Three-dimensional scaffold for regenerating biological tissues

Abstract

This disclosure pertains to the field of tissue regeneration to repair damaged tissue and in particular brain tissue. The scaffold (1) of the present disclosure comprises a set of horizontal layers (11, 12, 13, 14), each layer extending in a horizontal plane orthogonal to the vertical direction (Z), each layer (11, 12, 13, 14) comprising a plurality of openings (15), each opening (15) of one layer (11, 12, 13, 14) being connected to one opening (15) of the directly adjacent layer (11, 12, 13, 14) by at least one first pillar (16) extending along the vertical direction (Z). Thus, the scaffold (1) of the present disclosure is designed to promote a cell growth radially along each layer (11, 12, 13, 14) to reconstruct the grey matter portion of the damaged area and the white matter along the vertical pillars (16) extending through the different layer (11, 12, 13, 14).

Description

DescriptionTitle: Three-dimensional scaffold for regenerating biological tissuesTechnical Field

[0001] This disclosure pertains to the field of tissue regeneration to repair damaged tissue and in particular brain tissue. The present disclosure relates to a three-dimensional implant device acting as cellular supportive scaffold to facilitate cell infiltration and proliferation for producing engineered neural tissue directly in the damaged area for tissue repair and functional recovery, in human and animal subjects.Backg ound Art

[0002] The brain is a very sophisticated organ. It contains two regions, white matter composed mostly of axons and gray matter composed of neuron somas. Traumatic brain injury, brain tumors, and brain hemorrhages are common causes of brain damages. After injury, activation of the immune system and a poor spontaneous repair process makes it difficult to regenerate injured tissue.

[0003] Recent studies (Eduarda P Oliveira et al. “Advances in bioinks and in vivo imaging of biomaterials for CNS applications” Acta Biomater, 2019 Sep 1: 95:60-72) show that biodegradable and biocompatible scaffolds may be a promising strategy in the treatment of brain injury.

[0004] A biodegradable and biocompatible scaffold is a three-dimensional implant device, designed to have a 2D or 3D topological structure that can closely reproduce the topological, biochemical and mechanical properties of the extracellular matrix (ECM) in the central (CNS). After its implantation in the damaged area, the scaffold makes contact with peri-lesioned areas and serve as temporary ECM to provide a reservoir for cell infiltration and differentiation. It provides a support for the surrounding neural tissue and acts as a woven substrate for cell migration, cell growth, neurite formation, and axon sprouting. The structure of a biodegradable scaffold is obtained via a predefined series of microfabrication steps, such as optical lithography, nanoimprint, 3D printing, bio-printing. For instance, the additive manufacturing technology like digital light processing is used to realize 3D neural scaffold where the desired 3D structure is obtained, layer by layer, by photo-polymerizing a resin using a projected light source to cure the entire layer at once with a platform controlled remotely by a computer. After implantation, the scaffold should be progressively replaced by the regenerating tissue, while lasting long enough to permit cell infiltration and axon regrowth.

[0005] The ability of controlling the adhesion, proliferation, and differentiation of cells for a scaffold in reconstruction of brain tissue is characterized mainly by a combination of key parameters such as size, porosity, stiffness, inner architecture, interconnectivity and biodegradability.

[0006] Porosity is defined as the ratio of volumes of pores to the total volume of the material under consideration. It is a crucial parameter to let the cells infiltrate easily within the material and facilitate the regeneration. The porosity of the architecture is also directly correlated to other crucial parameters of the engineered architecture, which are its structural stability and mechanical properties.A too enhanced porosity can sensitively influence the stiffness of the scaffold and lead to collapse either during the cell proliferation or during the implantation. There is a fine balance in designing a scaffold architecture between porosity and stiffness.

[0007] Another important feature of cell scaffold is their ability to dissolve after a certain amount of time with minimal release of toxic or inflammatory byproducts, what is referred as biodegradability and biocompatibility. This characteristic strictly depends on the physical and biochemical properties of the material employed for scaffold fabrication. Most of the material employed for in-vivo brain and spinal cord regeneration are polymer-based which can be classified into two types, namely natural polymers and synthetic polymers. Naturally-derived polymers, such as collagen, hyaluronic acid, benzyl ester of hyaluronan, gelatin, chitosan and alginate offer biocompatibility and biodegradability, nevertheless they are still affected by several drawbacks, such as the limited sources of available material and the reproducibility. On the other hand, synthetic polymeric material, such as polylactic acid-PLA, polycaprolactone-PCL, polyethylene glycol) diacrylate-PEGDA, Poly(dimethyl siloxane- PDMS) can ensure a good level of reproducibility, as well an easy tunability of their mechanical properties. However, synthetic polymer materials do not often hold optimal degradability / biocompatibility, for which additional biochemical functionalization is required. In order to enhance the biocompatibility features of the materials employed for neural repair approaches, the scaffold may be coated with specific biomolecules.

[0008] The mechanisms of a biodegradable scaffold in promoting brain regeneration are also based on their ability in enhancing interaction with the microenvironment, guiding brain cells migration and axonal regrowth in the different directions, with the aim to optimize reconnections along the three- dimensional axes.

[0009] Current proposed designs do not allow a full reconstruction of brain tissue for both gray matter and white matter.

