Field of the Invention

[0001] The present invention relates to a biocompatible implantable scaffold. In some preferred embodiments, the scaffold is biphasic and has a first scaffold portion for periodontal ligament cells and a second scaffold portion for bone cells. Whilst the invention has been developed primarily for use as a biocompatible implantable scaffold for implanting in the periodontium of a subject for treating and/or repairing a periodontal defect, it will be appreciated that the invention is not limited to this particular field of use.

Background of the Invention

[0002] Periodontitis is a chronic inflammatory condition characterized by the structural deterioration of the periodontium leading to the destruction of the tooth supporting tissue structures, such as cementum, periodontal ligament (PDL) and alveolar bone, and in severe cases results in tooth loss. Regeneration of the destroyed tissues is characterized by fully restored and functional insertion of newly formed periodontal ligament into the adjacent bone and cementum structures.

[0003] However, predictable regeneration remains challenging, largely due to the periodontium's complex anatomical nature, which requires a highly coordinated and compartmentalised temporospatial interplay of the multiple soft and hard tissues (cementum, periodontal ligament, and alveolar bone) that comprise the periodontium.

[0004] In this context, the utilisation of tissue engineering strategies, combining advanced biomaterials with biological cues that promote the biological requirements for periodontal regeneration (wound stabilisation, space maintenance for bone ingrowth and selective cell repopulation) is scientifically sound.

[0005] A key feature of successful periodontal regeneration is the alignment and attachment of perpendicular and/or oblique periodontal ligament (PDL) fibres at the tooth-ligament interface. However, this has been difficult or impossible to achieve with prior art scaffolds and methods. [0006] While several investigations have reported that cementogenesis can enhance PDL-like attachment, it was achieved only in specific locations along the tooth surface and lacked reproducibility. This suggests that cementogenesis is an important, but not necessarily the sole requirement for regenerating the periodontal complex and achieving periodontal attachment.

[0007] A growing emphasis is being placed on tissue engineering approaches using biodegradable constructs, with varying structural or compositional phases, known as multiphasic scaffolds. Local physical environments, such as the modification of surface geometry, have been widely reported to influence cellular function, differentiation and morphology. Previous studies have also shown that precise control over 2D microscaled geometries, such as grooves, elicits predictable alignment of cells and extracellular matrix (ECM).

[0008] In the context of periodontal regeneration, the controlled alignment of the periodontal ligament in a perpendicular and/or oblique manner to the root surface has not yet been achieved despite previous attempts.

[0009] For instance, scaffolds known in the art fail to achieve perpendicular and/or oblique attachment of cells to the root surface of a tooth in order to effectively produce the necessary orientation and alignment of cells to promote ligament fibre alignment.

[0010] For example, Figure 1 shows an isometric view of a prior art scaffold having an array of spaced apart walls fabricated from a plurality of fibres, having a fibre spacing (along the height of the walls, i.e., in an axial direction) of approximately I Q- 40 pm, and with approximately square/rectangular channels (when viewed in cross section) wherein the walls of the channels are transversely spaced at greater than 100 pm intervals.

[0011] Figure 2 shows a cross-sectional side view of the prior art scaffold of Figure 1 , in which the seeded periodontal ligament cells are shown growing in between and around the fibres of the scaffold walls in a random manner. [0012] In other words, this random manner in which the cells grow and orient, fails to result in ligament fibres that have the necessary orientation and alignment to facilitate periodontal attachment.

[0013] The present invention thus seeks to provide a biocompatible implantable scaffold, which will overcome or substantially ameliorate at least some of the deficiencies of the prior art, or to at least provide an alternative.

[0014] It is to be understood that, if any prior art information is referred to herein, such reference does not constitute an admission that the information forms part of the common general knowledge in the art, in Australia or any other country.

Summary of the Invention

[0015] According to a first aspect, the present invention provides a biocompatible implantable scaffold comprising: a first scaffold portion comprising a first array of spaced walls having a first height, wherein the walls of the first array of spaced walls are connected by one or more transverse connectors, and are substantially impermeable to cell transport and/or growth therethrough, and spaced apart from each other by a predetermined distance to promote cell growth and orientation substantially aligned with the first height of the walls of the first array of spaced walls.

[0016] Figure 4 shows an isometric view of a first scaffold portion of the biocompatible implantable scaffold according to a preferred embodiment of the present invention, showing a first array of spaced walls (dark grey) having a first height extending substantially in an axial direction and a second array of spaced walls functioning as transverse connectors (light grey) interconnecting the walls of the first array of spaced walls. The first array of spaced walls has been fabricated according to a preferred deposition technique as will be described in more detail below, so as to be substantially impermeable to cell transport and/or growth therethrough.

[0017] The inventors have surprisingly found that when the first scaffold portion of the biocompatible implantable scaffold is affixed to the surface of a pre-treated dentin block, and then subsequently seeded with periodontal ligament cells, the impermeable nature of the walls of the first array of spaced walls, and the structural constraints imposed by the geometry of the plurality of open channels defined by the first array of spaced walls and plurality of transverse connectors, combine to promote cell growth and orientation substantially aligned with the height of the walls of the first array of spaced walls. In other words, the cells tend to be guided by virtue of these structural constraints to enter the open channels axially and fill the space within each of the open channels toward the corresponding part of the surface of the dentin block enclosed by each of the open channels.

[0018] By virtue of this arrangement, and as shown in the cross-sectional side view of the first scaffold portion shown in Figure 5, the ligament fibres produced as a result of the subsequent cell growth are substantially oriented in a perpendicular and/or oblique manner with respect to the dentin surface, thereby resulting in the formation of a plurality of substantially aligned and perpendicularly and/or obliquely oriented ligament fibres.

[0019] In some embodiments, the walls of the first array of spaced walls are substantially parallel.

[0020] Preferably, the predetermined distance is less than 500 pm.

[0021] In some embodiments, the predetermined distance is between about 60 pm to about 100 pm, preferably between about 60 pm to about 90 pm, even more preferably between about 60 pm to about 80 pm.

[0022] In one embodiment, the one or more transverse connectors comprises a plurality of transverse connectors that define a second array of spaced walls, wherein the first and second arrays of spaced walls intersect to form a first plurality of open channels through the first scaffold portion.

[0023] In one embodiment, the first and second arrays of spaced walls intersect at substantially right angles to each other.

[0024] Preferably, the walls of the second array of spaced walls are substantially impermeable to cell transport and/or growth therethrough, such that the first plurality of open channels of the first scaffold portion substantially preclude cell transport and/or growth in a plurality of transverse directions.

[0025] In some embodiments, the walls of the second array of spaced walls are spaced apart from each other by a distance of between about 150 pm to about 250 pm, preferably between about 180 pm to about 230 pm, even more preferably between about 190 pm to about 220 pm.

[0026] In some embodiments, the first height is between about 50 pm to about 200 pm, preferably between about 80 pm to about 180 pm, even more preferably between about 90 pm to about 160 pm.

[0027] In one embodiment, the biocompatible scaffold further comprises a second scaffold portion being connected to the first scaffold portion.

[0028] In one embodiment, the second scaffold portion comprises a third array of spaced walls and a fourth array of spaced walls which intersect substantially at right angles to form a second plurality of open channels through the second scaffold portion, wherein the third array of spaced walls and the fourth array of spaced walls have a second height.

[0029] In one embodiment, the third array of spaced walls and/or the fourth array of spaced walls are substantially permeable to cell transport and/or growth therethrough.

[0030] The inventors have observed that permeability is useful from the perspective of promoting vascularisation.

[0031 ] In another embodiment, the third array of spaced walls and/or the fourth array of spaced walls are substantially impermeable to cell transport and/or growth therethrough, thereby substantially promoting cell growth and orientation that is substantially aligned with the second height of each of the third array of spaced walls and the fourth array of spaced walls.

[0032] In one embodiment, the second plurality of open channels are substantially aligned with the first plurality of open channels. [0033] In one embodiment, the cross-sectional dimensions of the open channels of the second scaffold portion increases from about 100 pm to about 1200 pm through the height of the second scaffold portion, as defined by the second height of each of the third array of spaced walls and the fourth array of spaced walls.

[0034] In some embodiments, the second height of the third array of spaced walls and the second height of the fourth array of spaced walls is between about 500 pm to about 15,000 pm, preferably between about 1 ,000 pm to about 10,000 pm, even more preferably between about 2,000 pm to about 5,000 pm.

[0035] In one embodiment, the first, second, third and fourth arrays of spaced walls are formed from a plurality of stacked biocompatible polymer fibres.

[0036] Preferably, a spacing between adjacent biocompatible polymer fibres in the stack of biocompatible polymer fibres for at least the first, third and fourth arrays of spaced walls is less than a size of a periodontal ligament cell.

[0037] In one embodiment, the biocompatible polymer fibres have a diameter of from about 5 pm to about 40 pm.

[0038] In one embodiment, the biocompatible polymer fibres are produced by melt electrowriting (MEW) or melt electrospinning.

[0039] In one embodiment, the biocompatible polymer fibres are produced by fused deposition modelling or 3D printing.

[0040] In some embodiments, the biocompatible polymer fibres are selected from the group of biodegradable polymer fibres consisting of: polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polydioxanone (PDO), poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), trimethylene carbonate (TMC), polydiols, poly(l-lactide-co-E-caprolactone), and combinations thereof.

[0041] In some embodiments, the biocompatible polymer fibres are selected from the group of non-biodegradable polymer fibres consisting of: polyethylene, polyethylene oxide, polyethylene terephthalate, polyester, polymethylmethacrylate, polyacrylonitrile, silicone, polyurethane, polycarbonate, polyether ketone ketone, polyether ether ketone, polyether imide, polyamide, polystyrene, polyether sulfone, polysulfone, polyvinyl acetate, and combinations thereof.

[0042] According to a second aspect, the present invention provides a method of making a biocompatible implantable scaffold, the method comprising the step of: fabricating a first scaffold portion by laying a plurality of stacked biocompatible polymer fibres to form a first array of spaced walls having a first height, and laying a plurality of stacked biocompatible polymer fibres to provide one or more transverse connectors that connect the first array of spaced walls, wherein the walls of the first array of spaced walls are substantially impermeable to cell transport and/or growth therethrough and are spaced apart from each other by a predetermined distance to promote cell growth and orientation substantially aligned with the first height of the walls of the first array of spaced walls.

[0043] In some embodiments, the walls of the first array of spaced walls are substantially parallel.

[0044] Preferably, the predetermined distance is less than 500 pm.

[0045] In some embodiments, the predetermined distance is between about 60 pm to about 100 pm, preferably between about 60 pm to about 90 pm, even more preferably between about 60 pm to about 80 pm.

[0046] In one embodiment, the one or more transverse connectors connecting the first array of spaced walls defines a second array of spaced walls, wherein the first and second arrays of spaced walls intersect to form a first plurality of open channels through the first scaffold portion.

[0047] In one embodiment, the walls of the second array of spaced walls are substantially impermeable to cell transport and/or growth therethrough, such that the first plurality of open channels through the first scaffold portion substantially preclude cell transport and/or growth in a plurality of transverse directions.

[0048] In one embodiment, the method further comprises the step of: seeding the first scaffold portion with periodontal ligament cells and culturing the cells in a tissue culture medium.

[0049] Preferably, the method further comprises the step of: fabricating a second scaffold portion by laying a plurality of stacked biocompatible polymer fibres to form a third array of spaced walls having a second height, and laying a plurality of stacked biocompatible polymer fibres to form a fourth array of spaced walls having said same second height, wherein the second scaffold portion is connected to the first scaffold portion, and wherein the third array of spaced walls and/or the fourth array of spaced walls are substantially permeable to cell transport and/or growth therethrough.

[0050] Preferably, the method further comprises the step of: fabricating a second scaffold portion by laying a plurality of stacked biocompatible polymer fibres to form a third array of spaced walls having a second height, and laying a plurality of stacked biocompatible polymer fibres to form a fourth array of spaced walls having said same second height, wherein the second scaffold portion is connected to the first scaffold portion, and wherein the third array of spaced walls and/or the fourth array of spaced walls are substantially impermeable to cell transport and/or growth therethrough, thereby substantially promoting cell growth and orientation substantially aligned with the second height of each of the third array of spaced walls and the fourth array of spaced walls.

[0051] In one embodiment, the third array of spaced walls and the fourth array of spaced walls intersect to form a plurality of second of open channels through the second scaffold portion, and wherein the second plurality of open channels are substantially aligned with the first plurality of open channels. [0052] In one embodiment, the cross-sectional dimensions of the plurality of second open channels increases from about 100 pm to about 1200 pm through the height of second scaffold portion, as defined by the second height of each of the third array of spaced walls and the fourth array of spaced walls.

[0053] It will be appreciated by persons of skill in the relevant art that it is not absolutely necessary that the average pore size of the first few layers of the plurality of second open channels is seamless with the average pore size (100 pm) of the plurality of first open channels of the first scaffold portion. Indeed, in other embodiments, the cross-sectional dimensions of the plurality of second open channels may range from about 200 pm to about 1200 pm, or even from about 500 pm to about 1200 pm.

[0054] In one embodiment, a spacing between adjacent biocompatible polymer fibres in the stack of biocompatible polymer fibres for at least the first, third and fourth arrays of spaced walls is less than a size of a periodontal ligament cell.

[0055] In one embodiment, the method further comprises the step of: seeding the second scaffold portion with cells and culturing the cells in a tissue culture medium.

[0056] In one embodiment, the cells are osteogenic cells.

[0057] In one embodiment, the biocompatible polymer fibres have a diameter of from about 5 pm to about 40 pm.

[0058] In one embodiment, the wherein the biocompatible polymer fibres are produced by melt electrowriting (MEW) or melt electrospinning.

[0059] In one embodiment, the biocompatible polymer fibres are produced by fused deposition modelling or 3D printing.

[0060] In some embodiments, the biocompatible polymer fibres are selected from the group of biodegradable polymer fibres consisting of: polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polydioxanone (PDO), Poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), trimethylene carbonate (TMC), polydiols, poly(l-lactide-co-E-caprolactone), and combinations thereof.

[0061] In some embodiments, the biocompatible polymer fibres are selected from the group of non-biodegradable polymer fibres consisting of: polyethylene, polyethylene oxide, polyethylene terephthalate, polyester, polymethylmethacrylate, polyacrylonitrile, silicone, polyurethane, polycarbonate, polyether ketone ketone, polyether ether ketone, polyether imide, polyamide, polystyrene, polyether sulfone, polysulfone, polyvinyl acetate, and combinations thereof.

[0062] According to a third aspect, the present invention provides an implantable scaffold obtained or obtainable by the method according to the second aspect.

