Field of the invention

The invention relates to implant devices, particularly the invention relates to scaffolds produced by electrospinning which can be used as implant device either combined with a hydrogel or by itself. The scaffolds have an aligned oriented region and a random oriented region that allows for surgical retention methods. The invention further relates to methods of producing the implants and uses thereof. The invention also relates to custom electrospinning targets for the production of the scaffolds.

Introduction

Hydrogels are some of the most promising materials in tissue engineering and regenerative medicine, due to their hydrated, extracellular matrix (ECM)-like environment. However, many of the hydrogels manufactured cannot support surgical retention methods such as suturing techniques or current medical adhesives and there are no alternatives available to surgeons to secure them in vivo. This limits their use to the research laboratory regardless of their promising biologically influential properties.

Electrospinning is a common bioengineering technique, producing non-woven fibrous matrices that mimic the collagen fibres of the native extracellular matrix, and controlled porosity to allow the infiltration of cells. However, electrospinning is limited in the ‘shapes’ it can create - mainly being produced in large sheets and cut to shape - stretching and damaging the fibres. Aligned electrospun fibres can be manufactured to allow the passage of light through the matrices for use in applications such as corneal implants or for specific bio-mimicking purposes, such as the aligned ECM of the cardiac microenvironment; however, cutting aligned fibres can affect their alignment and alter their optical properties.

Therefore, improved implant devices are needed that allow surgical retention methods. Particularly there is a need for implants that can combine the advantages of increased structural integrity, allowing surgical retention methods, and hydrogel-like properties such as ability to grow cells and/or transparency. These, among others, objectives are achieved by the implant devices as defined in the appended claims. Description of the figures

Fig. 1 depicts different examples of custom-made electrospinning targets for used in the manufacturing of the scaffolds as described herein. The depicted scaffolds are used to electrospin corneal or tricuspid implant scaffolds. The targets comprise a conductive and a resistive part.

Fig. 2 depicts an exemplary corneal implant produced by electrospinning. The implant comprises a random region and an aligned region. The aligned region is optically translucent, while the random region allows for surgical retention methods such as suturing. A quarter of the scaffold was cut and placed on a black scanning electron microscopy chuck to visualise the aligned region with the naked eye and an enlarged picture is shown (bottom).

Fig. 3 depicts scanning electron microscopy images of the exemplary corneal implant at different magnifications, demonstrating detailed views of the aligned and random regions.

Fig. 4 depicts an exemplary electrospun corneal implant. Shown are from left to right the used electrospinning target, the electrospun scaffold, the scaffold embedded in a hydrogel, and scanning electron microscopy images of the random, aligned and border regions of the scaffold.

Fig. 5 depicts an exemplary electrospun tricuspid valve implant. Shown are from left to right the used electrospinning targets (one large and one smaller), the electrospun scaffolds, and scanning electron microscopy images of the random, aligned, border and centre regions of the scaffold.

Fig. 6 (top) depicts a graph plotting the suture retention of a radial UPy polymer construct. Displacement in mm is plotted on the x-axis, and force (in gram) is plotted on the y-axis. Fig. 6 (bottom) depicts a graph plotting the suture retention of a radial PLA construct. Displacement in mm is plotted on the x-axis, and force (in gram) is plotted on the y-axis.

Fig. 7 depicts a graph plotting the transmission of different wavelengths of light through the aligned part of a PLA construct embedded in 20% gelatine in comparison to tissue culture plastic. The PLA construct demonstrates equal opacity compared to the tissue culture plastic.

Fig. 8 depicts the computer aided design (CAD) exploded view for the electrospinning target for the tricuspid valve implant. The bottom structure depicts the resistive component of the target, while the middle and top structures depict the conductive components.

Fig. 9 depicts the CAD exploded view for the electrospinning target for the corneal implant. The middle structure depicts the resistive component of the target, while the top and bottom structures depict the conductive components.

Fig. 10 depicts the suture retention of different materials as indicated in the inset (top panel). The bottom panel compares the optical transparency of different electrospun materials.

Fig. 11 depicts the rheology of different fibres embedded in gels.

Fig. 12 depicts qPCR results for different marker genes of corneal cells grown in fibres alone or embedded in gels.

Summary of the invention

In a first aspect, the invention relates to a scaffold for an implant device, wherein the scaffold comprises:

- an aligned oriented region, and

- a random oriented region that allows surgical retention methods, and wherein the scaffold is constructed by charge based electrospinning of a biocompatible material, wherein the aligned oriented region and random oriented region are produced of a continuous fibre, and wherein the scaffold does not require cutting prior to use as an implant device.

In a second aspect, the invention relates to a hydrogel implant device comprising a biocompatible hydrogel and embedded therein the scaffold as defined in the first aspect of the invention.

In a third aspect the invention relates to a method of manufacturing a scaffold as defined in any one of claims 1 to 4, the method comprising: electrospinning a biocompatible polymer to a custom electrospinning target; wherein the custom electrospinning target comprises one or more areas with conductive material and one or more areas with resistive material, wherein the conductive material allows for random fibre generation, and wherein the one or more areas with conductive material are positioned such as to allow during electrospinning jumping of the electrospun material jet between different parts of the area or different areas thus creating an aligned area on the one or more resistive areas, and wherein the electrospinning target is not electrically charged or electrically connected to the electrospinning device.

