MEW tissue scaffold

Abstract

A fusion electrowriting soft tissue scaffold comprising: a first region having one or more sets of fibers; a second region having one or more sets of fibers; and an interface region joining the first region and the second region, the interface region being electrowritten together with the first region and the second region in a successive printing pass such that the fibers in the interface region are bonded between individual pairs of fibers in the first region and the second region, respectively.

Description

[Technical Field]

[0001] The present disclosure relates generally to implantable devices or scaffolds, such as those for engineered heart valves. More specifically, the present invention relates to an interface between a structural region and a gradient within the scaffold.

[0002] (Incorporated by reference) The present inventors have previously developed a novel method of providing scaffolds or implants using fused electrowriting (MEW), as disclosed in Patent Cooperation Treaty Application No. PCT / AU2020 / 210877, the contents of which are incorporated herein by reference. [Background technology]

[0003] Biological tissues are complex, multiphasic, heterogeneous, and hierarchical structures that ideally exhibit sophisticated properties that are well suited to their function. Furthermore, tissue interfaces not only physically connect heterogeneous regions, but also exhibit graded structural, cellular, and mechanical characteristics, and thus importantly, play a crucial role in contributing to the overall functionality of the tissue or organ. Therefore, interfaces pose a crucial yet challenging challenge when attempting to bioengineer scaffolds for these tissues.

[0004] The majority of research on tissue interface engineering concerns soft-hard tissue interfaces in areas such as ligaments and tendons, cartilage, and dental and maxillofacial implants. In these applications, heterogeneous scaffolds have been achieved through advanced manufacturing techniques such as multi-screw extrusion, varying cross-linking degrees, two-stage phase separation, multi-material bio-inks, and through controlled spatial deposition of biomaterials using advanced three-dimensional (3D) printing technologies.

[0005] Fibre scaffolds, particularly electrospun meshes, have been widely investigated in the field of soft tissue engineering for applications such as skin, nerve, vascular, or cardiac tissue. However, comparable research on interface and gradient design is scarce. Interfaces between domains have primarily been achieved layer-by-layer, either by modifying printing parameters or solution concentrations during the fabrication process, followed by cross-linking, or by individual layer-by-layer assembly. The absence of interface complexity and gradient structures for electrospun scaffolds may be due to the lack of precise control of fiber orientation when fabricating using this technique.

[0006] MEW is a highly precise additive manufacturing technique capable of printing complex fibrous scaffold structures at submicron resolution. The complexity and resolution characteristics of MEW make it beneficial for creating functional biomimetic soft tissue scaffolds for skin, nerve, myocardium, cartilage, and aortic heart valves. Similar to electrospinning, interfaces on a layer-by-layer basis have been demonstrated using MEW. More recently, the ability to fabricate heterogeneous fibrous structures within layers using MEW has been demonstrated, representing a significant advance in the field of fibrous scaffold fabrication. Nevertheless, the concept of heterogeneous MEW printing is still in its infancy, and as a result, designing interfaces between heterogeneous regions has not been a primary focus in previous studies.

[0007] Where any prior art is referred to herein, it will be understood that such reference does not constitute an admission that the prior art forms part of the common general knowledge in the art in Australia or any other country. Summary of the Invention [Means for solving the problem]

[0008] In a first aspect, disclosed herein is a fused electrowritten scaffold comprising a first region having one or more sets of fibers and a second region having one or more sets of fibers, the first region and the second region being joined by an interface region, the interface region being electrowritten together with the first region and the second region along successive printing paths within the same layer such that the fibers in the interface region bond between individual pairs of fibers in the first region and the second region, respectively.

[0009] The scaffolds may be used for the engineering of biodegradable or non-biodegradable implantable devices.

[0010] The first region and the second region can be heterogeneous in that they differ from one another in one or more spatial parameters.

[0011] The path for at least one of the fibers in the interface region can be defined manually or by a mathematical function.

[0012] The function can be such that the fiber has a complex shape as it transitions from one of the first region or the second region, through the interface region, to the other of the first region or the second region.

[0013] The second region can have a higher porosity than the first region.

[0014] The higher porosity in the second region can be formed, at least in part, by joining all of the two or more adjacent fibers in the first region as they transition into the second region.

[0015] The higher porosity in the second region can be created, at least in part, by causing the fibers to fan outward as they transition into the second region.

[0016] The higher porosity in the second region can be formed, at least in part, by setting successive printing passes to cause additional sets of fibers to be deposited that are offset from other fibers in the first region by a distance that is less than the size of the pore size.

[0017] The first region or the second region or both can include a first set of fibers arranged generally parallel to one another and a second set of fibers arranged generally parallel to one another, the second set of fibers arranged at an angle, preferably transverse to the first set of fibers, and each fiber in the second set of fibers has a serpentine arrangement with defined valleys and peaks.

[0018] The fiber arrangement in the interface region can be, at least in part, biomimetic. For example, the fiber arrangement can be based on a simplified form of collagen fiber organization observed in the interface region of a biological sample of soft tissue. The collagen fiber organization can be determined by orientation analysis.

[0019] The scaffold can have multiple layers of fibers.

[0020] A portion of the scaffold can have a different number of layers than another portion of the scaffold. The scaffold can be a heart valve scaffold. The first region can be a valve leaflet and the second region can be an inter-leaflet trigone.

[0021] In another aspect, disclosed herein is a method of providing a melt-electrowritten soft tissue scaffold having at least two structurally heterogeneous regions and an interfacial region between the at least two structurally heterogeneous regions, comprising providing a printing path during the melt-electrowriting process along which a polymer melt material is continuously extruded to form the heterogeneous regions and the interfacial region.

[0022] The method can include causing successive print paths to be defined piecewise, the successive print paths being defined by different functions in the heterogeneous regions and the interface regions.

[0023] In another aspect, also disclosed herein is a method for designing a MEW scaffold for soft tissue having multiple structural regions, the method including: imaging the soft tissue using an imaging modality capable of imaging collagen fiber structure within the soft tissue; identifying interface regions from the structural regions; applying orientation analysis to at least the image from the imaging step to indicate the interface regions; determining a simplified morphology of fiber distribution observed in the orientation analysis; and configuring a printing path for an interface gradient region of the scaffold corresponding to the interface region of the soft tissue based on the simplified morphology of fiber distribution.

[0024] The function or functions that define the continuous printing path as it traverses the interface gradient region are preferably mathematical functions that provide a curved or serpentine shape.

[0025] The present invention has scaffold-based biomedical applications because natural tissues are very rarely homogenous, i.e., gradients exist throughout any type of tissue.

[0026] In one aspect of the invention, the scaffold comprises a gradient of fibers comprising the scaffold. Gradient refers to a gradual spatial change in property. The change can be related to geometric characteristics (e.g., size, thickness, shape, density, orientation), mechanical properties (stiffness, elasticity), composition (cell / tissue type), chemical properties (pH), or other. The scaffold may be used to create multiple gradient properties within a single scaffold.

[0027] For example, disclosed herein is a fused electrowriting scaffold with gradient porosity. The term "gradient porosity" or "porosity gradient" refers to a change in the size and / or shape of pores within a scaffold across a region of the scaffold. The size of the pores may gradually increase or decrease in one or two directions spatially across the scaffold. The gradient change in porosity is determined by a mathematical function, and the shape of the pores within the scaffold can be, for example, rectangular, diamond-shaped, or serpentine.

[0028] Preferably, the scaffold comprises multiple layers of fibers. The multiple layers of fibers may comprise a gradient in geometric characteristics and / or mechanical properties, forming a gradient scaffold. Each fiber of the scaffold may comprise a gradient in geometric characteristics and / or mechanical properties, forming a gradient scaffold. For example, the fibers may be thicker, denser, and / or stiffer in one region and thinner, less dense, and / or more flexible in another region, along with the gradient. The function or functions defining the successive printing paths across the gradient region are mathematical functions that define the geometric characteristics and / or mechanical properties of the fibers within the region.

[0029] The scaffold gradient design may be used in either all or part of the area of ​​the scaffold.

[0030] Gradient functions may be generated for heart valve applications that incorporate the patient-specific anatomy of the heart valve as inspired by the orientation, distribution, and density of natural collagen fibers.

[0031] The scaffold fibers forming the porosity gradient may be intentionally aligned to be either parallel or perpendicular to the interface between the two regions. In the context of heart valves, this would mean aligning them with the interface between the valve leaflets and the inter-leaflet trigone / commissure / annulus.

[0032] The porosity in the gradient may be arranged to reflect areas of higher load, with less in the commissure regions and more in the center of the leaflets, or vice versa.

[0033] The fiber density gradient may be arranged to reflect areas of higher loading, higher in the commissure regions and central leaflet, above the annulus, and lower inside the leaflets, or vice versa.

[0034] Any of the above types of gradients (geometric features, mechanical properties, composition, chemical properties, etc.) may be combined or applied individually to engineer fused electrowriting scaffolds for heart valves or other tissues. [Brief explanation of the drawings]