[0063] According to a fourth aspect, the present invention provides a method of treating and/or repairing a periodontal defect in a subject in need thereof, the method comprising implanting an implantable scaffold according to the first aspect into the periodontium of said subject.

[0064] According to a fifth aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating and/or repairing a periodontal defect in a subject in need thereof.

[0065] According to a sixth aspect, the present invention provides a method of treating periodontal disease or degeneration in a subject in need thereof, the method comprising implanting an implantable scaffold according to the first aspect into the periodontium of said subject.

[0066] According to a seventh aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating periodontal disease or degeneration in a subject in need thereof.

[0067] According to an eighth aspect, the present invention provides a method of regenerating periodontal tissue in a subject comprising implanting an implantable scaffold according to the first aspect into the periodontium of said subject. [0068] According to a ninth aspect, the present invention provides an implantable scaffold according to the first aspect for use in regenerating periodontal tissue in a subject.

[0069] According to a tenth aspect, the present invention provides a method of treating and/or repairing musculoskeletal disorders and defects involving soft-hard tissue interfaces in a subject in need thereof, the method comprising implanting an implantable scaffold according to the first aspect into a soft-hard tissue interface of said subject.

[0070] According to an eleventh aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating and/or repairing musculoskeletal disorders and defects involving soft-hard tissue interfaces in a subject in need thereof.

[0071] According to a twelfth aspect, the present invention provides a method of treating and/or repairing any disorders and defects requiring soft tissue fibre guidance in a subject in need thereof, the method comprising implanting an implantable scaffold according to the first aspect into the soft tissue of said subject.

[0072] According to a thirteenth aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating and/or repairing any disorders and defects requiring soft tissue fibre guidance in a subject in need thereof.

[0073] According to a fourteenth aspect, the present invention provides a method of treating and/or repairing any disorders and defects requiring soft tissue fibre attachment and/or insertion in a subject in need thereof, the method comprising implanting an implantable scaffold according to the first aspect into the soft tissue of said subject.

[0074] According to a fifteenth aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating and/or repairing any disorders and defects requiring soft tissue fibre attachment and/or insertion in a subject in need thereof. [0075] According to a sixteenth aspect, the present invention provides a method of treating and/or repairing soft-hard tissue insertion comprising implanting an implantable scaffold according to the first aspect into and/or around an insertion previously inserted at a soft-hard tissue interface of said subject.

[0076] According to a seventeenth aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating and/or repairing soft-hard tissue insertion in a subject in need thereof.

[0077] According to an eighteenth aspect, the present invention provides a method of obtaining oblique and/or perpendicular fibre attachment and/or insertion for repairing soft-hard tissue insertion comprising implanting an implantable scaffold according to the first aspect into and/or around an insertion previously inserted at a soft-hard tissue interface of said subject.

[0078] According to a nineteenth aspect, the present invention provides an implantable scaffold according to the first aspect for use in obtaining oblique and/or perpendicular fibre attachment and/or insertion for repairing soft-hard tissue insertion in a subject in need thereof.

[0079] According to a twentieth aspect, the present invention provides a method of treating and/or repairing gingival recession in a subject in need thereof, the method comprising implanting an implantable scaffold according to the first aspect into the gingiva of said subject.

[0080] According to a twenty first aspect, the present invention provides an implantable scaffold according to the first aspect for use in treating and/or repairing gingival recession in a subject in need thereof.

[0081] Other aspects of the invention are also disclosed.

Brief Description of the Drawings

[0082] Notwithstanding any other forms which may fall within the scope of the present invention, preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: [0083] Figure 1 shows an isometric view of a prior art scaffold having an array of spaced apart walls fabricated from a plurality of fibres, having a fibre spacing in the axial direction of approximately 10-40 pm, and with approximately square/rectangular channels (when viewed in cross section) wherein the walls of the channels are transversely spaced at approximately >100 pm intervals;

[0084] Figure 2 shows a cross-sectional side view of the prior art scaffold of Figure 1 showing periodontal ligament cells growing in between and around the fibres of the scaffold walls in a random way;

[0085] Figure 3 shows a schematic overview of the fabrication and scaffold concept, (a): design concept for the biocompatible implantable scaffold according to a preferred embodiment of the present invention, wherein the biocompatible implantable scaffold comprises a first scaffold portion in the form of a periodontal ligament (PDL) compartment and a second scaffold portion in the form of a bone compartment, both fabricated by melt-electrowriting a plurality of biocompatible polymer fibres, thereby realising a biocompatible implantable scaffold, hereinafter referred to as a biphasic implantable scaffold; (b): photograph of the melt-electrowritten (MEW) biphasic implantable scaffold (Scale bar: 0.5 cm);

[0086] Figure 4 shows an isometric view of a first scaffold portion of the biocompatible implantable scaffold according to a preferred embodiment of the present invention, showing a first array of spaced walls (dark grey) having a first height extending substantially in an axial direction and a second array of spaced walls functioning as transverse connectors (light grey) interconnecting the walls of the first array of spaced walls, wherein the first array of spaced walls are substantially impermeable to cell transport and/or growth therethrough, to promote cell growth and orientation substantially aligned with the first height of the walls of the first array of spaced walls;

[0087] Figure 5 shows a cross-sectional side view of the first scaffold portion (referred to hereinafter as a periodontal ligament (PDL) compartment) of Figure 4 showing periodontal ligament cells growing with uniform orientation in substantial alignment with/along the first height of the walls of the first array of spaced walls, as promoted by the substantially impermeable nature of the first array of spaced walls; [0088] Figure 6 shows the morphology of the periodontal ligament (PDL) compartments and corresponding biphasic scaffolds, as seen by scanning electron microscopy (SEM): Figures 6(a)-(c) show the morphology of the PDL compartments (a): 100 pm channel (100CH-2D), (b): 200 pm Box (200BX-2D), (c): salt leached sponge (SP-2D); Figure 6(d) shows a plot depicting the pore size (pm) for the open channels for each of the three PDL compartments; Figure 6(e) shows a plot depicting the diameter (pm) of the polymer fibres used to form the 100CH and 200BX PDL compartments; Figures 6(f)-(h) show the morphology of the corresponding biphasic scaffolds; (f): The 100 pm channel biphasic scaffold (100CH), (g): 200 pm box biphasic scaffold (200BX), and (h): PCL sponge (SP). White arrows indicate the transverse fibres. Brackets indicate the periodontal (PDL) and bone compartments of the melt electrowritten (MEW) biphasic scaffolds;

[0089] Figure 7 shows micro-CT reconstructions of the following biphasic scaffolds: (a) 100CH; (b) 200BX; (c) SP; and (d) shows a porosity quantification (%) of the three biphasic scaffolds (Scale bar: 1 mm);

[0090] Figure 8 shows confocal laser microscopy images of human periodontal ligament (hPDL) cells seeded onto the following PDL compartments. Figures 8(a), (d) and (g): 100 pm channel (100CH-2D) at 3, 7 and 14-days post seeding, Figures 8(b), (e) and (h): 200 pm Box (200BX-2D) at 3, 7 and 14 days post seeding, and Figures 8(c), (f) and (i): salt leached sponge (SP-2D) at 3, 7 and 14 days post seeding. Blue: nuclei, red: actin, green: collagen-l. (Figure 10 displays unmerged channels for nuclei, actin and collagen-l);

[0091] Figure 9 shows cell alignment frequency distribution (%) of human periodontal ligament (hPDL) cells cultured onto the various PDL compartments (100 pm channel (100CH-2D), 200 pm Box (200BX-2D), salt leached sponge (SP-2D)) at 3, 7 and 14 days, measured from confocal laser microscopy images, (a): Nuclei directionality, (b): actin directionality, (c): collagen type I directionality (The 0° and 90° angles represent horizontal and vertical alignment, respectively);

[0092] Figure 10 shows confocal imaging of human periodontal ligament (hPDL) cells seeded on the following biphasic scaffolds at day 14: (a) 100CH; (b) 200BX; (c) SP. Blue: nuclei, red: actin, green: collagen-l. BN-C: bone compartment and PDL-C: periodontal compartment. (Figure 12 displays unmerged channels for nuclei, actin and collagen-l); Brackets indicate the periodontal (PDL) and bone compartments of the melt electrowritten (MEW) biphasic scaffolds;

[0093] Figure 11 shows (a): a timeline and schematic representation of a periodontal ligament attachment model and its assembly; Figures 11 (b)-(d): Dentin block characterisation; (b): polished and EDTA treated dentin block, (c): dentin wrapped cell sheet morphology, as seen by SEM after 4 weeks of in vitro culture (white arrows indicate mineralised nodules), (d): Immunofluorescence staining of cell sheets (red: actin filaments, blue: nuclei, green: CEMP1 ). Figures 11 (e)-(j): Hematoxylin and Eosin (H&E) staining of resin embedded tissue sections of biphasic scaffolds seeded with an osteogenic culture of human periodontal ligament (hPDL) cells; (e) and (h): Fibre-guiding 100CH biphasic scaffold, (f) and (i) 200BX, (g) and (j): Sponge (SP). The white arrows indicate the MEW PCL fibres. Figures 1 1 (h)-(j); (k)-(m) show nuclei alignment frequency distribution (%) cultured onto the various biphasic scaffolds: (k) Fibre-guiding 100CH biphasic scaffold, (I) 200BX, and (m) Sponge (SP), measured relative to the dentin block (0° represents parallel nucleus to the dentin and 90° represent perpendicular alignment). The Dentin block is represented by the symbol: D;

[0094] Figure 12 shows a histology overview of the dentin block/scaffolds 8 weeks post-implantation in rats, with H&E and Piero Sirius Red staining used to demonstrate the fibre-guiding reproducibility of the 100CH group, (a)-(f): Morphology of the dentin/scaffold interface or the various groups (cellularised or non-cellularised) postimplantation. Figures 12(a) and (d): non-cellularised control 100CH, Figures 12(b) and (e): non-cellularised control 200BX, Figures 12(c) and (f): non-cellularised control SP, Figures 12(g) and (j): cellularised 100CH+C, Figures 12(h) and (k): cellularised 200BX+C, Figures 12(i) and (I): cellularised SP+C. Dashed rectangles indicate higher magnification regions of the PDL compartment used in Figures 13 and 14. The Dentin block is represented by the symbol: D (Scale bar = 50 pm);

[0095] Figure 13 shows the histology of the dentin block/scaffolds 8 weeks postimplantation in rats, with H&E staining. Figures 13(a)-(f): Morphology of the dentin/scaffold interface or the various groups (cellularised or non-cellularised) prior to implantation, (a): non-cellularised control 100CH, (b): non-cellularised control 200BX, (c): non-cellularised control SP, (d): cellularised 100CH+C, (e): cellularised 200BX+C, (f): cellularised SP+C. The Dentin block is represented by the symbol: D; Figures 13(g)-(l) quantification of nuclei alignment frequency distribution (%) in reference to the dentin block. Figure 13(m): High magnification images used for demonstrating nuclei alignment categories; Figure 13(n): shows a chart depicting the frequency distribution (%) of the mean nuclei angle (°); and Figure 13(o) shows a chart depicting the frequency distribution (%) of alignment of nuclei with orientation between 30-60° (oblique), 60-90° (perpendicular). Bars represent a statistically significant difference between the groups at p < 0.05 (Scale bar = 50 pm);

[0096] Figure 14 shows the collagen fibre morphology at the interface between the biphasic scaffold and the dentin for the following groups, with Piero Sirius Red staining, (a): non-cellularised control 100CH, (b): non-cellularised control 200BX, (c): non- cellularised control SP; (d): cellularised 100CH+C, (e): cellularised 200BX+C, (f): cellularised SP+C; The Dentin block is represented by the symbol: D; Figures 14(g)- (i) shows plots that depict the quantification of nuclei alignment frequency distribution (%) in reference to the Dentin block. Figure 14(m): High magnification images of the PDL compartments to demonstrate collagen fibre alignment; Figure 14(n): shows a chart depicting the average collagen fibre angle (°); and Figure 14(o): shows a chart depicting the frequency distribution (%) of alignment of collagen fibres in the PDL compartments with orientation between 30-60° (oblique), and 60-90° (perpendicular). Bars represent statistical differences between the groups at p < 0.05 (Scale bar = 50 pm);

[0097] Figure 15 shows the morphology of the cross-section of the various biphasic scaffolds, as seen by scanning electron microscopy (SEM): (a) salt leached sponge (SP), (b) 200 pm box (200BX), (c) 100 pm box (100BX), (d) 100 pm channel (100CH), (e) 80 pm channel (80CH), (f) 60 pm channel (60CH), (g) micro-CT reconstructions of each of (a) to (f); and (h) a porosity quantification (%) of each of (a) to (f) (Scale bar: 1 mm). [BC: bone compartment, PDL-C: periodontal compartment];

[0098] Figure 16 shows PDL micro-architecture structural integrity evaluation, (a) Schematic diagram of the compressive testing methodology, (b) Cellscale testing rig with MEW biphasic scaffold submerged in PBS at 37°C, and (c) post-compression morphology of the various PDL compartments as seen by SEM, indicating the maintenance of the fibre guiding features;

[0099] Figure 17 shows the histology of the dentine slice/scaffolds 8 weeks postimplantation in a subcutaneous rodent model, high and low magnification H&E staining, (a): salt leached sponge (SP), (b) 200 pm boxes (200BX), (c) 100 pm boxes (100BX), (d) salt leached sponge with cells (SP+C), (e) 200 pm boxes with cells (200BX+C), (f) 100 pm boxes with cells (100BX+C), (g) 100 pm channels (100CH), (h) 80 pm channels (80CH), (i) 60 pm channels (60CH), (j) 100 pm channels with cells (100CH+C), (k) 80 pm channels with cells (80CH+C), (I) 60 pm channels with cells (60CH+C). [Voids created by the dissolution of the PCL during histology processing: *, dentine slice: D, (Scale bar: 50 pm)];

[00100] Figure 18 (a)-(d) shows the quantification of nuclei alignment frequency distribution in reference to the dentine block. A-c: functional alignment as measured by the percentage of nuclei with orientation between 0-30° (parallel, non-functional), 30-60° (oblique, functional), 60-90° (perpendicular, highly functional). [D: mean nuclei angle. Bars represent a statistically significant difference between the groups at p < 0.05];

[00101] Figure 19 shows the histology of the dentine slice/scaffolds 8 weeks postimplantation in a subcutaneous rodent model, high and low magnification Picrosirius Red staining, (a) salt leached sponge (SP), (b) 200 pm boxes (200BX), (c) 100 pm boxes (100BX), (d) salt leached sponge with cells (SP+C), (e) 200 pm boxes with cells (200BX+C), (f) 100 pm boxes with cells (100BX+C), (g) 100 pm channels (100CH), (h) 80 pm channels (80CH), (i) 60 pm channels (60CH), (j) 100 pm channels with cells (100CH+C), (k) 80 pm channels with cells (80CH+C), (I) 60 pm channels with cells (60CH+C). [Voids created by the dissolution of the PCL during histology processing: *, dentine slice: D, (Scale bar: 50 pm)];

[00102] Figure 20 (a)-(d) shows the quantification of collagen alignment frequency distribution in reference to the dentine block. A-c: functional alignment as measured by the percentage of collagen with orientation between 0-30° (parallel, non-functional), 30-60° (oblique, functional), 60-90° (perpendicular, highly functional). D: mean collagen angle. [Bars represent a statistically significant difference between the groups at p < 0.05];

[00103] Figure 21 shows 3D reconstruction images via micro-computed tomography, (a) Empty + Emdogain® (EMP + E), (b) sponge + Emdogain® (SP+E), (c) micro-boxes 200 pm + Emdogain® (200BX+E), (d) micro-channels 80 pm + Emdogain® (80CH+E). (e) Percentage of bone fill (bone volume/tissue volume) quantified. [Bars represent statistically significant difference between the groups at p < 0.05];

[00104] Figure 22 shows the tissue histology 4 weeks post-implantation in a rat periodontal defect model. H&E sections are represented at high and low magnification: (a) empty + Emdogain® (EMP+E), (b) sponge + Emdogain® (SP+E), (c) micro-boxes 200 pm + Emdogain® (200BX+E), (d) micro-channels 80 pm + Emdogain® (80CH+E). R1 : first molar, R2: second molar, NB: new bone. [Voids created by the dissolution of the PCL during histology processing: *]; and

[00105] Figure 23 shows Histomorphometry (a) new cementum formation, (b) Oblique PDL attachment, (c) periodontal regeneration (percentage of the tooth root featuring oblique PDL attachment and simultaneous insertion into new bone). [Bars represent a statistically significant difference between the groups at p < 0.05].

