A 3-d porous scaffold

Abstract

Described herein is a 3-D porous scaffold for biological and medical applications such as a tissue engineering construct, a scaffold for drug delivery, a tissue culture scaffold, a scaffold for growing tissue and organoids, and as a 3-D substrate for organ-on-chip applications. The 3-D scaffold is generally 3-D printed. The scaffold has a layered structure comprising one scaffold layer or a plurality of scaffold layers associated together. Each scaffold may be printed as a monolith, or the layers can be printed separately and adhered together post-printing. Each scaffold layer comprises an array of Modular Units (M.U) that interconnect to form inter-unit pores. Each Modular Unit of the scaffold comprises a plurality of concentric circular struts namely annuluses and struts connecting the annuluses (hereafter "connecting strut") and intra-unit pores between the circular struts. The scaffold layers thus have a defined porosity and compressive stiffness, and the porosity and compressive stiffness can be controlled by the number, dimension and arrangement of the Modular Units in each of the scaffold layers.

Description

[0001] TITLE

[0002] A 3-D Porous Scaffold

[0003] Technical Field

[0004] The present disclosure relates to a 3-D porous scaffold suitable for use in biological application, including use as a tissue engineering scaffold to treat bone or cartilage defects, a scaffold for culturing cells, tissues and organoids, an implantable drug delivery device, and as a 3-D substrate for organ on-chip applications.

[0005] Background

[0006] Scaffolds are three dimensional artificial constructs which provide cells with structural support to attach and proliferate. Scaffolds have been the focus of much interest to support the regeneration of tissues that may have been impaired due to disease or trauma. Alternatively, scaffolds can be used as platforms for drug release. More recently, there is interest in 3-D scaffold structures for cell culture studies to address the limitations associated with 2-D cell culture systems that poorly replicate the in vivo environment in terms of biology and physical structure and have poor prediction capability.

[0007] Among the different methods to produce 3-D scaffolds, additive manufacturing (AM) techniques (3-D printing) represent some of the more advanced approaches. AM guarantees to achieve superior control over the structure, geometrical patterns and the physical properties. However, despite the prospect for more intricate geometries and greater control over architecture, the 3-D scaffolds published in the literature to date poorly replicate the natural geometrical layout and hierarchical structure which characterises the native tissue e.g., bone. Most designs are generated automatically by software and are characterised by the multiple repetition of the same geometry (e.g., triangles, squares, hexagons etc.) throughout or tend to be generated by layering perpendicular, wavy struts or filaments orthogonally on top of one another making the scaffolds appear as a simple 3-D grid. As a direct consequence, there is a shortage of computer aided design (CAD) descriptives to easily reproduce scaffolds characterised by a biologically relevant intricacy of the geometries and structures as the bone tissue requires by nature.

[0008] So far, the methods used for the production of 3D scaffolds include conventional methods such as gas foaming, porogen leaching, electrospinning, lyophilisation etc. More recently additive manufacturing techniques (3-D printing) including fused deposition modelling (FDM), stereolithography (SLA), , and 2 photon polymerisation (2PP) have been employed. While the conventional methods are associated with limitations like poorer reproducibility, deficits in the scaffold geometries and high inter and intra batch variability, 3-D printing helps ensure superior control over the structure, the geometrical patterns and physical properties of the scaffolds, which have proven to influence the scaffold performance and impact the behaviour of cells cultured on these 3-D structures.

[0009] Currently, the scaffold designs proposed for FDM or SLA are simple and not complex from a geometrical point of view. The scaffolds generated with 3-D printing are often characterised by the multiple repetition of the same layer, involving struts or filaments layered orthogonally on top of one another and appear as a simple grid. Rarely intricate conceptual designs and geometries are seen.

[0010] Scaffolds manufactured using more traditional methods (e.g., gas foaming, particle leaching etc.) give rise to scaffolds with variable properties and lack of controlled structures or detailed design. Controlled architecture offers reproducibility, and reliability. So far, no such design has been disclosed.

[0011] It is an object of the invention to overcome at least one of the above-referenced problems. Summary

[0012] Described herein is a 3-D porous scaffold for biological and medical applications such as a tissue engineering construct, a scaffold for drug delivery, a tissue culture scaffold, a scaffold for growing tissue and organoids, and as a 3-D substrate for organ-on-chip applications. The 3-D scaffold is generally 3-D printed. The scaffold has a layered structure comprising one scaffold layer or a plurality of scaffold layers associated together. Each scaffold may be conceived and printed as a monolith, or the layers can be printed separately and adhered together post-printing. Each scaffold layer comprises an array of Modular Units (M.U) that interconnect to form inter-unit pores. Each Modular Unit of the scaffold comprises a plurality of concentric circular struts namely annuluses and struts connecting the annuluses (hereafter “connecting strut”) and intra-unit pores between the circular struts. The scaffold layers thus have a defined porosity and compressive stiffness, and the porosity and compressive stiffness can be controlled by the number, dimension and arrangement of the Modular Units in each of the scaffold layers.