[0010] Wong et al {“Brain cortex regeneration affected by scaffold architectures: laboratory investigation", Journal of Neurosurgery, vol.109, n. 4, pp.715-722, 2008) describes a cylindrical PCL scaffold comprising a plurality of longitudinal and orthogonal guiding channels. Thus, compared to mono-directional channels, the channels extending in the two directions allows the tissue reconstruction, infiltration and alignment along microgrooves or channels of immature neurons. However, this bi-directional architecture does not allow a complete tissue reconstruction with the appropriate interconnective architecture.

[0011] There thus remains a need for biodegradable and biocompatible scaffolds allowing efficient brain tissue regeneration. Such scaffolds should exhibit a high biodegradability and biocompatibility. They should have an appropriate porosity to let cells infiltrate within the material, allow the perfusion of nutrients and facilitate the vascularization of the restored tissue. The engineered architecture should have a large porosity while providing a high structural stability. Furthermore, the suitable design should have a complex architecture, providing guiding pathways with preferred orientationsto promote bundle reconstruction specific of lost brain functions and a full brain tissue reconstruction of the connections between brain areas, and rehabilitation of the impaired neural functions. In particular, the architecture should guide brain cells migration and axonal regrowth in the different directions, with the aim to optimize functional reconnections along the three-dimensional axes.

[0012] In addition, these scaffolds should enable rapid brain tissue regeneration to shorten recovery times and decrease risks of post lesional complications for patients. Favoring certain connections could also prevent aberrant connections which could lead to adverse events like epilepsy.Summary

[0013] This disclosure improves the situation.

[0014] It is proposed a scaffold for biological tissue regeneration, such as brain tissue regeneration, the scaffold comprising: a. an implantable body extending between a top end and a bottom end along a vertical direction A-A; b. said body comprising a first part extending between the top end and an intermediate end and a second part extending between said intermediate end and the bottom end: i. the first part comprising a set of horizontal layers of material positioned between the top end and the intermediate end and spaced apart from each other, each layer extending in a horizontal plane “P” orthogonal to the vertical direction, said horizontal layer of material being configured to a cell growth radially along each layer, each layer comprising a plurality of openings, each opening of one layer being connected to one opening of the directly adjacent layer by at least one first pillar extending along the vertical direction, said pillar comprising at least a portion not in contact with the edge of the opening; ii. the second part comprising a plurality of second pillars extending along the vertical direction between the intermediate end and the bottom end.

[0015] A remarkable feature is the presence of pillars guiding and traversing the structure from top to bottom. Thanks to the free portion of the pillars, cells are not blocked on each layer and can migrate from the top layer to the bottom layer through the structure. In other words, the vertical pillars enable a cell on the top layer to cross the structure in a straight line from top to bottom, or vice versa from bottom to top. They also enable cells on any layer to turn and grow on an intermediate layer.

[0016] The following features, can be optionally implemented, separately or in combination one with the others:

[0017] In one or more embodiments, the layers of the first part may be arranged such that the openings are aligned along the vertical direction.

[0018] In one or more embodiments, each second pillar of the second part may be an extension portion of the first pillar of the first part.

[0019] In one or more embodiments, the scaffold may further comprise a handle positioned on the top end of the body, vertically to the center of gravity of the body.

[0020] In one or more embodiment, the scaffold may further comprise a funnel element positioned between the handle (2) and the top end of the body configured to drop cells into the body.

[0021] In one or more embodiment, the second part may comprise a plurality of horizontal beams configured to interconnect two second pillars directly adjacent.

[0022] In one or more embodiment, the plurality of horizontal beams may be arranged such that at each level along the vertical direction, each second pillar is connected by at least one horizontal beam to its directly adjacent pillar.

[0023] In one or more embodiment, the openings may be equally spaced relative to each other to form an array of openings.

[0024] In one or more embodiment, the first and second pillars may be equally spaced relative to each other to form an array of pillars.

[0025] In one or more embodiment, the openings may have a diameter between 50 and 300 pm, preferably between 100 and 200 pm.

[0026] In one or more embodiment, the layers may have a thickness between 5 pm and 300 pm, preferably between 10 pm and 200 pm,

[0027] In one or more embodiment, the distance between two adjacent layers may be between 100 pm and 400 pm, preferably between 200 pm and 350 pm.

[0028] In one or more embodiment, the pillars may have a diameter between 10 pm and 350 pm, preferably between 50 pm and 100 pm.

[0029] In one or more embodiment, the implantable body may have a porosity between 25% and 99%, preferably between 60% and 90%.

[0030] In one or more embodiment, the body may be made of at least one biocompatible and biodegradable or bioeleminable (PEG) polymer selected from a group comprising: polylactic acid- PLA, polycaprolactone-PCL, polycaprolactone-PCL-DA, polyethylene glycol) PEGDA, Poly(dimethyl siloxane-PDMS), GelMA (Gelatin MethAcryloyl), poly(trimethylene carbonate)- trimethacrylate (PTMC-tMA ), PPF (polypropylene fumarate) and combination thereof.Brief Description of Drawings

[0031] Other features, details and advantages will be shown in the following detailed description and on the figures, on which:Fig. 1

[0032] [Fig. 1] Fig.1 is a perspective view of a biodegradable and biocompatible scaffold for brain regeneration according to an embodiment.Fig. 2

[0033] [Fig. 2] Fig.2 is a perspective view of the first part of the scaffold of Figure 1 according to an embodiment.Fig. 3

[0034] [Fig. 3] Fig.3 is a perspective and partial exploded view of two layers used to form the first part of Figure 2.Fig. 4

[0035] [Fig. 4] Fig.4 is a perspective view of an elementary pattern used to form the first part of Figure 3, the elementary pattern comprising two aligned openings connected by four pillars.Fig. 5

[0036] [Fig. 5] Fig. 5 is a top view of the first layer of the first part of Figure 2 showing an array of openings and the top end of four vertical pillars through each opening and physically in contact with the border of the opening.Fig. 6

[0037] [Fig. 6A] Fig. 6A is a top view of the first part of the scaffold of Figure 1 according to another embodiment.