[0035] Embodiments will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] FIG. 1 illustrates diagrammatically the components of a MEW device. [Figure 2] FIG. 2 is a digital microscope image of an exemplary MEW scaffold with two heterogeneous regions and a continuous interface between the heterogeneous regions. [Figure 3-1] Figure 3-1 is a schematic depiction of a scaffold with constant porosity throughout. [Figure 3-2] FIG. 3-2 is a schematic depiction of a scaffold in which pairs of fibers from the scaffold of FIG. 3-1 have been joined together, doubling the pore size. [Figure 3-3] FIG. 3-3 is a schematic depiction of "halving" a scaffold whereby another fiber layer is added to the scaffold to halve the pore size in one section of the scaffold. [Figure 3-4] Figures 3-4 are schematic depictions of "fanning" of the scaffold, whereby the fibers in the second (right) section are fanned out to gradually increase the pore size in that section. [Figure 3-5] Figures 3-5 are exemplary images of MEW-fabricated devices in which "halving" has been performed within a section of the scaffold. [Figure 3-6] 3-6 are exemplary scaffolds with sections in which the fibers are fanned out to reduce pore size. [Figure 3-7] 3-7 are exemplary scaffolds with sections in which the fibers are fanned out to increase pore size. [Figure 4-1] Figure 4-1 includes a schematic depiction of overlapping, sutured, and continuous interfaces. [Figure 4-2] Figure 4-2 shows digital microscope (top) and SEM (bottom) images of MEW PCL scaffolds with 1 mm pore square patterns and 0.5 mm pore diamond patterns connected using overlapping, stitching, and continuous methods (scale bar = 1 mm). [Figure 5-1] Figures 5-1 through 5-3 show images acquired during uniaxial tensile testing of biphasic MEW scaffolds connected using either overlap, stitching, or continuous techniques. Figure 5-1 shows time-lapse images throughout the tensile testing of continuously connected 1 mm pore serpentine and 0.5 mm pore square scaffolds (scale bar = 5 mm). Figure 5-2 shows a representative stress-strain plot of the continuously connected scaffold, with labels corresponding to regions of i) elastic deformation, ii) plastic deformation, and iii) scaffold fracture. Figure 5-3 shows representative combined stress-strain plots for the serpentine-square scaffold and the control scaffold with each type of interface. [Figure 5-2]Figures 5-1 through 5-3 show images acquired during uniaxial tensile testing of biphasic MEW scaffolds connected using either overlap, stitching, or continuous techniques. Figure 5-1 shows time-lapse images throughout the tensile testing of continuously connected 1 mm pore serpentine and 0.5 mm pore square scaffolds (scale bar = 5 mm). Figure 5-2 shows a representative stress-strain plot of the continuously connected scaffold, with labels corresponding to regions of i) elastic deformation, ii) plastic deformation, and iii) scaffold fracture. Figure 5-3 shows representative combined stress-strain plots for the serpentine-square scaffold and the control scaffold with each type of interface. [Figure 5-3] Figures 5-1 through 5-3 show images acquired during uniaxial tensile testing of biphasic MEW scaffolds connected using either overlap, stitching, or continuous techniques. Figure 5-1 shows time-lapse images throughout the tensile testing of continuously connected 1 mm pore serpentine and 0.5 mm pore square scaffolds (scale bar = 5 mm). Figure 5-2 shows a representative stress-strain plot of the continuously connected scaffold, with labels corresponding to regions of i) elastic deformation, ii) plastic deformation, and iii) scaffold fracture. Figure 5-3 shows representative combined stress-strain plots for the serpentine-square scaffold and the control scaffold with each type of interface. [Figure 5-4] Figure 5-4 shows the Young's modulus, yield strength, and ultimate tensile strength for the two-phase scaffolds and control scaffolds using each type of connection method (mean values ​​across n=3, error bars represent 1 standard deviation, significance assessed using one-way ANOVA with Tukey's multiple comparison test; when statistical significance is shown across a single column, the data had at least that level of significance compared to all other columns on the graph (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001)). [Figure 6-1] Figure 6-1 shows images of clamping distances of 2.5 mm, 4.5 mm, and 6.5 mm applied to MEW scaffolds to test the uniaxial tensile properties. [Figure 6-2]Figure 6-2 shows representative stress versus strain plots for applying strain to a serially connected serpentine-square scaffold at clamping distances of 2.5 mm, 4.5 mm, and 6.5 mm, respectively. [Figure 6-3] FIG. 6-3 shows the Young's modulus measured with applied strain at different clamping distances. [Figure 6-4] FIG. 6-4 shows the yield strength measured with applied strain at different clamping distances. [Figure 6-5] FIG. 6-5 shows the ultimate tensile strength measured with applied strain at different clamping distances. [Figure 7A] Figure 7-1 is a schematic depiction of the bending test apparatus, where θ represents the measured bending angle. Figure 7-2 is a close-up of a biphasic scaffold showing the location of the pivot line (scale bar = 1 mm). Figure 7-3 is an image of a test of a multiphasic scaffold with a serpentine pattern (scale bar = 10 mm). [Figure 7B] Figure 7-4 depicts the bending stiffness data (mean values ​​across n=3, error bars represent standard deviation; two-way ANOVA with Tukey's multiple comparison test showed p<0.0001 for pattern effect, p=0.0002 for interface effect, and no significant differences were observed between individual means). [Figure 8-1] FIG. 8-1 is an exemplary graphical user interface for generating continuous and spatially heterogeneous G-code. [Figure 8-2] Figure 8-2 is an exemplary scaffold designed using a graphical user interface (GUI). [Figure 8-3] FIG. 8-3 is another exemplary scaffold designed using a graphical user interface (GUI). [Figure 8-4] Figure 8-4 shows digital microscope images including an image of a MEW PCL scaffold having the design shown in Figure 8-2, a high-resolution image of the 1 mm pore auxetic star pattern in section "A", and a high-resolution image of the fanning interface showing the continuous transition from 1 mm pores to 2 mm pores. [Figure 8-5] Figure 8-5 shows digital microscope images of a MEW PCL scaffold with the design shown in Figure 8-3, including a high-resolution image of the 1 mm pore diagonal serpentine pattern in section "A" and a high-resolution image of the fanning interface showing the continuous transition from 1 mm pores to 2 mm pores (scale bar = 2 mm). [Figure 9-1] FIG. 9-1 is an image showing the aortic valve region. [Figure 9-2] Figure 9-2 depicts the 3D reconstructed valve architecture and cut planes viewed from i) coronal (external), ii) sagittal, iii) axial, and iv) coronal (internal) directions. [Figure 9-3] Figure 9-3 is a second harmonic generation (SHG) image of the aortic valve showing the commissures and the interface with the origins of the adjacent leaflets. [Figure 9-4] Figure 9-4 is an SHG image of a slice of a sample aortic valve showing the commissures. [Figure 9-5] Figure 9-5 is an SHG image of a slice of a sample aortic valve showing the intercusp trigone. [Figure 10] FIG. 10 is a schematic depiction of a hybrid immersion fixation arrangement for fixation of porcine aortic valve tissue samples. [Figure 11-1] Figure 11-1 is a second harmonic generation (SHG) image of the aortic valve commissure showing the location of the imaging plane selected for correlated focused ion beam scanning electron microscopy (FIBSEM) (scale bar = 1 mm). [Figure 11-2] FIG. 11-2 is a low-magnification FIBSEM image taken in the circumferential direction, showing the aortic valve fibrocellular microstructure in the superior region of the commissure (scale bar = 10 μm). [Figure 11-3] Figure 11-3 is an enlarged image of the area bounded by the dashed box in Figure 11-2, showing individual collagen fiber cross sections running in the circumferential (C) direction and longitudinal fiber cross sections extending in the radial (R) and longitudinal (L) directions (scale bar = 1 μm). [Figure 12-1]FIG. 12-1 depicts a color-mapped orientation analysis of SEG images of the intercusp trigone region (scale bar = 0.5 mm). [Figure 12-2] Figure 12-2 is a simplified schematic diagram of collagen fiber orientation in the intercusp trigone. [Figure 12-3] Figure 12-3 depicts the resulting MEW scaffold and color-mapped orientation analysis of the interfacial region (scale bar = 2 mm). [Figure 12-4] Figure 12-4 shows the overlaid orientation distribution from the intercusp trigone tissue sample and MEW scaffold. [Figure 12-5] Figure 12-5 shows the resulting MEW scaffold labeled with the entered G-code indicating the design parameters. [Figure 12-6] Figure 12-6 shows snapshots of the G-code path with corresponding timestamps at times = 1, 2, 6, 29, and 74 seconds after the start time, displaying the continuous print path at these times. [Figure 12-7] Figure 12-7 is an SEM image (scale bar = 100 μm) showing a close-up of the continuous fiber interface employing a joining technique in which the fibers are fused according to joining paths. [Figure 13-1] Figure 13-1 is an image of the biaxial tensile test setup for applying strain in the circumferential and longitudinal test directions (scale bar = 5 mm). [Figure 13-2] FIG. 13-2 is a graph of the results for the measured Young's modulus, yield strength, and yield strain for the circumferential and longitudinal tests. [Figure 13-3] Figure 13-3 depicts the hysteretic behavior from cyclic tests at constant strain, showing representative stress-strain plots in both directions. [Figure 13-4] FIG. 13-4 depicts the quantification of hysteresis, shown by the area under the unloaded curve divided by the area under the loaded curve, for the same cycle. [Figure 13-5]Figure 13-5 depicts representative relaxation behavior after four step increases in strain (2.5% and 5% in the longitudinal and circumferential directions, respectively), each step held for 1,000 seconds, followed by quantification of the relaxation percentage (all data show mean values ​​over n=3, error bars represent 1 standard deviation, significance was assessed using a parametric paired t-test (*p≦0.05, **p≦0.01, ***p≦0.001)). [Figure 13-6] FIG. 13-6 depicts the relaxation percentage versus the number of steps of applied strain. [Figure 14-1] FIG. 14-1 is a conceptual illustration of an example of a sinusoidal interface. [Figure 14-2] FIG. 14-2 is a conceptual illustration of an example of an angled interface. [Figure 14-3] FIG. 14-3 is a conceptual illustration of an example of an arrowhead-shaped interface. [Figure 14-4] Figure 14-4 illustrates a schematic of an exemplary scaffold for a layer, showing a sinusoidal interface transitioning between regions with a rectangular grid pattern and regions where the fibers are arranged in an "auxetic star" pattern. [Figure 14-5] Figure 14-5 illustrates a schematic of an exemplary scaffold for a layer, showing an "angled" interface that transitions between regions with a rectangular grid pattern and regions where the fibers are arranged in an "auxetic star" pattern. [Figure 14-6] Figure 14-6 illustrates a schematic of an exemplary scaffold for a layer, showing an arrow or arrowhead shaped interface transitioning between regions with a rectangular grid pattern and regions where the fibers are arranged in an "auxetic star" pattern. [Figure 15-1] FIG. 15-1 shows the parameters used to generate the schematic of the scaffold layer shown in FIG. 14-4. [Figure 15-2] FIG. 15-2 shows the parameters used to generate the schematic of the scaffold layer shown in FIG. 14-5. [Figure 15-3] FIG. 15-3 shows the parameters used to generate the schematic of the scaffold layer shown in FIG. 14-6. [Figure 16] 16 is a schematic diagram showing an exemplary gradient porosity scaffold and the underlying mathematical function governing the gradient porosity, where the values ​​of the initial pore size (p) and gradient coefficient (m) can be varied. The gradient function is applied in three different patterns (rectangular, diamond, or serpentine) to affect pore dimensions either parallel or orthogonal to the direction of the gradient (which is applied horizontally from left to right). [Figure 17] Figure 17 is an example preview of parallel and orthogonal gradient porosity scaffolds for rectangular, diamond, or serpentine patterns. All scaffolds have dimensions of 25 x 25 mm, an initial pore size (p) of 0.5 mm, and a gradient coefficient (m) of 0.5, 1.0, or 1.5. Note that for orthogonal gradient porosity scaffolds, the gradient coefficient refers to the steepest gradient line, and the rest of the line is interpolated between it and flat (zero gradient). [Figure 18] Figure 18 shows exemplary gradient porosity scaffolds fabricated using melt electrowriting using a 23G needle, 100 kPa pressure, a working distance of 3 mm, a potential difference of 3.8 kV, a printing speed of 400 mm / min, a syringe temperature of 75°C, a needle temperature of 85°C, and a bed temperature of 30°C. Each scaffold was made from polycaprolactone, had dimensions of 25 x 25 mm, contained five layers, and had an average fiber diameter of 20 μm. [Figure 19] Figure 19 shows local strain mapping of a gradient scaffold subjected to constrained biaxial tensile testing. The scaffold was clamped using 15 mm clamps and strained horizontally at 1% / s to 100% strain while remaining fixed vertically. Images were taken at 20% strain. The color map shows the heterogeneous distribution of the principal engineering strains, calculated using VIC-2D software (Correlated Solutions, USA). [Figure 20]Figure 20 shows the heterogeneous loading throughout different regions of the valve, particularly in the central and commissure regions. Figure adapted from Emmert, et al. Science Translational Medicine 10, no. 440 (2018) (https: / / doi.org / 10.1126 / scitranslmed.aan4587). [Figure 21] Figure 21 shows exemplary heart valve scaffold design aspects incorporating gradients. A) Design from PCT / AU2020 / 210877, featuring uniform meander amplitude / wavelength throughout, aligned in only two directions: circumferential (blue, left-to-right) or radial (red, top-to-bottom), with anisotropic fiber density / porosity (higher density circumferentially, lower density radially). B) Radial fibers cross perpendicular (or nearly perpendicular) to the leaflet line. C) Circumferential fibers move parallel to the leaflet line, to some extent. D) Serpentine wavelength and / or amplitude vary across the leaflet. E) Fiber density varies throughout the leaflet (at regions of higher load, e.g., the commissures and base of the mid-leaflet). [Figure 22] FIG. 22 is a schematic illustration of an exemplary heart valve scaffold design incorporating gradient porosity. [Figure 23] Figure 23 is a schematic illustration of a method for sequentially and gradually interlacing fibers between three leaflets. Note that the lines are drawn as straight lines for convenience, but they may also be meandering, sloped, and / or have other patterns. DETAILED DESCRIPTION OF THE INVENTION