[0013] Since the scaffold layers possess the ability to dissipate mechanical forces, given by the annuluses, the Modular Units and the connecting struts, the 3-D porous scaffold has the capability to dissipate applied mechanical forces from the top or the side of the scaffold to recapitulate the mechanical properties of human tissues such as the bone. The porosity and mechanical properties of the 3-D construct can be further controlled by (a) including scaffold layers of differing construction, for example a first scaffold layer having a first design arrangement of the Modular Units and a second scaffold layer having a second design arrangement that is different to the first (b) inclusion of different types of modular scaffold units in any given layer, for example Modular Units that have the same number and arrangement of concentric circles but different designs of connecting struts, (c) adjusting the position of one scaffold layer relative to an adjacent scaffold layer (linear or rotational adjustment) such that the Modular Units of one layer are out of phase (e.g. not in register) with those of another scaffold layer, (d) providing a first scaffold layer with an first array of Modular Units and a second scaffold layer with a second array of Modular Units, where the first array is out of register with the second array, or a combination of any of the foregoing. Each scaffold layer is typically circular, and the 3-D scaffold is cylindrical.

[0014] In a first aspect, there is provided a 3-D porous scaffold for tissue engineering, cell culture or drug delivery, comprising a plurality of scaffold layers, in which each scaffold layer comprises an array of closely packed, interconnecting, Modular Units, in which each Modular Unit comprises a plurality of annuluses and connecting struts, and in which the 3-D scaffold is typically 3-D printed.

[0015] In any embodiment, the intra-unit pores are arcuate. The intra-unit pores are generally defined by neighbouring concentric circular struts being spaced apart to define the intra-unit pores (or spaces). The space / pore between neighbouring annuluses is generally almost fully annular (and only interrupted by a diametrical or radial connecting strut).

[0016] In any embodiment, the circular annuluses are solid and monolithic.

[0017] In any embodiment, the struts are solid and monolithic.

[0018] In any embodiment, the Modular Units of at least one scaffold layer all have the same diameter.

[0019] In any embodiment, the Modular Units of all scaffold layers have the same diameter.

[0020] In any embodiment, at least one of the scaffold layers comprises a plurality of different Modular Units. The Modular Units may differ by comprising a different type of connecting strut.

[0021] In any embodiment, the 3-D scaffold comprises at least 1 , for example 2-20, scaffold layers. In any embodiment, the 3-D scaffold comprises at least 1 , and typically 2-5 scaffold layers.

[0022] In any embodiment, the Modular Units of each layer of the 3-D scaffold have the same diameter.

[0023] In any embodiment, the Modular Units of each layer of the 3-D scaffold all comprise 2, 3, 6 or more connecting struts.

[0024] In any embodiment, the Modular Units of at least one, or each, layer of the 3-D scaffold all comprise minimum 3 concentric circular annuluses, for example 3-10, 3- 8, 3-5, 4-10, 4-8 or 4-6 concentric circular annuluses.

[0025] In any embodiment, the Modular Units in one of the plurality of scaffold layers are not in register with the Modular Units of another of the plurality of scaffold layers. This may be achieved by translating one scaffold layer relative to an adjacent scaffold layer. The translation of the one scaffold layer may be along the X or Y axis of the array. The translation of one scaffold layer comprises moving an origin of a layer (a centre point of the centre Modular Unit) so that it is not in register with an origin of an adjacent layer. The translation of the origin of a layer may be along the X or Y axis of the array. This is described in more detail below with reference to Figure 9.

[0026] In any embodiment, a disposition of Modular Units in one of the plurality of scaffold layers differs from a disposition of the Modular Units of another of the plurality of scaffold layers

[0027] In any embodiment, each scaffold layer comprises a primary Modular Unit and six secondary Modular Units arranged around the primary Modular Unit, in which each secondary Modular Unit abuts the primary Modular Unit and two adjacent secondary Modular Units. In any embodiment, the 3-D porous scaffold includes a plurality of tertiary Modular Units arranged around the six secondary Modular Units, in which each tertiary Modular Unit abuts two secondary Modular Units and two adjacent tertiary Modular Units.

[0028] Where two Modular Units abut (e.g. interconnect), the outer annulus of the Modular Units may tangentially abut, or they may merge into a single outer annulus along a section of abutment.

[0029] In any embodiment, each Modular Unit comprises three concentric circular struts.

[0030] In any embodiment, the connecting struts comprise a diametrical strut or a plurality of radial struts.

[0031] In any embodiment, at least some of the Modular Units comprise one diametrical strut, three diametrical struts, or three radial struts arranged in a Y-shape.

[0032] In any embodiment, a scaffold layer may comprise a row of Modular Units in which each Modular Unit in the row comprises a diametrical connecting strut, and which the connecting struts of the row of Modular Units are co-linear and form an elongated linear strut extending fully or partially across the scaffold layer.

[0033] In any embodiment, the scaffold layers are circular, and the 3-D scaffold is cylindrical.

