HK1260555B - Systems and methods for producing gastrointestinal tissues - Google Patents

Description

Systems and methods for generating gastrointestinal tissue

Citation of Related Applications

This application claims priority to U.S. Provisional Patent Application No. 62/254,700, filed on November 12, 2015, and U.S. Provisional Patent Application No. 62/276,715, filed on January 8, 2016, the entire disclosures of which are hereby incorporated by reference into this application.

Technical Field

The present invention relates to engineered tissues for use in replacing or repairing damaged tissue.

Background Art

Engineered biological tissues for replacing or repairing damaged tissue are typically produced by seeding cells on a synthetic scaffold and exposing the cells to conditions that allow them to synthesize and secrete extracellular matrix components on the scaffold. Various techniques have been used to produce synthetic scaffolds, including nanofiber assembly, casting, printing, physical spraying (e.g., using a pump and syringe), electrospinning, electrostatic atomization, and other techniques for depositing one or more natural or synthetic polymers or fibers to form a scaffold of a suitable shape and size for transplantation into a subject (e.g., a human subject, e.g., an organ or region in which the engineered tissue is desired).

It is estimated that over 500,000 people worldwide are diagnosed with esophageal malignancies each year. Congenital malformations of the esophagus, such as esophageal atresia, have an average prevalence of 2.44 per 10,000 births. Chronicesophageal stricture following esophageal injury is also common. Although procedures such as endoscopic mucosal resection have been developed to minimize the extent of esophageal resection in early malignant disease, surgical esophagectomy remains the mainstay of treatment for many esophageal diseases. Traditionally, autologous conduits (e.g., stomach, small intestine, colon) are harvested and rerouted into the chest to restore gastrointestinal continuity. Many children with esophageal atresia or patients with traumatic or corrosive esophageal injury ultimately undergo similar reconstructions. However, these treatments are associated with significant morbidity and mortality.

Due to the complex structure of the esophagus, autologous conduits are commonly used. The layers of the esophagus are composed of stratified squamous epithelium, submucosa, outer circular muscle layer, and longitudinal muscle layer. These multilayered structures of the esophagus provide a barrier to oral ingestion and contamination from outside the gastrointestinal tract. Furthermore, this layer provides a physiological mechanism for propulsion and stress management during the passage of a bolus, or during swallowing or vomiting.

There is a need to provide structures that can support tissue regeneration and methods for making the structures.

Summary of the Invention

The present invention discloses an implementation method of a synthetic scaffold and its related systems that can produce gastrointestinal tissue (e.g., esophagus, stomach, intestine, colon or other hollow gastrointestinal tissue). In some embodiments, the scaffold provides guidance for the growth and regeneration of gastrointestinal tract (e.g., esophagus) tissue in a study subject. In some embodiments, the regenerated gastrointestinal tissue includes muscle tissue, nervous system tissue, or muscle tissue and nervous system tissue. In some embodiments, gastrointestinal tract (e.g., esophagus) tissue regenerates around the scaffold. In some embodiments, the scaffold is not fused into the final regenerated tissue (e.g., new esophageal tissue does not fuse the scaffold into the regenerated esophageal wall). Therefore, aspects of the present disclosure relate to guided tissue regeneration, wherein the scaffold provides support and/or signals that can promote host tissue regeneration, and there is no need for the scaffold to be fused into the regenerated tissue (e.g., without the need for the scaffold to provide structural or functional support in the final regenerated tissue).

In some embodiments, a gastrointestinal (eg, esophageal) scaffold comprises a biodegradable and/or resorbable material that is resorbed after gastrointestinal (eg, esophageal) tissue regeneration has begun (eg, after functional esophageal tissue has been regenerated).

In some embodiments, a gastrointestinal (eg, esophageal) scaffold includes one or more structures that can be used to assist in removing the scaffold after gastrointestinal (eg, esophageal) tissue regeneration has begun (eg, after functional esophageal tissue has been regenerated).

In some embodiments, prior to scaffold implantation, the scaffold is cellularized with one or more types of cells. In some embodiments, the cells are autologous cells. In some embodiments, the cells are progenitor cells or stem cells. In some embodiments, the cells are obtained from bone marrow, adipose tissue, esophageal tissue, or other suitable tissues. In some embodiments, the cells can be obtained from various allogeneic sources, including but not limited to sources such as amniotic fluid, umbilical cord blood, etc. In some embodiments, the cells are mesenchymal stem cells (MSCs).

In some embodiments, the scaffold is implanted in a position that can provide enough stem cell niches for tissue regeneration in the subject (e.g., an esophageal position or other gastrointestinal position that can provide a stem cell niche). In some embodiments, without wishing to be bound by theory, the scaffold and/or the cells provided on the scaffold help promote the growth and/or regeneration of gastrointestinal tissue from the host stem cells present at the scaffold implantation site.

In some aspects, the present disclosure relates to the discovery that esophageal tissue growth can be promoted or enhanced by the presence of a synthetic scaffold, wherein the synthetic scaffold can be modified to replace or repair the natural structural pattern and/or functional properties of a diseased or damaged tissue or organ without requiring the scaffold to be fully integrated into the final regenerated tissue. Therefore, in some aspects, the present disclosure provides a method for promoting or enhancing the growth of gastrointestinal tissue (e.g., esophagus), the method comprising: delivering a synthetic scaffold to a gastrointestinal region (e.g., esophagus) of a subject, wherein delivering the synthetic scaffold causes new gastrointestinal (e.g., esophageal) tissue to grow in that region of the subject. In some embodiments, the diseased or damaged gastrointestinal tissue is removed (e.g., surgically removed) before the scaffold is implanted. In some embodiments, the scaffold is an implanted, approximately tubular structure (e.g., sutured to the end of the remaining gastrointestinal tissue after the diseased or damaged tissue is removed). In some embodiments, the implanted scaffold is shorter than the removed tissue (e.g., 5-50% shorter). In some embodiments, when the tissue is attached (e.g., sutured) to both ends of the scaffold, the remaining gastrointestinal tissue adjacent to the implant site is stretched. In some embodiments, new gastrointestinal tract (such as esophagus) tissue is regenerated on the implanted scaffold, and does not completely fuse with the scaffold. In some embodiments, although the scaffold can be retained in the lumen (lumen) of the regenerated tissue, the wall of the regenerated tissue does not include the wall of the scaffold. 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.

In some embodiments, the growth of new gastrointestinal (eg, esophageal) tissue results in the formation of functional tissue (eg, a functional esophagus), which obviates the need for the continued presence of the functional scaffold.

In some embodiments, the synthetic scaffold is absorbable or dissolvable under physiological conditions. In some embodiments, after the functional esophagus is formed, the synthetic scaffold is removed from the subject's gastrointestinal tract region (eg, esophagus).

In some embodiments, the methods and compositions described herein can also be used to regenerate tracheal and/or bronchial tissue.

These and other aspects are described in greater detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is best understood by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, according to common practice, the various features in the drawings are not drawn to scale. Instead, the dimensions of the various features may be arbitrarily expanded or reduced for clarity.

FIG1A is a perspective view, partially in partial cross-section, of one embodiment of a synthetic stent disclosed herein;

FIG1B is a micrograph of the surface of a tube of one embodiment of a synthetic stent disclosed herein;

FIG1C is a side perspective view of a second embodiment of a synthetic stent disclosed herein;

FIG2 is a non-limiting depiction of a biofilm layer of the esophagus;

FIG3 shows a non-limiting example of regenerated esophageal tissue compared to corresponding native tissue;

FIG4A is a SEM micrograph of an outer surface region of one embodiment of a synthetic scaffold disclosed herein showing cell growth after 7 days of bioreaction at a magnification of 5000X;

FIG4B is a micrograph of an outer surface region of one embodiment of a synthetic scaffold disclosed herein showing cell growth after 7 days of bioreaction;

FIG5 is a flow chart of one embodiment of the regeneration method disclosed herein;

FIG6 is an overall research flow diagram of one embodiment of the method disclosed herein, including the generation and subsequent implantation of a cellularized scaffold;

FIG7A is a SEM image of an electrospun scaffold sample according to an embodiment disclosed herein, the SEM images being taken at magnifications of 1000X, 2000X, and 5000X, respectively;

FIG7B is a graphic representation of representative uniaxial mechanical testing loading of an electrospun scaffold before and after implantation according to the disclosure herein;

FIG7C is a table showing the uniaxial mechanical properties of a stent prepared according to an embodiment disclosed herein before and after implantation;

FIG8 is a graphic representation of flow cytometry of MSCs isolated from adipose tissue and expanded for up to 5 passages;

FIG9 is an overview of an implantation procedure according to one embodiment disclosed herein;

FIG10 is a timeline representation according to one embodiment of the method disclosed herein;

11A to 11C are photographs of regenerated tissue inside the regenerated tubular tissue at the esophageal resection site of a test subject at specified time intervals after surgery;

12A to 12E are photographs of regenerated tissue inside the regenerated tubular tissue at the esophageal resection site of a test subject at specified time intervals after surgery;

Figures 13A to F, I to K are photographs of histological analysis of esophageal tissue;

14A to K are photographs of histological analysis of tissue from porcine esophagus 2.5 months after implantation of one embodiment of the scaffold disclosed herein.

