System and method for generating an object having a conduit for specific vascular applications

Abstract

According to an embodiment of the present disclosure, a method for producing an object having conduits is disclosed. Material is deposited onto a sacrificial collector, which is then removed to produce an object having conduits. Both the object and the sacrificial collector can approximate the shape of a desired vessel. The method can also include 3D printing a mold of the desired shape and using gallium as the sacrificial collector. The sacrificial object is produced by inserting gallium into the mold. After removing the gallium from the mold, fibers are electrospun onto the gallium. The gallium is removed via melting, leaving behind a scaffold, the object having conduits. In addition to the gallium, an additional sacrificial layer of water-soluble material can be used.

Description

Technical Field

[0001] The present invention relates to a manufacturing technique for 3D structures. More specifically, the present disclosure is directed to a system and method for generating an object having a conduit for a specific vascular application.

[0002] Related applications / priority rights This application claims priority to U.S. Application No. 17 / 467,340, filed Sep. 6, 2021, and U.S. Provisional Application No. 63 / 075,242, filed Sep. 7, 2020. The application incorporates this provisional application by reference herein for all purposes.

Background Art

[0003] As the leading cause of death worldwide, cardiovascular diseases have a significant impact on society. Current treatments for these diseases consist of revascularization techniques such as angioplasty, stents, or surgical bypass. Vascular bypass surgery forms the basis of revascularization for ischemic heart disease and peripheral vascular disease. Autologous blood vessels such as the saphenous vein and internal thoracic artery are representative grafts for small-diameter blood vessels, but obtaining them poses additional risks. It requires an invasive procedure to be performed on already ill patients. Grafts need to be replaced every 10 to 15 years and are not readily available to patients with extensive systemic vascular diseases. In the United States alone, 1.4 million arterial bypass surgeries are performed annually, but approximately 100,000 patients do not have suitable autologous arteries or veins.

[0004] Due to the invasive and non-sustainable nature of autologous grafts, researchers have selected tissue-engineered vascular grafts (TEVGs). This technology is promising but still does not fully address its potential.

[0005] For a more complete understanding of the present disclosure and its features, reference is now made to the following description in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0006] [Figure 1] This is a diagram of the method according to the disclosed embodiment. [Figure 2-1] Figures 2A and 2B show computer-aided design (CAD) images and actual images of the 3D printed mold and the gallium pieces obtained therefrom. [Figure 2-2] Figure 2C is a close-up view of gallium after it has solidified overnight and been removed from the mold. [Figure 2-3] Figure 2D is an image of the scaffolding after the gallium has melted away. [Figure 3] Figures 3A, 3B, and 3C show the gallium residue. [Figure 4] Figure 4A shows the branched shape of gallium. Figure 4B shows the branched shape of gallium covered with fibers. [Figure 5] Figures 5A, 5B, and 5C show the mold in a combined shape, gallium from the mold, and gallium covered with fibers, respectively. [Overview of the project]

[0007] According to embodiments of the disclosure, a method for producing an object having conduits is disclosed. A material is deposited on a sacrificial collector, which is then removed to produce an object having conduits. Both the object and the sacrificial collector can approximate the shape of a desired blood vessel. The method may also include 3D printing of a mold of the desired shape and the use of gallium as a sacrificial collector. The sacrificial object is produced by inserting gallium into a mold. After the gallium is removed from the mold, fibers are electrospinned onto the gallium. The gallium is removed by melting, leaving behind a scaffold and an object having conduits. In addition to gallium, an additional sacrificial layer of water-soluble material may be used.

