Three dimensional printable biodegradable metal infused bone regenerative material utilizing additive manufacturing for bone tissue engineering

Abstract

A bioactive composite filament for fused-deposition modeling comprises 10 vol% to about 60 vol% biodegradable metal powder (about 10 µm to about 100 µm particle size) and about 40 vol% to about 90 vol% biocompatible thermoplastic polymer. The filament exhibits controlled rheology (viscosity ≤ 5.5×10⁵ Pa·s at 120 °C), enhanced mechanical properties (up to 3-fold increase in tensile modulus), and printability with high fidelity (print score ≥ 0.85). Three-dimensional scaffolds manufactured from the filament demonstrate pH-neutral degradation, composition-tunable metal ion release, and sustained bioactivity with greater than 95% cell viability over 21 days. The manufacturing process involves cryogenic milling, inert-atmosphere blending, screw extrusion, and layer-by-layer deposition to produce porous scaffolds or non-porous implants for bone tissue engineering and orthopedic applications.

Description

THREE DIMENSIONAL PRINTABLE BIODEGRADABLE METAL INFUSED BONE REGENERATIVE MATERIAL UTILIZING ADDITIVE MANUFACTURING FOR BONE TISSUE ENGINEERINGTECHNICAL FIELD

[0001] Composite materials for additive manufacturing, specifically bioactive metal-polymer composite filaments for fused-deposition modeling (FDM) of biodegradable scaffolds and implants for bone tissue engineering and orthopedic applications.BACKGROUND

[0002] Composite filaments incorporating metal, ceramic, or fiber reinforcements into polymer matrices have enabled additive manufacturing of functional parts with enhanced mechanical, thermal, and biological properties. Metal loadings in thermoplastic fused-deposition modeling (FDM) filaments rarely exceed 15% to 20 wt%. however, due to processing constraints including elevated melt viscosity, inadequate particle wetting, filament brittleness, and nozzle clogging. These limitations restrict achievable property enhancements and preclude applications requiring high metal content for bioactivity, conductivity, or mechanical reinforcement.

[0003] For bone tissue engineering applications, biodegradable metals, such as magnesium, offer advantageous properties including bone-matching elastic modulus (10 GPa to 20 GPa), promotion of osteogenesis via cellular signaling pathways, and controlled degradation compatible with bone remodeling timescales. Such Mg-polymer composites for FDM, however, have been limited to <20 wt% Mg due to the aforementioned processing barriers, thereby restricting their mechanical and biological performance.SUMMARY

[0004] In some aspects, the presently disclosed subject matter provides a fused-deposition modeling (FDM) printing filament comprising a composite having between about 10% to about 60% by volume of biodegradable metal powder having a mean particle size between about 10 pm and about 100 pm; and between about 40% to about 90% by volume of a biocompatible, biodegradable thermoplastic polymer powder having a melting temperature below 250 °C; wherein: a ratio of thermoplastic polymer powder particle size to metal powder particle size does144426.601_P18564-02not exceed about 10:1; the composite has a viscosity of about 1.6xl05to about 5.5x105Pa-s at 120 °C and a tensile modulus of about 100 MPa to 1000 MPa; and wherein particles comprising the metal powder are encapsulated beneath a continuous surface layer of the thermoplastic polymer.

[0005] In some aspects, the thermoplastic polymer comprises polycaprolactone (PCL) having a melting temperature between about 59 °C and about 60 °C and a crystallization enthalpy between about 14 J / g and about 17 J / g.

[0006] In some aspects, the biodegradable metal powder comprises a metal selected from magnesium, a magnesium alloy, iron, an iron alloy, zinc, a zinc alloy, and combinations thereof.

[0007] In some aspects, the filament comprises a cross-section, a periphery, and a center and wherein: particles comprising the metal powder are distributed throughout the filament crosssection with a particle number density between about 82 particles / mm2and about 385 particles / mm2; and a ratio of the particle number density at the filament periphery to a ratio of the particle number density at the filament center does not exceed 1.8:1.

[0008] In some aspects, the composite has one or more properties selected from: a yield stress that increases by a factor of about 2.5 to about 3.5 relative to neat thermoplastic polymer as metal volume fraction increases from about 0% to about 60%; a flow behavior index that decreases from about 0.92 to about 0.82 as metal volume fraction increases from about 0% to about 60%; and an apparent viscosity at about a 0.1 s’1shear rate that does not exceed about 5.5xl05Pa-s at about 120 °C.

[0009] In some aspects, the filament has an effective diameter between about 100 pm and about 4 mm, and wherein the effective diameter varies by less than about 10% over any 100-mm length of the filament after an initial stabilization distance of about 50 mm from extrusion initiation.

[0010] In some aspects, incorporation of metal powder into the thermoplastic polymer does not alter the polymer melting temperature by more than about 2 °C relative to neat thermoplastic polymer, and wherein a melting enthalpy remains within about 17 J / g to about 20 J / g across all metal volume fractions.

[0011] In some aspects of the presently disclosed filament, a maximum achievable metal volume fraction scales inversely with metal powder particle size; and about a 60 % by volume of metal powder loading with a mean particle size of about 44 pm.

[0012] In other aspects, the presently disclosed subject matter provides a three-dimensional porous scaffold comprising: a composite described hereinabove having a pore size between 100 pm and244426.601_P18564-021.2 mm configured to permit vascular ingrowth and interconnected porosity with isotropic or anisotropic pore architecture; a print fidelity score greater than 0.85, wherein the print fidelity is defined as:( Ave. Deviation . ,+Ave. Deviation \1 - — -1— j x Strut Completion0 / ); and wherein:

[0013] the scaffold exhibits controlled metal ion elution in aqueous media without pH deviation exceeding about 0.5 units; and the scaffold maintains structural integrity for at least 21 days in physiological media at body temperature, e.g., a temperature between about 37 °C to about 40 °C and a physiological pH between about 7 and about 7.8.

[0014] In some aspects, the scaffold comprises one or more properties selected from: a tensile Young’s modulus that increases by a factor from about 1 up to about 3 relative to a scaffold fabricated from neat polymer; an increase in the tensile Young’s modulus that scales linearly with metal volume fraction according to rule-of-mixtures predictions; and a compressive modulus having a range from about 2 MPa to about 100 MPa, which is a typical range of cancellous bone.

[0015] In certain aspects, the scaffold exhibits the following properties: one or more strut dimensions deviate from a design target by less than about 15%; one or more pore dimensions deviate from a design target by less than about 10% and a strut completion percentage exceeds about 95%.

[0016] In certain aspects, one or more strut surfaces exhibit gradual roughening with increasing metal content without evidence of interfacial failure, particle pull-out, or cracking; and particles comprising the metal powder are encapsulated beneath a continuous polymer layer, and remain that way during manufacture, as verified by pCT imaging and scanning electron microscopy.

[0017] In other aspects, the presently disclosed subject matter provides a method for manufacturing a bioactive composite filament, the method comprising: cryogenic milling a thermoplastic polymer to produce a polymer powder having a particle size distribution with a mean diameter of about 120 pm; providing biodegradable metal powder having a mean particle size between 10 pm and 100 pm; blending the polymer powder and metal powder in an inert atmosphere to produce a blended powder having a target metal volume fraction between about 10% and about 60%; extruding the blended powder through a heated die at a temperature not exceeding about 30 °C above a melting temperature of the thermoplastic polymer to produce an extruded filament; cooling the extruded filament by convection at an ambient temperature, e.g.. a temperature between about 15 °C to about 35 °C) and ambient humidity, e.g., a relative humidity 344426.601_P18564-02between about 0% to about 100%; and winding the cooled filament onto a spool for storage in a moisture-controlled environment.

[0018] In certain aspects, the cryogenic milling comprises: precooling thermoplastic polymer pellets in liquid nitrogen for about 3 minutes; grinding the precooled thermoplastic polymer pellets for about one minute at an impact frequency of about 10 cycles per second; repeating for 45 consecutive cycles, with cooling for about 3 minutes in between each cycle, to achieve a target particle size; and verifying particle size distribution by dynamic light scattering.

[0019] In certain aspects, the blending comprises: transferring a pre-weighed metal powder to a container; layering thermoplastic polymer powder on top of the metal powder to minimize premature mixing; sealing the container in an inert atmosphere to prevent metal oxidation; and agitating on a vortex mixer for at least 5 minutes to achieve homogeneous blending.

[0020] In certain aspects, the extruding comprises: feeding the blended powder into a heated screw extruder; maintaining barrel temperatures of the heated die between about 65 °C to about 80 °C across heating zones; controlling screw speed between about 1 rpm and about 5 rpm to achieve a target filament diameter; monitoring filament diameter in real-time by laser micrometry; and collecting the filament after diameter stabilization over a distance of at least 50 mm.

[0021] In certain aspects, the method further comprises storing the filament in an airtight container with desiccant to maintain moisture content below about 0.1 wt% prior to printing.

[0022] In other aspects, the presently disclosed subject matter provides a method for three-dimensional printing of a bioactive scaffold, the method comprising: providing a filament as described hereinabove; heating the filament to a processing temperature between a melting temperature of the thermoplastic polymer and about 30 °C above the melting temperature; extruding the heated filament through a nozzle having an orifice diameter between about 400 pm and about 1000 pm; depositing the extruded filament layer-by-layer onto a build platform at a predetermined print speed to form a three-dimensional structure; and controlling layer thickness, infill density, infill pattern, and raster orientation to achieve target mechanical properties and porosity.

[0023] In certain aspects, the nozzle is maintained at a temperature of 105°C ± 5°C; the build platform is maintained at a temperature between about 50 °C to about 70°C; the layer thickness is between about 100 pm and about 400 pm; the print speed is controlled between about 10 mm / s and about 40 mm / s to optimize bonding between layers; and the infill pattern is selected from a444426.601_P18564-02rectilinear, a honeycomb, a gyroid, and a custom pattern to achieve target porosity and mechanical anisotropy.

[0024] In certain aspects, the method further comprises: varying metal volume fraction across one or more regions of the three-dimensional structure; creating functionally graded scaffolds with spatially varying mechanical properties, degradation rates, or ion release kinetics; and / or coprinting multiple composite formulations in a single build to achieve heterogeneous material distribution.

[0025] In certain aspects, the method further comprises immersion of the scaffold in cell culture medium for 21 days resulting in: a pH stability within ±0.3 units of an initial pH; instantaneous metal ion release scaling with metal volume fraction up to about 30 vol%, followed by a decreased release at higher loadings; and a cumulative metal ion concentration demonstrating compositiondependent elution kinetics suitable for therapeutic delivery.

[0026] In certain aspects, the method further comprises culturing the scaffold with osteoblastlineage cells for 21 days resulting in: progressive cell proliferation peaking at metal volume fractions between about 30 vol% to about 45 vol%; an increased normalized calcium deposition (Ca / DNA ratio) with increasing metal fraction, indicating enhanced osteoconductivity and biomineralization; and cell viability exceeding about 95% at all metal loadings as assessed by live / dead fluorescence staining.

