integrated into a
pre-made synthetic scaffold.
[0091] In some embodiments, the end of a scaffold having an
enlarged diameter is configured to facilitate attachment of
regenerated tissue (e.g., regenerated esophageal tissue) to a
target tissue (e.g., stomach tissue) by anastomosis.
[0092] It has been found, quite unexpectedly, that embodiments of
the synthetic scaffold 200 as depicted in FIGS. 15 A and 15 B can
be advantageously employed to provide a scaffold that supports
regenerative growth of tubular organ tissue proximate
gastrointestinal organs such as the stomach and the like.
[0093] In some embodiments, the total length of scaffold 10, 200
can be shorter than the length of a gastrointestinal (e.g.,
esophageal) region being replaced. In some embodiments, the
scaffold 10 has a length that is 50-95% (for example, about 50-60%,
60-70%, 70-80%, 80-90%, about 80%, about 85%, about 90%, or about
95%) of the length of the tissue being replaced. Without being
bound to any theory, it is believed that certain regions of the
associated gastrointestinal region can respond positively to
traction force exerted on the associated organ tissue resulting the
generation of certain bio-organically mediated signals that
initiate or promote tissue growth and differentiation.
[0094] In certain embodiments, the length of scaffold 10, 200 can
have a length longer than the length of a gastrointestinal (e.g.,
esophageal) region being replaced. In some embodiments, the
scaffold 10 has a length that is between 100% and 150% (for
example, about 100-110%, 110-120%, 120-130%, 130-140%, about 100%,
about 105%, about 110%, or about 115%) of the length being
replaced. It is contemplated that the length of the scaffold will
be that necessary to effectively replace the effected region. In
certain situations, it is contemplated that a scaffold 10 will have
a length that is longer than the replaced gastrointestinal region
to effectively position the scaffold and reduce or minimize trauma
and ischemia in the effected or associated regions.
[0095] In some embodiments, a scaffold 10 can be composed of a
single layer of synthetic material. However, it is within the
purview of this disclosure that the scaffold 10 also can include
more than one layer of synthetic material.
[0096] Accordingly, in some embodiments, the synthetic scaffold 10
can be composed of multiple layers (e.g., 2 or more layers, for
example 2, 3, 4, 5, or more layers). In some embodiments, one or
more layers are made of the same material. In some embodiments, the
different layers are made of different materials (e.g., different
polymers and/or different polymer arrangements). Synthetic
scaffolds 10, 200 as disclosed herein may include two or more
different components that are assembled to form the scaffold as it
exists e.g., prior to cellularization and/or implantation. In some
embodiments, a synthetic scaffold 10 includes two or more layers
that are brought into contact with each other, for example by the
synthetic techniques that are used to manufacture the scaffold 10.
In some embodiments, a scaffold 10 may be synthesized using a
technique that involves several steps that result in two or more
layers being brought together (e.g., the application of a layer of
electrospun material onto a portion of the scaffold that was
previously made, such as an prior layer of electrosprayed material,
a prior layer of electrospun material, a surface of a different
component (e.g., a braided tube or mesh) that is being incorporated
into the scaffold, or a combination of two or more thereof).
[0097] In the embodiment as depicted in FIG. 1A, scaffold 10,
includes at least one outer layer 18 that defines the outer surface
14 of the scaffold body 12. The scaffold 10, 200 includes at least
one additional inwardly oriented layer 20. In the embodiment as
illustrated, the at least one additional inwardly oriented layer 20
is in direct contact with an inwardly oriented face of the outer
layer 18. Where desired or required, the at least one inwardly
oriented layer 20 can be configured to provide structural support
to the associated scaffold body 12. In the embodiment depicted, in
FIG. 1 A, the at least one inwardly oriented layer 20 can be
configured as a suitable mesh or braid positioned circumferentially
around at least a portion of the longitudinal length of the
scaffold body 12. In other embodiments, it is contemplated that the
at least one inwardly oriented layer 20 can be composed of a
suitable polymeric layer. In the embodiment as illustrated in FIG.
1A. the body 12 of scaffold 10 includes at least one layer 22 that
is located interior to the mesh or braid layer 20.
[0098] Where desired or required, the scaffold 10 can have a wall
thickness that is generally uniform. However, in some embodiments,
the wall thickness can vary at specific regions of the body 12. In
some embodiments, the wall thickness at one or both ends 24, 26 of
the body 12 of scaffold 10 is different (e.g., thicker) than the
walls of the central portion 28 of the scaffold 10 (not shown). In
some embodiments, the thicker wall regions are stronger and provide
greater support for sutures that are connected to one or both ends
24, 26 of the scaffold 10 when the scaffold is connected to
surrounding gastrointestinal tissue. The thicker wall region(s) can
also include discrete configurations that facilitate suturing.
Non-limiting examples of such configurations include tubes, wholes,
etc.
[0099] In certain embodiments, at least the exteriorly oriented
surface 14 defined on the outwardly oriented layer 18 can be
composed of an electrospun polymeric material. In certain
embodiments, it is contemplated that the outwardly oriented wall 18
can be composed of electrospun polymeric material. In certain
embodiments, the electrospun outwardly oriented layer can be in
direct contact with a suitable braid material layer 20.
[0100] In some embodiments, the wall thickness at one or both ends
of a scaffold is different (e.g., thicker) than the walls of the
central portion of the scaffold. In some embodiments, the thicker
wall regions are stronger and provide greater support for sutures
at the ends of the scaffold.
[0101] In some embodiments, synthetic scaffolds comprise an
anti-reflux system. As used herein, "anti-reflux system" refers to
a system for preventing reflux (e.g. restricting flow in a single
direction). For example, in the context of regenerated
gastrointestinal tissue (e.g., esophagus tissue) that has been
anastomosed to a target tissue (e.g., stomach tissue), it is
desirable, in some embodiments, to prevent reflux by restricting
the flow of gastric contents toward the regenerated esophageal
tissue. In some embodiments, an anti-reflux system comprises a
valve (e.g., a one-way valve, check valve, flapper valve, cusp
valve, a semilunar cusp valve) configured to minimize or prevent
the flow of gastric juices or acidic fluids out from the stomach,
e.g., into the esophagus.
[0102] In some embodiments, an anti-reflux system is a hollow
structure extending beyond an end having an enlarged diameter. In
some embodiments, the hollow structure has a lumen that is
contiguous with the lumen of the synthetic scaffold (e.g. allows
the passage of contents through the scaffold and into the lumen of
an anastomosed tissue, for example a stomach). The hollow structure
can be any suitable shape, including but not limited to
cylindrical, elliptical, cuboidal, pyramidal, bell-shaped, and
trumpet-shaped. In some embodiments, the inner diameter of the
anti-reflux system is about 0 cm (e.g., identical size), 0.1 cm,
0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0
cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm,
1.9 cm, or 2.0 cm larger than the non-enlarged end of the scaffold.
In some embodiments, the inner diameter of the anti-reflux system
is about 0 cm (e.g., identical size), 0.1 cm, 0.2 cm, 0.3 cm, 0.4
cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm,
1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, or 2.0 cm
smaller than the non-enlarged end of the scaffold.
[0103] A synthetic scaffold comprising an enlarged end can be
fitted onto a target tissue (e.g., a stomach) in a manner such that
the enlarged end rests on top of the target tissue and the hollow
structure (e.g., anti-reflux system) extends beyond the edges of
the enlarged end and into the lumen of the target tissue (e.g.,
stomach) without substantially contacting the walls of the target
tissue. In some embodiments, an anti-reflux system is formed from a
compressible or deformable (e.g., malleable) material, such as a
soft plastic or other polymer.