[0008] As used herein, the terms “include” and “equip” and their derivatives mean unrestricted inclusion; the term “or” is inclusive and means and / or; the phrases “associated with” and “associated with it” and their derivatives may mean include, contained within, interconnected with, contained within, connected to or with, joined to or with, communicable with, cooperating with, alternating, juxtaposed with, adjacent to, linked to or with, possessing, etc. The phrase “at least one of” when used with an item list means that one or more different combinations of the listed items may be used, but only one item in the list may be required. For example, “at least one of A, B, and C” includes any of the following combinations: A;B;C;A and B;A and C;B and C; and A, B, and C. Definitions of specific words and phrases are provided throughout this patent document, and those skilled in the art should understand that, in most, if not most, such definitions apply to the prior and future use of such defined words and phrases. [Modes for carrying out the invention]

[0009] The figures described below, and the various embodiments used to illustrate the principles of the disclosure of this patent document, are for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way. Those skilled in the art will understand that the principles of the invention of this disclosure can be implemented in any type of appropriately configured device or system. In addition, the drawings are not necessarily drawn to scale.

[0010] While specific advantages of particular embodiments are described, it should be understood that some embodiments may have some or all of the advantages described, or none of them. Furthermore, some embodiments may have additional advantages described.

[0011] A brief overview As the leading cause of death worldwide, cardiovascular disease has a significant impact on society. Current treatments for these diseases consist of revascularization techniques such as angioplasty, stenting, or surgical bypass. Vascular bypass surgery is fundamental to revascularization for ischemic heart disease and peripheral vascular disease. Autologous vessels, such as the saphenous vein and internal mammary artery, are typical grafts for small-bore vessels, but obtaining them carries additional risks. It requires an invasive procedure performed on patients who are already diseased. Grafts must be replaced every 10 to 15 years and are not readily available to patients with widespread systemic vascular disease. In the United States alone, 1.4 million arterial bypass surgeries are performed annually, but approximately 100,000 patients do not have suitable autologous arteries or veins.

[0012] Due to the invasive and non-sustainable nature of autologous grafts, researchers are opting for tissue-engineered vascular grafts (TEVG). While this technique is promising, its potential is still not fully realized.

[0013] Among its many drawbacks, TEVG attempts to mimic the complexity and composition of natural blood vessels. Currently, the technology is limited to flat or cylindrical vessels, which only partially resemble human physiology.

[0014] Considering these difficulties, an ideal vascular graft (and method of production) is disclosed that possesses mechanical strength, adapts to natural biological conditions, and can be manufactured in various forms. The graft mimics the complex shapes and characteristics of the human body and can be personalized for individual patients. The unique and versatile graft production method disclosed herein is a valuable tool for medical researchers and physicians. As a non-limiting example, vascular mimetic bodies can be used in preclinical testing of various devices aimed at treating cardiovascular diseases, reducing the needs (and costs) associated with animal testing. In addition, the artificial blood vessels disclosed herein can be implanted in patients to directly treat cardiovascular diseases.

[0015] In summary, specific embodiments of this disclosure involve 3D printing a mold and casting gallium therein. The shape of the cast gallium resembles a solid version of a desired vascular shape. After curing, the cast gallium is removed and used as a collector during electrospinning of fibers to generate a fibrous scaffold on the gallium surface. The gallium is then melted, leaving a hollow fibrous scaffold. Endothelial cells, smooth muscle cells, and / or mesenchymal stem cells can then be attached to the fibrous scaffold. Over time, the cells rapidly proliferate, generating an extracellular matrix (ECM) and resulting in a vascular (or vascular mimetic) of the desired shape.

[0016] Gallium is selected as a collector material because, in certain embodiments, it can be cast into a wide variety of desired shapes. Gallium can function as a collector in electrospinning because it can melt at a lower temperature than the melting point of PCL (30°C vs. 60°C) and is conductive. The lower melting point of gallium is important because if PCL nanofibers are deformed at high temperatures close to the melting point of PCL, they lose their excellent affinity for cell adhesion. In experiments involving polymers with higher melting points rather than PCL nanofibers, gallium can melt away at higher temperatures, accelerating the gallium removal process. While gallium is described in the specific embodiments described herein, it should be understood that other embodiments may use other materials—some of which function substantially similarly to gallium and other materials that differ slightly in properties from gallium.

[0017] Other substances that may be included, but are not limited to, wax, as well as conductive particles, plus ice, graphene oxide solution, graphite, carbon (crushed or ground), sand / particles / fine powder / powder, and wax with added conductive polymers. Gallium alloys, or other metals and their alloys, plus, but not limited to, bismuth or indium-based alloys.

