A METHOD OF PRODUCING A BIOACTIVE POLYMER FILAMENT, THE BIOACTIVE POLYMER FILAMENT AND PRINTING METHODS USING THE SAME

Abstract

There is provided a method of producing a bioactive polymer filament, the method comprising: providing a base polymer powder and a bioactive copolymer; mixing the base polymer powder with the bioactive copolymer to obtain a mixture; and extruding a bioactive polymer filament from the mixture at an extrusion temperature profile that is based on a predetermined melt/softening temperature and a predetermined onset degradation temperature of the bioactive polymer; and performing a post-extrusion thermal analysis on the extended bioactive polymer filament to assess onset degradation of the bioactive copolymer in the filament. There is also provided a bioactive polymer filament obtained from said method and a fused filament fabrication (FFF) or fused deposition modelling (FDM) based three-dimensional printing method.

Claims

1. A method of producing a bioactive polymer filament, the method comprising: providing a base polymer powder and a bioactive copolymer; mixing the base polymer powder with the bioactive copolymer to obtain a mixture; and extruding a bioactive polymer filament from the mixture at an extrusion temperature profile that is based on a predetermined melt/softening temperature and a predetermined onset degradation temperature of the bioactive polymer; and performing a post-extrusion thermal analysis on the extruded bioactive polymer filament to assess onset degradation of the bioactive polymer in the filament.

2. The method of claim 1, wherein the bioactive copolymer is acellular.

3. The method of claim 1 or 2, wherein the bioactive copolymer is obtained by ring-opening metathesis polymerisation (ROMP).

4. The method of any one of the preceding claims, wherein the bioactive copolymer is a bioactive synthetic copolymer with a poly(norbornene) backbone comprising one or more repeating units represented by general formula (I) and one or more repeating units represented by general formula (II): ##STR00009## wherein R.sup.1 is optionally substituted alkyl; R.sup.2 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl; R.sup.3 is selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; L is heteroalkylene; X comprises a bioactive moiety selected from the group consisting of proteins, peptides, carbohydrates, collagen, hyaluronic acid, therapeutic/drug molecules and derivatives thereof; Y.sup.1 comprises a synthetic polymer or parts thereof; and Z.sup.1 and Z.sup.2 are each independently selected from CR.sup.aR.sup.b, O, NR.sup.c, SiR.sup.aR.sup.b, PR.sup.a or S, wherein R.sup.a, R.sup.b and R.sup.c are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

5. The method of claim 4, wherein Y.sup.1 is represented by general formula (III): ##STR00010## wherein A is selected from a single bond, oxy, carbonyl, oxycarbonyl, carboxyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl, optionally substituted alkoxycarbonylalkyl, N or NR.sup.c wherein R.sup.c is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; B is optionally present as a ring selected from 1,2,3-triazole or succinimide; R.sup.5 is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl; Y.sup.2 is selected from the group consisting of polypropylene (PP), polyesters, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylates, poly(meth)acrylates, polyamides (PA), polyurethane (PU), and parts thereof; and T is a terminal group selected from the group consisting of hydrogen, halogen, hydroxyl, amino, acyl, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl, optionally substituted alkylcarbonylalkyl, optionally substituted carboxyalkyl, optionally substituted oxycarbonylalkyl, optionally substituted alkylcarboxylalkyl or optionally substituted alkoxycarbonylalkyl.

6. The method of claim 5, wherein Y.sup.1 is selected from the following general formulae (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), or (IIIg): ##STR00011## wherein R.sup.y is selected from an alkyl, aryl or biaryl; R.sup.z is alkyl; A is 0 or NR.sup.c wherein R.sup.c is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; T is a terminal group selected from the group consisting of hydrogen and methyl; n1; and m1.

7. The method of any one of the preceding claims, wherein the base polymer powder is obtained from cryogenic milling of base polymer pellets.

8. The method of any one of the preceding claims, wherein the base polymer powder has an average particle size of no more than 1 mm.

9. The method of any one of the preceding claims, wherein the post-extrusion thermal analysis comprises application of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC).

10. The method of any one of the preceding claims, wherein the extruding is performed using an extruder having one or more rotating screws.

11. The method of any one of the preceding claims, wherein the extruded bioactive polymer filament has a filament diameter falling in the range of from 1.5 mm to 4.0 mm.

12. The method of any one of the preceding claims, further comprising performing a pre-extrusion thermal analysis on the base polymer and/or bioactive copolymer to determine the melt temperature and the onset degradation temperature of the bioactive polymer.

13. The method of any one of the preceding claims, wherein the base polymer and bioactive copolymer have been vacuum dried prior to mixing.

14. The method of any one of the preceding claims, wherein the mixture of base polymer and bioactive copolymer comprises 60.0 wt % to 99.9 wt % of the base polymer and 0.1 wt % to 40.0 wt % of the bioactive copolymer.