In a fourth aspect the invention relates to a method for manufacturing a hydrogel implant device, the method comprising: manufacturing a scaffold according to the method of the third aspect of the invention, or obtaining a scaffold as defined by the first aspect of the invention, and embedding the scaffold in a biocompatible hydrogel, preferably wherein the biocompatible hydrogel is selected from the list consisting of alginate, gelatine, collagen, poly-ethylene glycol (PEG), hyaluronic acid, methacrylated gelatine, methacrylated collagen, methacrylated hyaluronic acid, norbornene, acrylate-siloxane, cellulose, chitosan, polyvinyl alcohol (PVA), elastin, fibroin, fibrinogen, fibronectin, albumin, human serum albumin, poly(acrylic acid) and it’s salts, poly(vinylpyrrolidone), polyacrylamide, starch and other polysaccharides, decellularized extracellular matrix and silk.

In a fifth aspect the invention relates to the scaffold as defined by the first aspect of the invention or the hydrogel implant device according to the second aspect of the invention or obtained or obtainable according to fourth aspect of the invention for use in a surgical method.

In a sixth aspect the invention relates to a custom electrospinning target comprising one or more areas with a conductive material and one or more areas with a resistive material, wherein the conductive material has a minimum resistivity of 0 p (Q m) and the resistive material has a minimum resistivity of 1 .5x103 p (Q m) at 20°C, and wherein the one or more areas with conductive material are positioned such as to allow, during electrospinning, jumping of the electrospun material jet between different parts of the area or different areas thus creating an aligned area on the one or more resistive areas, and wherein the electrospinning target is not electrically charged or electrically connected to the electrospinning device. Detailed description of the invention

The inventor has overcome the above referenced shortcomings and problems posed in the prior art. Particularly, the present invention describes a scaffold for an implant that allows surgical retention methods such as suturing, and its production by electrospinning. To achieve this the inventor custom 3D printed and milled exemplary aluminium parts as components of an electrospinning target which can be placed in any standard electrospinner. The scaffolds can be made in any electrospinnable, biocompatible material. The design was optimised in these exemplary cases for corneal tissue engineering and cardiac implants, but is customizable to any other shape (e.g. spirals for cartilage engineering, leaflets for vein or artery valve engineering, tympanic membrane, tendon engineering, intervertebral disc engineering, pelvic mesh or oesophageal implants).

This invention describes the first optically transparent corneal prosthesis that may incorporate hydrogels and is manufactured from electrospun fibres as described herein. It can be used either acellularly or with donor cells such as primary cells or iPSC-derived cells. It was further found that optional cells may implanted directly in the scaffold, and the scaffold thus may be used in the absence of hydrogel, or the optional cells may be implanted together with a hydrogel in the scaffold. Whether the use of hydrogel and/or cells is desirable depends on the intended application of the implant, the type of material used and the intended cell type.

This invention addresses the fracture strength and elastic modulus that is required to support the expected load in tissue engineered constructs subjected to surgical retention methods such as suturing. It allows the use of promising materials otherwise incompatible with common surgical techniques. This invention’s main application is in the field of tissue engineering and medical implants. As an example is provided a corneal implant and a tricuspid valve implant, however it could be used to stabilize any hydrogel like material for implantation and can have further applications (optionally with customization) such as in but not limited to cartilage, tympanic membrane and the tendon tissue engineering field. It may also be used as a filter for cells in microfluidic devices, and used in biosensors.

For example, current corneal tissue engineered solutions rely on two part constructs - a ‘window’ of PMMA, acrylic or glass to allow vision and a ‘skirt’ region made of materials such as titanium, human tooth/bone, electrospun PCL and acrylic. Incompatibility with the mechanical characteristics of these rigid materials within the human eye leads to stromal melting, rejection and loss of vision - they are reserved as last ditch attempts to preserve patient vision. Complications such as intraocular disassembly are reported. Additionally, these implants are highly visible in the eye, and patients report low satisfaction. Therefore, improved implants that allow surgical retention methods are needed.

EP 3287149A1 discloses a wound dressing; the dressing may also be used for cornea treatment. The document describes creating with electrospinning a surface with aligned fibres. Attached to this aligned area is a frame to provide support. The described product is thus a two-part construct.

KR 101966932B1 describes a method of electrospinning, where domes are used on a rotating electrospinning target, which is electrically connected to the electrospinning device to charge the electrospinning target. The fibres deposited on the dome are aligned; the structure may be used as a corneal implant.

WO 2017/052054A1 describes an implementation of gap spinning using an electrospinning target, which is electrically connected to the electrospinning device to charge the electrospinning target.

Therefore, in a first aspect the invention relates to a scaffold for an implant device, wherein the scaffold comprises:

- an aligned oriented region, and

- a random oriented region that allows surgical retention methods, and wherein the scaffold is constructed by charge based electrospinning of a biocompatible material, wherein the aligned oriented region and random oriented region are produced of a continuous fibre, and wherein the scaffold does not require cutting prior to use as an implant device. It is further envisioned that instead of solely allowing surgical retention methods, the random oriented region provides structural support for the scaffold.

The present invention further provides a method where in a single electrospinning step is used to construct the scaffold. This means that the scaffold is essentially produced from a single fibre. An advantage of this is that it greatly enhances the strength of the resulting product and reduces risk of issues such as delamination in situ. Further, producing the scaffold in a single electrospinning step allows for automation of the process. The scaffold can be produced by using custom electrospinning targets as detailed below, resulting in several advantages: Equipment does not have to be devoted to the manufacture of these scaffolds as the targets are simply placed in existing electrospinning set ups, and targets can be transferred between electrospinning equipment easily. Targets can be customised for dimensions easily. Further, degradation, biocompatibility and mechanical properties are fully customisable via the materials used.

