Systems and methods for producing gastrointestinal tissue

Abstract

The present invention relates to methods for regenerating gastrointestinal tissue (e.g., esophageal tissue) and synthetic scaffolds.

Description

[0001] This application is a divisional application of Chinese application No. 201680065115.6, entitled "System and Method for Generating Gastrointestinal Tissue," filed on November 14, 2016, which claims priority dates of November 12, 2015 and January 8, 2016, filed with the U.S. Patent and Trademark Office. Technical Field

[0002] This invention relates to engineered tissues for replacing or repairing damaged tissues. Background Technology

[0003] Engineered biological tissues used to replace or repair damaged tissue are typically produced by seeding cells onto 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 jetting (e.g., using pumps and syringes), electrospinning, electro-nebulization, and other techniques for depositing one or more natural or synthetic polymers or fibers to form a scaffold with suitable shape and size for transplantation into test subjects (e.g., human test subjects, such as organs or regions requiring engineered tissue).

[0004] It is estimated that more than 500,000 people worldwide are diagnosed with esophageal malignancies each year. The prevalence of congenital esophageal malformations, such as esophageal atresia, is 2.44 per 10,000 newborns. Chronic esophageal stricture following esophageal injury is also common. Although the extent of esophageal resection for early malignancies has been minimized, such as through endoscopic mucosal resection, surgical esophagectomy remains the primary treatment for many esophageal diseases. Traditionally, an autologous catheter (e.g., from the stomach, small intestine, or colon) is harvested and routed into the chest cavity to restore gastrointestinal continuity. Many children with esophageal atresia or those with traumatic or corrosive damage to the esophagus eventually undergo similar reconstruction. However, these treatments have high morbidity and mortality rates.

[0005] Due to the complex structure of the esophagus, autologous conduits are typically used. The layers of the esophagus consist of stratified squamous epithelium, submucosa, an outer circular muscle layer, and a longitudinal muscle layer. These multi-layered structures of the esophagus provide a barrier against oral intake and contamination escaping from the gastrointestinal tract. Furthermore, this layered structure provides physiological mechanisms for propulsion and stress management during the passage of the bolus, or during swallowing or vomiting.

[0006] It is necessary to provide structures that can help support tissue regeneration and methods for manufacturing such structures. Summary of the Invention

[0007] This invention discloses implementations of synthetic scaffolds and related systems capable of generating 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 (e.g., esophageal) tissue in a subject. In some embodiments, the regenerated gastrointestinal tissue includes muscle tissue, nervous system tissue, or both. In some embodiments, the gastrointestinal (e.g., esophageal) tissue regenerates around the scaffold. In some embodiments, the scaffold does not fuse into the final regenerated tissue (e.g., the new esophageal tissue does not fuse the scaffold into the regenerated esophageal wall). Therefore, aspects of this disclosure relate to guided tissue regeneration in which the scaffold provides support and / or signaling that can promote host tissue regeneration, without requiring the scaffold to fuse into the regenerated tissue (e.g., the scaffold does not provide structural or functional support in the final regenerated tissue).

[0008] In some embodiments, the gastrointestinal (e.g., esophageal) stent comprises a biodegradable and / or reabsorbable material that is reabsorbed after the initiation of gastrointestinal (e.g., esophageal) tissue regeneration (e.g., after functional esophageal tissue regeneration).

[0009] In some embodiments, a gastrointestinal (e.g., esophageal) stent includes one or more structures that can be used to assist in the removal of the stent after the initiation of gastrointestinal (e.g., esophageal) tissue regeneration (e.g., after functional esophageal tissue regeneration).

[0010] In some embodiments, the scaffold is cellularized with one or more types of cells prior to implantation. 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 derived from bone marrow, adipose tissue, esophageal tissue, or other suitable tissues. In some embodiments, the cells can be derived from various allogeneic sources, including but not limited to sources such as amniotic fluid and umbilical cord blood. In some embodiments, the cells are mesenchymal stem cells (MSCs).

[0011] In some embodiments, the scaffold is implanted into a site that provides sufficient stem cell niches for tissue regeneration in the subject (e.g., an esophageal site or other gastrointestinal site that can provide stem cell niches). In some embodiments, it is not desirable to be bound by theory that the scaffold and / or the cells provided on the scaffold contribute to promoting the growth and / or regeneration of gastrointestinal tissue from host stem cells present at the scaffold implantation site.

[0012] In some aspects, this disclosure relates to the finding that the presence of a synthetic scaffold can promote or enhance esophageal tissue growth, wherein the synthetic scaffold can be modified to replace or repair the natural structural patterns and / or functional characteristics of diseased or damaged tissue or organs without requiring the scaffold to fully fuse into the final regenerated tissue. Therefore, in some aspects, this disclosure provides a method for promoting or enhancing the growth of gastrointestinal tissue (e.g., the esophagus), the method comprising: delivering a synthetic scaffold to a gastrointestinal region (e.g., the esophagus) of a subject, wherein delivery of the synthetic scaffold results in the growth of new gastrointestinal (e.g., esophageal) tissue in that region of the subject. In some embodiments, diseased or damaged gastrointestinal tissue is removed (e.g., surgically removed) prior to implantation of the scaffold. In some embodiments, the scaffold is an implanted, approximately tubular structure (e.g., sutured to the end of the remaining gastrointestinal tissue after removal of diseased or damaged tissue). In some embodiments, the implanted scaffold is shorter than the removed tissue (e.g., 5-50% shorter). In some embodiments, when tissue is attached (e.g., sutured) to both ends of the scaffold, the remaining gastrointestinal tissue adjacent to the implantation site is stretched. In some embodiments, new gastrointestinal (e.g., esophageal) tissue regenerates on the implanted stent without completely fusing with it. In some embodiments, although the stent may remain within the lumen of the regenerated tissue, the walls of the regenerated tissue do not include the walls of the stent. In some embodiments, the stent can be removed from the lumen formed by the regenerated tissue at an appropriate point during the tissue regeneration process.

[0013] In some implementations, the growth of new gastrointestinal (e.g., esophagus) tissue leads to the formation of functional tissue (e.g., functional esophagus), thus eliminating the need for the functional scaffold to continue.

[0014] In some embodiments, the synthetic scaffold is absorbable or soluble under physiological conditions. In some embodiments, the synthetic scaffold is removed from the gastrointestinal region (e.g., the esophagus) of the subject after functional esophageal formation.

[0015] In some embodiments, the methods and compositions described herein can also be used for the regeneration of tracheal and / or bronchial tissues.

[0016] These and other aspects will be described in more detail in this article. Attached Figure Description

[0017] This disclosure can be best understood by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features in the drawings are not drawn to scale. Instead, for clarity, the dimensions of the various features may be arbitrarily enlarged or reduced.

[0018] Figure 1A This is a perspective view of one embodiment of the synthetic scaffold disclosed herein, in which a partial cross-section is shown;

[0019] Figure 1B This is a micrograph of the tube surface of one embodiment of the synthetic scaffold disclosed herein;

[0020] Figure 1C This is a side perspective view of the second embodiment of the synthetic scaffold disclosed herein;

[0021] Figure 2 This is a non-limiting description of the biofilm layer of the esophagus;

[0022] Figure 3 A non-limiting example of regenerated esophageal tissue compared with corresponding natural tissue is shown;

[0023] Figure 4A This is a SEM micrograph of the outer surface region of one embodiment of the synthetic scaffold disclosed herein, showing cell growth after 7 days of biological reaction at a magnification of 5000X.

[0024] Figure 4B This is a micrograph of the outer surface region of one embodiment of the synthetic scaffold disclosed herein, showing cell growth after 7 days of biological reaction;

[0025] Figure 5 This is a flowchart of one implementation of the regeneration method disclosed herein;

[0026] Figure 6 This is the overall research process of one implementation of the method disclosed herein, which includes the generation and subsequent implantation of cellularized scaffolds;

[0027] Figure 7A The images are SEM images of an electrospinning support sample according to one embodiment disclosed herein, taken at magnifications of 1000X, 2000X and 5000X respectively.

[0028] Figure 7B The illustrations are based on the representative uniaxial mechanical test loadings before and after implantation of the electrospun scaffold disclosed in this article.