[0018] Additionally, while certain embodiments describe "melting" a substance to remove such material, other embodiments may alternatively dissolve the material to remove such material.

[0019] As前述の非限定的な例として, other methods of removing an object (which may be referred to as a sacrificial object or sacrificial collector) include, but are not limited to, dissolving with a solvent (e.g., water, PBS, blood, acetone, HFIP, ethanol, acid, etc.), melting (e.g., at not only atmospheric pressure but also sub-atmospheric pressure), scraping, deflating, evaporating (e.g., including in a vacuum), decomposing (e.g., enzymatic decomposition, organism-based decomposition, cell-based decomposition, network cleavage, chain cleavage, or chelation), centrifugal force, mechanical separation (connected together and disconnecting the connection, pieces made separately), and use of stimuli-responsive materials (e.g., pH, ionic strength, electrically induced decomposition, magnetism, radiation-based decomposition, and ultrasonic decomposition). Also, any of the above may include a washing step.

[0020] Regarding sub-atmospheric pressure (or vacuum), the decrease in pressure lowers the melting point or evaporation point and enables a temperature decrease for the necessary phase change.

[0021] Specific descriptions of creating / forming a sacrificial object or a mold for a sacrificial object are described below. While specific descriptions are given, other methods can be used including, but not limited to, 3D printing (e.g., SLA, FDM, sintering, etc.), soft lithography, molding / casting, vacuum forming, growth, glass blowing, weaving, carving, stamping, spin coating, curving, CNC, sintering, freezing / freeze drying / rapid freezing, other additional techniques, other subtractive techniques, and any combination of those previously mentioned.

[0022] It should be noted that there are some inaccuracies in the original Chinese text. I have tried my best to translate it according to the content you provided. If you have any further questions or need more accurate translations, please feel free to let me know.The mold itself for the sacrificial collector can also be sacrificial. Due to the high value of the complex shapes in the collector, shapes and forms that cannot be removed from conventional molds may be desirable. A 3D printed mold that is then chemically removed by a process that does not interact with the collector material is an envisioned embodiment. The sacrificial collector material and the sacrificial mold material are selected such that removal of the mold is possible without significantly affecting the shape or other important properties of the sacrificial collector.

[0023] As non-limiting examples of the foregoing, other methods of removing the sacrificial mold include, but are not limited to, dissolving using a solvent (e.g., water, PBS, blood, acetone, HFIP, ethanol, acid, etc.), melting (e.g., at not only atmospheric pressure but also sub-atmospheric pressure), scraping, deflating, evaporating (e.g., including in a vacuum), decomposing (e.g., enzymatic degradation, organism-based degradation, cell-based degradation, network cleavage, chain cleavage, or chelation), centrifugal force, mechanical separation (connected integrally and disconnecting the connection, pieces made separately), and the use of stimuli-responsive materials (e.g., pH, ionic strength, electrically induced degradation, magnetism, radiation-based degradation, and ultrasonic degradation). Also, any of the above may include a washing step.

[0024] Specific details As suggested above, cardiovascular diseases are the leading cause of death worldwide, and their incidence is predicted to increase in the coming decades. The impact of these diseases is significant, costing the US healthcare system $219 billion annually, including the cost of medical services, medications, and productivity losses due to death. Heart diseases are often associated with narrowing or blockage of blood vessels, reduced blood flow, restricted nutrient supply, and tissue damage. They can exist in many forms, including coronary artery disease, cerebrovascular disease, peripheral artery disease, and deep vein thrombosis.

[0025] Bypass grafting is a common treatment for cardiovascular disease. Most involve replacing diseased vessels with grafts of the patient's own artery, or more commonly, the saphenous vein. While such autoreplacements are effective in some respects, they have limited availability, require invasive procedures for harvesting, and must be replaced every 10 years. These natural autoreplacements are not always available in patients with severe vascular disease. For pediatric patients, bypass grafting every 10 years would represent a very challenging, lifelong prospect. Furthermore, bifurcated vascular grafts are needed for several indications, including reconstruction of the superficial palmar arch of the hand, reconstruction of congenital vascular / cardiac malformations, and coronary artery bypass grafting. Using available autoreplacement grafts to reconstruct bifurcated vessels can prolong surgery and increase the risk of postoperative complications.