15. The method of any one of the preceding claims, wherein the base polymer is selected from the group consisting of polypropylene (PP), polyesters, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), polystyrene (PS), polyacrylates, poly(meth)acrylates, polyamides (PA), polyurethane (PU) and combinations thereof.

16. A bioactive polymer filament obtained from the method of any one of claims 1 to 15.

17. A fused filament fabrication (FFF) or fused deposition modelling (FDM) based three-dimensional printing method, the method comprising: feeding a bioactive polymer filament of claim 16 into a FFF or FDM based three-dimensional printing apparatus; applying heat to bioactive polymer filament to obtain a molten form of the bioactive polymer; and depositing the molten bioactive polymer on a print bed to form a printed three-dimensional part or structure.

18. The method of claim 17, further comprising performing one or more of post-printing analysis of the printed three-dimensional part or structure, the post-printing analysis selected from the group consisting of: i. a mechanical analysis of the printed three-dimensional part or structure to assess its mechanical properties; ii. a biocompatibility analysis of the printed three-dimensional part or structure to assess its biocompatibility with living cells; iii. a thermal analysis on the printed three-dimensional part or structure to assess onset degradation of the bioactive polymer in the printed three-dimensional part or structure; and iv. a spectrometric analysis of the printed three-dimensional part or structure to assess the presence of bioactive copolymer in the printed three-dimensional part or structure.

19. The method of claim 17 or claim 18, wherein the step of applying heat is at a temperature that is based on a predetermined melt/softening temperature and a predetermined onset degradation temperature of the bioactive polymer.

20. The method of any one of claims 17-19, wherein the FFF or FDM based three-dimensional printing apparatus is configured for filament feedstock having filament diameters falling in the range of from 1.5 mm to 4.0 mm.

Description

BRIEF DESCRIPTION OF FIGURES

[0225] FIG. 1 shows simultaneous thermal analysis (STA) results of PA6-GPHP in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PA6 refers to polyamide-6 and GPHP refers to (GPHyp).sub.3.

[0226] FIG. 2 shows simultaneous thermal analysis (STA) results of PA6-PHPG in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PA6 refers to polyamide-6 and PHPG refers to (PHypG).sub.3.

[0227] FIG. 3 shows a photograph taken for a PA12-collagen filament in accordance with various embodiments disclosed herein. Bioactive polymer: PA6-collagen; additive content: 10%; and filament diameter: 2.850.1 mm. FIG. 4 shows thermogravimetric analysis (TGA) results of PA12-PHPG samples of 3D printed sheet and its filament in accordance with various embodiments disclosed herein. In the figure, PA12-PHPG 3DP sheet is represented by graph [2]; dashed line and its filament is represented by graph [1]; solid line. PA12 refers to polyamide-12 and PHPG refers to (PHypG).sub.3.

[0228] FIG. 5 shows thermogravimetric analysis (TGA) results of PA12-GPHP samples of 3D printed sheet and its filament in accordance with various embodiments disclosed herein. In the figure, PA12-GPHP 3DP sheet is represented by graph [2]; dashed line and its filament is represented by graph [1]; solid line. PA12 refers to polyamide-12 and GPHP refers to (GPHyp).sub.3.

[0229] FIG. 6 is a graph showing Young's modulus (on the left axis) and yield strength (on the right axis) of PA12-based specimens, namely PA12-PHPG and PA12-GPHP in accordance with various embodiments disclosed herein.

[0230] FIG. 7 is a graph showing biocompatibility test results (i.e. % cell viability count) of sheet samples, namely (1) untreated sample; (2) pure PA12 sheet; (3) PA12+10% PA6-GPHyp; and (4) PA12+10% PA6-PHypG in accordance with various embodiments disclosed herein. The untreated sample acts as a control.

[0231] FIG. 8 shows simultaneous thermal analysis (STA) results of PCL-GPHP in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PCL refers to poly(caprolactone) and GPHP refers to (GPHyp).sub.3.

[0232] FIG. 9 shows simultaneous thermal analysis (STA) results of PCL-RGD in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PCL refers to poly(caprolactone) and RGD refers to arginine-glycine-aspartic acid.

[0233] FIG. 10 shows thermogravimetric analysis (TGA) results of PCL-GPHP samples of 3D printed sheet and its filament in accordance with various embodiments disclosed herein. In the figure, PCL-GPHP 3DP sheet is represented by graph [2]; dashed line and its filament is represented by graph [1]; solid line. PCL refers to poly(caprolactone) and GPHP refers to (GPHyp).sub.3.

[0234] FIG. 11 shows simultaneous thermal analysis (STA) results of PLA-GPHP in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PLA refers to poly(lactic acid) and GPHP refers to (GPHyp).sub.3.