[0038] [Fig. 6B] Fig. 6B is a zoomed view of a region of Figure 6A showing another example of pillarsFig. 7

[0039] [Fig. 7] Fig. 7 is a perspective view of the second part of the scaffold of Figure 1 according to an embodiment.Fig. 8

[0040] [Fig. 8] Fig.8 is a perspective view of a group of four second pillars of Figure 7 connected by a plurality of horizontal beams according to an embodiment.Fig. 9

[0041] [Fig. 9] Fig. 9 is a top view of an array of second pillars showing an example of pattern of the disposition of the horizontal beams that connect two directly adjacent second pillars according to an embodiment.Fig. 10

[0042] [Fig. 10] Fig.10 is a perspective view of a group of four second pillars connected together by four horizontal beams according to another example.Fig. 11

[0043] [Fig. 1 1 A] Fig.1 1 A is a perspective view of a CAD design used for PEGDA-200 and PEGDA- 500 according to an embodiment.

[0044] [Fig. 1 1 B] Fig.1 1 B is an example of printed structure after the yellow photo-absorber was washed away, the width of the second part is 5 mm.

[0045] [Fig. 1 1 C] Fig.1 1 C is a microscopic view of the implantable body, showing the pillars in one direction and the layers.Fig. 12

[0046] [Fig. 12] Fig.12 represents longitudinal MRI follow-up before and after implantation of a scaffold made with different biomaterials in a brain lesion: (A) PEG DA-500, (B) PEGDA-200 and a rat served as a brain lesioned control.Description of Embodiments

[0047] Spatially relative terms, such as "top," "bottom," "inner," "outer," "beneath," "below," "lower," "above," "upper," and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the figures.

[0048] Although the terms first, second, third, etc. may be used herein to describe various elements, components, and / or layers, these elements, components, and / or layers should not be limited by these terms, unless otherwise indicated. These terms may be only used to distinguish one element, component, layer from another element, component, layer. Terms such as "first," "second," and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component or layer discussed below could be termed a second element, component, layer without departing from the teachings of the example embodiments.

[0049] “Tissue regeneration scaffold” refers to a porous structure suitable for implantation in the body for the purpose of regeneration and repair of tissue, especially soft tissue like brain tissue.

[0050] “Biocompatible” refers to a material biocompatible with respect to cells if it is nontoxic to the cells and does not cause an immunological or inflammatory reaction.

[0051] “Biodegradable” or “bioeliminable” refers to a material that can be eliminated within cells or within the body of a subject, by natural biological processes such as the action of enzymes present within cells or within the body. In general, a biodegradable or bioeliminable material is biocompatible.

[0052] The scaffold is formed from additive manufacturing printed layers that are printed on each other in an iterative manner to build-up the scaffold. The scaffold may be printed to dimensionally match a damaged area.

[0053] The additive manufacturing method is a high-resolution additive manufacturing method with the capability to produce pillars in the micrometer range.

[0054] The central nervous system (CNS) has a limited capacity for cell regeneration, and in particular a very limited axon growth ability which induces a poor functional regeneration if axons are injured. There is a need to provide an implantable scaffold with a specific architecture for promoting neuronal regeneration and axonal growth in a subject. The proposed scaffold for brain tissue regeneration allows an effective integration, a crosstalk between new cells and surrounding microenvironment to establish a new and functional neuronal network.

[0055] The present scaffold is configured to reproduce the architecture of a cerebral cortex. In particular, the scaffold can be configured to allow axonal growth.

[0056] The cerebral cortex is the outer grey matter that covers the surface of the two cerebral hemispheres. It is about 2 to 4 mm thick and contains an aggregation of nerve cell bodies. The cerebral cortex is organized into six cellular layers. Neurons are connected through the layer by axons.

[0057] The scaffold of the present disclosure can be used for repairing a damaged area in a cerebral cortex of a subject. The subject may be an animal with a complex nerve system, such as a mammal, like a human, or animal.

[0058] As the scaffold may be produced in a variety of shapes and sizes, it can be applied in a similar manner to repair any other biological tissue.

[0059] Figure 1 is a perspective view of a scaffold used for repairing biological tissue according to one embodiment.

[0060] The scaffold 1 is configured to repair a damaged area localized in a brain, for instance a damaged area localized in a cerebral cortex.

[0061] The cerebral cortex is the outer grey matter that covers the surface of the two cerebral hemispheres. It is about 2 to 4 mm thick and contains an aggregation of nerve cell bodies. The cerebral cortex is organized into six cellular layers. Neurons are connected through the layer by axons. The cortex displays a poor ability to self-repair, whether by generating new cells and / or long- range connection through the layers.

[0062] The scaffold of the present disclosure is designed to promote the cell growth in the three directions in the space to reconstruct the grey matter portion of the damaged area along a set of horizontal layers and the white matter along a set of vertical pillars extending through the different layer.