DETAILED DESCRIPTION

Aspects of the present 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 requiring the scaffold to be fully integrated into the regenerated tissue. Accordingly, in some embodiments, the present disclosure provides a method for promoting or enhancing the growth of gastrointestinal tissue (e.g., esophagus), the method comprising: delivering a synthetic scaffold to the gastrointestinal region (e.g., esophagus) of a subject, wherein delivering the synthetic scaffold results in the growth of new gastrointestinal (e.g., esophageal) tissue in the region of the subject.

The tissue that can be regenerated using the methods disclosed herein can be any gastrointestinal tissue, such as the esophagus, stomach, intestines, colon, rectum, or other hollow gastrointestinal tissue. In some aspects, the present disclosure is based, to some extent, in part on the surprising discovery that the methods described herein result in the regeneration of gastrointestinal tissue comprising muscle tissue, nervous system tissue, or both muscle tissue and nervous system tissue.

In some embodiments, the synthetic scaffold is resorbable or dissolvable under physiological conditions (e.g., over a period of time roughly corresponding to the time required for tissue regeneration). In some embodiments, at least a portion of the scaffold is resorbable or dissolvable under suitable physiological conditions.

In some embodiments, after the regenerated functional tissue (eg, esophagus or portion of esophagus) is formed, the synthetic scaffold is removed from the subject.

In some embodiments, the stent is designed to be easily retrievable, which can be achieved by having a) one or more reversible attachments that are easier to remove than sutures, such as to help separate the stent from surrounding tissue (e.g., esophagus) after tissue regeneration; and/or b) one or more features that can be used to help retrieve the stent, such as after the stent is separated from surrounding tissue (e.g., adjacent esophageal tissue).

Non-limiting examples of reversible attachments include mechanical devices (e.g., hooks and loops, connectors such as stents, or other detachable mechanical attachments) and/or chemomechanical devices (e.g., 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, the absorbable staples comprise, for example, a copolymer of polylactic acid and polyglycolide or any other absorbable material blend.

In some embodiments, surgical implantation and/or retrieval of the stent can be performed with thoracoscopic assistance.

Non-limiting examples of structural features that can help retrieve or remove the support (e.g., after the support is separated from the surrounding gastrointestinal tissue) include holes, indents, protrusions, or other structural features or any combination thereof, which are only located on the outer surface of the support. One or more of these structural features can be used to help grasp or hold the tool (e.g., grasper) that is being used to retrieve the support. In some embodiments, one or more of these structural features can only be located at one end of the support (e.g., located near the end of the subject's mouth). In some embodiments, one or more of these structural features can be located at both ends of the support or spread over the entire support length. In some embodiments, one or more of these structural features are only located on the outer surface of the support. In some embodiments, one or more of these structural features are located on the outer and inner surfaces of the support. In some embodiments, the support is reinforced at or near the position of one or more structural features for retrieving the support (e.g., making the support thicker and/or the support include a more solid material).

In some embodiments, the detached stent can be removed endoscopically via the airway lumen to the esophagus. In some embodiments, the detached stent can be removed surgically.

In some embodiments, the subject has diseased or damaged gastrointestinal tissue that needs to be replaced. In some embodiments, the subject is a human (e.g., a human patient).

In some embodiments, present disclosure provides the engineered stent that can be used for replacing or repairing esophagus or part esophagus.In some embodiments, esophageal stent as herein described can be used for promoting tissue regeneration (such as regenerated esophagus or part esophagus) to replace the tissue in study subject (such as people).For example, the study subject (such as people) suffering from some cancer (such as esophageal cancer) can benefit from the replacement of tissue or organ affected by cancer.Without wishing to be bound by any particular theory, synthetic stent as herein described promotes the growth of new tissue (such as esophageal tissue) in study subject, and therefore provides therapeutic benefit to study subject.

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 fuse the scaffold into the regenerated esophageal wall. In some embodiments, the scaffold is designed and manufactured to be absorbable and/or easily recyclable after esophageal tissue regeneration. In some embodiments, the scaffold is designed to be at least partially absorbable.

In some embodiments, the size and shape of the synthetic scaffold approximates the size and shape of the diseased or damaged gastrointestinal region (eg, the esophagus) to be replaced.

In some embodiments, the stent will have at least two layers. In certain embodiments, the stent can have an approximately tubular structure. Figure 1A shows a non-limiting embodiment of a stent 10, which has an approximately tubular body 12, the body 12 having an outer surface 14 and an inner surface 16. In some embodiments, a cross section of the stent 10 is roughly circular. In some embodiments, the cross section is roughly "D" shaped. However, stents 10 with other cross-sectional shapes can be used. Depending on the size of the corresponding tissue to be regenerated, the stent 10 can have any suitable length and diameter, depending on the size of the corresponding regenerated tissue. In some embodiments, in certain embodiments, the length of the stent 10 can be about 1-10 cm (e.g., 3-6 cm, e.g., about 4 cm), or in other embodiments, a length of 10-20 cm. However, it is conceivable that, depending on the specific application, the needs of the patient and/or the location of the gastrointestinal tract to be treated, a shorter or longer stent 10 can be used. In some embodiments, the stent 10 can have an inner diameter of 0.5 to 5 cm. However, depending on the specific application, the needs of the patient, and/or the location of the gastrointestinal tract requiring treatment, stents having smaller or larger inner diameters may be used.

In some embodiments, the length of the stent 10 can be shorter than the length of the replaced gastrointestinal tract (e.g., esophagus) region. In some embodiments, the length of the stent 10 is 50-95% (e.g., about 50-60%, 60-70%, 70-80%, 80-90%, about 80%, about 85%, about 90%, or about 95%) of the length of the replaced tissue. Without being bound by any theory, it is believed that certain regions of the relevant gastrointestinal region can respond positively to the traction applied to the relevant organ tissue, resulting in the generation of certain bio-organically mediated signals that can induce or promote tissue growth and differentiation.

In some embodiments, the length of the stent 10 can be longer than the length of the gastrointestinal tract (e.g., esophagus) region being replaced. In some embodiments, the length of the stent 10 is 100%-150% (e.g., about 100-110%, 110-120%, 120-130%, 130-140%, about 100%, about 105%, about 110%, or about 115%) of the length of the tissue being replaced. It is conceivable that the length of the stent will be the length necessary to effectively replace the affected area. In some cases, it is conceivable that the stent 10 will have a longer length than the gastrointestinal tract region being replaced to effectively position the stent and reduce or minimize trauma and ischemia in the affected area or related areas.

In some embodiments, the stent 10 can be composed of a single layer of synthetic material. However, it is also within the scope of the present disclosure that the stent 10 can include more than one layer of synthetic material.

Thus, in some embodiments, the synthetic scaffold 10 can be composed of multiple layers (e.g., 2 or more layers, such as 2, 3, 4, 5 or more layers). In some embodiments, one or more layers are made of the same material. In some embodiments, different layers can be made of different materials (e.g., different polymers and/or different polymer arrangements). The synthetic scaffold 10 as disclosed herein can comprise two or more different components that, when present, can be assembled to form the scaffold (e.g., before cellularization and/or implantation). In some embodiments, the synthetic scaffold 10 comprises two or more layers that are in contact with each other, such as by the synthetic technique used to manufacture the scaffold 10, causing the layers to be in contact with each other. In some embodiments, the scaffold 10 can be synthesized using a technique comprising multiple steps that results in two or more layers being brought together (e.g., applying a layer of electrospun material to a portion of a pre-made scaffold, such as a prior layer of electrospun material, a prior layer of electrospun material, one or more of the surfaces of different components incorporated into the scaffold (e.g., a braided tube or mesh).