Therefore, in an embodiment the invention relates to a scaffold for an implant device, wherein the scaffold comprises: an aligned oriented region, and a random oriented region that allows surgical retention methods, and wherein the scaffold is constructed by charge based electrospinning of a biocompatible material, wherein the aligned oriented region and random oriented region are produced of a continuous fibre, and wherein the scaffold does not require cutting prior to use as an implant device, wherein the electrospinning is performed using a custom electrospinning target, the electrospinning target comprising one or more areas with a conductive material and one or more areas with a resistive material, wherein the conductive material has a minimum resistivity of 0 p (Q m) and the resistive material has a minimum resistivity of 1 .5X 10 ³ p (Q m) at 20°C, and wherein the one or more areas with conductive material are positioned such as to allow, during jumping of the electrospun material jet between different parts of the area or different areas thus creating an aligned area on the one or more resistive areas, and wherein the electrospinning target is not electrically charged or electrically connected to the electrospinning device.

When used herein, the term cutting, when referring to the scaffold is not intended to refer to cutting of off target fibres, which may occur in any electrospinning process and need to be removed from the final product. The term cutting is intended to refer to removing excess material, for example because multiple scaffolds are produced in a single process which need to be cut out, or for example because the electrospinning process or dimensions cannot be controlled properly and excess material is deposited around the edges of the scaffold. Therefore, the term cutting is also not intended to cover the creation of structural features such as gaps or openings in the scaffold. The term cutting may refer to any process used to remove the excess material, such as but not limited to punching out the scaffold, cutting with a sharp object or using a laser.

An advantage of using the herein described custom electrospinning targets is that they allow very high accuracy and control over the final product. One advantageous effect thereof is that the very precisely manufactured scaffolds do not require any additional cutting or modification before use, e.g. to remove excess material or uneven parts. This has the advantage that no additional steps are needed in the procedure and the strength of the scaffold is much higher, as cutting will inadvertently cut the fibre and reduce strength of the product.

An additional advantage of the electrospinning targets described herein is that that the target can be transferred between electrospinnners without altering the target or the process, and requires no alteration of electrospinning machinery.

It was found that the scaffolds described herein may be used alone, or to support biologically compatible but un-handleable hydrogels. Mechanical testing confirms suture retention at loads multiple-fold times higher than those applied in a surgical setting. An exemplary construct was assessed by an expert and found to be handle-able in clinical setting, in dry, hydrated and embedded in hydrogel forms. It can be sutured and handled in a surgical setting.

It was found that using electrospinning target having conductive and resistive areas a construct can be produced where parts of the construct have aligned fibres and parts of the construct have randomized fibres. There are several advantages for this. For example, aligned fibres are optically transparent when hydrated which may for example be beneficial for corneal implants. Random oriented fibres on the other are not optically transparent but have much higher tensile strength and can thus serve for surgical retention methods like suturing.

By defining areas in the electrospinning target that are conductive and are resistive, the deposition of the jet of the electrospun material can be controlled. It was found by the inventor that the jet of material “jumps” between different areas of conductive area - depositing random oriented fibre on the conductive areas. Because the jet of material “jumps” from conductive area to conductive area over the resistive area, inherently the fibres deposited on the resistive area by the jet of material are much more aligned, while the material is deposited in a more or less random manner on the conductive area. Thus by controlling the shape and area of the conductive and resistive areas on the electrospinning targets, the orientation of the fibres in the electrospun construct can be controlled. It is understood that in order to provide the envisioned functionality, the conductive areas of the electrospinning target are preferably grounded.

When used herein, the terms conductive and resistive refer to electrical conductivity (or lack thereof).

When used herein, the term electrospinning refers to the process of drawing continuous polymeric fibre from either a polymer solution or polymer melt, based on an electrohydrodynamics phenomenon, using electrostatic force in a liquid-jet form to fabricate polymer composites.

Electrospinning is a technology well known to the skilled person. Different types of electrospinning can be distinguished and each are suitable for use in the invention:

Basic Needle Based Electrospinning - Vertical and horizontal setups are known. A typical setup consists of three parts: a) a syringe needle (associated to a syringe and a syringe pump) with a droplet of polymer solution hanging at the tip, b) a ground electrode used as the fibre collector, and c) a high voltage source which creates an electrical potential difference between syringe needle and collector. Polymer solution is pumped through a syringe at a constant rate, forming a droplet at the end of the needle. A high voltage (commonly from 5 to 30 kV, but can be as high as 100 kV) is applied between the polymer solution feed and a collecting electrode. Electric field is subjected to the end of a capillary tube that contains the polymer fluid held by its surface tension. This induces a charge on the surface of the liquid. Mutual charge repulsion causes a force directly opposite to the surface tension. As the intensity of the electric field increases, the hemispherical surface of the solution at the tip of the capillary tube elongates to form a conical shape known as Taylor cone. When the electric field reaches a critical value at which the repulsive electric force overcomes the surface tension force, a charged jet of the solution is ejected from the tip of the cone. The fluid jet is accelerated and stretched by columbic force exerted the external electric field and becomes dramatically thin while travelling toward the collector, leading to the formation of a continuous solid fibre as the solvent evaporates. Decreasing the jet diameter, the surface charge density increases and the resulting high repulsive forces split the jet into several smaller jets. The jet is seriously elongated by a bending and whipping processes caused by electrostatic repulsion initiated at small bends in the fibre, until it is finally deposited on the collector. Two types of collector may be used, either stationary or rotary/moving collector.

Other types of electrospinning known and suitable for the invention are: Coaxial Electrospinning, Tri-axial Electrospinning, Bi-component Electrospinning, Multineedle Electrospinning, Electro-blowing/Gas-assisted/Gas jet Electrospinning, Magnetic Field Assisted Electrospinning, Conjugate Electrospinning, Centrifugal Electrospinning, Needleless Electrospinning, such as Bubble Electrospinning, Two Layer Fluid Electrospinning, Splashing Electrospinning, Melt Differential Electrospinning, Gas Assisted Melt Differential Electrospinning, Rotary Cone Electrospinning, Rotating Roller Electrospinning/ Nano Spider Technology, Edge/Bowl Electrospinning and Blown Bubble Electrospinning.