[0029] Figure 7C This is a table of uniaxial mechanical properties of a stent prepared according to one embodiment disclosed herein, before and after implantation;

[0030] Figure 8 This is a flow cytometry illustration of MSCs isolated from adipose tissue and proliferated up to 5 generations.

[0031] Figure 9 This is an overview of an implantation procedure based on one embodiment disclosed herein;

[0032] Figure 10 This is a timeline representation based on one embodiment of the method disclosed herein;

[0033] Figure 11A C represents photographs of the regenerated tubular tissue located at the esophagectomy site of the test subject within a specified postoperative time interval.

[0034] Figure 12A E represents photographs of the regenerated tubular tissue located at the esophagectomy site of the test subject within a specified postoperative time interval.

[0035] Figure 13A Photographs of the histological analysis of esophageal tissue are shown in image I.

[0036] Figure 14A Image K is a histological analysis photograph of tissue from the porcine esophagus 2.5 months after implantation of one embodiment of the stent disclosed herein. Detailed Implementation

[0037] A significant aspect of this disclosure relates to the finding that inserting a synthetic scaffold into the esophageal region of a subject can promote or enhance the regeneration of new esophageal tissue (e.g., an intact and functional esophagus) in the subject without requiring complete fusion of the scaffold into the regenerating tissue. Therefore, in some embodiments, this disclosure provides a method for promoting or enhancing the growth of gastrointestinal tissue (e.g., the esophagus), the method comprising: delivering a synthetic scaffold to a gastrointestinal region (e.g., the esophagus) of a subject, wherein delivery of the synthetic scaffold results in the growth of new gastrointestinal (e.g., esophageal) tissue in that region of the subject.

[0038] The tissue that can be regenerated using the methods disclosed herein can be any gastrointestinal tissue, such as the esophagus, stomach, intestine, colon, rectum, or other hollow gastrointestinal tissue. In some respects, this disclosure is based, in part, on the surprising finding that the methods described herein result in the regeneration of gastrointestinal tissue comprising muscle tissue, nervous system tissue, or both muscle and nervous system tissue.

[0039] In some embodiments, the synthetic scaffold is reabsorbable or soluble 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 reabsorbable or soluble under suitable physiological conditions.

[0040] In some implementations, the synthetic scaffold is removed from the subject after the regenerated functional tissue (e.g., the esophagus or part of the esophagus) has formed.

[0041] In some implementations, the stent is designed to be easily retrieved, which can be achieved by having a) one or more reversible attachments that are easier to remove than sutures, for example, to help the stent separate from surrounding tissue (e.g., the esophagus) after tissue regeneration; and / or b) having one or more features that can be used to help retrieve the stent, for example after the stent has separated from surrounding tissue (e.g., adjacent esophageal tissue).

[0042] Non-limiting examples of reversible attachments include mechanical devices (e.g., hooks and rings, connectors such as stents, or other detachable mechanical attachments) and / or chemical-mechanical 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 may be used. In some embodiments, the absorbable staples comprise, for example, a copolymer of polylactic acid and polyglycolic acid or a blend of any other absorbable material.

[0043] In some implementations, the surgical implantation and / or retrieval of the stent can be performed with thoracoscopic assistance.

[0044] Non-limiting examples of structural features that can aid in the retrieval or removal of a stent (e.g., after the stent has separated from surrounding gastrointestinal tissue) include holes, indentations, protrusions, or other structural features or any combination thereof, located solely on the outer surface of the stent. One or more of these structural features can be used to aid in grasping or holding the tool (e.g., a gripper) being used to retrieve the stent. In some embodiments, one or more of these structural features may be located only at one end of the stent (e.g., at the end near the subject's mouth). In some embodiments, one or more of these structural features may be located at both ends of the stent or throughout the entire length of the stent. In some embodiments, one or more of these structural features are located solely on the outer surface of the stent. In some embodiments, one or more of these structural features are located on both the outer and inner surfaces of the stent. In some embodiments, the stent is reinforced at or near the location of one or more structural features used for stent retrieval (e.g., making the stent thicker and / or the stent comprising a more robust material).

[0045] In some embodiments, the dissected stent can be removed endoscopically via an airway leading to the esophagus. In some embodiments, the dissected stent can be removed surgically.

[0046] In some embodiments, the subject has diseased or damaged gastrointestinal tissue that needs to be replaced. In some embodiments, the subject is a human being (e.g., a human patient).

[0047] In some embodiments, this disclosure provides engineered stents that can be used to replace or repair the esophagus or a portion of the esophagus. In some embodiments, the esophageal stents described herein can be used to promote tissue regeneration (e.g., regenerated esophagus or a portion of the esophagus) to replace tissue in a subject (e.g., a human). For example, a subject (e.g., a human) suffering from certain cancers (e.g., esophageal cancer) may benefit from replacement of tissue or organ affected by the cancer. Without wishing to be bound by any particular theory, the synthetic stents described herein promote the growth of new tissue (e.g., esophageal tissue) in a subject and thus provide a therapeutic benefit to the subject.

[0048] In some embodiments, the growth of new esophageal tissue leads to the formation of a functional esophagus in the subject. In some embodiments, the new esophageal tissue does not fuse the stent into the regenerated esophageal wall. In some embodiments, the stent is designed and manufactured to be absorbable and / or easily recyclable after esophageal tissue regeneration. In some embodiments, the stent is designed to be at least partially absorbable.

[0049] In some implementations, the size and shape of the synthetic stent are similar to the size and shape of the diseased or damaged gastrointestinal region (e.g., the esophagus) to be replaced.

[0050] In some embodiments, the stent will have at least two layers. In some embodiments, the stent may have an approximately tubular structure. Figure 1A A non-limiting embodiment of the stent 10 is shown, the stent 10 having a nearly tubular body 12 having an outer surface 14 and an inner surface 16. In some embodiments, a cross-section of the stent 10 is generally circular. In some embodiments, the cross-section is generally "D"-shaped. However, stents 10 with other cross-sectional shapes can be used. Depending on the size of the corresponding regenerated tissue, the stent 10 can have any suitable length and diameter, depending on the size of the corresponding regenerated tissue. In some 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, it can be 10-20 cm. However, it is contemplated that shorter or longer stents 10 can be used depending on the specific application, the patient's needs, and / or the location of the gastrointestinal tract to be treated. In some embodiments, the stent 10 can have an inner diameter of 0.5 to 5 cm. However, depending on the specific application, the patient's needs, and / or the location of the gastrointestinal tract to be treated, stents with smaller or larger inner diameters can be used.

[0051] In some embodiments, the length of the scaffold 10 may be shorter than the length of the replaced gastrointestinal tract (e.g., esophagus) region. In some embodiments, the length of the scaffold 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 tract are capable of actively responding to traction forces applied to the relevant organ tissue, resulting in the generation of certain bio-organic mediated signals that can initiate or promote tissue growth and differentiation.

[0052] In some embodiments, the length of the stent 10 may 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% of the length of the tissue being replaced (e.g., about 100-110%, 110-120%, 120-130%, 130-140%, about 100%, about 105%, about 110%, or about 115%). It is contemplated that the length of the stent will be the length necessary for effective replacement of the affected area. In some cases, it is contemplated that the stent 10 will have a length longer than the gastrointestinal tract region being replaced to effectively position the stent, reducing or minimizing trauma and ischemia in the affected or related areas.

[0053] In some embodiments, the stent 10 may consist of a single layer of synthetic material. However, the stent 10 may also include more than one layer of synthetic material, which is also within the scope of this disclosure.

[0054] Therefore, in some embodiments, the synthetic scaffold 10 may consist of multiple layers (e.g., two 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 may be made of different materials (e.g., different polymers and / or different polymer arrangements). The synthetic scaffold 10 disclosed herein may contain two or more different components that, when present, can be assembled to form the scaffold (e.g., prior to cellularization and / or implantation). In some embodiments, the synthetic scaffold 10 comprises two or more layers in contact with each other, for example, by bringing the layers into contact with each other using a synthetic technique for manufacturing the scaffold 10. In some embodiments, the scaffold 10 may be synthesized using a technique comprising multiple steps that results in two or more layers agglomerated together (e.g., applying an electrospun material layer to a pre-made portion of the scaffold, such as a prior electrospun material layer, a previous electrospun material layer, or one or more of the surfaces of different components incorporated into the scaffold (e.g., braided tubes or meshes).