[0034] In any embodiment, the Modular Units have a diameter of at least 500 microns, for example a diameter of 500 microns to 10 mm.

[0035] In any embodiment, the struts have a thickness of at least 50 microns, for example a thickness of 50 microns to 1000 microns.

[0036] In any embodiment, the annuluses have a minimum thickness of 50 microns. In any embodiment, the 3-D porous scaffold has a diameter of at least 6 mm, for example 6 mm to 100 mm.

[0037] In any embodiment, the layers of the 3-D porous scaffold have a height of at least 100 microns, for example 100 microns to 100 mm.

[0038] In any embodiment, each scaffold layer comprises a plurality of tertiary Modular Units with the struts directed to the origin (centre of the primary Modular Unit) of the layer.

[0039] In any embodiment, the 3-D porous scaffold includes a plurality of tertiary Modular Units surrounding a hexagonal core comprising primary Modular Units and secondary Modular Units.

[0040] In any embodiment, the concentric circular annuluses are spaced apart to define annular pores between neighbouring circular annuluses.

[0041] In any embodiment, the annuluses and / or struts have a thickness of 50-1000 microns.

[0042] In any embodiment, neighbouring annuluses are spaced apart by 50-1000 microns.

[0043] The 3-D scaffolds described herein are generally fabricated by additive manufacturing (3-D printing), for example by fused deposition modelling (FDM), stereolithography (SLA), and 2 photon polymerisation (2PP). Each scaffold layer may be 3-D printed individually, and then the scaffold layers adhered together postprinting to form the 3-D scaffold, or the whole scaffold may be 3-D printed as a monolith.

[0044] Also described is a construct comprising a 3-D porous scaffold of the disclosure and biologically or pharmaceutically active agent disposed within pores or on the struts of the 3-D porous scaffold. In any embodiment, the 3-D porous scaffold is 3-D printed with an ink comprising the biologically or pharmaceutically active agent.

[0045] In any embodiment, the biologically or pharmaceutically active agent is selected from the group consisting of: cells; a pharmaceutical; and a cellular growth factor.

[0046] In any embodiment, the cells are selected from human cells, cell lines (e.g. osteoblasts) and cancer cells.

[0047] In any embodiment, the cells are disposed within pores of the porous 3-D scaffold.

[0048] Also described in a device comprising a plurality of wells, and a 3-D porous scaffold disposed in the plurality of wells.

[0049] The device may be a cell culture plate, or a lab-on-chip plate.

[0050] Also described is a drug eluting implant comprising a 3-D porous scaffold as described herein and a drug disposed in pores or the structure of the 3-D porous scaffold.

[0051] Also described is a method of forming a 3-D porous scaffold according to the invention, comprising 3-D printing a plurality of scaffold layers, and adhering the scaffold layers together to form the 3-D porous scaffold.

[0052] In any embodiment, the method comprises 3-D printing the 3-D porous scaffold as a monolith.

[0053] Also described is a scaffold layer comprising an array of closely packed, interconnecting, Modular Units, in which each Modular Unit comprises a plurality of spaced apart concentric circular annuluses defining annular pores between neighbouring circular annuluses, and struts connecting the annuluses, and in which the scaffold layer is generally 3-D printed. In any embodiment, the annuluses and / or struts have a thickness of 50-1000 microns.

[0054] In any embodiment, neighbouring annuluses are spaced apart by 50-1000 microns.

[0055] The invention offers an innovative scaffold design concept based on the Euclidean geometry, inclusive of i) sketching method and ii) relative designs which can be adapted to obtain custom scaffolds for deployment in diverse applications. The invention addresses the limitations of the scaffolds manufactured with more conventional methods and surpasses the status quo by providing scaffolds characterised by controlled structures and detailed design in antithesis with the ones produced so far. The invention presents a scaffold design which has not been either conceived, used, or patented before. In addition, the design of the layers has been conceived to facilitate the dissipation of the mechanical forces applied either vertical or laterally to the scaffold since the Modular Units and layers it is composed of are arranged in a radial pattern with respect to the centre or origin of each layer. Basically, each layer has been designed similarly to the wheel rim of a car to accentuate the mechanical resistance of the scaffold layers.

[0056] Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

[0057] Brief Description of the Figures

[0058] Fig. 1 - Schematic flow of the process of compartmentation of the core spaces of Modular Unit 0.

[0059] Fig. 2 - Representation of the different M.U originating from the Modular Unit 0. A) Modular Unit 0 and the definition of its core design; B) Modular Unit 1 -3. Red represents M.U 1 ; orange represents M.U 2 and blue represents M. U 3. Fig. 3 - Schematic representation of the process to generate the grid used for the tangential design to delineate the design of each scaffold layer (presented layer 1 for reference). A) Sketch of the equilateral triangle with M.U 0 centered at the vertexes and equations used to extrapolate the distances separating the grid lines on the x and y axes. B) Grid used to design the scaffold’s layers displaying the expansion of M.U 0 in all the directions. M.U 0 is at the vertexes of the adjacent triangles, and identified by dash lines intersecting the lines of the grid. Red circle and blue circle, Modular Unit 0 (M.U 0); red circle identifying the origin of the axes in layer 1 in xo;yo ; fine dash lines representing the sides of adjacent triangles. Note: (H) refers to the height of the equilateral triangle, (I) is the length of the side of the triangle, while (b) is the length of the base of the triangle.