[0063] The scaffold 1 comprises an implantable body 10 extending between a top end 31 and a bottom end 33 along a vertical axis direction AA’. The body has a generally cylindrical shape with a circular section. It should be noted that although figures show a body with a circular section, different geometric shape may be provided, such as a square, rectangle or oval, or a combination of various shapes. The scaffold may be printed to dimensionally match a damaged area. The design of the scaffold is composed of small unities that can be repeated in three dimensions to match the volume of the damaged area.

[0064] The body is formed from biocompatible and biodegradable polymers, such as polyester polymers. Suitable biodegradable and biocompatible polymers for forming the body include a polylactic acid, polylactic acid-PLA, polycaprolactone (PCL), polycaprolactone-PCL-DA, polyglycolic acid, poly(lactide-co-glycolide polymer (PLGA), polyethylene glycol) PEGDA, Poly(dimethyl siloxane-PDMS), GelMA (Gelatin MethAcryloyl), PTMC-tMA (poly(trimethylene carbonate)- trimethacrylate, PPF (polypropylene fumarate) and copolymers, derivatives, and mixtures thereof. In certain preferred aspects, the biocompatible and biodegradable material is selected in the group of polymers consisting of: polycaprolactone (PCL), polyethylene glycol) PEGDA, and copoplymers, derivatives, and combinations thereof.

[0065] The implantable body 10 comprises a first part 4 extending between the top end 31 and an intermediate end 32 and a second part 5 extending between the intermediate end 32 and the bottom end 33.

[0066] In an embodiment not illustrated, the scaffold may comprise a peripheral wall surrounding the implantable body. The peripheral wall may have a cylindrical shape. The use of the peripheral wall permits to avoid the risks of the mechanical deformation of the body contained within the wall. The peripheral wall is porous. The pore size and the porosity are selected to promote the lateral cell growth. The wall and the body 10 may be printed by two different methods. The wall may take the form of a hollow sleeve dimensioned to receive the body.

[0067] The scaffold further includes a handle 2 for holding the implantable body and positioning the body within the damaged area during surgery. The handle 2 is positioned on top end of the body 10. The center of the handle is aligned with the center of gravity of the implantable body 10. The handle facilitates the displacement of the body and reduce the risk of damage of the body during the operation. In a preferred example, the handle has an ogive shape, and the tip of the handle is positioned vertically to the center of gravity so that the implantable body may stand vertically within the damaged area.

[0068] The scaffold 1 further includes a funnel 3 positioned between the handle 2 and the top end 31 of the body 10. The funnel 3 is configured to facilitate the deposit of the cells into the body 10.

[0069] The handle 2 and the funnel 3 will be removed from the implantable body 10 during surgery. It is not necessary to use biodegradable material for forming the handle and the funnel.

[0070] With reference to figures 2 to 5, the first part of the body according to embodiments will now described.

[0071] As shown in Figure 2, the first part stands in a coordinate system XYZ. The first comprises a plurality of layers extending in the horizontal plane containing orthogonal axes X and Y, orthogonal to the axe Z. The layers are spaced apart from each other in the Z-axis direction. The distance between the layers nearest from each other is indicated by “L”. The distance “L” between two directly adjacent layers can be in a range between 100 pm and 600 pm, optionally in a range between 100 pm and 400 pm, in certain variations, preferably in a range between 200 pm and 350 pm. The layers can be of the same or different thickness. The thickness “e” of the layers can be in a range between 5 pm and 300 pm, preferably between 10 pm and 200 pm.

[0072] Although Figure 2 shows the layers with rectangular shape, other shape may be appropriated such as circular shape as shown in Figure 1 , or oval shape. Thus, although the first part of the body 10 is shown as a cylindrical shape with a circular cross-sectional shape in Figurel , however, the first part of the body 10 may have a variety of other shapes. The implantable body can be adjusted in dimension and in shape to match damaged areas of subjects.

[0073] The first part may comprise two or more layers of material that enables cells to colonize and grow radially across the surface of the layer. In other words, the layers are solid layers of polymers. In case where the implantable body 10 is used to be implanted in a cerebral cortex to repair a damaged area, the body is designed to reproduce the architecture of the cortex. The first part may reproduce the grey matter and may comprise six (6) layers. The first part has a height between 1 .5 mm and 2.5 mm, preferably close to 2 mm. Figure 2 illustrates an example of the first part of a scaffold. It comprises four layers 1 1 , 12, 13, 14, equally spaced from each other by a distance L. The layers have the same thickness “e”.

[0074] The horizontal layers have the function to host the cell bodies and to promote horizontal cell growth radially along each layer. The horizontal layers are used to reconstruct the gray matter portion of the lesion.

[0075] In addition, and as shown in figure 2, each layer comprises a plurality of openings 15 for porosity. The openings may vary in number, size and / or shape within a given layer. In one example, a layer may comprise a plurality of openings that vary in size, shape from those of the adjacent layer. In another example and as can be seen in figure 2, the openings are of the same size and the same shape for all layers.

[0076] It will be appreciated that although all figures show round openings, other shapes of openings may be appropriate. The shape can be circular, oval, rectangular or square of any other suitable geometric form. The diameter of the opening can be in a range between 50 pm and 300 pm, preferably between 100 pm and 200 pm.

[0077] In one embodiment, the layers are arranged such that the openings are opposite and aligned with each other along the vertical Z axis direction. Each opening of one layer can be aligned with theopenings of the directly adjacent layers. The density of the opening of all layers along a vertical direction defines an axial porosity.