[0055] In such Figure 1A In the illustrated embodiment, the support 10 includes at least one outer layer 18 that defines the outer surface 14 of the support body 12. The support 10 includes at least one additional inwardly oriented layer 20. In the illustrated embodiment, the at least one additional inwardly oriented layer 20 is in direct contact with an inner surface of the outer layer 18. Where desired or required, the at least one additional inwardly oriented layer 20 can be configured to provide structural support to the associated support body 12. Figure 1AIn the embodiments described herein, the at least one additional inwardly oriented layer 20 may be configured as a suitable mesh or braid circumferentially arranged to surround at least a portion of the longitudinal length of the support body 12. In other embodiments, it is conceivable that the at least one additional inwardly oriented layer 20 may be composed of a suitable polymer layer. Figure 1A In the embodiment shown, the body 12 of the support 10 includes at least one layer 22 located inside the mesh 20 or the woven layer 20.

[0056] 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 regions 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 differs from the wall thickness at the middle portion 28 (not shown) of the stent 10 (e.g., the wall thickness at ends 24, 26 is thicker than that at the middle portion 28). In some embodiments, the thicker wall region is more robust when the stent is attached to the surrounding gastrointestinal tissue and provides better support for sutures attached to one or both ends 24, 26 of the stent 10. The thick-walled region may also include discrete configurations that facilitate suturing. Non-limiting examples of such discrete configurations include tubes, orifices, etc.

[0057] In some embodiments, the outer surface 14, at least defined on the outer layer 18, may be made of an electrospun polymer material. In some embodiments, it is conceivable that the externally oriented wall 18 may be made of an electrospun polymer material. In some embodiments, the externally oriented electrospun layer may be in direct contact with a suitable braided material layer 20.

[0058] Fiber orientation

[0059] 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. Furthermore, fiber orientation can be varied in each layer of a composite scaffold or sandwich scaffold.

[0060] In some embodiments, scaffolds with different porosities can be used. In some embodiments, one or more layers of the scaffold substantially allow for complete cell penetration and uniform seeding. In some embodiments, one or more layers of the scaffold can be constructed to prevent the penetration of one or more cell types, for example, by densely packing fibers. Since porosity varies with fiber diameter, controlling the fiber diameter can be used to alter the scaffold porosity. Optionally, blends of different polymers can be electrospun together, with one polymer preferentially dissolved to increase scaffold porosity. Fiber properties can be controlled to optimize fiber diameter, fiber spacing or porosity, and the morphology of each fiber, such as fiber porosity or aspect ratio, changing the fiber shape from circular to ribbon-like. In some embodiments, the mechanical properties of each fiber can be controlled or optimized, for example, by altering the fiber composition and / or degradation rate.

[0061] In some embodiments, electrospun fiber materials can provide, for example... Figure 1B The contoured surface is shown. In some embodiments, at least one electrospun layer in the scaffold 10 may 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 rotary drying.

[0062] The spacing and porosity of electrospun fiber materials can be such that cells seeded on the scaffold surface can adhere between the fibers in a suspended, layered relationship, allowing the seeded cell material to form on it as... Figure 4A and 4B The thin slice shown.

[0063] Formation of synthetic scaffolds

[0064] This disclosure relates to methods for producing synthetic stents. In some embodiments, tubular synthetic stents (e.g., synthetic esophageal stents) can be produced on a mandrel (e.g., by depositing material via electrostatic atomization and / or electrospinning).

[0065] In some embodiments, one or more layers of the synthetic scaffold provide structural support and impart desired mechanical properties. In some embodiments, a braided material (e.g., braided tubing, nitinol braid, PET braid, or braid of other metallic or non-metallic materials) can be inserted between two different layers of the scaffold to provide structural support. The pressure of the braided material (e.g., the force the braid can exert on the next layer of material (e.g., the outer electrospun layer of the material)) can be controlled by controlling the pick count. In some embodiments, the braid can be coated (e.g., by impregnation or other techniques) in an organic solvent to help it adhere to one or more other layers of the scaffold 10. In some embodiments, the length of the braid 20 does not extend to the end 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, while the middle portion 28 of the scaffold body 12 includes an additional braided layer.

[0066] In some embodiments, one or more layers of the synthetic scaffold provide a barrier within the scaffold, creating a space (e.g., a relatively impermeable space) between the internal space (e.g., a cavity space) and the external space. In some embodiments, the barrier may be an electrostatically atomized polyurethane (PU) layer.

[0067] In some embodiments, the different layers of the support 10 may comprise one or more polymers (e.g., polyethylene terephthalate (PET), PU, ​​or blends thereof). In some embodiments, the support 10 may comprise a nitinol braid sandwiched between an inner PU layer (e.g., an inner PU layer electrostatically atomized to or electrospun onto a mandrel) and an outer PU layer (e.g., an outer PU layer electrostatically atomized onto a braided material).

[0068] In some embodiments, the support 10 may be formed using a support member or a mandrel. In some embodiments, the support member or mandrel may be coated with a material (e.g., PLGA or other polymers) before depositing one or more layers of PU, PET, or a combination thereof.

[0069] In some embodiments, the material in the woven or mesh layer may be composed of an absorbable polymer material.

[0070] Scaffold Production - Fiber Materials

[0071] In some embodiments, one or more layers of the scaffold may be constructed using fibrous materials. In some embodiments, the scaffold comprises one or more types of fibers (e.g., nanofibers). In some embodiments, the scaffold 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 (e.g., different fibers) can be used in the methods and compositions (i.e., scaffolds) described herein. In some embodiments, the material is biocompatible and therefore can promote cell growth. In some embodiments, the material is permanent, semi-permanent (e.g., persisting for years after implantation in a host) or rapidly degradable (e.g., absorbed within weeks or months after implantation in a host).

[0072] In some embodiments, the scaffold comprises or is composed of an electrospun material (e.g., microfibers or nanofibers). In some embodiments, the electrospun material comprises or is composed of PET (polyethylene terephthalate). In some embodiments, the electrospun material comprises or is composed of polyurethane (PU). In some embodiments, the electrospun material comprises PET and PU, or is composed of PET and PU.

[0073] In some embodiments, the artificial scaffold may consist of or include one or more of the following materials: elastic polymers (e.g., one or more polyurethanes (PUs), such as polycarbonate and / or polyester), acrylamide polymers, nylon, and absorbable polysulfone polymers. In some embodiments, the scaffold may consist 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), and polyvinyl alcohol in crosslinked and non-crosslinked forms with varying degrees of hydrolysis (e.g., 87%-99.5%). In some embodiments, the polymer may also include other compounds or methods that can increase the hydrophilicity of the polymer. In some embodiments, this may involve introducing compounds such as block copolymers based on ethylene oxide and propylene oxide. It is also contemplated that the hydrophilicity of the polymer can be improved by suitable plasma treatment, if desired or required.

[0074] In some embodiments, the scaffold may consist of or contain block copolymers. In some embodiments, addition polymers such as polyvinylidene fluoride, syndiotactic polystyrene, copolymers of vinylidene fluoride and hexafluoropropylene, polyvinyl alcohol, and polyvinyl acetate, such as polyacrylonitrile and its copolymers with acrylic acid and methacrylates, polystyrene, poly(vinyl chloride) and its various copolymers, poly(methyl methacrylate) and its various copolymers, and amorphous addition polymers of PET (polyethylene terephthalate), can be solution spun or electrospun and combined with any other materials disclosed herein to produce the scaffold. In some embodiments, highly crystalline polymers such as polyethylene and polypropylene can be solution spun or combined with any other materials disclosed herein to produce the scaffold.

[0075] In some implementations, one or more polymers may be modified after scaffold synthesis but before scaffold cellularization and / or implantation to reduce their hydrophobicity and / or increase their hydrophilicity.

[0076] In some embodiments, the electrospun fiber may have a diameter of no more than 10 μm. In some embodiments, the electrospun fiber may have a diameter of 3-10 micrometers. In some embodiments, the electrospun fiber may have a diameter of 3-5 micrometers.

[0077] In some embodiments, it is conceivable that the material in the braided layer may be made wholly or partially of a bioabsorbable material such as PLGA. It is also conceivable that in some configurations, the braided material may be a load material and 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 substances: antibiotics, growth factors, etc.