[0036] (Detailed explanation) In the following detailed description, reference is made to the accompanying drawings, which form a part of the detailed description. The illustrative embodiments described in the detailed description and depicted in the drawings are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the presented subject matter. In general, it will be readily understood that the aspects of the present disclosure as described herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are contemplated by the present disclosure.

[0037] Gradient structures are abundant in biological tissues and play an important role in their functionality. The ability to engineer structures that replicate these gradients and interfaces could be beneficial for achieving functional structures for tissue engineering applications.

[0038] Gradient scaffolds have been investigated in the context of tissues such as bone, tendon, vasculature, and cardiac muscle, to name a few. These scaffolds have been fabricated using techniques that control properties such as temperature, pH, or concentration of the material spatially throughout the material. Fiber fabrication techniques such as MEW have also been applied to create gradients, but are limited to simple rectangular geometries. In addition, gradient scaffolds can be used to create disease or tissue models and elicit gradient cellular responses, using gradient stiffness hydrogels, for example.

[0039] New approaches to fabricating microfibrous scaffolds, such as MEW, may enable more sophisticated interface or gradient designs. MEW has the potential to enable complex and tunable interface and gradient structures, which may be beneficial for a wide range of tissue engineering applications or disease modeling.

[0040] Fused electrowriting (MEW) is a highly precise additive manufacturing technique with the potential to exploit specific aspects of microstructural features observed in natural tissues, such as collagen orientation, in the design of complex biomimetic scaffolds. MEW scaffold printing primarily relies on precise control of five process parameters: temperature, pressure, voltage, working distance, and collector speed, resulting in highly controllable molten polymer jets with micrometer-level resolution that are deposited onto the collector in direct writing mode. However, unlike other 3D printers, MEW does not allow for stopping and starting of extrusion during the printing process, as this would disrupt the Taylor cone and cause print defects. Therefore, traditionally, MEW printing is performed region by region with similar spatial features in one uninterrupted printing process. Tissues with spatially heterogeneous regions are therefore fabricated by printing each region separately and then joining them together, such as by stitching.

[0041] Disclosed herein is an inventive method for using MEW to engineer continuous, user-defined interfaces between distinct structural regions of implantable devices, where the interfacial and structural regions are printed sequentially on each layer. Design strategies for connecting the different architectures of multiphase MEW scaffolds require careful consideration, as this can affect their ultimate mechanical and biological functionality.

[0042] The continuous interface disclosed herein has applications in MEW printing for different implantable devices. An exemplary application is the fabrication of MEW scaffolds for aortic valves.

[0043] The complexity and heterogeneity of the aortic valve make it an excellent case for validating the technical capabilities of MEW in fabricating complex scaffolds for soft-tissue interfaces. However, it should be understood that the method also has applicability for engineering tissues other than heart valve scaffolds.

[0044] The average human aortic valve undergoes over 30 million cycles per year, equating to over 2 billion cycles over a 70-year lifespan. Such extraordinary hemodynamic properties are made possible by the combined biomechanical behavior resulting from the cooperation of each of the valve's structurally distinct regions and the interfaces between them. Using a bioinspired design approach, we have previously demonstrated MEW scaffolds with mechanical properties similar to those of physiological heart valve leaflets. This was made possible by available data on the relationship between leaflet mechanical properties and the collagen mobilization mechanism under load.

[0045] In one application, the disclosed method relates to regions of the heart valve other than the leaflets and the interfaces between them, potentially opening up additional applications of MEW and improving valve functionality. Regions such as the commissures, inter-leaflet trigone, and the interfaces between them all play critical roles in the biomechanical behavior of the heart valve.

[0046] Heart valves are heterogeneous structures with highly variable structural and mechanical properties. These gradient properties exist not only in the valve leaflets but also in surrounding structures such as the commissures, interleaflet trigone, and annulus, each of which plays an important role in valve function. Therefore, in the context of heart valve engineering, the ability to create gradient scaffolds and thereby control local mechanical properties is invaluable.

[0047] The present invention therefore applies the gradient scaffold concept to aspects of fibrous heart valve scaffold design (Figs. 22, 23).

[0048] PCT / AU2020 / 210877 identified the benefits of continuously joining fused electrowritten fibers between leaflets for improved printing and bending properties. However, in previous disclosures, the change in orientation between regions, while continuous, was abrupt. The present invention provides a method for gradually and continuously combining fibers within and between regions.

[0049] Figure 1 diagrammatically illustrates components of a MEW device 10, including a nozzle 12 for extruding a polymer melt onto a collector 14. For clarity, the "x," "y," and "z" directions mentioned herein are used to refer to the orientations shown in Figure 1. Directional labeling may vary and is not intended to limit the scope of the invention.

[0050] The nozzle 12 is positioned above the collector 14 at a working distance (height), which is defined here as the "z" direction. Relative movement between the nozzle 12 and the collector 14 in the "x" and "y" directions, or a combination thereof, while the nozzle 12 extrudes the polymer allows for the formation of a 2D MEW scaffold across layers; i.e., multi-layered 3D structures can be created by adding relative movement between the nozzle and the collector along the "z" direction. The translation of the nozzle 12 relative to the collector 14 in the "y" direction, or vice versa, defines the translation speed.

[0051] The polymer jet extruded from the nozzle 12 is deposited onto a collector 14 as fibers 16. The fibers 16 follow a predefined printing path to form the desired structure. The scaffold will have multiple layers of fibers at the end of the MEW printing process. The number of layers will depend on the MEW settings and configuration required for the scaffold.

[0052] Some, but not necessarily all, scaffolds that can be fabricated using the methods described herein are heterogeneous. Heterogeneous scaffolds include at least two regions that are spatially heterogeneously structured. The spatial heterogeneity may reside in differences in one or more parameters, such as, but not limited to, pore size, fiber orientation, and fiber density, pattern, length, etc., in one or more dimensions.

[0053] According to the present invention, adjacent structural regions are each formed contiguously in one or more layers of the scaffold with an interface therebetween.

[0054] 2 is a digital microscope image of a MEW scaffold layer 20. Layer 20 includes two structural regions 22, 24 and a continuous interface region (which may also be simply referred to as an "interface") 26 that joins regions 22, 24. In this example, the two regions 22, 24 are spatially heterogeneous. However, the interfaces described herein can also be used to join regions that are not spatially heterogeneous.

[0055] In this example, region 22 has larger pore sizes than region 24, and the shape of the pores in region 22 more closely resembles a rectangle, while the shape of the pores in region 24 more closely resembles a diamond. A continuous interface 26 exists between heterogeneous regions 22, 24. The exact configuration of the fibers shown does not represent a critical element of the invention. Rather, they are provided as examples only and serve to illustrate the concept of a "continuous interface" as intended in this disclosure, where the interface joining two structural regions is printed in a continuous manner, with the structural regions on the same layer.

[0056] For example, as can be seen in FIG. 2 , starting from region 22, the fiber follows a path defining a grid pattern within region 22, but then deviates at the boundary between region 22 and interface region 26, following an interface path governed by the interface function as the fiber enters interface region 26. At the boundary between interface region 26 and region 24, the fiber again deviates from its current path (which is the interface path) and follows a path defining a diamond pattern within region 24. This deviation between the path defining the local pattern and the interface path occurs throughout the entire print, printing the entire scaffold layer in one continuous print. The overall printing path thus remains continuous, but at the boundary of the region with the interface region, it switches to follow different paths defined for the various regions, respectively. This arrangement, in contrast to conventional MEW printing, where different structural regions are printed separately and intended for the scaffold, avoids deviations in the printing path. The regions are then typically joined together either by stitching or overlapping or a combination of both.

[0057] In this method, during printing, layer by layer, the fibers of the layer are formed by depositing polymer along a printing path that traverses the layer 20 several times until the layer is formed, and the MEW printer then continues to form further layers. The printing for the further layers may follow the same printing path as the first layer, or may follow a different printing path if the scaffold to be fabricated is configured to have variations across the layer (i.e., across the thickness of the sheet along the "z" dimension).