[0078] The figure 3 shows a perspective and partial exploded view of two layers 1 1 , 12 used to form the first part of Figure 2. The figure 3 shows a specific configuration in which the layers 1 1 , 12 are arranged such that the openings 15 are aligned along the vertical z axis direction. Each opening 15 of the layer 1 1 is aligned with one opening of the layer 12. The aligned openings 15 are connected by four first pillars 16 extending along the vertical z axis direction. The aligned openings 15 are interconnected by four first pillars 16 extending along the vertical direction from the layer 1 1 to the directly adjacent layer 12.

[0079] In case where the body comprises a plurality of layers, the aligned openings 15 are interconnected by four first pillars extending along the vertical direction from the first layer of the first part to the last layer of the first part. Thus, each first pillar 16 interconnects all aligned openings along a vertical direction. As shown in Figures 4 and 5, each pillar 16 comprises a portion that is not in contact with the layer. These free portions enable cells to grow in a straight line from the top to the bottom of the structure, allowing cells to migrate from one layer to another, even far from the starting layer. The first pillars create a plurality of pathways or a plurality of vertical guides extending vertically from one layer to the directly adjacent layer for cell growth along the pillars, from the first layer to the last layer. In other words, the openings and the pillars promote linear axonal tissue growth along the longitudinal axis of each first pillar through the different layers. The pillars form a plurality of vertical guides extending from a layer to the directly adjacent layer, e.g., the layer positioned above said layer and the layer beneath said layer, to guide axonal growth.

[0080] In the case where the scaffold is used to repair a damaged area of a cerebral cortex, the first part of the scaffold intended to reconstruct the gray matter comprises six layers. Each layer comprises a plurality of openings. The scaffold comprises a set of vertical first pillars interconnecting the openings from the first layer to the sixth layer. In other words, the openings of one layer interconnect with the openings of the adjacent layers, e.g., the layer positioned above said layer and the layer beneath said layer. The height of the first part can be about 2 mm, corresponding to the cortical thickness.

[0081] In the example of Figure 2, each layer comprises an array of sixteen openings 15 equally spaced from each other. The openings 15 are aligned along the vertical direction by four pillars extending through the four layers 1 1 , 12, 13, 14, from the first layer 1 1 of the first part to the last layer 14 of the first part.

[0082] As alternative variants and not illustrated, the layers are arranged along the vertical direction such that the openings are opposite but not aligned with each other. Each opening of one layer may be slightly offset vertically with respect to the opening of the directly adjacent layer so that there is a partial overlap between the openings along one vertical direction.

[0083] The figure 4 shows a perspective view of an elementary pattern which can be repeated to form the structure of the figure 3. The opening 15.1 of the elementary layer 1 1 .1 is aligned with the opening 15.2 of the layer 12.2. along the vertical axis BB’ parallel to the Z axis. In addition, each opening is connected to one opening of the directly adjacent layer by four first pillars 16.1 , 16.2, 16.3, 16.4 extending along the vertical z axis direction.

[0084] The figure 5 shows a top view of the body 10 of the figure 2, from the first layer 1 1 of the layered structure. The openings 15 are equally spaced relative to each other to form an array of openings. As variant, the openings of a layer can be non-equally spaced relative to each other. The density of the openings within each layer defines a radial porosity. The openings of each layer participate to the porosity of the scaffold. The openings form an open passage for the cell bodies from one layer to another that permits axonal growth along the vertical first pillars which connect the aligned openings. Figure 5 shows that the openings 15 aligned along a vertical direction are connected by four first pillars 16.1 , 162, 16.3, 16.4. The first pillars within a layer are equally spaced relative to each other to form an array of pillars. Each pillar has a cylindrical shape, with a circular section. The diameter of the first pillar can be in a range between 10 pm and 350 pm, preferably between 50 pm and 100 pm.

[0085] It will be appreciated that although all figures show circular section, other shapes of pillars may be appropriate. The shape can be circular, oval, rectangular or square of any other suitable geometric form. Like the openings, the pillars can vary in size and shape.

[0086] As can be shown in Figure 5, the end of the four first pillars 16.1 , 16.2, 16.3, 16.4 are equally spaced relative to each other and their positions form substantially a square. The positions and the number of the first pillars per opening may be adjusted to the form and the size of the opening. As variant, the aligned openings may be interconnected by two or three first pillars equally spaced relative to each other.

[0087] In one embodiment and as illustrated in Figure 2, the first pillars extend from the first layer 1 1 of the stack of layers to the last layer 14 of the stack of layers. Each pillar is formed by a single cylindrical solid rod trough the four layers.

[0088] In another embodiment, each pillar extends only between two directly adjacent layers to interconnect the aligned openings. Each pillar is formed by a succession of cylindrical solid rods to interconnect the aligned openings between two layers along the vertical direction. For the example of Figure 2, each first pillar may formed by three rods.

[0089] Figures 6A and 6B illustrate a scaffold 20 according to another embodiment. In this example, the pillars are concave, instead of convex in the previous example. They can still guide cells and axons from the top to the bottom. Depending on the biomaterial and its printable features, porosity can be modulated.

[0090] The Figure 6A shows a top view of the first layer of the scaffold 20 comprising an array of openings. The openings are equally spaced relative to each other. They have identical in size and inshape which is circular. The layers are arranged such that the openings of a layer are aligned with the openings of the directly adjacent layer. The aligned openings 25 are interconnected by a single first pillar 26.