[0078] electrospinning

[0079] In some embodiments, a scaffold is manufactured comprising one or more layers (e.g., 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 the following literature: Doshi and Reneker. Electrospinning process and application of electrospun fibers. J Electrostat. 1995; 35:151–60.; Reneker DH, ChunI. 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 content relating to electrospinning is incorporated herein by reference. Electrospinning is a versatile technique that can be used to produce fibers with arbitrary orientations or arrangements, which can have virtually any chemical composition and diameter ranging from the nanoscale (e.g., about 15 nm) to the microscale (e.g., about 10 micrometers).

[0080] In some embodiments, the electrospinning and electrostatic atomization techniques used herein involve charging a polymer solution (or melt) with a high-voltage electric field, which is delivered (e.g., as a polymer solution stream) through a nozzle and deposited onto a target surface. The target surface may be the surface of an electrostatic plate, the surface of a rotating drum (e.g., a mandrel), or other form of collector surface that is both conductive and electrically grounded, such that the charged polymer solution moves toward said surface.

[0081] In some embodiments, the electric field used is typically on the order of several kilovolts, 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) during the process of leaving the nozzle and reaching the target surface. This results in polymer fibers being deposited on the surface. Typical fiber diameters range from a few nanometers to a few micrometers. The movement of the target surface relative to the nozzle affects the relative orientation of the fibers. For example, if the target surface is the surface of a rotating mandrel, then the fibers will align (at least partially align) on the surface along the direction of rotation. In some cases, the nozzle may scan back and forth between the two ends of a rotating mandrel.

[0082] In some embodiments, the size and density of the polymer fibers, the degree of fiber alignment, and other physical properties of the electrospun material are 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 speed), and other factors that may affect solvent evaporation and polymer deposition.

[0083] Electrospinning and electro-atomization processes can be used to produce interconnected polymer fiber scaffolds (e.g., hollow synthetic scaffolds) on a mandrel.

[0084] Support / Mandrel

[0085] In some embodiments, the scaffold 10 (e.g., a scaffold having two or more layers) can be manufactured using a support member (e.g., a solid or hollow support member) on which the scaffold 10 can be formed. For example, the support member can be an electrospinning collector, such as a mandrel, tube, or any other shape of support member. It is understood 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 scaffold 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 is understood 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.

[0086] We have quite unexpectedly discovered that placing a suitable braided material to integrate it into the formed support 10 near the mandrel surface can help facilitate the removal of the formed support 10 from contact with the mandrel.

[0087] Support performance

[0088] It should be understood that aspects of this application can be used to enhance the physical and functional properties of any scaffold (e.g., scaffolds based on electrospun and / or electrosprayed fibers). In some embodiments, one or more scaffold components can be sheets, cylinders, thick ribs, solid blocks, branched networks, etc., or any combination thereof, of different sizes. In some embodiments, the dimensions of the integral and / or assembled scaffold are similar to or the same as the dimensions of the tissue or organ being replaced. In some embodiments, the individual components or layers of the scaffold have small dimensions. For example, the thickness of the nanofiber layer can range from a few nanometers to 100 nanometers, 1-1000 micrometers, or even a few millimeters. However, in some embodiments, the dimensions of one or more scaffold components can range from about 1 mm to 50 cm. However, as described herein, larger, smaller, or medium-sized structures can be fabricated.

[0089] In some embodiments, the scaffold is formed as a tubular structure, which can be seeded with cells to form a tubular tissue region (e.g., the esophagus or other tubular region). 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 (e.g., including portions with different diameters along the length of the tubular region). The tubular region may also include one branch or a series of branches. In some embodiments, tubular scaffolds with one open end, two open ends, or multiple open ends (e.g., in the case of branched scaffolds) are prepared. However, the tubular scaffold can be closed at one, two, or all ends, and aspects of the invention are not limited in this respect. It is also understood that, because the invention is not limited in this respect, aspects of the invention can be used to produce scaffolds of any type or organ (including hollow and solid organs). In some embodiments, aspects of the invention can be used to enhance the stability of scaffolds or other structures comprising two or more non-physically connected fibrous regions or fibrous layers (e.g., electrospun nanofibers).

[0090] In some embodiments, the scaffold is designed to have a porous surface with pores of a diameter ranging from about 10 nm to about 100 μm that promote cellification. In some embodiments, the average diameter of the pores is less than 50 μm, less than 40 μm, less than 30 μm, less than 20 μm, or less than 10 μm (e.g., about 5 μm, about 10 μm, or about 15 μm). In some embodiments, the average diameter of the pores is between 20 and 40 μm. In some embodiments, the pore size is selected to prevent or reduce immune responses or other unwanted host responses in the subject. The pore size can be estimated using computational and / or experimental techniques (e.g., using mercury porosimetry). However, it is understood that the porous surface of the scaffold may also include pores of other sizes.

[0091] In some embodiments, a surface layer of a fiber-based scaffold is used, the fibers comprising one or more soluble particles that can dissolve during or after synthesis (e.g., by exposure to a solvent, aqueous solution, such as water or a buffer), leaving pores the size of the soluble particles. In some embodiments, the particles are contained in a polymer mixture pumped into a nozzle of an electrospinning apparatus. 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., with an average diameter of several micrometers, about 10 μm, and larger). In some embodiments, if the fibers are deposited in a dense pattern, one or more fibers will merge before curing to form larger microstructures (e.g., 10-100 micrometers thick or more). In some embodiments, these microstructures may be wound around two or more fiber layers 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 a microstructure is formed at one or more stages of the scaffold synthesis process (e.g., the stage used to connect two or more layers and / or components) (e.g., by electrospinning as described herein), the surface of the microstructure may be treated (e.g., etched, or made porous with soluble particles as described herein) to provide a surface suitable for cellularization.

[0092] In some implementations, the amount of flexible scaffold material (e.g., relaxation) 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, flexural strength, resistance, compliance, degrees of freedom, elasticity, or any other mechanical property, or combinations thereof).

[0093] In some embodiments, the scaffold 10 may further include a cell sheath derived from cells seeded on the outer surface of the scaffold during incubation. The cell sheath attaches to and overlaps with the outer surface of the scaffold. It is contemplated that a majority of the 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.

[0094] In some embodiments, the cell sheath may have a thickness sufficient to provide structural integrity to the sheath layer. In some embodiments, the cell sheath will consist of a plurality of cells in contact with the outer surface of the scaffold, the plurality of cells being sufficient to guide regenerating cells into contact with the sheath to generate a tissue wall covering the sheath but not fusing with it. In some embodiments, the sheath may consist of a lining with an average thickness between 1 and 100 cells. In some embodiments, the lining may have a cell thickness between 10 and 100, 10 and 30, 20 and 30, 20 and 40, 20 and 50, 10 and 20, 30 and 50, 30 and 60, 40 and 60, 40 and 70, or 70 and 90 cells.

[0095] The scaffold 10 with associated cell sheaths provides a movable, insertable device that can be placed at a suitable gastrointestinal resection site. The scaffold 10 with associated cell sheaths in contact with it can be transported to the desired resection site for implantation. In some embodiments, the scaffold 10 is configured to be removed from the implantation site after appropriate regeneration of the resected organ. In some embodiments, the removed scaffold will include some or all of the cell sheaths connected thereto.

[0096] This application also discloses various embodiments of a method for regenerating tubular organs, such as gastrointestinal organs. In some embodiments, method 100 includes a resection step, which includes removing a portion of a tubular organ in the subject, as indicated by reference numeral 110. The organ to be resected may be a tubular organ of the gastrointestinal tract that has been damaged or impaired due to disease, trauma, or congenital conditions. In some embodiments, non-limiting examples of suitable organs include one of the esophagus, rectum, etc. In some embodiments, suitable organs include at least one of the esophagus, small intestine, colon, and rectum.

[0097] Resection can be achieved through any suitable surgical procedure, resulting in a resected organ portion that remains connected to the gastrointestinal tract and retained within the subject after resection. In some embodiments, the resection operation can produce suitable resection margins.

[0098] After resection, the synthetic scaffold is implanted into the resection site, as shown by reference numeral 120. In some embodiments, implantation may include the step of connecting a corresponding end of the resected organ remaining in the subject to a corresponding end of the synthetic scaffold, such that the synthetic scaffold and the resected organ can achieve a suitable connection between the corresponding components. This can be achieved using one or more sutures, bio-organic tissue adhesives, etc.