[0027] In certain aspects, quality of the scaffold is verified by: measuring dimensional deviation for each strut and pore relative to CAD design targets; calculating average deviations separately for struts and pores; assessing strut completion percentage as the fraction of fully formed struts; computing an overall print score according to:( Avg. Deviation . ,+Ave. Deviation \1 - — -1— j x Strut Completion0 / ); and

[0028] accepting only scaffolds with print scores exceeding 0.85.

[0029] In other aspects, the presently disclosed subject matter provides a non-porous biomedical implant structure comprising a filament described hereinabove or prepared by the methods described herein, the implant structure comprising one or more of: a bone fixation screw having a customizable thread geometry, pitch, head design, and length; a fixation plate having a customizable thickness, hole pattern, and curvature; an intramedullary rod having a customizable diameter and length; and / or an orthopedic fixation device requiring mechanical strength, ductility, and biodegradability.544426.601_P18564-02

[0030] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0031] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0032] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0033] FIG. 1 is an illustration of the presently disclosed magnesium (Mg) three-dimensional (3D) printed scaffolds: 3D-printed custom-designed scaffolds for craniomaxillofacial bone regeneration comprise magnesium microparticles embedded within poly-e-caprolactone (PCL).

[0034] FIG. 2 is a schematic of material processing and characterization for Bespoke Bone showing the multi-material additive manufacturing of scaffolds and representative characterization tests.

[0035] FIG. 3 shows particle sizing via dynamic light scattering of cryomilled particles. DCB particles rapidly reduce in size, reaching a limit of roughly 100 pm after 45 cycles and plateauing.

[0036] FIG. 4 is a plot of the polycaprolactone (Plastic) and magnesium (Mg) composite tensile modulus of the PCL-Mg composite material. The addition of Mg increased the tensile modulus significantly, suggesting that inclusion of Mg strengthens the matrix of the polymer.

[0037] FIG. 5 shows the thermal properties of Mg-PCL composite material.

[0038] FIG. 6A and FIG. 6B show shear stress (FIG. 6A) and viscosity (FIG. 6B) at increasing shear rate up to 16 / s of PCL-Mg at 120 °C. We observe that the addition of Mg has the fluid behave in a shear-thickening behavior, which is not ideal in 3D printing. This observation is consistent with our printing speed limits determined experimentally.

[0039] FIG. 7A and FIG. 7B show SEM and SEM-EDX of PCL-Mg scaffold. To see the distribution of magnesium within the scaffold, pCT was conducted, and particle distribution was mapped.644426.601_P18564-02

[0040] FIG. 8A and FIG. 8B are pCT of PCL-Mg filament and a particle distribution map. (FIG.8A) pCT scans reveal that Mg particles are trapped under a very thin layer of PCL and exhibit a lack of surface presentation of biodegradable metal on the surface, which might have burned off with the beam in prolonged SEM-EDX resulting in surface presentation. FIG. 8B shows the magnesium particle distribution in composite polycaprolactone (plastic) and magnesium (metal) material.

[0041] FIG. 9A and FIG. 9B show the magnesium elution as a function of magnesium concentration 3D surface plots with various DCB weight percentage and particle size.

[0042] FIG. 10 shows magnesium elution as a function of surface area and surface area / volume ratio. We observed that while adding more material does slightly increase the release, cutting the material doubles the release in the same amount of time. As observed in pCT, Mg particles are trapped under a thin layer of hydrophobic PCL. By cutting the material, we are directly overcoming the lag time observed in FIG. 9 due to the material needing to “wet” as the water diffuse into the matrix of the material.

[0043] FIG. 11 A and FIG. 1 IB is a material production and characterization pipeline. (FIG. 11A) Material design rationale. Mg particles are suspended then trapped when pre-polymerized PCL pellets are melted then solidified by air convection at room temperature (25 °C). The Mg can then be eluted from the material via water entering the mesh matrix in vivo, reacting with Mg to create Mg(OH)2 which ionizes into Mg2+and OH’. (FIG. 11B) Production pipeline of novel composite material. (FIG. 11C) Materials characterizations performed at the various production stages.

[0044] FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, FIG. 12F, FIG. 12G, FIG. 12H, FIG.121, and FIG. 12J show the thermophysical properties of composite material as a function of Mg volume %. (FIG. 12A) Sample diameter readout from 3DEVO filament extruder. Red box indicates discarded filament, green box indicates collected filament. (FIG. 12B) Average diameters as Mg volume % increases. 0% (*) is commercially available filament at 2.86 mm ±0.1 mm. (FIG.12C) Melting temperature. (FIG. 12D) Crystallization temperature. (FIG. 12E) Enthalpy of melting. (FIG. 12F) Enthalpy of crystallization. (FIG. 12G) Shear stress vs shear rate at 120 °C. (FIG. 12H) Shear stress vs shear rate at 120 °C. (FIG. 121) Herschel Buckley modelled yield stress at 120 °C. (FIG. 12 J) Herschel Buckley modelled flow behavior index at 120 °C.

[0045] FIG. 13A, FIG. 13B. FIG. 13C, FIG. 13D, FIG. 13E. and FIG. 13F demonstrate that addition of Mg volume % does not affect particle distribution profile. (FIG. 13A) pCT of filament744426.601_P18564-02as a function of Mg volume %. Scale bars are 500 pm. (FIG. 13B) Particle counts as we go further from the center. (FIG. 13C) Average particle count per slice as a function of Mg volume %. (FIG.13D) Particle distribution per mm2as function of Mg volume %. (FIG. 13E) Representative XRD of 0%, 30% and 60% Mg volume %. (FIG. 13F) 30-402theta region of XRD of 0%, 30% and 60% Mg volume %.

[0046] FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D. FIG. 14E. FIG. 14F, FIG. 14G, FIG. 14H, FIG.141, and FIG. 14J demonstrate that addition of Mg changes mechanical properties compared to PCL only scaffolds. (FIG. 14A) Tensile ASTM 638 type V 3D Sample Model. (FIG. 14B) Tensile Young’s modulus as a function of Mg volume %. (FIG. 14C) Representative tensile curves. (FIG.14D) Peak tensile stress as a function of Mg volume %. (FIG. 14E) Peak tensile load as a function of Mg volume %. (FIG. 14F) Compressive Isotropic 1.2-mm pore Sample Model. (FIG. 14G) Compressive Young’s modulus as a function of Mg volume %. (FIG. 14H) Representative compression curves. (FIG. 141) Peak compressive stress as a function of Mg volume %. (FIG. 14J) Peak compressive load as a function of Mg volume %.

[0047] FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, and FIG. 15E demonstrate that print quality is relatively conserved as the Mg volume % increases. (FIG. 15 A) SEM of filament as a function of Mg volume %. Scale bars are 500 pm. (FIG. 15B) Representative stereoscope images of 2-layer scaffolds as a function of Mg volume %. Scale bars are 500 pm. (FIG. 15C) Strut dimensions in the vertical and horizontal direction compared to the design target of 800 pm (Red). (FIG. 15D) Pore dimensions compared to the design target of 1200 pm (Red). (FIG. 15E) Print quality as a function of Mg volume %.

[0048] FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, and FIG. 16E demonstrate that scaffold degradation releases Mg ions but does not affect pH. (FIG. 16 A) pH change in DMEM as a function of Mg volume %. (FIG. 16B) Mg concentration (PPM) released in DMEM as a function of Mg volume %. (FIG. 16C) Cumulative Mg concentration released as a function of Mg volume %. (FIG. 16D) SEM of 21 days degraded scaffolds. Scale bars are 500 pm. Scale bars are 500 pm. (FIG. 16E) pCT of 21 days degraded scaffolds. Scale bars are 500 pm.

[0049] FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, FIG. 17F show that scaffolds exhibit bioactivity in addition to degradation over the course of 21 days. (FIG. 17A) pH change in DMEM as a function of Mg volume %. (FIG. 17B) Mg concentration (PPM) released in DMEM as a function of Mg volume %. (FIG. 17C) Cumulative Mg concentration released as a function of Mg844426.601_P18564-02volume %. (FIG. 17D) DNA content of scaffolds at 7, 14 and 21 days (FIG. 17E) Calcium per DNA content of scaffolds at 21 Days (FIG. 17F) Live-dead staining of scaffolds after 7 days. Scale bars are 100 pm. s.

[0050] FIG. 18 is a MATLAB image processing pipelines for print score. Pipeline to process raw stereoscope images into print score. Algorithm uses MATLAB’s regionprops computer vision package to detect polygons by binarizing images, filling in holes and finding 4 sided polygons with 90 degree angles.

[0051] FIG. 19 is a MATLAB image processing pipelines for particle distribution. Pipeline to process reconstructed pCT images and map particles. Algorithm uses MATLAB’s regionprops computer vision package to detect particles from the background, tag, and map it relative to the detected center of the circle.

[0052] FIG. 20 is a pCT of scaffold degradation, demonstrating scaffold degradation over the course of 21 days for all study groups.

[0053] FIG. 21 shows SEMs of degraded scaffolds. SEM at various magnifications of 2 ID degraded scaffolds of all study groups.DETAILED DESCRIPTION

[0054] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0055] As provided in more detail herein below, the presently disclosed subject matter overcomes limitations currently known in the art by providing composite filaments with unprecedented metal loadings (up to about 60 vol%, about 69 wt%) that maintain printability, dimensional accuracy, and biological performance.

[0056] Certain embodiments of the presently disclosed subject matter include a cryogenic powder processing and inert- atmosphere blending protocol that achieves homogeneous metal particle dispersion with controlled size ratios; identification of rheological windows (viscosity <5.5xl05944426.601_P18564-02Pa-s, shear-thinning behavior) enabling extrusion and printing at extreme loadings; encapsulation of reactive metal particles beneath a continuous polymer surface layer, conferring environmental stability; demonstration of print fidelity >0.85 across all metal loadings, with linear mechanical reinforcement (up to 3-fold modulus increase) and composition-tunable degradation kinetics; and validation of sustained bioactivity including pH-neutral ion release, enhanced mineralization, and >95% cell viability over 21 days. Accordingly, the presently disclosed subject matter enables manufacturing of bioactive scaffolds and implants with independently tunable mechanical properties, degradation rates, and therapeutic ion delivery for bone regeneration applications.