[0235] FIG. 12 shows simultaneous thermal analysis (STA) results of PLA-RGD in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PLA refers to poly(lactic acid) and RGD refers to arginine-glycine-aspartic acid.

[0236] FIG. 13 shows simultaneous thermal analysis (STA) results of PLA-HA in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PLA refers to poly(lactic acid) and HA refers to hyaluronic acid.

[0237] FIG. 14 shows thermogravimetric analysis (TGA) results of PLA-GPHP samples of 3D printed sheet and its filament in accordance with various embodiments disclosed herein. In the figure, PLA-GPHP 3DP sheet is represented by graph [2]; dashed line and its filament is represented by graph [1]; solid line. PLA refers to poly(lactic acid) and GPHP refers to (GPHyp).sub.3.

[0238] FIG. 15 shows simultaneous thermal analysis (STA) results of PLGA-GPHP in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PLGA refers to poly(lactic-co-glycolic acid) and GPHP refers to (GPHyp).sub.3.

[0239] FIG. 16 shows simultaneous thermal analysis (STA) results of PLGA-RGD in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PLGA refers to poly(lactic-co-glycolic acid) and RGD refers to arginine-glycine-aspartic acid.

[0240] FIG. 17 shows simultaneous thermal analysis (STA) results of PLGA-HA in accordance with various embodiments disclosed herein. In the figure, thermogravimetric analysis (TGA) graph is represented by solid line (see y-axis on the left) and differential scanning calorimetry (DSC) is represented by dashed line (see y-axis on the right). PLGA refers to poly(lactic-co-glycolic acid) and HA refers to hyaluronic acid.

[0241] FIG. 18 shows thermogravimetric analysis (TGA) results of PLGA-HA samples of 3D printed sheet and its filament in accordance with various embodiments disclosed herein. In the figure, PLGA-HA 3DP sheet is represented by graph [2]; dashed line and its filament is represented by graph [1]; solid line. PLGA refers to poly(lactic-co-glycolic acid) and HA refers to hyaluronic acid.

[0242] FIG. 19 is a graph showing biocompatibility test results (i.e. % cell viability count) of sheet samples, namely (1) untreated sample; (2) pure PLGA sheet; (3) PLGA-RGD; (4) PLGA-HA; (5) untreated sample; (6) pure PLA sheet; (7) PLA-RGD; (8) PLA-HA; and (9) PLA-GPHP in accordance with various embodiments disclosed herein. The untreated sample acts as a control.

[0243] FIG. 20 and FIG. 21 show a comparison of bio-implanted murine skin tissues using PA-based materials in accordance with various embodiments disclosed herein.

[0244] FIG. 20A shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 2+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0245] FIG. 20B shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 3+ for PA12. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0246] FIG. 20C shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 3+ for PA12-(PA6-GPHP). Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0247] FIG. 21A shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 2+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0248] FIG. 21B shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 3+ for PA12. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0249] FIG. 21C shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 3+ for PA12-(PA6-GPHP). Bioactive nylon filament PA12-(PA6-GPHP) consists of PA12 as base polymer and PA6-GPHP as the bioactive copolymer. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0250] FIG. 22 and FIG. 23 show a comparison of bio-implanted murine skin tissues using PCL-based materials in accordance with various embodiments disclosed herein.

[0251] FIG. 22A shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0252] FIG. 22B shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for PCL. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0253] FIG. 22C shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for PCL-RGD. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0254] FIG. 22D shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for PCL-GPHP. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0255] FIG. 23A shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0256] FIG. 23B shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for PCL. Image shown is representative with at least 4 C.sub.57BL/6 mice per group. Scale bar=50 m.

[0257] FIG. 23C shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for PCL-RGD. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0258] FIG. 23D shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Skin tissues from negative control mice and bio-implanted mice elicited comparable level of immune response. Pathological assessment reports 1+ for PCL-GPHP. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0259] FIG. 24 and FIG. 25 show a bio-implanted murine skin tissues using PLA-based materials in accordance with various embodiments disclosed herein.

[0260] FIG. 24A shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 1+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0261] FIG. 24B shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 1+ for PLA. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0262] FIG. 24C shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 1+ for PLA-HA. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0263] FIG. 24D shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 0+ for PLA-RGD. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0264] FIG. 24E shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 1+ for PLA-GPHP. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0265] FIG. 25A shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 1+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0266] FIG. 25B shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 1+ for PLA. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0267] FIG. 25C shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 1+ for PLA-HA. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0268] FIG. 25D shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 0+ for PLA-RGD. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0269] FIG. 25E shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 1+ for PLA-GPHP. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0270] FIG. 26 and FIG. 27 show a bio-implanted murine skin tissues using PLGA-based materials in accordance with various embodiments disclosed herein.