When used herein the term scaffold refers to a plastic fibre construct obtained by electrospinning, which can be used in the construction of an implant or can serve as an implant. The scaffold may be implanted by itself, i.e. serve as the implant, or it may be embedded in a hydrogel to provide structural integrity and means for surgical retention of the hydrogel implant device. The scaffold or scaffold imbedded in hydrogel may further serve to hold cells.

When used herein the terms implant and implant device are used interchangeably and refer to a device to be implanted in a subject by means of surgical retention. The implant device may thus refer to a scaffold or it may refer to a scaffold embedded in a hydrogel, in which case it may also be referred as hydrogel implant device.

When used herein the term biocompatible implies that a material is not harmful or toxic to living tissue, cells, organs or organisms. Preferably, it implies the material is not harmful or toxic to cells intended to be embedded in the implant device and/or not harmful or toxic to the tissue or organ of the subject it is intended to be implanted in, and/or it is not harmful or toxic to the organism itself.

When used herein subject refers to a human or an animal such as a non-human mammal in which the implant device is intended to be implanted.

When used herein, the term construct refers to the electrospun object, preferably the scaffold without any additional components such as hydrogel or cells. When used herein the term surgical retention method refers a method for retaining a device or implant in the desired location in a subject or object. Preferred surgical retention methods may be suturing or gluing, but other surgical retention methods are known to the person skilled in the art and may be applied. In general, any surgical retention method will put a strain on the implant device. For this reason, hydrogels are not suitable for implanting, as they lack the necessary rigidity for surgical retention. For example, to be suitable for suturing the implant device should be able to tolerate a tensile strength of up to 0.2 Newton (N). Therefore, in an embodiment, the random region has a suture retention load tolerance of at least 0.05 N, preferably at least 0.1 N, more preferably at least 0.2 N. It is however understood that the minimal suture retention load will vary and depend on the envisioned application of the implant.

When used herein the terms aligned oriented region and random oriented region refer to a region in the electrospun scaffold with substantially aligned or randomly oriented electrospun fibres. Preferably, the orientation is determined by analysing the orientation of each fibre (in degrees) in a square millimetre. Thus when used herein, an aligned oriented region refers to a region wherein in a square millimetre at least 50% of the fibres fall within an orientation bandwidth of 60 degrees, meaning of all the different determined angles (e.g. from a potential 0 to 180 or -90 to 90 degrees) at least 50% fall within a range of 60 degrees from each other. Alternatively, when fibres are randomly distributed this should be reflected in the distribution of angles determined, which should be roughly even, meaning that approximately 33% of fibres should fall within each range of 60 degrees. Therefore when less than 50% of the fibres fall within an orientation bandwidth of 60 degrees in a region (e.g. a square millimetre) the region is considered random oriented. Methods for determining fibre angle and distribution are known to the skilled person and for example described in Walser and Ferguson (2015) Oriented nanofibrous membranes for tissue engineering applications: Electrospinning with secondary field control. Journal of the Mechanical Behavior of Biomedical Materials. 58. 10.1016/j.jmbbm.2015.06.027, which is hereby incorporated by reference in its entirety.

Therefore, in an embodiment, the aligned region has a fibre orientation such that per square millimetre, at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80% or 85%, of the fibres are oriented within a range of 60 degrees, preferably 55, 50, 45, 40, 35 or 30 degrees, or less from each other.

It is understood that the thickness of the electrospun fibre depends on the material used and further may be varied depending on desired physical characteristics of the final construct (e.g. rigidity). Therefore, in an embodiment the biocompatible material has a fibre thickness of 0.15 nm to 10 pm, for example between 0.25 nm and 5 pm, between 0.4 nm and 2.5 pm, between 1 nm and 1 pm or between 2.5 nm and 400 nm.

It is understood that any material that can be used for electrospinning can produce the scaffolds described herein. Because one of the envisioned purposes of the scaffold is to implant the device in a subject, preferably a biocompatible material is used. Therefore in an embodiment, the biocompatible material (used for electrospinning) is selected from the list consisting of polycaprolactone (PCL), polylactic acid (PLA), ureido-pyrimidone (UPy) polymers, polyvinyl alcohol (PVA), poly(DTE carbonate), polyurethane, polycarbonate, poly-L-lactic acid (PLLA), Poly(DL-lactide-co- caprolactone, Poly(ethylene-co-vinyl acetate) vinyl acetate, Poly(methyl methacrylate), Poly (propylene carbonate), Poly(vinylidene fluoride), Polyacrylonitrile, Polycarbomethylsilane, Polystyrene, Polyvinylpyrrolidone, polyethylene oxide (PEO), polyvinyl chloride (PVC), hyaluronic acid (HA), gelatine, collagen, chitosan, cellulose, alginate, polyhydroxybutyrate and its copolymers, Nylon 11 , Cellulose acetate, hydroxyapatite, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), decellularized extracellular matrix, silk, AE-941 (shark cartilage) and mixtures thereof.

It was found that the scaffold may be particularly suitable to provide a hydrogel with improved structural integrity and means for surgical retention such as suturing. Therefore, in a second aspect the invention relates to a hydrogel implant device comprising a biocompatible hydrogel and embedded therein the scaffold as defined in the first aspect of the invention.

Hydrogels are known to the skilled person. As the intended purpose of the implant device is to implant in a subject, the hydrogel is preferably biocompatible. It is understood that any biocompatible hydrogel may in principle be used in the invention, however certain hydrogels may be particularly suitable. Therefore in an embodiment, the biocompatible hydrogel is selected from the list consisting of alginate, gelatine, collagen, poly-ethylene glycol (PEG), hyaluronic acid, methacrylated gelatine, methacrylated collagen, methacrylated hyaluronic acid, norbornene, acrylate-siloxane, cellulose, chitosan, polyvinyl alcohol (PVA), elastin, fibroin, fibrinogen, fibronectin, albumin, human serum albumin, poly(acrylic acid) and it’s salts, poly(vinylpyrrolidone), polyacrylamide, starch and other polysaccharides, decellularized extracellular matrix, silk and combinations thereof.