[0058] A printing path may also be considered to include several round trip passes, which may include one or more passes across a full or partial extent of the width of the layer (e.g., along the "y" direction), one or more passes across a full or partial extent of the height of the layer (e.g., along the "x" direction), one or more passes across a full or partial extent of the diagonal extent of the layer, or a combination thereof.

[0059] More generally, the printing path will be determined by a MEW printing algorithm (e.g., written in G-code), which defines the device (e.g., scaffold) to be fabricated. By "continuous printing path" here is meant the path along which polymer is continuously extruded. The pass for printing the entire sheet will be part of the continuous printing path. The printing path may include both curved and straight sections, again depending on what is being fabricated.

[0060] Thus, successive printing paths will pass through different regions. To facilitate this, the algorithm defining the paths for the different regions will switch between these paths when the fiber encounters the boundary between the two regions to form one continuous printing path that transitions between two regions within a layer without the MEW printing process for the layer being paused.

[0061] In some embodiments, the printing path that dictates the fiber deposition site or path within interface region 26 will be an interface function, which may refer to a single or possibly complex mathematical function that defines a particular shape, rather than simply connecting fiber locations within regions 22, 24, while still allowing the region boundaries to continuously transition from one to the other. Examples include, but are not limited to, mathematical functions that form sinusoidal, parabolic, arrowhead, and angled shapes. Conceptual illustrations of examples of a sinusoidal interface 1401, an angled interface 1402, and an arrowhead-shaped interface 1403 are shown in Figures 14-1, 14-2, and 14-3, respectively. Schematics of exemplary scaffolds of layers with sinusoidal, "angled," and "arrowhead"-shaped interfaces that transition between regions with a rectangular grid pattern and regions in which fibers are arranged in an "auxetic star" pattern are shown in Figures 14-4, 14-5, and 14-6, respectively. The parameters used to generate the scaffold schematics in Figures 14-4, 14-5, and 14-6 are shown in Figures 15-1, 15-2, and 15-3, respectively.

[0062] The shapes for the interfaces may be manually defined instead of or in addition to being defined by a function or composite function (i.e., "function-defined"). For example, a combination of functions or manual definitions for fibers may be present in one or more of the layers. All of the interface fibers in one or more of the layers may be manually defined. All of the interface fibers in one or more of the layers may be function-defined.

[0063] Furthermore, depending on the fiber arrangement provided for the region to be joined by the interface, different print paths intended for different interface fibers, each extending between corresponding fiber pairs from neighboring structural regions, can have different shapes or interface functions, where the different interface functions defining a series of adjacent print paths within the interface region preferably define gradual, rather than abrupt, changes in shape.

[0064] The choice of interface shape or function will depend on various factors, such as the desired printing time and any mechanical properties (e.g., specific bending or flexing requirements) required for the interface region or the overall tissue scaffold structure. The choice may also depend on the values ​​of the MEW process parameters. For example, a shorter time to traverse the interface region (and therefore a shorter printing path) may be required if any fusion or bonding is intended to occur between previously deposited and later deposited fiber portions (e.g., during return passes).

[0065] A continuous interface for joining two structural regions, when the interface is further defined by a function as described above, has been demonstrated in experiments performed by the inventors to provide the greatest degree of bending compared to conventional techniques for joining two structures. This is particularly useful for applications such as heart valve scaffolds, where bending or flexing within the implanted valve is required to function in a hemodynamic environment, in providing the opportunity to engineer valves that can better maintain their performance over time.

[0066] Optionally, two or more of the printing paths may be defined such that the fibers deposited along the paths are directly adjacent to one another, forming bundles. The bundles can fuse or bond with one another under the correct MEW settings. This allows the fabrication to provide scaffold parts that mimic anatomical features found in biology. For example, in test porcine heart valve samples, the inventors observed splitting of collagen fiber bundles and also joining of separate collagen fibers into larger bundles. This is described later in this specification.

[0067] Pore ​​size may be varied between different sections of a printed structure when providing an interface through which two structural regions transition between each other. Three techniques for varying pore size between sections are proposed: bonding, halving, and fanning. The same techniques may also be applicable to varying porosity within individual structural regions instead of, or in addition to, the transition area (i.e., interface) between different regions.

[0068] Figures 3-1 through 3-4 conceptually depict the "bonding," "halving," and "fanning" concepts. Figure 3-1 diagrammatically shows a scaffold with consistent pore size that does not vary between the left and right portions of the scaffold. In the "bonding" example in Figure 3-2, each pair of adjacent fibers from the left section bonds together, resulting in a doubling of pore size on the right side. This may be a way to create a transition from one structural region to a second structural region with a larger pore size. This would also result in a doubling of the number of fiber layers in the second region. This technique mirrors observations from natural tissues (see, e.g., Figure 9-5), where small collagen bundles gradually combine to form thicker bundles, as observed within interfaces anywhere in the body. The "bonding" concept can be generalized so that two or more fibers are bonded. Potentially, different numbers of fibers may be bonded in different areas within the scaffold in this process, creating bonded bundles of different sizes.

[0069] The halving method deposits an additional layer of scaffold into the first (left) section at an offset equal to half the pore size. This results in a halving of the overall pore size within the first section while maintaining equal fiber layers between the sections. Figure 3-3 depicts a schematic depiction of the pore size halving where an additional fiber layer 32 is added. Figure 3-5 provides an example image of where this occurs within a MEW-fabricated device.

[0070] The "halving" method can be generalized to reduce pore size in areas where additional layers are deposited in amounts other than half. For example, "halving" can be performed twice, at 1 / 3 pore size offset and 2 / 3 pore size offset, to reduce pore size to 1 / 3 of the original pore size. Thus, the "halving" concept can be generalized to the "splitting" or "fractionation" concepts.

[0071] The fanning method creates a gradient change in porosity. The gradient change can be set to occur over a "fanning height" that is predefined or selected by the MEW scaffold designer. Fanning can occur as a gradual decrease in pore size (Figures 3-4, 3-6) or a gradual increase in pore size (Figures 3-7).

[0072] (experiment) Example 1 Comparison of sequential connections with other methods of connecting multiphase MEW scaffolds The influence of the design of the interface region on the behavior of printed scaffolds was systematically investigated. Besides the continuous interface described above, two other connection methods, including overlapping and stitching, were also investigated. See Figure 4-1, which includes schematic depictions of a continuous interface, a stitching interface, and an overlapping interface.

[0073] Three different patterns (square, diamond, and serpentine) were selected to be combined using three interfacial printing strategies to produce biphasic MEW scaffolds. The scaffolds were fabricated from medical-grade poly(ε-caprolactone) (PCL) to create five-layer 10 mm x 40 mm scaffolds with two 20 mm patterns and an interface region. The size of the interface region depended on the connection method used: overlap was 1 mm, suture was 2 mm, and the continuous method resulted in a gradual, undefined transition region. To demonstrate the ability of the three methods to connect spatially heterogeneous scaffolds, pore size was varied from 1 mm to 0.5 mm for each pattern. The square pattern was selected to alternate pore sizes, while the diamond and serpentine pore sizes were kept constant at 0.5 mm and 1 mm, respectively. The pore sizes were selected to facilitate ease of printing and differentiation of the effect of the connection method, independent of the scaffold's applicability for tissue engineering. Control scaffolds (40 mm x 10 mm single-pattern scaffolds with no interfaces) were also printed to test each pattern and pore size independently of any interfaces. The average fiber diameter was 26.09 ± 1.90 μm. Figure 4-2 shows exemplary images of each of the interface types for square / diamond scaffolds.

[0074] Next, the similarity of each connection method to the programmed print path was assessed by comparing their morphologies using light microscopy and scanning electron microscopy (SEM). Fiber bridging, a known defect in which fibers deviate from their intended path onto adjacent fibers due to electrostatic repulsion caused by residual charges, was identified in varying amounts across all scaffolds. Overlapping scaffolds exhibited the highest density interfacial regions and, in some cases, bridging. The stitching method was more customizable but, for the selected design, exhibited a slightly lower fiber density and a comparable amount of bridging to overlapping scaffolds. The sequential printing technique best matched the planned print path, with fewer fiber bridging defects and the lowest density. These results are consistent with the literature; the amount of printing defects in MEW scaffolds is expected to increase in areas of higher fiber density and areas where jetting changes direction abruptly. Thus, the continuous interface produced a more precise print, exhibiting a gradual transition in density between spatially heterogeneous regions, as opposed to a denser band containing more fibers.

[0075] Example 2 Effect of interface printing method on the mechanical properties of biphasic MEW scaffolds Uniaxial Tensile Test To assess the effect of the interface printing method on mechanical properties, uniaxial tensile tests were performed on the two-phase MEW scaffolds with a load perpendicular to the interface. Figure 5-1 includes time-lapse images taken at different extension stages: no load, elastic deformation, plastic deformation, and scaffold fracture, from left to right. Figure 5-2 depicts a representative stress-strain plot of a continuously connected scaffold, with labels corresponding to regions of i) elastic deformation, ii) plastic deformation, and iii) scaffold fracture. Figure 5-3 depicts representative combined stress-strain plots for serpentine-square scaffolds with each type of interface and a control scaffold. Figure 5-4 depicts the Young's modulus, yield strength, and ultimate tensile strength for each type of biphasic scaffold using the connection method and the control scaffold (mean values ​​across n=3, error bars represent 1 standard deviation; significance was assessed using one-way ANOVA with Tukey's multiple comparison test; when statistical significance is shown across a single column, the data had at least that level of significance compared to all other columns on the graph (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001)).

[0076] Figure 6-1 depicts images of a serially connected square / serpentine scaffold to which uniaxial strain forces were applied at different clamping distances. The effect of clamping distance from the interface on mechanical properties was first investigated, and no significant differences were observed in the calculated stress. Stress parameters, including Young's modulus, yield strength, and ultimate tensile strength, are depicted in Figures 6-3, 6-4, and 6-5. In each of Figures 6-3, 6-4, and 6-5, the results are depicted sequentially from left to right and correspond to results obtained using clamping distances of 2.5 mm, 4.5 mm, and 6.5 mm, respectively.