[0057] Fused-deposition Modeling (FDM) Printing Filament

[0058] In some embodiments, the presently disclosed subject matter provides a fused-deposition modeling (FDM) printing filament comprising a composite having between about 10% to about 60% by volume of biodegradable metal powder, including about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42. 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54. 55, 56, 57, 58, 59, and 60%, having a mean particle size between about 10 pm and about 100 pm, including about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 pm; and between about 40% to about 90% by volume of a biocompatible, biodegradable thermoplastic polymer powder, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76. 77, 78, 79, 80, 81, 82, 83, 84, 85, 86. 87, 88, 89, and 90% by volume, having a melting temperature below 250 °C; wherein: a ratio of thermoplastic polymer powder particle size to metal powder particle size does not exceed about 10:1; the composite has a viscosity of about 1.6xl05to about 5.5xl05Pa-s at 120 °C and a tensile modulus of about 100 MPa to 1000 MPa, including about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 MPa; and wherein particles comprising the metal powder are encapsulated beneath a continuous surface layer of the thermoplastic polymer.

[0059] Without wishing to be bound to any one particular theory, the metal powder particle size is maintained between about 10 pm and about 100 pm to avoid spontaneous combustion of fine particles (less than 10 pm) and nozzle clogging of large particles (greater than 100 pm);

[0060] As used herein, fused-deposition modeling (FDM) printing refers to a manufacturing process for creating three-dimensional objects. The process utilizes a thermoplastic filament, which is unwound from a coil and fed through an extrusion nozzle. The nozzle is heated to a1044426.601_P18564-02temperature that allows the filament material to become semi-liquid. As the filament is extruded, the nozzle deposits it layer by layer to form the desired geometry.

[0061] During the printing process, the thermoplastic filament is heated in the extrusion nozzle to a temperature slightly above its melting point, making it pliable enough to be extruded. The extruder head lays down the filament on a build platform according to a programmed path for each layer. Once a layer is deposited, the platform may be lowered (or the nozzle raised) to allow for the deposition of the next layer. This process continues iteratively until the object is fully formed.

[0062] Thermoplastics suitable for use in FDM printing include, but are not limited to, PLA (Polylactic Acid), ABS (Acrylonitrile Butadiene Styrene), PETG (Polyethylene Terephthalate Glycol), Nylon (Polyamide), PC (Polycarbonate), TPU (Thermoplastic Polyurethane), PEEK (Polyether Ether Ketone), ULTEM (Polyetherimide), and ASA (Acrylonitrile Styrene Acrylate. Of these materials, PLA, PETG, and Nylon are generally considered to be biocompatible.

[0063] In the presently disclosed application, in certain embodiments, the biocompatible, biodegradable synthetic polymer is adapted for use in FDM printing and does not require lubrication to print. In particular embodiments, the biocompatible, biodegradable synthetic polymer comprises a thermoplastic. In more particular embodiments, the thermoplastic is selected from polycaprolactone (PCL), polyglycolic acid (PGA), and poly(lactic-co-glycolic acid) (PLGA). In even more particular embodiments, the thermoplastic comprises PCL.

[0064] In some embodiments, the thermoplastic polymer comprises polycaprolactone (PCL) having a melting temperature between about 59 °C and about 60 °C and a crystallization enthalpy between about 14 J / g and about 17 J / g.

[0065] In some embodiments, the biodegradable metal powder comprises a metal selected from magnesium, a magnesium alloy, iron, an iron alloy, zinc, a zinc alloy, and combinations thereof. In particular embodiments, the biodegradable metal powder comprises magnesium.

[0066] In some embodiments, the filament comprises a cross-section, a periphery, and a center and wherein: particles comprising the metal powder are distributed throughout the filament crosssection with a particle number density between about 82 particles / mm2and about 385 particles / mm2; and a ratio of the particle number density at the filament periphery to a ratio of the particle number density at the filament center does not exceed 1.8:1. One of ordinary skill in the art would appreciate that in such embodiments, the metal phase exhibits characteristic X-ray diffraction peaks confirming crystalline metal structure.1144426.601_P18564-02

[0067] In some embodiments, the composite has one or more properties selected from: a yield stress that increases by a factor of about 2.5 to about 3.5 relative to neat thermoplastic polymer as metal volume fraction increases from about 0% to about 60%. including about 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60%; a flow behavior index that decreases from about 0.92 to about 0.82 as metal volume fraction increases from about 0% to about 60%, indicating enhanced shear-thinning behavior; and an apparent viscosity at about a 0.1 s ' shear rate that does not exceed about 5.5xl05Pa-s at about 120 °C.

[0068] The “volume fraction loading” is a function of particle size. The smaller the particle size, the more volume can be loaded. For example, in certain embodiments, no more than 60% by volume fraction could be loaded with about 40-pm Mg particles. In other representative embodiments, about 100-pm Mg particles results in a loading of only 30% volume fraction. Without wishing to be bound to any one particular theory, it is thought that if the particle size is reduced to about 10 pm, the volume fraction could be even higher (for example, about 70%).

[0069] In some embodiments, the filament has an effective diameter between about 100 pm and about 4 mm, including about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2000, 3000, and 4000 pm, and wherein the effective diameter varies by less than about 10% over any 100-mm length of the filament after an initial stabilization distance of about 50 mm from extrusion initiation.

[0070] In some embodiments, incorporation of metal powder into the thermoplastic polymer does not alter the polymer melting temperature by more than about 2 °C relative to neat thermoplastic polymer, i.e., a thermoplastic polymer substantially free (less than 0.1% of metal powder) and wherein a melting enthalpy remains within about 17 J / g to about 20 J / g including about 17, 18, 19, and 20 J / g, across all metal volume fractions.

[0071] In some embodiments of the presently disclosed filament, a maximum achievable metal volume fraction scales inversely with metal powder particle size; and about a 60 % by volume of metal powder loading with a mean particle size of about 44 pm. In certain embodiments, the filament comprises materials approved by the FDA for biomedical applications. In certain embodiments, the filament is substantially free (less than about 0.1%) of additional lubricant additives and exhibits a printability without requiring additional lubrication additives.1244426.601_P18564-02

[0072] Three-dimensional Porous Scaffold

[0073] In other embodiments, the presently disclosed subject matter provides a three-dimensional porous scaffold comprising: a composite described hereinabove having a pore size between 100 pm and 1.2 mm, including about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, and 1200 pm, configured to permit vascular ingrowth and interconnected porosity with isotropic or anisotropic pore architecture; a print fidelity score greater than 0.85, wherein the print fidelity is defined as:Print Score = (1 - - - —J x Strut Completion0 / ); and wherein:

[0074] the scaffold exhibits controlled metal ion elution in aqueous media without pH deviation exceeding about 0.5 units; and the scaffold maintains structural integrity for at least 21 days in physiological media at body temperature, e.g.. a temperature between about 37 °C to about 40 °C, including about 37, 38, 39, and 40 °C, and a physiological pH between about 7 and about 7.8, including about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, and 7.8.

[0075] In some embodiments, the scaffold comprises one or more properties selected from: a tensile Young’s modulus that increases by a factor from about 1 up to about 3 relative to a scaffold fabricated from neat polymer; an increase in the tensile Young’s modulus that scales linearly with metal volume fraction according to rule-of-mixtures predictions; and a compressive modulus having a range from about 2 MPa to about 100 MPa, including about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50. 55, 60, 65, 70. 75, 80, 85, 90. 95, 99, and 100 MPa, which is a typical range of cancellous bone.

[0076] In certain embodiments, the scaffold exhibits the following properties: one or more strut dimensions deviate from a design target by less than about 15%; one or more pore dimensions deviate from a design target by less than about 10% and a strut completion percentage exceeds about 95%.

[0077] In certain embodiments, one or more strut surfaces exhibit gradual roughening with increasing metal content without evidence of interfacial failure, particle pull-out, or cracking; and particles comprising the metal powder are encapsulated beneath a continuous polymer layer, and remain that way during manufacture, as verified by CT imaging and scanning electron microscopy.

[0078] In certain embodiments, the scaffold further comprises one or more cells and / or therapeutic agents, wherein the therapeutic agents have an elution rate from the scaffold.1344426.601_P18564-02

[0079] Method of Manufacturing a Bioactive Composite Filament

[0080] In other embodiments, the presently disclosed subject matter provides a method for manufacturing a bioactive composite filament, the method comprising: cryogenic milling a thermoplastic polymer to produce a polymer powder having a particle size distribution with a mean diameter of about 120 pm; providing biodegradable metal powder having a mean particle size between 10 pm and 100 pm; blending the polymer powder and metal powder in an inert atmosphere to produce a blended powder having a target metal volume fraction between about 10% and about 60%; extruding the blended powder through a heated die at a temperature not exceeding about 30 °C above a melting temperature of the thermoplastic polymer to produce an extruded filament; cooling the extruded filament by convection at an ambient temperature, e.g., a temperature between about 15 °C to about 35 °C, including about 15, 20, 25, 30, and 35 °C) and ambient humidity, e.g., a relative humidity between about 0% to about 100%; and winding the cooled filament onto a spool for storage in a moisture-controlled environment.

[0081] In certain embodiments, the cryogenic milling comprises: precooling thermoplastic polymer pellets in liquid nitrogen for about 3 minutes; grinding the precooled thermoplastic polymer pellets for about one minute at an impact frequency of about 10 cycles per second; repeating for 45 consecutive cycles, including cooling for about 3 minutes between grinding cycles, to achieve a target particle size; and verifying particle size distribution by dynamic light scattering. In some embodiments, the cryomilling comprises between about 15 to about 75 cycles, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72. 73. 74. and 75 cycles.

[0082] In certain embodiments, the blending comprises: transferring a pre- weighed metal powder to a container; layering thermoplastic polymer powder on top of the metal powder to minimize premature mixing; sealing the container in an inert atmosphere to prevent metal oxidation; and agitating on a vortex mixer for at least 5 minutes to achieve homogeneous blending.

[0083] In certain embodiments, the extruding comprises: feeding the blended powder into a heated screw extruder; maintaining barrel temperatures of the heated die between about 65 °C to about 80 °C across heating zones; controlling screw speed between about 1 rpm and about 5 rpm to achieve a target filament diameter; monitoring filament diameter in real-time by laser micrometry; and collecting the filament after diameter stabilization over a distance of at least 50 mm.1444426.601_P18564-02

[0084] In certain embodiments, the method further comprises storing the filament in an airtight container with desiccant to maintain moisture content below about 0.1 wt% prior to printing.

[0085] Three-dimensional Printing of a Bioactive Scaffold

[0086] In other embodiments, the presently disclosed subject matter provides a method for three-dimensional printing of a bioactive scaffold, the method comprising: providing a filament as described hereinabove; heating the filament to a processing temperature between a melting temperature of the thermoplastic polymer and about 30 °C above the melting temperature; extruding the heated filament through a nozzle having an orifice diameter between about 400 pm and about 1000 pm; depositing the extruded filament layer-by-layer onto a build platform at a predetermined print speed to form a three-dimensional structure; and controlling layer thickness, infill density, infill pattern, and raster orientation to achieve target mechanical properties and porosity.