The design was optimised in an exemplary case for corneal tissue engineering, and in another exemplary case for tricuspid valve engineering, but is customizable to any other shape. Non-limiting examples are cardiac leaflet or valve, vein or arterial implant, tympanic membrane, cartilage, tendon, intervertebral disc, pelvic mesh or oesophageal implant. For example and useful for each of these examples, it was found that cells are preferentially grown in areas with aligned oriented fibres, while random oriented fibres can be incorporated in the implant device at the locations where surgical retention (e.g. suturing) is intended. Thus, implants can be constructed with aligned areas suitable for seeding cells and random oriented areas that provide structural integrity and allow surgical retention methods. An example of this application is provided by the tricuspid valve implant. Therefore, in a preferred embodiment the region with aligned oriented fibres of the scaffold or hydrogel implant device comprises cells.

In an embodiment, the invention relates the scaffold according to the first aspect of the invention or the hydrogel implant device according to the second aspect of the invention, wherein the scaffold or hydrogel implant device is a corneal implant device, wherein the aligned region is centrally located in the implant device and wherein the random region partially or fully surrounds the centrally aligned region.

It was found that the electrospinning target can be designed such that a construct may be produced with a roughly disk shaped centre of substantially aligned oriented fibres, surrounded by a region of random oriented fibres. In this construct, the central (aligned) area is optically transparent when hydrated, while the non-optically transparent edge (random oriented region) provides the structural integrity that allows surgical retention method such as suturing. Such construct is particularly suitable for use in corneal implants, and may be used as such or embedded in a hydrogel. Cells may be included in the scaffold of hydrogel implant device. In order to construct desirable constructs for corneal implantation electrospinning targets were designed from a resistive material which include a conductive roughly circular rim. Optionally a central core of conductive material may be present allowing radial patterning of the aligned region towards the core. Exemplary electrospinning targets are shown in figure 4 and schematically represented by Figure 9.

In order to construct desirable constructs for cardiac, venous or arterial valve implantation, electrospinning targets were designed from a resistive material which include a conductive roughly circular rim and further having two or more, for example two, three, four, five, six or more, central spokes of conductive material may be present. Preferably, when the construct is intended as a tricuspid valve implant, the electrospinning target has three spokes of conductive material. Preferably, when the construct is intended as a bicuspid valve implant, the electrospinning target has two spokes of conductive material. Exemplary electrospinning targets are shown in figure 5 and schematically represented by Figure 8.

The scaffold for a corneal implant according to the invention is intended as a corneal prosthesis to address the shortage of donor human corneas. The scaffold according to the invention:

1. Is optically transparent by way of patterned aligned fibres;

2. Contains a mechanically stable random-fibre region into which surgeons can suture - securing the scaffolds in place;

3. Can be easily embedded into hydrogels without negatively/drastically affecting their optical properties to support them in vivo, and

4. Is directly manufactured in the desired format - minimizing post processing and scaffold damage. For example, the scaffold does not require cutting.

5. Is constructed from a single continuous fibre to ensure structural integrity.

It was found that scaffolds thus produced are optically translucent when hydrated. Further, scaffolds are compatible with human donor corneal primary keratocytes and human donor primary corneal endothelial cells in culture. Therefore, in an embodiment, the scaffold or hydrogel implant device further comprises cells, preferably corneal primary keratocytes and/or human donor primary corneal cells and/or differentiated induced pluripotent stem cells (iPSCs). It was found that the random region is wide enough for suturing, and provides sufficient strength for doing so. Without wishing to be bound by theory, it is speculated that the structural strength is at least attributed to the fact that the finished scaffold, that is the scaffold in a state ready for implanting, is produced from a single continuous fibre by electrospinning. This is due to the method described herein below, allow high control over the shape and aligned I random regions of the scaffold, and due to the fact that the scaffold does not require cutting (which inherently leads to the fibres not being continuous anymore). It is understood that the sporadic off target fibre may be removed from the scaffold, therefore when used herein the term single continuous fibre is to be interpreted as an essentially continuous single fibre, taking into account that some off target fibres need to be removed from the scaffold.

In a third aspect, the invention relates to a method of manufacturing a scaffold as defined in the first aspect of the invention, the method comprising: electrospinning a biocompatible polymer to a custom electrospinning target; wherein the custom electrospinning target comprises one or more areas with conductive material and one or more areas with resistive material, wherein the conductive material allows for random fibre generation, and wherein the one or more areas with conductive material are positioned such as to allow during electrospinning jumping of the electrospun material jet between different parts of the area or different areas thus creating an aligned area on the one or more resistive areas, and wherein the electrospinning target is not electrically charged or electrically connected to the electrospinning device.

When used herein, the term electrospinning target is interchangeable used with collector, and refers to the object on which the jet of electrospun material is deposited. The shape and composition of the target may be varied as described herein. It was found that by using an electrospinning target with conductive and resistive areas, the construct can be controlled to have areas of fibres with aligned orientation and areas with fibres with random orientation. In an embodiment, the conductive material has a minimum resistivity of 0 p (Q m) at 20°C and the resistive material has a minimum resistivity of 1.5x 10 ³ p (Q m) at 20°C. Preferably the resistive material has a minimum resistivity of 2.0x10 ³ p (Q m) more preferably 5.0X 10 ³ p (Q m) even more preferably 1.0X 10 ⁴ p (Q m) or more at 20°C. Preferably the conductive material has a maximum resistivity of 1.5x 10 ³ p (Q m) or less at 20°C, more preferably 1.0x 10 ³ p (Q m) or less, more preferably 5.0X 10 ² p (Q m) or less, most preferably 1.0X 10 ² p (Q m) or less.