Detailed Description of a Preferred Embodiment of the Invention

[00106] It should be noted in the following description that like or the same reference numerals in different embodiments denote the same or similar features.

[00107] The present invention is predicated on the fabrication of a biocompatible implantable scaffold for use in encouraging the attachment of oblique and/or perpendicular periodontal ligament fibres to the root surface of a tooth for the purpose of achieving periodontal regeneration.

[00108] To encourage periodontal ligament attachment in an oblique and/or perpendicular manner, the inventors have recognised that it is not simply a matter of fabricating a biocompatible implantable scaffold seeded with the requisite cells and implanting it into the periodontium of a subject and expecting the periodontal ligament cells to grow to produce ligament fibres with the desired orientation to facilitate oblique and/or perpendicular attachment of the ligament fibres to the root surface of a tooth.

[00109] Rather, as will be described in more detail below, the structural constraints imposed by the geometry of a plurality of open channels in a first scaffold portion of the implantable scaffold, as defined by the first array of spaced walls and plurality of transverse connectors, combine to guide cells to enter the open channels axially and fill the space within each of the open channels toward the corresponding part of the root surface of a tooth enclosed by each of the open channels.

Biphasic Implantable Scaffold

[00110] With the above in mind, the inventors have fabricated a biocompatible biphasic implantable scaffold according to a preferred embodiment of the present invention, in which the biphasic implantable scaffold is configured to promote periodontal ligament cell growth and orientation in substantial alignment with the height of the walls of the biphasic implantable scaffold so that the resulting ligament fibres that are formed, are uniformly oriented and aligned to necessitate oblique and/or perpendicular attachment to the root surface of a tooth.

[00111] An outline of the architecture of the biphasic implantable scaffold is shown in Figure 3(a) and the morphology of the biphasic implantable scaffold is shown in Figure 3(b).

[00112] With reference to Figure 3(a), the biphasic implantable scaffold as fabricated comprises a first scaffold portion and a second scaffold portion, in which the two scaffold portions are seamlessly interconnected by superimposing the second scaffold portion on top of the first scaffold portion. As will be described in more detail below, both scaffold portions comprise a plurality of open channels, such that when the two portions are interconnected, the open channels of each scaffold portion are substantially aligned thereby defining a plurality of open channels that extend substantially through the biphasic implantable scaffold.

[00113] It will be appreciated by persons of skill in the relevant art that it is not absolutely necessary that the cross-sectional dimensions of the plurality of open channels for the two scaffold portions is the same. Indeed, in some embodiments, the cross-sectional dimensions of the plurality of open channels for the second scaffold portion may be larger than those of the first scaffold portion, and may even increase in pore size with increasing height of the second scaffold portion.

[00114] In one particular application, the biphasic implantable scaffold is configured to be implanted into the periodontium of a subject, with the first scaffold portion being positioned adjacent the root surface of a tooth, and the second scaffold portion being positioned adjacent the alveolar bone and/or the gingival tissue of the subject.

[00115] What follows is a detailed description of the first and second scaffold portions.

[00116] Periodontal ligament (PDL) compartment

[00117] As shown in Figure 3, the first scaffold portion, hereinafter referred to as the periodontal ligament (PDL) compartment, is fabricated by laying a plurality of biocompatible polymer fibres by stacking the fibres on top of each other to form a first array of spaced walls having a first height and laying a plurality of biocompatible polymer fibres, again by stacking the fibres, to form a second array of spaced walls that function as transverse connectors that connect the first array of spaced walls.

[00118] The walls of the first array are substantially parallel and are spaced apart from each other by a predetermined distance, which the inventors have identified as being desirable for the purpose of promoting cell growth and subsequent ligament fibre orientation in substantial alignment with the height of the walls of the first array of spaced walls.

[00119] The predetermined distance between the spaced apart walls of the first array is less than 500 pm.

[00120] Specifically, the inventors have observed good results when the predetermined distance between the spaced apart walls of the first array is set between about 60 pm and about 100 pm, preferably between about 60 pm to about 90 pm, even more preferably between about 60 pm to about 80 pm. [00121] As will be described in more detail below, in a preferred embodiment, good results have been achieved with respect to aligned periodontal ligament fibre formation when the walls of the first array are spaced apart by a distance of about 60 pm.

[00122] As indicated in Figure 3(a), the first array of spaced walls are produced by a melt deposition technique, in which polymer fibres are deposited with an orientation of 0°/0° to form stacking layers of aligned polymer fibres.

[00123] Importantly, the polymer fibres are deposited on top of the other in a stacking arrangement in such a way as to preclude the formation of voids between the stacked layers of aligned polymer fibres that are large enough for periodontal ligament cells to pass through. By virtue of the stacking arrangement disclosed herein, the spaced walls of the first array are substantially impermeable to cell transport and/or growth therethrough. In other words, the fibre spacing between the stacked layers of aligned polymer fibres of the first array of spaced walls is less than the diameter of a periodontal ligament cell, to prevent the passage of cells through the wall.

[00124] As a result, the inventors have observed that the combination of the predetermined distance between the spaced walls of the first array, and the impermeable nature of these walls is conducive for substantially promoting cell growth and subsequent ligament fibre orientation in substantial alignment with the height of the walls of the first array of spaced walls.

[00125] In other words, the constraints imposed by the structure of the first array of spaced walls of the PDL compartment typically preclude cell transport and/or growth in one or more transverse directions.

[00126] The walls of the second array of spaced walls interconnecting the walls of the first array, act as transverse connectors to provide mechanical support and stabilisation of the first array of spaced walls, and of the PDL compartment itself.

[00127] The walls of the second array may be spaced apart from each other by a distance of between about 150 pm to about 250 pm, preferably between about 180 pm to about 230 pm, and even more preferably between about 190 pm to about 220 pm. [00128] In a preferred embodiment, and as described in more detail below, the walls of the second array are spaced apart from each other by a distance of about 200 pm.

[00129] The second array of spaced walls are also produced by melt depositing a plurality of polymer fibres, but this time, at an orientation of 90° relative to the orientation of the plurality of aligned polymer fibres forming the walls of the first array of spaced walls.

[00130] In a preferred embodiment, the polymer fibres for the two arrays of spaced walls are deposited in an alternating manner, with two layers of polymers fibres with an orientation of 0°/0° being deposited on top of each other as part of the first array of spaced walls, followed by a single layer of polymer fibres being deposited at an orientation of 90° across the top of the already deposited layers of polymer fibres of the first array as part of the second array.

[00131] As a result of this particular patterning process, the second array of spaced walls intersect the first array of spaced walls at substantially right angles to each other, to form a plurality of open channels extending substantially through the PDL compartment.

[00132] The patterning process is then repeated until a sufficient number of stacked layers of aligned polymer fibres for each array have been deposited to attain a desirable height to define the PDL compartment; this height corresponding to the desired height of the walls of the first array of spaced walls.

[00133] The spaced walls of the first array of the PDL compartment may be fabricated to a height of between about 50 pm to about 200 pm, preferably between about 80 pm to about 180 pm, even more preferably between about 90 pm to about 160 pm.

[00134] In a preferred embodiment, and as described in more detail below, the walls of the second array are spaced apart from each other by a distance of between about 190 pm to about 220 pm. Or by placing the second array of walls at a ratio of 2:1 or greater in relation spacing of the first array of walls, as to create a cell impermeable wall in the first array of walls. [00135] Specifically, Figure 4 shows an isometric view of the PDL compartment produced according to this method, showing a first array of spaced walls (dark grey) having a first height, and a second array of spaced walls functioning as transverse connectors (light grey) interconnecting the walls of the first array of spaced walls.

[00136] The first array of spaced walls shown in this figure have a fibre diameter of about 9-10 pm, with a fibre spacing that is typically less than the size of a periodontal ligament cell. The walls of the first array of spaced walls are spaced apart from each other by a predetermined distance of between about 60 pm and about 100 pm.

[00137] As such, the first array of spaced walls are substantially impermeable to cell transport and/or growth therethrough.

[00138] Indeed, with reference to the cross-sectional side view of the PDL compartment shown in Figure 5, which has been fabricated on the surface of a dentine substrate, after the PDL compartment has been seeded with periodontal ligament cells, the cells are confined by the structural constraints imposed by the substantially impermeable first array of spaced walls to grow in substantial alignment with the height of the walls of the first array of spaced walls, so that the resulting ligament fibres that are formed, are uniformly oriented and aligned to necessitate oblique and/or perpendicular attachment to the root surface of a tooth.

[00139] Unlike the first array of spaced walls, the inventors have observed that the walls of the second array of spaced walls do not need to be substantially impermeable to cell transport and/or growth therethrough in order to promote cell growth and subsequent ligament fibre orientation in substantial alignment with the height of the walls of the first array of spaced walls.

[00140] Thus, the second array of spaced walls play little or no part in guiding the alignment of the PDL fibres, but merely act as transverse connectors to provide mechanical support and stabilisation of the first array of spaced walls, and of the PDL compartment itself.

[00141] That said, in one embodiment, the walls of the second array may be fabricated to preclude the formation of voids between the stacked layers of aligned polymer fibres that are large enough for periodontal ligament cells to pass through. The plurality of open channels formed as result of this arrangement are expected to substantially preclude cell transport and/or growth in a plurality of transverse directions, thereby leaving only the axial direction, as defined by the height of the walls of the first array of spaced walls, for such cell growth and ligament fibre alignment to occur.

[00142] In this regard, irrespective of whether the walls of the second array of spaced walls are configured to allow cell transport and/or growth therethrough, it is the combination of the predetermined distance between the walls of the first array of spaced walls, and the impermeable nature of these said same walls, which is conducive for promoting cell transport and/or growth and subsequent ligament fibre orientation in substantial alignment with the height of the walls of the first array of spaced walls.

[00143] Bone compartment

[00144] As shown in Figure 5, the second scaffold portion of the biphasic implantable scaffold, hereinafter referred to as the bone compartment, has a very similar structure to the PDL compartment, in that it comprises two arrays of intersecting spaced walls, hereinafter referred to as the third and fourth arrays to avoid confusion with the first and second arrays of the PDL compartment.

[00145] The third and fourth arrays of spaced walls each extend substantially from an uppermost portion of the corresponding first and second arrays of spaced walls of the PDL compartment.

[00146] Thus, by virtue of this arrangement, the third and fourth arrays of spaced walls of the bone compartment also intersect each other, preferably at substantially right angles, to form a second plurality of open channels, which are substantially aligned with the corresponding open channels of the PDL compartment.

[00147] The third and fourth arrays of spaced walls for the bone compartment are deposited using the same process as described for the PDL compartment, only this time, around 500 to 600 layers of aligned polymer fibres are deposited in a stacked arrangement to provide the plurality of second open channels having cross-section dimensions that define an average pore size that increases in the range of from about 100 pm to about 1200 pm.

[00148] In other words, the third and fourth arrays of spaced walls of the bone compartment are each fabricated to a second height of between about 500 pm to about 15,000 pm, preferably between about 1000 pm to about 10,000 pm, even more preferably between about 2,000 pm to about 5,000 pm.

[00149] By virtue of the same fabrication method being employed to deposit the polymer fibres for the third and/or fourth arrays of spaced walls, it will be appreciated by a person of skill in the relevant art that the third array and/or the fourth array of spaced walls may be permeable or substantially impermeable to cell transport and/or growth therethrough, depending on the conditions used to deposit the polymer fibres.

[00150] The inventors have observed that permeability is useful from the perspective of promoting vascularisation.

[00151] In a preferred embodiment, however, the polymer fibres used to fabricate the third array and/or the fourth array of spaced walls are deposited under conditions that ensure that the fibre spacing between adjacent biocompatible polymer fibres in the stack of biocompatible polymer fibres for the third and/or fourth arrays of spaced walls is less than a size of a periodontal ligament cell.

[00152] In other words, the fibre spacing render the third array and/or the fourth array of spaced walls substantially impermeable to cell transport and/or growth therethrough thereby substantially promoting cell growth and orientation substantially aligned with the second height of each of the third array of spaced walls and the fourth array of spaced walls.

[00153] Polymer fibres

[00154] It will be appreciated by persons of skill in the relevant art that a range of polymer fibres may be used to fabricate the biocompatible implantable scaffold shown in Figure 3 by a melt deposition technique. Such a deposition technique may include melt electrospinning and melt electrowriting (MEW). [00155] In a preferred embodiment, and as described in more detail below, the polymer fibres are biocompatible polymer fibres produced and deposited by melt electrowriting (MEW).

[00156] Such biocompatible polymer fibres may include biodegradable or non- biodegradable polymer fibres.