[0060] Fig. 4 - Schematic representation of the degree of misalignment adopted to generate the scaffold layers 1-5. A) equilateral triangle (green) with M.U 0 centered at the vertexes of the triangle and tangential to each other. The origin of the figure is in xo;yo; B) Scaffold layer 1 : the origin of the axes is xo;yo , at the vertex of the equilateral triangle; C) Scaffold layer 2: the origin of the axes is x.2 / 3H;yo , and displaced at -2 / 3 of the triangle’s height; D) Scaffold layer 3: the origin of the axes is x.3 / 3H;yo , at -3 / 3 of the triangle’s height and it coincides with the base of the triangle; E) Scaffold layer 4: the origin of the axes is x.i / 3H;yo , at -1 / 3 of the triangle’s height; F) Scaffold layer 5: the origin of the axes is x.i / 2H;yo , at -1 / 2 of the triangle’s height. Red circle and blue circle, Modular Unit 0 (M.U 0); fine dash lines representing the sides of adjacent triangles; green triangle and blue arrow indicating the equilateral triangle and the shifts of the origin occurred.

[0061] Fig. 5 - Schematic representation of the process followed from the definition of the dimensions of the scaffold layers to the positioning of the different M.U in each scaffold layer: A,D,G,J,M) Definition of the dimension of the scaffold layers through the sketching of a bigger circle (blue) of 1 .2 cm in diameter in the grid generated. B,E,H,K,N) Compartmentation of the spaces in the scaffold layers though the generation of the external wall, thickness 200 pm (black circle and green circle in B,E,H,K,N and C,F,l,L,0 respectively) and positioning of the Modular Units in the different layers. Red circle, Modular Unit 0 (M.U 0); green triangle indicating the equilateral triangle to identify the shifts of the origin across the grid. Pink represents M.U 1 ; Orange, M.U 2 and Blue, M.U. 3. Black dot represents the centre of the figure (layers) where the origin of the axes of the figure has been positioned.

[0062] Fig. 6 - Representation of the different layers (following either the tangential or overlap design) producing a ‘star-like’ effect arising from the struts designed to dissipate the mechanical forces applied to the layers. A,D,G,J,M) Tangential (light grey) and B,E,H,K,N) Overlap (dark grey) designs. C,F,I,L,O) ‘Star-like’ effect characterising the layers. Yellow: represents channels of dissipation of the mechanical forces. Black dot in A,D,G,J,M and B,E,H,K,N represents the centre of the figure (layers) where the origin of the axes of the figure has been positioned.

[0063] Fig. 7 - Schematic representation of the process to generate the grid for the overlap design (presented for layer 1 only). A) Sketch of the new equilateral triangle (shown in green; side length: 1600 pm) with M.U 0 (outer diameter: 1700 pm) at the vertices. B) Grid used to design the scaffold’s layers displaying the expansion of M.U 0 in all directions. Red circle and blue circle identify Modular Unit 0 (M.U 0); and M.U 0 in red identifies the origin of layer 1 in xo;yo. Fine dash lines representing the sides of adjacent triangles. Note: M.U 0 is presented with outer circle only to display the overlap within the units.

[0064] Fig. 8 - Example of rotation applied in layer 1 in both A) tangential (light grey in top panel) and B) overlap (dark grey in bottom panel) design. The rotation applied was 30° (red arrow) Note: only layer 1 is presented. The rotated layers appear in red on the right.

[0065] Fig. 9 - In order to get the layers out of phase (not in register) with each other and create the intricate porous network when iMARS is assembled, the origin of each layer was shifted a fixed distances along the x axis corresponding to -1 / 3, -1 / 2, -2 / 3 and -3 / 3. These coordinates indicate specific points on the triangle’s height (H; 1472.243186 pm) which lies on the x axis and has fixed vertex in x0;y0. To obtain the 5 layers, the origin of the layers shifted towards a negative direction on the x axis meaning moving from x0;y0 (layer 1 ; L1 ), to x-2 / 3H;yO (layer 2; L2), x-3 / 3H;yO (layer 3; L3), x-1 / 3H;yO (layer 4; L4) and x-1 / 2H;yO (layer 5; L5).

[0066] N.B: Layer Coordinates

[0067] (layer 2; L2) x-2 / 3H;yO x -981.495;y0

[0068] (layer 3; L3) x-3 / 3H;yO x -1472.243;y0

[0069] (layer 4; L4) x-1 / 3H;yO x -490.747;y0

[0070] (layer 5; L5) x-1 / 2H;yO x -736.121 ;y0

[0071] The type of shift that has occurred in layers 2-3 and layers 4 and 5 is different. The shift in layers 2-3 involves the origin of the axes only moving towards the negative direction on the x axis to find the coordinates on the triangle, while the established grid was kept fixed. The shift in layers 4 and 5, rather involves the shift of the whole CAD grid towards a negative direction on the x axis and the subsequent repositioning of the origin of the axes in x-1 / 3H;yO (layer 4; L4) and x-1 / 2H;yO (layer 5; L5).