[0026] Due to the difficulties associated with autologous grafts, many researchers view TEVGs as a promising alternative. Early versions of these TEVGs utilized synthetic materials such as ePTFE, Dacron®, and polyurethane. These materials offer ease of adjustment and flexibility in graft design and have proven effectiveness for replacing large and medium-diameter arteries such as the carotid or common femoral artery. However, these materials are limited by poor elasticity, low compliance, and thrombus formation at the synthetic surface, making them unsuitable for grafts smaller than 6 mm in diameter.

[0027] To address these issues with synthetic TEVGs, many researchers are currently using biodegradable polymers as a scaffold for growing cell layers. As the scaffold degrades, it is replaced and reshaped by ECM secreted by cells. ECM is the main component of the outer layer of blood vessels and contains elastin and collagen fibers. The fibrous structure of ECM contributes not only to mechanical integrity but also to the elasticity of blood vessels. For example, the recoil of the elastic fibrous arterial wall during cardiac diastole promotes the continuation of blood flow. In addition, elastic fibers in the elastic layering of blood vessels contribute to the concentric arrangement of smooth muscle cells.

[0028] Currently, electrospinning is a preferred method for generating nanofiber vascular scaffolds. However, the shape of electrospinned nanofiber scaffolds is limited to flat or cylindrical shapes that do not resemble the shapes of natural human blood vessels. When the desired scaffold has a complex shape, researchers and manufacturers must employ redundant post-processing methods, which still have shape limitations, resulting in inaccurate shapes and / or insufficient fibrous properties.

[0029] The inadequacy of artificial blood vessel grafts in mimicking the complexity and diversity of organic biological forms is a major challenge facing the field. The U.S. Food and Drug Administration recommends that medical devices, such as stents, designed specifically for coronary arteries, should simulate the worst-case scenario of tortuous tissue. They should be adaptable to bends or bifurcations in vessels and should be tested in simulated vessels representing the most clinically challenging tissues observed.

[0030] Vascular grafts, according to specific embodiments, are permanent and functionally effective synthetic blood vessels designed to mimic the biomorphology of natural blood vessels while maintaining and promoting biological function at the micro level. In certain configurations, such techniques possess mechanical strength and can withstand prolonged hemodynamic stress. They are non-toxic, non-immunogenic, biocompatible, thrombosis-resistant, and available in various sizes and forms for emergency medicine.

[0031] Embodiments of this disclosure also provide a more versatile method for developing artificial vascular grafts for the treatment of cardiovascular disease. The specific method has the ability to generate vascular mimics that replicate the shape and complexity of blood vessels and a wide variety of natural vascular shapes. A non-limiting example of an outline of the steps follows. Such general-level steps are described, but more or fewer steps may be provided. Such steps, along with further details corresponding to such steps, can be grasped in whole with reference to Figure 1 below. Step 1. Custom 3D printing 2. Molten gallium is cast into a mold to create the desired vascular shape composed of gallium, allowing the gallium to cool and solidify. 3. Remove the solidified cast gallium from the mold. 4. Electrospinning nanofibers onto cast gallium. 5. Dissolve and remove the gallium, leaving behind a nanofiber scaffold in the desired shape. 6. Seed cells onto the scaffold. 7. Allows cells to rapidly proliferate over several weeks to form the extracellular matrix (ECM).

[0032] After these steps, the resulting vascular mimetic becomes suitable for testing devices such as diverters, stents, and aneurysm coils. And, in specific configurations, the vessel or scaffold may be suitable for implantation.