[0077] However, strain values ​​were observed to vary significantly, and therefore, relevant measurements such as yield strain cannot be compared. Subsequently, due to the limited strain test range of the instrument, the scaffold was clamped closer to the interface on the weaker scaffold side. Figure 5-2 shows a representative stress-strain plot 501 of a continuously connected serpentine-square scaffold. The initial elastic deformation (i) of the scaffold, followed by plastic deformation (ii), is almost entirely due to the weaker pattern, in this case, the serpentine. The fibers of the weaker scaffold then began to neck, resulting in strain hardening, indicated by a gradual increase in stress. When the scaffold reached its ultimate tensile strength (UTS), the scaffold broke, as indicated by a sudden decrease in stress (iii), corresponding to the strain at which the two patterns separated from each other. Similar stress-strain curves were also observed for the rest of the connected scaffolds, as shown in Figure 5-3. Stress-strain plots and scaffold testing for serpentine-square, square-diamond, and serpentine-diamond scaffolds showed similar behavior.

[0078] The Young's modulus of the connected scaffolds was dominated by the more elastic pattern, i.e., the pattern with the lower modulus, of the two connected patterns (Figure 5-4). With one exception (continuous serpentine-square), the elasticity of the connected scaffolds was either equal to or greater than that of the more elastic pattern. For both the square-diamond and serpentine-diamond scaffolds, the Young's modulus was unaffected by the connection method. Interestingly, connecting the square-diamond scaffold resulted in a significantly more elastic scaffold than either of its configurations, regardless of the connection method. However, for the serpentine-square scaffold, the stitching method was the only technique that resulted in a more elastic scaffold. These results suggest that, despite the majority of the elastic behavior resulting from the more elastic pattern, both patterns contribute in some way to the final scaffold elasticity. Yield strength indicates the level of stress at which the scaffold will begin to permanently deform. The results showed that yielding was entirely dictated by the weaker pattern, meaning the pattern with the lower yield strength. Similarly, the UTS of the scaffolds was primarily determined by the weaker of the two patterns, with one exception (sutured square-diamond). Overall, the connection method did not significantly affect the uniaxial mechanical properties, due to the dominance of the weaker scaffold. However, other mechanical properties associated with tissue-engineered scaffolds may be affected.

[0079] (bending test) Next, the extent to which the printing method used to connect the two different patterns would alter the bending properties of the resulting biphasic scaffold was investigated. To that end, a bending test apparatus 700, shown diagrammatically in Figure 7-1, was constructed in accordance with ASTM D1388. Figure 7-2 shows a close-up of a biphasic scaffold 701, with an imaginary dashed line 702 representing the location of the pivot line (scale bar = 1 mm). Figure 7-2 shows an image of a test of a multiphasic scaffold with a serpentine pattern. Bending stiffness data are shown in Figure 7-4 (mean values ​​across n = 3, error bars represent standard deviation; two-way ANOVA with Tukey's multiple comparison test showed p < 0.0001 for the effect of pattern and p = 0.0002 for the effect of interface; no significant differences were found between individual means).

[0080] During testing, each scaffold was immediately pivoted on one side of the interface, while the other pattern remained fixed, and one side was bent. From this, the bend angle was measured, and then the bending stiffness (measured in Nm, G) was calculated. The G value for the control scaffold was also calculated. A lower G value indicates a more flexible scaffold. The bending properties were primarily dependent on the bending scaffold pattern, as can be seen in Figure 7-4. Of the patterns tested, the serpentine scaffold was the most flexible (lowest bending stiffness), followed by the diamond, 1 mm square, and 0.5 mm square. Note that as the bend angle θ tends toward 0°, G tends toward infinity, meaning that very stiff scaffolds that are barely bendable have exponentially higher values ​​for G. This was observed for both square patterns, which had significantly higher values ​​for G. Interestingly, the type of interface significantly affected bending stiffness. In particular, for the stiffer patterns, both the stitched and continuous interfaces enabled higher bending relative to the stiffer square pattern, with the continuous interface enabling the greatest increase in bending. In contrast, overlapping interfaces often had a negative effect on bending. For already flexible patterns such as serpentine and diamond, the effect of the interface was negligible. Comparing across interface techniques, a correlation can be observed: the continuous interface provided the greatest degree of bending, followed by stitching and then overlapping. Interestingly, a similar trend was also observed when previously visually analyzing the fiber density in the interface region between methods. Therefore, this may be a possible mechanism behind the observed bending behavior, according to which interfaces with a higher density and more sites of fiber fusion would provide greater bending stiffness.

[0081] In summary, the test results indicate that, in most cases, the interconnection of MEW scaffolds does not weaken the mechanical performance of the scaffold and often results in elasticity equal to or greater than that of the individual components. Furthermore, the use of a continuous interconnection approach is recommended when attempting to achieve greater bending properties. This is valuable in the context of interfacial tissue engineering, as specific bending properties may be desired across the interface between two regions. In the context of tissue-engineered heart valve scaffolds, a high degree of bending is required for the interface between the valve leaflets and the interleaflet trigone, where a large amount of cyclic bending will occur. This strategy may therefore continue to be exploited to expand the potential capabilities of MEW scaffolds.

[0082] Example 3 User-generated MEW scaffolds with complex continuous interfaces Writing G-code for serial printing of MEW scaffolds can be time-consuming, especially for complex scaffold architectures involving multiple regions. Graphical user interfaces (GUIs) can be written to take advantage of advances in GUI technology and computing to more quickly generate MEW scaffolds, including tunable interfaces, using serial printing.

[0083] A screenshot of GUI 800 is shown in Figure 8-1 for designing a scaffold with an interface between two sections "A" and "B." We have previously proposed a novel MEW scaffold structure in which each region comprises intersecting sets of fibers, each set containing fibers aligned parallel or nearly parallel to each other. This results in pore shapes that are identical or nearly identical within each section.

[0084] For convenience, a GUI 800 is provided for designing scaffolds of the type described above. The scaffolds further have a continuous interface, as discussed herein. The GUI 800 provides selectable fields 810, 820 in which the pore pattern (i.e., shape) for each segment can be selected. Rectangular, serpentine, auxetic cube, and auxetic star are included as examples, but are not intended to be limiting. Additional fields for selection of segment length, pore size, and path type are also included within the GUI 800. In this example, the "path type" options are "grid," in which the fibers are aligned in horizontal and vertical lines to form a rectangular grid; "diamond," in which the fibers are aligned diagonally; and "junction," in which two or more fiber junctions occur. It should be understood that other path types may be defined and presented as options. In GUI field 830, the interface function for the continuous interface is further set to be a mathematical function by selecting one of the function options and allowing the boundary between sections "A" and "B" to be dictated by the mathematical function while still being continuous. Non-limiting exemplary functions here include sine wave, parabola, arrowhead, and angled shapes. GUI field 840 also allows selection of methods for varying the Y-pore size between sections, including halving and fanning, as described above in connection with FIG. 3.

[0085] Figures 8-2 and 8-3 show two exemplary MEW scaffold designs and input parameters used in the design in Figure 8-1, respectively: scaffold 841 is designed to employ a joining and fanning method. In Figure 8-2, scaffold 842 is designed to employ a diagonally oriented path where fanning occurs in one section (i.e., the path type is selected to be "diamond"), and is fabricated using PCL. Digital microscopy images of the MEW scaffolds are shown in Figures 8-4 and 8-5.

[0086] The method of the present invention has the capability to generate continuous, user-defined MEW scaffolds. The novel method and its implementation, e.g., using a GUI, opens up the possibility for the rapid iterative design of a wide range of continuously connected scaffolds, leading to a wide range of tunability of mechanical and morphological properties that may be highly beneficial in MEW-based interfacial tissue engineering applications.

[0087] Example 4 Morphological analysis of the aortic heart valve interface In the experiments conducted, multimodal imaging was applied to examine the morphological structure of the interface region of an aortic heart valve as a basis for establishing a functional bioinspired design for MEW-scaffold interfaces. Subsequently, three methodologies for connecting heterogeneous scaffolds were investigated to understand their effects on tensile and bending properties. Combining studies on native tissue investigations and connection methods, an integrated biomimetic heart valve interface was designed, fabricated, and tested.

[0088] The aortic valve leaflets are responsible for the majority of the valve's overall functionality and have been the focus of previous research. Furthermore, other regions of the aortic valve are also known to contribute significantly to valve functionality. These regions have been classified in different ways. For the purposes of this specification, the regions are divided into the leaflets, sinuses of Valsalva, annulus, commissures, and intercusp trigone, as shown in Figure 9-1. Each region has different concentrations and orientations of extracellular matrix (ECM) components, including collagen and elastin fibers, which merit particular attention when considering the mechanical functionality of the valve. Collagen fibers are the primary load-bearing component of the ECM and are responsible for the tissue's anisotropic mechanical properties and J-shaped stress-strain response. Elastin fibers play a role in the valve's low-strain performance but, more importantly, regulate collagen fiber orientation, ensuring that they return to their preload state between cycles. Therefore, although all components of the ECM play important roles in valve functionality, our investigation focuses on collagen fiber orientation to guide MEW scaffold design. It is therefore important to understand what is already known about collagen fiber orientation in the heterogeneous regions of the aortic valve, which is briefly outlined below.

[0089] The aortic valve cusp contains three distinct layers: the fibrous layer, the spongy layer, and the ventricular surface (from the aorta toward the ventricle). The tensile load-bearing layers are the fibrous layer and the ventricular surface, which are primarily composed of circumferentially and radially aligned collagen fibers, respectively. The sinuses of Valsalva are similar in composition to the aortic wall and consist of three distinct layers: the inner (intima) and outer (adventitia) layers are primarily composed of longitudinally aligned collagen fibers, while the middle (media) layer is primarily composed of circumferentially aligned fibers. The annulus is a fibrous structure that connects the cusps and sinus wall within the left ventricle and is primarily composed of very dense, circumferentially aligned collagen bundles, providing relatively stiff mechanical properties. The commissures are located at the points where the free edges of the two cusps meet. By helping to transmit forces between the valve leaflets and the surrounding aortic root, the commissures serve a critical function both in supporting the bending properties required to open the valve during diastole and in withstanding high tensile loads as the valve closes during systole.

[66] Regarding microarchitecture, collagen fibers primarily extend radially from the leaflets, interlacing through the commissure region and anchoring into the aortic wall, allowing for the transmission of forces between the leaflets and the root. The intercusp trigone is dominated by regions below the commissures, between the leaflets, and above the annulus. The orientation of the fiber microarchitecture is poorly understood in the intercusp trigone; however, it is thought to primarily follow that of the annulus, i.e., circumferentially. Notably, while the collagen microarchitecture in these regions is known, the specific nature of how collagen fibers orient at the interface between these regions remains to be investigated. This may provide valuable insight into the design of scaffolds that seek to mimic native fiber morphology. Therefore, we began the problem-solving process by investigating whether high-resolution multimodal imaging of porcine tissue could provide insight into some of these unknowns.