[0271] FIG. 26A shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 2+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0272] FIG. 26B shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 2+ for PLGA. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0273] FIG. 26C shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 2+ for PLGA-RGD10%. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0274] FIG. 26D shows an image obtained from immunohistochemistry (IHC) staining for CD3.sup.+ cells, with cell nuclei (dark coloured spots) and CD3 (representatively circled) differentially labelled. Pathological assessment reports 1+ for PLGA-RGD20%. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0275] FIG. 27A shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 2+ for negative control. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0276] FIG. 27B shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 2+ for PLGA. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0277] FIG. 27C shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 2+ for PLGA-RGD10%. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0278] FIG. 27D shows an image obtained from Hematoxylin and Eosin (H&E) staining, with nuclear component (hematoxylin) and cytoplasmic components (eosin) differentially stained. Pathological assessment reports 1+ for PLGA-RGD20%. Image shown is representative with at least 4 C57BL/6 mice per group. Scale bar=50 m.

[0279] FIG. 28 shows Hematoxylin and Eosin (H&E) staining image of skin tissue around implantation site. PLGA was observed to be intact despite repeated washing and attempts to detach material from tissues. Skin tissues were also observed to fill up the void left behind by degraded PLGA (circled area).

[0280] FIG. 29 shows alkaline phosphatase (ALP) activity of C2C12 cells cultured on PCL coupons pre-incubated with or without BMP-2 after 3 days. Data are represented as the mean ALP activity (n=2) relative to no treatment.

[0281] FIG. 30 shows alkaline phosphatase (ALP) activity of C2C12 cells cultured on PLA coupons pre-incubated with or without BMP-2 after 3 days. Data are represented as the mean ALP activity (n=2) relative to no treatment.

EXAMPLES

[0282] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, biological and/or chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0283] Fused filament fabrication (FFF) or fused deposition modelling (FDM) allows for end-to-end precise manufacturing of medical devices with biological and mechanical properties using suitable filament feedstock. Creation of filaments made of appropriate polymers is paramount to the utilization of FFF or FDM printing technology. The following examples present the technical process of creating filament made of bioactive polymers for FFF-based or FDM-based 3D printers with 2.85 mm filament diameter configuration. Twin-screw extruder (TSE) was utilized in the examples to produce bioactive polymer filaments as it allows good mixing of base polymer and bioadditive while reducing residence time in the heated sections to avoid thermal degradation of material. Thermal analysis, mechanical test and biocompatibility tests were performed to determine the material properties for its mechanical and biological competence.

[0284] The following examples describe the conversion of bioactive polymer formulation (comprising a base polymer and a bioadditive) into filament feedstock which can be used with FFF-type or FDM-type 3D printers for medical device manufacture. That is, filament feedstock are created which are made of bioactive polymers comprising a copolymer of biological molecules (e.g., oligopeptide, collagen and/or sugar, oligosaccharides, hyaluronic acid) and a synthetic polymer. The filament feedstock to be fed into the printhead is made of bioactive polymers. A method of producing bioactive polymer filaments with bioadditive homogenously distributed throughout the base polymer matrix is also described.

Example 1: Base Polymers and Bioadditives

[0285] Fused filament fabrication (FFF) or fused deposition modelling (FDM) is a preferred manufacturing method due to its technological advantages. Advantageously, the filament extrusion process offers significant material blending that enables the use of developed bioactive polymers. The base polymer and bioadditive may be chosen depending on the biological and physical requirements of the desired application. The term bioadditive refers to a copolymer of synthetic polymer and biological molecule prepared by ring-opening metathesis polymerisation (ROMP). It is an acellular material which does not contain any live cells but is able to stimulate host cells to proliferate which promotes tissue growth. Scheme 1 below shows examples of different base polymers and bioadditives that may be used.

[0286] Base polymers can be various thermoplastic polymers or free radical polymers to be used with the bioadditive composition to produce the bioactive polymer disclosed herein. Examples of polymers are polyamide, poly(lactic-co-glycolic) acid, polycaprolactone, poly(lactic) acid, polyacrylate, polystyrene and polyurethane. Bioadditive is a copolymer of biological molecules and synthetic polymer prepared by ring-opening metathesis polymerisation (ROMP) method. Synthetic polymer herein may or may not be similar to that of the base polymer disclosed. Examples of biological molecules are collagen, oligopeptide, oligosaccharides, sugar and hyaluronic acid. In various embodiments, the method disclosed herein comprises incorporating at least one bioadditive composition with one base polymer into the monofilament required as the feedstock for FFF or FDM method of manufacturing.