[0033] While other techniques are limited to simple shapes, this disclosure enables the creation of blood vessels that conform to the complex structure of natural blood vessels. This means that the disclosed design can be used in a wide range of cardiovascular applications. Where multiple grafts are required (e.g., double, triple, and quadruple bypass grafts), the disclosed method enables the creation of a single branched graft, which reduces surgical time, limits the number of incisions (where the graft is sutured), and eliminates the need to obtain one or more veins / arteries from other parts of the human body. This is just one example of several possible applications. Further applications requiring complex fibrous scaffolds to which this disclosure is applicable include the formation of complex biological structures such as human or animal fibers and tissues. Further additional applications will be apparent to those skilled in the art who have read this disclosure.

[0034] The global tissue engineering and regeneration market is projected to reach $109.9 billion by 2023, with a compound annual growth rate of 34.8%. Due to its cost-effectiveness, 3D printing is seen as a novel tissue engineering strategy and has become one of the top drivers of the market.

[0035] The global tissue engineering market is expected to grow further due to the increasing attention on tissue engineering-based therapies. The application of nanotechnology, specifically the 3D design of nanofibers to aid in cell regeneration, has driven the growth of tissue engineering products. As a result, the nanofiber materials market was valued at $3.6 billion in 2018.

[0036] A gap exists between the need for bioengineered blood vessels and the limited range of clinically available options. Approximately 100,000 patients each year who require grafts lack suitable autologous arteries or veins. Nevertheless, the demand for vascular tissue repair persists due to the persistence of ischemic diseases such as atherosclerosis. In the United States alone, 1.4 million patients require arterial replacement annually, a treatment costing a total of $25 billion.

[0037] Currently, companies are forced to conduct expensive initial device testing on animals because there are not enough vascular mimetic devices available. The disclosed vascular mimetic devices would offer an alternative, less expensive option for device testing that does not involve animals. This would improve animal welfare while helping companies save thousands of dollars and identify problems earlier before spending time and money on complex animal experiments.

[0038] Another market available through this disclosure is the implantation of artificial blood vessels into patients for the treatment of heart diseases such as peripheral artery disease and coronary heart disease. An estimated 340,000 coronary artery bypass grafting (CABG) procedures are performed annually in the United States to treat these diseases. CABG is one of the most commonly performed cardiovascular surgeries, and therefore there is ample room to introduce our proprietary technology into this market. The projected cardiovascular regeneration market is expected to reach $4.14 billion by 2023.

[0039] Currently, the most common CABG procedure uses the patient's own blood vessels, such as the saphenous vein. However, obtaining autologous grafts requires additional surgery, which often makes them unsuitable for use. This disclosure describes a personalized graft design that possesses the structural and functional characteristics of natural blood vessels without the complications associated with autologous grafts.

[0040] The disclosed technology aims to closely replicate the microstructure of natural blood vessels by generating nanofiber scaffolds with complex macromorphologies. Existing alternatives are unable to achieve these complex nanofiber structures.

[0041] The disclosed innovation is a simple alternative to current methods for manufacturing nanofiber vascular scaffolds having complex, irregular, or customized shapes.

[0042] Figure 1 discloses a method according to an embodiment of the disclosure. Referring to label A, two pieces of a mold are illustrated. In a particular configuration, the pieces of the mold may be 3D printed using a wide variety of techniques that will become apparent to those skilled in the art who have read this specification. In non-limiting examples, both addition and removal techniques may be used to produce an object of a desired shape that will be used as a sacrifice. Further examples are described below. Two pieces of the mold are shown here, but more than two pieces may be used in other configurations.

[0043] Referring to label B, a casting of molten gallium is illustrated. While gallium is described in this particular configuration, a wide variety of other materials may also be used, as described below.

[0044] Referring to label C, after gallium coagulation, its shape resembles the desired vascular shape (in this case, a branched aneurysm with complex curvature and variable diameter). In certain configurations, in addition to gallium, another substance may be added as an additional sacrificial layer, as described below. For example, gallium may be coated with another sacrificial layer that functions to protect the fibers described below from direct contact and exposure to the gallium. Non-limiting illustrative methods for depositing the sacrificial layer include electrospraying or electrospinning, or any other spraying method, or immersion coating. Other methods will become apparent to those skilled in the art who have read this specification. The sacrificial layer may be any kind of water-soluble substance, such as PEG (polyethylene glycol), water-soluble wax, etc. In certain configurations, gallium provides a structural basis for a particular desired design, while the additional sacrificial layer provides protection against specific interactions between gallium and fibers. Thus, the use of different substances can both be utilized—either of which will be sacrificially removed after fiber coating.