[0104] In the embodiment depicted in FIGS. 15 A and B, scaffold 200
has a body section 210. The body section 210 has a first end 212
and a second end 214 opposed to the first end. The body section 210
further has a least one portion configured as a tubular member 216
and at least one portion configured as a flared member 218. The
flared member 218 is contiguously connected to the tubular member
216 and is located proximate to either the first end 212 or the
second end 214 of the body section 210. In certain embodiments, the
body section 210 comprises an outwardly oriented surface 220. The
outwardly oriented surface 210 has at least one region that is
composed of spun polymeric fibers. In certain embodiments, the spun
polymeric fibers have an average fiber diameter between 15 nm and
10 microns with at least a portion of the spun polymeric fibers
interlinked to form pores having an average diameter less than 50
microns.
[0105] In certain embodiments, the synthetic scaffold 200 further
comprises a tubular member region 222 that extend outward from an
interior region defined by the flared member 218. In certain
embodiments, the tubular member region 222 can be coaxially
positioned relative to the tubular member 216. In some embodiments,
the tubular member region 222 and can extend extends within the
interior region defined by the flared member 218.
[0106] Where desired or required, the spun polymeric fibers are
electropsun. The polymeric fibers can be interconnected and can
form an outer layer of the body section. In certain embodiments,
the body section can comprises at least one inner layer. In certain
embodiments, the inner layer can be composed of at least one of a
polymeric mesh, a polymeric braided support material, a solid
polymeric member, an electrospun layer with the outer layer in
overlying contact with the inner layer. The electrospun material
can have an average fiber diameter of 3 to 10 micrometers and is
composed of at least one of one of the following polymeric
materials: polyvinylidene fluoride, syndiotactic polystyrene,
copolymer of vinylidene fluoride and hexafluoropropylene, polyvinyl
alcohol, polyvinyl acetate, poly(acrylonitrile), copolymers of
polyacrylonitrile and acylic acid, copolymers of polyacrylonitrile
and methacrylates, polystyrene, poly(vinyl chloride), coploymeris
of poly(vinyl chloride), poly(methyl methacrylate), copolymers of
poly(methyl methacrylate), polyethylene terephthalate,
polyurethane.
[0107] In certain embodiments, at least one layer is a polymeric
material containing polyethylene terepthalate, polyurethane, blends
of polyethylene terepthlatae and polyurethane. In certain
embodiments, a brad or mesh layer can be present. In certain
embodiments, the braid or mesh layer can be composed of at least
one of polyethylene terepthalate, polyurethane, nitinol and
mixtures thereof.
[0108] The synthetic scaffold 210 can be transportable and can be
composed of at least one sheath layer, the sheath layer composed of
cellular material, the cellular material composed of mesenchymal
cells and stem cells present in a defined layer the defined layer
being between 1 and 100 celled thick. The cellular material sheath
layer can overlay the the electrospun fibers present on the outer
surface such that the cellular material is contained on the outer
surface and spans pores defined therein.
[0109] The synthetic scaffold 210 can also include at least one
hole, indent, protrusion, or a combination thereof defined
proximate to at least one of the first or second ends that is
adapted to assist in at least one of the retrieval of the scaffold
from a subject after tissue regeneration has occurred around the
scaffold at the site of implantation in the subject or implanting
the synthetic scaffold in a location in the body of a subject.
Fiber Orientation
[0110] Electrospun fibers can be isotropic or anisotropic. In some
embodiments, fibers in different layers can have different relative
orientations. In some embodiments, fibers in different layers can
have substantially the same orientation. Fiber orientation can be
altered in each layer of a composite or sandwich scaffold in
addition.
[0111] In some embodiments, scaffolds with different porosities can
be used. In some embodiments, one or more layers of a scaffold
permit substantially complete cellular penetration and uniform
seeding. In some embodiments, one or more layers of the scaffold
may be constructed to prevent the penetration of one or more cell
types, for example by densely packing the fibers. Controlling fiber
diameter can be used to change scaffold porosity as the porosity
scales with fiber diameter. Alternatively, blends of different
polymers may be electrospun together and one polymer preferentially
dissolved to increase scaffold porosity. The properties of the
fibers can be controlled to optimize the fiber diameter, the fiber
spacing or porosity, the morphology of each fiber such as the
porosity of the fibers or the aspect ratio, varying the shape from
round to ribbon-like. In some embodiments, the mechanical
properties of each fiber may be controlled or optimized, for
example by changing the fiber composition, and/or the degradation
rate.
[0112] In certain embodiments, the electrospun fiber material can
provide a contoured surface such as a that depicted in FIG. 1B. In
certain embodiments, at least one electrosupn layer in scaffold 10
can be a polymeric fiber material such as polycarbonate
polyurethane and can be produced by dissolving
polycarbonate-polyurethane in a suitable solvent such as hexa
fluoroisopropanol (HFIP) that is spun and dried.
[0113] The spacing and porosity of the electrospun fiber material
can be that such that cells seeded on the scaffold surface can
adhere in suspended overlying relationship between respective
fibers to permit the seeded cellular material to form sheets
thereon as illustrated in FIGS. 4A and 4B.
Layering of Synthetic Scaffolds
[0114] Aspects of the disclosure relate to methods for producing
synthetic scaffolds. In some embodiments, tubular synthetic
scaffolds (e.g., a synthetic esophageal scaffold) are produced on a
mandrel (e.g., by depositing material via electrospraying and/or
electrospinning).
[0115] In some embodiments, one or more layers of a synthetic
scaffold provide structural support to the scaffold, conferring a
desired mechanical property to the scaffold. In some embodiments, a
braided material (e.g., a braided tube, for example a nitinol
braid, a PET braid, or a braid of other metallic or non-metallic
material) can be inserted between two different layers of a
scaffold to provide structural support. The compression force of
the braided material (e.g., the force that the braid can exert on
the next layer of material, for example the outer electrospun layer
of material) can be controlled by controlling the pick count of the
braid. In some embodiments, a braid can be coated (e.g., by dipping
or other technique) in an organic solvent to help attach it to one
or more other layers of the scaffold 10. In some embodiments, the
length of the braid 20 does not extend to the ends of the scaffold
body 12. In some embodiments, one or both ends of the scaffold 10
consist of two or more layers of material without a braided layer,
whereas the central portion 28 of the scaffold body 12 includes an
additional braided layer.
[0116] In some embodiments, one or more layers of a synthetic
scaffold provide a barrier in the scaffold, creating a separation
(e.g., a relatively impermeable separation) between an inner space
(e.g., a lumenal space) and an external space. In some embodiments,
a barrier can be an electrosprayed polyurethane (PU) layer.
[0117] In some embodiments, different layers of a scaffold 10 can
include one or more polymers (e.g., polyethylene terephthalate
(PET), PU, or blends thereof). In some embodiments, a scaffold 10
can include a nitinol braid sandwiched between an inner PU layer
(e.g., that was electrosprayed or electrospun onto a mandrel) and
an outer PU layer (e.g., that was electrosprayed onto the braided
material).
[0118] In certain embodiments the scaffold 10 can be formed using a
scaffold support or mandrel. In some embodiments, a scaffold
support or mandrel may be coated with a material (e.g., PLGA or
other polymer) prior to depositing one or more layers of PU, PET,
or a combination thereof.
[0119] In certain embodiments, the material in the braid or mesh
layer can be composed of absorbable polymeric material.
Scaffold Production
[0120] In some embodiments, tubular scaffolds (e.g., a synthetic
esophageal scaffold) are produced by nanofiber assembly, casting,
printing (e.g., 3D printing), physical spraying (e.g., using pumps
and syringes), extrusion molding, electrospinning, or
electrospraying. Other appropriate methods may be used.