[0099] In some embodiments, the implanted synthetic scaffold may be a tubular member having an outer polymer surface and a cellular 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 some embodiments, the synthetic scaffold will include a first end, a second end opposite the first end, an outer polymer surface located between the first and second ends, and a cellular sheath covering at least a portion of the outer polymer surface. In some embodiments, the implantation step may be a step of bringing at least a portion of the cellular sheath into direct contact with at least one resection edge of the resected organ portion.

[0100] In some embodiments, the method disclosed herein further includes the step of holding 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 some embodiments, the guided tissue growth originates from and contacts tissue present in the resected organ portion retained within the subject's body. In some embodiments, the guided tissue growth will be adjacent to a relevant region of the resected organ. In some embodiments, the guided tissue growth will exhibit differentiated tissue. In some embodiments, at a location outside the cell sheath, the guided tissue growth is parallel to the outer surface of the cell sheath. In some embodiments, the guided tissue growth originates from and contacts tissue present in the resected organ portion retained within the subject's body and will be adjacent to a relevant region of the resected organ. The guided tissue growth will exhibit differentiated tissue growth and may be parallel to the outer surface of the cell sheath at a location outside the cell sheath.

[0101] After the guided tissue growth has been achieved, the method 100 disclosed herein may include a step of removing the synthetic scaffold, as indicated by reference numeral 140. In some embodiments, the removal step is performed in such a manner that the guided tissue growth remains in contact with a resected portion of a tubular organ retained in the subject. In some embodiments, the removal process may include endoscopically removing the synthetic scaffold from inside the guided tissue growth.

[0102] In some embodiments, the synthetic scaffold may be constructed wholly or partially of a bioresorbable polymer material. In this case, the method disclosed herein may include the steps of maintaining contact between the synthetic scaffold and the resection margin for sufficient time intervals 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 sufficient time to achieve guided tissue growth along the synthetic scaffold. In some embodiments where the scaffold is entirely composed of a bioresorbable material, the scaffold will be configured to maintain structural integrity during guided tissue growth. In some embodiments, where the synthetic scaffold is composed of a bioresorbable material in selected areas, it is conceivable that, after the guided tissue growth has been achieved, the remaining portion of the scaffold may be removed with appropriate steps.

[0103] The guided tissue growth can be monitored by appropriate means. In some embodiments, tissue growth can be monitored endoscopically.

[0104] In some embodiments of the method disclosed herein, the method may further include the steps of introducing cellular material onto the polymer surface of a synthetic scaffold and allowing the cellular material to grow to form a cell sheath, wherein the introduction step and the cell material growth step occur prior to the excision step.

[0105] In some embodiments, the synthetic scaffold used in the methods disclosed herein is a tubular member whose outer surface comprises spun polymer fibers. In some embodiments, the spun fibers can be obtained by electrospinning using suitable methods such as those described herein. In some embodiments, the cellularized sheath extends across at least a portion of the externally located electrospun fibers. The cellularized sheath may be composed of cellular material, including mesenchymal cells, stem cells, and pluripotent cells. The cellular material may be autologous or allogeneic derived from the subject.

[0106] Unbound by any particular theory, it is believed that implantation of various synthetic scaffolds, such as those disclosed herein, particularly synthetic scaffolds seeded with a covering cell sheath, promotes the growth, regeneration, and differentiation of subject tissue in contact with or adjacent to the implanted synthetic scaffold. The growing regenerating tissue is guided by the synthetic scaffold and the associated sheath to generate a tubular cell body that is integrally connected to the excised end of the remaining tubular organ and expands outward to encapsulate the synthetic scaffold and the associated cell sheath. It is believed that the scaffold and the associated cell sheath can promote or stimulate the regenerative growth of the excised tissue while minimizing tissue rejection. We also believe that the presence of the cell sheath can reduce or minimize the penetration of the sheath into the regenerating tissue during growth and differentiation. In some embodiments, tissue regeneration proceeds from the extremities toward the center. Once the regenerated tissue is in place, the synthetic scaffold can be removed. In some embodiments, upon removal of the synthetic scaffold, the regenerated tissue structure will lack an inner epithelial layer. Figure 11A , 11B As shown in Figure 11C, endothelial regeneration was observed at 2 months and 3 months post-stent removal, respectively.

[0107] To further understand this disclosure, please refer to the following embodiments. These embodiments are included for illustrative purposes and are to be considered as an explanation of the invention set forth in this disclosure and the claims.

[0108] Example

[0109] Example 1: Esophageal stent

[0110] like Figure 1A As shown, a synthetic esophageal stent comprising three layers of material was fabricated. A first layer of polyurethane (PU) was deposited onto a metal mandrel using electrostatic atomization, followed by the deposition of a braided material onto the first PU layer. A second PU layer was then deposited using electrospinning. The resulting stent was then removed from the mandrel. Each stent defines a tubular structure with walls comprising three layers: an inner electrostatic atomization layer, an outer electrospinned layer, and a braided layer sandwiched between the inner and outer electrostatic atomization layers. The physical dimensions of the stent were determined using scanning electron microscopy (SEM). The average stent wall thickness was approximately 500 micrometers. Figure 1B The image shows a non-restrictive SEM view of a cross-section of the wall. Figure 1C The figure shows a non-limiting view of a cross-section of a tubular support. The cross-section is shown to be approximately "D"-shaped. This can be achieved by using a mandrel with a "D"-shaped cross-section.

[0111] The outer electrospun layer is a defined porous polymer fiber layer. The average fiber diameter in the outer layer is approximately 3-6 micrometers. The average pore size is approximately 15-20 micrometers, and the median pore size is approximately 25-45 micrometers.

[0112] The support can be attached to a rotatable member within a liquid medium bath inside the bioreactor chamber. The rotation mechanism may include a magnetic actuator that allows the support, together with the attached support, to rotate about its longitudinal axis within the liquid bath.

[0113] Cells (e.g., MSCs or other stem cells) can be seeded onto the scaffold by depositing a cell solution onto its outer surface. The seeded scaffold is then incubated in a liquid culture medium supporting cell growth by rotating the scaffold in a liquid culture bath within a bioreactor chamber for approximately one revolution. The resulting scaffold comprises a cell sheath overlapping the outer surface of the scaffold. In some embodiments, this cell sheath may have a thickness sufficient to provide structural integrity for the sheath layer. In some embodiments, the cell sheath will consist of a plurality of cells in contact with the outer surface of the scaffold, said plurality of cells being sufficient to guide regenerating cells into contact with the sheath to generate a tissue wall covering the sheath but not fusing with it. In some embodiments, the sheath may consist of a lining with an average thickness between 1 and 100 cells. In some embodiments, the membrane may 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, or between 70 and 90.

[0114] The scaffold 10 with seeded cell sheaths can be implanted into the excision site and placed in an appropriate position. It is conceivable that the seeded cells within the sheath can continue to grow after implantation. In this case, the seeded cells within the sheath will maintain and support a structure that is separate from and connected to the regenerating tissue at the implantation site.

[0115] The appropriate stent was then implanted into the esophagus of the pig. Approximately 5 cm of the esophagus was removed and replaced with the stent portion, which was then sutured to the end of the remaining esophageal tissue in the subject.

[0116] Endoscopic monitoring of esophageal tissue regeneration for several weeks.

[0117] The esophagus is a long, muscular tube that has a neck, thoracic, and abdominal section. Figure 2This diagram shows 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 upper striated muscle, lower smooth muscle, and a mixture of striated and smooth muscle in the middle. Therefore, in some embodiments herein, a multilayered synthetic scaffold is provided that can promote the repair and regeneration of esophageal tissue with two or more layers, corresponding to the natural esophageal tissue layers.

[0118] Figure 3 The image shows stained cross-sections of natural and regenerated esophageal tissue 1–2 weeks after esophageal stent implantation in pigs. The cross-sections reveal regeneration of virtually all layers of esophageal tissue, including distinct muscle and glandular layers. Further analysis of the regenerated tissue shows that the stent itself did not fuse to the regenerated esophageal wall. The stent remains within the esophagus, but it appears to act as a guide to stimulate esophageal regeneration rather than becoming an integral part of the regenerated esophagus.