[0091] The Figure 6B is a zoomed view of an area of the layer of Figure 6A and illustrates in detail the shape of each pillar. The pillar 26 is centered in each opening 25. The external surface of the pillar is formed by four concave surfaces 26.1 , 26.2, 26.3, 26.4. The ends 27.1 , 27.2, 27.3, 27.4 of each concave surface are in contact with the internal wall of the opening 25 in which the pillar is positioned. The number of the concave surfaces may vary to adjust the porosity of the scaffold.

[0092] Figures 7 to 10 illustrate the second part 5 of the scaffold 1 which extends from the intermediate end 32 to the bottom end 33.

[0093] As shown in Figure 7, the second part 5 of the implantable body 10 of the scaffold 1 comprises a plurality of second pillars 18 extending in a vertical direction between the intermediate end 32 and the bottom end 33.

[0094] In case where the scaffold is used to reconstruct the cortical tissue, the second part is used to regenerate the white matter. The second part has a height H2 in a range between 2 mm and 100 mm. The height of the second part may vary to be adjusted to the injured area.

[0095] In one embodiment, the second pillars 18 are the extension portion of the first pillars 16. In other words, the first pillar 16 and the second pillar 18 form a single cylindrical solid rod extending from the top end 31 to the bottom end 33 of the implantable body 10.

[0096] In alternative variants, the second pillars 18 are distinct from the first pillars 16. Each second pillar 18 of the second part 5 may be aligned with each first pillar 16 of the first part 4. As result, the first part and the second part have the same number of pillars. The first pillars and the second pillars may have the same shape and the same diameter.

[0097] Each second pillar has a cylindrical shape, with a circular section. The diameter of the second pillar can be in a range between 10 pm and 350 pm, preferably between 50 pm and 100 pm. In one embodiment, the second pillars 18 are equally spaced relative to each other to form an array of pillars. Figure 7 shows an example of array comprising six columns of second pillars and six rows of second pillars. The second pillars create a plurality of vertical guides to regenerate the white matter.

[0098] In some embodiments, the second part 5 comprises a plurality of horizontal beams 17 configured to connect the second pillars 18 to prevent the implantable body 10 from collapsing under its weight or during implantation. The horizontal beams 17 connect the second pillars along the vertical direction, at a plurality of levels.

[0099] In one embodiment, the second pillars 18 are interconnected by the horizontal beams 17 according to a specific pattern that maximizes the rigidity of the implant structure while minimizing the number of horizontal beams 17.

[0100] Figure 8 shows four second pillars 18 indicated by the reference numerals 1 .1 , 1 .2, 2.1 , 2.2. At first level, only the second pillar 18(1 .1 ) and the second pillar 18(1 .2) are interconnected by a horizontal beam 17. At second level, only the second pillar 18(1 .2) and the second pillar 18(2.2) are interconnected by a horizontal beam 17. At third level, only the second pillar 18(2.2) and the second pillar 18(2.1 ) are interconnected by a horizontal beam 17. At fourth level, only the second pillar 18(2.1 ) and the second pillar 18(1 .1 ) are interconnected by a horizontal beam 17. As result, at each level, among the four second pillars, each second pillar is connected to its directly adjacent pillar by a single horizontal beam 17.

[0101] Figure 9 show an example of an array of pillars comprising six columns and four rows. The second pillars 18 are interconnected by four levels of horizontal beams indicated respectively by the reference numerals 1 , 2, 3 and 4. The second pillars are indicated by their matrix position in the array 18 (i = 1 -6, j = 1 -4). The pattern for connecting a group of four directly adjacent second pillars is similar to that of Figure 8. At first level, only the second pillar 18(1 .1 ) and the second pillar 18(1 .2) are interconnected by a horizontal beam 17. At second level, only the second pillar 18(1 .2) and the second pillar 18(2.2) are interconnected by a horizontal beam 17. At third level, only the second pillar 18(2.2) and the second pillar 18(2.1 ) are interconnected by a horizontal beam 17. At fourth level, only the second pillar 18(2.1 ) and the second pillar 18(1 .1 ) are interconnected by a horizontal beam 18. This connection pattern is repeated to connect a next group of four second pillars as illustrated in Figure 9. As result, for each level, each second pillar is connected to its directly adjacent second pillar by a single horizontal beam 17. This configuration is believed to be particularly advantageous to maximize the rigidity of the implant structure while minimizing the number of horizontal beams 17.

[0102] In another embodiment and with reference to Figure 10, each second pillar 18 is connected to its directly adjacent pillar by a horizontal beam 17. For instance, the second pillar 18(1 .1 ) is connected to its directly adjacent second pillars by a horizontal beam, e.g., the two directly adjacent second pillars 18(1 .2), and 18(2.1 ). In a similar manner, the second pillar 18(1 .2) is connected to its directly adjacent second pillars by a horizontal beam, e.g., the two directly adjacent second pillars 18(1 .1 ), and 18(2.2). In a similar manner, the second pillar 18(2.2) is connected to its directly adjacent second pillars by a horizontal beam, e.g., the two directly adjacent second pillars 18(2.1 ), and 18(1 .2). In a similar manner, the second pillar 18(2.1 ) is connected to its directly adjacent second pillars by a horizontal beam, e.g., the two directly adjacent second pillars 18(1 .1 ), and 18(2.2).