[0087] In certain embodiments, the nozzle is maintained at a temperature of 105°C ± 5°C; the build platform is maintained at a temperature between about 50 °C to about 70°C; the layer thickness is between about 100 pm and about 400 pm; the print speed is controlled between about 10 mm / s and about 40 mm / s to optimize bonding between layers; and the infill pattern is selected from a rectilinear, a honeycomb, a gyroid, and a custom pattern to achieve target porosity and mechanical anisotropy.

[0088] In certain embodiments, the method further comprises: varying metal volume fraction across one or more regions of the three-dimensional structure; creating functionally graded scaffolds with spatially varying mechanical properties, degradation rates, or ion release kinetics; and / or co-printing multiple composite formulations in a single build to achieve heterogeneous material distribution.

[0089] In certain embodiments, the method further comprises immersion of the scaffold in cell culture medium for 21 days resulting in: a pH stability within ±0.3 units of an initial pH; instantaneous metal ion release scaling with metal volume fraction up to about 30 vol%, followed by a decreased release at higher loadings; and a cumulative metal ion concentration demonstrating composition-dependent elution kinetics suitable for therapeutic delivery. One of ordinary skill in the art would appreciate that preservation of the scaffold architecture can be confirmed by micro-CT imaging.1544426.601_P18564-02

[0090] In certain embodiments, the method further comprises culturing the scaffold with osteoblast-lineage cells for 21 days resulting in: progressive cell proliferation peaking at metal volume fractions between about 30 vol% to about 45 vol%: an increased normalized calcium deposition (Ca / DNA ratio) with increasing metal fraction, indicating enhanced osteoconductivity and biomineralization; and cell viability exceeding about 95% at all metal loadings as assessed by live / dead fluorescence staining.

[0091] In certain embodiments, quality of the scaffold is verified by: measuring dimensional deviation for each strut and pore relative to CAD design targets; calculating average deviations separately for struts and pores; assessing strut completion percentage as the fraction of fully formed struts; computing an overall print score according to:( Ave. Deviationf4- Ave. Deviation1 - - - — j x Strut Completion%; and

[0092] accepting only scaffolds with print scores exceeding 0.85.

[0093] Non-porous Biomedical Implant Structure

[0094] In other embodiments, the presently disclosed subject matter provides a non-porous biomedical implant structure comprising a filament described hereinabove or prepared by the methods described herein, the implant structure comprising one or more of: a bone fixation screw having a customizable thread geometry, pitch, head design, and length; a fixation plate having a customizable thickness, hole pattern, and curvature; an intramedullary rod having a customizable diameter and length; and / or an orthopedic fixation device requiring mechanical strength, ductility, and biodegradability.

[0095] In certain embodiments, the structure is selected from a scaffold, a braid, a screw, a stent, a plate, a rod, and a bar. In certain embodiments, the composite, filament, or structure is adapted for bone tissue regeneration.

[0096] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,1644426.601_P18564-02e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0097] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -10%, + / -5%, + / -4%, + / -3%, + / -2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0098] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments may be readily combined, without departing from the scope or spirit of the presently disclosed subject matter.

[0099] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references. i.e„ “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.1744426.601_P18564-02EXAMPLES

[0100] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 1

[0101] 3D-Printed Magnesium Isotropic Porous Scaffolds Via Multi-Material Additive Manufacturing For Bone Tissue Engineering

[0102] 1.1 Magnesium as a biomaterial

[0103] Blended multiple material processes multi-material additive manufacturing presents an attractive solution to addressing inherent material characteristics of magnesium, specifically with thermoplastics due to their versatility, and low cost allowing for ease of adoption. By utilizing 3D-printing and polycaprolactone fused filament fabrication, we are able to overcome the constraints of requiring inert environments and other safety concerns and print magnesium at room temperature and normal conditions. See FIG. 1.

[0104] / .2 Manufacturing process

[0105] In this Example, we characterized the presently disclosed scaffolds throughout a three-step manufacturing process: raw materials processing, filament production, and scaffold printing. See FIG. 2.

[0106] Raw materials are first cryo-milled separately, and sizing was correlated with different durations using dynamic light scattering (DLS). Particles are then mixed, extruded into 2.85-mm filaments and characterized mechanically via tensile testing and micro-hardness; thermally via differential scanning calorimetry (DSC). Scaffolds are then 3D-printed via fused deposition and topographically characterized using scanning electron microscopy (SEM). Particle distribution within the scaffold and thermal expansion was examined by micro computed tomography (pCT) and SEM energy-dispersive X-ray spectroscopy (EDX). After production, scaffolds were1844426.601_P18564-02immersed in cell culture media (DMEM, 10% FBS, 1 % Pen / Strep). Fluid was collected at different timepoints and magnesium concentration was analyzed via inductively coupled plasma optical emission spectroscopy (ICP-OES).

[0107] 1.3 Cryo-milling and particle mixing

[0108] Before production can start, PCL was processed via cryo-milling. Particles were then sized via dynamic light scattering after every 15 cycles, with water as the suspension liquid. It is observed that there is a limitation with our equipment as the particles reaches 120 pm. See FIG. 3.

[0109] After cryo-milling, PCL was mixed with magnesium particles for uniform distribution. Due to inherent spontaneous combustibility of magnesium, powders were mixed in an inert environment to avoid fires. Based on the breakdown rate of PCL particles in cryo-milling, appropriate- sized magnesium powder was procured to match the best time to particle size ratio of PCL particles. After mixing, the PCL acts as a heatsink, and the mixture is no longer combustible, allowing for it to be melt-extruded into 3D printing filament.

[0110] 1.4 Thermomechanical characterization

[0111] ASTM D638 dog bone standards were printed at 100% infill, 800-pm strut at various magnesium concentrations. Tensile testing was conducted (see FIG. 4). To understand the change in thermal properties with the addition of metallic powders in PCL, DSC was conducted to determine the melting and crystallization temperature. Referring now to FIG. 5, the data show that addition of Mg increases the crystallization temperature, and decreases melting temperature of the composite material. Further addition of Mg, however, does not show any trend.

[0112] Viscometry was conducted at 120 °C, our 3D printing temperature, on the melted composite material to determine viscosity. Data were collected up to 16 / s shear rate as that is correlates to our maximum viable printing speed. See FIG. 6.

[0113] 7.5 Scaffold characterization

[0114] Scaffolds composed of PCL or PCL-Mg were sputter coated with 8 nm of Au / Pd and imaged using SEM. SEM-EDX shows that Mg particles are present on surface of the scaffold. See FIG. 7 and FIG. 8.

[0115] 1.6 Magnesium elution

[0116] Mg scaffolds were submerged in a solution mimicking physiological conditions, comprising DMEM supplemented with 10% FBS and 1% penicillin. The medium were collected at specific intervals (0, 1, 3, 7, and 14 days) and subjected to analysis using inductively coupled1944426.601_P18564-02plasma mass spectrometry (ICP-MS). Results show that there was an increase in elution for 30 v% scaffolds. See FIG. 9.

[0117] To study the effect of surface area and volume on magnesium elution, 3-mm (H) 2.85-mm (D) and 6-mm (H) 2.85-mm (D) rods, uncut and cut down the middle lengthwise were immersed in physiological mimicking solution. The medium will be collected at specific intervals (0, 1, 3, 7, and 14 days) and subjected to analysis using inductively coupled plasma mass spectrometry (ICP-M S). See FIG. 10.

[0118] 7.7 Summary

[0119] The findings unveiled that addition of magnesium to scaffolds is limited to 30 v% due to the increase in viscosity as Mg percentage in the material increases and printing becomes too challenging. We observed increased crystallization temperature by 4 °C, decreased melting temperature by 4 °C, and enhanced shear thickening behavior (by 10X for every 10%). Furthermore, the presence of Mg in scaffolds correlates with superior tensile strength. SEM-EDX and pCT results shows successful incorporation of magnesium in scaffolds, however the lack of Mg presentation on the surface of the scaffolds correlates with the delayed release of Mg. This Example verifies the possibility of printing PCL with magnesium microparticles, as well as provides valuable insights into PCL / Mg additive manufacturing, paving the way for superior biodegradable implant materials for 3D printed scaffolds.EXAMPLE 2

[0120] 2. Background

[0121] The development of custom composite filaments for material extrusion additive manufacturing has accelerated over the past decade, driven by the demand for structural and functional parts with finely tuneable properties that exceed those of neat thermoplastics. Ma et al., 2024. By incorporating micro- and nano-scale reinforcements, such as metals, ceramics, carbon fibers, or natural fibers, into polymer matrices like polylactide (PLA), acrylonitrile-butadiene-styrene (ABS), or polycaprolactone (PCL), composite filaments can exhibit increases in tensile strength, stiffness, thermal stability, and wear resistance of 20-50% or more relative to the corresponding unfilled polymers For example, Fe-reinforced PLA filaments have been reported to show improvements in tensile strength approaching 50% compared with neat PLA, while recycled carbon-fiber PLA composites and bio-based fiber blends similarly enhance flexural and tensile performance through improved load transfer and stiffness contributions from the reinforcement2044426.601_P18564-02phase. These property gains, combined with the geometric freedom of fused filament fabrication (FFF), have positioned composite filaments as enabling materials for aerospace, automotive, energy, and biomedical applications that require lightweight, mechanically robust, and functionally graded components.

[0122] Within this broader context, porous scaffolds fabricated from composite filaments have emerged as a particularly promising class of structures for bone tissue engineering, regenerative medicine, and lightweight lattice architectures. Additively manufactured scaffolds can be designed with pore sizes, connectivity, and overall porosity tailored to promote cell adhesion, migration, vascularization, and nutrient transport, while simultaneously achieving compressive strengths in the range of cancellous bone (approximately 2-12 MPa), which is critical for load-bearing and load-sharing applications in orthopaedics. Studies using polymer / metal and polymer / ceramic composites — such as PCL loaded with amorphous calcium phosphate or hydroxyapatite, or polymer / metal hybrids — demonstrate that tuning composition and internal geometry allows largely independent control of mechanical response and biological performance, including osteoconductivity and degradation behavior. Mohammadi Zerankeshi et al., 2022.

[0123] Magnesium offers key advantages for bone tissue engineering scaffolds, including biodegradability, a bone-matching elastic modulus (10-20 GPa), osteogenesis promotion via AMPK / mTORCl signaling, hydroxyapatite nucleation for mineralization, and M2 macrophage polarization to reduce inflammation. Staiger et al., 2006. Incorporating Mg into polycaprolactone (PCL) scaffolds via fused deposition modeling (FFF) 3D printing overcomes pure polymer drawbacks in mechanics and bioactivity, allowing tailored porosity, sustained ion release, and reinforcement. For example, incorporating Mg into PCL scaffolds via FFF 3D printing yields composites with tunable degradation, ion release matching bone remodeling (months), improved hydrophilicity, and bioactivity, as demonstrated in rotator cuff (10% Mg / PCL boosting osteogenesis / angiogenesis) and skull defect models. Wang et al„ 2024; Lei et al.. 2022, while Mg / PCL composites boosted mechanical strength and skull defect healing, Wang et al., 2024, Mg phosphate / PCL hybrids further promoted in situ mineralization and osteogenesis.