Scaffold Production-Fiber Materials
[0121] In some embodiments, one or more layers of a scaffold may be
constructed from fibrous material. In some embodiments, scaffolds
comprise one or more types of fiber (e.g., nanofibers). In some
embodiments, scaffolds comprise one or more natural fibers, one or
more synthetic fibers, one or more polymers, or any combination
thereof. It should be appreciated that different material (e.g.,
different fibers) can be used in methods and compositions described
herein. In some embodiments, the material is biocompatible so that
it can support cell growth. In some embodiments, the material is
permanent, semi-permanent (e.g., it persists for several years
after implantation into the host), or rapidly degradable (e.g., it
is resorbed within several weeks or months after implantation into
the host).
[0122] In some embodiments, the scaffold comprises or consists of
electrospun material (e.g., macro or nanofibers). In some
embodiments, the electrospun material contains or consists of PET
(polyethylene terephthalate (sometimes written poly(ethylene
terephthalate)). In some embodiments, the electrospun material
contains or consists of polyurethane (PU). In some embodiments, the
electrospun material contains or consists of PET and PU.
[0123] In some embodiments, the artificial scaffold may consist of
or include one or more of any of the following materials: elastic
polymers (e.g., one or more polyurethanes (PU), for example
polycarbonates and/or polyesters), acrylamide polymers, Nylon,
resorbable materials (e.g. PLGA, PLA, PGA, PCL), synthetic or
natural materials (e.g., silk, elastin, collagen, carbon, gelatin,
chitosan, hyaluronic acid, etc.) or any combination thereof. In
some embodiments, the scaffold may consist of or include addition
polymer and/or condensation polymer materials such as polyolefin,
polyacetal, polyamide, polyester, cellulose ether and ester,
polyalkylene sulfide, polyarylene oxide, polysulfone, modified
polysulfone polymers and mixtures thereof. In some embodiments, the
scaffold may consist of or include polyethylene, polypropylene,
poly(vinylchloride), polymethylmethacrylate (and other acrylic
resins), polystyrene, and copolymers thereof (including ABA type
block copolymers), poly(vinylidene fluoride), poly(vinylidene
chloride), polyvinylalcohol in various degrees of hydrolysis (e.g.,
87% to 99.5%) in cross-linked and non-cross-linked forms. In
certain embodiments, the polymeric compound can also include
compounds or processes to increase the hydrophilic nature of the
polymer. In certain embodiments, this can involve incorporating
compounds such as block copolymers based on ethylene oxide and
propylene oxide. It is also contemplated that the hydrophilic
nature of the polymer can be increase by suitable plasma treatment
if desired or required.
[0124] In some embodiments, the scaffold may consist of or include
block copolymers. In some embodiments, addition polymers like
polyvinylidene fluoride, syndiotactic polystyrene, copolymer of
vinylidene fluoride and hexafluoropropylene, polyvinyl alcohol,
polyvinyl acetate, amorphous addition polymers, such as
poly(acrylonitrile) and its copolymers with acrylic acid and
methacrylates, polystyrene, poly(vinyl chloride) and its various
copolymers, poly(methyl methacrylate) and its various copolymers,
and PET (polyethylene terephthalate (sometimes written
poly(ethylene terephthalate))) can be solution spun or electrospun
and combined with any other material disclosed herein to produce a
scaffold. In some embodiments, highly crystalline polymers like
polyethylene and polypropylene may be solution spun or combined
with any other material disclosed herein to produce a scaffold.
[0125] In some embodiments, one or more polymers are modified to
reduce their hydrophobicity and/or increase their hydrophilicity
after the scaffold synthesis, but before scaffold cellularization
and/or implantation.
[0126] The electrospun fibers can have a diameter less than 10
micrometers in certain embodiments. In certain embodiments, the
electrospum fibers. In certain embodiments, the electrospun fibers
can have a diameter between 3 and 10 micrometers. The electrospun
fibers can have a dimeter between 3 and 5 micrometers in certain
embodiments.
[0127] In certain embodiments, it is contemplated that the material
in the braid layer can be made in whole or in part of bioabsorbable
materials such as PLGA and the like. it is also contemplated that,
in certain configurations, the braid material can be loaded
materials and compounds that can promote and/or support tissue
growth and regeneration. Non-limiting examples of such compounds
and materials include one or more of the following: antibiotics,
growth factors and the like.
Electrospinning
[0128] In some embodiments, scaffolds are produced that include one
or more layers (e.g., of PU and/or PET) produced via
electrospinning. Electrospun material can be used for a variety of
applications, including as a scaffold for tissue engineering.
Appropriate methods of electrospinning polymers may include those
described in Doshi and Reneker. Electrospinning process and
application of electrospun fibers. J Electrostat. 1995; 35:151-60;
Reneker D H, Chun I. Nanometer diameter fibers of polymer produced
by electrospinning. Nanotechnology. 1996; 7:216-23; Dzenis Y.
Spinning continuous fibers for nanotechnology. Science. 2004;
304:1917-19; or Vasita and Katti. Nanofibers and their applications
in tissue engineering. Int J. Nanomedicine. 2006; 1(1): 15-30, the
contents of which relating to electrospinning are incorporated
herein by reference. Electrospinning is a versatile technique that
can be used to produce either randomly oriented or aligned fibers
with essentially any chemistry and diameters ranging from nm scale
(e.g., around 15 nm) to micron scale (e.g., around 10 microns).
[0129] In some embodiments, electrospinning and electrospraying
techniques used herein involve using a high voltage electric field
to charge a polymer solution (or melt) that is delivered through a
nozzle (e.g., as a jet of polymer solution) and deposited on a
target surface. The target surface can be the surface of a static
plate, a rotating drum (e.g., mandrel), or other form of collector
surface that is both electrically conductive and electrically
grounded so that the charged polymer solution is drawn towards the
surface.
[0130] In some embodiments, the electric field employed is
typically on the order of several kV, and the distance between the
nozzle and the target surface is usually several cm or more. The
solvent of the polymer solution evaporates (at least partially)
between leaving the nozzle and reaching the target surface. This
results in the deposition of polymer fibers on the surface. Typical
fiber diameters range from several nanometers to several microns.
The relative orientation of the fibers can be affected by the
movement of the target surface relative to the nozzle. For example,
if the target surface is the surface of a rotating mandrel, the
fibers will align (at least partially) on the surface in the
direction of rotation. In some cases, the nozzle can be scanned
back and forth between both ends of a rotating mandrel.
[0131] In some embodiments, the size and density of the polymer
fibers, the extent of fiber alignment, and other physical
characteristics of an electrospun material can be impacted by
factors including, but not limited to, the nature of the polymer
solution, the size of the nozzle, the electrical field, the
distance between the nozzle and the target surface, the properties
of the target surface, the relative movement (e.g., distance and/or
speed) between the nozzle and the target surface, and other factors
that can affect solvent evaporation and polymer deposition.
[0132] Electrospinning and electrospraying processes may be used
for producing interlinked polymer fiber scaffolds (e.g., hollow
synthetic scaffolds) on a mandrel.
Support/Mandrel
[0133] In some embodiments, scaffold 10 (e.g., a scaffold having
two or more layers) can be produced using a support (e.g., a solid
or hollow support) on which the scaffold 10 can be formed. For
example, a support can be an electrospinning collector, for example
a mandrel, or a tube, or any other shaped support. It should be
appreciated that the support can have any size or shape. However,
in some embodiments, the size and shape of the support is designed
to produce a scaffold that will support an artificial tissue of the
same or similar size as the gastrointestinal tissue (or portion
thereof) being replaced or supplemented in a host. It should be
appreciated that a mandrel for electrospinning should have a
conductive surface. In some embodiments, an electrospinning mandrel
is made of a conductive material (e.g., including one or more
metals). However, in some embodiments, an electrospinning mandrel
includes a conductive coating (e.g., including one or more metals)
covering a non-conductive central support.