Resistive materials suitable for use in an electrospinning target are known to the skilled person. Resistive materials may be solid or liquids. As a non-limiting example, suitable solid resistive materials are silicone, glass, rubber, wood, diamond, porcelain, ceramic or plastics such as acrylonitrile butadiene styrene (ABS). Therefore, in an embodiment, the resistive material is selected from the group consisting of silicone, glass, rubber, wood, diamond, porcelain, ceramic, cardboard, or plastics such as acrylonitrile butadiene styrene (ABS). As a non-limiting example, suitable liquid resistive materials are aqueous solutions of copper sulphate, ammonium chloride, sodium chloride, sodium thiosulphate, gallium liquid metals, gallium-indium liquid metals, and weak acids/bases, or combinations thereof.

Conductive materials suitable for use in an electrospinning target are known to the skilled person, and may for example be selected from different metals. Non-limiting examples are aluminium or alloys thereof, brass, silver, gold, copper, KCI solutions, Phosphate buffered saline, or combinations thereof. Therefore, in an embodiment the conductive material is selected from aluminium or alloys thereof, brass, silver, gold, copper, KCI solutions, Phosphate buffered saline, or combinations thereof.

The fibre material is dissolved during the electrospinning process. Suitable solvents are known to the skilled person. In an embodiment, the solvent is selected from the group consisting of hexafluoroisopropanol (HFIP), tetrahydrofuran (THF), dichloromethane (DCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), pyridine, hexane, chloroform, acetone, water, ethanol, isopropanol, methanol, and combinations thereof.

When used herein, the term “electrically charged or electrically connected to the electrospinning device” when referring to the electrospinning target implies that the electrospinning target has no charge. Typically, the target is charged, for example by connecting the target to the electrospinning device. The methods described herein use an electrospinning target which is not connected to the electrospinning device nor charged, this has the advantage that the target does not need to be an integral part of the system, and can easily be exchanged. This is particularly useful as electrospinning devices are costly, and this allows the device to be used in many different applications (meaning many different shapes and objects can be produced), as opposed to having a dedicated electrospinner for a single shape. An additional advantage is that targets can be rapidly manufactured in custom dimensions (less than 12 hours) so custom dimension scaffolds can be rapidly made

In an embodiment the biocompatible material is selected from the list consisting of polycaprolactone (PCL), polylactic acid (PLA), ureido-pyrimidone (UPy) polymers, polyvinyl alcohol (PVA), poly(DTE carbonate), polyurethane, polycarbonate, poly-L- lactic acid (PLLA), Poly(DL-lactide-co- caprolactone, Poly(ethylene-co-vinyl acetate) vinyl acetate, Poly(methyl methacrylate), Poly (propylene carbonate), Poly(vinylidene fluoride), Polyacrylonitrile, Polycarbomethylsilane, Polystyrene, Polyvinylpyrrolidone, polyethylene oxide (PEO), polyvinyl chloride (PVC), hyaluronic acid (HA), gelatine, collagen, chitosan, cellulose, alginate, polyhydroxybutyrate and its copolymers, Nylon 11 , Cellulose acetate, hydroxyapatite, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), decellularized extracellular matrix, silk, AE-941 (shark cartilage) and mixtures thereof.

In a fourth aspect the invention relates to a method for manufacturing a hydrogel implant device, the method comprising: manufacturing a scaffold according to the method of the third aspect of the invention, or obtaining a scaffold as defined by the first aspect of the invention, and embedding the scaffold in a biocompatible hydrogel, preferably wherein the biocompatible hydrogel is selected from the list consisting of alginate, gelatine, collagen, poly-ethylene glycol (PEG), hyaluronic acid, methacrylated gelatine, methacrylated collagen, methacrylated hyaluronic acid, norbornene, acrylate-siloxane, cellulose, chitosan, polyvinyl alcohol (PVA), elastin, fibroin, fibrinogen, fibronectin, albumin, human serum albumin, poly(acrylic acid) and it’s salts, poly(vinylpyrrolidone), polyacrylamide, starch and other polysaccharides, decellularized extracellular matrix and silk.

In an embodiment, the scaffold or the hydrogel implant device is a corneal implant device.

In an embodiment, the scaffold or the hydrogel implant device is a tricuspid valve implant.

In a fifth aspect the invention relates to the scaffold as defined by the first aspect of the invention or obtained or obtainable according to the thirds aspect of the invention or the hydrogel implant device according to second aspect of the invention or obtained or obtainable according to the fourth aspect of the invention for use in a surgical method.

It is envisioned that the implant device is implanted in a subject. Therefore, in an embodiment, the surgical method comprises implanting the scaffold or hydrogel implant device in a subject in need thereof. In an embodiment, the implanting uses a surgical retention method. In an embodiment, the surgical retention method is suturing.

It is further envisioned that the implant device is used in the treatment or prevention of a disease or disorder. Therefore in an alternative embodiment, the invention relates to the scaffold as defined by the first aspect of the invention or obtained or obtainable according to the thirds aspect of the invention or the hydrogel implant device according to second aspect of the invention or obtained or obtainable according to the fourth aspect of the invention for use in the treatment or prevention of a disease or disorder in a subject. In an alternative embodiment, the invention relates to a method of treatment comprising implanting the invention relates to the scaffold as defined by the first aspect of the invention or obtained or obtainable according to the thirds aspect of the invention or the hydrogel implant device according to second aspect of the invention or obtained or obtainable according to the fourth aspect of the invention in a subject in need thereof.