[0045] Referring to label D, nanofibers are electrospinned onto solidified gallium, causing the gallium to dissolve and disappear. If additional layers are used, these additional layers can also be removed. As mentioned below, electrospinning is described for certain configurations, but other configurations may employ other techniques to add the fibers to a sacrificial collector—in this case, gallium and potentially another layer.

[0046] After the removal of gallium (and any additional layers, if used), what remains is a nanofiber scaffold of the desired shape, as illustrated with reference to label E. Electrospinning requires a conductive collector that needs to be removed after the nanofibers are positioned. This is why gallium is useful. Gallium is conductive (and therefore can function as a good collector in the electrospinning process) and can be easily removed from the electrospinned scaffold by melting at temperatures above 30°C and below the melting point of common polymers used in tissue engineering (such as PCL).

[0047] Referring to label F, cells are then seeded onto a nanofiber scaffold, which rapidly proliferates in a bioreactor, generating an extracellular matrix (ECM) over time. After several weeks, the blood vessels are ready for use—including for research and development purposes. Depending on their shape and suitability, the scaffold or grown blood vessels may also be used for medical purposes (transplantation).

[0048] The disclosed techniques overcome the morphological limitations of other procedures using 3D-printed metal, machined collectors, molded aluminum foil, or machined molds. In one technique, a grounded spinning rod to which small compressed pieces of aluminum foil are attached is used as an electrospinning collector. The metal rod represents blood vessels, and the aluminum foil represents the growth of aneurysms. Nanofibers are simultaneously electrospinned onto the rod and aluminum foil while rotating.

[0049] The main problem with this method arises when the rod is removed. The aluminum foil is trapped inside the shape of the aneurysm and secured in place by electrospinned nanofibers. An incision is made in the nanofiber scaffold and the foil is removed. The incision leaves an undesirable opening in the scaffold. The rod is reinserted into the scaffold and electrospinning is started again. This covers the incision with more nanofibers, but leaves an undesirable thickness where the incision was. The method also cannot smooth out the incision inside the scaffold. This is a laborious process, resulting in undesirable shape defects and limiting the form to very simple shapes. Workarounds exist, but they result in limited shapes and require laborious post-processing.

[0050] Existing and inadequate methods for producing custom-molded TEVGs demonstrate the need for the disclosed technology. In particular, this technology has the potential to produce shapes that cannot be achieved by other methods and that minimize the number of post-processing steps.

[0051] A key factor in the success of transplanted TEVGs is their ability to mimic the natural microenvironment of the extracellular matrix (ECM). Electrospinning is considered one of the most flexible scaffold fabrication methods. Electrospinning allows for a wide range of flexibility in designing the composition of nanoscale fibers and their associated mechanical and biological functions to mimic the natural microenvironment of the extracellular matrix. Electrospinning enables the formation of 3D networks made of fibers with diameters ranging from 50 to 500 nm, a size range found in natural tissues. Our use of cast gallium in 3D printed molds further improves this technique by allowing collector shapes to be more complex than plates or cylinders. This invention creates the ability to generate nanofiber scaffolds in virtually unlimited shapes.

[0052] Attempts have been made to mimic more complex structures, with varying degrees of success. Tiffany W. Shen et al. developed and tested scaffolds for fusiform and saccular aneurysm-shaped vascular mimics. Cells were seeded into these scaffolds and used to test diverters. While this method successfully generated aneurysm vascular mimics of multiple shapes, it was time-consuming due to the need for CNC machining processes, and the method could not be adapted to a wide range of shapes.