[0090] Experiments and analyses using porcine tissue have been widely adopted in cardiovascular research due to the wider availability of tissue with anatomical and hemodynamic similarities to humans. When analyzing collagen microarchitecture in these studies, regional fiber orientation was consistent between human and porcine species. Therefore, porcine tissue has been used for biomechanical studies and to elucidate fiber alignment using microscopy.

[0091] As depicted diagrammatically in Figure 10, porcine aortic valve tissue 1001 was fixed using a hybrid immersion fixation and hydrostatic expansion method to preserve near-physiologic diastolic conditions. First, micro-computed tomography (microCT) was used for 3D reconstruction of the valve architecture, facilitating macroscale identification of regions of interest. Next, the tissue was sectioned longitudinally through the wall of the aortic root, parallel to the direction of blood flow, at a thickness of 250 μm, for further analysis via second-harmonic generation (SHG) imaging. Figure 9-2 depicts the 3D reconstructed valve architecture 901 and cut planes 902 viewed from i) coronal (external), ii) sagittal, iii) axial, and iv) coronal (internal) directions. This cut orientation allowed imaging of the commissures and intercusp trigone, their interfaces, and the interfaces with the valve leaflets, sinuses, and annulus. Acquisition of three slices relative to the aortic wall allowed confirmation of orientation information in three dimensions. SHG is an optical microscopy technique that is ideally suited for imaging collagen from tissue to molecular scales due to the second-order nonlinear properties of collagen fibers.

[0092] The microstructure of specific cardiac valve regions, including the commissures and the fibrous and ventricular layers of two adjacent valve leaflets, was identified in SHG images (e.g., Figure 9-3). At the commissures, a large number of collagen fibers were densely intertwined and interwoven between vertical and horizontal directions. Three distinct groups of collagen fibers progressed from the top of the commissure into the valve leaflets. The left and right of these groups merged into the fibrous layers of two adjacent valve leaflets, which could be identified by their distinctly rounded structures, where collagen fibers progress in and out of the page, and were indicated by a decreasing SHG signal. Interestingly, we observed that the central group of fibers progressed downward from the top of the commissure for approximately 500 μm before splitting into the ventricular layers of each leaflet, identifiable by vertically aligned collagen. To our knowledge, the orientation and extent of the commissure's connection with the fibrous and ventricular layers of two adjacent valve leaflets was unknown until now. In deeper images of the commissure, we observed that the highly aligned collagen fibers progressed horizontally from the sinus before redirecting to progress vertically downward and joining larger bundles, at which point the fibers again became strongly aligned and tightly bundled (Figure 9-4).

[0093] SHG imaging of the intercusp trigone revealed horizontally oriented bundles of collagen fibers emerging from the leaflets and sinuses on both sides (see Figure 9-5). The fibers are regularly intertwined as they transition from horizontal to diagonal to a predominantly vertical orientation. Vertically aligned bundles exit the top of the image and continue into the commissure region. The change in fiber orientation is gradual, with fibers recruiting into individualized bundles as they pivot before reaching vertical or diagonal alignment.

[0094] To verify the SHG collagen fiber orientation data, correlated focused ion beam scanning electron microscopy (FIBSEM) imaging was performed on the same sample. A specific region from the SHG image was selected and co-registered to the FIBSEM slice (Figure 11-1). At low magnification, the FIBSEM slice shows collagen fiber bundles filling the spaces between valve fibroblasts (Figure 11-2). Figure 11-3 is a higher magnification image of the boxed area 1101 shown in Figure 11-2. At higher magnification, individual collagen fiber cross sections can be seen grouped together into bundles, progressing primarily in the circumferential direction (C), with one bundle changing orientation from radial to longitudinal (R, L). This is consistent with the observation from the SHG imaging in Figure 11-1, where the collagen fibers progress primarily in the circumferential direction, perpendicular to the plane of the slice, before gradually changing direction and extending longitudinally, parallel to the plane of the slice.

[0095] From multimodal imaging analysis of porcine aortic valves, we can summarize the following findings: 1) the commissures consist of a complex, interwoven network of collagen fibers from multiple directions, which then fuse into a predominantly vertical alignment at the core of the commissures; 2) in the intercusp trigone region, collagen fiber bundles exhibit a regular diagonal weave before fusing toward a predominantly vertical alignment and becoming more bundled as they transition toward the commissures; and 3) fiber orientation changes across the interface are gradual, often characterized by a transition from a uniformly aligned large-fibrillar sheet to a more discrete bundle-like structure as fibers change direction at more acute angles. Due to the complexity associated with developing a sample preparation method compatible with precise cross-modal correlation imaging and the extensive time required to perform this, a limitation of this study was n = 1 sample. Observations from this high-resolution multimodal imaging analysis performed on the aortic heart valve interface were used in this study as inspiration for connecting fibrous heart valve scaffold designs.

[0096] (Tissue procurement and preparation) The collection and use of porcine animal tissue for this study was approved by the University of Western Australia Institutional Biosafety Committee (F 69199). Pig hearts were procured from the University of Western Australia Large Animal Facility, and tissue was harvested within 2 hours of euthanasia. Immediately after removal, the hearts were dissected, leaving the aortic root with its valves, sinuses, the first few millimeters of the coronary arteries, and a portion of the ascending aorta.

[0097] Primary fixation of tissue (n = 1) was performed in freshly prepared 4% paraformaldehyde solution (Cat. #C007, ProSciTech, Australia) in 0.1M phosphate buffer (PB) (pH 7.4). Fixation was performed at room temperature for 1 hour at equivalent diastolic pressure (80 mmHg) using the hybrid immersion fixation and hydrostatic expansion apparatus shown in Figure S1 (Supplementary Information). Secondary fixation was performed via overnight immersion in 2.5% glutaraldehyde (Cat. #EMS16400; ProSciTech, Australia) in 0.1M PB (pH 7.4). Excess tissue was removed by dissection before storage in glutaraldehyde fixative solution at 4°C.

[0098] (micro-computed tomography) During imaging, tissue samples were kept moist under paper towels soaked in glutaraldehyde solution and placed inside sealed Ziploc bags. MicroCT imaging (Skyscan 1176, Bruker-microCT, Kontich, Belgium) was performed at a source voltage of 45 kV, a source current of 556 μA, an exposure time of 86 ms, and a pixel resolution of 34.81 μm, using a 0.2 mm aluminum filter at 0.7° intervals over a 360° rotation with 2x image frame averaging enabled. MicroCT data were segmented and reconstructed using in-house software before being imported into STAR-CCM+ (v16.04.012-R8, Siemens) for further smoothing and processing.

[0099] (Second Harmonic Generation Imaging) A 5% agarose (Agarose LE (analytical grade), Promega, Australia) embedding solution was used at 65°C. Tissue samples were removed from the fixative solution and embedded in custom-sized (approximately 30 mm) embedding media designed to accommodate the entire tissue volume and minimize the amount of agarose solution used. 3 The tissue was blotted dry before being placed into a Lego (The Lego Group, Denmark) mold. Agarose solution was poured around the tissue, ensuring minimal bubbles remained. The embedded tissue was allowed to cool to room temperature and then stored overnight at 4°C. For sectioning, the solid gel / tissue block was removed from the mold and fixed with superglue to the stage of a vibratome (Vibratome 3000, The Vibratome Company, St. Louis, MO, USA). After sectioning, the block was cut to the region of interest, and three serial sections were obtained at 250 μm thickness. Optical imaging was then performed on these slices using an inverted A1RMP multiphoton microscope (Nikon) equipped with a 10x / 0.45NA objective lens (Nikon) and a tunable laser (10 mW output; 900 nm emission wavelength). All images were captured using NIS-Elements AR software (v5.30.02; Nikon).

[0100] (Electron microscopy of tissues) Electron microscopy samples were prepared from sections by microwave-assisted processing using a BioWave Pro microwave system (Pelco) after SHG imaging. Briefly, samples were osmotic stained using the R-OTO method, block stained with aqueous uranyl acetate and lead aspartate, and then dehydrated through a graded ethanol series (80%, 90%, 95%, 100%, 100% (v / v)) and propylene oxide (100%, 100% (v / v)). Sample infiltration with Araldite 502 / Embed 812 was performed under vacuum through a graded concentration series in propylene oxide (25%, 50%, 75%, 100%, 100% (v / v)). Samples were polymerized at 60°C for 48 hours and sectioned on an ultramicrotome (Leica UC6, Leica Biosystems) in preparation for imaging on a FE1 Helios Nanolab G3 CX DualBeam FIB-SEM (Thermo Fischer Scientific). Target areas were milled using a gallium FIB current of 65 nA at 30 kV before backscattered electron imaging of the block face at an accelerating voltage of 2 kV in magnetic immersion mode.

[0101] Example 5 Design, fabrication, and testing of a bioinspired aortic heart valve interface scaffold By leveraging novel microstructural information of the aortic valve interface obtained from multimodal imaging studies, a bioinspired MEW scaffold for the aortic heart valve interface region was designed using a complex continuous interface. First, orientation analysis of the collagen structure from Figure 9-5 enabled a quantitative understanding of microfibrillar collagen orientation. See, for example, Figures 12-1, 12-2, and 12-3, which show color-mapped orientation analysis (each scale bar = 0.5 mm) of SHG images of the intercusp trigone region. In the tissue sample, a gradual change in fiber distribution is observed from 0° (horizontal) to a peak at ±50° (diagonal), then again to a gradual change in fiber alignment at ±90° (vertical). Interestingly, some fibers were perfectly vertically oriented, while the majority were interwoven in a regular diagonal pattern with a slight vertical tilt.

[0102] The elaborate and complex interwoven fibrillar structure of the intercusp trigone region was simplified to a schematic form (Figure 12-2) to facilitate scaffold design. The pattern within the leaflet region was based on our previously published cardiac valve leaflet design (Saidy et al. (2019) Small 15(24), https: / / doi.org / 10.1002 / smll.201900873). For the intercusp region 1201, a gradient diamond pattern was utilized, as can be seen in Figure 12-3, as opposed to the distinction between the commissures, intercusp trigone, and annulus. The gradient transitions from a predominantly horizontal alignment at the base, intended to mimic the circumferential fibers present within the annulus, to a predominantly vertical alignment at the top, mimicking the longitudinal fibers present in the commissures (as can be seen in Figures 9-3 and 9-4).