In the embodiment shown in Figure 1A, the stent 10 includes at least one outer layer 18, which defines the outer surface 14 of the stent body 12. The stent 10 includes at least one additional inwardly oriented layer 20. In the embodiment shown, the at least one additional inwardly oriented layer 20 is in direct contact with an inner surface of the outer layer 18. If desired or required, the at least one additional inwardly oriented layer 20 can be configured to provide structural support to the associated stent body 12. In the embodiment depicted in Figure 1A, the at least one additional inwardly oriented layer 20 can be configured as a suitable mesh or braid that is circumferentially disposed around at least a portion of the longitudinal length of the stent body 12. In other embodiments, it is contemplated that the at least one additional inwardly oriented layer 20 can be comprised of a suitable polymer layer. In the embodiment shown in Figure 1A, the body 12 of the stent 10 includes at least one layer 22 positioned internal to the mesh 20 or braid layer 20.

Where desired or required, the stent 10 may have a generally uniform wall thickness. However, in some embodiments, the wall thickness may vary in specific areas of the body 12. In some embodiments, the wall thickness at one or both ends 24, 26 of the body 12 of the stent 10 is different from the wall thickness of the middle portion 28 (not shown) of the stent 10 (e.g., the wall thickness at the ends 24, 26 is thicker than the wall thickness of the middle portion 28). In some embodiments, when the stent is connected to the surrounding gastrointestinal tissue, the thicker wall areas are stronger and can provide better support for the sutures connected to one or both ends 24, 26 of the stent 10. The thick wall areas may also include discrete configurations that are conducive to suturing. Non-limiting examples of such discrete configurations include tubes, holes, etc.

In some embodiments, at least the outer surface 14 defined on the outer layer 18 can be composed of an electrospun polymer material. In some embodiments, it is contemplated that the outer oriented wall 18 can be composed of an electrospun polymer material. In some embodiments, the outer oriented electrospun layer can be in direct contact with a suitable woven material layer 20.

Fiber orientation

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. Additionally, fiber orientation can vary within each layer of a composite or sandwich scaffold.

In some embodiments, a scaffold with different porosities can be used. In some embodiments, one or more layers of the scaffold allow complete cell infiltration and uniform inoculation. In some embodiments, one or more layers of the scaffold can be constructed to prevent the infiltration of one or more cell types, for example, by densely packing the fibers. Since porosity varies with the change in fiber diameter, controlling fiber diameter can be used to change the scaffold porosity. Alternatively, a blend of different polymers can be electrospun together, and a polymer can be preferentially dissolved to increase the scaffold porosity. The properties of the fiber can be controlled to optimize the morphology of fiber diameter, fiber spacing or porosity, each fiber, such as the porosity or aspect ratio (aspectratio) of the fiber, the shape of the fiber is changed from circular to ribbon-like. In some embodiments, the mechanical properties of each fiber can be controlled or optimized, for example, by changing fiber composition and/or degradation rate.

In some embodiments, the electrospun fiber material can provide a contoured surface as shown in Figure 1B. In some embodiments, at least one electrospun layer in the scaffold 10 can be a polymer fiber material, such as polycarbonate polyurethane, which can be prepared by dissolving polycarbonate-polyurethane in a suitable solvent, such as hexafluoroisopropanol (HFIP), after spin drying.

The spacing and porosity of the electrospun fiber material can be such that cells seeded on the scaffold surface can adhere in a suspended, layered relationship between the fibers to allow the seeded cell material to form a sheet thereon as shown in Figures 4A and 4B.

Formation of synthetic scaffolds

Aspects of the present disclosure relate to methods for producing synthetic scaffolds. In some embodiments, a tubular synthetic scaffold (eg, a synthetic esophageal scaffold) can be produced on a mandrel (eg, by depositing material by electrostatic atomization and/or electrospinning).

In some embodiments, one or more layers of the synthetic stent provide structural support for the stent, giving the stent the desired mechanical properties. In some embodiments, a braided material (e.g., a braided tube, such as a nitinol braid, a PET braid, or a braid of other metal or non-metallic materials) can be inserted between two different layers of the stent to provide structural support. The pressure of the braided material (e.g., the force that the braid can exert on the next layer of material (e.g., the outer electrostatic spinning layer of the material)) can be controlled by controlling the pick count of the braid. In some embodiments, the braid can be coated (e.g., by dipping or other techniques) in an organic solvent to help the braid adhere to one or more other layers of the stent 10. In some embodiments, the length of the braid 20 does not extend to the end of the stent body 12. In some embodiments, one or both ends of the stent 10 are made of two or more layers of material and do not have a braided layer, and the middle portion 28 of the stent body 12 includes an additional braided layer.

In some embodiments, one or more layers of the synthetic stent provide a barrier in the stent, creating a separation (e.g., a relatively impermeable separation) between the interior space (e.g., the luminal space) and the exterior space. In some embodiments, the barrier can be an electrostatically atomized polyurethane (PU) layer.

In some embodiments, the various layers of stent 10 can include one or more polymers (e.g., polyethylene terephthalate (PET), PU, or blends thereof). In some embodiments, stent 10 can include a nitinol braid sandwiched between an inner PU layer (e.g., an inner PU layer electrostatically atomized or electrospun onto a mandrel) and an outer PU layer (e.g., an outer PU layer electrostatically atomized onto a braided material).

In certain embodiments, the stent 10 can be formed using a stent support or mandrel. In some embodiments, the stent support or mandrel can 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.

In certain embodiments, the material in the braid or mesh layer may be composed of an absorbable polymeric material.

Stent production-fiber materials

In some embodiments, one or more layers of support can be made of fibrous materials. In some embodiments, support comprises one or more types of fibers (such as nanofibers). In some embodiments, support comprises one or more types of natural fibers, one or more synthetic fibers, one or more polymers or any combination thereof. It should be noted that different materials (such as different fibers) can be used in the methods and compositions (i.e., support) described herein. In some embodiments, the material is biocompatible, so it can promote cell growth. In some embodiments, the material is permanent, semi-permanent (such as, after implanting in the host, continuing for several years) or rapidly degradable (such as, after implanting in the host, being absorbed in a few weeks or months).

In some embodiments, the scaffold comprises or consists of an electrospun material (e.g., microfibers or nanofibers). In some embodiments, the electrospun material comprises or consists of PET (polyethylene terephthalate (sometimes written as poly(ethylene terephthalate)). In some embodiments, the electrospun material comprises or consists of polyurethane (PU). In some embodiments, the electrospun material comprises or consists of PET and PU.

In some embodiments, the artificial scaffold can be composed of or include one or more of the following materials: elastomeric polymers (e.g., one or more polyurethanes (PU), such as polycarbonate and/or polyester), acrylamide polymers, nylon, and absorbable polysulfone polymers. In some embodiments, the scaffold can be composed of or include the following materials: polyethylene, polypropylene, poly(vinyl chloride), polymethyl methacrylate (and other acrylic resins), polystyrene and its copolymers (including ABA-type block copolymers), poly(vinylidene fluoride), poly(vinylidene chloride), polyvinyl alcohol in cross-linked and non-cross-linked forms and at varying degrees of hydrolysis (e.g., 87%-99.5%). In certain embodiments, the polymer can also include other compounds or methods that increase the hydrophilicity of the polymer. In certain embodiments, this can involve the introduction of compounds such as block copolymers based on ethylene oxide and propylene oxide. It is also contemplated that the hydrophilicity of the polymer can be increased by suitable plasma treatment, if desired or necessary.

In some embodiments, the scaffold can be composed of or comprise a block copolymer. In some embodiments, addition polymers such as polyvinylidene fluoride, syndiotactic polystyrene, copolymers of vinylidene fluoride and hexafluoropropylene, polyvinyl alcohol, polyvinyl acetate, such as polyacrylonitrile and its copolymers with acrylic acid and methacrylate, polystyrene, poly(vinyl chloride) and its various copolymers, poly(methyl methacrylate) and its various copolymers, amorphous addition polymers of PET (polyethylene terephthalate (sometimes written as 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 such as polyethylene and polypropylene can be solution spun or combined with any other material disclosed herein to produce a scaffold.

In some embodiments, one or more polymers may be modified after scaffold synthesis but prior to scaffold cellularization and/or implantation to reduce its hydrophobicity and/or increase its hydrophilicity.

In certain embodiments, the electrospun fibers may have a diameter of no more than 10 μm. In certain embodiments, the electrospun fibers may have a diameter of 3-10 microns. In certain embodiments, the electrospun fibers may have a diameter of 3-5 microns.

In certain embodiments, it is contemplated that the material in the braided layer may be made, in whole or in part, of a bioresorbable material such as PLGA. It is also contemplated that, in certain configurations, the braided material may be a carrier material or compound that promotes and/or supports 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

In some embodiments, a scaffold is produced that includes one or more layers (eg, PU and/or PET) produced by electrospinning. Electrospun materials can be used in a variety of applications, including as scaffolds for tissue engineering. Suitable methods for electrospinning polymers may include those described in Doshi and Reneker. Electrospinning process and application of electrospun fibers. J Electrostat. 1995; 35: 151–60.; Reneker DH, 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 arbitrarily oriented or aligned fibers with essentially any chemical composition and diameter ranging from the nanometer scale (eg, about 15 nm) to the micrometer scale (eg, about 10 microns).