[0053] In a study by Chavez et al., cells were cultured on an ePTFE scaffold in a bioreactor. The scaffold had a complex shape with several branching and curving options on which the cells were consistently positioned throughout. Although successful, this method was limited by the use of ePTFE. ePTFE does not exhibit the same elastic modulus as natural arteries, is not bioabsorbable, and has low patency over time.

[0054] Fukunishi et al. 3D printed stainless steel into the shape of a desired blood vessel and then electrospinned nanofibers onto it. This resulted in a simple cylindrical shape that is slightly bendable and, like other techniques, limited in shape due to the fact that the researchers had to remove the 3D printed steel after electrospinning without allowing it to melt away.

[0055] This disclosure provides an improved electrospinning method using a gallium-based collector, which can be used to generate nanofiber scaffolds with complex shapes that are impossible or difficult to achieve using conventional electrospinning methods. Furthermore, the disclosed method can be used to manipulate nanofiber scaffolds from a variety of biomaterials and potentially biocompatible polymers.

[0056] experiment The applicant conducted experiments to obtain preliminary data regarding the process for producing a gallium-based collector. The applicant 3D printed a mold and cast molten gallium into it (Figure 2A). The cavity in the mold was shaped like a 4 mm blood vessel with a small aneurysm. The gallium was removed after solidification overnight (Figure 2B).

[0057] The applicant electrospinned polycaprolactone (PCL) onto cast gallium using gallium as the collector. The PCL was 14% w / v and electrospinned at a flow rate of 0.1 mL / min over a distance of 6 inches. The voltage started at 12 kV and was increased to 18 kV after 10 minutes. The gallium was held at 0 volts (grounded) and rotated slowly (60 RPM). A needle was moved over the gallium and back and forth to spread the nanofibers along the length of the gallium collector. The total time taken to complete this process was 20 minutes.

[0058] The gallium and scaffold were placed in a rotating oven at 38°C, and by manual stirring, a large amount of gallium melted away after 30 minutes. The process was repeated twice until all the gallium had melted and been removed, leaving a nanofiber vascular scaffold (Figure 2D). Representative images of the steps for producing a gallium-based collector according to embodiments of this disclosure are shown in Figures 2A-2D. To create a columnar vessel with an aneurysm, first a CAD file for the mold was designed (Figure 2A), then the mold was 3D printed (Figure 2B), and molten gallium was cast into the mold. The mold was placed in a pressure chamber to remove air bubbles and solidify the gallium. The gallium vessels can be removed seamlessly from the mold (Figures 2B and 2C). One common challenge during the casting step is flushing. Flashing, also known as casting burrs or flash, can be described as unwanted and excess material adhering to the casting in contact with the mold pieces. To minimize flushing, the mold pieces were fastened together with bolts and nuts rather than elastic bands. Furthermore, the 3D-printed mold was produced using a flexible material, which, compared to a non-flexible mold, allowed the compressive force to completely seal the space between the mold surfaces and made the removal of the solidified gallium much easier. Small vents were also designed on the mold to prevent the formation of trapped air bubbles within the gallium, allowing air to escape while the gallium was being cast. In addition, by placing the mold and gallium in a pressurized chamber, the size of any remaining bubbles was reduced to a microscopic scale. The gallium collector was then placed on our EHD printing press and rotated at a speed of 60 rpm. Polycaprolactone (PCL) was electrospinned onto the collector over a distance of 6 inches. The print head was connected to 15kV, and the collector was grounded. A needle was moved over the gallium and back and forth to spread the nanofibers along the length of the gallium collector.

[0059] Gallium was removed by placing the gallium collector and scaffold in an oven at 38°C for 1 hour. The nanofiber scaffold after gallium removal is illustrated in Figure 2D.