Example 2: Experimental Process Workflow

[0287] Scheme 2 shows the complete process workflow. Material preparation includes bioadditive synthesis, cryogenic grinding of base polymer, vacuum drying of materials and blending of bioadditive with base polymer.

[0288] Bioactive polymer filament extrusion constitutes of cryogenic milling of base polymer pellets into powder form, vacuum drying of base polymer and bioadditive, blending of base polymer and bioadditive into a single formulation and filament extrusion of formulation into monofilaments.

[0289] Base polymer pellets were cryogenically grinded to powder form that is less than 1 mm in particle size using SPEX 6770 Freezer/Miller or SPEX 6875 Freezer/Miller. Each base polymer (e.g., polyamide-12 (PA12), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL)) was subjected to different cryogenic grinding parameters as shown in Table 1. The vacuum drying parameters are shown in Table 2. Subsequently, the base polymer and bioadditive were mixed using ThinkyMixer ARE-250 or SPEX 8000D Mixer/Mill for at least 10 to 12 minutes and this mixture is referred to as the formulation.

[0290] Filament extrusion was performed using ThermoScientific Process 11 Twin-Screw Extruder (TSE) which comprises extruder, melt pump with a nozzle of 3 mm, water bath and haul unit. The filament extrusion parameters are shown in Table 3 and the filament extrusion result is shown in Table 4. Filament diameter needs to range between 2.5 mm to 3.1 mm as the FFF printer is configured for 2.85 mm filament. It is also able to manufacture filament suitable for FFF printer with 1.75 mm filament configuration.

[0291] In the following examples, simultaneous thermal analysis (STA) comprising thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were carried out at different points to obtain the thermal properties of the bioadditive, bioactive polymer filament and 3D printed sheet. These results would provide necessary data required for filament extrusion and allow a side-by-side comparison of onset degradation of the materials after undergoing two high temperature processes, namely filament extrusion and 3D printing.

[0292] Mechanical tests were carried out according to American Society for Testing and Materials (ASTM) standards to determine the relevant mechanical properties of the materials tested.

[0293] Samples were also sent for biocompatibility tests with various human cell lines which the materials are designed to interact with.

TABLE-US-00001 TABLE 1 Cryogenic grinding parameters for base polymers Number Precool Cycle Cool Impactor Speed of Cycles Time (min) Duration (min) Time (min) (cycles/second) PA12, PLA 3 1 3 2 14 PCL, PLGA 2 1 3 2 14

TABLE-US-00002 TABLE 2 Vacuum drying parameters Material Drying Temperature ( C.) Duration (Hrs) PA12 80 8-12 PA12 bioadditives 45 8-12 PCL 40 <12 PCL bioadditives 42.5 <12 PLA 45 <12 PLA bioadditives 45 <12 PLGA 40 <12 PLGA bioadditives 42.5 <12

TABLE-US-00003 TABLE 3 Filament extrusion parameters Melt Melt Screw Pump Feed Heating Die Pump Spool Speed Speed Rate Zone 2 Zone 3 Zone 4 Zone 5 Zone 6 Zone 7 Zone 8 Temp. Temp. Speed (RPM) (RPM) (%) ( C.) ( C.) ( C.) ( C.) ( C.) ( C.) ( C.) ( C.) ( C.) (m/min) Material(s): PA12, PA12-GPHP, PA12-PHPG/Cooling medium: Water 105 5 14 180 200 210 220 240 240 230 230 220 0.4 Material(s): PCL, PCL-GPHP, PCL-RGD/Cooling medium: Water 275-360 4.8-5.sup. 18-25 40 60 70 80 90 100 100 95 85 0.4 Material(s): PLA, PLA-GPHP, PLA-RGD, PLA-HA/Cooling medium: Air 220-280 10-12 12 100-180 140-190 180-200 200-210 210-220 210-230 210-220 210-220 205-215 0.4 Material(s): PLGA, PLGA-RGD, PLGA-HA/Cooling medium: Air 220-260 4.5-4.7 11-15 60-100 100-140 120-180 140-190 160-200 180-200 180-190 170-180 160-170 0.4

Example 3: Polyamide (PA)-Based Bioactive Polymer Filament

3.1. Bioactive Polymer Filament

(e.g., Produced from Polyamide (PA) and Collagen)

[0294] In this example, the collagen-like peptides used for synthesis were (Pro-Hyp-Gly).sub.3, abbreviated as PHPG, and (Gly-Pro-Hyp).sub.3, abbreviated as GPHP. Bioadditives synthesized are chemically similar and comprised poly(norbornene) dicarboximide brush polymers with polyamide-6 and poly(ethylene glycol)-collagen side chains which are referred to as PA6-PHPG and PA6-GPHP. The bioactive polymers produced by filament extrusion are referred to as PA12-PHPG and PA12-GPHP. Scheme 3 shows chemical structure of an example of PA6-[GPHyp]3 brush copolymer bio additive.