[0119] Example II: Esophageal Implantation

[0120] like Figure 1A As shown, a synthetic esophageal stent comprising three layers was prepared, featuring an outer electrospun polycarbonate-polyurethane layer deposited from a 12% w / v polycarbonate-type polyurethane solution dissolved in hexafluoroisopropanol (HFIP, DuPont, Wilmington, Delaware, USA). The electrospinning apparatus used was purchased from IME Technologies, Heldrop, Netherlands. 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, producing a stent with an average wall thickness of 500 μm. The stent was dried in a vacuum to remove residual solvent. It was then subjected to plasma treatment using a low-pressure plasma system (Diener Tetra 150-LF-PC-D) with two forward cycles of ethylene and oxygen gas. The stent was then gamma sterilized (STERIS, Northborough, Massachusetts). The gamma dose range used was 25–35 kGy.

[0121] The resulting tubular structure is a polymer scaffold composed of electrospun polyurethane, with a uniform outer diameter (OD) of 22 mm and a length of 11 cm.

[0122] Scanning electron microscopy (Zeiss-EVO MA10) was used to analyze the morphology of the electrospun fibers. Sputtering coating was performed using a sputtering system (Cressington-208HR, TED PELLA, Inc., Reading, California) at a pressure of 8 x 10⁻⁶. -2 The support sample was sputter-coated with platinum and palladium films for two minutes at a voltage of 300V and a pressure of mbar. Porosity was calculated using gravity measurement. Porosity ε was defined based on the apparent density ρAPP of the fiber felt and the bulk density ρPU of the polymer, where ε = 1 - ρAPP / ρPU. The apparent support density ρAPP is measured as the mass-to-volume ratio on a 10 mm dry disc: ρAPP = Mass / VPU. Pore size was measured using a mercury porosity system (Micromeritics AutoPore IV). Tensile tests were performed on 10 mm × 40 mm samples mounted on an electromechanical load frame (Instron 5943 device) using a 1 kN pressure gauge, 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. Figure 7A As shown, the high-magnification scanning electron microscope image reveals the isotropic fiber arrangement of the electrospun synthetic scaffold. The smooth surface and isotropic properties of the fibers ensure that the scaffold exhibits uniform strength and elasticity in all directions.

[0123] Tensile tests with uniaxial mechanical loads were performed on three pre-implantation and three post-implantation stents. Figure 7B All six samples showed similar in vivo loading values. The consistency of the six samples under in vivo loading indicates that the stent has very low variability after manufacturing and implantation. Figure 7B , Figure 7C The mean (±SD) tensile strain of the six stents ranged from 119.5±1.61 mm to 124.5±3.44 mm. Regarding fracture tensile strain, the strain before implantation reached 397.38%±5.52%, and after implantation reached 408.61%±17.64%. The strain value exceeding 400% indicates the reliability of the fabrication process and its relative in vivo stability. The fracture tensile stress at failure before and after stent implantation was 7.25±0.59 MPa and 4.43±0.77 MPa, respectively. Therefore, the Young's modulus before implantation was larger than that after implantation, although the elasticity of the two groups was comparable in terms of in vivo strain. Figure 7B , Figure 7CThe trend of the failure load is the same as that of Young's modulus; that is, the failure load value before implantation is greater than the failure load value after implantation.

[0124] Autologous porcine adipose-derived mesenchymal stem cells (aMSCs) were isolated from eight pigs following open fat biopsy and their characterization was performed. Before aseptic, open adipose tissue biopsy from the lateral ventral wall, eight Yucatan mini-pigs were subjected to general anesthesia and chlorhexidine preparation. A 5 cm incision was made next to the linea alba, and hemostasis was achieved using electrocautery. Approximately 30–50 g of adipose tissue was isolated and transferred to 50 mL conical tubes containing α-minimum essential medium (MEM) / glutamax medium (Thermo Fisher Scientific, Waltham, Massachusetts) and 1% penicillin / streptomycin (Thermo Fisher Scientific).

[0125] Abdominal adipose tissue of 20-60 g was excised from each anesthetized Yucatan miniature pig (50-60 kg). The tissue sample was washed three times in α-minimum 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 fragments smaller than 5 mm. The tissue fragments were dissociated for 55 min at 37°C and 5% CO2 in digestion buffer (300 IU / mL type II collagenase, 0.1% bovine serum albumin (7.5%, fraction V), 1% penicillin / streptomycin, α-MEM / glutamax). After terminating dissociation in complete growth medium (Stem XVivo medium (R&D Systems, Minneapolis, ND) and 1% penicillin / streptomycin), the cells were centrifuged at 1500 rpm for 15 min. Resuspend the cell clusters in 5 mL of growth medium and filter through a 70 μm filter. Centrifuge the cell filtrate at 1500 rpm for 15 minutes, resuspend the cell clusters in 5 mL of growth medium, and seed the cells according to tissue weight (3 g of ex vivo adipose tissue per T75 flask containing 20 mL of growth medium).

[0126] Cells were washed twice with calcium- or magnesium-free PBS (Thermo Fisher Scientific) and dissociated using TrypLe (Thermo Fisher Scientific). Dissociation was terminated with growth medium, and cells were centrifuged at 1000 rpm for 5 minutes. Cell pellets were resuspended in 1% bovine serum albumin diluted with PBS. Aliquots containing 1 million cells were incubated with antibody in the dark at 4°C for 30 minutes (see Supplementary Table 1). Labeled cells were washed three times with buffer, and if necessary, with secondary antibody (Life Technologies, Carlsbad, CA) for 30 minutes in the dark at 4°C. After three more washes, the cell suspension was placed in 96-well plates for flow cytometry analysis (Guava easyCyte HT, EMD Millipore, Bill Ricardo, Massachusetts). Based on viability measurements, gating of events representing live cells using forward and side-scatter values ​​was performed (ViaCount, EMD Millipore). Cell type analysis was performed on unstained samples and antibody-stained samples of isotype controls using fluorescence compensation events. The obtained data were exported and analyzed using standalone software (FlowJo version 10, FlowJo, LLC, Ashland, Oregon).

[0127] To assess colony formation, adipocytes were isolated as described above, homogenized into a single-cell suspension, and diluted to 10 cells / mL of growth medium. 100 μL of this cell suspension was added to each well of a 96-well plate (Corning Incorporated, Corning, NY). Cell counts were visually checked the next day. Colonies were visible after 5–7 days, and the medium was changed every 3 days until each colony contained at least 50 cells. The wells with colonies were counted and expressed as a percentage of the total number of wells.

[0128] The pluripotency of isolated adipocytes was determined by their ability to be chemically induced to form adipocytes and osteoblasts. Cells were seeded separately in 6-well tissue culture plates and cultured in complete growth medium, allowing them to grow to either 60% confluence for adipogenic differentiation or 100% confluence for osteogenic differentiation. Upon reaching confluence, the medium was replaced with either adipogenic or osteogenic differentiation medium (CCM007, R&D Systems, Minneapolis, NU). The medium was changed every 2 days for 14 days. Cells cultured in adipogenic differentiation medium were stained with Oil Red O (American MasterTech, Lottie, CAN) to identify adipocytes, and cells cultured in osteogenic differentiation medium were stained with Alizarin Red (EMD Millipore) to identify calcium deposition.

[0129] The concentrations of glucose and lactate were measured in the conditioned medium of the bioreactor at inoculation and at 2, 5 and 7 days after inoculation (iSTAT, Abbott, Princeton, New Jersey).

[0130] Cell supernatants were analyzed to determine the production of porcine cytokines and growth factors. This could be done using multiplex analysis on a Luminex 200 platform, or by ELISA analysis using commercially available kits at the University of Minnesota Cytokine Reference Laboratory, following the manufacturer's instructions. 13-plex porcine-specific beads (EMD Millipore) were used to determine porcine VEGF, GM-CSF, IL-1ra, IL-6, and IL-8 levels. Values ​​were interpolated from the standard curves generated from each plate. Standard curves could be generated using BioPlex software (BioRad, Hercules, CA) for the Luminex platform, or using microplate reader management software for ELISA plates read on a BioRad 550 reader. All samples were tested repeatedly.