In some embodiments, the electrospinning and electrostatic atomization 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 stream of polymer solution) and deposited onto a target surface. The target surface can be the surface of an electrostatic plate, the surface of a rotating drum (e.g., a mandrel), or other forms of collector surfaces that are both conductive and electrically grounded, such that the charged polymer solution moves toward the surface.

In some embodiments, the electric field used is typically on the order of several thousand volts, and the distance between the nozzle and the target surface is typically several centimeters or more. The solvent of the polymer solution evaporates (at least partially evaporates) between leaving the nozzle and arriving at the target surface. This causes the polymer fibers to be deposited on the surface. Typical fiber diameters vary from a few nanometers to a few microns. The movement of the target surface relative to the nozzle can affect the relative orientation of the fibers. For example, if the target surface is the surface of a rotating mandrel, the fibers will be arranged neatly (at least partially arranged) on the surface along the direction of rotation. In some cases, the nozzle can scan back and forth between the two ends of the rotating mandrel.

In some embodiments, the size and density of the polymer fibers, the degree of fiber alignment, and other physical properties of the electrospun material can be affected by factors including, but not limited to, the properties of the polymer solution, the size of the nozzle, the electric field, the distance between the nozzle and the target surface, the properties of the target surface, the relative motion between the nozzle and the target surface (e.g., distance and/or velocity), and other factors that can affect solvent evaporation and polymer deposition.

Electrospinning and electrostatic atomization processes can be used to produce interconnected polymer fiber scaffolds (eg, hollow composite scaffolds) on a mandrel.

Support/mandrel

In some embodiments, the stent 10 (e.g., a stent having two or more layers) can be produced using a support member (e.g., a solid or hollow support member) on which the stent 10 can be formed. For example, the support member can be an electrospinning collector, such as a core shaft, or a tube, or a support member of any other shape. It will be appreciated that the support member can have any size or shape. However, in some embodiments, the size and shape of the support member are designed to produce a stent that will support an artificial tissue of the same or similar size as the gastrointestinal tissue (or a portion thereof) that is replaced or supplemented in the host. It will be appreciated that the mandrel used for electrospinning should have a conductive surface. In some embodiments, the electrospinning mandrel is made of a conductive material (e.g., a conductive material comprising one or more metals). However, in some embodiments, the electrospinning mandrel includes a conductive coating (e.g., a conductive coating comprising one or more metals) covering a non-conductive central support member.

We have surprisingly discovered that positioning a suitable braiding material so as to be integrated into a formed stent 10 near the surface of a mandrel can serve to help facilitate removal of the formed stent 10 from contact with the mandrel.

Bracket performance

It should be understood that aspects of the present application can be used for enhancing the physical properties and functional properties of any support (for example, support based on electrospinning and/or electrospraying fibers). In some embodiments, one or more support components can be thin slices, cylinders, thick ribs, solid blocks, branched networks, etc. or any combination thereof with different sizes. In some embodiments, the size of the support of one-piece and/or assembly is similar or identical to the size of the tissue or organ replaced. In some embodiments, each component or layer of support has less size. For example, the thickness of the nanofiber layer can be from several nanometers to 100 nanometers, 1-1000 micron or even several millimeters. However, in some embodiments, the size of one or more support components can be approximately from 1mm to 50cm. However, as described herein, larger, smaller or medium-sized structures can be manufactured.

In some embodiments, the support is formed into a tubular structure, which can be seeded with cells to form a tubular tissue region (such as the esophagus or other tubular regions). It is understood that the tubular region can be a cylinder with a uniform diameter. However, in some embodiments, the tubular region can have any suitable tubular shape (for example, including parts with different diameters along the length direction of the tubular region). The tubular region can also include a branch or a series of branches. In some embodiments, a tubular support with one end opening, two ends opening or multiple ends opening (for example, in the case of a branched support) is prepared. However, the tubular support can be closed at one end, both ends or all ends, and the various aspects of the present invention are not limited in this respect. It is also understood that since the present invention is not limited in this respect, the aspects of the present invention can be used to produce the support of any type or organ (including hollow and solid organs). In some embodiments, the various aspects of the present invention can be used to enhance the stability of the support or other structures, and the other structures include two or more fiber regions or fiber layers (such as electrospinning nanofibers) that are not physically connected.

In some embodiments, support is designed to have porous surface, and described porous surface has the hole of diameter in the range of about 10nm to about 100 microns, can promote cellularization.In some embodiments, the average diameter of hole is less than 50 microns, less than 40 microns, less than 30 microns, less than 20 microns or less than 10 microns (for example, about 5 microns, about 10 microns or about 15 microns).In some embodiments, the average diameter of hole is at 20-40 micron.In some embodiments, pore size is selected to prevent or reduce the immune response or other unwanted host response in study subject.Aperture can be estimated using computing technology and/or experimental technique (for example, using mercury porosimetry (Porosimetry)).But, it is to be understood that the porous surface of described support also can comprise the hole of other sizes.

In some embodiments, the surface layer of the scaffold is synthesized using fibers, the fibers comprising one or more soluble particles that can be dissolved during or after synthesis (e.g., by exposure to a solvent, an aqueous solution, such as water or a buffer), leaving pores the size of the soluble particles. In some embodiments, the particles are included in a polymer mixture that is pumped into the nozzle of an electrospinning device. As a result, the particles are deposited together with the fibers. In some embodiments, the electrospinning process is configured to deposit thick fibers (e.g., having an average diameter of several microns, an average diameter of about 10 μm and greater). In some embodiments, if the fibers are deposited in a dense pattern, one or more fibers will merge before solidification to form a larger microstructure (e.g., 10-100 microns thick or thicker). In some embodiments, these microstructures can be wrapped around two or more fiber layers and/or parts (e.g., fibers) of two or more different components from a scaffold, thereby increasing the mechanical integrity of the scaffold. In some embodiments, when such microstructures are formed (e.g., by electrospinning as described herein) at one or more stages of the scaffold synthesis process (e.g., a stage used to connect two or more layers and/or components), the surface of the microstructure can be treated (e.g., etched, or made porous with soluble particles as described herein) to provide a surface suitable for cellularization.

In some embodiments, the amount of flexible scaffold material (e.g., slack) between two or more structural components (e.g., rings), between structural members of a single continuous structural component (e.g., arcuate members), and/or between braided support materials can be used to determine the mechanical properties of the composite scaffold (e.g., tensile strength, elongation, rotation, compression, range of motion, bending, resistance, compliance, degrees of freedom, elasticity, or any other mechanical property, or combinations thereof).

In certain embodiments, the scaffold 10 may further comprise a cell sheath derived from cells seeded onto the outer surface of the scaffold during incubation. The cell sheath adheres to and overlaps the outer surface of the scaffold. It is envisioned that the majority of cells present in the cell sheath will be attached to the outermost surface of the outer surface and will span the pores defined herein to form a continuous or substantially continuous surface.

In some embodiments, the cell sheath can have a thickness that is enough to provide structural integrity for the sheath layer. In some embodiments, the cell sheath will be made up of a plurality of cells contacted with the support outer surface, and the plurality of cells are enough to guide the regenerative cells to contact with the sheath to produce a tissue wall covering the sheath but not merging with it. In some embodiments, the sheath can be made up of a film (lining) with an average thickness between 1 and 100 cells. In some embodiments, the film 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.

The support 10 with the relevant cell sheath provides a removable insertable device, which can be placed on a suitable gastrointestinal tract resection site. The support 10 with the relevant cell sheath in contact with it can be transported to the desired resection site and implanted. In some embodiments, the support 10 is configured to remove from the implantation site after the suitable regeneration of the excised organ. In some embodiments, the support of the removal will comprise some or all of the cell sheaths connected thereto.

The application also discloses various embodiments of the regeneration method of the tubular organ such as gastrointestinal organ. In certain embodiments, method 100 includes a resection step, and the resection step includes a part for the tubular organ in the subject, as shown in reference numeral 110. The organ to be resected can be a tubular organ of the gastrointestinal tract that is damaged or impaired due to disease injury, trauma or congenital condition. In certain embodiments, the non-limiting example of suitable organ includes one in esophagus, rectum etc. In certain embodiments, suitable organ includes at least one in esophagus, small intestine, colon and rectum.