[0124] High Mg loadings (>20 wt%) in thermoplastic FFF filaments, such as PLA / PCL, however, remain uncommon primarily due to processing constraints that compromise filament extrudability and print quality. Kalva et al., 2023; PascuaLGonzdlez et al., 2022. Key limitations include sharply elevated melt viscosity from Mg particles, which impedes flow through nozzles and dies;2144426.601_P18564-02inadequate particle wetting causing voids and porosity; filament brittleness leading to buckling or breakage; and thermal expansion mismatches promoting agglomeration and nozzle wear. Kalva et al., 2023; Mora-Castano et al., 2022.

[0125] Beyond 15-20 wt%, these effects render filaments unprintable. Kalva et al., 2023. Mitigation via particle size reduction or additives rarely pushes past this threshold without sacrificing mechanical integrity or resolution. Although composite filaments containing metallic fillers have been widely explored, printable metal loadings in extrusion-based systems are typically limited by excessive viscosity, particle agglomeration, and filament brittleness, often restricting filler content to below approximately 20 wt%. Angelopoulos et al„ 2019; Rajendran et al„ 2023.

[0126] 2.2 Scope

[0127] This Example addresses these challenges by presenting a technically rigorous, standardized method for producing custom composite filaments and for additively manufacturing porous scaffolds with controlled geometries and properties suitable for structural and biomedical applications. The proposed framework integrates (1) systematic filament compounding and extrusion workflows, including control of filler loading, dispersion, and moisture management; (2) a structured printing protocol that links filament rheology and thermal behaviour to printer settings, infill architecture, and target porosity; and (3) a comprehensive characterization suite encompassing dimensional analysis, porosity quantification, thermal characterization, mechanical testing and bioactivity under application-relevant loading conditions.

[0128] 2.3 Materials and Methods

[0129] 2.3.1 Raw material Processing, Filament production and FDM 3D Printing Raw Material Processing and mixing

[0130] Raw materials were subjected to cryogenic milling using a 6875 Freezer / Mill equipped with a large polyvial set and corresponding vial opener (SPEX SamplePrep, USA) to achieve homogenous particle dispersion. Spherical Mg powder with a mean particle size of approximately 44 pm (ThermoFisher Scientific, USA) was used as received. Commercial PCL pellets (approximately 4 mm diameter; Sigma-Aldrich, USA) were converted to powder by cryogenic milling. Milling was performed in a SPEX 6870 Freezer / Mill® for 45 consecutive cycles; each cycle is comprised of a 3-minute precooling phase, 1 -minute grinding cycle, and a 3-minute intermediate cooling phase at an impact frequency of 10 cycles per second (CPS). Milling was conducted under liquid nitrogen to maintain cryogenic conditions, with operational parameters set2244426.601_P18564-02to a 3 Milling was performed iteratively until target particle size distributions were achieved, approximately 120 pm, as verified by dynamic light scattering.

[0131] PCL-Mg composites were prepared with nominal Mg volume fractions of 0%, 15%. 30%, 45%, and 60%, corresponding to 0%, 21%, 39%, 55%, and 69% weight fraction. The corresponding weight of each constituent was determined from the rule-of-mixtures expression (Egbo, 2021):Additive w%Additive densityAdditive w% 100 — Additive w%Additive density Polymer density

[0132] We assume densities of 1.74g / cm3for Mg, 1.14 g / cm3for PCL, resulting in:X1.74X 100 - 1.74+1.14

[0133] The calculated weights were used to obtain the required mass of each powder prior to blending. All weighing and blending operations were earned out inside a ventilated fume hood. The pre-weighed Mg powder was first transferred to a 0.47 L stainless- steel paint can (VWR International), after which the appropriate mass of PCL powder was gently layered on top to minimize premature mixing. The can was sealed by hammer-crimping the lid and then agitated on a vortex mixer (Vortex-Genie 2, Scientific Industries) for 5 min to achieve a homogeneous blend. The blended powders were stored in sealed containers under ambient laboratory conditions until further processing.

[0134] 2.3.2 Filament production

[0135] Mixed powder was added to the hopper of 3DEVO Precision 350. Screw speed 2 rpm, target diameter 2.85 mm, mixing zone temperatures l->4 is 65, 80, 75, 65 in that order. Filament is extruded and diameter is recorded. Filament was cooled by convection cooling at room temperature, then spooled and stored in an airtight container with desiccant beads.

[0136] 2.3.3 FDM 3D Printing

[0137] Spooled filament is fed into an Ultimaker S2+ FDM 3D printer. Prints were printed with a 800 pm diamond insert type V6 nozzle (DiamondBack) at 105 °C onto a glass build plate. Doublesided tape was used for bed adhesion.

[0138] 2.3.4 Material mechanophysical Properties Evaluation2344426.601_P18564-02

[0139] 2.3.4.1 Rheometry

[0140] Rheometry was performed on a Anton Parr MCR 102e. Extruded filament were placed onto the stage heated to 120 °C until fully melted. A 25-mm Sandblasted plate was utilized to shear the fluid from 1-100 s'1.

[0141] 2.3.4.2 X-ray Diffraction

[0142] X-ray diffraction (XRD) patterns were acquired using a PANalytical diffractometer operating at 40 kV and 40 mA with Cu Ka radiation (A = 1.5406 A). Bulk filament samples were mounted on a flat sample stage in reflection geometry, and scans were performed over a 29 range of 5-90° at a step size of 0.017° 20 and scan speed of 2-57min, with sample spinning enabled to minimize preferred orientation effects. Raw data were processed using HighScore Plus.

[0143] 2.3.4.2 Tensile

[0144] Samples were 3D printed following ASTM D638 Type V standard design at 100% infill. Testing was conducted at room temperature on a MTS (Model name) at a static strain rate of approximately 0.01 s'1.

[0145] 2.3.4.3 DSC

[0146] Differential Scanning Calorimetry was performed utilizing aluminum Tzero pans on a TA Instalments DSC 25. Samples were heated to 120 °C and cooled to -80 °C at a rate of 10 °C / min. Melting and crystallization temperature, as well as enthalpies of melting and crystallization, was obtained using a custom python script using trapezoidal Riemann sum.

[0147] 2.3.5 Characterization and Quantification of Printed Product

[0148] 2.3.5.7 Print Quality

[0149] To assess dimensional deviations in printed products, we devised the following formula:Actual measure — Design TargetDeviation; =Design Target

[0150] For every measured geometrical feature (i) either a strut thickness or a pore width, the dimensional error was expressed as an absolute, normalized deviation from the computer-aided design (CAD) target value.„ . „ Avq. Deviation^frl,r+Avg. Deviationnnrp^ „ „ .

[0151] Print Score(%) = (1 - - -2- ■ Strut Completion%

[0152] The individual deviations obtained for all struts in a specimen were averaged to give Avg. Deviation_strut, and the same procedure was applied to all pores to obtain Avg. Deviation_pore.2444426.601_P18564-02In addition to dimensional accuracy, the presence of missing or discontinuous struts was recorded. The strut-completion factor was defined as the percentage of struts that were fully formed relative to the number specified in the CAD model. Overall build quality was expressed as a “print score”, which couples dimensional accuracy with structural completeness

[0153] A perfect build (zero average deviation and 100 % strut completion) yields a print score of 1, whereas any increase in dimensional error or loss of struts proportionally decreases the score.

[0154] 2. .5.2 MicroCT and Particle Distribution

[0155] pCT was performed with a RX Solutions EasyTom 150 / 160 pCT for both filament and printed structure. Scans were ran using 260 kV x-ray at 52 kV and 128 pA, with a resulting voxel size of 5-6 pm. Particle distribution analysis performed using a custom MATLAB script for the reconstructed Y slices of the scans.

[0156] 2.3.5.3 SEM and surface properties

[0157] We employed Scanning Electron Microscopy (SEM) in Secondary Electron (SE) and Backscattered Electron (BSE) modes along with Energy Dispersive Spectroscopy (EDS). A JEOL IT700HR InTouchScope™SEM with EDAX EDS was utilized for imaging and EDS analysis, processed with APEX EDS software to image the surface of the material.

[0158] 2.3.6. Immersion and Elution Testing

[0159] 2.3.6.1 Sample Immersion and pH measurement

[0160] Samples were immersed in a 24-well cell culture plate with 1 mL of cell culture media (Low Glucose DMEM, 10% Fetal Bovine Serum, 1% Penicillin / Streptomycin, 10-mM 0-Glycerophosphate, 50-pM Ascorbic Acid and 100-nM Dexamethasone). Fluid was changed every 2-3 days to mimic in-vitro cell-culture protocols. Upon change, fluid was collected and pH was measured 3 times using a pH probe.

[0161] 2.3.6.2 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES)

[0162] An Agilent 5800 ICP-OES was utilized for ICP-OES to detect and quantify magnesium ion concentrations in the collected fluids. Samples were diluted step diluted from lOx to lOOx in 3% HNO3 for benchtop digestion for 4 hours before samples were run due to machine measurement range. Measured concentrations were calculated to find fluid concentration.

[0163] 2.3.7 Cell proliferation and Differentiation Testing

[0164] 2.3.7.1 DN A Assay2544426.601_P18564-02

[0165] Cellular DNA content was quantified using a PicoGreen-based fluorescence assay adapted from the Quant-iT PicoGreen dsDNA kit (Invitrogen, Molecular Probes Pl 1496). Cells grown in 24-well plates were washed with PBS and lysed in 250 pL of lysis buffer (10 mM Tris base, 1 mM EDTA, 0.1% Triton X-100, 0.1 mg / mL proteinase K) with proteinase K added fresh on the day of use, followed by overnight incubation at 50 °C and subsequent vortexing and brief centrifugation prior to analysis. Double-stranded DNA standards were prepared from DNA in TE buffer to generate a standard curve ranging from 0 to 1000 ng / mL, and all standards and samples were assayed in duplicate in black 96-well plates to minimize well-to-well fluorescence bleed-through. After incubation at room temperature for at least 3 min in the dark, fluorescence measurement was taken (excitation 480 nm, emission 520 nm) on a microplate reader. Background-corrected fluorescence values were averaged for technical replicates, and total DNA per sample was calculated from the standard curve using the linear regression slope and intercept, adjusted for sample dilution and harvested lysate volume to obtain DNA content per sample.

[0166] 2.3.7.2 Calcium Assay

[0167] Samples, were washed with PBS without Ca2+, harvested in 0.5 M HC1, agitated overnight at 4 °C in the cold room, and stored at -20 °C until analysis. Prior to assay, samples were thawed at room temperature, vortexed, and centrifuged at 1000 g for 1 min to pellet debris. Supernatant was then collected, and calcium concentrations were read using ICP-OES following ICP-OES sample prep.