[0072] Detailed Description of the Invention

[0073] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0074] Definitions and general preferences Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0075] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0076] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps.

[0077] As used herein, the term “ 3-D porous scaffold” or “scaffold” refers to a 3-D structure comprising at least one, and ideally a plurality of, scaffold layers, in which the or each scaffold layer comprises an array of closely packed, interconnecting, Modular Units, in which each Modular Unit comprises a plurality of concentric annuluses (ideally concentric circular annuluses) and one or more struts connecting the annuluses, and in which the 3-D scaffold is typically 3-D printed. The concentric annuluses are generally spaced apart to define the intra-unit pores (or spaces) between neighbouring annuluses. The pores are typically incomplete annuluses (interrupted by one or more struts) comprising at least two arcuate pores or spaces. The intra- unit arcuate pores or spaces defined by the spaced apart annuluses, together with inter-unit pores defined by the array of interconnected Modular Units, provide porosity to the 3-D scaffold. The porosity can be tuned by modifying the spacing between the annuluses, and by the number of annuluses in each Modular Unit. The scaffold is typically suitable for use as a tissue engineering, cell or tissue culture, drug delivery construct, or may be employed in 3-D cell culture plates, lab-on-chip devices, drug eluting implants, and tissue engineering constructs, in particular implants for bone defects. The scaffold typically has a diameter of at least 6 mm (e.g. 6 mm to 10 cm). The scaffold typically has a height of at least 100 microns (e.g. 100 microns to 10 cm). Typically, each Modular Unit has a diameter of at least 500 microns (e.g. 500 microns to 10 mm).

[0078] As used herein, the term “interconnected” as applied to the Modular Units means that each Modular Unit abuts at least one other modular unit forming an array of interconnected Modular Units. Where two Modular Units abut (e.g. interconnect), the outer annulus of the Modular Units may tangentially abut, or they may merge into a single outer annulus along a section of abutment (e.g. overlap).

[0079] Exemplification

[0080] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0081] EXAMPLE 1 - DESIGN OF THE MODULAR UNITS

[0082] The fundamental Modular Unit 0 (M.U 0) is a circle of 1700 pm in diameter (radius (r) = 850 pm). The compartmentation of the core spaces of M.U 0 arises due to inserting three annuluses, each 100 pm in thickness, separated by 200 pm circular and hollow spaces (Figure 1).

[0083] The definition of the final diameter and the design of the M.U 0 result from the compartmentation of the core spaces by generating a series of 3 concentric circular layers (100 micron thick). Starting from the centre of the M.U 0: 300 pm (inner, hollow) (r = 150 pm), 500 pm (r = 150 pm + 100 pm (annulus)), 900 pm (r = 250 pm + 200 pm (hollow space)), 1100 pm (r = 450 pm + 100 pm (annulus)), 1500 pm (r = 550 pm + 200 pm (hollow space)) and 1700 pm (outer) (r = 750 pm + 100 pm (annulus)). The diameter of 1700 m will dictate the dimensions of the side of the equilateral triangle subsequently used to generate the grid for each of the layers in the tangential design.

[0084] Once defined, the concentric circles of M.U 0 were connected with 100 pm thick struts to produce specific configurations, M.U 1 -3. Where, Modular Unit 1 , is referred to as M.U 1 , Modular Unit 2 as M.U 2 and Modular Unit 3 as M.U 3. Six struts separated by 60° were used to generate M.U 1 . Two struts separated by 180° were used to generate M.U 2. In the case of M.U 3, 3 struts are used to create a Y shape whereby 2 struts are separated by 60° angle and the third strut is at an 150° angle from the other struts (Figure 2)

[0085] EXAMPLE 2 - TANGENTIAL DESIGN - DESIGN OF THE SCAFFOLD’S LAYERS

[0086] STEP 1 - Definition of the sketching area (“grid”)

[0087] The sketching area or ‘grid’ has been generated by application of Euclid’s geometry starting with an equilateral triangle of side 1700 pm, whereby 3 circles (or Modular Unit 0) of 1700 pm in diameter were centered and disposed at the vertices of the triangle and tangential to each other. The origin of one of the M.U 0 (herein referred as in xo;yo) has been placed at one of the vertices, highlighted in red (Figure 3). The distances separating the grid lines on the x and y axis were then determined through application of the Pythagoras’ theorem, which is of the side (I) of the triangle (on the y axis), thus 850 pm.

[0088] Equation 1.

[0089] The distance separating grid lines running parallel to the y axis, can be calculated using Pythagoras' theorem, Equation 2 and corresponds to 1472.243186 pm or the height (H) of the triangle.

[0090] Equation 2.