[0060] To minimize the remaining traces of gallium, a thin layer (50 μm) of water-soluble polyethylene glycol (PEG) polymer was first electrosprayed onto the gallium collector, followed by electrospinning of PCL. The PEG served as a protective layer to reduce direct exposure of the PCL fiber layer to gallium and was dissolved in water after gallium removal. Our results showed that when the protective PEG layer was used, there were no visually detectable gallium residues on the surface of the nanofiber layer (Figure 3B). Figure 3A shows gallium and PCL, while Figure 3C shows stainless steel, PCL, and no PEG. In addition, the temperature and length of the post-treatment step in the oven were increased to 40°C and 5 hours, respectively, to optimize the gallium removal process. After optimizing the process for generating simple cylindrical shapes with aneurysms (Figures 2A-D), the method was used to generate nanofiber structures with more complex branched shapes. One of the initial observations of branched shapes was the possibility of fiber webbing due to the long fiber length and electric field orientation in the space between two gallium branches (Figure 4A). When the molecular weight of the PCL polymer being formed was reduced from 80 kDa to 24 kDa, webbing was minimized, while a uniform coating of gallium with continuous fibers was present (Figure 4B).

[0061] Using the optimized method described above with reference to Figures 2A-2D, structures with a bifurcated shape containing an aneurysm were generated (Figures 5A, 5B, and 5C). More specifically, Figure 5A shows a mold of the bifurcated shape. Figure 5B shows the bifurcated shape of gallium from the mold. Figure 5C shows the bifurcated shape of gallium covered with fibers.

[0062] While this disclosure describes specific embodiments and commonly associated methods, variations and modifications of these embodiments and methods will be apparent to those skilled in the art. Other variations, alternatives, and modifications are also possible without departing from the spirit and scope of this disclosure as defined by the following claims.

Claims

1. A method for generating a fibrous scaffold having conduits, Electrospinning fibers onto a sacrificial collector that includes a conductive sacrificial material and a sacrificial layer on top of the sacrificial material, This includes generating the fibrous scaffold having the conduits by removing the sacrificial collector from the electrospun fibers, The sacrificial layer is deposited on top of the sacrificial material by electrospray or electrospinning. The method wherein the material of the sacrificial layer is different from the sacrificial material.

2. The method according to claim 1, wherein both the fibrous scaffold and the sacrificial collector approximate the shape of a blood vessel.

3. The method according to claim 1, wherein the sacrificial material includes gallium, a gallium alloy, bismuth, a bismuth alloy, indium, or an indium alloy.

4. The method according to claim 1, further comprising generating the sacrificial collector by depositing the sacrificial material into a mold.

5. The method according to claim 4, further comprising generating the mold for the sacrificial material.

6. The method according to claim 5, wherein the mold is 3D printed.

7. The method according to claim 1, wherein the sacrificial collector is removed from the electrospun fibers by melting the sacrificial material.

8. The method according to claim 7, further comprising reducing the environment in which the melting occurs to subatmospheric pressure before the melting, thereby lowering the melting point of the sacrificial material.

9. The method according to claim 1, wherein the sacrificial layer is deposited on top of the sacrificial material to prevent the sacrificial material from directly interacting with the electrospun fibers deposited on the sacrificial collector.

10. The sacrificial material is gallium, The method according to claim 9, wherein the sacrificial layer is a water-soluble material.

11. The sacrificial collector is generated by depositing the sacrificial material into a mold, Removing the aforementioned sacrificial material from the mold, The method according to claim 1, further comprising depositing the sacrificial layer on the sacrificial material.

12. The method according to claim 10, wherein the water-soluble material is polyethylene glycol or a water-soluble wax.

13. The method according to claim 1, further comprising rotating the sacrificial collector while electrospinning the fibers onto the sacrificial collector.

14. Seeding cells onto the fibrous scaffold, and The method according to claim 2, further comprising maturing the seeded fibrous scaffold in a bioreactor to generate a vascular mimetic.

15. The method according to claim 14, wherein the cells are endothelial cells, smooth muscle cells, or mesenchymal stem cells.

16. The method according to claim 1, wherein the fiber is a polycaprolactone fiber.

17. The method according to claim 1, wherein the fiber has a diameter in the range of 50 nanometers (nm) to 500 nm.

18. The method according to claim 1, wherein the conductive sacrificial material comprises a material selected from a conductive polymer, a wax, a wax with conductive particles added, graphene oxide particles, graphite particles, and carbon particles.

Images