[0168] 2.3.7.3 Live / Dead Staining

[0169] Cell viability was assessed using a live / dead fluorescence staining assay with calcein AM and ethidium homodimer-1 (EthD-1; Invitrogen C1430 and El 169). Working solution was freshly formulated in Ca2+-containing DPBS (2 pM calcein AM at 1:2000 dilution; 4 pM EthD-1 at 1:500 dilution), protected from light, vortexed, and applied to samples in sufficient volume to cover following medium removal. Samples were incubated at 37 °C for 20 mins, washed once with Ca2+-PBS, maintained in Ca2+-PBS, and immediately imaged via fluorescence microscopy, with live cells appearing green (calcein) and dead cells red (EthD-1), minimizing excitation to prevent calcein photobleaching.

[0170] 2.4 Results and Discussion

[0171] 2.4.1 Material / Filament Fabrication and characterization

[0172] 2.4. / . / Filament-forming behavior during screw extrusion2644426.601_P18564-02

[0173] Real-time laser micrometry showed that the diameter of the extruded strand stabilized after an initial start-up phase of approximately 50 mm (FIG. 12A, red box). Beyond this distance (green box) the profile levelled off and fluctuated around a constant mean value, indicating that steadystate processing conditions had been reached. The average steady- state diameter of neat PCL filaments was 2.74 ± 0.05 mm, whereas the addition of Mg systematically reduced the strand size to 2.54 ± 0.06 mm at 60 vol % Mg (FIG. 12B). One-way ANOVA followed by Tukey’s post-hoc test confirmed that every Mg-containing formulation produced significantly thinner filaments than the polymer control (p < 0.0001), but no significant pair- wise differences were detected among the different composite levels.

[0174] Thermal transitions are largely preserved after incorporation of Mg Differential scanning calorimetry revealed no measurable influence of Mg on the melting transition of PCL. The melting temperature remained within 59-60 °C for all compositions (FIG.12C) and the associated melting enthalpy varied only slightly between 17 and 20 J g1(FIG. 12D). In contrast, Mg noticeably modified the crystallization behavior. The crystallization onset temperature decreased from 33.3 ± 0.8 °C for neat PCL to 26.3 ± 1.1 °C at 15 vol % Mg (p < 0.0001) and then gradually recovered to ~30 °C at >45 vol % Mg (FIG. 12E). Despite this shift in temperature, the crystallization enthalpy (FIG. 12F) remained statistically unchanged (14-17 J g ')• suggesting that the overall degree of crystallinity was maintained.

[0175] Composite formation had a pronounced effect on the flow response of the molten feedstock. Steady-shear measurements demonstrated a composition-dependent increase in shear stress across the investigated shear-rate window (FIG. 12G). Accordingly, the apparent viscosity at low shear (0.1 s ') rose from 1.6 x 105Pa s for neat PCL to 5.5 x 105Pa s for the 60 vol % composite (FIG. 12H). This rise in zero-shear viscosity was accompanied by a progressive growth in yield stress (FIG. 121), which increased almost three-fold between 0 and 60 vol % Mg (p < 0.05, p < 0.01 and p < 0.0001 for successive comparisons). Concomitantly, the flow-behavior index (n) obtained from the power-law fit decreased from 0.92 ± 0.01 (PCL) to 0.82 + 0.02 (60 vol % Mg) (FIG. 12J), signifying stronger shear-thinning with increasing particulate loading.

[0176] Collectively, these results establish that incorporation of Mg particles (i) produces slightly thinner yet dimensionally stable extruded filaments, (ii) preserves PCL melting characteristics while transiently depressing the crystallization onset, and (iii) increases both viscosity and yield2744426.601_P18564-02stress while simultaneously enhancing shear-thinning. These processing-relevant changes must be considered when selecting extrusion parameters for additive manufacturing of Mg-PCL scaffolds.

[0177] 2.4.2 Spatial distribution of Mg particles in the extruded filaments

[0178] High resolution pCT provides a non-destructive approach for quantifying particle size distributions and spatial heterogeneity in composite materials, including subtle segregation effects that are difficult to detect via surface based methods. Behnsen et al., 2025. Representative pCT cross-sectional slices (FIG. 13 A) show that Mg particles were readily discernible as bright, nearly spherical inclusions within the darker PCL matrix for all loading levels examined (15-60 vol %). At the lowest concentration (15 vol % particles were well separated and no large agglomerates were detected. As the nominal Mg fraction increased to 30 and 45 vol % the inter-particle distance decreased and a contiguous, yet still discrete, Mg phase became apparent. At 60 vol % the polymer matrix was largely filled with densely packed Mg particles, although a continuous PCL phase was still visible, indicating that complete percolation of the metal phase had not been reached.

[0179] To quantify the radial homogeneity of particle dispersion, every filament cross-section was divided into concentric annuli (0.1 mm width) and the absolute number of Mg particles in each zone was counted (FIG. 13E-FIG. 13H). For all formulations the particle count increased gradually from the center towards the outer surface, revealing a mild particle-rich “shell” and a particle-lean “core”. The skew, however, was small: even at the highest loading the peripheral zone contained only about 1.8-fold more particles than the filament center. When the data were normalized and expressed as particle number density (particles mm'2, FIG. 131), the overall trend scaled with composition, rising from 82 ± 14 mm'2at 15 vol % to 385 + 32 mm-2at 60 vol %. Two-way ANOVA confirmed (i) a significant main effect of Mg content on particle density (p < 0.0001) and (ii) a modest but significant effect of radial position (p < 0.01), while the interaction term was not significant, indicating that the extent of center-to-edge variation was similar for all compositions. X-ray diffraction was performed on the bulk filament with and without Mg. The characteristic major peaks of Mg (100, 002, 101) between was observed in the 30-40° region in sample with Mg incorporated, and missing from the neat PCL filament. It is also observed that 60% sample results in a slightly stronger peak indicating presence of more Mg.

[0180] Collectively, these observations demonstrate that (i) the cryomilling-blending protocol yields a macroscopically homogeneous particle dispersion, (ii) the extrusion process does not induce pronounced particle segregation, and (iii) the intended Mg volume fraction is reliably2844426.601_P18564-02translated into a proportional and spatially uniform particle number density within the final filament.[0018112.4.3. Mechanical characterization ofMg-PCL scaffolds

[0182] Incorporation of magnesium (Mg) into polycaprolactone (PCL) scaffolds significantly altered tensile and compressive mechanical properties, as detailed in FIG. 14. Tensile testing per ASTM D638 Type V (FIG. 14A) revealed a pronounced increase in Young's modulus with rising Mg volume fraction (FIG. 14B), accompanied by representative stress-strain curves showing enhanced stiffness (FIG. 14C); peak tensile stress (FIG. 14D) and load (FIG. 14E) likewise trended upward, indicating improved tensile strength. Complementary compressive testing on isotropic 1.2 mm pore structures (FIG. 14F) demonstrated mixed results in Young's modulus (FIG. 14G), with stress-strain profiles (FIG. 14H) following classical plastic behavior but with no clear correlation with Mg volume fraction. The same was observed for peak compressive stress (FIG. 141) and load (FIG. 14J). Variations in effective relative density arising from printed structure likely dominate compressive behavior, masking compositional effects. This behavior is consistent with established models for cellular solids, in which compressive stiffness and strength are primarily governed by relative density and architecture rather than constituent material properties. Maconachie et al., 2019; Gibson and Ashby, 1997.

[0183] 2.4.4. Print fidelity and macroscopic build quality ofMg-PCL scaffolds

[0184] Scanning electron micrographs of individual strands (FIG. 15A) revealed that neat PCL exhibited a smooth exterior characteristic of plastics. Introduction of 15 vol % Mg led to shallow surface bumps, while 30 vol % Mg resulted in more pronounced bumps. At 45 and 60 vol % Mg, the strand surface became uniformly rough with larger bumps, consistent with the higher particulate load, but no cracks or regions of particle pull-out were detected, indicating good interfacial adhesion.|00185|2.4.-Z. / Geometric accuracy of two-layer lattices

[0186] Representative stereomicroscope images of 20 x 20 mm, two-layer grids printed from each formulation are shown in FIG. 15B. Visually, all scaffolds maintained rectilinear architecture, yet the 15 vol % grids appeared marginally less regular, exhibiting occasional interruptions along horizontal rasters. Quantitative dimensional analysis confirmed these impressions. Horizontal struts were consistently undersized (540-670 pm) for every group compared to the design target of 800 pm (red line), whereas vertical struts were much closer to the nominal value (770-830 pm).2944426.601_P18564-02Two-way ANOVA found a significant effect of raster orientation (p < 0.0001) but no main effect of Mg content (p = 0.27) and no orientation x composition interaction, demonstrating that metal loading did not compromise strut formation. The intended pore measured 1,200 pm x 1,200 pm. Measured major-axis values ranged from 1,180 + 60 pm (0 vol %) to 1,280 + 70 pm (60 vol %), whereas minor-axis dimensions were consistently smaller (1,050-1,120 pm). Again, no statistically significant dependence on Mg level was detected.

[0187] 2.4.4.2 Overall print quality

[0188] The composite print-quality metric that combines dimensional error and strut completeness (see Methods) is summarized in FIG. 15E. Neat PCL achieved the highest score (0.94 + 0.03). A transient dip was observed at 15 vol % Mg (0.82 + 0.04; p < 0.01 vs. control), primarily attributable to the interrupted horizontal rasters noted above. Beyond this concentration the score recovered, reaching 0.90 + 0.02, 0.92 + 0.03 and 0.93 + 0.02 for 30, 45 and 60 vol % Mg, respectively; none of these values differed statistically from the PCL baseline.

[0189] Taken together, these findings indicate that (i) filament surface roughness increases gradually with particulate loading but without evidence of interfacial failure, (ii) Mg incorporation does not adversely affect macroscopic dimensional fidelity of printed lattices, and (iii) overall build quality remains high (print score >0.85) for composites containing Mg, demonstrating the suitability of highly filled Mg-PCL feedstocks for extrusion-based additive manufacturing.