[0091] To delineate the design of the layers, expansion of M.U 0 in the grid occurred in all the directions starting from the equilateral triangle with the centre of each M.U 0 at the vertices and tangential to each other. The established grid was used to design all the scaffold layers.

[0092] To achieve misalignment of the spatial geometries between the scaffold layers and with respect to the previous layer, the origin of each layer was shifted a fixed distance along the x axis corresponding to -1 / 3, -1 / 2, -2 / 3 and -3 / 3 of the triangle’s height (H), relative to the origin in xo;yo. The degree of the shift was defined by Euclid’s theorem by which, in an equilateral triangle, the circumcentre, the orthocentre and barycentre all converge in a focal point, which divides the triangle’s height in two segments in a ratio of 2:1 (2 / 3H and 1 / 3H). Additionally, points which divide the height in 1 / 3H and in >2 were also chosen as easy-to-identify points in the geometry of the equilateral triangle used. The origin of the layers shifted towards a negative direction on the x axis from xo;yo (layer 1 ; L1 ) to x.2 / 3H;yo (layer 2; L2), x.3 / 3H;yo (layer 3; L3), x.i / 3H;yo (layer 4; L4) and x.i / 2H;yo (layer 5; L5) to generate a total of 5 different layers (Figure 4).

[0093] The shifts that occurred in layers 2-3 and layers 4 and 5 are substantially different. The shift in layers 2-3 involves the origin of the axes only moving towards the negative direction on the x axis, while the grid was kept in a fixed position. On the other hand, the shift in layers 4 and 5, involve the shift of the whole grid towards a negative direction on the x axis and the subsequent re-positioning of the origin of the axes in x.i / 3H;yo (layer 4; L4) and x.i / 2H;yo (layer 5; L5) respectively.

[0094] STEP 2 - Determination of the dimensions of the scaffold layers

[0095] Starting from the origin of the axes in CAD, a bigger circle (diameter 12000 pm) is drawn. This circle delineates the dimensions and resultant geometry of each scaffold layer (Figure 5). The diameter of the layers and scaffold was established a priori to be 1 .2 cm (12,000 pm) based on the diameter of a well in a 24 well-plates and also for the potential deployment of the printed scaffold for inclusion in lab on chip devices. The external wall of each layer was established to be 200 pm thick therefore the compartmented space in the interior of the scaffold’s layers is 11 600 pm. After determining the dimensions of the layers, the external wall of each scaffold’s layer was constructed to delimitate the core space in the layers prior to replacing M.U 0 with M.U 1 -3.

[0096] STEP 3 - Arrangement of the core design of the scaffold’s layers and disposition of the Modular Units

[0097] An important factor in the design was to confer the scaffold’s layers with the ability to sustain high mechanical loads and dissipate the mechanical forces applied either from the top or laterally, reminiscent with human tissue e.g., bone. The overall design of each of the scaffold’s layers depends on the subsequent positioning of the Modular Units in the layers. The Modular Units (M.U 1 -3) were disposed in the spaces occupied by the M.U 0 to generate ‘star-like’ patterns in each of the 5 layers (Figure 6). The composition proposed for the layers was conceived with the aim to increase the scaffold’s resistance to the mechanical forces applied and confer the capability to dissipate the forces. These attributes could potentially be obtained with the stratification of different layers or the same layer to achieve either anisotropic (non- uniform) or isotropic (uniform) scaffold. Isotropic or anisotropic scaffolds depend on the combination of layers, rotations, and Modular Units used per strata in the attempt to artificially reproduce the biomechanics of the targeted tissue e.g., bone.

[0098] EXAMPLE 3 - OVERLAP DESIGN - DESIGN OF THE SCAFFOLD’S LAYERS

[0099] STEP 1 - Definition of the sketching area (“grid”)

[0100] The sketching area to obtain the scaffold’s layers with overlapping design was again generated by application of the Euclid’s geometry as before. Once again, the starting point to generate the grid was the equilateral triangle but, to obtain the overlap of the Modular Units (1700 pm in diameter), the length of the triangle’s side was established as 1600 pm instead of 1700 pm. At the vertices of the triangle, three M.U 0, each of 1700 pm in diameter were disposed, leading to an overlap of 100 pm. As per the tangential design previously described, the origin was fixed at one of the vertices of the triangle, in xo;yo.

[0101] The distance separating the grid lines on the x and y axis was then determined through application of the Pythagoras’ theorem (Equation 1 and Equation 2).

[0102] The distance separating lines running parallel to the x axis is geometrically of the triangle’s side, thus 800 pm, while the distance separating the parallels to the y axis is 1385.64064606 pm or the height (H) of the triangle. The new grid can be used to design the scaffold’s layers, as with the tangential design. However, the key difference is the overlapping of the outer annulus of the Modular Units. To delineate the design of the layers, the M.U 0 is expanded across the new grid in all the directions starting from the new equilateral triangle with M.U 0 centred at the vertices and overlapping of the M.U (Figure 7).