[00157] For instance, suitable biodegradable polymer fibres may be selected from the group consisting of: polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polydioxanone (PDO), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), trimethylene carbonate (TMC), polydiols, poly(l-lactide-co-£-caprolactone), and combinations thereof.

[00158] Alternatively, suitable non-biodegradable polymer fibres may be selected from the group consisting of: polyethylene, polyethylene oxide, polyethylene terephthalate, polyester, polymethylmethacrylate, polyacrylonitrile, silicone, polyurethane, polycarbonate, polyether ketone ketone, polyether ether ketone, polyether imide, polyamide, polystyrene, polyether sulfone, polysulfone, polyvinyl acetate, and combinations thereof.

[00159] In some embodiments, the first and second arrays of spaced walls for each of the PDL compartment and the bone compartment may be formed using the same biocompatible polymer fibres (having the same or different diameters), or different biocompatible polymer fibres (having the same or different diameters), as desired.

[00160] In a preferred embodiment, however, the first and second arrays of spaced walls for the PDL compartment and the third and fourth arrays of spaced walls for the bone compartment are formed using polymer fibres produced from polycaprolactone (PCL), with a diameter of from about 5 pm to about 40 pm.

[00161] Seeding

[00162] As will be described in more detail below, the PDL compartment of the biphasic implantable scaffold is seeded with periodontal ligament cells prior to implantation of this biocompatible implantable scaffold into the periodontium, and these cells are then cultured in a tissue culture medium according to standard procedures.

[00163] Summary

[00164] In short, the inventors have developed a micro-scaled polycaprolactone (PCL) melt electrowritten (MEW) biocompatible biphasic implantable scaffold comprising of two integrated compartments: a periodontal ligament (PDL) and a bone compartment. The PDL compartment is comprised of a parallel fibre arrangement forming a plurality of typically 100 pm spaced open channels, while the bone compartment is comprised of a plurality of open channels with an average pore size gradient ranging from about 100 pm to about 1200 pm, where the open channels for the two compartments are substantially aligned.

RESULTS

[00165] Scaffold Morphology

[00166] The present study investigated the efficacy of cellular and tissue guidance of a biphasic implantable scaffold fabricated using melt electrowriting, consisting of a periodontal ligament (PDL) compartment composed of a first plurality of 100 pm open channels (100CH) and a bone compartment (Figure 3) having a second plurality of open channels with an average pore size gradient ranged from about 100 pm to about 1200 pm.

[00167] The biphasic implantable scaffold was compared to a non-fibre-guiding 200 pm box configuration manufactured by melt electrowriting (hereinafter referred to as “200BX”) and to an amorphous sponge (SP) fabricated by porogen leaching as negative controls. The three scaffolds (100CH, 200BX and SP) were each manufactured using medical grade polycaprolactone (PCL).

[00168] The morphologies of the three different scaffolds (100CH, 200BX and SP) and their associated periodontal ligament (PDL) compartments are shown in Figure 6. The PDL compartment of the fibre-guiding scaffold (100CH) was composed of 100 mm channels created by the stacking of melt electrowritten (MEW) PCL fibres (Figure 6a). This demonstrated that the selected printing parameters enabled accurate and reproducible deposition of highly homogenous channels. The incorporation of the transverse 200 pm spaced PCL fibres enabled the maintenance of the structural integrity of the channels upon handling (Figure 6a) and did not interfere with the stacking of the PCL fibres. The PDL compartment for the 200BX scaffold consists of a 0-90° layering pattern with a 200 pm fibre interspacing, as shown in Figure 6b.

[00169] The PCL sponge (SP) displayed the typical morphology of a porogen leached scaffold, featuring an irregular pore shape with limited pore interconnection (Figure 6c). As a measure of manufacturing accuracy and reproducibility, the width of the open channels (100CH-2D) and the pore size of the 200BX-2D were measured. This revealed that the open channels had an average width of 87.5 ± 2.4 pm, exemplifying the high precision and reproducibility of the deposition. The 200BX-2D has a plurality of open channels with a pore size of 208.1 ± 14.0 pm, also indicating an effective reproducibility. The SP-2D has channels with an average pore size of 398.8 ± 159.4 pm and was characterized by large variation originating from the porogen particle size distribution (Figure 6d). Although there was a statistically significant difference in the fibre diameters between 100CH (8.7 ± 0.4 pm) and 200BX (9.2 ± 0.5 pm), this change did not greatly impact the microstructural architecture of the biphasic implantable scaffolds.

[00170] Figures 6 f-i displays the morphology of the scaffolds for periodontal regeneration, including the 100 pm channel fibre-guiding biphasic scaffold (100CH), the 200 pm box biphasic scaffold (200BX) and the PCL salt leached sponge (SP). The bone compartment in the 100CH and 200BX biphasic scaffolds, which consists of a plurality of second open channels having an average pore size gradient ranging from 100 pm to 1200 pm, was electrowritten over the respective PDL compartments. Figures 6f and h show the first pores of 200 and 400 pm positioned over the periodontal compartment. This resulted in the creation of a fully porous interface between the bone and the periodontal compartments, which is an essential feature for enabling periodontal ligament attachment to the newly formed bone. In contrast to the highly organised architecture of the 100CH and 200BX biphasic scaffolds, the SP exhibited a randomised internal porous structure (Figure 6). [00171] Micro-CT data confirmed the high porosity of the biphasic implantable scaffolds regardless of the fabrication technique. The 100CH and 200BX MEW biphasic scaffolds had the highest porosities of 91.7 ± 0.1 % and 89.1 ± 0.1 % respectively and no statistical difference was observed between these groups. The SP group was slightly less porous with 84.8 ± 0.1 % porosity, which was significantly lower only when compared to the 100CH group (Figure 7).

[00172] 2D and 3D in vitro fibre-guiding efficacy

[00173] The in vitro study assessed the performance of the various PDL compartments (100CH-2D, 200BX-2D and SP-2D) for inducing cellular alignment in 2D of human periodontal ligament (hPDL) cells, using immunofluorescence at 3, 7 and 14-days. Further to this, cellular alignment and organisation were also investigated in 3D for the biphasic implantable scaffolds (100CH and 200BX) and the sponge (SP).

[00174] Cells attached to all three periodontal compartments and deposited ECM over the 14-day culture period, as shown in Figure 8. The 100CH-2D group demonstrated a high capacity to align the cells and ECM (more specifically collagen type I) along the main direction of the channels. Cell nuclei and collagen type-l alignment was already observed at day 3 and were maintained throughout the culture period.

[00175] The 200BX-2D scaffold also displayed some level of nuclei and collagen alignment at the early time points (day 3 and 7 cells), although this was restricted to the direct vicinity of the MEW fibres, as a result of their contact guidance (Figures 8b and e). However, the cells at the centre of the pores were randomly oriented. At day 14, the cells had grown over the 200BX-2D and formed a cell sheet, which resulted in the orientation of the cell and ECM in a direction unrelated to the 200BX-2D architectural features (Figure 8i) (Figure 10 displays unmerged fluorescence channels). The SP group demonstrated a completely random cellular organisation at all time points (Figures 8c, f and j).

[00176] Cellular orientation was further quantified by assessing the nuclei, actin and collagen type I directionality. Nuclei directionality demonstrated that the 100CH-2D resulted in alignment of the cell nuclei along the channels as early as day 3 and that the alignment was further enhanced at 7 and 14 days, as shown by the increased frequency of the angles between 60-90° (Figure 9a).

[00177] Interestingly, the 200BX-2D displayed some level of nuclei alignment, albeit to a lesser extent than the 100CH-2D, as demonstrated by the small peak observed for the 90° angle at days 3 and 14, confirming the contact guidance of the electrowritten fibres. In contrast, the SP-2D displayed an even distribution of the nuclei angles, indicating a random orientation of the cells at all time points.

[00178] A similar trend was observed when actin or collagen fibres were analysed, with the highest alignment seen for the 100CH-2D, which was maintained up to day 14. 200BX-2D only displayed a modest impact on actin and collagen type 1 alignment. The SP-2D showed a random distribution. However, the 200BX-2D presented an increased actin alignment at day 14 when compared to the previous time points. This may have resulted from the proliferation of the cells over the PDL compartment once the pores were filled leading to the formation of a cell sheet which adopted a certain degree of alignment. While this was well reflected in the actin alignment, the nuclei and collagen fibres remained randomly distributed.

[00179] Confocal laser microscopy imaging conducted at 14 days on the entire biphasic constructs demonstrated an architecturally induced guidance of the hPDL cells in both the bone and PDL compartments (Figure 10), whereas the sponge did not display any specific cell arrangement. In addition, cell infiltration was also limited in the sponge, and this was in contrast to the MEW constructs (100CH and 200BX), which were fully infiltrated at 14 days.

[00180] In both of the biphasic scaffolds, the cells were seeded over the bone compartment and spontaneously aligned along the direction of the MEW fibres, creating a convolutional downwards spiral arrangement, cascading from the bone to the PDL compartment. It was further confirmed that an identical effect was still observed when initially seeding the cells on the periodontal compartment and culturing the construct with the bone compartment facing down (Figure 11). This demonstrated that the cellular guidance seen in the bone compartment was not just from the influence of gravity. [00181] Interestingly, the 100CH and 200BX scaffolds revealed a homogeneous infiltration of cells within both the bone and the PDL compartments, confirming that a porous interface between the compartments enabled abundant cellular communication and integration. In addition, the cells in the periodontal compartment of the 100CH were obliquely and/or perpendicularly aligned to the channels (Figure 10a), whereas the cells and their ECM adopted a parallel orientation in the 200BX (Figure 10b). The SP resulted in predominantly orientating the cells and ECM in a parallel manner although some regions of oblique alignment were observed.

[00182] 3D In vitro periodontal ligament attachment model

[00183] The inventors further investigated the fibre-guiding and periodontal ligament attachment capacity of the different constructs in a 3D setting using a 3D In vitro periodontal attachment model. In this model, cementogenesis was simulated by the placement of mineralised hPDL cell sheets over the dentin block, which was subsequently placed in contact with the various scaffolds (100CH, 200BX, SP) previously cultured with hPDL cells (Figure 11a) and hence represented the newly forming periodontal ligament.

[00184] Cell sheet/dentin characterisation

[00185] Surface morphology via SEM validated the removal of the cementum layer via the cutting and polishing technique (Figure 11 b). In addition, the treatment of EDTA resulted in the successful exposure of the dentin tubules.

[00186] SEM imaging demonstrated the cell sheet formed a dense homogenously distributed layer over the dentin block and displayed evidence of mineralisation as indicated by the presence of numerous mineralised nodules embedded throughout the ECM, ranging between 1 to 7 pm. CEMP1 immunostaining confirmed that the protein was expressed after 4 weeks of culture in osteogenic media. The cell sheets displayed CEMP-1 clusters resembling the morphological appearance of the mineralised nodules observed by SEM. This was confirmed to be clusters of calcium phosphate via energy-dispersive X-ray spectroscopy (Figure 13). [00187] Fibre-guiding capacity of PCL biphasic scaffolds in a 3D in vitro periodontal model

[00188] The assembled construct was further cultured for 1 week, and then resin embedded to investigate 3D in vitro periodontal ligament attachment and cellular orientation. The H&E-stained sections demonstrated that in all groups there was some level of attachment to the dentin, although better integration was observed for the 100CH and 200BX in comparison to the SP group. Interestingly, the architecture of the channels in the 100CH specimens was maintained, despite the slight compression caused by suturing during assembly. Nevertheless, the various scaffold configurations displayed distinct differences when cellular and ECM orientations were considered (Figures 11 b-g). Indeed, the 100CH architecture resulted in oblique and/or perpendicular alignments of the cells to the dentin block, whereas the cells were parallelly orientated for the two other groups (200BX and SP).

[00189] This was further confirmed by the quantification of the nuclei orientation in relation to concave-shaped dentin. Indeed, nuclei alignment followed a bimodal distribution for the 100CH, with the majority of the cells forming a 40-70 ° angle, while about 16% of the cells were perpendicularly aligned (Figure 11 h). While the 200BX also resulted in perpendicular alignment of nuclei due to the presence of the staked melt electrowritten fibres, this feature was limited to only 7% of the cells (Figure 11 i). Most of the nuclei formed angles in the range of 0 to 40°, indicating that the cells predominantly adopted an orientation parallel to the dentin.

[00190] A similar pattern was observed for the SP group with the great majority of the cell nuclei forming angles in the range of 0-40°, hence parallel to the dentin (Figure 11j).

[00191] In summary, the 3D In vitro model confirmed the capacity of the 100CH scaffold to obliquely and/or perpendicularly orientate cells to a dentin surface, hence mimicking the 3D attachment of the native periodontal tissue on the root surface. [00192] In vivo study

[00193] To determine the fibre-guiding capacity of the MEW biphasic scaffolds, the constructs were subcutaneously implanted using an established ectopic periodontal regeneration model in immunocompromised rats. In order to recapitulate the biological events leading to periodontal regeneration, including cementogenesis and periodontal ligament attachment, three mineralised cell sheets mimicking newly formed cementum were placed on the periodontal compartment and further incubated overnight to allow attachment. Prior to implantation, the scaffolds were placed onto the dentin block and secured using sutures similarly to the 3D In vitro periodontal model (Figure 11a). The post-operative course was uneventful, and sample retrieval was performed at 8 weeks post-implantation. All scaffolds were infiltrated with tissue and were well integrated with the surrounding host subcutaneous tissues.

[00194] Hematoxylin and Eosin (H&E) and Piero Sirius Red staining showed cell infiltration and collagen deposition in all PDL regions after 8 weeks (Figure 12). The MEW scaffolds demonstrated high levels of cell infiltration and tissue integration between the bone and PDL compartments (Figures 13a, b, d, e).

[00195] Similar to the 3D In vitro data, periodontal-like tissue attachment to the dentin was observed for all groups regardless of the scaffold configuration or the presence of cells prior to implantation. Indeed, cell sheet placement had no notable bearing on tissue alignment and/or attachment, however a differential tissue alignment was observed in the scaffold periodontal compartments.

[00196] The 100CH and 100CH + C scaffolds demonstrated cellular and tissue orientation in an oblique and/or perpendicular manner over the entire dentin surface (Figure 12 and Figures 13a and d), whereas this feature was present to a lesser extent in the 200BX groups. As expected, the SP groups did not display any specific cell or tissue alignment.

[00197] The quantification of the nuclei alignment within the periodontal ligament (PDL) cell compartments of the various scaffold configurations confirmed these findings and demonstrated that the 100CH groups had the highest percentage of nuclei perpendicularly orientated to the dentin when compared to all other groups. [00198] In addition, the representation of nuclei alignment frequency resulted in the formation of a tri-modal bell-shaped distribution centred around 90° (with a sharp peak) and centred at 40° and 130° (with a flattened bell curve) as shown in Figures 13g and j for the 100CH groups. In contrast, the 200BX and SP groups displayed concave shaped distribution curves with a high percentage of nuclei forming at 10° with the dentin (hence parallel orientated) while only a low percentage of nuclei were perpendicularly aligned (Figures 13h, k, i and I).