3.2. Simultaneous Thermal Analysis (STA

[0295] STA was carried out for each bioadditive component prior to filament extrusion. From FIG. 1 and FIG. 2, DSC graphs featured PA6-GPHP and PA6-PHPG melting at 213.2 C. and 211.5 C. respectively which are higher than the melting temperature of PA12 (180 C.). Without being bound by theory, it is believed that this was largely due to the presence of PA6 which is known to have higher melting temperature than PA12. TGA graphs of PA6-GPHP and PA6-PHPG highlights onset degradation temperatures of 336.5 C. and 313.9 C. respectively at 95% weight percentage. These results establish the working temperature range that should be adhered to. This facilitates in determining the suitable temperature profile to be used on twin screw extruder (TSE) for a good blending process during the filament extrusion of PA12-collagen.

##STR00008##

3.3. Filament Extrusion and PA12-Collagen Filament

[0296] Filament extrusion was carried out using the parameters as shown in Table 3. It was observed that thermoplastic polymers exhibit good melt viscosity when heated at 15 C. to 40 C. above the melting temperature which enables good material flow for filament extrusion. Melt pump temperature of 220 C. was specifically used as the molten extrudate to allow more time for it to be manipulated into the water bath, measuring unit and haul unit for spooling process to occur. In addition to the above, a desired filament diameter can be achieved by adjusting the speed-based parameters such as screw speed, melt pump and spool speed. Molten extrudate which has cooled down is then referred to as the filament.

[0297] FIG. 3 shows a photograph of the obtained PA12-collagen filaments, which appeared to be in a translucent tangerine colour.

3.4. Thermogravimetric Analysis (TGA)

[0298] FIG. 4 shows two graphs stacked together, featuring PA12-PHPG samples of 3D printed sheet (dashed line) and its filament (solid line) while FIG. shows two graphs stacked together, featuring PA12-GPHP samples of 3D printed sheet and filament. It was observed that PA12-PHPG filament and 3D printed samples have similar thermal degradation profile whereby the difference in degradation temperature at various weight percentages ranged between 0.09 to 0.8% only. Similarly, PA12-GPHP exhibited similar thermal degradation profile for filament and 3D printed samples whereby the difference in degradation temperature at various weight percentages ranged between 0.4 to 1.5%. This finding substantiates the thermal stability of the bioactive copolymers tested and shows that the biological molecule is not lost after filament extrusion and 3D printing.

3.5. Mechanical Properties

[0299] Mechanical test was carried out to determine the tensile properties of the materials according to ASTM D638 (Standard Test Method for Tensile Properties of Plastics). The test specimens were dimensioned to the specifications of Type V specimen. Results from the test are compiled and shown in the bar graphs in FIG. 6.

[0300] It can be observed that the Young's modulus and yield strength of PA12-PHPG and PA12-GPHP were reduced as compared to pure PA12 which implies that the addition of the bioadditive resulted in those reduction. This was due to the pegylated peptide in the bioactive polymers as the biological molecules are amorphous with no significant mechanical advantage which compromises the tensile test results.

[0301] From the Young's modulus result, PA12 has an average of 1.68 GPa while PA12-GPHP and PA12-PHPG have an average of 1.44 GPa and 1.14 GPa, which translate to a reduction of 14.3% and 32.1% respectively.

[0302] From the yield strength result, PA12 has an average of 16.53 MPa while PA12-GPHP and PA12-PHPG have an average of 12.79 MPa and 12.16 MPa, which translate to a yield strength reduction of 22.6% and 26.4% respectively.

[0303] It was observed that the specimens did not fracture cleanly as a whole because some layers of the specimens fractured earlier than others and this was observed in all of the materials tested. However, PA12 test specimens displayed less of this phenomenon as compared to PA12-PHPG and this was largely due the nature of FFF method of manufacturing. In addition, two of the PA12-PHPG specimens also exhibited premature layer delamination during the test which could have compromised the mechanical properties to a certain degree. This was due to a printing issue known as under-extrusion whereby very little material was extruded which created gaps between the infill lines and inconsistent layer height.

3.6. Biocompatibility Tests

[0304] Biocompatibility test was performed using cell viability test assays with human fibroblasts Hs27 on 3D printed sheet samples. The result indicates that each of the bioactive material tested was biocompatible in comparison to untreated and virgin PA12 groups, as shown in FIG. 7.