[0103] As before, to achieve misalignment of the spatial geometries across the scaffold’s layers and with respect to the previous layer, the origin of each layer is shifted a fixed distance corresponding to -1 / 3, -1 / 2, -2 / 3 and -3 / 3 of the triangle’s height (H). The origin of the new scaffold layers shifted again from xo;yo (layer 1 ; L1 ) to x.2 / 3H;yo (layer 2; L2), x.3 / 3H;yo (layer 3; L3), x.i / 3H;yo (layer 4; L4) and x.i / 2H;yo (layer 5; L5) for a total number of 5 layers.

[0104] STEP 2 - Determination of the dimensions of the scaffold layers

[0105] The design of the 5 layers in the overlap design follows the same principle described for the tangential design. Again, the diameter of the scaffold is 1 .2 cm (12,000 pm) and the external wall of each layer is 200 pm thick, thereby delineating the interior (11 600 pm). M.U 0 is replaced with the M.U 1-3.

[0106] STEP 3 - Arrangement of the core design of the scaffold’s layers and disposition of the Modular Units

[0107] Similarly, to the ‘tangential’ design, the positioning of the M.U generates a “star like” effect in the design. It was also desirable to confer the scaffold’s layers with the ability to sustain high mechanical loads and dissipate the mechanical forces. However, the rationale underpinning the overlap of the M.U was to create a more consolidated structure (Figure 6 A,D,G,J,M ‘tangential’ vs Figure 6 B,E,H,K,N ‘overlap’), with the potential to sustain greater forces compared to constructs generated using the tangential design.

[0108] EXAMPLE 4 - ROTATION OF THE SCAFFOLD’S LAYERS

[0109] Each of the 5 different scaffold layers, regardless of whether the tangential or overlap design is employed, can also be rotated around their own axis. By rotating each layer (Figure 8), it is possible to potentially modify the scaffold geometry and subsequently influence parameters such as the porosity by altering the pore dimensions and interconnectivity. Many rotations are possible since the degree of rotation (a) lies between 0° and 360°.

[0110] EXAMPLE 5 - COLLATERAL PROPERTIES INCLUDED OR DERIVED FROM THE DESIGN AND USE OF THE SCAFFOLD

[0111] Based on the generation of the sketching grid of both the tangential and overlap design, the origin and the grid itself can be physically moved around the CAD worksheet identifying other points to position the origin of the layers. The repositioning can happen on the x axis, as proposed, on the y axis or other points in the working space identified by x;y coordinates.

[0112] The sketching grid can be modified by changing the dimensions of the side in the equilateral triangle used herein as we demonstrate to generate the overlap design.

[0113] The disposition of the Modular Units across the layers determines the uniqueness of the core design of each of the 5 layers. The proposed designs of the layers can be modified positioning the Modular Units differently from the original design. The Modular Units can be increased or decreased in dimensions based on the target cell line. Additional connecting structs can be added to the Modular Units depending on the target tissue. The sophistication in the geometries which defines the design of each layer ensures a specific definition of the physical properties e.g., pore geometries, pore dimensions and consequently porosities of the scaffold, and influences the capability to dissipate the forces applied. The ability to dissipate the forces is achieved since the arches generated in the annuluses characterising the Modular Units have the intrinsic property of dispersing the forces applied either laterally or orthogonally through the connecting struts within the Modular Units of the scaffold layers.

[0114] The design of the layers forms a three-dimensional (3-D) network to entrap the cells within the intricate structure derived once the scaffold is assembled. The scaffold can be assembled in toto in CAD and printed as a monolith or the layers can be printed separately and assembled to construct the scaffold once the printing of the layers has occurred.

[0115] The number of layers proposed herein is 5. But establishing a height for the layer does not exclude the possibility to print them at a different height (e.g., 20 pm, 200 pm, 400 pm, 1 mm etc.) from the one we printed (600 pm).

[0116] The number of layers proposed can increase by disposing the Modular Units differently from the design proposed, thus generating other layers. Tangential and overlap design can be mixed generating gradient scaffolds.

[0117] Depending on the number of layers considered, it is possible to use less layers or increase the number of layers from the ones proposed and combine them in multiple arrangements such as layer 1 -2-3-4-5; 2-3-1 -5-4; 1-1 -1-1 -1 etc. The number of combinations exponentially increases when the number of layers in the scaffold also increases.

[0118] The scale of the design of the scaffold can be increased or decreased uniformly.

[0119] The versatility in the design of the layers, in the layer combinations and rotations, in the possibility to adapt the design proposed based on the requirements of the target, make the scaffold of this disclosure more sophisticated than the standard scaffold designs in use, and consequently advances the current state of the art in the fields of 3-D tissue culture models and tissue engineering. Specifically, the scaffold of the disclosure finds application in the field of metastatic prostate cancer in the bone which lacks appropriate in vitro 3D models to replicate disease pathology and conduct drug development studies. According to the latest updates in the literature, none of the scaffolds currently described or used reflect a scaffold consisting of several layers that can be shaped, rotated and combined to produce archetypes with a variety of tuneable pore geometries and porosities in response to the different requirements of different cell populations.