[00199] Figure 14m displays higher magnification image utilised for determining morphological mimicry to the native periodontal ligament.

[00200] The 100CH groups, with or without cells, had a mean nuclei angle of 46 ± 0.7° and 48 ± 2.3° respectively, and both of these groups were significantly higher compared to all other groups (200BX = 22 ± 2.8°, 200BX + C= 27 ± 2.5°, SP= 18 ± 1 .9°, SP + C= 18 ± 1 .4°). Here again, there was no difference with the cellularised and non-cellularised groups indicating that nuclei alignment was mainly dependent on micro-architecture rather than on the presence of cells (Figure 13n).

[00201] The guiding potential of the various configurations was further analysed by assessing based on the morphological alignment observed in the native periodontal ligament. Hence, three categories were selected; parallel orientation with angles ranging from 0-30° (as shown in Figure 14), oblique 30-60°, perpendicular 60-90° (Figure 13m & o).

[00202] Consistent with the previous data, the 100CH and 100CH + C scaffolds demonstrated the highest level of alignment (60-90°) with over 30% of cells displaying a highly aligned, perpendicular-like orientation and this was significantly increased compared to the other groups. This trend was also again observed for oblique angles (30-60°) in the 100CH groups compared to the other groups (with the exception of the comparison between 100CH+C and 200BX, which did not reach statistical significance).

[00203] The 100CH biphasic scaffold also displayed the lowest number of parallel nuclei, thus demonstrating a superior capacity for cellular guidance when compared to the other groups. On the other hand, the 200BX and SP configurations, regardless of the presence of cells, displayed the highest frequency of nuclei with a parallel orientation to the dentin. Overall, the 100CH architecture appeared to topographically favour oblique and/or perpendicular tissue orientation to the dentin.

[00204] Collagen fibres were densely deposited throughout all PDL compartments, irrespective of the architecture of the scaffolds or the presence of cells prior to implantation (Figures 14a-f).

[00205] Fibres at close proximities to the PCL material experienced a higher degree of fibre guidance, as previously observed with the nuclei alignment quantification. This contact guidance was particularly evident for the 200BX configuration, whereby the collagen fibres near the PCL material were mostly obliquely aligned, whereas those in the centre of the box were mainly parallel to the dentin (Figures 14b & e).

[00206] The analysis revealed minor alignment distribution differences for collagen fibres between the 100CH and the 100CH + C groups, as both displayed convex profiles associated with more perpendicular fibre orientations (Figures 14g & j).

[00207] In contrast, the 200BX and SP compartments, irrespective of prior cell seeding, resulted in concave distribution profiles with high frequencies at parallel orientations, and consequently low frequencies at perpendicular orientations (Figures 14h, i, k, I).

[00208] High magnification images enabled the categorisation of the collagen fibre angle. This demonstrated that mean fibre angles were significantly higher for scaffolds with 100 pm channel architecture (54.35° with cells and 53.35° without cells), when compared to all other groups (Figure 14n). While significant differences between the 200BX and 200BX + C groups were found, all other groups displayed similar mean fibre angles regardless of cell seeding.

[00209] The degree of collagen alignment was further analysed into different categories based on specific ranges, parallel (0-30°) (parallel; as shown in Figure 14), oblique (30-60°) and perpendicular (60-90°) (Figure 14m). This further demonstrated the superior fibre-guiding capacity of the 100CH architecture. These groups (100CH and 100CH + C) possessed the least number of parallel fibres and the highest percentage of oblique and/or perpendicular fibres, and this was significantly different to all other groups (Figure 14o).

[00210] In addition, the 100CH groups, regardless of cell seeding, displayed approximately half the amount of parallel collagen fibres, and almost three times more perpendicular fibres in comparison to the other groups. Once again, hPDL cell seeding appeared to have little influence on alignment across the groups. The morphologies of a range of different sized scaffolds (SP, 200BX, 100BX, 100CH, 80CH and 60CH) and their associated bone compartment (BC) and periodontal ligament compartment (PDL-C) are shown in Figures 15a-f.

[00211] A close inspection of the morphological observations of the various PDL compartments demonstrated that the printing parameters resulted in the manufacturing of highly homogenous micro-structures, ranging from 200 - 60 pm spacing (Figures 15a-f). The SP-2D displayed a typical porogen leached internal pore composition consisting of irregular voids with variable interconnectivity. The 200BX possessed a very consistent 0-90° layering pattern with a 200 pm pores size. The micro-channel groups and 100BX arrangements also displayed organised morphologies throughout the entire 18-layer stacking. The transverse fibre at 200 pm spacing did not impede the fibre stacking or organisation for the micro-channel or micro-box groups. The fusion between the layers appeared consistent and uniform for all MEW configurations, regardless of the pore spacing or arrangement.

[00212] Quantification of pore size demonstrated high control over the fibre placement with little deviation from the theoretical fibre inter-distances. (200BX (195 ± 0.3 pm), 100BX (101 ± 1 pm) 100CH (97 ± 0.1 pm), 80CH (79 ± 0.5 pm) and 60CH (62 ± 2.4 pm). As expected, the SP (259 ± 1 10 pm) displayed the greatest variation in pore size compared to all groups. Fibre diameter remained stable during printing of the various PDL compartments averaged around 8 pm (200BX = 8.1 ± 0.2 pm, 100BX = 8.3 ± 0.5 pm, 100CH = 8.3 ± 0.4 pm, 80CH = 8.6 ± 0.2 pm and 60CH = 6.9 ± 0.4 pm).

[00213] The bone compartment consisting of a pore size gradient from 200 pm to 1200 pm, was accurately superimposed to the PDL compartments (Figures 15b-f). This established an interconnected interface within the MEW biphasic design with a graded structure, ideal for enabling tissue integration between the bone and PDL compartments.

[00214] Regardless of fabrication method, the biphasic scaffolds exhibited high porosity as confirmed via micro-CT. (Figure 15g). No statistical difference was observed between the MEW fabricated scaffolds, with all exhibiting around 97% porosity (200BX-2D = 97 ± 0.5%, 100BX-2D = 97 ± 0.9 %, 100CH-2D = 97 ± 0.4 %, 80CH-2D = 97 ± 0% and 60CH-2D = 97 ± 0% pm). The lowest porosity, albeit not significantly different was found for the SP (93 ± 2.4 %) (Figure 15h).

[00215] Micro-channel Stability after mechanical compression

[00216] The methodology for compression testing is displayed in Figure 16A-B. After 50% compression, the structural integrity of the scaffold’s PDL compartments were observed under high magnification SEM (Figure 16c). The SEM images revealed that the various PDL micro-architectures displayed no signs of damage or collapse. In addition, subsequent to compression, all constructs regained their original shape.

[00217] Ectopic periodontal attachment model - Histology

[00218] The histology of the dentine slice/scaffolds 8 weeks post-implantation in a subcutaneous rodent model demonstrated that all groups were infiltrated with cells and tissue and all groups presented tissue attachment onto the dentine interface regardless of the presence of cells in the scaffolds, as shown in Figure 17. The biphasic scaffolds with a 100 pm or less inter-fibre spacing induced consistent oblique and perpendicular alignment of the cell nuclei and ECM (Figure 17c-l) with the greatest influence on cellular guidance occurring at close proximities to the MEW fibres. The porous interface between the PDL and bone compartment resulted in excellent tissue integration of these compartments, an essential attribute for multiphasic tissue regeneration (Figure 17b-l).

[00219] The guiding capacity was further quantified by measuring nuclei directionality (Figure 18a-d) and three alignment categories were created: parallel 0-30 ° (nonfunctional), oblique 30-60° (functional), perpendicular 60-90° (highly functional). Approximately 50% of cells measured within the PDL compartments of the SP (56 ± 7.1 %), SP+C (48 ± 7.5%), 200BX (53 ± 15.9%) and 200BX+C (63 ± 7.2%) were parallelly aligned to the dentine slice. In contrast, the fibre-guiding biphasic scaffolds displayed only a low percentage of cells parallel to the dentine (100CH: 12 ± 2.2%, 100CH+C: 13 ± 4.7%, 80CH: 7 ± 2.6%, 80CH+C: 9 ± 3.2%, 60CH: 7 ± 1.1 %, 60CH+C: 9 ± 1.9%), (Figure 18a) regardless of hPDL seeding. Oblique alignment was not substantially different across all groups, and as unrelated to the presence or absence of cells previously seeded in the scaffold (Figure 18b). However, perpendicular alignment was significantly improved by the fibre-guiding micro-architectures with over 50% of the cells adopting this orientation (100CH: 62 ± 2.8%, 100CH + C: 56 ± 6.4% , 80CH: 65 ± 3.2%, 80CH+C: 64 ± 4.9%, 60CH: 65 ± 4.8%, 60CH + C: 65 ± 4%) which was in contrast to the other configurations (SP: 13 ± 1 %, SP+C: 14 ± 6.1 %, 200BX: 17 ± 9.4%, 200BX+C: 8 ± 4.7%, 100BX: 34 ± 6.2%, 100BX+C: 37 ± 3) (Figure 18c). The effect of channel width had no significant effect on the global nuclei alignment and all channel groups from 60 to 100 pm performed very similarly (Figure 18d). Nevertheless, the qualitative evaluation indicates that the degree of alignment in the centre of the channels seemed to increase when the channel width was below 80 pm. Interestingly, the regular closure of the 100 pm channel in the 100BX group resulted in a decreased fibre-guiding capacity when compared to the corresponding 100CH group. However, the 100BX still presented some level of enhanced guidance since this group significantly outperformed the 200BX for perpendicular alignment. The quantification of the mean nuclei angle (Figure 18d) demonstrated that the channel configuration performed in a similar manner with a mean angle around 60°, which was significantly higher to the SP and 200BX groups with or without cells that ranged between 26-33°. The channels configuration also outperformed the 100BX group (with the exception of the 100CH+C). The 100BX had a mean angle of around 45° and was significantly higher than the SP and 200BX groups.

[00220] In terms of the histology of the dentine slice/scaffolds 8 weeks postimplantation in a subcutaneous rodent model, the collagen fibres (stained by Picrosirius Red) densely populated the PDL compartments of all the groups as shown in Figure 19. The fibre alignment and organisation were dependent on the presence of the fibre-guiding features similarly to the nuclei alignment. The control groups SP, SP+C, 200BX and 200BX+C, although attached to the dentine, displayed high levels of parallel collagen fibres (Figure 19a-b). In contrast, the fibre-guiding groups with 100 pm or less spacing demonstrated a reproducible and consistent oblique and perpendicular collagen alignment to the dentine (Figure 19c-l). [00221] The highest amount of parallel collagen alignment was observed in the control groups SP (57 ± 19.1 %), SP+C (71 ± 16.5%), 200BX (64 ± 7.3%) and 200BX+C (65 ± 6.7%), and this was statistically significant when compared to all fibre guiding configurations. The next best performance was by the 100BX (42 ± 6.5%) and 100BX + C (43 ± 1.1 %) groups with around 40% parallel fibres, reaching significance to all groups besides 200BX and 200BX+C. The channel configurations displayed a significantly lower number of parallel fibres of around 20 to 30% (100CH: 20 ± 3.6%, 100CH+C: 29 ± 7.2%, 80CH: 28 ± 6.3%, 80CH+C: 24 ± 4.8%, 60CH: 23 ± 3.7%, 60CH+C: 24 ± 5%) (Figure 20a). In terms of oblique (30-60°) alignment frequency, there was a slight increase for all MEW biphasic groups with a 100 pm or less spacing, although it did not reach statistical significance in comparison to the other groups (Figure 20b). The frequency of collagen fibres perpendicularly (60-90°) aligned towards the dentine was significantly higher in the channels regardless of width or presence of cells (100CH: 51 ± 4.3%, 100CH+C: 41 ± 8.3% , 80CH: 43 ± 8.1 %, 80CH+C: 47 ± 4.5%, 60CH: 49 ± 2.1%, 60CH+C: 45 ± 8.7%) (Figure 20c) when compared to the other groups (100BX (28 ± 6.1 %), 100BX+C (27 ± 2.8%), SP (25 ± 18.3%), SP+C (12 ± 7.4%), 200BX (14 ± 2.7%) and 200BX+C (16 ± 6.5%)). Mean collagen angle (Figure 20d), further validated the fibre-guiding effect of the microchannel configurations as these groups (100CH: 64 ± 2.5%, 100CH+C: 59 ± 6.7%, 80CH: 58 ± 4.4%, 80CH+C: 62 ± 3.7%, 60CH: 64 ± 1.6%, 60CH+C: 62 ± 4.3%), performed similarly and had a mean collagen angle significantly higher when compared to the control SP and 200BX ((SP: 41 ± 14.2%) , SP+C (31 ± 10.3%), 200BX (36 ± 4.2%) and 200BX+C (36 ± 4.7%)). The mean collagen angle for 100BX and 100BX+C was less than all channel groups, although it did not reach significance.

[00222] Overall, the micro-channels were found to have the least parallel alignment and had a vastly significant effect on perpendicular alignment.

[00223] Periodontal defect model

[00224] The regenerative potential of the fibre guiding scaffold was further assessed in a surgically created periodontal defect in rat. The animal model involving bilaterally surgically created periodontal defects. The following periodontal defect model received Animal ethics approval from the Animal Ethics Committee of The University of Queensland (Ethics approval number: 408/19). Eight groups (n=5) were implanted 1 ) Empty (EMP), 2) Empty + Emdogain® (EMP+E), 3) Sponge (SP), 4) Sponge + Emdogain® (SP+E), 5) micro-boxes 200 gm (200BX), 6) micro-boxes 200 gm + Emdogain® (200BX+E), 7) micro-channels 80 gm (80CH) and 8) micro-channels 80 gm + Emdogain® (80CH+E). The four groups without Emdogain were part of a study outlined in Chapter 5 and are utilised for comparative purposes. Power analysis was conducted using EPITOOLS software (with the following parameters based on past literature: SD: 12.5, confidence level: 0.95 and acceptable error: 15) to determine the minimum number of repeats for this type of model to be three. Therefore, ten (five male and five female), twelve-week-old Sprague Dawley rats (Animal Resources Centre, Canning Vale, WA, Australia) were used.