[0103] The proposed scaffold with preferred orientations, aims at replacing brain lesioned structures with a complex architecture, like the six-layer cortex with orthogonal connections. Owing to the presence of layers interconnected by the pillars, the proposed scaffold of the present disclosure allows an extensive brain tissue reconstruction. It may support neurons, and guide the development of axon and neurite connection, providing thus an efficient integration and differentiation of the new cells within the damaged nerve tissue. The scaffold would also help the arrangement of correct connections, avoiding the formation of aberrant neuronal pathways.

[0104] The provided structure, thanks to a specific combination between layers and pillars, permits a fast regeneration of brain tissue, with the result that the scaffold displays enhanced healing compared with a conventional scaffold.Examples

[0105] Preparation of hydrogels

[0106] Implants were designed with Fusion 360® (Autodesk®), a computer assisted design software. The geometry aims at maximizing porosity, cell infiltration and axonal guidance. Geometric fidelity, thickness of the structure and 1 D swelling of the hydrogel were assessed via microscopic observation. Before printing the actual implants, the difference between the 3D model and what was actually printed from the designs was quantified. Post-injury MRI images were used to measure the lesion volume and dimensions. The size of the implants needed to pass through the 5mm-diameter hole drilled in the skull and to fit into the lesion cavity.

[0107] Preparation of the PEGDA implants

[0108] Two types of Polyethylene glycol) diacrylate (PEGDA) were tested: PEGDA-200 PhotolnkTM and PEGDA-500 PhotolnkTM (from Cellink Inc., USA). PEGDA is a biocompatible and transparent hydrogel that can be photo-crosslinked. PEGDA-200 has a molecular weight over 2000 Da and is called 200 because modulus at end of exposure is 200 ± 20 kPa. PEGDA-500 is composed of low (<2000 Da) and high (>2000 Da) molecules is stiffer and less transparent, and has a modulus at end of exposure of 500 ± 25 kPa. The implants were printed with a LumenXTM (Cellink Inc.). This printer works in digital light processing technology (DLP), with a wavelength of 405nm (violet) and a horizontal resolution of 50 pm (x, y). We used the recommended printing parameters with a 50 pm layer height and a light power of 20 mW / cm2. Respectively for PEGDA-200 and PEGDA-500: an exposure time for first layers of 9 sec and 6 sec and an exposure time of 3 seconds and 2 seconds. An example of PEGDA implant is shown in Figure 1 1 .

[0109] Implant sterilization

[0110] The printed scaffold was then removed from the printer and rinsed three times with sterile Dulbecco's phosphate-buffered saline and antibiotics (1 % Pen Strep). The day before implantation, the implants were sterilized with 70% ethanol filtered at 0.22 pm for 2h in a multi-well plate. Three washes of 10 min each with sterile PBS and Pen Strep were performed after the ethanol sterilization step. The sterilized implants were stored in the refrigerator overnight. The day of implantation, the implants were placed under UV light for 30 minutes and kept in closed PBS+PenStrep medium until the time of implantation. Ethanol sterilization, PBS washes, and UVs were performed under a microbiological safety station (MSS) in a level 2 safety laboratory.

[0111] Handle of hydrogels

[0112] Since no dedicated manipulation tools were available or described in the literature for fragile hydrogels, tools were 3D printed or sculpted from PTFE rod and autoclaved. We made a tool with a thin and narrow “blade” to remove the fragile structure from the LumenX build plate. Due to the low elongation at break, the hydrogels tended to break after repeated use. To avoid direct handling, handle on top of the implants to pick up them with a hook is provided, as shown in Figure 1 1 . The handle has an ogive shape. The tip was positioned vertically to the center of gravity so that the implant naturally stands vertically. The handle and the funnel were removed during surgery.

[0113] Animals

[0114] Five female Sprague-Dawley rats (280-320 g, 1 1 -weeks-old, Janvier, France) were used in this study. There were housed two per cage (30 cm length, 18 cm height, 32 cm width), in a regulated environment (20°C) under a 12h / 12h light / dark cycle with free access to food and water. Animals were treated according to the Council of the European Communities guidelines (EU Directive 2010 / 63). This protocol was approved by the “Direction Departementale de la Protection des Populations de la Haute - Garonne” and the “Comite d’ethique pour I’experimentation animale MidiPyrenees” (protocol n° APAFIS#22419-2019101 1 15259327v5). According to 3R recommendation, this pilot study used the minimize number of animals. Enrichment was utilized and all efforts was made to reduce the suffering.

[0115] Results

[0116] A scaffold with a complex shape could be successfully printed with a spatial resolution and a porosity adapted to brain reconstruction. Using an optical microscope, we measured a 1 D swelling of 30% for PEGDA-200 and 10% for PEGDA-500 in water. Layer thickness was respectively 210 pm and 250 pm, pillars width 150 pm and 160 pm, and distance between consecutive layers 360 pm and 250 pm. Based on those measurements, a porosity of 67% and 54% was estimated respectively. The PEGDA implant width measured 5 mm before implantation (microscopic observation), and again 5 mm after implantation based on MRI data. The term “porosity” of an implant is the ratio of the volume of the pores or spaces over the total volume of the implant. The volume of the pores of the implant corresponds to the difference between the total volume of the implant and the volume of the layers and the pillars.