In an embodiment, the invention relates to the corneal implant device as defined herein or obtained or obtainable by the method described herein for use in the treatment of a subject with a corneal disorder, wherein the treatment comprises transplanting the corneal implant or part thereof in the eye of the subject, preferably wherein the corneal disorder is selected from thinning of the cornea, scarring of the cornea, a disease of the cornea or an injury of the cornea.

In an embodiment, the invention relates to the cardiac or aortic valve implant device as defined herein or obtained or obtainable by the method described herein for use in the treatment of a subject with a cardiac or aortic disorder, wherein the treatment comprises transplanting the tricuspid valve implant or part thereof in the heart or aorta of the subject. Preferably wherein the disorder is a cardiac disorder, more preferably wherein the valve implant is a bicuspid valve implant or a tricuspid valve implant.

In a sixth aspect the invention relates to a custom electrospinning target comprising one or more areas with a conductive material and one or more areas with a resistive material, wherein the conductive material has a minimum resistivity of 0 p (Q m) and the resistive material has a minimum resistivity of 1 ,5x10 ³ p (Q m) at 20°C, and wherein the one or more areas with conductive material are positioned such as to allow, during electrospinning, jumping of the electrospun material jet between different parts of the area or different areas thus creating an aligned area on the one or more resistive areas, and wherein the electrospinning target is not electrically charged or electrically connected to the electrospinning device. Preferably the resistive material has a minimum resistivity of 2.0x10 ³ p (Q m) more preferably 5.0X 10 ³ p (Q m) even more preferably 1 .0X 10 ⁴ p (Q m) or more at 20°C. Preferably the conductive material has a maximum resistivity of 1.5X 10 ³ p (Q m) or less at 20°C, more preferably 1 .0X 10 ³ p (Q m) or less, more preferably 5.0X 10 ² p (Q m) or less, most preferably 1 .0X 10 ² p (Q m) or less. In a further embodiment, the invention relates to the use of the electrospinning target according to the sixth aspect of the invention in electrospinning an object. Preferably, the electrospun object has an aligned oriented region and a random oriented region.

Materials and Methods

Target design and manufacture

Targets were designed using SolidEdge 2020 professional CAD system. The resistive parts were 3D printed in Acrylonitrile-butadiene-styrene [ABS], and the conductive parts milled from Aluminium 6080 alloy by the IDEE department at Maastricht University. Exemplary targets are depicted in Figure 1.

Electrospinning solutions

17.5% polylactic acid w/w [PLA 80kDa, Goodfellow] was dissolved in a 50:50 mix of Hexafluoroisopropanol [HFIP, Sigma Aldrich] and Dichloromethane [DCM, VWR], 10% w/w UPy SPEY09 (proprietary, Suprapolix) was dissolved in HFIP, 17.5% w/w polycaprolactone [PCL 32kDa, Sigma Aldrich] was dissolved in chloroform [Sigma Aldrich] overnight on a rotary mixer at room temperature. All polymers successfully electrospun patterned scaffolds.

Electrospinning conditions

Solutions were placed into a 12 ml syringe and pumped using syringe pump EP-H11 [Harvard Apparatus] into an EC-DIG electrospinning system [IME technologies] via a 27G [0.4mm] bore needle at 22°C and 40% humidity under the following parameters [Table 1];

Table 1 ; Electrospinning parameters

Volume per Total Targetneedle Positive Negative hour volume distance charge charge

0.5ml 0.1 ml 10cm 18kV -1 kV/0kV

Scanning Electron Microscopy

SEM was used to characterise fibre architecture and size. Samples were fully dried overnight in a freeze drier [Labconco], mounted onto SEM chucks using double sided carbon tape and coated with a thin layer of gold and palladium alloy [Polaron Sputter coater]. All images were captured using a Hitachi S-4700 SEM.

Scaffold embedding

For tensile and optical testing, electrospun samples were sterilised for 60 seconds in 30% Ethanol [Sigma Aldrich] and embedded in 20% bovine gelatine [Sigma Aldrich], or a proprietary hydrogel at 40°C and cross-linked using UV light. Samples were allowed to equilibrate to room temperature for 30 minutes before mechanical testing.

Tensile Testing

Tensile testing was performed using a TA Electroforce Model: 3230-ES series III. Constructs were sutured using a 10-0 vicryl Ethicon 20 ophthalmic suture [Johnson and Johnson] with a 6.5mm diameter, 3/8c spatulated needle. The construct was secured at 50% depth, and the suture tied in a simple surgeons knot and secured in the equipment grippers. Extension was applied at a rate of 1 mm/min. The system was fitted with side action grips and a 3N load cell. Results are depicted in Figure 6. A minimum of three successful tests results were obtained for each sample. A successful test was defined visually by observation of the sample failing away from the edge of the grippers and there being minimal slipping of the sample within the grippers.

Optical testing

Optical transmittance was tested in a Clariostar Plus [BMG Labtech] plate reader and absorbance read at wavelengths between 0 and 1000nm. The results for a PLA based construct are depicted in Figure 7, and for a variety of polymers in Figure 10.

Rheology

A DHR-2 rheometer from TA instruments equipped with a Peltier-heating element and solvent trap was used for the rheological measurements. A 20mm cone-plate geometry (truncation of 2.002° with gap =53pm) was used with UV (365nm) attachment. All measurements were performed at 25 °C with enough water in the solvent trap to minimize dehydration. Material was loaded on the rheometer, the cone and plate lowered to geometry gap, and the gel left to stabilise before any measurements were performed on each sample. Stability was measured through a time sweep which recorded the increasing G’ of the hydrogel during calcium crosslinking. For irradiated materials, the UV light was activated for the allotted time to begin the crosslinking reaction. Typically 4000 s was enough to achieve stable G’ after which a stress relaxation test was performed to measure the relaxation of the material with a strain of 0.01 and rise time of 0.1s. A time sweep proceeded the relaxation test to monitor stability of the hydrogel’s rheological properties and then followed by an oscillatory frequency sweep (0.1% strain, 100-0.01 rad/s, 5 points/decade). Lastly, an amplitude sweep was performed on the samples to ensure measurements were performed in the LVE region.