[00225] The animals were anaesthetized with Isoflurane at 3.5% via inhalation. Preemptive multi-modal analgesia (buprenorphine 0.01 -0.05 mg/kg, meloxicam 1 mg/kg) and prophylaxis (Kefzol (20mg/kg) and Gentamicin (5mg/kg)) was provided by subcutaneous administration. The surgical site was clipped and disinfected using Povidone-iodine, 10% antiseptic solution. A scalpel blade was used to create a skin incision along the inferior border of the mandible. Thereafter, the masseter muscle and periosteum were elevated as a flap to reveal the buccal surface. Round dental burrs in combination with saline irrigation were used to remove the alveolar bone and cementum tissues covering the roots of the first molars. The defect size was approximately 3 x 1 .5 mm ² . Before the placement of the scaffold, the defect area was washed with saline. The MEW scaffolds were orientated with the PDL compartment facing the tooth root and collagen membrane (Bio-Gide, Geistlich, Switzerland) was utilised to cover the defect consistently with guided tissue regeneration protocol. The wound was closed by suturing the masseter muscle using Vicryl 5/0 and then skin using Vicryl 4/0 to allow healing by primary intention. Four weeks post-surgery the rats were sacrificed, and the mandibles were harvested and fixed in 4% paraformaldehyde for 24hrs at 4°C and then washed in PBS.

[00226] After defect exposure, the site was irrigated with saline and Pregel® (Straumann, Basel, Switzerland) was applied to the tooth root for 2 min. The defect was again irrigated with saline, followed by the application of 10 pL of Emdogain® (Straumann, Basel, Switzerland) evenly distributed over root surfaces. The MEW scaffold was gently positioned against the root with the PDL compartment facing downwards. 4 weeks post-surgery the rats were sacrificed, and the mandibles were harvested and fixed in 4% paraformaldehyde for 24hrs at 4°C and washed in PBS. [00227] Bone Volume

[00228] From the 3D reconstruction images via micro-computed tomography shown in Figure 21 , 4 weeks post-implantation new bone formation was detected in all the groups via Micro-computed tomography (micro-CT) (Figure 21). Micro-CT segmentation demonstrated that no ankylosis was present across any of the specimens.

[00229] Quantification revealed that overall, the groups performed equally with bone fill ranging from 40 to 70% and there was no remarkable effect of Emdogain on bone formation. The only statistical difference observed was between the EMP+E and SP+E groups. The quantification of the defect bone fill was also characterised by large variation between specimens across the groups with the exception of the 80CH group with or without Emdogain® which presented a narrower distribution.

[00230] Histology and Histomorphometry

[00231] From the tissue histology 4 weeks post-implantation in a rat periodontal defect model shown in Figure 22, the histological observations demonstrated that the EMP control group displayed full tissue colonisation and the presence of newly formed bone was noted in all specimens (Figure 22a). In addition, all the PCL scaffolds showed bone formation and were well integrated with the host tissues and presented the characteristic voids associated with the dissolution of the PCL materials during the histology processing voids (Figure 22c-d Black asterisk). The histology also demonstrated that melt electrowritten scaffolds (200BX and 80CH) provided sufficient space maintenance and preserved their structural integrity during the 4 weeks implantation. This enabled excellent tissue colonisation throughout the bone and the periodontal compartment. In comparison the SP+E displayed larger voids with a more random tissue organisation (Figure 22b). The implanted scaffolds maintained direct contact with the root surface with PDL attachment and no ankylosis. Newly formed bone and cementum deposition were observed in all groups. While all the groups revealed some areas of oblique PDL attachment, a consistent and reproducible effect was present in the 80CH and 80CH+E groups and to a lesser extent although still present in the 200BX and the 200BX+E group. The EMP+E and SP+E groups displayed new bone and cementum formation however it was mostly accompanied by an inconsistent PDL orientation. [00232] The quantitative assessment revealed that the groups with Emdogain resulted in significantly higher cementum formation when compared to their corresponding groups without Emdogain®, which did not present with obvious new cementum formation. The application of Emdogain® in this study showed minor differences and high variability across the various groups. Non-significant differences were found between the EMP+E (56.2% ± 27), 200BX+E (56.2% ± 27) and the 80CH+E (56.2% ± 27) groups (Figure 23b). The 80CH+E group however did display significantly more cementum coverage than the SP+E group (SP+E; 40% ± 35).

[00233] The highest amounts of oblique PDL attachment were observed for the 80CH and 80CH+E groups, around 70% ± 21 and 85% ± 9 respectively (Figure 23a) which was significantly different compared to EMP (30% ± 18), EMP+E (23% ± 29), SP+E (22% ± 19). Although not reaching statistical significance, the application of Emdogain® increased the average oblique attachment for both MEW scaffolds, 80CH and 200BX.

[00234] Periodontal regeneration was identified as areas of oblique PDL fibre attachment onto the root surface and simultaneous insertion into newly formed bone. The 80CH+E group elicited the highest periodontal regeneration with around 85% ± 9, which was significant to all groups (EMP; 17.5% ± 17, EMP+E; 27.4% ± 38, SP; 29.4 % ± 14, SP+E: 2.8% ± 5), at the exception of 200BX+E (40.7% ± 29) and 80CH (58.3% ± 29) (Figure 23c).

[00235] DISCUSSION

[00236] This study described the development of a highly porous biodegradable scaffold with highly controlled and reproducible fibre-guiding capacity fabricated by melt electrowriting. The fibre-guiding concept relied on the capacity of microchannels to influence the alignment of the soft tissue via these topographical cues during periodontal attachment formation.

[00237] While channel-like or groove architecture has been shown to induce alignment in 2D in vitro culture, the systematic and perpendicular orientation of cells and tissues to a mineralized surface has not yet been reported. This present study demonstrated cell and collagen alignment along the direction of the channel when evaluated in 2D, in line with previous reports. [00238] More importantly, however, a systematic and perpendicular orientation to the dentin block was obtained when the fibre guidance was assessed in a 3D in vitro periodontal attachment model. This was achieved by the migration and proliferation of the cells through the walls of channels in a perpendicular manner as opposed to along the direction of the channels parallel to the dentin. Indeed, previous research used a statically fabricated melt electrospun scaffold which featured concentrically arranged rings, inducing tissue alignment in an unsystematic manner.

[00239] Perpendicular topographical guidance had also been previously explored using soft lithography, via the creation of pillars possessing perpendicularly orientated micro-grooves. This resulted in perpendicular alignment of cells and collagen fibres in a similar fashion to the present study. Interestingly, the microgrooves were reported to maintain efficient contact guidance at a 35 mm distance on average, whereas the 100CH scaffolds in the present study displayed high alignment in the centre of the channels suggesting a more potent topographical guidance from the walls of the channels.

[00240] Previous research in the development of fibre-guiding scaffolds has utilised a variety of techniques or combination of techniques, ranging from thermally induced phase separation (TIPS), selective laser sintering, high precision 3D printing/TIPS, soft lithography, the stacking solution electrospinning mats, and solvent-non solvent exchange. These techniques either lacked reproducibility or did not enable efficient cross-compartment periodontal-bone communication and tissue integration. Indeed, the stacking and gluing of aligned solution electrospun fibrous mats using chitosan has been proposed as a method for inducing perpendicular and/or oblique periodontal ligament attachment to the root surface. This scaffold design however employed manual fabrication and therefore was prone to large batch-to-batch and operator variations. This is overcome in the present study using the melt electrowriting (MEW) fabrication technique, which is automated, highly reproducible and up-scalable.

[00241] In a similar manner, the utilisation of thermally induced phase separation for the manufacturing of fibre-guiding channels using gelatine did not allow extensive control over the dimensions of the guiding features or their placement within the scaffold. In addition, TIPS is also subject to significant variations caused by small changes in solution concentration and cooling rates.

[00242] In this aspect, the melt electrowriting (MEW) technology utilising a computer- controlled manufacturing process enables high reproducibility of the fibre-guiding component and control over fibre diameter and channel spacing. As a result, the 100CH scaffold displayed an unprecedented, systematic, and perpendicular alignment of both cell and collagenic tissue at the dentin surface, resembling the tooth-ligament interface.

[00243] Another limitation of current techniques for the fabrication of guiding features is the lack of tissue integration between the newly formed bone and periodontal ligament. Indeed, several studies demonstrated promising periodontal guidance but with a limited porosity at the interface between the bone and the periodontal ligament compartments, restricting cross-compartment tissue infiltration and therefore the formation of functional periodontal attachment on the bone side.

[00244] In contrast, the integrated and continuous fibre-guiding scaffold fabrication method used in the present study resulted in a fully interconnected and open periodontal-bone compartment, reinforcing the importance of a truly porous interface for promoting successful cross-compartment tissue infiltration and integration, an essential, yet challenging feature for multi-tissue interface healing. In addition, the high fibre resolution (8.7 pm ± 0.4 pm) and the layer-by-layer fabrication method created a high level of porosity and interconnectivity within the pores of the scaffold. This is essential for vascularisation and tissue regeneration, especially between the bone and PDL phases, and is crucial for functional attachment of the new formed ligament to the regenerated bone, a feature which is mostly absent from previous designs.

[00245] Another critical aspect of scaffold design for periodontal regeneration is the reduction in the number of synthetic materials present in the construct in order to increase tissue integration. A precedent clinical case demonstrated that the utilisation of a low porosity polycaprolactone scaffold manufactured by selective laser sintering utilised for the treatment of a periodontal defect became exposed and was removed after 14 months. Post-explantation analysis revealed only limited bone formation and tissue integration within the scaffold. This was attributed to the bulkiness of the construct, lack of porosity and pore interconnection, subsequently impeding vascularisation, osteogenesis and tissue integration.

[00246] In addition, the rigidity of this particular scaffold in comparison to the adjacent soft tissue may also have caused mechanical mismatch leading to soft tissue dehiscence.

[00247] While in this clinical case the slow degradation of the PCL combined with the stiffness of the scaffold may have caused wound dehiscence, the utilisation of a long lasting biodegradable polymer may still be advantageous as it can provide long-term stability for supporting bone maintenance, the prevention of resorption and enabling several cycles of bone turn-over ensuring that bone is mature once the scaffold has fully degraded.

[00248] Similar observations can be made for the periodontal fibre-guiding compartment, whereby the minimal amount of polymer is preferable for ensuring periodontal ligament attachment over the largest possible surface area of dentin. In the case of the 100CH scaffold, only 8% of the dentin surface was covered by PCL, leaving 92% of the surface available for periodontal ligament attachment.

[00249] While the creation of fibre-guiding channels by MEW provides significant advantages in terms of reproducibility and efficacy, the accurate placement of the polymers at a 100 pm distance was challenging and presented at the boundary of the machine’s spatial resolution. Existing limitations of the MEW printer, such as jet instabilities and dielectric interferences, have commonly prevented accurate fibre placement at close inter-fibre distances.

[00250] A recent fundamental investigation on MEW demonstrated that the minimal fibre inter distance was in the 60 pm range for a given fibre diameter of 13 pm, and that a closer spacing resulted in fibre attraction. Here, the inventors exploited this attraction effect between layers in order to accurately stack the fibres resulting in the formation of the channels. Therefore, newly deposited fibres were attracted by residual charge effects of the pre-existing fibres, thus enabling the manufacturing of highly organised and reproducible 100 pm spacing geometry. [00251] In comparison to other constructs, MEW PCL fibres exhibit flexible and forgiving handling, despite being made from PCL, which is an otherwise a relatively rigid polymer. The use of more traditional fabrication methods such as 3D-printing or TIPS will result in a product of higher rigidity and poor defect conformity. In contrast, the flexibility of the 100CH biphasic implantable scaffold would result in a better ability to conform to defect irregularities. Interestingly, the 100CH biphasic scaffold maintained high fibre-guiding efficacy despite the various degrees of compression it experienced during both the placement of the surgical suture applied for mechanical fixation on the dentin and the physiological subcutaneous in vivo forces. This suggests that surgical handling and fixation does not drastically interfere with the structural integrity of the channels and hence with the fibre-guiding capacity of the construct, which is a significant consideration for clinical translation. In addition, the present study demonstrated that a non-cellullarised scaffold may be clinically as advantageous as a cellularised scaffold - which reduces both regulatory burden and cost. The effects of cellularised scaffolds were perhaps under-represented in this study, due to the use of a single human donor per experiment. Utilising cells sourced from a single donor does not account for the variation in regenerative capacity from patient to patient. The use of additional donors as biological repeats for each experimental stage would eliminate some variation experienced across the donor groups.

[00252] While the fibre guiding biphasic scaffold demonstrated promising results for periodontal ligament attachment, its capacity for promoting bone regeneration was not assessed in this study, as bone regeneration usually requires long-term osteogenic differentiation of cellularised constructs. That said, the inventors believe that prolonged implantation of the biphasic scaffold 100CH will address this issue.

[00253] In addition, no cementogenesis was observed despite the placement of an osteogenically induced cell sheet at the dentine interface. However, the fibre guiding biphasic scaffold can be combined with commercially available dental products such as Emdogain® in order to increase its cementogenic capacity and therefore circumvent this shortcoming.

[00254] Another limitation of this study originated from the ectopic model used in this study which lacked the inflammatory and microbiome component usually present in periodontal wound healing. Therefore, further testing in a larger animal model possibly featuring a chronic periodontal inflammatory environment is necessary to further confirm the present findings.

[00255] Conclusion

[00256] This study shows that highly controlled manufacturing of fibre-guiding channels in biphasic scaffolds resulted in systematic perpendicular alignment of cells and collagen fibres, resembling the attachment of the native periodontal ligament onto a dentin surface. Using a periodontal regeneration ectopic model, it was further demonstrated that the presence of previously seeded and in vitro cultured cells within the fibre-guiding features was not a pre-requisite for obtaining perpendicular attachment. Since collagen fibre orientation appeared solely dependent upon the topographical cues provided by the 100 pm channels, the fibre-guiding scaffold could be utilised as an off-the-shelf product, which is a significant advantage for clinical translation.

[00257] MATERIALS AND METHODS

[00258] Scaffold fabrication

[00259] Fibre-guiding biphasic scaffold fabrication

[00260] The biphasic scaffold (Figure 5) was manufactured in a continuous process using an in-house built melt electrowriting device. PCL polymer pellets (PC12, Corbion, Amsterdam, The Netherlands) were loaded into a 2 ml syringe with a 23 G needle tip. The PDL compartment was composed of 12 stacked layers of aligned fibres with an inter-distance of 100 pm. 2 layers of fibres with an orientation of 0°/0° were deposited onto the collector at a speed of 1200 mm/min, a pressure of 0.9 bar and a 7.4 mm tip to collector distance (See Figure 5 for layering schematic). Subsequently, one layer with a 200 pm inter-fibre distance at an orientation of 90° was deposited in order to provide mechanical support and stabilisation to the compartment. Thereafter, the process was repeated another 6 times, resulting in the creation of a PDL compartment with 100 pm channels and an overall height of around 100 pm. All fibres were electrowritten at 9.5 kV, as well as temperatures set at the cartridge and needle tip of 74°C and 83°C, respectively.