[0117] For PEGDA-200 and based on MRI data, no modification of the outer dimensions of the implants during the in vivo experiment was observed, i.e. , no swelling was observed in vivo. Cutting off the extra parts of the implants during surgery (handle and funnel) was easy since the material is very soft.

[0118] Figure 12 shows longitudinal MRI follow-up before and after implantation of a scaffold according to an embodiment of the present disclosure in a brain lesion: coronal and axial T2 MR images of the lesion and the implants. Implants were made with different biomaterials: PEGDA-500 (A), PEGDA-200 (B) and a rat (C) served as a brain lesioned control. The implants and the lesion are shown in three states.

[0119] Thanks to the specific design of the scaffold of the present disclosure, it is possible to have a large porosity while providing a high structural stability. Furthermore, thanks to the presence of the pillars and the layers, the scaffold of the present disclosure provides guiding pathways with preferred orientations to promote bundle reconstruction specific of lost brain functions and a full brain tissue reconstruction of the connections between brain areas, and rehabilitation of the impaired neural functions. In particular, the architecture allows axonal regrowth in the different directions, in order to optimize functional reconnections along the three-dimensional axes.

[0120] In general, the scaffold of the present disclosure can be used to repair any injured neural tissue. Such injury may occur as a result of surgery, trauma, stroke, tumor or neurodegenerative disease such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. The scaffold can be used to restore anatomical and / or functional integrity to the neural tissue. The scaffold of the present disclosure may be introduced at a site of injury and allows axon growth from the site injury to a location distal to the site of injury.

[0121] For example, in a case where the neural tissue has been lost, e.g., due to a stroke, the scaffold allows the regeneration of new neural tissue that replaces lost neural tissue.

[0122] As described above, the scaffold may be used to repair injured nerves localized in the central nervous system (CNS).

[0123] In the present examples, the scaffold is described for the brain tissue reconstruction. Nevertheless, the invention encompasses every alternative that a person skilled in the art would envisage when reading this text and the implant described may be adapted to other soft tissue reconstruction.

Claims

Claims

1. Scaffold (1 ) for biological tissue regeneration, such as brain tissue regeneration, the scaffold comprising: a. an implantable body (10) extending between a top end (31 ) and a bottom end (33) along a vertical direction A-A; b. said body comprising a first part (4) extending between the top end (31 ) and an intermediate end (32) and a second part (5) extending between said intermediate end (32) and the bottom end (33): i. the first part comprising a set of horizontal layers of material (1 1 , 12, 13, 14) positioned between the top end and the intermediate end and spaced apart from each other, each layer extending in a horizontal plane “P” orthogonal to the vertical direction, said horizontal layer of material being configured to a cell growth radially along each layer, each layer of material comprising a plurality of openings (15), each opening of one layer being connected to one opening of the directly adjacent layer by at least one first pillar (16) extending along the vertical direction, said pillar comprising at least a portion not in contact with the edge of the opening; ii. the second part comprising a plurality of second pillars (18) extending along the vertical direction between the intermediate end (32) and the bottom end (33).

2. Scaffold according to claim 1 , wherein the layers of the first part are arranged such that the openings are aligned along the vertical direction.

3. Scaffold according to claim 1 or 2, wherein each second pillar (18) of the second part (5) is an extension portion of the first pillar (16) of the first part (4).

4. Scaffold according to any one of claims 1 to 3, further comprising a handle (2) positioned on the top end of the body (10), vertically to the center of gravity of the body (10).

5. Scaffold according to claim 4, further comprising a funnel element (3) positioned between the handle (2) and the top end of the body (10) configured to drop cells into the body (10).

6. Scaffold according to any one of claims 1 to 5, wherein the second part (5) comprises a plurality of horizontal beams (17) configured to interconnect two second pillars (18) directly adjacent.

7. Scaffold according to claim 6, wherein the plurality of horizontal beams (17) is arranged such that at each level along the vertical direction, each second pillar (18) is connected by at least one horizontal beam to its directly adjacent pillar.

8. Scaffold according to any one of claims 1 to 7, wherein the openings (15) are equally spaced relative to each other to form an array of openings.

9. Scaffold according to any one of claims 1 to 8, wherein the first (16) and second pillars (18) are equally spaced relative to each other to form an array of pillars.

10. Scaffold according to any one of claims 1 to 9, wherein the openings (15) have a diameter between 50 and 300 pm, preferably between 100 and 200 pm.

11. Scaffold according to any one of claims 1 to 10, wherein the layers have a thickness between 5 pm and 300 pm, preferably between 10 pm and 200 pm,

12. Scaffold according to any one of claims 1 to 11 , wherein the distance between two adjacent layers is between 100 pm and 400 pm, preferably between 200 pm and 350 pm.

13. Scaffold according to any one of claims 1 to 12, wherein the pillars have a diameter between 10 pm and 350 pm, preferably between 50 pm and 100 pm.

14. Scaffold according to any one of claims 1 to 13, wherein the implantable body has a porosity between 25% and 99%, preferably between 60% and 90%.

15. Scaffold according to any one of claims 1 to 14, wherein the body is made of at least one biocompatible and biodegradable polymer selected from a group comprising: polylactic acid-PLA, polycaprolactone-PCL, polycaprolactone-PCL-DA, polyethylene glycol) PEGDA, Poly(dimethyl siloxane-PDMS), GelMA (Gelatin MethAcryloyl), poly(trimethylene carbonate)-trimethacrylate (PTMC-tMA ), PPF (polypropylene fumarate) and combination thereof.

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