[0120] In summary, there are many areas whereby the versatility of the scaffold of the disclosure may help and advance the state of the art: first, the exceptional design of the Modular Units, layers and scaffold, combined with the possibility to place differently the Modular Units, increase or decrease the dimensions, add different layer combinations and rotations make the scaffold outstanding compared to the scaffolds used for bone cancer research but then it is also represents an exceptional design for other areas: i) bone tissue engineering and regeneration, ii) organ on-chip technology iii) 3D inserts for well plates to culture cells in 3D environments to recapitulate the bone and (iv) drug delivery applications. The invention also does not exclude any further use for different cell types other than osteoblasts and prostate cancer cells, other microenvironments beyond bone tissue or any other use in alternative fields of research.

[0121] Equivalents

[0122] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A 3-D porous scaffold for tissue engineering, cell culture or drug delivery, comprising at least one, ideally a plurality of scaffold layers, in which each scaffold layer comprises an array of closely packed, interconnecting, Modular Units, in which each Modular Unit comprises a plurality of concentric circular annuluses with intraunit pores between the circular annuluses and struts connecting the annuluses, and in which the 3-D scaffold is 3-D printed.

2. A 3-D porous scaffold according to Claim 1 , in which the intra-unit pores are arcuate.

3. A 3-D porous scaffold according to Claim 1 or 2, in which the circular annuluses are solid and monolithic.

4. A 3-D porous scaffold according to any of Claims 1 to 3, in which the struts are solid and monolithic.

5. A 3-D porous scaffold according to any of Claims 1 to 4, in which the Modular Units of at least one scaffold layer all have the same diameter.

6. A 3-D porous scaffold according to any of Claims 1 to 4, in which the Modular Units of all scaffold layers have the same diameter.

7. A 3-D porous scaffold according to any preceding Claim, in which at least one of the scaffold layers comprises a plurality of different Modular Units, in which the Modular Units differ by comprising a different type of connecting strut.

8. A 3-D porous scaffold according to any preceding Claim, in which the Modular Units of each layer of the 3-D scaffold all comprise at least three concentric circular annuluses.

9. A 3-D porous scaffold according to any preceding Claim, in which the Modular Units of each layer of the 3-D scaffold comprise from three to five concentric circular annuluses.

10. A 3-D porous scaffold according to any preceding Claim, in which the array of Modular Units in one of the plurality of scaffold layers is not in register with the array of Modular Units in another of the plurality of scaffold layers.11 . A 3-D porous scaffold according to any preceding Claim, in which each scaffold layer comprises a primary Modular Unit and six secondary Modular Units arranged around the primary Modular Unit, in which each secondary Modular Unit abuts the primary Modular Unit and two adjacent secondary Modular Units.

12. A 3-D porous scaffold according to Claim 11 , including a plurality of tertiary Modular Units arranged around the six secondary Modular Units, in which each tertiary Modular Unit abuts two secondary Modular Units and two adjacent tertiary Modular Units.

13. A 3-D porous scaffold according to any preceding Claim, in which the connecting strut comprises a diametrical strut or a plurality of radial struts.

14. A 3-D porous scaffold according to any preceding Claim, in which one or more of the Modular Units comprise one diametrical strut, three diametrical struts, or three radial struts arranged in a Y-shape.

15. A 3-D porous scaffold according to any preceding Claim, in which each scaffold layer is circular and the 3-D scaffold is cylindrical.

16. A 3-D porous scaffold according to any preceding Claim, in which the Modular Units have a diameter of 500 microns to 10 mm.

17. A construct comprising a 3-D porous scaffold according to any preceding Claim and a biologically or pharmaceutically active agent disposed within pores or on / in the struts of the 3-D porous scaffold.

18. A construct according to Claim 17, in which the biologically or pharmaceutically active agent is selected from the group consisting of: cells; a pharmaceutical; a biopharmaceutical; a cellular growth factor.

19. A device comprising a plurality of wells and a 3-D porous scaffold of any of Claims 1 to 16 or a construct of Claim 17 or 18 disposed in the wells.

20. A method of forming a 3-D porous scaffold of any of Claims 1 to 16, comprising 3-D printing a plurality of scaffold layers, and adhering the scaffold layers together to form the 3-D porous scaffold.21 . A method of forming a 3-D porous scaffold of any of Claims 1 to 16, comprising 3-D printing the 3-D porous scaffold as a monolith.

22. A scaffold layer comprising an array of closely packed, interconnecting, Modular Units, in which each Modular Unit comprises a plurality of spaced apart concentric circular annuluses defining annular pores the neighbouring circular annuluses and struts connecting the annuluses, and in which the scaffold layer is 3-D printed.

23. A scaffold layer according to Claim 22, in which the annuluses and / or struts have a thickness of 50-1000 microns.

24. A scaffold layer according to Claim 22, in which neighbouring annuluses are spaced apart by 50-1000 microns.

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