[0134] It has been found quite unexpectedly that positioning
suitable braid material to be integrated in the resulting scaffold
10 at a location proximate to the surface of the mandrel can serve
as an aid to facilitate removal of the resulting scaffold 10 from
contact with the mandrel.
Scaffold Properties
[0135] It should be appreciated that aspects of the disclosure are
useful for enhancing the physical and functional properties of any
scaffold, for example a scaffold based on electrospun and/or
electro sprayed fibers. In some embodiments, one or more scaffold
components can be thin sheets, cylinders, thick ribs, solid blocks,
branched networks, etc., or any combination thereof having
different dimensions. In some embodiments, the dimensions of a
complete and/or assembled scaffold are similar or identical to the
dimension of a tissue or organ being replaced. In some embodiments,
individual components or layers of a scaffold have smaller
dimensions. For example, the thickness of a nanofiber layer can be
from several nm to 100 nm, to 1-1000 microns, or even several mm.
However, in some embodiments, the dimensions of one or more
scaffold components can be from about 1 mm to 50 cms. However,
larger, smaller, or intermediate sized structures may be made as
described herein.
[0136] In some embodiments, scaffolds are formed as tubular
structures that can be seeded with cells to form tubular tissue
regions (e.g., esophageal, or other tubular regions). It should be
appreciated that a tubular region can be a cylinder with a uniform
diameter. However, in some embodiments, a tubular region can have
any appropriate tubular shape (for example, including portions with
different diameters along the length of the tubular region). A
tubular region also can include a branch or a series of branches.
In some embodiments, a tubular scaffold is produced having an
opening at one end, both ends, or a plurality of ends (e.g., in the
case of a branched scaffold). However, a tubular scaffold may be
closed at one, both, or all ends, as aspects of the invention are
not limited in this respect. It also should be appreciated that
aspects of the invention may be used to produce scaffolds for any
type or organ, including hollow and solid organs, as the invention
is not limited in this respect. In some embodiments, aspects of the
invention are useful to enhance the stability of scaffold or other
structures that include two or more regions or layers of fibers
(e.g., electrospun nanofibers) that are not physically
connected.
[0137] In some embodiments, a scaffold is designed to have a porous
surface having pores ranging from around 10 nm to about 100 micron
in diameter that can promote cellularization. In some embodiments,
pores have an average diameter of less than 50 microns, less than
40 microns, less than 30 microns, less than 20 microns or less than
10 microns (e.g., approximately 5, approximately 10, or
approximately 15 microns). In some embodiments, pores have an
average diameter of 20-40 microns. In some embodiments, pore size
is selected to prevent or reduce an immune response or other
unwanted host response in the subject. Pore sizes can be estimated
using computational and/or experimental techniques (e.g., using
porosimetry). However, it should be appreciated that pores of other
sizes also can be included.
[0138] In some embodiments, a surface layer of a scaffold is
synthesized using fibers that include one or more dissolvable
particles that can be dissolved during or after synthesis (e.g., by
exposure to a solvent, an aqueous solution, for example, water or a
buffer) to leave behind pores the size of the dissolvable
particles. In some embodiments, the particles are included in the
polymer mix that is pumped to the nozzle of an electrospinning
device. As a result, the particles are deposited along with the
fibers. In some embodiments, the electrospinning procedure is
configured to deposit thick fibers (e.g., having an average
diameter of several microns, about 10 microns, and thicker). In
some embodiments, if the fibers are deposited in a dense pattern,
one or more fibers will merge prior to curing to form larger
macrostructures (e.g., 10-100 microns thick or more). In some
embodiments, these macrostructures can entangle two or more layers
of fibers and or portions (e.g., fibers) from two or more different
components of a scaffold thereby increasing the mechanical
integrity of the scaffold. In some embodiments, when such
macrostructures are formed (e.g., via electrospinning as described
herein) at one or more stages during scaffold synthesis (e.g., to
connect two or more layers and/or components), the surface of the
macrostructure(s) can be treated (e.g., etched or made porous using
dissolvable particles as described herein) in order to provide a
surface suitable for cellularization.
[0139] In some embodiments, the amount of flexible scaffold
material (e.g., the slack) between two or more structural
components (e.g., rings), between structural members (e.g., arcuate
members) of a single continuous structural component, and/or of a
braided support material can be used to determine the mechanical
properties (e.g., tensile strength, elongation, rotation,
compression, range of motion, bending, resistance, compliance,
degrees of freedom, elasticity, or any other mechanical property,
or a combination thereof) of a synthetic scaffold.
[0140] In certain embodiments, the scaffold 10 can also include a
cellular sheath derived from cells seeded on the outer surface of
the scaffold during incubation. The cellular sheath adheres to and
is in overlying relationship to the outer surface of the scaffold.
It is contemplated that a major portion of the cells present in the
cellular sheath will be connected to the outermost surface of the
outer surface and will span pores defined therein to form a
continuous or generally continuous surface.
[0141] In certain embodiments, the cellular sheath can have a
thickness sufficient to provide structural integrity to the sheath
layer. In certain embodiments, the cellular sheath will be composed
of a number of cells which are in contact with the external surface
of the scaffold sufficient to direct regenerating cells in contact
with the sheath to produce a tissue wall that overlays the sheath
but does not integrate therewith. In certain embodiments, the
sheath can be composed of a lining that is between 1 and 100 cells
thick on average. Certain embodiments can have a cell thickness
between 10 and 100; between 10 and 30; between 20 and 30, between
20 and 40; between 20 and 50; between 10 and 20; between 30 and 50;
between 30 and 60; between 40 and 60; between 40 and 70; between 70
and 90.
[0142] The scaffold 10 with the associated cellular sheath provides
a moveable insertable device that can be positioned in a suitable
gastrointestinal resection site. The scaffold 10 with the
associated cellular sheath in contact therewith can be transported
to the desired resection site for implantation. In certain
embodiments, the scaffold 10 is configured to be removable from the
implantation site after suitable regeneration of the resected
organ. In certain embodiments, the removed scaffold will include
some or all of the cellular sheath connected thereto.
[0143] Also disclosed is are various embodiments of method of
regenerating a tubular organ such as a gastrointestinal organ. In
certain embodiments, the method 100 includes the step of resecting
a that comprises resecting a portion of a tubular organ in a
subject as at reference numeral 110. The organ to be resected can
be a tubular organ of the gastrointestinal tract that has been
damaged or compromised by disease injury, trauma or congenital
conditions. In certain embodiments, non-limiting examples of
suitable organs include one of the esophagus, rectum and the like.
In certain embodiments, suitable organs include at least one of the
esophagus, small intestines, colon, rectum.
[0144] The resection can be achieved by any suitable surgical
procedure and produced a resected organ portion that remains
connected to the gastrointestinal tract and remains in the subject
after resection. The resection operation can yield suitable
resection edges in certain embodiments.
[0145] After resection is completed, a synthetic scaffold is
implanted at the site of the resection as at reference numeral 120.
In certain embodiments, implantation can include the step of
connecting the respective ends of the resected organ as it remains
in the subject to respective ends of the synthetic scaffold such
that the synthetic scaffold and at the resected organ can achieve a
suitable junction between the respective members. This can be
achieved by one or more of sutures, bioorganic tissue glue,
etc.