Example 4: Polycaprolactone (PCL)-Based Bioactive Polymer Filament

[0305] In this example, simultaneous thermal analysis (STA) consisting of thermogravimetric (TGA) and differential scanning calorimeter (DSC) analyses were performed on PCL-based bioadditives and bioactive PCLs after extrusion and 3D printing (3DP) processes. From FIG. 8 and FIG. 9, it can be derived that bioadditives, PCL-GPHP and PCL-RGD, melted between 59 C. to 60 C. while onset degradation occurred at 257.9 C. and 282 C. respectively at weight percentage of 95%. These results enabled for process optimization of extrusion and 3DP processes whereby processing temperatures should be between 90 C. to 240 C. ensuring good melt flow. The parameters used for filament extrusion process are shown in Table 3 above. Additionally, FIG. 10 shows the thermographs of bioactive PCL, PCL-GPHP, after filament extrusion and 3DP processes. It can be seen that the degradation at various weight percentages were similar with a difference ranging between 0.2 to 0.5%. This exhibits the thermal stability of the bioadditive where there was no loss of biological molecule after two high heat processes.

Example 5: Poly(Lactic Acid) (PLA)-Based Bioactive Polymer Filament

[0306] In this example, simultaneous thermal analysis (STA) consisting of thermogravimetric (TGA) and differential scanning calorimeter (DSC) analyses were performed on PLA-based bioadditives.

[0307] FIG. 11, FIG. 12 and FIG. 13 respectively show the STA graphs of PLA-based bioadditives, namely PLA-GPHP, PLA-RGD and PLA-HA whereby no specific melting points were identified which corresponded to an amorphous structure of the bioadditives. Onset degradation of the bioadditives occurred between 224 C. to 254 C. at 95% weight percentage. PLA-HA attained the highest temperature resisting thermal degradation due to the longer chain length of hyaluronic acid as compared to peptide and collagen. However, pure semi-crystalline PLA used as the base polymer exhibited a melting point of 190 C., resulting in a narrow processing temperature range, namely 190 C. to 220 C. for PLA-GPHP & PLA-RGD and 190 C. to 250 C. for PLA-HA. Table 3 shows the parameters used for filament extrusion process. From FIG. 14, TGA thermographs of PLA-GPHP filament and 3D printed samples exhibited similar degradation points at different weight percentages with the difference ranging between 2.7% to 3.5%. This shows the thermal stability of the bioadditive whereby no biological molecule was lost after undergoing extrusion and 3DP processes at elevated temperatures.

Example 6: Poly(Lactic-Co-Glycolic Acid) (PLGA)-Based Bioactive Polymer Filament

[0308] In this example, simultaneous thermal analysis (STA) consisting of thermogravimetric (TGA) and differential scanning calorimeter (DSC) analyses were performed on PLGA-based bioadditives.

[0309] As shown in FIG. 15, FIG. 16 and FIG. 17, the melting point and onset degradation of PLGA bioadditives were established by STA. Melting points of PLGA-GPHP, PLGA-RGD and PLGA-HA were not clearly identified and this highlighted the amorphous nature of the bioadditives' crystal structure. This is also similar to the pure PLGA used as the base polymer. Nevertheless, the onset degradation of these bioadditives ranged between 243 C. to 281 C. while pure PLGA degrades at 309 C. Table 3 shows the parameters used for filament extrusion process. In addition, FIG. 18 shows the TGA thermographs comparing the degradation points at different weight percentages of PLGA-HA filament and 3DP samples. The difference of degradation points between two samples ranged between 5 to 10% only.

Example 7: Biocompatibility Test of PLGA-Based and PLA-Based Materials

[0310] Material biocompatibility was tested by cell viability assay method using chondrocytes (CHON-001) cell line on 3DP samples. FIG. 19 shows the cell viability percentage after 72 hours incubation period. For PLGA-based materials, it can be observed that PLGA-HA has the best performance as compared to the other PLGA materials. Alternately, PLA-based materials experienced lower cell viability. PLA-GPHP performed similar to PLA at an estimated of 75% cell viability while PLA-RGD and PLA-HA exhibited inferior performance.

Example 8: In Vivo Biocompatibility Analysis

[0311] In vivo biocompatibility analysis of 3D printed coupons was performed using murine models. 8 weeks old female C.sub.57BL/6 wild type mice were purchased from InVivos Pte Ltd and randomly assigned to the material groups. All groups had at least 4 mice. Immunohistochemistry staining of murine skin tissues from study groups was performed using PA12-based (FIG. 20A, FIG. 20B, FIG. 20C and FIG. 21A, FIG. 21B, FIG. 21C), PCL-based (FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D and FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D), PLA-based (FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D, FIG. 24E and FIG. 25A, FIG. 25B, FIG. 25C, FIG. 25D, FIG. 25E) and PLGA-based (FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D and FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D) materials. Briefly, each mice was anaesthetized with ketamine/xylazine and a small incision was made on the upper dorsal back. Coupons were then inserted into the subcutaneous space under the skin. 4 weeks post implantation, all mice were sacrificed. Mouse skin surrounding the implant was harvested, fixed in formalin and embedded in paraffin for histological studies. Haematoxylin and Eosin (H&E) staining, as well as immunohistochemistry staining for CD3 (clone SP162) was performed on these skin sections. Appropriate controls were included and images were captured using Zeiss Axio Scan Z1 slide scanner. Analyses was carried out by a trained histopathologist, Dr Joe Yeong of Institute of Molecular Cell Biology, A*STAR. All animal handling procedures were approved by the Institutional Animal Care and Use Committee and conformed to the National Advisory Committee for Laboratory Animal Research Guidelines (IACUC #201550).