Data analysis

Raw mechanical data was corrected for load and extension before converting data into stress and strain using equations 1 and 2 respectively. Load

Stress = - (1)

Area

Strain = AL/L (2)

Young’s modulus was obtained from the line of best-fit equation applied to the stressstrain graphs plotted to show each incremental strain band.

Raw absorbance data was converted to transmittance using the following formula;

%T = antilog (2 - absorbance)

To determine the T1/2, first the data was normalized to the initial modulus of the gels and then the time was recorded where the modulus relaxed to half its initial value.

Statistical analysis

All statistical analyses were performed in IBM SPSS and GraphPad Prism software. Where n is used n = number of biological replicates. All experiments were performed with a minimum of 3 biological replicates to allow for statistical analysis. Multiple comparisons tests were been used following the Ryan Joiner test for normality and Bartlett’s test for the homogeneity of variances. Tukey and Games-Howell post hoc tests following analysis of variance (ANOVA) were performed as appropriate.

RT-QPCR

A reverse transcription real-time polymerase chain reaction (RT-qPCR) was used to quantify the levels of RNA for markers of interest expressed by cells. Quantification was performed and standard deviation was propagated using the 2-AACt method.

RT-qPCR was performed in a two-step process. Cells and scaffolds were homogenised using Trizol® (Life Technologies). RNA was isolated following chloroform mediated phase separation and purified using an RNeasy kit (Qiagen) as per manufacturer’s instructions. cDNA was synthesised from a reverse transcription reaction using an iScript™ cDNA Synthesis Kit (Biorad), according to manufacturer’s instructions. Gene expression levels were normalised using expression of the housekeeping gene Peptidylprolyl isomerase A (PPIA) and presented as a relative expression. The 2-AACt method was used to calculate relative RNA levels of the housekeeping gene Peptidylprolyl isomerase A (PPIA) , Aldehyde Dehydrogenase 3 Family Member A1 (ALDH3A1), Cluster of Differentiation 34 molecule (CD34) , Keratocan (KER), Aggrecan (AGG), Alpha smooth muscle actin (a-SMA) and Cluster of Differentiation 90 molecule (CD90) were investigated. Exon spanning forward and reverse primers (Sigma) were designed using the free to access NCBI Primer BLAST

Results

Using different custom-made electrospinning targets, two different types of implants were made;

Ophthalmic constructs

Electrospun scaffolds for ophthalmic use were made to be used as corneal implants. Embedded scaffolds demonstrate optical properties when hydrated. The implants are depicted in Figure 4, and comprise an optically translucent centre of aligned fibres and an opaque circumference of random aligned fibres, details of the random, aligned and border region shown in the scanning electron micrographs.

Cardiac constructs

Electrospun scaffolds for cardiac use were made to be used as tricuspid valve implants. Two different sizes and a complex design demonstrates translatability of this work. The tricuspid valve implants are depicted in Figure 5, details of the random, aligned and border region shown in the scanning electron micrographs.

Suture retention in hybrid constructs

Suture retention was determined for the corneal implant as described above. The results for a PLA and UPy made construct are plotted in Figure 6. Results for PLA, UPy and PCL made constructs embedded in hydrogel, compared to hydrogel alone are plotted in Figure 10. Radial constructs retain sutures at loads of over 20g, in comparison to hydrogel only which cannot be sutured. One-way ANOVA with Games Howell and Tukey post hoc testing, * = p<0.05, ** = p<0.005, *** = p<0.001 (Figure 10) Optical transmittance

Optical transmittance was determined for the corneal implant as described above. The results are described in Figure 7 and in Figure 10. Radial constructs for ophthalmic use are as transparent as tissue culture plastic. N = 3.

Rheology

Embedding the electrospun scaffolds in hydrogel materials does not significantly influence rheological properties of the gels. One-way ANOVA with Games Howell post hoc testing, * = p<0.05, ** = p<0.005, *** = p<0.001 , n=3. Error = Std Dev (not shown). (Figure 11)

RT-QPCR Gene expression

Gene expression relative to tissue culture plastic, normalised to housekeeping gene PPIA, One-way ANOVA with Games Howell and Tukey post hoc testing, * = p<0.05, ** = p<0.005, *** = p<0.001. AACT method, error = std dev. Samples were tested for; A. Aldehyde dehydrogenase 3A1 (ALDH3A1), a protective corneal crystalline. B. Keratocan, one of the major proteoglycans of the stroma. C. Vimentin a major structural intermediate filament (type III) protein expressed in injured corneal fibroblasts and in low levels in normal corneal keratocytes. D. CD90, a stem cell marker indicative of a switch to fibroblastic phenotype. E. Alpha-smooth muscle actin, a marker of a myofibroblast. F. CD34, an alternate keratocyte marker down regulated in injured corneas. G. Lumican regulates collagenous matrix assembly as a keratan sulphate proteoglycan Desirable keratocyte markers ALDH3A1 and KERATOCAN are expressed on both radial fibres alone and radial constructs within gel. Undesirable fibroblast markers VIMENTIN and ALPHA-SMOOTH MUSCLE ACTIN are both expressed at lower levels than on tissue culture plastic. CD90 is downregulated on fibres alone. This indicates that the electrospun constructs are capable of maintaining a keratocyte like phenotype in vitro (Figure 12).