[0146] In certain embodiments, the synthetic scaffold that is
implanted can be a tubular member that has an outer polymeric
surface and a cellularized sheath layer overlying at least a
portion of the of the outer polymeric surface. Various embodiments
of the synthetic scaffold have been discussed and can be employed
and utilized in the method disclosed herein. In certain
embodiments, the synthetic scaffold will include a first end and a
second end opposed to the first end, an outer polymeric surface
positioned between the first end and the second end and a
cellularized sheath layer overlying at least a portion of the outer
polymeric surface. In certain embodiments, the implantation step
can be one that brings at least a portion of the cellularized
sheath layer into proximate contact with to at least one of the
resection edges of the resected organ portion.
[0147] In certain embodiments, the method as disclosed herein also
includes the step of maintaining the synthetic scaffold at the
resection site for a period of time sufficient to achieve guided
tissue growth along the synthetic scaffold as at reference numeral
130. In certain embodiments, the guided tissue growth is derived
from and is in contact with the tissue present in the resected
organ portion remaining in the subject. In certain embodiments, the
guided tissue growth will be contiguous with the associated regions
of the resected organ. In certain embodiments, the guided tissue
growth will exhibit differentiated tissue. In certain embodiments,
the guided tissue growth will parallel the outer surface of the
cellularized sheath layer at a position outward thereof. In certain
embodiments, the guided tissue growth is derived from and is in
contact with the tissue present in the resected organ portion
remaining in the subject and will be contiguous with the associated
regions of the resected organ. The guided tissue growth will
exhibit differentiated tissue growth and can be parallel the outer
surface of the cellularized sheath layer at a position outward
thereof.
[0148] After the guided tissue growth has been achieved, the
process 100 as disclosed herein can include step of removing the
synthetic scaffold as at reference numeral 140. In certain
embodiments, the removing step occurs in a manner such that the
guided tissue growth remains in the contact with the resected
portion of the organ remaining in the subject. In certain
embodiments, the removal process can include intrascopically
removing the synthetic scaffold from the interior of the guided
tissue growth.
[0149] In certain embodiments, the synthetic scaffold can be
constructed in whole or in part from bioabsorbable polymeric
material. In such situations, the method as disclosed herein can
include the step of maintaining contact between the synthetic
scaffold and the resection edge for an intervals sufficient to
achieve guided tissue growth along the synthetic scaffold such that
at least a portion of the synthetic scaffold is absorbed at the
site of resection within a period of time sufficient to achieve
guided tissue growth along the synthetic scaffold. In certain
embodiments where the scaffold is composed entirely of bioabsorbale
material, the scaffold will be configured to maintain structural
integrity during guided tissue growth. In certain embodiments,
where the synthetic scaffold is composed of bioabsorbable material
in selected regions, it is contemplated that the remainder of the
scaffold can be removed by suitable procedures after the guided
tissue growth has been achieved.
[0150] Guided tissue growth can be monitored by suitable means. In
certain embodiments, tissue growth can be monitored
endoscopically.
[0151] In certain embodiments of the method as disclosed herein,
the method can also include the step of imparting cellular material
onto the polymeric surface of the synthetic scaffold and allowing
the cellular material to grow to form the cellular sheath layer,
the imparting and allowing steps occurring prior to the resecting
step.
[0152] In certain embodiments, the synthetic scaffold that is
employed in the method disclosed herein a tubular member where the
outer surface includes spun polymeric fibers. In certain
embodiments, the spun fibers can be electrospun by suitable methods
such as those described in this disclosure. The cellularized sheath
layer spans at least a portion outwardly positioned electrospun
fibers in certain embodiments. The cellularized sheath layer can is
composed of cellular material, the cellular material including at
least one of mesenchymal cells, stem cells, pluripotent cells. The
cellular material can be autologously derived from the subject or
can be allogenically derived.
[0153] Without being bound to any theory, it is believed that
implanting a synthetic scaffold such as those as variously
disclosed herein, particularly one seeded with an overlying
cellular sheath, promotes growth, regeneration and differentiation
of the subject tissue in contact with or proximate to the location
of the implanted synthetic scaffold. The growing regenerating
tissue is guided by the synthetic scaffold and associated sheath to
produce a tubular cellular body that is integrally connected to the
resected ends of the remaining tubular organ and outwardly flaring
to encapsulate the synthetic scaffold and associated cellular
sheath layer. It is believed that the scaffold and associated
cellular sheath layer may promote or stimulate regenerative growth
of the resected tissue while minimizing tissue rejection responses.
It is also believed that the presence of the cellular sheath layer
can reduce or minimize penetration of the regenerated tissue into
the sheath layer during growth and differentiation. In certain
embodiments, tissue generation proceeds from the respective ends
toward the middle. Once the regenerated tissue is in position, the
synthetic scaffold can be removed. In certain embodiments,
immediately after the removal of the synthetic scaffold, the
regenerated tissue structure will lack the inner epithelial layer.
This layer has been seen to regenerate after removal of the
scaffold.
[0154] In order to further understand the present disclosure,
reference is made to the following Examples. These Examples are
included for purposes of illustration and are to be considered
illustrative of the present disclosure and the invention as set
forth in the claims.
EXAMPLES
Example I: Esophageal Scaffolds
[0155] Synthetic esophageal scaffolds were produced containing
three layers of material as illustrated in FIG. 1A. A first layer
of polyurethane (PU) was deposited onto a metallic mandrel via
electrospraying. A braided material was then deposited on the first
PU layer. A second PU layer was then deposited via electrospinning.
The resulting scaffolds were then removed from the mandrel. Each
scaffold defined a tubular structure having a wall that included
three layers (a braided layer sandwiched between and inner
electrosprayed layer and an outer electrospun layer). Physical
dimensions of the scaffold were determined by scanning electron
microscopy (SEM). The average scaffold wall thickness was
approximately 500 microns. A non-limiting SEM view of a
cross-section of the wall is shown in FIG. 1B. A non-limiting
visual image of a cross-section of the tubular scaffold is shown in
FIG. 1C. This image shows that the cross-section is approximately
"D" shaped. This can be obtained by using a mandrel that has a "D"
shaped cross section.
[0156] The outer electrospun layer was a layer of polymer fibers
defining pores. The average fiber diameter in the outer layer was
approximately 3-6 microns The average pore size was approximately
15-20 microns, and the median pore size was approximately 25-45
microns.
[0157] Scaffolds were attached to a support capable of rotating in
a bath of liquid medium within a bioreactor chamber. The rotating
mechanism can include magnetic drives that allow the support along
with the attached scaffold to be rotated around its longitudinal
axis within the liquid bath.
[0158] Scaffolds were seeded with cells (e.g., MSCs or other stem
cells) by depositing cell solutions on the external scaffold
surface. The seeded scaffolds were then incubated in liquid media
that supports cell growth by rotating the scaffolds in a bath of
the liquid media within a bioreactor chamber for approximately one
week. The resulting scaffolds include a cellular sheath that is in
overlying relationship to the outer surface of the scaffold. In
certain embodiments, the cellular sheath can have a thickness
sufficient to provide structural integrity to the sheath layer. In
certain embodiments, the cellular sheath will be composed of a
number of cells which are in contact with the external surface of
the scaffold sufficient to direct regenerating cells in contact
with the sheath to produce a tissue wall that overlays the sheath
but does not integrate therewith. In certain embodiments, the
sheath can be composed of a lining that is between 1 and 100 cells
thick on average. Certain embodiments can have a cell thickness
between 10 and 100; between 10 and 30; between 20 and 30, between
20 and 40; between 20 and 50; between 10 and 20; between 30 and 50;
between 30 and 60; between 40 and 60; between 40 and 70; between 70
and 90.
[0159] The scaffold 10 having the seeded cellular sheath can be
implanted in to the resection site and can be positioned in place.
It is contemplated that the seeded cells present in the sheath can
continued to grow post implantation. In such situations, the seeded
cells present in the sheath will maintain and support a structure
that is separate from and tandem to the tissue regenerating at the
implantation site.