[00261] The bone compartment was subsequently superimposed on top of the PDL compartment, albeit using slightly different parameters. After the fabrication of the PDL compartment was completed, the electrowriting printer head was positioned away from the PDL compartment to enable the adjustment of parameters suitable for the superimposition of the bone compartment. The pressure in the syringe was increased to 1 .1 bar, the collector velocity was decreased to 1080 mm/min and the manufacturing process was allowed to equilibrate for 5 min until a stable electrowriting jet was observed. The bone compartment consisting of 530 layers of increasing pore sizes of 200, 400, 800, 1200 pm with a 0°/90° pattern was electrowritten over the PDL compartment.

[00262] To assess the biological impact of the channels on 2D in vitro cellular behaviour, the PDL compartment was also fabricated without the bone compartment and this group is herein referred to as 100CH-2D.

[00263] Non-fibre-guiding biphasic scaffold fabrication

[00264] The non-fibre-guiding melt electrowritten scaffold (200BX) was composed of a periodontal component comprising of 12 layers with a 0°/90° pattern at a 200 pm fibre inter-distance. Fibres were deposited at 1080mm/min with a pressure of 1.1 bar. The bone compartment was electrowritten over this layer using the conditions described above. Similar to the fibre-guiding scaffold, the first 12 layers of the 200BX were also electrowritten without the bone compartment and this group is herein referred to as 200BX-2D.

[00265] Sponge scaffold fabrication

[00266] The sponge scaffold was fabricated using a solvent casting salt leaching technique. The PCL pellets were dissolved in 25 wt/v% chloroform and salt particles were added to create a mixture with a 90/10 wt/wt ratio. The mixture was poured on a glass petri-dish and the solvent was evaporated in a fume hood, then the scaffolds were sectioned into square blocks of 5x5 mm ² and immersed in distilled water. The porogen was leached out in an excess of distilled water for 14 days with the water being replaced every 2 days. The resulting PCL sponge was air-dried and kept in a desiccator until use. The biological impact of the sponge on cellular alignment was also assessed in 2D and is herein referred to as SP-2D.

[00267] Scaffold characterisation

[00268] Scanning electron microscopy

[00269] Scanning electron microscopy (SEM) was used to assess scaffold morphology. The scaffolds were soaked in 100% ethanol and sectioned with a scalpel blade (whilst still submerged in ethanol) to enable a clean cut for visualization of the cross-section. The samples were iridium coated for 3 min and observed with a Hitachi SU3500 microscope at an accelerating voltage of 5 kV. Pore size and fibre diameter were assessed (n=15 per group) from three different samples per group using Imaged (National Institutes of Health (NIH), Bethesda, MD, USA).

[00270] Micro-CT Analysis

[00271] Porosity was determined using a microcomputed tomography (micro-CT) scanner (mCT40, SCANCO Medical AG, Bruttisellen, Switzerland). A silver nitrate coating was applied to the scaffolds as a contrast enhancer. The scaffolds were sectioned into 5x5 mm ² samples and submerged in silver nitrate (1 % w/v in 100% ethanol) for 30 mins, followed by incubation for 30 mins in L-ascorbic acid solution (10 mM) (Sigma -Aldrich, Darmstadt, Germany). After gentle rinsing in water, this process was repeated, and the samples were incubated overnight. Then, the samples (n=3) were washed in 80% ethanol and dried before scanning. Micro-CT scans were performed at a resolution of 6 pm. Three-dimensional (3D) images of implants were reconstructed from the scans by the micro-CT system software package. [00272] In vitro study

[00273] Cell isolation and culture

[00274] Periodontal ligament (PDL) explants were obtained from redundant dental tissues. Tissue collection was approved by the University of Queensland human ethics committee (approval number: 2019000134).

[00275] Third molars were extracted and placed into a 50 mL tube containing Dulbecco’s Modified Eagle Medium (DMEM) with 2% penicillin/streptomycin and fungizone (4 pg/mL). The middle third of the PDL was subsequently gently removed from the root surface with a scalpel and further sectioned into approximately 1 x 1 mm ² pieces. The PDL tissues were placed into a 25 cm ² culture flask which was left standing upright in an incubator at 37°C and 5% CO2 atmosphere for 30 min to allow tissue adhesion.

[00276] After this incubation period, DMEM (3 mL) containing 10% FBS, 1 % of penicillin/streptomycin and fungizone (0.1 pg/mL) was added, and the flask was carefully laid flat in the incubator. The first medium change occurred 4 days postextraction, and the culture medium used for any subsequent changes was only composed of 10% FBS and 1 % of penicillin/streptomycin (basal medium). After one week of culture, periodontal ligament (PDL) cells started migrating outwards from the PDL tissues, and generally reached confluence after 2-3 weeks of culture. The PDL cells were passaged using 0.25% trypsin and further expanded until P3. Multiple donors were used throughout this study, although a single donor was utilised per experiment. The 2D and 3D in vitro study used the same donor, however, the 3D periodontal attachment model and periodontal ectopic regeneration model used separate donors (a sum total of three donors).

[00277] 2D and 3D in vitro fibre-guiding efficacy

[00278] The guidance efficacy of the PDL compartments (100CH-2D, 200BX-2D and SP-2D) was investigated in vitro via a 2D setting (hence, uniquely using only the initial 12 layers that form the PDL compartment). The PDL compartments were soaked in 70% ethanol and sectioned with a scalpel blade into 5 x 5 mm ² constructs. A 1 M sodium hydroxide treatment was performed at 37°C for 15 min followed by multiple washes in distilled water in order to enhance the hydrophilicity of the PDL compartments. The PDL compartments were sterilized using an overnight immersion in 100% ethanol, followed by a 20 min-UV-irradiation and subsequently dried in a biosafety cabinet. Prior to seeding the PDL compartments, a 2-hour incubation in 100% FBS at 37°C was performed in order to enhance cell attachment.

[00279] The seeding was performed with a single donor, as follows: 20,000 cells in media (20 pL) were seeded onto the PDL compartments. The cells were allowed to adhere for 4 hours, after which 1 mL of basal medium was added. After 12 hrs of culture, the basal medium was substituted with a ligament differentiation medium made of basal medium supplemented with ascorbic acid (100 pg/mL ascorbate-2- phosphate). The medium was changed every 3 days, and the cells were cultured for 3, 7 and 14 days.

[00280] For characterization purposes, entire scaffolds were seeded in vitro to assess their guidance capacity in a more 3D context. To this end, the fibre-guiding biphasic scaffold (100CH), the non-fibre-guiding (200BX) and the sponge and (SP) scaffolds were seeded with 20,000 cells according to the protocol described above and cultured for 14 days in ligament differentiation medium.

[00281 ] Immunofluorescence and confocal laser microscopy

[00282] Cellular and ECM alignment in the various scaffolds (100CH-2D, 200BX-2D and SP-2D) was assessed by measuring nucleus and collagen type-l orientation. The 2D and 3D constructs were fixed using a 4% paraformaldehyde (PFA) in PBS solution for 15 mins at room temperature and subsequently rinsed three times in 0.1 % tween/PBS. The samples were then incubated in blocking buffer solution (5% BSA, 1 % v/v goat serum, 3M glycine, 2% tween/PBS, and PBS) for 2 hours to block nonspecific binding of antibodies. The primary antibody for collagen-l (ab34710,1 :400) (Abeam, Melbourne, Australia) in blocking buffer was incubated at 4°C overnight. The constructs were washed again in tween/PBS solution prior to incubating the secondary antibody (goat anti-rabbit IgG H&L, Alexa Fluor®, ab150077, Abeam, excitation: 488, emission ranging from 500-550) at a 1 :500 dilution, DAPI (1 :1000, excitation: 405, emission ranging from 417-477) and Phalloidin-iFluor™ 568 Conjugate (1 :400, excitation: 561 , emission ranging from 570-1000) in blocking buffer for 60 min. The PDL compartments were rinsed in PBS and imaged with a Nikon Elipse Ti microscope (Nikon, Tokyo, Japan).

[00283] 2D nuclei, actin and collagen fibre alignments were quantified using the directionality vO.92.4 plugin on Imaged software. Directionality vO.92.4 was assessed from representative images taken at 40x (n = 3 from 3 different samples). RGB image channels were split to separate the nuclei (blue), actin (red) and collagen (green) so directionality could be calculated without interference from the other channels. Images were then converted to 8-bit file format and the directionality vO.92.4 plugin was used on each channel individually with the horizontal direction (0 ^s ) as a reference for measuring angles.

[00284] Similarly, the cellular organisation and alignment were qualitatively investigated in the entire biphasic scaffold at 14 days post-seeding. To this end, the scaffolds were sectioned with a scalpel blade to image their cross-sectional area by confocal microscopy using the aforementioned protocol.

[00285] 3D periodontal attachment model: in vitro

[00286] The efficacy of cell and tissue guidance was assessed using a 3D in vitro periodontal attachment model. This model involved the attachment of a mineralized PDL cell sheet (previously cultured in osteogenic media) onto a dentin block, prior to placing a scaffold previously seeded with PDL cells. Therefore, this 3D in vitro periodontal model mimicked the anatomy of the native tissue with the dentin block, the mineralised PDL cell sheet and the PCL scaffold representing the dentin, cementum and periodontal ligament respectively. All cells utilised were in the 3D periodontal attachment model: in vitro were sourced from a single donor.

[00287] Dentin block preparation

[00288] One millimetre thick dentin blocks were prepared from sheep teeth and adjusted to the size of the biphasic scaffolds (approximately 5x5 mm ² ) using an EXACT Diamond Band Pathology Saw (EXACT Technologies, Oklahoma City, USA) (see Figure 6 for a schematic of preparation). The resulting blocks were subsequently polished using an 800 grit sandpaper to remove the native cementum and to expose the dentin using an EXACT 4000CS Micro Grinding System (EXACT technologies, Germany). Dentin blocks were then sterilised with gamma radiation (25kGy, Steritech, Narangba, QLD, Australia) and treated with 14% EDTA for 5 mins at 37°C and thereafter, washed 5 times with sterile PBS.

[00289] Harvesting of cell sheets

[00290] PDL cells were seeded at 50,000 per well in a 24-well plate and cultured in osteogenic medium (ascorbate-2-phosphate (50 mg/mL), b-glycerophosphate (10 mM) and dexamethasone (0.1 mM)). Spontaneous contraction occurred around 21 days, with the cells detaching from the plate walls enabling mechanical harvesting using fine tweezers. The sheet was subsequently wrapped around the dentin block and cultured for another one week in osteogenic medium to allow for adhesion.

[00291 ] 3D attachment model assembly

[00292] The various scaffolds (100CH, 200BX and SP) were treated with sodium hydroxide (1 M) for 15 mins at 37°C followed by multiple rinses, then sterilized using 100% ethanol and ultraviolet C irradiation for 20 min. Prior to seeding, they were soaked in FBS at 37°C for 2 hrs in order to increase cell seeding efficacy. The scaffolds were seeded with 50,000 PDL at P6 in medium (30 pL). Cell adhesion was allowed for 4 hours. Thereafter, DMEM (1 mL) supplemented with ascorbate-2-phosphate (100 pg/mL) was added, and the scaffolds were cultured for 2 weeks, with a medium change every 3 days.

[00293] The scaffolds were then positioned over the cell sheet wrapped dentin, and a suture was used to mechanically secure the construct onto the dentin block. The assembled constructs were further cultured for another week in ligament differentiation medium (basal medium supplemented with L-ascorbate-2-phosphate (100 pg/mL)), before being fixed in 4% PFA for 30 min, prior to transferring the tissue to PBS.

[00294] The samples were progressively dehydrated in multiple changes of ethanol and then placed in graded blends of ethanol mixed with resin (methyl methacrylate I glycol methacrylate, Tecknovit 7200, Heraeus Kulzer, Wehrheim, Germany). The concentration of resin was increased with each change until reaching 100%. Samples were then prepped for curing under UV light for 2 days. The resin blocks were glued onto a sample slide for grinding to expose the sample using the EXAKT 400 CS microgrinding system. Sections were taken at approximately 150 pm thickness with the EXAKT 300 cutting system (Exact Apparatebau, GmbH Norderstedt Germany). Slides were polished using abrasive paper (ranging from grit 800 to 4000) to a 25 pm thickness and were subsequently stained with H&E and imaged with a Leica DM IL LED microscope (Leica, Wetzlar, Germany).

[00295] Scanning electron microscopy

[00296] After 4 weeks in osteogenic in vitro culture, the cell sheet wrapped dentin blocks were fixed and stored in 3.4% glutaraldehyde until dehydration. Dehydration occurred in three repeated changes of increasing ethanol baths for 10mins each (30:70, 50:50, 70:30, 90:10,95:5, 100) for both the cell sheet wrapped dentin block and a dentin block with removed cementum layer. The samples were carbon coated for 3 min and observed with a Hitachi SU3500 microscope at a voltage of 10kV.

[00297] 3D attachment model immunofluorescence and confocal microscopy

[00298] The presence of cementum enamel matrix protein 1 (CEMP1 ) was assessed by immunofluorescence using the staining protocol described in section 2.4.1 . CEMP1 antibody (ab134231 ,1 :50) (Abeam, Melbourne, Australia) was incubated on the sample in blocking buffer at 4°C overnight. The dentin was washed again in tween/PBS solution prior to being incubated with the secondary antibody (goat antirabbit IgG H&L, Alexa fluor® 488, excitation: 488, emission: 500-550) (Abeam; ab150077) at a 1 :500 dilution, DAPI (1 :1000, excitation: 405, emission: 417-477) and Phalloidin-iFluor™ 561 Conjugate (1 :400, excitation: 561 , emission: 570-1000) in blocking buffer for 60 min at room temperature. The dentin blocks were then rinsed in PBS and imaged with a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan). A negative control without primary antibody but with the secondary antibody was also prepared (Figure 7). [00299] 3D attachment model nuclei alignment quantification

[00300] The fibre-guiding capacity of the scaffolds was assessed by quantifying nuclei orientation within the PDL compartment relative to the dentin block. Nuclei orientations were measured using Imaged software, ObjectJ version 1.05d. Directionality was assessed from representative images taken at 20x (n=5 from a single sample per group). The direction of the nuclei was measured across the long axis and the dentin surface was treated as 0 ^s (Figure 8). In order to prevent doublecounting originating from the same nucleus, all measurements were constrained to the 0-90° range, to then provide a distribution of angles from 0 to 180°, the angle frequencies were halved and mirrored on either side of the 90° angle.