[0190] 2.4.5. Degradation in vitro of Mg-PCL Scaffolds

[0191] Degradation of Mg-incorporated PCL scaffolds in DMEM released Mg2+ions proportionally to Mg volume fraction without perturbing medium pH, as illustrated in FIG. 16. Panel 6A shows stable pH across all Mg compositions (0-60 vol%), confirming no acidification from degradation products. Instantaneous Mg release (panel 6B) and cumulative release (panel 6C) scaled with Mg content until 30% volume fraction, then decreased, demonstrating controlled, composition-dependent ion elution suitable for therapeutic delivery. The absence of measurable pH changes is consistent with prior reports showing that buffered culture media and polymer encapsulation significantly moderate Mg corrosion kinetics in vitro. Li et al., 2017. Buffered culture media such as DMEM are known to significantly stabilize pH during Mg degradation, moderating alkalization effects observed in simpler immersion environments. Raguraman et al., 2025. Scanning electron microscopy (SEM; panel 6D, 500 pm scale bars) of 21-day degraded scaffolds revealed surface erosion with higher Mg levels, while micro-CT (pCT; panel 6E, 5003044426.601_P18564-02pm scale bars) highlighted internal microstructural preservation amid degradation. These results indicate Mg integration promotes predictable scaffold resorption and ion release while maintaining architectural stability over 21 days.

[0192] 2.4.6. In-vitro bioactivity ofMg-PCL scaffolds

[0193] Mg-incorporated PCL scaffolds demonstrated sustained bioactivity over 21 days of degradation, coupling ion release with enhanced cellular infiltration and mineralization, per FIG.17. Degradation profiles recapitulated stable pH across Mg volume fractions (0-60%; FIG. 17A) even in the presence of cells, with Mg release showing both instantaneous (FIG. 17B) and cumulative (FIG. 17C) increases in presence of Mg content, affirming controlled elution. DNA content quantification (FIG. 17D) indicated progressive cell from days 7 to 21, peaking at intermediate Mg levels (e.g., 30-45 vol%). directly correlating with Mg ion concentration. Normalized calcium deposition (Ca / DNA; FIG. 17E) rose markedly with Mg fraction, evidencing Mg-promoted osteoconductivity or biomineralization. Live / dead staining at day 7 (panel FIG. 17F; 100 pm scale bars) revealed predominantly viable (green) cells throughout scaffolds, with minimal red (dead) staining even at high Mg loadings, confirming cytocompatibility amid bioactivity.

[0194] 2.5 Discussion

[0195] From a materials processing perspective, this Example expands the printable composition space of FFF feedstocks and address key limitations associated with highly filled thermoplastic filaments. Enriconi et al.. 2025. More particularly, this Example demonstrates successful incorporation of unprecedented Mg loadings (up to 60 vol%, 70 wt%) into PCL scaffolds via FDM 3D printing, overcoming well-documented processing barriers that typically limit metal-polymer composites to <20 wt% filler.

[0196] 2.5.7 Manufacturing Innovation

[0197] Cryomilling and vortex-blending enabled homogeneous Mg dispersion (82-385 particles / mm2) with minimal radial segregation (1.8-fold center-to-periphery gradient), while extrusion preserved thermal stability (Tm = 59-60°C, AHm = 17-20 J / g). The observed three-fold yield stress increase and shear-thinning enhancement (n: 0.92^-0.82) reflect classical suspension rheology, where particle-particle interactions elevate viscosity while reducing inter-particle friction under shear — critical insights explaining filament extrudability at extreme loadings previously deemed impossible.3144426.601_P18564-02

[0198] 2.5.2 Mechanical Reinforcement

[0199] Tensile properties scaled linearly with Mg content (3-fold Young's modulus increase), following rule-of-mixtures predictions for particulate composites where metal stiffness dominates load transfer. Egbo, 2021. The lack of compressive property correlation likely reflects geometric factors — plateau stress in open-cell foams depends more on relative density (approximately 20% here) than matrix reinforcement, consistent with Gibson-Ashby models for polymer foams. Gibson and Ashby, 1997.

[0200] 2.5.3 Print Fidelity Preservation

[0201] Print scores > 0.85 across compositions confirm that Mg incorporation does not compromise FDM dimensional accuracy (strut deviations <15%, pore accuracy ±70 pm), despite 3.5-fold viscosity increases. The transient 15 vol% print quality dip corresponds to maximum particle-matrix decohesion risk during cooling-induced shrinkage, after which higher loadings create a self-stabilizing particulate network. Further, print fidelity was not majorly affected by particle loading as similar mild radial particle gradients have been reported in highly metal-filled FFF filaments and are not typically detrimental to print fidelity or mechanical performance. Strugova et al., 2025.

[0202] 2.5.4 Controlled Degradation and Bioactivity

[0203] pH-neutral Mg2+elution scaling with composition (peaking at 30 vol%) enables precise dosing for osteogenic signaling, while 21-day bioactivity data (elevated Ca / DNA ratios, 95%+ cell viability) confirm therapeutic windows without cytotoxicity, consistent with the literature of Mg2+release has been shown to promote osteogenesis and angiogenesis through modulation of osteoblast activity and vascularized bone formation pathways. Hu et al., 2023. Frequent media exchange and the buffering capacity of DMEM likely mitigated localized alkalization. Unlike pure Mg implants causing rapid H2evolution, polymer encapsulation here throttles corrosion kinetics to match bone remodeling (months), representing a major advance over existing Mg / PCE systems limited to 10-20 wt%. The longitudinal pCT imaging from pre-immersion to post-degradation suggesting a process-dependent upper limit for effective Mg flow from the nozzle under the present printing conditions, with 30% volume fraction being the peak for the particular particlenozzle diameter combination chosen in this experiment (300 mesh particle - 0.8 mm nozzle)3244426.601_P18564-02

[0204] In summary, this manufacturing platform unlocks high-Mg PCL composites with tunable mechanics, degradation, and bioactivity suitable for bone tissue engineering, establishing new performance thresholds for extruded composite scaffolds.

[0205] 2.6 Summary

[0206] This Example successfully addresses a critical manufacturing bottleneck by developing a robust method to incorporate unprecedented levels of magnesium (up to 60 vol%, 70 wt%) into polycaprolactone scaffolds via FDM 3D printing while maintaining print fidelity and biological performance. The cryomilling and blending protocol achieved homogeneous Mg particle dispersion with minimal segregation during extrusion, yielding filaments with stable thermal characteristics and systematically enhanced rheological properties (viscosity, yield stress, shearthinning) that remained compatible with extrusion-based printing despite significantly increased filler loading. Tensile mechanical properties scaled linearly with Mg content, achieving up to 3-fold improvements in Young's modulus over neat PCL, while print quality scores remained consistently high (>0.85) across all compositions, demonstrating that Mg addition did not compromise dimensional accuracy or structural completeness. In vitro degradation studies revealed pH-stable Mg2+ion release, supporting controlled, composition-dependent elution suitable for therapeutic delivery, while bioactivity assays confirmed that Mg-laden scaffolds promoted osteogenic differentiation, mineralization, and cell viability over 21 days. These findings establish Mg-PCL composites as a viable platform for bone tissue engineering applications requiring load-bearing capacity and biomineralization, with broader implications for engineering degradable, multifunctional scaffolds via high-filler-content composite filament extrusion.REFERENCES

[0207] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications,3344426.601_P18564-02patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art,

[0208] Hung, B. P.: Naved, B. A,; Nyberg, E. L.; Dias. M.; Holmes, C. A.; Elisseeff, J. H.; Dorafshar, A. H.; Grayson, W. L. Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration. ACS Biomater. Sci. Eng. 2016, 2 (10), 1806-1816.

[0209] T. Ma et al., “Advances in 3D printing for polymer composites: A review,” Jun. 01. 2024, John Wiley and Sons Inc. doi: 10.1002 / inf2.12568.

[0210] M. Mohammadi Zerankeshi, R. Bakhshi, and R. Alizadeh, “Polymer / metal composite 3D porous bone tissue engineering scaffolds fabricated by additive manufacturing techniques: A review,” Mar. 01, 2022, Elsevier B.V. doi: 10.1016 / j.bprint.2022.e00191.

[0211] M. P. Staiger, A. M. Pietak, J. Huadmai, and G. Dias, “Magnesium and its alloys as orthopedic biomaterials: A review,” Mar. 2006. doi: 10.1016 / j. biomaterials.2005.10.003.

[0212] T. Wang et al., “3D-printed Mg-incorporated PCL-based scaffolds improves rotator cuff tendon-bone healing through regulating macrophage polarization,” Front. Bioeng. Biotechnol., vol. 12, 2024, doi: 10.3389 / fbioe.2024.1407512.

[0213] B. Lei, X. Gao, R. Zhang, X. Yi, and Q. Zhou, “In situ magnesium phosphate / polycaprolactone 3D-printed scaffold induce bone regeneration in rabbit maxillofacial bone defect model,” Mater. Des., vol. 215, Mar. 2022, doi: 10.1016 / j.matdes.2022.110477.

[0214] S. N. Kalva, F. Ali, C. A. Velasquez, and M. Kog, “3D-Printable PLA / Mg Composite Filaments for Potential Bone Tissue Engineering Applications,” Polymers (Basel). , vol. 15, no. 11, Jun. 2023, doi: 10.3390 / polyml5112572.

[0215] C. Pascual-Gonzalez et al., “Processing and properties of PLA / Mg filaments for 3D printing of scaffolds for biomedical applications,” Rapid Prototyp. J., vol. 28, no. 5, pp. 884-894, May 2022, doi: 10.1108 / RPJ-06-2021-0152.

[0216] G. Mora-Castano. M. Millan-Jimenez, V. Linares, and I. Caraballo. “Assessment of the Extrusion Process and Printability of Suspension-Type Drug-Loaded Affinisol™ Filaments for 3D Printing,” Pharmaceutics, vol. 14, no. 4, Apr. 2022, doi: 10.3390 / pharmaceuticsl4040871.

[0217] P. M. Angelopoulos, M. Samouhos, and M. Taxiarchou, “Functional fillers in composite filaments for fused filament fabrication: A review,” in Materials Today: Proceedings, Elsevier Ltd. 2019, pp. 4031-4043. doi: 10.1016 / j.matpr.2020.07.069.3444426.601_P18564-02

[0218] S. Rajendran et al., “Metal and Polymer Based Composites Manufactured Using Additive Manufacturing — A Brief Review,” Jun. 01, 2023, MDPI. doi: 10.3390 / polyml5112564.

[0219] M. K. Egbo, “A fundamental review on composite materials and some of their applications in biomedical engineering,” Dec. 01, 2021, King Saud University. doi: 10.1016 / j.jksues.2020.07.007.

[0220] J. G. Behnsen, J. W. Roberts. O. J. Rogan, J. M. Mcardle, and K. Black, “Micro-X-ray-CT for analysis of particle size segregation during powder spreading in Binder Jet Printing,” 2025, doi: 10.17638 / datacat.

[0221] T. Maconachie et al., “SLM lattice structures: Properties, performance, applications and challenges,” Dec. 05, 2019, Elsevier Ltd. doi: 10.1016 / j.matdes.2019.108137.

[0222] L. J. Gibson and M. F. Ashby, Cellular Solids. Cambridge University Press. 1997. doi: 10.1017 / CBO9781139878326.

[0223] X. Li et al., “In vitro degradation kinetics of pure PLA and Mg / PLA composite: Effects of immersion temperature and compression stress,” Acta Biomater., vol. 48, pp. 468-478, Jan. 2017, doi: 10.1016 / j.actbio.2016.11.001.