[0160] The respective scaffolds were then implanted into esophageal
sites in pigs. An approximately 5 cm section of esophagus was
removed and replaced with a scaffold section that was sutured to
the ends of the remaining esophageal tissue in the subject.
[0161] The regeneration of esophageal tissue was monitored
endoscopically for several weeks.
[0162] The esophagus is a long muscular tube that has cervical,
thoracic, and abdominal parts. FIG. 2 is a diagram that illustrates
a cross-section of an esophagus in a human. In an adult human the
esophagus can be 18 cm to 25 cm in length. An esophagus wall is
composed of striated muscle in the upper part, smooth muscle in the
lower part, and a mixture of the two in the middle. Accordingly,
provided herein, in some embodiments, are multilayered synthetic
scaffolds that can promote repair and regeneration of esophageal
tissue having two or more layers corresponding to natural
esophageal tissue layers.
[0163] FIG. 3 shows stained cross-sections of native and
regenerated esophageal tissue 1-2 weeks after an esophageal
scaffold implant in a pig. The cross section shows regeneration of
essentially all the esophageal tissue layers (including different
muscle and gland layers). Further analysis of the regenerated
tissue revealed that the scaffold itself was not incorporated into
the regenerated esophageal wall. The scaffold was still present
within the esophagus, but appeared to have acted as a guide that
stimulated esophageal regeneration as opposed to becoming an
integral part of the regenerated esophagus.
Example II: Esophageal Implant
[0164] Synthetic esophogeal scaffolds were produced that contained
three layers as illustrated in FIG. 1A with the outer electrospun
layer of poly-carbonate-polyurethane being deposited as a solution
of polycarbonate polyurethane dissolved in Hexafluoroisopropanol
(HFIP) (DuPont, Wilmington, Del., USA) at 12% w/v. The
electrospinning apparatus used was commercially available from IME
Technologies, Geldrop, Netherlands. The electrospun fibers were
collected on a target aluminum mandrel rotating at 800 rpm and
placed at a distance of 22 mm from the syringe tip to deposit an
isotropic fiber to produce a scaffold having an average wall
thickness of 500 microns. The scaffolds were dried in a vacuum to
remove residual solvent. The scaffolds were then plasma treated
with 2 consequent cycles of ethylene and oxygen gases using a low
pressure plasma system (Diener Tetra 150-LF-PC-D). Scaffolds were
gamma sterilized (STERIS, Northborough, Mass.). The applied dose
range was 25-35 KGy.
[0165] The resulting tubes were polymeric scaffolds composed of
electrospun polyurethane having a consistent outer diameter (OD) of
22 mm and a length of 11 cm.
[0166] The morphology of the electrospun fibers was analyzed by
scanning electron microscopy (Zeiss-EVO MA10). Samples of the
scaffolds were sputter coated with Platinum and Palladium using a
sputter coater for two minutes (Cressington-208HR, TED PELLA, Inc,
Redding, Calif.) under a pressure of 8.times.10.sup.-2 mbar and an
electric potential of 300 V. Porosity was calculated using
gravimetric measurements. Porosity, .epsilon., is defined in terms
of the apparent density of the fiber mat, .rho.APP and bulk density
of the polymer, .rho.PU of which it is made:
.epsilon.=1-.rho.APP/.rho.PU. The apparent scaffold density
.rho.APP was measured as mass to volume ratio on 10 mm dry disks:
.rho.APP=Mass/VPU. Pore size measurements were taken using a
mercury porosimeter system (Micromeritics AutoPore IV). Tensile
tests on were performed consistent with ASTM D638 guidelines on 10
mm.times.40 mm samples that were mounted on an electromechanical
load frame (Instron 5943 Apparatus) using a 1 kN load cell. The
testing parameters were the same for all samples, at a 100 Hz data
acquisition rate, a gauge length of 30 mm, and a test speed of 1
mm/sec. Scanning electron microscopy at increasing magnifications
as illustrated in FIG. 7A demonstrated the isotropic fiber
arrangement aspects of the electrospun synthetic scaffold. The
smooth surface and isotropic nature of the fibers insures strength
and elasticity of the scaffold is uniform in all directions.
[0167] Tensile testing via uniaxial mechanical loading was
performed on three pre-implantation and three post-implantation
scaffolds (FIG. 7B), which all showed similar results at in vivo
loading values. Consistency between the six samples at in vivo
loading shows that the scaffolds have a low degree of variability
present after fabrication and in vivo implantation (FIG. 7B, C).
The mean (.+-.SD) tensile strain ranged between 119.5.+-.1.61 mm
and 124.5.+-.3.44 mm across the six scaffolds. At failure, the
tensile strain for the samples pre-implantation reached
397.38%.+-.5.52% and post-implantation 408.61%.+-.17.64%. Strain
values above 400% suggest the reliability of the fabrication
process and relative in vivo stability. Tensile stress at failure
was 7.25.+-.0.59 MPa and 4.43.+-.0.77 MPa for pre- and
post-implantation scaffolds, respectively. Consequently, the
Young's modulus was larger in the pre-implantation samples than the
post-implantation samples, though both groups were comparable in
elasticity at in vivo strains (FIG. 7B, C). The load at failure
followed the same trend as the Young's modulus, with the
pre-implantation values being greater than the post-implantation
values.
[0168] Autologous porcine adipose-derived mesenchymal stem cells
(aMSCs) were isolated from 8 pigs following an open adipose biopsy
and analyzed for characterization. The 8 Yucatan mini-pigs
underwent general anesthesia and chlorhexidine skin preparation
prior to a sterile, open adipose tissue biopsy taken from the
lateral abdominal wall. A 5 cm incision was performed next to the
linea alba with hemostasis achieved using electrocautery.
Approximately 30-50 g of adipose tissue was isolated, and
transferred to a 50 mL conical tube containing alpha Minimal
Essential Medium (MEM)/glutamax (Thermo Fisher Scientific, Waltham,
Mass.) and 1% penicillin/streptomycin (Thermo Fisher
Scientific).
[0169] 20-60 g of abdominal adipose tissue was surgically excised
from each anesthetized Yucatan mini pig (50-60 kg body weight). The
tissue samples were washed 3 times in alpha Minimal Essential
Medium (MEM)/glutamax (Thermo Fisher Scientific) and 1%
penicillin/streptomycin (Thermo Fisher Scientific). The washed
tissue was trimmed to remove lymph nodes and blood vessels and
minced into pieces smaller than 5 mm. The tissue pieces were
dissociated in digestion buffer (300 IU/mL collagenase type II,
0.1% bovine serum albumin (7.5%, fraction V), 1%
penicillin/streptomycin, alpha MEM/glutamax) for 55 minutes at
37.degree. C., 5% CO.sub.2. After quenching in complete growth
medium (StemXVivo, R&D Systems, Minneapolis, Minn.) and 1%
penicillin/streptomycin), the cells were centrifuged for 15 minutes
at 1500 rpm. The cell pellet was re-suspended in 5 mL of growth
medium and filtered through a 70 .mu.m filter. The cell filtrate
was centrifuged for 5 minutes at 1500 rpm. The cell pellet was
re-suspended in 5 mL of growth medium and cells were plated
according to tissue weight (3 g of adipose tissue isolate per T75
flask containing 20 mL growth medium).
[0170] Cells were washed twice in PBS without calcium or magnesium
(Thermo Fisher Scientific) and dissociated using TrypLe (Thermo
Fisher Scientific). The dissociation was quenched with growth
medium and the cells were centrifuged at 1000 rpm for 5 minutes.