[0131] Cells were washed in PBS and fixed with 10% formalin for 15 minutes at room temperature. Cells were gently washed three 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. Rabbit anti-nesin antibody (Biolegend, 1:100) was diluted in 10% normal goat serum and PBS-T and incubated overnight at 4°C. Cells were washed twice in PBS-T and incubated for 1 hour at room temperature in fluorescently labeled goat anti-rabbit antibody (Alexa Fluor 594, Thermo Fisher Scientific). Cells were washed twice and counterstained with 4′,6-diamidinyl-2-phenylindole (DAPI).

[0132] After counterstaining at 37°C for 48 hours, the cells were washed twice in phosphate-buffered saline (Thermo Fisher Scientific) containing calcium and magnesium, and then replaced with fresh growth culture. The culture medium was then changed every two days until the flasks reached 70%–80% confluence. At passage, the cells were dissociated (TrypLe, Thermo Fisher Scientific), counted (Countess, Thermo Fisher Scientific), and reseeded into T175 flasks at a rate of 200,000 cells / flask.

[0133] Each 11 cm long scaffold was placed in a bioreactor and seeded with 32 million cells (viability >70%, trypan blue staining, Countess, Thermo Fisher Scientific) in growth medium supplemented with 0.1875% sodium bicarbonate, MEM (Lonza), and 0.01 M hydrochloric acid containing 1.19 mg / mL bovine collagen (organogenesis). The cells were incubated at 37°C and 5% CO2 for 5 minutes, and then 200 mL of growth medium was slowly added to the bioreactor. The bioreactor was incubated for 7–8 days before scaffold implantation. The culture medium was changed every 2 days, and various analyses described below were performed.

[0134] Porcine aMSCs were inoculated onto characterized scaffolds and then incubated in a bioreactor. The inoculated scaffolds were then implanted into Yucatan miniature pigs that had undergone esophagectomy, and the scaffolds were removed after 3 weeks. Figure 6 The cells were repeatedly stained using known MSC markers for positive staining (anti-pig CD44, CD73, CD90, CD105, and CD146 antibodies) and for negative staining (CD14, CD45, CD106, CD271, and SLA Class II DR). Over 95% of the cultured cells were positively stained with nestin and αSMA, indicating that stem cell characteristics were maintained during culture. Pluripotency was determined by the ability of the porcine MSC isolates to form adipocytes and osteoblasts, respectively, through chemical induction. These aMSCs were routinely expanded and characterized from passages 1 to 5, exhibiting consistent phenotypic and functional characteristics.

[0135] Second-generation porcine aMSCs were seeded onto polymer scaffolds and incubated in a bioreactor at 37°C for 7 days (+ / - 1 day). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of cytokines and growth factors to determine whether the seeded aMSCs cultured on the scaffold secreted factors that may contribute to angiogenesis and immune regulation. Cell secretions 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 high levels in the conditioned medium. Figure 4A However, other cytokines such as TNF-α, IL-1α, IL-1β, INF-γ, IL-10, IL-12, IL-18, platelet-derived growth factor (PDGF), regulatory factors for the expression of activated normal T cells, and chemokines (RANTES) were measured but were not detected.

[0136] At the end of the 7-day culture period, sectionings of the inoculated grafts were subjected to punch biopsies to assess cell health and scaffold permeability. Cell health was assessed using immunofluorescence staining with calcein (live cells) and ethidium bromide (dead cells). Cell permeability of the scaffold was assessed using ethidium bromide for cell identification. The predominance of calcein staining in the biopsy specimens indicated a live cell population attached to the scaffold. In the cross-section of the scaffold biopsy, most cells were attached to the scaffold surface, although some evidence suggested cell proliferation and growth within the scaffold. Metabolic activity of the grafts was measured every 48 hours during bioreactor incubation to determine glucose uptake and lactate production. Measurements of the conditioned medium consistently showed a decrease in glucose levels and an increase in lactate levels over time, two indicators of sustained metabolic cell growth. Furthermore, cell expansion in the bioreactor over 7 days was quantified by total DNA content, revealing a several-fold increase in total DNA content during bioreactor cell inoculation. Further identification of cell phenotypes on the scaffold after 7 days of culture showed that the cells continuously expressed α-smooth muscle actin (αSMA) and nestin.

[0137] After endotracheal intubation and induction of general anesthesia, the animals were placed in the left lateral decubitus position. Hair was clipped, skin was prepared with chlorhexidine or povidone-iodine, and the animals were aseptically covered. A standard right-sided thoracotomy was performed at the fourth intercostal level in each animal, and access to the thoracic cavity was made. One-lung ventilation was achieved using a double-lumen endotracheal tube. A 4–4.5 cm segment of the esophagus located in the mid-thoracic region (posterior to the right hilum) was circumferentially moved and resected to create a 6 cm defect (tissue contraction at both 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 absorbable sutures of polydioxanone (PDS, Johnson & Johnson Ethicon, Somerville, NJ). Following implantation, a commercially available esophageal stent (WallFlex M00516740, Boston Scientific) was placed under direct endoscopic guidance (Storz Video Gastroscope Silver Scope 9.3mm x 110cm, Tuttlingen, Germany). Stent placement was performed under both endoscopic and surgical visualization. The esophageal stent was secured to normal esophageal tissue using absorbable sutures, positioned at both the proximal and distal stent sites.

[0138] Postoperatively, the animals were supported by gastrostomy feeding and kept on a liquid diet via feeding tube for 2 weeks, followed by a mashed diet for another 2 weeks, and then allowed to eat solid food orally, after which the study continued.

[0139] Approximately 21 days after stent implantation, the stent was retrieved endoscopically and perfused with platelet-rich plasma (PRP) gel infused with aMSCs to enhance the healing process of the newly formed esophageal duct. Following PRP application, a new full-coverage esophageal stent (WallFlex™, 12 cm long x 23 mm outer diameter, Boston Scientific) was placed around the implantation site to prevent stenosis and maintain anatomical structure during regeneration. Animals were sedated every two weeks, and assessments of esophageal anastomosis and stent replacement were performed to allow direct visualization of esophageal regeneration progress. Subsequent endoscopic observation was performed (Storz Video Gastroscope SilverScope 9.3 mm x 110 cm, Tuttlingen, Germany).

[0140] Endoscopic examination was also used to assess regeneration progress. The implantation site was observed endoscopically for approximately 3-4 weeks after stent removal; two representative animals were shown. Figure 10 (See Figure 11). Three to four weeks after implantation, only partial regeneration of the mucosal layer was completed. However, initial ridges formed at the proximal and distal ends of the mucosal layer before the two layers fused, and complete mucosal regeneration indicated that the esophageal healing process continued over time. Early reconstruction of esophageal continuity and integrity, and subsequent growth from the resected submucosa on both opposite sides, were consistent in all eight animal species; two animals maintained this state until 8 and 9 months post-surgery, and both animals showed no signs of esophageal stenosis at 2 and 3 months, respectively, and had sustained oral intake and significant weight gain.

[0141] To determine the histological similarity between the regenerated and native esophageal tissues, tissue samples were taken from representative porcine esophagus 2.5 months post-surgery, and histological examination was performed on tissues including the surgical site and adjacent distal and proximal sections. Figure 13A (The dashed box indicates a histological analysis specimen). Representative images of histological sections stained with hematoxylin-eosin (Fig. 13B and Fig. 13D) and Masson's trichrome stain (Fig. 13C and Fig. 13E) show the morphology of intact multilayered esophageal epithelial cells, submucosa, and normal inner muscular layer.