[0224] S. Raguraman, B. Ulugun, T. Ayodeji, R. Chowdhury, A. Griebel, and T. Weihs, “Evaluating In-Vitro Corrosion Testing of ECAP-Processed Lean Magnesium Alloys: The Critical Role of Degradation Media Composition, Buffering, and Volume,” JOM, vol. 77, no. 6, pp. 4374-4383, Jun. 2025, doi: 10.1007 / sl 1837-025-07176-7.

[0225] M. Enriconi et al., “A Comprehensive Review of Fused Filament Fabrication: Numerical Modeling Approaches and Emerging Trends,” Jun. 01, 2025, Multidisciplinary Digital Publishing Institute (MDPI). doi: 10.3390 / appl5126696.

[0226] D. Strugova et al., “Polymer blends as a tool to improve mechanical properties and printability of metal-filled polymer filaments for material extrusion additive manufacturing in the context of sustainable manufacturing,” J. Manuf. Process., vol. 156. pp. 611-626, Dec. 2025, doi: 10.1016 / j.jmapro.2025.11.002.

[0227] J. Hu, J. Shao, G. Huang, J. Zhang, and S. Pan, “In Vitro and In Vivo Applications of Magnesium-Enriched Biomaterials for Vascularized Osteogenesis in Bone Tissue Engineering: A Review of Literature,” Jun. 01, 2023, Multidisciplinary Digital Publishing Institute (MDPI). doi: 10.3390 / jfbl4060326.3544426.601_P18564-02

[0228] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.3644426.601_P18564-02

Claims

WHAT IS CLAIMED IS:

1. A fused-deposition modeling (FDM) printing filament comprising a composite having between about 10% to about 60% by volume of biodegradable metal powder having a mean particle size between about 10 pm and about 100 pm; and between about 40% to about 90% by volume of a biocompatible, biodegradable thermoplastic polymer powder having a melting temperature below 250 °C; wherein:a ratio of thermoplastic polymer powder particle size to metal powder particle size does not exceed about 10:1;the composite has a viscosity of about 1.6*105to about 5.5 x 105Pa s at 120 °C and a tensile modulus of about 100 MPa to 1000 MPa; andwherein particles comprising the metal powder are encapsulated beneath a continuous surface layer of the thermoplastic polymer.

2. The filament of claim 1, wherein the thermoplastic polymer comprises polycaprolactone (PCL) having a melting temperature between about 59 °C and about 60 °C and a crystallization enthalpy between about 14 J / g and about 17 J / g.

3. The filament of claim 1, wherein the biodegradable metal powder comprises a metal selected from magnesium, a magnesium alloy, iron, an iron alloy, zinc, a zinc alloy, and combinations thereof.

4. The composition of claim 1, wherein the filament comprises a cross-section, a periphery, and a center and wherein:particles comprising the metal powder are distributed throughout the filament crosssection with a particle number density between about 82 particles / mm2and about 385 particles / mm2; anda ratio of the particle number density at the filament periphery to a ratio of the particle number density at the filament center does not exceed 1.8:1.3744426.601_P18564-025. The filament of claim 1 , wherein the composite has one or more properties selected from:a yield stress that increases by a factor of about 2.5 to about 3.5 relative to neat thermoplastic polymer as metal volume fraction increases from about 0% to about 60%;a flow behavior index that decreases from about 0.92 to about 0.82 as metal volume fraction increases from about 0% to about 60%; andan apparent viscosity at about a 0.1 s ' shear rate that does not exceed about 5.5xl05Pa s at about 120 °C.

6. The filament of claim 1, wherein the filament has an effective diameter between about 100 pm and about 4 mm, and wherein the effective diameter varies by less than about 10% over any 100-mm length of the filament after an initial stabilization distance of about 50 mm from extrusion initiation.

7. The filament of claim 1, wherein incorporation of metal powder into the thermoplastic polymer does not alter the polymer melting temperature by more than about 2 °C relative to neat thermoplastic polymer, and wherein a melting enthalpy remains within about 17 J / g to about 20 J / g across all metal volume fractions.

8. The filament of claim 1, wherein:a maximum achievable metal volume fraction scales inversely with metal powder particle size; andabout a 60 % by volume of metal powder loading with a mean particle size of about 44 pm.

9. A three-dimensional porous scaffold comprising:a composite of claim 1 having a pore size between 100 pm and 1.2 mm configured to permit vascular ingrowth and interconnected porosity with isotropic or anisotropic pore architecture;a print fidelity score greater than 0.85, wherein the print fidelity is defined as:( Avg. Deviation . ,+Avg. Deviation \1 - — -!— j x Strut Completion0 / ); and wherein:3844426.601_P18564-02the scaffold exhibits controlled metal ion elution in aqueous media without pH deviation exceeding about 0.5 units; and the scaffold maintains structural integrity for at least 21 days in physiological media at a temperature between about 37 °C to about 40 °C and a pH between about 7 and about 7.8.

10. The scaffold of claim 9. wherein the scaffold comprises one or more properties selected from:a tensile Young’s modulus that increases by a factor from about 1 up to about 3 relative to a scaffold fabricated from neat polymer;an increase in the tensile Young’s modulus that scales linearly with metal volume fraction according to rule-of-mixtures predictions; anda compressive modulus having a range from about 2 MPa to about 100 MPa.

11. The scaffold of claim 9. wherein:one or more strut dimensions deviate from a design target by less than about 15%; one or more pore dimensions deviate from a design target by less than about 10% and a strut completion percentage exceeds about 95%.

12. The scaffold of claim 9, wherein:one or more strut surfaces exhibit gradual roughening with increasing metal content without evidence of interfacial failure, particle pull-out, or cracking; andparticles comprising the metal powder are encapsulated beneath a continuous polymer layer as verified by pCT imaging and scanning electron microscopy.

13. A method for manufacturing a bioactive composite filament, the method comprising:cryogenic milling a thermoplastic polymer to produce a polymer powder having a particle size distribution with a mean diameter of about 120 pm;providing biodegradable metal powder having a mean particle size between 10 pm and 100 pm;3944426.601_P18564-02blending the polymer powder and metal powder in an inert atmosphere to produce a blended powder having a target metal volume fraction between about 10% and about 60%;extruding the blended powder through a heated die at a temperature not exceeding about 30 °C above a melting temperature of the thermoplastic polymer to produce an extruded filament;cooling the extruded filament by convection at a temperature between about 15 °C to about 35 °C) and a relative humidity between about 0% to about 100%; andwinding the cooled filament onto a spool for storage in a moisture-controlled environment.

14. The method of claim 13, wherein the cryogenic milling comprises: precooling thermoplastic polymer pellets in liquid nitrogen for about 3 minutes; grinding the precooled thermoplastic polymer pellets for about one minute at an impact frequency of about 10 cycles per second;repeating for 45 consecutive cycles, with cooling for about 3 minutes between cycles, to achieve a target particle size; andverifying particle size distribution by dynamic light scattering.

15. The method of claim 13, wherein the blending comprises:transferring a pre-weighed metal powder to a container;layering thermoplastic polymer powder on top of the metal powder to minimize premature mixing;sealing the container in an inert atmosphere to prevent metal oxidation; and agitating on a vortex mixer for at least 5 minutes to achieve homogeneous blending.

16. The method of claim 13, wherein the extruding comprises:feeding the blended powder into a heated screw extruder;maintaining barrel temperatures of the heated die between about 65 °C to about 80 °C across heating zones;controlling screw speed between about 1 rpm and about 5 ipm to achieve a target filament diameter;4044426.601_P18564-02monitoring filament diameter in real-time by laser micrometry; andcollecting the filament after diameter stabilization over a distance of at least 50 mm.

17. The method of claim 13, further comprising storing the filament in an airtight container with desiccant to maintain moisture content below about 0.1 wt% prior to printing.

18. A method for three-dimensional printing of a bioactive scaffold, the method comprising:providing a filament of claim 1;heating the filament to a processing temperature between a melting temperature of the thermoplastic polymer and about 30 °C above the melting temperature;extruding the heated filament through a nozzle having an orifice diameter between about 400 pm and about 1000 pm;depositing the extruded filament layer-by-layer onto a build platform at a predetermined print speed to form a three-dimensional structure; andcontrolling layer thickness, infill density, infill pattern, and raster orientation to achieve target mechanical properties and porosity.

19. The method of claim 18, wherein:the nozzle is maintained at a temperature of 105°C ± 5°C;the build platform is maintained at a temperature between about 50 °C to about 70°C; the layer thickness is between about 100 pm and about 400 pm;the print speed is controlled between about 10 mm / s and about 40 mm / s to optimize bonding between layers; andthe infill pattern is selected from a rectilinear, a honeycomb, a gyroid, and a custom pattern to achieve target porosity and mechanical anisotropy.

20. The method of claim 18, further comprising:varying metal volume fraction across one or more regions of the three-dimensional structure;4144426.601_P18564-02creating functionally graded scaffolds with spatially varying mechanical properties, degradation rates, or ion release kinetics; and / orco-printing multiple composite formulations in a single build to achieve heterogeneous material distribution.

21. The method of claim 18, further comprising immersion of the scaffold in cell culture medium for 21 days resulting in:a pH stability within ±0.3 units of an initial pH;instantaneous metal ion release scaling with metal volume fraction up to about 30 vol%, followed by a decreased release at higher loadings; anda cumulative metal ion concentration demonstrating composition-dependent elution kinetics suitable for therapeutic delivery.

22. The method of claim 18, further comprising culturing the scaffold with osteoblastlineage cells for 21 days resulting in:progressive cell proliferation peaking at metal volume fractions between about 30 vol% to about 45 vol%;an increased normalized calcium deposition (Ca / DNA ratio) with increasing metal fraction, indicating enhanced osteoconductivity and biomineralization; andcell viability exceeding about 95% at all metal loadings as assessed by live / dead fluorescence staining.

23. The method of claim 18, wherein quality of the scaffold is verified by: measuring dimensional deviation for each strut and pore relative to CAD design targets; calculating average deviations separately for struts and pores;assessing strut completion percentage as the fraction of fully formed struts; computing an overall print score according to:( Avg. Deviation . +Avg. Deviation1 - - - — j x Strut Completion0 / ); andaccepting only scaffolds with print scores exceeding 0.85.4244426.601_P18564-0224. A non-porous biomedical implant structure comprising a filament of claim 1 or prepared by the method of claim 18, the implant structure comprising one or more of:a bone fixation screw having a customizable thread geometry, pitch, head design, and length;a fixation plate having a customizable thickness, hole pattern, and curvature;an intramedullary rod having a customizable diameter and length; and / oran orthopedic fixation device requiring mechanical strength, ductility, and biodegradability.4344426.601_P18564-02

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