The cell pellet was re-suspended in 1% bovine serum albumin diluted
with PBS. Aliquots of 1 million cells were incubated in antibody at
4.degree. C. for 30 minutes in the dark (Supplemental Table 1). The
labeled cells were washed 3 times in buffer and secondary
antibodies (Life Technologies, Carlsbad, Calif.) were applied as
necessary at 4.degree. C. for 30 minutes in the dark. After a
further 3 washes, the cell suspensions were placed into a 96 well
plate for flow cytometry (Guava easyCyte HT, EMD Millipore,
Billerica, Mass.). Events representative of live cells were gated
on forward and side scatter values, based upon measurements of
viability (ViaCount, EMD Millipore). Cell type analysis was
performed using fluorescent events compensated against unstained
and isotype control antibody stained samples. Acquired data was
exported and analyzed using standalone software (FlowJo version 10,
FlowJo, LLC, Ashland, Oreg.).
[0171] To assess colony formation, adipose-derived cells were
isolated as described, triturated to a single cell suspension and
diluted to 10 cells/mL of growth medium. 100 .mu.L of the cell
suspension was added to each well of a 96 well plate (Corning,
Inc., Corning, N.Y.) and visually inspected for cell number the
following day. After 5-7 days, colonies of cells became visible and
medium was changed every 3 days until the colonies contained at
least 50 cells. Wells were counted for the presence of colonies and
expressed as a percentage of total wells analyzed.
[0172] Pluripotency of isolated adipose-derived cells were
determined by their ability to undergo adipogenesis and
osteogenesis by chemical induction. Cells were plated in 6-well
tissue-culture plates, cultured in complete growth medium, and
allowed to grow to 60% or 100% confluency for adipogenic and
osteogenic differentiation, respectively. Upon reaching confluence,
medium was changed to either adipogenic or osteogenic
differentiation medium (CCM007, R&D Systems, Minneapolis,
Minn.). Medium was changed every 2 days until 14 days in culture.
Cells cultured in adipogenic differentiation medium were stained
with Oil Red O (American MasterTech, Lodi, Calif.) to identify
lipids and cells cultured in osteogenic medium were stained with
Alizarin Red (EMD Millipore) for calcium deposition.
[0173] Concentrations of glucose and lactate were measured in
conditioned medium from bioreactors at the time of seeding and 2, 5
and 7 days post-seeding (iSTAT, Abbott, Princeton, N.J.).
[0174] Cell supernatants were analyzed for the production of
porcine cytokines and growth factors either by multiplex assay on
the Luminex 200 platform or by ELISA at the University of Minnesota
Cytokine Reference Laboratory using commercially available kits and
performed according to manufacturers' directions. A 13-plex
porcine-specific bead-set panel (EMD Millipore) was used to
determine levels of porcine VEGF, GM-CSF, IL-1RA, IL-6 and IL-8.
Values were interpolated from standard curves generated on each
plate using BioPlex software (BioRad, Hercules, Calif.) for the
Luminex platform, or Microplate Manager software for ELISA plates
read on a BioRad 550 plate reader. All samples were assayed in
duplicate.
[0175] Cells were rinsed in PBS and fixed with 10% formalin for 15
minutes at room temperature. The cells were gently rinsed 3 times
in PBS containing 0.1% Triton X-100 (PBS-T) and incubated for 1
hour at room temperature in 10% normal goat serum (Vector) diluted
in PBS-T. The rabbit anti-nestin antibody (Biolegend, 1:100) was
diluted in 10% normal goat serum and PBS-T and incubated overnight
at 4.degree. C. The cells were rinsed twice in PBS-T and incubated
in fluorescent goat anti-rabbit antibody (Alexa Fluor 594, Thermo
Fisher Scientific) at room temperature for 1 hour. The cells were
rinsed twice and counterstained with 4',6-diamidino-2-phenylindole
(DAPI).
[0176] After 48 hours at 37.degree. C., the cells were washed twice
in phosphate buffered saline containing calcium and magnesium
(Thermo Fisher Scientific) and replaced with fresh growth medium.
Thereafter, culture medium was replaced every 2 days until the
flasks were 70%-80% confluent. At passaging, the cells were
dissociated (TrypLe, Thermo Fisher Scientific), counted (Countess,
Thermo Fisher Scientific) and replated at 200,000 cells per T175
flask. The cells were passaged twice prior to seeding of
scaffolds.
[0177] Each 11 cm long scaffold was placed in a bioreactor and
seeded with 32 million cells (viability >70%, trypan blue dye
exclusion, Countess, Thermo Fisher Scientific) in growth medium
supplemented with 0.1875% sodium bicarbonate (Thermo Fisher
Scientific), MEM eagle (Lonza) and 1.19 mg/mL bovine collagen
(Organogenesis) in 0.01M hydrochloric acid. The cells were
incubated for 5 minutes at 37.degree. C., 5% CO.sub.2 before 200 mL
of growth medium was slowly added to the bioreactor. The bioreactor
was incubated for 7-8 days prior to scaffold implantation. Culture
media was changed every 2 days and taken for various assays
described below.
[0178] The porcine aMSCs were seeded onto a previously
characterized scaffold and subsequently incubated in a bioreactor.
Seeded scaffolds were then implanted following esophagus resection
in Yucatan mini-pigs until scaffold removal at 3 weeks (FIG. 6) and
reproducibly stained positive for known MSC markers using
anti-porcine CD44, CD73, CD90, CD105, and CD146, antibodies and
were negative for CD14, CD45, CD106, CD271, and SLA Class II DR.
Greater than 95% of the cultured cells stained positive for nestin
and aSMA, indicating stem cell characteristics are maintained in
culture. Pluripotency was determined by chemically inducing the
porcine MSC isolates to undergo adipogenesis and osteogenesis,
respectively. These aMSCs were routinely expanded and characterized
from passage 1 to 5, and showed consistent phenotypic and
functional characteristics.
[0179] Porcine aMSCs grown from passage 2 were seeded onto a
polymeric scaffold and incubated in a bioreactor for 7 days (+/-1
day) at 37.degree. C. A number of cytokines and growth factors were
measured using enzyme-linked immunosorbent assay (ELISA) to
determine if the seeded aMSCs cultured on the scaffold secrete
factors that may assist in angiogenesis and immunomodulation. Cell
secretion of vascular endothelial growth factor (VEGF),
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin (IL)-6, IL-8, and IL-1RA was detected in conditioned
medium at levels significantly above medium alone (FIG. 4A).
However, additional cytokines, TNF-.alpha., IL-1.alpha.,
IL-1.beta., INF-.gamma., IL-10, IL-12, IL-18, platelet-derived
growth factor (PDGF), and regulated on activation, normal T
expressed and secreted (RANTES), were measured but not
detected.
[0180] Punch biopsies of sections of the seeded graft were taken at
the end of the incubation time at 7 days, to assess cell health and
penetration into the scaffold. Cellular health was assessed by
immunofluorescence staining using calcein (live cells) and ethidium
bromide (dead cells). Cellular penetration of the scaffold was
assessed using ethidium bromide for cell identification. The
populations of live cells attached to the scaffold are indicated by
the predominance of calcein staining of the biopsy samples. On
cross sections of the scaffold biopsies the majority of cellular
attachment was present at the surface of the scaffold. While there
was some evidence of cellular proliferation and ingrowth within the
scaffold. Metabolic activity of the implant graft during bioreactor
incubation was measured every 48 hours for glucose uptake and
lactate production. Measurements of conditioned medium consistently
indicated decreased glucose and increased lactate levels over time,
both indicators of continued metabolic cell growth. In addition,
cell expansion over 7 days in the bioreactor was quantified by
total DNA content which increase several fold over the course of
bioreactor cell seeding. Further characterization of cell phenotype
on the scaffold following 7 days incubation shows cells continue to
express alpha smooth muscle actin (aSMA) and nestin.