[00301 ] Periodontal ectopic regeneration

[00302] The ability of the biphasic scaffold to promote periodontal attachment and alignment on the dentin block was assessed using an ectopic periodontal regeneration model in athymic rats. Animal ethics approval for this study was granted by the Animal Ethics Committee of The University of Queensland (Ethics approval number: 408/19). Six different groups were implanted in each animal 1 ) Sponge (SP), 2) Sponge with cells (SP + C), 3) Box 200 pm (200BX), 4) Box 200 pm with cells (200BX + C), 5) Biphasic fibre-guiding scaffold (100CH), 6) Biphasic fibre-guiding scaffold with cells (100CH + C).

[00303] The cellularised scaffolds (SP + C, 200BX + C, 100CH + C) were seeded with 50,000 PDL cells from one donor as described above, and further cultured for 4 weeks in the ligament differentiation medium. Thereafter, a 3-layer PDL cell sheet cultured in osteogenic medium for 21 days as described above was placed on the scaffold periodontal compartment. The scaffolds were then placed on a dentin block prepared according to the protocol described above and mechanically secured using a suture. The assembled constructs were immersed in ligament differentiation medium for another 24 hr to allow attachment of the cell sheet onto the dentin block prior to implantation. The control groups without cells (SP, 200BX, and 100CH) were placed directly onto the EDTA treated dentin and secured using a suture. [00304] Five 8-week-old male rats (Animal Resources Centre, Canning Vale, WA, Australia) were used. The animals were anaesthetized with isoflurane. Six small incisions were performed longitudinally along the central line of the shaved dorsal area for one technical replicate of each of the six experimental groups (SP, 200BX, 100CH, SP + C, 200BX + C, 100CH + C), approximately 2 cm apart, and subcutaneous pockets were made on each side of the incision with a pair of surgical scissors. Each pocket held one scaffold, and thus there were 5 biological replicates for each group. The incisions were closed with surgical staples. The animals were sacrificed at eight weeks and the implants retrieved and fixed in 4% paraformaldehyde in PBS at pH 7.4 for 24 hours prior to being transferred in PBS for further analysis.

[00305] Histology

[00306] The samples were decalcified in 10% ethylene diaminetetraacetic acid (EDTA) at pH 7.4 for 3 months at room temperature, with a weekly change in solution, and subsequently embedded in paraffin. Sections near the central area of the implants were stained with H&E and Piero Sirius Red (collagenous ECM).

[00307] The fibre-guiding capacity of the scaffolds was assessed by quantifying nuclei orientation within the PDL compartment relative to the dentin block. Nuclei orientations were measured using Imaged software, ObjectJ version 1.05d. Directionality was assessed from representative images taken at 20x (n=5 from a single sample per group). The direction of the nuclei was measured across their long axis and the dentin surface was treated as 0 ^s (Figure 8). In order to prevent doublecounting originating from the same nucleus, all measurements were constrained to the 0-90° range, to then provide a distribution of angles from 0 to 180°, the angle frequencies were halved and mirrored on either side of the 90° angle. The average nuclei was calculated and the average percentage of nucleus orientated with angle ranging from 30°-60° and 60°-90° was also calculated to further assess the capacity of the different scaffolds to perpendicularly aligned the cells.

[00308] Collagen fibres were observed using picrosirius staining in order to stain a broad range of collagen types similarly to the composition of the native periodontal ligament. Fibre orientations were measured using the Imaged software plugin, directionality vO.92.4. Representative images were taken at 20x (n=3) from three techniques and biological repeats per group. The split channels feature was used on these images to remove any background from the cell nuclei. Before collagen directionally was measured, segments were cropped from the image removing the bone compartment and the dentin tissues (Figure 9), leaving only the remaining collagen tissues within the PDL compartment. The results were expressed as a frequency distribution across 0-180° similarly to the protocol described above. The analysis was further refined to determine the degree of aligned attachment in an attempt to determine morphological mimicry by categorizing the angles into one of the following: parallel (0°-30°), oblique (30°-60°) and perpendicular (60°-90°), providing an overall assessment of guidance capacity. This was performed by calculating both the average collagen fibre angle and the average percentage of fibre orientated with angle ranging from 30°-60° and 60°-90° (similarly to cells present in the native tissue).

[00309] Statistical analysis

[00310] Quantification for all data was represented by the mean standard deviation of the mean. Scaffold evaluations check for normality was conducted using Jamovi 1.8.1 (The Jamovi Project, Sydney, Australia). Normality was confirmed for this data set therefore statistical significance was evaluated utilising a parametric one-way analysis of variance (ANOVA), followed by a Tukey Post-hoc Test. For the in vivo data, generalized estimating equations using SPSS Statistics 25 (IBM) were employed using individual animals as the clustering variable. Treatments were used as explanatory variables and a pairwise comparison with an LSD Post-Hoc test was utilised. A p < 0.05 was considered to represent statistically significant differences.

[00311] Embodiments:

[00312] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[00313] Similarly, it should be appreciated that in the above description of example embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the Detailed Description of Specific Embodiments are hereby expressly incorporated into this Detailed Description of Specific Embodiments, with each claim standing on its own as a separate embodiment of this invention.

[00314] Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[00315] Other Embodiments:

[00316] While the preferred embodiments of the invention described above relate to the fabrication of a biphasic implantable scaffold by melt electrowriting (MEW) a plurality of biocompatible polymer fibres to produce at least a PDL compartment having walls that define a plurality of open channels with a configuration that imposes a structural constraint on the growth and orientation of cells to produce aligned ligament fibres, and configuring the biphasic implantable scaffold for use in, for example:

(i) treating and/or repairing a periodontal defect,

(ii) regenerating periodontal tissue, or

(Hi) treating periodontal disease or degeneration, [00317] it will be appreciated by persons of ordinary skill in the relevant art that the biphasic scaffold may be utilised in treating and/or repairing the gingiva of a subject.

[00318] Treating and/or repairing gingival recession

[00319] According to one embodiment, the present invention provides a method of treating and/or repairing gingival recession in a subject in need thereof, wherein the method comprises the step of implanting the biphasic implantable scaffold into the gingiva of said subject.

[00320] In addition, it will also be appreciated by persons of skill in the relevant art that with little or no modification, the biphasic implantable scaffold may also be implanted into subjects to treat and/or repair an altogether different set of conditions.

[00321] For instance, the biphasic implantable scaffold may also be configured for use in one or more of the following applications:

[00322] Treating and/or repairing musculoskeletal disorders and defects involving soft-hard tissue interfaces

[00323] According to one embodiment, the present invention may be configured to provide a method of treating and/or repairing musculoskeletal disorders and defects involving soft-hard tissue interfaces in a subject in need thereof, wherein the method comprises implanting the biphasic implantable scaffold into a soft-hard tissue interface of said subject, which is formed between soft tissue such as tendon, ligament, gingiva, connective tissue, muscle, and hard tissue such as bone, cartilage or tooth.

[00324] Treating and/or repairing any disorders and defects requiring soft tissue fibre guidance

[00325] According to one embodiment, the present invention may be configured to provide a method of treating and/or repairing any disorders and defects requiring soft tissue fibre guidance in a subject in need thereof, wherein the method comprises implanting the biphasic implantable scaffold into such soft tissue as ligament, tendon and/or muscle. [00326] Treating and/or repairing any disorders and defects requiring soft tissue fibre attachment and/or insertion

[00327] According to one embodiment, the present invention may be configured to provide a method of treating and/or repairing any disorders and defects requiring soft tissue fibre attachment and/or insertion in a subject in need thereof, wherein the method comprises implanting the biphasic implantable scaffold into such soft tissue as ligament, tendon and/or muscle.

[00328] Treating and/or repairing soft-hard tissue insertion

[00329] According to one embodiment, the present invention may be configured to provide a method of treating and/or repairing soft-hard tissue insertion, wherein the method comprises the step of implanting the biphasic implantable scaffold into and/or around an insertion previously inserted at a soft-hard tissue interface of said subject.

[00330] Obtaining oblique and/or perpendicular fibre attachment and/or insertion for repairing soft-hard tissue insertion

[00331] According to one embodiment, the present invention may be configured to provide a method of obtaining oblique and/or perpendicular fibre attachment, wherein the method comprises the step of implanting the biphasic implantable scaffold of the invention into a hard-soft tissue interface of said subject.

[00332] It will also be appreciated by skilled persons in the art that the method of manufacturing the biphasic implantable scaffold is not limited to a melt technique such as melt electrowriting (MEW) or melt electrospinning.

[00333] For example, in other embodiments, techniques such as for example, fused deposition modelling (FDM) or 3D printing, may be used to produce a biphasic implantable scaffold having corresponding dimensions to those described above for 100CH. [00334] Different Instances of Objects

[00335] As used herein, unless otherwise specified the use of the ordinal adjectives "first", "second", "third", etc., to describe a common object, merely indicate that different instances of like objects are being referred to, and are not intended to imply that the objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner.

[00336] Specific Details

[00337] In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[00338] Terminology

[00339] In describing the preferred embodiment of the invention illustrated in the drawings, specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar technical purpose. Terms such as "forward", "rearward", "radially", "peripherally", "upwardly", "downwardly", and the like are used as words of convenience to provide reference points and are not to be construed as limiting terms.

[00340] Definitions

[00341] As used herein, "cellularised" refers to an entity (e.g., an implantable scaffold) that has been seeded with cells and cultured in a suitable medium under appropriate cell growth conditions.

[00342] As used herein, “non-cellularised” refers to an entity (e.g., an implantable scaffold) that has not been seeded with cells and cultured in a suitable medium under appropriate cell growth conditions. [00343] As used herein, "polymer" means a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions, or combinations of compositions.

[00344] The polymers described herein may be prepared by synthetic or natural methods. However, the method preferably provides the desired polymer in a form sufficiently pure for use as an implantable material. The polymer is preferred not to contain any undesirable residues or impurities which could elicit an undesirable response either in vitro in the case of a cell-seeded construct, or in vivo.

[00345] The polymers may be prepared from any combination of monomeric units. These units may be capable of biodegrading in vivo to nontoxic compounds, which can optionally be excreted or further metabolized. The combination of units in the polymer should be biocompatible to minimise or avoid any undesirable biological response upon implantation. The polymer may be biodegraded in vivo by any means, including hydrolysis, enzymatic attack, a cell-mediated process, or by any other biologically mediated process. It is considered desirable for tissue engineering applications that a polymer scaffold as described herein can serve as a transitional construct, and thus be fully degraded once the new tissue is able to take over the function of the scaffold. Since the rates at which different new tissues are likely to be able to assume their new function will vary, it is desirable to have polymers with a range of degradation rates as well as a range of different properties. Generally, however, preferred polymers may degrade in a matter of weeks to months, preferably less than one year, rather than several years.

[00346] As used herein, the terms "extracellular matrix" and "ECM" refer to a scaffolding for cell growth, which is produced, secreted, supplied or deposited by the cell from which the ECM is said to derive. For example, an osteoblast-derived extracellular matrix refers to an ECM which has been produced, secreted, supplied or deposited by one or more osteoblasts. A periodontal ligament cell-derived extracellular matrix refers to an ECM which has been produced, secreted, supplied or deposited by one or more PDL cells. The ECMs may include mixtures of structural and non- structural biomolecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemo-attractants, cytokines, and growth factors.

[00347] As used herein, "porosity" means the ratio of the volume of interstices of a material to a volume of a mass of the material.

[00348] As used herein, the term "cell" refers to a membrane-bound biological unit capable of replication or division.

[00349] As used herein, "osteoblast" shall mean a bone-forming cell which forms an osseous matrix in which it becomes enclosed as an osteocyte. It may be derived from mesenchymal stem or progenitor cells with osteogenic potential (e.g., osteoprogenitor cells). The term may also be used broadly to encompass osteoblast-like, and related, cells, such as osteocytes and osteoclasts. An "osteoblast-like cell" means a cell that shares certain characteristics with an osteoblast (such as expression of certain proteins unique to bones) but is not an osteoblast. "Osteoblast-like cells" include preosteoblasts and osteoprogenitor cells. Preferably the cells are from a compatible human donor. More preferably, the cells are from the patient (i.e., autologous cells).

[00350] As used herein, "periodontal ligament cell" means a cell that is isolated from the periodontal ligament. Such a periodontal ligament cell can include differentiated cells and periodontal ligament stem cells.

[00351] As used herein, “periodontium” means a complex structure composed of the gingiva, periodontal ligament (PDL), cementum, and alveolar bone. The primary functions of the periodontium are to allow the tooth to be attached to the bone and to provide a barrier for the underlying structures from the oral microflora.

[00352] As used herein, a "biocompatible" material is a synthetic or natural material used to replace part of a living system or to function in intimate contact with living tissue.

[00353] Biocompatible materials are intended to interface with biological systems to evaluate, treat, augment, or replace any tissue, organ or function of the body. The biocompatible material has the ability to perform with an appropriate host response in a specific application and preferably does not have toxic or injurious effects on biological systems.

[00354] As used herein, a "biodegradable" implantable scaffold is a scaffold that once implanted into a host, will begin to degrade. The implantable scaffolds of the present invention may be used in open surgical procedures as may be required and determined by a dental surgeon. Preferably, the implantable scaffold is biomimetic and biodegradable. The rate of biodegradation may be engineered into the scaffolds based on the polymers used, the ratio of copolymers used, and other parameters well known to those of skill in the art. Moreover, in certain embodiments of the present invention, the rate of biodegradation of each phase may be separately engineered according to the needs of the particular surgery to be performed.

[00355] As used herein, the terms "treatment" or "treating" mean: (1 ) improving or stabilizing the subject's condition or disease or (2) preventing or relieving the development or worsening of symptoms associated with the subject's condition or disease.

[00356] As used herein, the terms "subject" and "patient" are used interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse, or primate) that can be afflicted with or is susceptible to a disease or disorder but may or may not have the disease or disorder. In certain embodiments, the subject is a human being.

[00357] Comprising and Including

[00358] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

[00359] Any one of the terms: including or which includes or that includes as used herein is also an open term that also means including at least the elements/features that follow the term, but not excluding others. Thus, including is synonymous with and means comprising.

[00360] Scope of Invention

[00361] Thus, while there has been described what are believed to be the preferred embodiments of the invention, those skilled in the art will recognize that other and further modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications as fall within the scope of the invention. For example, any formulas given above are merely representative of procedures that may be used. Steps may be added or deleted to methods described within the scope of the present invention.

[00362] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

[00363] Industrial Applicability

[00364] It is apparent from the above, that the arrangements described are applicable to the biomedical healthcare industry.