Resection can be achieved by any suitable surgical procedure and produces a resected organ portion that remains connected to the gastrointestinal tract and retained within the subject after resection.In certain embodiments, the resection procedure can produce suitable resection margins.

After the resection is complete, the synthetic scaffold is implanted into the resection site, as indicated by reference numeral 120. In certain embodiments, implantation may include the step of connecting the respective ends of the resected organ retained within the subject to the respective ends of the synthetic scaffold, such that the synthetic scaffold and the resected organ are suitably connected between the respective components. This may be accomplished using one or more sutures, bio-organic tissue glue, and the like.

In certain embodiments, the implanted synthetic scaffold can be a tubular member having an outer polymer surface and a cellularized sheath covering at least a portion of the outer polymer surface. Various embodiments of synthetic scaffolds have been discussed and can be used and utilized in the methods disclosed herein. In certain embodiments, the synthetic scaffold will include a first end, a second end opposite the first end, an outer polymer surface positioned between the first end and the second end, and a cellularized sheath covering at least a portion of the outer polymer surface. In certain embodiments, the implanting step can be a step of placing at least a portion of the cellularized sheath in direct contact with at least one resected edge of the resected organ portion.

In certain embodiments, the methods disclosed herein further comprise the step of maintaining the synthetic scaffold at the resection site for a sufficient period of time to achieve guided tissue growth along the synthetic scaffold, as indicated by reference numeral 130. In certain embodiments, the guided tissue growth originates from tissue present in the portion of the organ after resection retained in the subject and in contact therewith. In certain embodiments, the guided tissue growth will be adjacent to the relevant region of the organ after resection. In certain embodiments, the guided tissue growth will exhibit differentiated tissue. In certain embodiments, at a position outside the cell sheath, the guided tissue growth is parallel to the outer surface of the cell sheath. In certain embodiments, the guided tissue growth originates from tissue present in the portion of the organ after resection retained in the subject and in contact therewith and will be adjacent to the relevant region of the organ after resection. The guided tissue growth will exhibit differentiated tissue growth and may be parallel to the outer surface of the cell sheath at a position outside the cell sheath.

After the guided tissue growth has been achieved, the method 100 disclosed herein can include a step of removing the synthetic scaffold, as indicated by reference numeral 140. In certain embodiments, the removal step is performed in a manner such that the guided tissue growth remains in contact with the resected portion of the tubular organ remaining in the subject. In certain embodiments, the removal process can include endoscopically removing the synthetic scaffold from within the guided tissue growth.

In certain embodiments, the synthetic scaffold may be constructed in whole or in part from a bioabsorbable polymer material. In such cases, the methods disclosed herein may include the steps of maintaining contact between the synthetic scaffold and the resection edge for a period of time sufficient to achieve guided tissue growth along the synthetic scaffold, such that at least a portion of the synthetic scaffold is absorbed at the resection site for a period of time sufficient to achieve guided tissue growth along the synthetic scaffold. In certain embodiments where the scaffold is comprised entirely of bioabsorbable material, the scaffold will be constructed to maintain structural integrity during the guided tissue growth. In certain embodiments where the synthetic scaffold is comprised of bioabsorbable material in selected areas, it is contemplated that after the guided tissue growth is achieved, the remainder of the scaffold may be removed in appropriate steps.

The guided tissue growth can be monitored by suitable means. In certain embodiments, the tissue growth can be monitored endoscopically.

In certain embodiments of the methods disclosed herein, the method may further comprise the steps of introducing cell material onto the polymer surface of the synthetic scaffold and allowing the cell material to grow to form a cell sheath, wherein the introducing step and the allowing the cell material to grow step occur before the cutting step.

In certain embodiments, the synthetic scaffold used in the methods disclosed herein is a tubular member whose outer surface comprises a spun polymer fiber. In certain embodiments, the spun fiber can be obtained by electrostatic spinning by suitable methods such as those described in the present disclosure. In certain embodiments, the cellularized sheath spans at least a portion of the electrostatically spun fiber located outside. The cellularized sheath can be composed of cell material, and the cell material includes one of mesenchymal cells, stem cells, and pluripotent cells. The cell material can be derived from the subject's own body or can be derived from an allogeneic body.

Without being bound by any theory, it is believed that the implantation of various synthetic scaffolds such as those disclosed herein, particularly those seeded with a covering cell sheath, promotes the growth, regeneration, and differentiation of tissues of the subject in contact with or adjacent to the position of the implanted synthetic scaffold. The regenerated tissue that grows is guided by the synthetic scaffold and the associated sheath to produce a tubular cell body that is integrally connected to the resected end of the remaining tubular organ and expands outward to encapsulate the synthetic scaffold and the associated cell sheath layer. It is believed that the scaffold and the associated cell sheath layer can promote or stimulate the regenerative growth of the resected tissue while minimizing tissue rejection. It is also believed that the presence of the cell sheath layer can reduce or minimize the penetration of the regenerated tissue into the sheath layer during growth and differentiation. In certain embodiments, tissue regeneration proceeds from each end toward the middle. Once the regenerated tissue is in place, the synthetic scaffold can be removed. In certain embodiments, once the synthetic scaffold is removed, the regenerated tissue structure will lack the inner epithelial layer. As shown in Figures 11A, 11B, and 11C, after the scaffold is removed, regeneration of the inner epithelial layer has been observed 2 months and 3 months after the scaffold is removed, respectively.

To further understand the present disclosure, reference is made to the following examples, which are included for illustrative purposes and are to be considered illustrative of the inventive content of the present disclosure and claims.

Example

Example I: Esophageal Stent

As shown in Figure 1A, a synthetic esophageal stent comprising three layers of material is prepared. A first layer of polyurethane (PU) is deposited onto a metal core shaft by electrostatic atomization, and then a braided material is deposited on the first PU layer. A second PU layer is then deposited by electrostatic spinning. The formed stent is then removed from the core shaft. Each stent defines a tubular structure having a wall comprising three layers (an inner electrostatic atomization layer, an outer electrostatic spinning layer, and a braided fabric layer sandwiched between the inner electrostatic atomization layer and the outer electrostatic spinning layer). The physical dimensions of the stent were determined by scanning electron microscopy (SEM). The average stent wall thickness was approximately 500 microns. A non-limiting SEM view of a cross section of the wall is shown in Figure 1B. A non-limiting view of a cross section of the tubular stent is shown in Figure 1C. The figure shows that the cross section is roughly "D" shaped. This can be obtained by using a core shaft with a "D" shaped cross section.

The outer electrospun layer is a layer of polymer fibers defining pores. The average fiber diameter in the outer layer is about 3-6 microns. The average pore size is about 15-20 microns, and the median pore size is about 25-45 microns.

The scaffold may be attached to a support that is rotatable in a bath of liquid medium within the bioreactor chamber. The rotation mechanism may include a magnetic actuator that allows the support with the attached scaffold to rotate about its longitudinal axis within the bath of liquid medium.

In some embodiments, the support of the present invention can be inoculated with cells (such as MSCs or other stem cells) by depositing the cell solution on the support outer surface. The support is then rotated about one week in the liquid culture medium bath in the bioreactor chamber, and the support of inoculation is hatched in the liquid culture medium supporting cell growth. The support obtained comprises a cell sheath overlapping with the outer surface of the support. In some embodiments, the cell sheath can have a thickness that is enough to provide structural integrity for the sheath layer. In some embodiments, the cell sheath will be made up of a plurality of cells contacted with the support outer surface, and the plurality of cells are enough to guide the regenerative cells to contact with the sheath to produce the tissue wall covering the sheath but not merging with it. In some embodiments, the sheath can be made up of a film (lining) with an average thickness between 1 and 100 cells. In certain embodiments, the membrane can have a thickness of 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, or between 70 and 90 cells.

The scaffold 10 with the seeded cell sheath can be implanted into the resection site and allowed to sit in place. It is conceivable that the seeded cells present in the sheath can continue to grow after implantation. In this case, the seeded cells present in the sheath will maintain and support a structure separate from and in series with the regenerated tissue at the implantation site.

The corresponding stents were then implanted into the esophagus of the pigs. Approximately 5 cm of the esophagus was removed and replaced with the stent portion, which was sutured to the end of the remaining esophageal tissue in the subjects.

Regeneration of esophageal tissue was monitored endoscopically for several weeks.

The esophagus is a long muscle tube with a neck, chest and abdomen. FIG2 is a diagram showing a cross-section of the esophagus in the human body. In adults, the esophagus can be 18 to 25 centimeters long. The esophageal wall is composed of a mixture of striated muscle at the top, smooth muscle at the bottom, and both striated and smooth muscle at the middle. Therefore, in some embodiments herein, a multilayer synthetic scaffold is provided that can promote the repair and regeneration of esophageal tissue corresponding to a natural esophageal tissue layer, with two or more layers.