[0181] After endotracheal intubation and induction of general
anesthesia, animals were placed in a left lateral decubitus
position. Hair was clipped and Chlorhexidine or povidone iodine was
used for skin preparation and the animal was sterilely draped. A
standard right thoracotomy at the level of the 4.sup.th intercostal
space on each animal was performed and the thoracic cavity was
entered. Single lung ventilation was achieved through the use of a
double lumen endotracheal tube. A 4-4.5 cm segment of the
esophagus, located in the mid thoracic region (posterior to the
right lung hilum, was circumferentially mobilized and resected to
generate a 6 cm defect (tissue retraction proximally and distally).
The seeded scaffold (6 cm length) was then implanted using
polydioxanone (PDS, Ethicon Inc., Somerville, N.J.) absorbable
sutures with anastomosis to the proximal and distal esophagus.
After the implantation, a commercially available esophageal stent
(WallFlex M00516740, Boston Scientific) was inserted under direct
endoscopic guidance (Storz Video Gastroscope Silver Scope 9.3
MM.times.110 CM, Tuttlingen, Germany). Stent deployment was
performed under endoscopic and surgical visualization. The
esophageal stent was fixed in place to the normal esophageal tissue
using absorbable suture, at both the proximal and distal stent
flares.
[0182] Postoperatively the animals were adjunct supported by
gastrostomy feeding and maintained on a liquid diet through a
feeding tube for 2 weeks, a mashed diet for a period of 2 more
weeks, and then allowed to eat an oral diet of solid food after for
the continuation of the study.
[0183] At approximately 21 days following the implantation, the
scaffolds were retrieved endoscopically and aMSC impregnated
platelet rich plasma (PRP) gel was applied to improve the healing
process of the newly formed esophageal conduit. After PRP
application, a new fully covered esophageal stent (WallFlex.TM., 12
cm long.times.23 mm outer diameter, Boston Scientific Corporation)
was placed across the implant zone to prevent stricture formation
and to maintain anatomy during regeneration. Every two weeks the
animals underwent sedation and assessment of the esophageal
anastomosis and esophageal stent exchange to allow direct
visualization and progression of esophageal regeneration. Follow-up
observations were conducted endoscopically (Storz Video Gastroscope
Silver Scope 9.3 MM.times.110 CM, Tuttlingen, Germany).
[0184] Regeneration progression was also assessed by endoscopic
inspection. Following scaffold removal. the implant zone was
visualized endoscopically at approximately 3-4 week intervals; 2
representative animals are shown (FIGS. 11 and 12). At 3-4 weeks
post-implantation, regeneration of the mucosal layer was only
partially complete as can be seen in FIGS. 11 A and 12 A. However,
the process of esophageal healing continued with time, indicated by
the proximal and distal ends of the mucosal layers forming an
initial ridge (FIG. 11B and FIGS. 12B, 12C and 12D before fusion of
the 2 layers and complete mucosal regeneration (FIGS. 11C and 12E).
The early reconstitution of the esophageal continuity and integrity
and the subsequent growth of the submucosa from the two opposite
edges of the resection have been consistent across all eight
animals; 2 animals have been maintained to 8 and 9 months
post-surgery and have been without esophageal stent respectively
for 2 and 3 months without evidence of stricture or stenosis and
have had durable oral intake, with noteworthy weight gain.
[0185] In order to ascertain histological similarities of the
morphologies of regenerated and native esophogeal tissue. Samples
of tissue were excised from a representative pig esophagus at 2.5
months post-implantation, and include both the site of surgery and
adjacent distal and proximal tissues for histology. (FIG. 13A,
dotted box indicates the histological analysis specimens).
Representative images of hematoxylin and eosin (FIGS. 13B and D)
and Masson's trichrome (FIGS. 13C and E) stained tissue sections
show histologically intact multi-layered esophageal epithelia and
submucosa and normal inner muscular layer morphology.
[0186] Representative immunohistochemical analysis from the
regenerated region demonstrates immunoreactivity for Ki67 (FIG.
14F) at 2.5 months suggests continued proliferation of mucosal and
submucosal cells, CD31 (FIG. 14G)), CD3.epsilon. (FIG. 14H, aSMA
FIG. 14I, transgelin/SM22a (FIG. 14J) and a relative absence of
striated myosin heavy chain (FIG. 14K) in tissue at the site of
surgery. The predominance of aSMA, SM22a, and relative absence of
myosin heavy chain suggest that smooth muscle proliferation
precedes skeletal muscle growth.
[0187] The synthetic matrix seeded with autologously derived
mesenchymal cells (aMSCs) resulted in full longitudinal
regeneration of the resected esophagus with minimal mucosal
ulcerations or perforations (Table 1). All animal experienced a
full 100% of longitudinal regeneration from 2-9 weeks after graft
removal with 1 out of 6 animals experiencing both mucosal
ulceration or perforation. No animals experienced leaks over the
course of the study.
TABLE-US-00001 TABLE I Scaffold Longitudinal Pig Time length
regeneration Mucosal Contained No (status) Stent (cm) (%)
ulceration perforation Leak 1 2 weeks No 4.5 100 No No (euthanized)
2 2 weeks No 4.5 100 No No (euthanized) 3 6 weeks Yes 6 100 No No
(euthanized) 4 7 weeks Yes 6 100 No No (euthanized) 5 9 weeks Yes 6
100 No Yes (euthanized) 6 9 weeks Yes 6 100 Yes No (euthanized) 7 7
months Yes 6 (alive) 8 7 months Yes 6 (alive)
Example III--Tubular Scaffold with Enlarged End
[0188] A tubular scaffold such as is illustrated in FIGS. 15 A and
B comprising an enlarged end is implanted a gastro-esophageal
junction in a pig. FIG. 16 shows regenerated tissue that grew
around the scaffold inside the pig. FIG. 16 shows the regenerated
tissue extends over the enlarged end of the scaffold. FIG. 17
demonstrates that the inner diameter of the regenerated tissue
grown on the scaffold is approximately 2.2 cm.
Example IV--Other Gastrointestinal Applications
[0189] The process as outlined in Examples I and II is implemented
replacing gastrointestinal regions localized to the rectum. Results
are similar to the results outlined previously.
[0190] Having thus described several embodiments with respect to
aspects of the inventions, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
[0191] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0192] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified unless clearly
indicated to the contrary. Thus, as a non-limiting example, a
reference to "A and/or B," when used in conjunction with open-ended
language such as "comprising" can refer, in one embodiment, to A
without B (optionally including elements other than B); in another
embodiment, to B without A (optionally including elements other
than A); in yet another embodiment, to both A and B (optionally
including other elements); etc.
[0193] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0194] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0195] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," and the like are to
be understood to be open-ended, i.e., to mean including but not
limited to. Only the transitional phrases "consisting of" and
"consisting essentially of" shall be closed or semi-closed
transitional phrases, respectively, as set forth in the United
States Patent Office Manual of Patent Examining Procedures, Section
2111.03.
[0196] Use of ordinal terms such as "first," "second," "third,"
etc., in the claims to modify a claim element does not by itself
connote any priority, precedence, or order of one claim element
over another or the temporal order in which acts of a method are
performed, but are used merely as labels to distinguish one claim
element having a certain name from another element having a same
name (but for use of the ordinal term) to distinguish the claim
elements.
[0197] While the disclosure has been described in connection with
certain embodiments, it is to be understood that the disclosure is
not to be limited to the disclosed embodiments but, on the
contrary, is intended to cover various modifications and equivalent
arrangements included within the scope of the appended claims,
which scope is to be accorded the broadest interpretation so as to
encompass all such modifications and equivalent structures as is
permitted under the law.
* * * * *