[0103] The fiber design completely avoided horizontal or vertical orientation because a linear MEW fiber pattern is stiffer and weaker than a diamond-shaped pattern of the same porosity, as can be seen from the data shown in Figures 5-4. A similar distribution of fiber orientation was achieved when comparing the fibers in the MEW scaffold design with those previously analyzed in native tissue, as shown in Figures 12-4. Thus, the resulting scaffold design combined native tissue observations with a continuous interface to create a biomimetic scaffold design with gradient porosity, region-specific layer counts, tailored fiber orientation, and spatially heterogeneous regions.

[0104] The above-described process for designing MEW heart valve scaffolds can be generalized to design MEW scaffolds for other soft tissues.

[0105] Scaffolds 1201 of the configuration shown in Figure 12-5 were successfully fabricated from PCL with an average fiber diameter of 27.69 ± 4.66 μm. Figure 12-6 shows snapshots of the deposited fibers taken at 1, 2, 6, 29, and 74 seconds after the start of MEW printing to illustrate the sequential printing path.

[0106] Sequential printing passes were designed to employ a bonding technique, employing a continuous transition from five layers in the leaflet region to ten layers in the remainder of the scaffold. In some cases, where dual printed layers were deposited due to the bonding method, fiber fusion occurred simultaneously, resulting in a thicker fiber instead of two layers of fiber. The fused, thicker fiber 1202 can be seen in Figure 12-7. The average fiber diameters for unfused and fused fibers were 24.34 ± 0.75 μm and 33.27 ± 2.30 μm, respectively. Comparison of the cross-sectional area of ​​fused fibers to that of unfused fibers confirmed the fiber fusion phenomenon, with fused fibers having approximately twice the area (187 ± 14%). This is likely due to the sustained high temperature of the deposited fibers caused by the very short time between fiber depositions on the same pass. This was reminiscent of the morphological arrangement of collagen fibrils found in native tissue, where smaller collagen bundles bond together to form larger, densely packed bundles as they change orientation.

[0107] The scaffolds were then mechanically characterized in the "circumferential" and longitudinal directions under biaxial, physiologically relevant strains, as depicted in Figure 13-1. Relative strain-related metrics, used to understand tissue-like properties such as anisotropy and viscoelasticity, were studied. Furthermore, it is important to note that the present scaffolds contained all leaflet, interleaflet trigone, annulus, and commissure regions, meaning that comparisons with region-specific data are not strictly physiologically accurate. Attempts were made to assess similar loading conditions in porcine tissue; however, due to the difficulty of clamping the tissue in the mechanical tester and the variable cross-sectional area, reliable, quantifiable data were not available for use as a comparison. Interestingly, across all samples of porcine tissue (n = 12), it was observed that failure never occurred in the interface region. Therefore, comparisons were made between the scaffolds and values ​​reported for native tissue in regions as similar as possible to the scaffolds. In future studies, hemodynamic testing in a flow loop setting will ensure appropriate physiological loading of such scaffolds.

[0108] The ratio of strain rate in the circumferential direction to that in the longitudinal direction was selected as approximately 2:1. When tested to failure, the scaffolds exhibited similar Young's moduli in both directions, whereas the yield strength in the circumferential direction was approximately twice that in the longitudinal direction (see Figure 13-2). Scaffold strength can be improved by adjusting the number of layers or fiber diameter. Strain-displacement vector mapping revealed complex, locally anisotropic deformation behavior, with circumferential and longitudinal strains primarily due to the leaflet and intercusp trigone regions, respectively. However, the strain-displacement vectors showed gradual changes in direction and magnitude between these regions, indicating a smooth transfer of load across the interface. Notably, the yield strains in the circumferential and longitudinal directions were 27.7 ± 6.9% and 16.6 ± 5.4%, respectively, both of which were higher than the average maximum strains reported in the literature across different regions of the aortic valve. This implies that the scaffold will remain within the elastic region and will not plastically deform under physiological strain.

[0109] The hysteresis of the scaffolds was then determined by cyclically testing the samples to constant strains of 20% and 10% in the circumferential and radial directions, respectively, and calculating the ratio of the area under the unloaded curve to the area under the loaded curve. The stress-strain plots are shown in Figure 13-3, with both plots for circumferential strain 1301 and longitudinal strain 1302. A consistent, low level of hysteresis (approximately 20% energy loss per cycle) was observed in both directions. Our data is consistent with the 17% hysteresis reported for porcine aortic heart valves in the literature. The relaxation behavior of the scaffolds was characterized using 5% and 2.5% strain increments in the circumferential and radial directions, respectively, as shown in Figure 13-6. The scaffolds were then allowed to relax for 1,000 seconds after each strain step, a reasonable time frame for them to exhibit the majority of their relaxation behavior. Stress-time graphs depicting relaxation behavior are shown in Figure 13-5. At all strain levels, the scaffolds exhibited a rapid initial relaxation before stabilizing to an asymptote, which was then reported as a relaxation percentage. A consistent relaxation of 27.6 ± 1.5% was maintained in the circumferential direction, while the radial direction exhibited a large initial relaxation before stabilizing to 30.6 ± 1.7%. These values ​​were notably similar to those reported for porcine aortic heart valve relaxation. In summary, the bioinspired aortic heart valve interface scaffold thus demonstrated properties reminiscent of native tissue, including anisotropy, hysteresis, and yield strain within those of native tissue.

[0110] In the above, a continuous interface across the structural regions of a MEW scaffold is provided. Complex functions are utilized to define the printing path between discrete endpoints within the structural regions, which are connected by the interface regions. Thus, fibers assume complex shapes as they transition from one region through the interface region to the next, structurally related to the first. A simplified view of the microstructural organization of collagen fibers in the interface regions of biological tissues may be exploited in designing MEW tissue scaffolds. For example, during testing and analysis, the microstructural organization of collagen fibers in the interface region of an aortic heart valve was captured using high-resolution correlative multimodal imaging. This was utilized to conceive the functional design of a MEW heart valve scaffold, including a biomimetic interface region for heart valve tissue engineering applications. Systematic morphological and mechanical studies of three different methods of connecting biphasic MEW scaffolds revealed that weaker regions dominate the tensile mechanical response. With regard to bending stiffness, a property often overlooked in scaffold design, continuously connecting biphasic scaffolds demonstrated improved flexibility of the resulting construct. Crucially, the novel software and accompanying GUI enabled the rapid design and G-code generation of not only an array of complex MEW scaffold designs, but also the continuous interfacial boundaries across which these regions are connected. This software opens up new capabilities of MEW for the field of interfacial tissue engineering. Subsequently, we designed, fabricated, and tested bioinspired MEW scaffolds for the aortic heart valve interface region, demonstrating for the first time scaffolds containing continuous interfaces, gradient porosity, region-specific layer counts, and tailored fiber orientation in a single biomimetic design. When tested under physiologically relevant biaxial conditions, the scaffolds exhibited promising tissue-like behavior, including strain yielding, hysteresis, and relaxation, all similar to native tissue.

[0111] (MEW scaffolding processing) Scaffolds were fabricated from medical-grade poly(ε-caprolactone) (PCL) using an in-house constructed MEW device as previously reported. The specific processing parameters used were air-driven extrusion at 100 kPa, passing a 23 G metal needle, a 3 mm working distance, a 400 mm / min translation speed, a 4.4 ± 0.1 kV voltage applied to the spinneret and grounded collector plate, an 86 °C ring heater, and a 31.5 °C bed heater. The exact processing parameters used varied slightly depending on the environmental conditions of the day. All two-phase scaffolds used for tensile and flexural testing had dimensions of 10 mm height x 40 mm width (20 mm per width pattern). This processing setup was found to allow for fiber fusion within the same layer and also proper bonding between different layers. Complete fusion between layers may not be necessary in some cases if the layers are not of the same composition or desired depending on the degree of flexibility required for the overall layered structure.

[0112] MEW scaffold imaging and measurements MEW scaffolds were first imaged at a resolution of 4.51 μm / pixel using a Hirox RH-2000 digital microscope (Hirox, Europe). Accompanying software was used to measure fiber diameter at three locations across n = 3 scaffolds of each type. Scaffolds were then cut using scissors and fixed to SEM stubs using double-sided carbon tape. The stubs were then sputter-coated with gold, two at a time, for 60 seconds using a JEOL Smart Coater (JEOL, USA). SEM images were then obtained using a JCM-6000 Benchtop SEM (JEOL, USA) at an accelerating voltage of 10 kV, 30x magnification, high probe current, and high vacuum mode.

[0113] (Uniaxial tensile test) Scaffolds were mechanically tensile tested uniaxially across the interface using a CellScale Biotester (CellScale, Waterloo, Canada) equipped with a 1.5 N load cell. Samples (n = 3 per scaffold type) were clamped using custom 3D-printed miniature clamps and suspended in air at room temperature. Uniaxial testing used a displacement rate of 1% length / min. Stress-strain curves were plotted using the generated force-displacement data, and strain was defined as the engineering strain (change in length / initial length). The initial length was kept constant at 15 mm for all calculations. Cross-sectional area was quantified as scaffold wall height, as opposed to peak height. Six measurements were taken across n = 3 samples of each biphasic scaffold. Cross-sectional area was observed to vary within individual scaffolds between the two patterns and the interface. However, because the majority of deformation occurred within the weaker pattern, as confirmed by visual analysis, the cross-sectional area of ​​the weaker pattern was used to calculate the associated stress. The average value for each pattern is meandering = 1.06 ± 0.03 mm 2 , rhombus=1.25±0.20mm 2 , 1mm square=1.40±0.21mm 2 , 0.5mm square=1.16±0.04mm 2 The Young's modulus was calculated from the slope of the stress-strain curve in the steepest linear region, and the strain range depended on the sample / pattern. The yield strength was calculated as the point where the stress-strain curve plateaued. The ultimate tensile strength was determined for each sample as the maximum stress was reached.