Fig. 3 shows the dyeing cross section of natural esophageal tissue and regenerated esophageal tissue after 1-2 week of esophageal stent implantation in pig.This cross section shows the regeneration of basically all esophageal tissue layers (comprising different muscle layers and glandular layers).Further analysis of regenerated tissue shows that the stent itself is not fused to the regenerated esophageal wall.Stent still exists in esophagus, but stent seems to serve as a guide to stimulate esophageal regeneration, rather than becoming an integral part of regenerating esophagus.

Example II: Esophageal Implantation

As shown in FIG1A , a three-layer synthetic esophageal stent was prepared with an outer electrospun polycarbonate-polyurethane layer deposited from a 12% w/v polycarbonate-polyurethane solution dissolved in hexafluoroisopropanol (HFIP, DuPont, Wilmington, DE, USA). The electrospinning apparatus used was purchased from IME Technologies, Geldrop, Netherlands. The electrospun fibers were collected on a target aluminum mandrel rotating at 800 rpm and positioned 22 mm from the syringe tip to deposit isotropic fibers, resulting in a stent with an average wall thickness of 500 μm. The stent was dried in vacuo to remove residual solvent. The stent was then plasma-treated using a low-pressure plasma system (Diener Tetra 150-LF-PC-D) with two co-current cycles of ethylene and oxygen. The stent was gamma-sterilized (STERIS, Northborough, MA). The gamma radiation dose used ranged from 25 to 35 kGy.

The tubular object formed was a polymer scaffold composed of electrospun polyurethane with a uniform outer diameter (OD) of 22 mm and a length of 11 cm.

Electrospun micromorphology was analyzed using a scanning electron microscope (Zeiss-EVO MA10). Scaffold samples were sputter-coated with platinum ^and palladium films for two minutes using a sputter coater (Cressington-208HR, TED PELLA, Inc., Redding, CA) at a pressure of 8 x 10-2 mbar and a potential of 300 V. Porosity was calculated gravimetrically. Porosity ε is defined as the apparent density ρAPP of the fiber mat and the bulk density ρPU of the polymer, where ε = 1 - ρAPP/ρPU. The apparent scaffold density ρAPP is measured as the mass-to-volume ratio on a 10 mm dry plate: ρAPP = Mass/VPU. Pore size measurements were performed using a mercury porosimetry system (Micromeritics AutoPore IV). Tensile tests were performed on 10 mm × 40 mm samples mounted on an electromechanical load frame (Instron 5943 apparatus) using a 1 kN load cell according to ASTM D638 guidelines. All samples were tested using the same parameters: a data acquisition rate of 100 Hz, a gauge length of 30 mm, and a test speed of 1 mm/s. As shown in Figure 7A, scanning electron micrographs at increased magnification reveal the isotropic fiber alignment of the electrospun scaffolds. The smooth surface and isotropic nature of the fibers ensure uniform strength and elasticity in all directions.

Tensile tests under uniaxial mechanical loading were performed on three stents before and after implantation (Figure 7B), all showing similar in vivo load values. The consistency of the six samples under in vivo loading indicates that the stent has very low variability during manufacturing and after in vivo implantation (Figures 7B and 7C). The mean (±SD) tensile strain of the six stents ranged from 119.5±1.61mm to 124.5±3.44mm. In terms of tensile strain at break, the tensile strain of the samples reached 397.38%±5.52% before implantation and 408.61%±17.64% after implantation. Strain values above 400% indicate the reliability of the preparation process and relative in vivo stability. The tensile stress at failure before and after stent implantation was 7.25±0.59MPa and 4.43±0.77MPa, respectively. Thus, the Young’s modulus of the samples before implantation was greater than that after implantation, even though the elasticity of the two groups with respect to in vivo strain was comparable (Figure 7B, Figure 7C). The load at failure followed the same trend as the Young’s modulus, i.e., the load at failure values before implantation were greater than those after implantation.

Autologous porcine adipose-derived mesenchymal stem cells (aMSCs) were isolated and characterized from eight pigs following open fat biopsy. Eight Yucatan mini-pigs were anesthetized and skin prepared with chlorhexidine before sterile, open adipose tissue biopsy from the flank wall. A 5-cm incision was made adjacent to the linea alba, and hemostasis was achieved using electrocautery. Approximately 30–50 g of adipose tissue was isolated and transferred to a 50 mL conical tube containing α-minimum essential medium (MEM)/glutamax medium (Thermo Fisher Scientific, Waltham, MA) and 1% penicillin/streptomycin (Thermo Fisher Scientific).

20-60 g of abdominal adipose tissue was excised from each anesthetized Yucatan minipig (50-60 kg body weight). The tissue samples were rinsed three times in α-minimum essential medium (MEM)/glutamax medium (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). The rinsed tissue was trimmed to remove lymph nodes and blood vessels and minced into fragments less than 5 mm. The tissue fragments were dissociated for 55 minutes in digestion buffer (300 IU/mL type _II collagenase, 0.1% bovine serum albumin (7.5%, fraction V), 1% penicillin/streptomycin, α-MEM/glutamax) at 37° C., 5% CO 2. After dissociation was stopped in complete growth medium (Stem XVivo medium (R&D Systems, Minneapolis, Minnesota) and 1% penicillin/streptomycin), the cells were centrifuged at 1500 rpm for 15 minutes. The cell mass was resuspended in 5 mL of growth medium and filtered through a 70 μm filter. The cell filtrate was centrifuged at 1500 rpm for 15 minutes and the cell mass was resuspended in 5 mL of growth medium and inoculated with cells (3 g of isolated adipose tissue/each T75 flask containing 20 mL of growth medium) according to tissue weight.

The cells were washed twice with PBS (Thermo Fisher Scientific) without calcium or magnesium and dissociated with TrypLe (Thermo Fisher Scientific). Dissociation was terminated with growth medium and the cells were centrifuged at 1000 rpm for 5 minutes. The cell pellet was resuspended in 1% bovine serum albumin diluted with PBS. An aliquot containing 1 million cells was incubated in the antibody for 30 minutes at 4°C in the dark (see Supplementary Table 1). The labeled cells were washed three times in buffer and, if necessary, used with secondary antibodies (Life Technologies, Carlsbad, CA) for 30 minutes at 4°C in the dark. After three more washes, the cell suspension was placed in a 96-well plate for flow cytometric analysis (Guava easyCyte HT, EMD Millipore, Billerica, MA). Based on the measurement of viability, the events representing live cells were gated for forward scatter and side scatter values (ViaCount, EMD Millipore). Cell type analysis was performed using fluorescence compensation events for unstained samples and samples stained with isotype controls. The acquired data were exported and analyzed using stand-alone software (FlowJo version 10, FlowJo, LLC, Ashland, OR).

To assess colony formation, adipocytes were isolated by the method described above, crushed into a single cell suspension, and diluted to a growth medium of 10 cells/mL. 100 μL of this cell suspension was added to each well of a 96-well plate (Corning, Corning, New York), and the cell count was visually inspected the next day. After 5-7 days, visible cell colonies formed, and the culture medium was replaced every 3 days until the colony contained at least 50 cells. The holes with colonies were counted and expressed as a percentage of the total number of pores.

The pluripotency of the adipocyte separated is to be measured by their ability of being subjected to chemical induction to form fat and osteogenesis.Cell is seeded in 6 hole tissue culture plates respectively, cultivates in complete growth medium, and allows cell to grow to 60% confluence for cell fat differentiation or 100% confluence for cell osteogenic differentiation respectively.When reaching confluence, culture medium is replaced by adipogenic differentiation culture medium or osteogenic differentiation culture medium (CCM007, R＆D Systems, Minneapolis, Minnesota).Every 2 days, culture medium is changed once, until cultivate 14 days.Carry out oil red O (American MasterTech, Lottie, California) dyeing to identify fat to the cell that is cultured in adipogenic differentiation culture medium, and carry out alizarin red (EMD Millipore) dyeing to identify calcium deposition to the cell that is cultured in osteogenic differentiation culture medium.

Glucose and lactate concentrations were measured in the conditioned medium of the bioreactor at the time of inoculation and 2, 5, and 7 days after inoculation (iSTAT, Abbott, Princeton, NJ).