[0312] PA12 samples showed increased inflammatory response relative to sham (no implantation) for both pure PA12 samples and PA12-GPHyp samples. PA12-GPHyP represents bioactive PA12 that is PA-6 brush copolymer with [GPHyp).sub.3] peptide, blended in PA12 base polymer. PCL samples showed no increase in inflammatory response after implantation, as compared to sham, for both pure PCL and bioactive PCL (PCL brush copolymers with RGD or [(GPHyp).sub.3] peptides). Interesting findings were observed for PLA and PLGA samples in that both bioactive PLA and bioactive PLGA were observed to reduce inflammatory response in vivo. Pure PLA, PLA-HA (PLA brush copolymer with hyaluronic acid of MW 3,000-5,000) and PLA-GPHyp showed slight reduction in inflammatory response in vivo. However, PLA-RGD showed negligible inflammatory response in vivo, relative to sham. This is exciting as it showed the ability of PLA-RGD brush copolymers being able to reduce inflammatory response in vivo, making it a useful material for 3DP implants such as fixation devices ad biodegradable sutures. Likewise, 20% bioactive PLGA (PLGA-RGD brush copolymer) in base PLGA, was also observed to reduce inflammatory response in vivo. PLGA was observed to be intact despite repeated washing and attempts to detach material from tissues. Skin tissues were also observed to fill up the void left behind by degraded PLGA (FIG. 28). This makes bioactive PLGA a good material for applications such as 3DP skin scaffolds.

[0313] As can be seen, the results also advantageously show retention of bioactivity.

Example 9: Alkaline Phosphatase (ALP) Activity

[0314] Alkaline phosphatase (ALP) activity of C2C12 cells cultured on PCL coupons pre-incubated with or without BMP-2 after 3 days are measured and presented in FIG. 29.

[0315] Alkaline phosphatase (ALP) activity of C2C12 cells cultured on PLA coupons pre-incubated with or without BMP-2 after 3 days are measured and presented in FIG. 30.

[0316] Alkaline phosphatase (ALP) assay test was performed on both 3DP PCL and PLA sheets using C2C12 murine myoblast cells. Base polymers used were eSun 800C and Resomer L210S respectively. Bioactive copolymers used were PCL-RGD, PCL-GPHP, PLA-RGD and PLA-GPHP where RGD refers to arginine-glycine-aspartic acid and GPHP refers to (GPHyp).sub.3 peptide. In the absence of BMP-2 for PCL, it was observed that PCL-GPHP material had greater ALP activity as compared to the no treatment group, pure PCL and PCL-RGD materials. This strongly suggests that PCL-GPHP may have osteoinductive properties which can be attributed to the inclusion of (GPHyp).sub.3 peptide in its copolymer. However, the same trend could not be observed when the materials were pre-incubated with BMP-2 as pure PCL exhibited the best performance with a marginal advantage over PCL-GPHP in ALP activity. As for PLA without BMP-2, it can be observed that PLA-GPHP samples have slight increase in ALP activity over pure PLA sample and no treatment group. When the PLA samples are pre-incubated with BMP-2, ALP activity increased significantly as compared to no treatment group with PLA-GPHP sample exhibiting the best result. This finding suggests that PLA-GPHP may also have osteoinductive property which is induced by the (GPHyp).sub.3 peptide in its copolymer. GPHyp is a common motif present in fibrilla collagen, including collagen l2, the predominant protein in bone and GPHyp is known to enhance bone formation. Hence, the ability to observe ALP activities from 3DP sheets containing GPHyp-bearing bioadditive is indicative of the possibility of using bioactive polymer filaments if such materials are made available for orthopaedic implants such as 3DP bone grafts.

Example 10: Technical Features of Bioactive Polymer Filament

[0317] Advantageously, various embodiments of the bioactive polymer filament disclosed herein possess thermal stability, mechanical/physical competence/characteristics and biological competence/characteristics that makes its suitable for use as a feedstock in printing medical-related structures using FFF or FDM 3D printing technologies. Scheme 4 shows some of the key technical features of the bioactive polymer filament produced in accordance with various embodiments disclosed herein.

[0318] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.