[0142] Figure 14 illustrates a representative immunohistochemical analysis of the regenerated region, describing the histological analysis of porcine esophageal tissue 2.5 months after implantation of the cellularized scaffold as described herein. Figure 14A Macroscopic images of the resected esophagus (near the left end sutured) are described. Tissue samples were excised to include the surgical site, and the tissue samples and adjacent distal and proximal tissues (dashed boxes) were monitored endoscopically for histological examination. Figures 14B-14E For hematoxylin-eosin ( Figure 14B and Figure 14D ) staining and Masson's trichrome staining ( Figure 14C and Figure 14E Representative images of tissue sections of Ki67. Representative immunohistochemical analyses demonstrate the immunoreactivity of Ki67. Figure 14F This suggests that CD31 in mucosal cells and submucosal cells... Figure 14G ), CD3ε ( Figure 14H ), αSMA ( Figure 14I ), transgelin / SM22α Figure 14J The continued proliferation of striated myosin heavy chain (striated MHC) in the tissue at the surgical site, and the presence of striated myosin heavy chain (striated MHC) Figure 14K The relative absence of Ki67. Scale bar: FK = 200 μm. Immunoreactivity of Ki67 at 2.5 months. Figure 14F This suggests the presence of mucosal cells and submucosal cells, CD31 ( Figure 14G ), CD3ε ( Figure 14H ), αSMA ( Figure 14I ), transaminase / SM22α ( Figure 14J The continued amplification of ) and the presence of rhabdomyosin heavy chains in the tissue at the surgical site ( Figure 14K The relative absence of αSMA and SM22α, and their relative absence compared to myosin heavy chain, indicate that smooth muscle proliferation precedes skeletal muscle growth.

[0143] Inoculation with a synthetic matrix containing autologous-derived mesenchymal stem cells (aMSCs) resulted in adequate longitudinal regeneration of the resected esophagus with minimal mucosal ulceration or perforation (Table 1). Following implant removal, all animals experienced 100% adequate longitudinal regeneration from 2–9 weeks, with 1 in 6 animals experiencing mucosal ulceration or perforation. No animals experienced leakage during the study.

[0144] Table 1

[0145]

[0146] Example III – Other Gastrointestinal Implants

[0147] The procedures described in Examples I and II can be performed by replacing the gastrointestinal tract region confined to the rectum. The results are similar to those previously summarized.

[0148] Several embodiments of the invention have been described herein, and it will be understood that various changes, modifications, and improvements will be readily apparent to those skilled in the art. These changes, modifications, and improvements are also considered part of this disclosure and are covered within the spirit and scope of the invention. Therefore, the foregoing description and drawings are merely examples.

[0149] The terms “a” and “an” as used in this specification and claims shall be understood as “at least one”, unless the opposite is explicitly stated.

[0150] The phrase “and / or” as used in this specification and claims should be understood as “one or both” of two connected elements, meaning that in some cases the two elements exist in combination, and in others they exist separately. In addition to the elements explicitly indicated by the “and / or” clause, other elements may optionally exist, whether or not they are related to the explicitly indicated elements, unless the opposite is explicitly stated. Thus, as a non-limiting example, “A and / or B,” when used in conjunction with open-ended language (such as “comprising”), may in one embodiment mean “A without B” (optionally including elements other than B), in another embodiment mean “B without A” (optionally including elements other than A), in yet another embodiment mean “A and B” (optionally including elements other than A and B), and so on.

[0151] The word “or” as used in this specification and claims shall be understood to have the same meaning as “and / or” as defined above. For example, when distinguished from an item in a group, “or” or “and / or” shall be interpreted as encompassing, that is, encompassing at least one of several elements or groups of elements, but also including more than one, and optionally including other items not listed. Only terms that explicitly indicate the opposite meaning, such as “only one” or “exact one”, or “comprising” as used in claims, will refer to exactly one element from several elements or groups of elements. Generally, when the term “or” as used herein precedes exclusive terms (e.g., “or,” “one of,” “only one of,” or “only one of”), it is interpreted only to indicate a proprietary alternative (i.e., one or another format, but not both). When used in claims as “consisting substantially of,” it shall have the general meaning used in the field of patent law.

[0152] The phrase "at least one" as used in this specification and claims, referring to a group of one or more elements, should be understood as at least one element selected from any one or more elements in the group, but not necessarily including every element specifically listed in the group, and does not exclude any combination of elements in the group. In addition to the elements expressly indicated in the group, this definition also allows for the optional presence of other elements, whether related to or unrelated to those expressly indicated elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalent to "at least one of A or B", or equivalent to "at least one of A and / or B") may, in one embodiment, mean "at least one, optionally including more than one A but no B (optionally including elements other than B)"; in another embodiment, it may mean "at least one, optionally including more than one B but no A (optionally including elements other than A)"; in yet another embodiment, it 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.

[0153] In the claims and the aforementioned description, all transitional phrases, such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “accommodating,” etc., should be understood as open-ended, that is, meaning “including but not limited to.” As set forth in Section 2111.03 of the Patent Examination Procedure Manual of the United States Patent and Trademark Office, only the transitional phrases “consisting of…” and “substantially composed of…” should be understood as closed or semi-closed transitional phrases, respectively.

[0154] 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, or chronological order of performing a method, but is merely used as a label to distinguish one claim element with a specific name from another claim element with the same name.

[0155] While this disclosure has been described in conjunction with certain embodiments, it is to be understood that this disclosure is not limited to the disclosed embodiments, but rather is intended to cover various modifications and equivalent variations within the scope of the appended claims, the scope of which should be interpreted in the broadest possible sense to include all such modifications and equivalent structures permitted by law.

Claims

1. A synthetic scaffold, comprising the following: A polymeric body having a first end and a second end opposite to the first end, at least a portion of the polymeric body being configured as a tubular member, the polymeric body including an inner surface, an outer surface and a central portion defined between the inner surface and the outer surface, the outer surface having at least one region composed of spun polymer fibers with 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 of less than 50 micrometers, and the inner surface, outer surface and central portion being made of a polymer material; The synthetic scaffold further includes at least one aperture, serration, protrusion, or combination thereof, said aperture, serration, protrusion, or combination thereof being located near at least one of the first or second ends, such that, after tissue regeneration occurs around the scaffold at the implantation site on the subject, said aperture, serration, protrusion, or combination thereof is adapted to assist in the retrieval of the scaffold from the subject.

2. The synthetic scaffold as described in claim 1, characterized in that, The spun polymer fibers are interconnected to form an outer layer of the polymeric body. The polymeric body also includes at least one inner layer, which is composed of at least one of a polymer mesh, a polymer braided support material, a solid polymer component, and an electrospun layer. The outer layer covers and contacts the inner layer.

3. The synthetic scaffold as described in claim 2, characterized in that, The spun polymer fiber is an electrospun polymer fiber.

4. The synthetic scaffold as described in claim 3, characterized in that, The electrospun polymer fiber has an average fiber diameter of 3-10 micrometers and is composed of at least one of the following polymer materials: polyvinylidene fluoride, syndiotactic polystyrene, polyvinylidene fluoride-hexafluoropropylene copolymer, polyvinyl alcohol, polyvinyl acetate, copolymer of polyacrylonitrile and acrylic acid, copolymer of polyacrylonitrile and methacrylate, polystyrene, polyvinyl chloride, polyvinyl chloride copolymer, polymethyl methacrylate, polymethyl methacrylate copolymer, polyethylene terephthalate, and polyurethane.

5. The synthetic scaffold as described in claim 2, characterized in that, At least one layer is a polymer material containing polyethylene terephthalate, polyurethane, or a blend of polyethylene terephthalate and polyurethane.

6. The synthetic scaffold as described in claim 2, characterized in that, The polymer braided support material is composed of at least one of polyethylene terephthalate and polyurethane.

7. The synthetic scaffold as described in claim 6, characterized in that, The polymer braided support material also includes nickelitanol.

8. The synthetic scaffold as described in claim 1, 2, or 3, characterized in that, The synthetic scaffold further includes at least one sheath layer composed of cellular material consisting of mesenchymal cells and stem cells present in a defined layer, the defined layer being between 1 and 100 cell thicknesses.

9. The synthetic scaffold as described in claim 8, characterized in that, 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.

10. The synthetic scaffold as described in claim 1, characterized in that, The synthetic scaffold is a tubular component, wherein the outer surface includes spun polymer fibers and a cellular layer, wherein the cellular layer spans at least a portion of the spun polymer fibers located on the outside.

11. The synthetic scaffold as described in claim 10, characterized in that, The cellular layer is composed of cellular material derived from the test subject.

12. The synthetic scaffold as described in claim 1, 2, or 3, characterized in that, The synthetic stent is configured as an esophagus.

13. Use of the synthetic scaffold as described in claim 1 in the preparation of a structure for promoting tissue regeneration in a subject / connecting tubular organs in a subject.

14. The use as described in claim 13, characterized in that, Introducing cellular material onto the polymer surface of the synthetic scaffold; and allowing the cellular material to grow to form a cell sheath.

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