Cell supernatants were analyzed to determine the production of porcine cytokines and growth factors, either by multiplex analysis on the Luminex 200 platform or by ELISA analysis using commercially available kits at the University of Minnesota Cytokine Reference Laboratory according to the manufacturer's instructions. A 13-plex porcine bead plate (EMD Millipore) was used to determine porcine VEGF, GM-CSF, IL-1ra, IL-6, and IL-8 levels. Standard curves were interpolated from the standard curves generated for each plate using BioPlex software (BioRad, Hercules, CA) for the Luminex platform or using the microplate reader management software for ELISA plates read on a BioRad 550 plate reader. All samples were tested in duplicate.

The cells were washed in PBS and fixed with 10% formalin for 15 minutes at room temperature. The cells were gently washed three times in PBS containing 0.1% Triton X-100 (PBS-T) and incubated in 10% normal goat serum (Vector) diluted in PBS-T for 1 hour at room temperature. Rabbit anti-nestin antibody (Biolegend, 1:100) was diluted in 10% normal goat serum and PBS-T and incubated overnight at 4°C. The cells were rinsed twice in PBS-T and incubated in fluorescently labeled goat anti-rabbit antibody (Alexa Fluor 594, Thermo Fisher Scientific) for 1 hour at room temperature. The cells were rinsed twice and counterstained with 4′,6-diamidino-2-phenylindole (DAPI).

After counterstaining for 48 hours at 37°C, cells were washed twice in phosphate-buffered saline (PBS) containing calcium and magnesium (Thermo Fisher Scientific) and replaced with fresh growth medium. Thereafter, the medium was changed every 2 days until the flasks reached 70%-80% confluence. At the time of passage, cells were dissociated (TrypLe, Thermo Fisher Scientific), counted (Countess, Thermo Fisher Scientific), and reseeded into T175 flasks at 200,000 cells/flask.

Each 11cm long scaffold is placed in a bioreactor and inoculated with 32 million cells in a growth medium supplemented with 0.1875% sodium bicarbonate, MEM (Lonza) and 1.19mg/mL bovine collagen (Organogenesis) in 0.01M hydrochloric acid (viability>70%, trypan blue exclusion method, Countess, Thermo Fisher Scientific). The cells are incubated at 37°C, 5% CO2 for 5 minutes, and then 200mL of growth medium is slowly added to _the bioreactor. The bioreactor is incubated for 7-8 days before the scaffold is implanted. The culture medium is replaced every 2 days and various analyses are performed as described below.

Porcine aMSCs were seeded onto characterized scaffolds and subsequently incubated in bioreactors. The seeded scaffolds were then implanted into Yucatan miniature pigs following esophagectomy until scaffold removal 3 weeks later (Figure 6) and replicated with antibodies against porcine CD44, CD73, CD90, CD105, and CD146, known MSC markers, for positive staining, and CD14, CD45, CD106, CD271, and SLA Class II DR for negative staining. Over 95% of the cultured cells stained positively for nestin and αSMA, indicating that stem cell properties were maintained during culture. Multipotency was determined by the ability of chemically induced porcine MSC isolates to form adipocytes and osteoblasts, respectively. These aMSCs were routinely expanded and characterized from passages 1 to 5, exhibiting consistent phenotypic and functional characteristics.

Porcine aMSCs at passage 2 were seeded onto polymer scaffolds and incubated in a bioreactor at 37°C for 7 days (± 1 day). Cytokine and growth factor levels were measured by enzyme-linked immunosorbent assay (ELISA) to determine whether the seeded aMSCs cultured on the scaffolds secreted factors that may contribute to angiogenesis and immunomodulation. Cellular secretions of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, and IL-1RA were detected at only moderate to moderate levels in the conditioned medium (Figure 4A). However, the other cytokines, TNF-α, IL-1α, IL-1β, INF-γ, IL-10, IL-12, IL-18, platelet-derived growth factor (PDGF), and regulated chemokine expressed on activated normal T cells (RANTES), were not detected.

At the end of the 7-day culture period, punch biopsies were taken from sections of the seeded grafts to assess cell health and scaffold penetration. Cell health was assessed by immunofluorescence staining with calcein (live cells) and ethidium bromide (dead cells). Cell permeability of the scaffolds was assessed using ethidium bromide for cell identification. The predominance of calcein staining in the biopsy specimens indicated a viable cell population attached to the scaffold. In cross-sections of the scaffold biopsies, the majority of cells were attached to the scaffold surface, although there was some evidence of cell proliferation and growth within the scaffold. During bioreactor incubation, graft metabolic activity was measured every 48 hours to determine glucose uptake and lactate production. Measurements of conditioned medium consistently demonstrated decreasing glucose levels and increasing lactate levels over time, two indicators of sustained metabolic cell growth. In addition, cell expansion over the 7-day bioreactor period was quantified using total DNA content, which increased several-fold during the bioreactor cell seeding process. Further characterization of the cell phenotype on the scaffold after 7 days of culture showed that the cells continued to express α-smooth muscle actin (αSMA) and nestin.

After endotracheal intubation and induction of general anesthesia, the animals were placed in the left lateral decubitus position. Hair was cut, skin was prepared with chlorhexidine or povidone-iodine, and the animals were covered with sterile cloth. Standard right thoracotomy was performed at the height of the fourth intercostal space of each animal, and the chest cavity was entered. Single lung ventilation was achieved by using a double-lumen endotracheal tube. A 4-4.5 cm esophageal segment located in the mid-thoracic segment (behind the right hilum) was moved circumferentially and removed to produce a 6 cm defect (tissue contraction at the proximal and distal ends). The inoculated stent (6 cm long) was then implanted and anastomosed to the proximal and distal ends of the esophagus using polydioxanone (PDS, Johnson & Johnson Ethicon, Somerville, New Jersey) absorbable sutures. After implantation, a commercially available esophageal stent (WallFlex M00516740, Boston Scientific Corporation) was placed under direct endoscopic guidance (Storz Video Gastroscope Silver Scope 9.3MM x 110CM, Tuttlingen, Germany). Stent placement was performed under endoscopic and surgical visualization. The esophageal stent was secured to the normal esophageal tissue with absorbable sutures at the proximal and distal stent locations.

Postoperatively, animals were supported with gastrostomy feeding and maintained on a liquid diet via feeding tube for 2 weeks, a mashed diet for an additional 2 weeks, and then allowed to eat an oral diet of solid food before continuing on the study.

Approximately 21 days after stent implantation, the stent was retrieved endoscopically and platelet-rich plasma (PRP) gel infused with aMSCs was used to enhance the healing process of the new esophageal conduit. After PRP was applied, a new fully covered esophageal stent (WallFlex ^™ , 12 cm long x 23 mm outer diameter, Boston Scientific) was placed around the implanted area to prevent stricture formation and maintain the anatomical structure during regeneration. Animals were sedated and esophageal anastomosis and esophageal stent exchange were assessed every two weeks to allow direct visualization of esophageal regeneration progress. Subsequent endoscopic observations were performed using a Storz Video Gastroscope SilverScope 9.3MM x 110CM, Tuttlingen, Germany.

The progress of regeneration was also assessed by endoscopic examination. After the stent was removed, the implantation area was observed endoscopically for nearly 3-4 weeks; two representative animals are shown (Figures 10, 11A, 11B and 11C). After 3-4 weeks of implantation, only partial regeneration of the mucosal layer was completed. However, before the two layers merged, initial ridges formed at the proximal and distal ends of the mucosal layer, and complete mucosal regeneration indicated that the esophageal healing process continued over time. The early reconstruction of esophageal continuity and integrity, and the subsequent growth of the submucosa from the two opposite sides of the resected portion were consistent in all eight animals; two animals were maintained until 8 and 9 months after surgery, and these two animals were free of the esophageal stent and had no signs of stenosis within 2 and 3 months, respectively, and they had persistent oral intake and significant weight gain.

To determine the histological similarity of regenerated and native esophageal tissue morphology. Tissue samples were removed from representative porcine esophagus 2.5 months after surgery, and tissues including the surgical site and adjacent distal and proximal ends were histologically examined (Figure 13A, dotted box indicates histological analysis specimen). Representative images of tissue sections stained with hematoxylin-eosin (Figures 13B and 13D) and Masson's trichrome staining (Figures 13C and 13E) showed intact multilayered esophageal epithelial cells (esophageal epithelia), submucosa, and normal inner muscular layer morphology.