[0114] (biaxial tensile test) Biaxial tensile tests were performed using the same equipment as the uniaxial tests, with a 5N load cell. A 50mN preload was applied before every test to ensure a consistent initial state of the scaffolds. Young's modulus and yield strength were calculated using the same protocol as for the uniaxial tests. Cyclic tests were performed for one preload cycle (to establish the in vivo state of the material) and nine subsequent load cycles, which were then plotted as stress-strain. Hysteresis was calculated for each cycle as the ratio of the area under the unloaded curve to the area under the loaded curve. The relaxation behavior of the scaffolds was characterized using 5% and 2.5% strain increments in the circumferential and radial directions, respectively (Figure 8D). The scaffolds were then allowed to relax for 1,000 seconds, an appropriate time frame for the scaffolds to exhibit the majority of their relaxation behavior. The relaxation percentage was reported as the difference between the maximum and minimum stress for each step.

[0115] (bending test) Bending tests were performed using the Peirce cantilever test method (ASTM D1388). A custom test fixture was designed in Fusion360 (Autodesk, USA) and printed using a Prusament PLA (Prusa Research, Czech Republic) in conjunction with a Prusa MK3s+ (Prusa Research, Czech Republic). Angles were measured six independently per bending pattern: three times in one direction, and then three more times with the scaffold inverted to account for the natural warping of the printed scaffold.

[0116] (Complex G-code generator GUI and path visualization) An in-house program was developed using Python (Python Software Foundation, USA) to incorporate the continuous interface design into spatially heterogeneous G-code for MEW printing. A script was created to generate a GUI into which the desired parameters of the scaffold could be input. Once input, the interface would execute another script that performed the calculations necessary to plot the data for the desired print path. This data was then converted by another script into a G-code Notepad file for input into the MEW printer software. To enable visualization of the G-code, another custom Python-based software was built to import the raw G-code and generate both static and dynamic renderings of the print path with customizable scale, color, line width, and path speed.

[0117] (Orientation Mapping) Orientation analysis of SHG images and MEW scaffolds was performed using the OrientationJ plugin

[90] for the software Fiji (National Institute of Health, USA)

[91] . Using the OrientationJ Analysis module, hue, saturation, and brightness color measurements were calculated using a 2-pixel cubic spine. The OrientationJ Distribution module was then used to plot the distribution of orientation.

[0118] (statistical analysis) All data are presented as mean ± standard deviation for n = 3 samples unless otherwise stated. For uniaxial tensile test data, significance was assessed using one-way ANOVA combined with Tukey's multiple comparison test. For flexural stiffness data, significance was assessed using two-way ANOVA combined with Tukey's multiple comparison test. For biaxial tensile test data, significance was assessed using a parametric paired t-test. Alpha was 0.05 for all tests. All statistical analyses were performed using GraphPad Prism software (GraphPad, Sandiego, USA).

[0119] As mentioned, MEW printing as described above contrasts with traditional MEW printing, in which layers with distinct structural regions would not be printed in one continuous step. Rather, the regions of the layer would be printed separately and then stitched together to form the layer. The mechanical advantage of continuous printing through the regions was not anticipated. The contrast with conventional thinking is even greater in embodiments where fibers are intentionally made to converge into a thicker fiber. This is considered "fiber bridging," which conventional wisdom has always avoided as a flaw. This is because MEW has a limited ability to reduce pore size due to electrostatic fiber repulsion from the trapped charge inherent in the MEW printing process, where high voltage electrostatically charges the melt jet, trapping the charge as it cools. This phenomenon is still being understood by those skilled in the art and would be expected to allow for fiber convergence, which is normally avoided. However, in some embodiments of the present method, "fiber bridging" is intentionally induced in a controlled manner. The MEW printing parameters presented above allow for control of the bond between two merging fibers to form one single, thicker fiber. This opens up the possibility of utilizing data on fiber distribution, which can be acquired using imaging modalities, to create biomimetic designs for MEW scaffolds. Scaffolds designed using this methodology can be used for the fabrication of soft tissue implants involving biodegradable materials or for the fabrication of non-biodegradable implantable devices.

[0120] Example 6 Gradient porosity scaffolds G-code is a language used by most 3D printers that specifies Cartesian (xyz) coordinates for the printer to move in a specific sequence. A G-code generator was created using Python that allows users to input desired design parameters, generate output G-code, and control the MEW printer as well as an image preview of the scaffold architecture. The novelty of this generator is its ability to specify gradient changes in porosity.

[0121] The gradient can be mathematically defined and customized (FIG. 16). The gradient function is a linear function determined by: [ka] In the formula, p n is the pore size (any positive number in mm) and m is the gradient coefficient (any non-zero number where a positive m will increase the pore size and a negative m will decrease it).

[0122] There are two dimensions per pore, length (X) and width (Y), and gradients can be applied to either one or both of these dimensions. These are referred to as either X-gradients or Y-gradients, respectively, and indicate the varying pore dimensions.

[0123] The overall scaffold architecture itself is also two-dimensional, and therefore the pore size can be varied in either one or both of these directions (horizontally or vertically). For convenience, in this example, the gradient is applied only in the horizontal direction.

[0124] This gives two types of gradients: X-gradient: applied horizontally. This can be seen as varying the pore size parallel to the direction of the gradient, hence the name parallel gradient. Y-gradient: applied horizontally, which can be considered as varying the pore size perpendicular to the direction of the gradient, hence the name orthogonal gradient.

[0125] Here, the gradient was applied in three different patterns / shapes (rectangular, diamond, or serpentine). Note that in the case of the serpentine scaffold, either the wavelength, the amplitude, or both can be varied.

[0126] Gradient refers to a gradual spatial change in properties (geometric features, mechanical properties, composition, chemical properties, or other). The present scaffolds create multiple gradient properties within a single scaffold. Geometrically, this is already evident (Figures 16-19), however, gradient mechanical properties are also present (Figure 20). Note that, although not shown here, other properties such as gradient tissue / cell composition could potentially be enabled / achieved through the use of these scaffolds.

[0127] To understand the effect of gradients on mechanical properties, constrained biaxial tensile tests were performed. In this, the scaffold was clamped on its four edges and then pulled horizontally while held (constrained) vertically. This allows for observation of local changes in strain throughout the scaffold, where strain is the amount of deformation that occurs in the object. Figure 20 demonstrates the highly heterogeneous mechanical properties produced as a result of the gradient scaffold architecture. Note that a completely uniform / homogenous scaffold design would exhibit the same color throughout. The novelty here is the ability to create gradients of mechanical properties throughout a single scaffold. The present invention provides the ability to create gradients not only in one direction, but also in complex arrays, which may be beneficial for tissue-related applications.

[0128] Variations and modifications may be made in some of the foregoing without departing from the spirit or scope of the disclosure.

[0129] The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only, and not by way of limitation. While particular embodiments have been illustrated and described, it will be apparent to those skilled in the art that changes and modifications may be made thereto without departing from the broader aspects of the inventors' contribution. The actual scope of protection sought is intended to be defined in the following claims when viewed in their proper perspective based on the prior art.

[0130] In the claims that follow and in the preceding description of the invention, unless the context requires otherwise, either by express terminology or by necessary implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., to specify the presence of stated features but do not exclude the presence or addition of further features in various embodiments of the invention.

Claims

1. A fused electrowriting soft tissue scaffold, a first region having one or more sets of fibers; and a second region having one or more sets of fibers. an interface region joining the first region and the second region, the interface region being electrowritten together with the first region and the second region in successive printing passes such that fibers in the interface region are bonded between individual fiber pairs in the first region and the second region, respectively; Scaffolding.

2. 10. The scaffolding of claim 1, wherein a path for at least one of said fibers within said interface region is defined manually or by a mathematical function.

3. 3. The scaffold of claim 2, wherein the function is defined such that the fiber has a complex shape as the fiber transitions from one of the first region or the second region, through the interface region, to the other of the first region or the second region.

4. 10. The scaffolding of any preceding claim, wherein the second region has a higher porosity than the first region, or vice versa.

5. 5. The scaffold of claim 4, wherein the higher porosity in the second region is formed, at least in part, by joining all of two or more adjacent fibers in the first region as the two or more adjacent fibers in the first region transition into the second region.

6. 6. The scaffold of claim 4 or claim 5, wherein the higher porosity in the second region is formed at least in part by causing the fibers to fan outward as they transition into the second region.

7. 7. The scaffold of any one of claims 4-6, wherein the higher porosity in the second region is formed at least in part by configuring the successive printing paths to cause additional sets of fibers to be deposited that are offset from other fibers in the first region by a distance that is less than the size of a pore size.

8. 10. The scaffolding of any preceding claim, wherein the first region or the second region or both comprise a first set of fibers arranged generally parallel to each other and a second set of fibers arranged generally parallel to each other, the second set of fibers being arranged at an angle, preferably transverse to the first set of fibers, and each fiber in the second set of fibers has a serpentine arrangement with defined valleys and peaks.

9. 10. The scaffold of any preceding claim, wherein the first region and the second region are heterogeneous in that they differ from each other in one or more spatial parameters.

10. 10. A scaffolding according to any preceding claim, comprising multiple layers of fibres.

11. 11. The scaffold of claim 10, having different portions and portions with different numbers of layers.

12. 10. The scaffold according to any of the preceding claims, which is a heart valve scaffold.

13. 13. The scaffolding of claim 12, wherein the first region is a valve leaflet and the second region is an inter-leaflet trigone.

14. 1. A method for providing a melt-electrowritten soft tissue scaffold having at least two structurally heterogeneous regions and an interface region between said at least two structurally heterogeneous regions, the method comprising: providing a printing path during a melt-electrowriting process, along which a polymer melt material is continuously extruded, to form said heterogeneous structural regions and said interface region in a single printing step.

15. The method of claim 14 , comprising causing the successive printing paths to be defined segment by segment, the successive printing paths being defined by different functions in the heterogeneous region and the interface region, respectively.

16. The method of claim 15 , wherein the function or functions defining the continuous printing path as it traverses the interface region are mathematical functions that provide a curved or serpentine shape.

17. 13. The scaffold of any one of claims 1-12, comprising gradient porosity, wherein the size of the pores gradually increases or decreases spatially across the scaffold in either one or two directions.

18. 18. The scaffold of claim 17, wherein the shape of the pores in the scaffold is selected from rectangular, diamond, or serpentine.

19. The scaffold of any one of claims 1-12, wherein the scaffold comprises multiple layers of fibers.

20. 20. The scaffolding of claim 19, wherein the multiple layers of fibers comprise gradients in geometric characteristics and / or mechanical properties.

21. 21. The scaffold according to any of claims 17-20, wherein said scaffold with gradient porosity is used for the manufacture of a heart valve.

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