Figure 14A-Figure 14K have described the representative immunohistochemical analysis of regeneration area, and it has described the histological analysis of pig esophageal tissue after implanting cellularized scaffold as described herein for 2.5 months.Figure 14A has described the macroscopic image of esophagus (near left end suture) after resection.Tissue sample is excised to include surgical site, and endoscopic monitoring tissue sample and adjacent distal and proximal tissue (dashed line frame) are used for histological examination.Figure 14B-Figure 14E is the representative picture of the tissue section of hematoxylin-eosin (Figure 14B and Figure 14D) staining and Masson's trichrome staining (Figure 14C and Figure 14E). Representative immunohistochemical analysis demonstrated Ki67 immunoreactivity (Figure 14F), suggesting continued proliferation of mucosal and submucosal cells, CD31 (Figure 14G), CD3ε (Figure 14H), αSMA (Figure 14I), and transgelin/SM22α (Figure 14J), as well as relative loss of striated muscle myosin heavy chain (striated MHC) (Figure 14K) in tissues at the surgical site. Scale bars: F-K = 200 μm. Ki67 immunoreactivity at 2.5 months (Figure 14F) demonstrated continued proliferation of mucosal and submucosal cells, CD31 (Figure 14G), CD3ε (Figure 14H), αSMA (Figure 14I), and transgelin/SM22α (Figure 14J), as well as relative loss of striated muscle myosin heavy chain (striated MHC) in tissues at the surgical site (Figure 14K). The predominance of αSMA and SM22α and the relative absence of myosin heavy chain indicate that smooth muscle proliferation precedes skeletal muscle growth.

Synthetic matrices seeded with autologous mesenchymal stem cells (aMSCs) resulted in complete longitudinal regeneration of the resected esophagus with minimal mucosal ulceration or perforation (Table 1). All animals experienced 100% complete longitudinal regeneration from 2 to 9 weeks after implant removal, with 1 of 6 animals experiencing mucosal ulceration or perforation. No animals experienced leakage during the study.

Table 1

Example III - Other Gastrointestinal Implants

The procedures described in Examples I and II can be performed in an area of the gastrointestinal tract that is alternatively confined to the rectum. The results are similar to those outlined previously.

While several embodiments of aspects of the present invention have been described herein, it is understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are considered part of this disclosure and are encompassed within the spirit and scope of the present invention. Therefore, the foregoing description and accompanying drawings are intended to be illustrative only.

The terms "a" and "an" as used in this specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used in this specification and claims should be understood to mean "one or both" of the two elements that are connected together, that is, in some cases the two elements are present in combination and in other cases separately. In addition to the elements explicitly indicated by the "and/or" clause, other elements may optionally be present, regardless of whether they are related or unrelated to the elements explicitly indicated, unless the contrary meaning is explicitly indicated. Thus, as a non-limiting example, "A and/or B", when used in conjunction with open language (such as "comprising"), may refer to "A without B" (optionally including elements other than B) in one embodiment, "B without A" (optionally including elements other than A) in another embodiment, "A and B" (optionally including elements other than A) in yet another embodiment, and so on.

As used in this specification and claims, "or" should be understood to have the same meaning as "and/or" defined above. For example, when distinguished from an item in a group, "or" or "and/or" should be interpreted as including, that is, including at least one of several elements or groups of elements, but also including more than one, and optionally including other unlisted items. Only terms that clearly indicate the opposite meaning, such as "only one" or "exactly one", or "comprising" when used in a claim, will refer to including exactly one element of several elements or groups of elements. In general, the term "or" as used herein, when preceding an exclusive term (such as "or", "one of...", "only one of...", or "only one of..."), is only to be interpreted as indicating exclusive alternatives (i.e., one or the other format, but not both). When used in a claim, "essentially consisting of..." should have the general meaning used in the field of patent law.

As used in this specification and claims, the phrase "at least one" with respect to a group of one or more elements should be understood to mean at least one element selected from any one or more elements in the group of elements, but does not necessarily include every element specifically listed in the group of elements, and does not exclude any combination of elements in the group of elements. In addition to the elements explicitly specified in the group of elements, this definition also allows for the optional presence of other elements related or unrelated to those explicitly specified elements. 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"), in one embodiment, may mean "at least one, optionally including more than one A but no B (optionally including elements other than B)"; in another embodiment, may mean "at least one, optionally including more than one B but no A (optionally including elements other than A)"; in yet another embodiment, may mean "at least one A (optionally including more than one A) and at least one B (optionally including more than one B)" (optionally including elements other than A and B), etc.

In the claims and the foregoing description, all transitional phrases, such as "comprises," "comprising," "carrying," "having," "containing," "involving," "containing," etc., should be construed as open-ended, i.e., meaning "including but not limited to." As set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" should be construed as closed or semi-closed transitional phrases, respectively.

The use of ordinal numbers (such as "first," "second," "third," etc.) in the claims to modify claim elements does not, in itself, imply any priority, advantage, or order of one claim element relative to another claim element, or a temporal order for performing a method, but serves merely as a label to distinguish a claim element having a specific name from another claim element having the same name.

While the present disclosure has been described in conjunction with certain embodiments, it should be understood that the present disclosure is not limited to the disclosed embodiments. On the contrary, the present disclosure is intended to cover various modifications and equivalent variations within the scope of the appended claims, the scope of which should be interpreted broadly so as to include all such modifications and equivalent structures as permitted by law.

Claims (12)

1. A removable synthetic scaffold, comprising: a tubular body portion having a first end and a second end opposite to the first end, at least a portion of the body portion being configured as a tubular member, the body portion including an outer surface having at least one region composed of non-woven spun polymer fibers having an average fiber diameter between 15 nm and 10 micrometers, at least a portion of the spun polymer fibers being interconnected to form pores with an average diameter less than 50 micrometers; the body portion further comprising at least one sheath covering the spun polymer fibers in the outer surface, the sheath being composed of cellular material having a thickness between 1 and 100 cells, at least a portion of cells being adhered to and spanning the pores in a suspended layered relationship between the fibers, the cellular material of the sheath comprising one of mesenchymal cells, stem cells, or pluripotent cells. 2. The synthetic scaffold as claimed in claim 1, wherein the spun polymer fiber is an electrospun polymer fiber, the electrospun polymer fibers are interconnected and form an outer layer of the main body, the main body further includes at least one inner layer, the inner layer being composed of at least one of a polymer mesh, a polymer braided support material, a solid polymer component, and an electrospun layer, and the outer layer covers and contacts the inner layer. 3. The synthetic scaffold as described in claim 2, characterized in that the average fiber diameter of the electrospun polymer fiber material is 3-10 micrometers, and the electrospun polymer fiber material is composed of at least one of the following polymer materials: polyvinylidene fluoride, syndiotactic polystyrene, polyvinylidene fluoride-hexafluoropropylene copolymer, polyvinyl alcohol, polyvinyl acetate, poly(acrylonitrile), copolymer of polyacrylonitrile and acrylic acid, copolymer of polyacrylonitrile and methacrylate, polystyrene, poly(vinyl chloride), poly(vinyl chloride) copolymer, polymethyl methacrylate, polymethyl methacrylate copolymer, polyethylene terephthalate, and polyurethane. 4. The synthetic scaffold as claimed in claim 2, characterized in that at least one layer is a polymer material, said polymer material containing polyethylene terephthalate, polyurethane, or a blend of polyethylene terephthalate and polyurethane. 5. The synthetic scaffold as claimed in claim 2, wherein the polymer braided support material is composed of at least one of polyethylene terephthalate and polyurethane. 6. The synthetic scaffold as claimed in claim 5, wherein the polymer braided support material further comprises nitinol. 7. The synthetic scaffold as claimed in claim 1, 2 or 3, wherein at least one of the sheath layers is configured as a cell sheath, the cell sheath being between 1 and 100 cells thick, the cells stimulating and directing the growth of gastrointestinal tissue. 8. The synthetic scaffold of claim 1, wherein the sheath of the cell material covers the spun polymer fibers in the outer surface such that the cell material is disposed on the outer surface and the cell material spans the pore. 9. The synthetic scaffold of claim 1, wherein the synthetic scaffold further comprises at least one aperture, serration, protrusion or combination thereof, the aperture, serration, protrusion or combination thereof being located near at least one of the first end or the second end, and the aperture, serration, protrusion or combination thereof being adapted to assist in the retrieval of the scaffold from the subject after tissue regeneration occurs around the scaffold at the implantation site on the subject. 10. The synthetic scaffold of claim 1, wherein the synthetic scaffold is a tubular member whose outer surface includes the spun polymer fibers, wherein a cellular sheath spans at least a portion of the spun polymer fibers located on the outside. 11. The synthetic scaffold as described in claim 1, 2 or 3, wherein the average fiber diameter of the spun polymer fiber is 3-6 micrometers, the average pore size is 15-20 micrometers, and the median pore size is 25-45 micrometers. 12. The synthetic scaffold of claim 1, wherein the cellular material is derived from the test subject.